Car Crashes and Central Disorders of Hypersomnolence: A French Study

Background

Drowsiness compromises driving ability by reducing alertness and attentiveness, and delayed reaction times. Sleep-related car crashes account for a considerable proportion of accident at the wheel. Narcolepsy type 1 (NT1), narcolepsy type 2 (NT2) and idiopathic hypersomnia (IH) are rare central disorders of hypersomnolence, the most severe causes of sleepiness thus being potential dangerous conditions for both personal and public safety with increasing scientific, social, and political attention. Our main objective was to assess the frequency of recent car crashes in a large cohort of patients affected with well-defined central disorders of hypersomnolence versus subjects from the general population.

Methods

We performed a cross-sectional study in French reference centres for rare hypersomnia diseases and included 527 patients and 781 healthy subjects. All participants included needed to have a driving license, information available on potential accident events during the last 5 years, and on potential confounders; thus analyses were performed on 282 cases (71 IH, 82 NT2, 129 NT1) and 470 healthy subjects.

Results

Patients reported more frequently than healthy subjects the occurrence of recent car crashes (in the previous five years), a risk that was confirmed in both treated and untreated subjects at study inclusion (Untreated, OR = 2.21 95%CI = [1.30–3.76], Treated OR = 2.04 95%CI = [1.26–3.30]), as well as in all disease categories, and was modulated by subjective sleepiness level (Epworth scale and naps). Conversely, the risk of car accidents of patients treated for at least 5 years was not different to healthy subjects (OR = 1.23 95%CI = [0.56–2.69]). Main risk factors were analogous in patients and healthy subjects.

Conclusion

Patients affected with central disorders of hypersomnolence had increased risk of recent car crashes compared to subjects from the general population, a finding potentially reversed by long-term treatment.     

Utilization of Benchtop Next Generation Sequencing Platforms Ion Torrent PGM and MiSeq in Noninvasive Prenatal Testing for Chromosome 21 Trisomy and Testing of Impact of In Silico and Physical Size Selection on Its Analytical Performance

Objectives

The aims of this study were to test the utility of benchtop NGS platforms for NIPT for trisomy 21 using previously published z score calculation methods and to optimize the sample preparation and data analysis with use of in silico and physical size selection methods.

Methods

Samples from 130 pregnant women were analyzed by whole genome sequencing on benchtop NGS systems Ion Torrent PGM and MiSeq. The targeted yield of 3 million raw reads on each platform was used for z score calculation. The impact of in silico and physical size selection on analytical performance of the test was studied.

Results

Using a z score value of 3 as the cut-off, 98.11% - 100% (104-106/106) specificity and 100% (24/24) sensitivity and 99.06% - 100% (105-106/106) specificity and 100% (24/24) sensitivity were observed for Ion Torrent PGM and MiSeq, respectively. After in silico based size selection both platforms reached 100% specificity and sensitivity. Following the physical size selection z scores of tested trisomic samples increased significantly—p = 0.0141 and p = 0.025 for Ion Torrent PGM and MiSeq, respectively.

Conclusions

Noninvasive prenatal testing for chromosome 21 trisomy with the utilization of benchtop NGS systems led to results equivalent to previously published studies performed on high-to-ultrahigh throughput NGS systems. The in silico size selection led to higher specificity of the test. Physical size selection performed on isolated DNA led to significant increase in z scores. The observed results could represent a basis for increasing of cost effectiveness of the test and thus help with its penetration worldwide.     

The comparative antimalarial properties of weak base and neutral synthetic ozonides

Thirty-three N-acyl 1,2,4-dispiro trioxolanes (secondary ozonides) were synthesized. For these ozonides, weak base functional groups were not required for high antimalarial potency against Plasmodium falciparum in vitro, but were necessary for high antimalarial efficacy in Plasmodium berghei-infected mice. A wide range of Log P/D_{pH 7.4} values were tolerated, although more lipophilic ozonides tended to be less metabolically stable.     

Genetic engineering of the major timothy grass pollen allergen, Phl p 6, to reduce allergenic activity and preserve immunogenicity

On the basis of IgE epitope mapping data, we have produced three allergen fragments comprising aa 1-33, 1-57, and 31-110 of the major timothy grass pollen allergen Phl p 6 aa 1-110 by expression in Escherichia coli and chemical synthesis. Circular dichroism analysis showed that the purified fragments lack the typical alpha-helical fold of the complete allergen. Superposition of the sequences of the fragments onto the three-dimensional allergen structure indicated that the removal of only one of the four helices had led to the destabilization of the alpha helical structure of Phl p 6. The lack of structural fold was accompanied by a strong reduction of IgE reactivity and allergenic activity of the three fragments as determined by basophil histamine release in allergic patients. Each of the three Phl p 6 fragments adsorbed to CFA induced Phl p 6-specific IgG Abs in rabbits. However, immunization of mice with fragments adsorbed to an adjuvant allowed for human use (AluGel-S) showed that only the Phl p 6 aa 31-110 induced Phl p 6-specific IgG Abs. Anti-Phl p 6 IgG Abs induced by vaccination with Phl p 6 aa 31-110 inhibited patients' IgE reactivity to the wild-type allergen as well as Phl p 6-induced basophil degranulation. Our results are of importance for the design of hypoallergenic allergy vaccines. They show that it has to be demonstrated that the hypoallergenic derivative induces a robust IgG response in a formulation that can be used in allergic patients.     

Immunotherapy of renal cell carcinoma

Carcinomas of the kidney generally have a poor prognosis and respond minimally to classical radiotherapy or chemotherapy. Immunotherapy constitutes an interesting alternative to these established forms of treatment, and indeed, cytokine-based therapies have been used for many years, leading to favorable clinical responses in a small subset of patients. During the past few years, immunotherapeutical trials targeting renal cell tumor-associated antigens have also been reported, with diverse passive or active approaches using antibodies or aimed at activating tumor-directed T lymphocytes. The following review presents the results and the progress made in the field, including classical cytokine treatments, non-myeloablative stem cell transplantation and antigen specific-based trials, with special focus on T-cell studies. In consideration of the few specific molecular targets described so far for this tumor entity, current strategies which can lead to the identification of new relevant antigens will be discussed. Hopefully these will very soon contribute to an improvement in renal cell carcinoma specific immunotherapy and its evaluation.     

Weak base dispiro-1,2,4-trioxolanes: potent antimalarial ozonides

Thirty weak base 1,2,4-dispiro trioxolanes (secondary ozonides) were synthesized. Amino amide trioxolanes had the best combination of antimalarial and biopharmaceutical properties. Guanidine, aminoxy, and amino acid trioxolanes had poor antimalarial activity. Lipophilic trioxolanes were less stable metabolically than their more polar counterparts.     

Activation of cardiac AMP-activated protein kinase by LKB1 expression or chemical hypoxia is blunted by increased Akt activity

AMP-activated protein kinase (AMPK) plays a major role in the regulation of cardiac energy substrate utilization and can be negatively regulated by Akt activation in the heart. It has recently been shown that Akt directly phosphorylates AMPKalpha(1)/alpha(2) on Ser(485/491) in vitro and prevents the AMPK kinase (AMPKK) LKB1 from phosphorylating AMPKalpha at its primary activation site, Thr(172) (S Horman, D Vertommen, R Heath, D Neumann, V Mouton, A Woods, U Schlattner, T Wallimann, D Carling, L Hue, and MH Rider. J Biol Chem 281: 5335-5340, 2006). To determine whether this is also the case in the cardiac myocyte, neonatal rat cardiac myocytes (NRCM) were infected with a recombinant adenovirus expressing a constitutively active mutant of Akt1 (myrAkt1) and then with or without adenoviruses expressing the active LKB1 complex. Expression of myrAkt1 blunted LKB1-induced phosphorylation of AMPKalpha at Thr(172), which resulted in a dramatic decrease in phosphorylation of AMPK's target, acetyl CoA-carboxylase. This decrease in AMPK activity was associated with prior Akt1-dependent phosphorylation of AMPKalpha(1)/alpha(2) at Ser(485/491). To investigate whether Akt1 activation was also able to prevent other AMPKKs from phosphorylating AMPKalpha, we subjected NRCM to chemical hypoxia and noted a marked increase in phosphorylation of AMPKalpha at Thr(172), despite no change in LKB1 activity. NRCM expressing myrAkt1 demonstrated increased phosphorylation of AMPKalpha(1)/alpha(2) at Ser(485/491) and a complete inhibition of chemical hypoxia-induced phosphorylation of AMPKalpha at Thr(172). Taken together, our data show that activation of Akt1 is able to prevent activation of cardiac AMPK by LKB1 and at least one other AMPKK, likely by prior phosphorylation of AMPKalpha(1)/alpha(2) at Ser(485/491).     

The sulfur oxygenase reductase from the mesophilic bacterium Halothiobacillus neapolitanus is a highly active thermozyme

A biochemical, biophysical, and phylogenetic study of the sulfur oxygenase reductase (SOR) from the mesophilic gammaproteobacterium Halothiobacillus neapolitanus (HnSOR) was performed in order to determine the structural and biochemical properties of the enzyme. SOR proteins from 14 predominantly chemolithoautotrophic bacterial and archaeal species are currently available in public databases. Sequence alignment and phylogenetic analysis showed that they form a coherent protein family. The HnSOR purified from Escherichia coli after heterologous gene expression had a temperature range of activity of 10 to 99°C with an optimum at 80°C (42 U/mg protein). Sulfite, thiosulfate, and hydrogen sulfide were formed at various stoichiometries in a range between pH 5.4 and 11 (optimum pH 8.4). Circular dichroism (CD) spectroscopy and dynamic light scattering showed that the HnSOR adopts secondary and quaternary structures similar to those of the 24-subunit enzyme from the hyperthermophile Acidianus ambivalens (AaSOR). The melting point of the HnSOR was ≈20°C lower than that of the AaSOR, when analyzed with CD-monitored thermal unfolding. Homology modeling showed that the secondary structure elements of single subunits are conserved. Subtle changes in the pores of the outer shell and increased flexibility might contribute to activity at low temperature. We concluded that the thermostability was the result of a rigid protein core together with the stabilizing effect of the 24-subunit hollow sphere.     

Availability, not respiratory capacity governs oxygen consumption of solid tumors

Contrary to conventional belief, the mitochondria of most cancer cells usually function normally, i.e., their respiratory capacity is not fundamentally impaired as compared to normal cells. Strong evidence against the misconception of mitochondrial dysfunction is provided by in vivo data clearly showing that O(2) availability is the major determinant of the O(2) consumption rate of cancer cells, independent of the means for increasing availability (e.g., by increasing blood flow or by elevating arterial O(2) content, the latter being accomplished either by an increase in the hemoglobin level and/or arterial hyperoxia). Additional support against the Warburg effect in its original concept comes from normal temperature coefficients (Q(10)) for O(2) consumption rates of malignant cells. Thus, the Warburg hypothesis postulating that mitochondrial dysfunction in cancer cells forces them to generate energy with a poor ATP yield through glycolysis appears to be elusive. Instead, due to a "reprogrammed" cancer cell metabolism, glycolysis is used to produce intermediates as building blocks for various biosynthetic pathways of cancer cells.     

High-density genetic linkage maps with over 2,400 sequence-anchored DArT markers for genetic dissection in an F2 pseudo-backcross of Eucalyptus grandis × E. urophylla

Traits that differentiate cross-fertile plant species can be dissected by genetic linkage analysis in interspecific hybrids. Such studies have been greatly facilitated in Eucalyptus tree species by the recent development of Diversity Arrays Technology (DArT) markers. DArT is an affordable, high-throughput marker technology for the construction of high-density genetic linkage maps. Eucalyptus grandis and Eucalyptus urophylla are commonly used to produce fast-growing, disease tolerant hybrids for clonal eucalypt plantations in tropical and subtropical regions. We analysed 7,680 DArT markers in an F2 pseudo-backcross mapping pedigree based on an F1 hybrid clone of E. grandis and E. urophylla. A total of 2,440 markers (31.7%) were polymorphic and could be placed in linkage maps of the F1 hybrid and two pure-species backcross parents. An integrated genetic linkage map was constructed for the pedigree resulting in 11 linkage groups (n = 11) with 2,290 high-confidence (LOD ≥ 3.0) markers and a total map length of 1,107.6 cM. DNA sequence analysis of the mapped DArT marker fragments revealed that 43% were located in protein coding regions and 90% could be placed in the recently completed draft genome assembly of E. grandis. Together with the anchored genomic sequence information, this linkage map will allow detailed genetic dissection of quantitative traits and hybrid fitness characters segregating in the F2 progeny and will facilitate the development of markers for molecular breeding in Eucalyptus.