Plasma RNA viral load in human immunodeficiency virus type 2 subtype A and subtype B infections

Human immunodeficiency virus type 2 (HIV-2) is much less pathogenic than HIV-1, and HIV-2 infection is associated with plasma viral loads significantly lower than those found in HIV-1 infection. We have developed a real-time quantitative PCR method for measuring the HIV-2 RNA load that covers the range of genetic diversity of HIV-2 isolates and that detects extremely low viral loads. Samples from 49 patients were studied. Proviral DNA was first detected and quantified. The strains that were detected were then genotyped: 21 patients were infected with HIV-2 subtype A and 15 patients were infected with HIV-2 subtype B; 1 patient was infected with a highly divergent strain. Env PCR failed for the remaining 12 patients, so subtypes could not be determined. For viral RNA quantification, a stock of HIV-2 strain NIHZ, which was counted by electron microscopy, was used as the standard. Several primer sets targeting the highly conserved gag region were evaluated. Various primer combinations failed to amplify subtype B strains. With the final primer pair selected, which detected both subtype A and subtype B strains, the sensitivity of the assay was 100% at a viral load of 250 copies/ml and 66% at a viral load of 125 copies/ml. We found a correlation between the CD4(+)-cell count, the clinical stage, and the plasma HIV-2 RNA level. The median plasma HIV-2 RNA value for the 33 asymptomatic patients was 2.14 log(10), whereas it was 3.1 log(10) for the 16 patients with AIDS (P < 0.01). Proviral DNA was detectable in 18 symptom-free patients with high CD4(+)-cell counts, in whom viral RNA was undetectable.     

Clinical and molecular overlap in overgrowth syndromes

Here, we report the clinical and molecular analysis of 75 patients with overgrowth and mental retardation, including 45 previously reported cases [Rio et al., 2003; Baujat et al., 2004]. Two groups are distinguished: group I corresponding to patients with recognizable overgrowth syndromes (Sotos syndrome (SS), Weaver syndrome (WS), Beckwith-Wiedemann syndrome, Simpson-Golabi-Behmel syndrome (SGBS), and del(22)(qter) syndrome) (60 cases) and group II corresponding to unclassified cases (15 patients). We investigated NSD1 and GPC3 deletions or mutations, 11p15 abnormalities, and 22qter deletions. Surprisingly, in Group I, two SS patients had 11p15 abnormalities and two patients with Beckwith-Wiedemann syndrome had NSD1 aberrations. In group II, two cases of del(22)(qter) were identified but neither NSD1, 11p15, nor GPC3 abnormalities were detected. These results emphasize the clinical and molecular overlap in overgrowth conditions.     

Morphofunctional ontogeny of the urinary system of the European sea bass Dicentrarchus labrax

European sea bass (Dicentrarchus labrax) are euryhaline fish that tolerate wide salinity fluctuations owing to several morphofunctional adaptations. Among the osmoregulatory sites (tegument, branchial chambers, digestive tract, urinary system), little is known about the kidney and the urinary bladder. The present study describes the ontogeny of the urinary system (kidney and urinary bladder) and focuses on the progressive expression of the Na⁺/K⁺-ATPase in the cells of these ion-transporting epithelia. A structural approach has shown that two pronephric urinary tubules are already present at hatching while the urinary bladder starts to differentiate. The glomus, an ultrafiltration site, occurs at day 5 (D5). The opisthonephros differentiates at D19/25 from the pronephric collecting tubules, then it rapidly grows longer and becomes folded. Na⁺/K⁺-ATPase immunolocalization and transmission electron microscopy show that ionocyte-like cells line the urinary tubules and the dorsal wall of the urinary bladder from D2/D5 on. Tubule ionocytes present a basolateral-localized fluorescence. Ionocytes of the collecting ducts and of the dorsal wall of the bladder present a fluorescence distributed in the whole cytoplasm. Fluorescence becomes stronger in later stages, suggesting a progressively increasing functionality of the urinary system in active ion transports. This observation is closely correlated with the ontogeny of osmoregulatory abilities. In juvenile and preadult fish kept in seawater, osmolality measurements demonstrate that urine is isotonic to blood. At low salinity, urine is hypotonic to blood in both stages. The capacity to produce hypotonic urine increases during ontogeny, a fact that suggests an increasing involvement of the urinary system in osmoregulation. The occurrence and the progressive functionality of the urinary system during the ontogeny, along with those of other osmoregulatory sites, are major adaptations allowing the sea bass to live in habitats of variable salinity such as lagoons and estuaries.     

Chronic Psychophysiological Insomnia: Hyperarousal and/or Inhibition Deficits? An ERPs Investigation

STUDY OBJECTIVES: Chronic primary insomnia has been hypothesized to result from conditioned arousal or the inability to initiate normal sleep processes. The event-related potentials (ERPs) N1, P2, and N350 are useful indexes of arousal. The objective is to compare these ERPs in primary chronic psychophysiological insomniacs (INS) and good sleepers (GS) during multiple recordings. PARTICIPANTS: Participants were 15 INS (mean age = 46 years, SD = 7.5) and 16 GS (mean age = 37 years, SD = 10.1). METHODS AND PROCEDURE: Following a multistep clinical evaluation, INS and GS participants underwent 4 consecutive nights of PSG recordings (N1 to N4). ERPs were recorded on the 3rd and 4th nights in the sleep laboratory (N3 and N4). ERPs recordings were made during wake on both nights (in the evening and upon awakening), with the addition of sleep-onset recordings on N4. Auditory stimuli consisted of "standard" and "deviant" tones. STATISTICAL ANALYSIS: Repeated measures ANOVAs were computed for each ERP for each recording for each type of stimulus. RESULTS: The amplitude of P2 and N350 was greater for the deviant than for the standard stimulus in both groups. The amplitude of N1 was larger in INS than GS in the morning and the evening. While the amplitude of N350 was larger in GS than in INS at sleep onset, the amplitude of P2 was greater in INS than in GS at that time. CONCLUSION: Signs of greater cortical arousal in psychophysiological insomnia individuals are observed, especially upon awakening in the morning. However, at sleep onset, difficulties from disengaging from wake processes and some inability at initiating normal sleep processes appear also present in individuals with insomnia compared to good sleepers.     

Dendronized iron oxide nanoparticles for multimodal imaging

The synthesis of small-size dendrons and their grafting at the surface of iron oxide nanoparticles were achieved with the double objective to obtain a good colloidal stability with a mean hydrodynamic diameter smaller than 100 nm and to ensure the possibility of tuning the organic coating characteristics including morphology, functionalities, physico-chemical properties, grafting of fluorescent or targeting molecules. Magnetic resonance and fluorescence imaging are then demonstrated to be simultaneously possible using such versatile superparamagnetic iron oxide nanocrystals covered by a dendritic shell displaying either carboxylate or ammonium groups at their periphery which could be further labelled with a fluorescent dye. The grafting conditions of these functionalized dendrons at the surface of SPIO NPs synthesized by co-precipitation have been optimized as a function of the nature of the peripheral functional group. The colloidal stability has been investigated in water and osmolar media, and in vitro and in vivo MRI and optical imaging measurements have been performed showing encouraging biodistribution.     

From Lowe syndrome to Dent disease: correlations between mutations of the OCRL1 gene and clinical and biochemical phenotypes

Mutations of OCRL1 are associated with both the Lowe oculocerebrorenal syndrome, a multisystemic and Dent-2 disease, a renal tubulopathy. We have identified a mutation in 130 Lowe syndrome families and 6 affected by Dent-2 disease with 51 of these mutations being novel. No founding effect was evidenced for recurrent mutations. Two mutations initially reported as causing Dent-2 disease were identified in patients, including two brothers, presenting with Lowe syndrome thus extending the clinical variability of OCRL1 mutations. mRNA levels, protein content, and PiP(2) -ase activities were analyzed in patient's fibroblasts. Although mRNA levels were normal in cells harboring a missense mutation, the OCRL1 content was markedly lowered, suggesting that enzymatic deficiency resulted mainly from protein degradation rather than from a catalytic inactivation. Analysis of a splicing mutation that led to the elimination of the initiation codon evidenced the presence of shortened forms of OCRL1 that might result from the use of alternative initiation codons. The specific mapping of the frameshift and nonsense mutations, exclusively identified in exons 1-7 and exons 8-23, respectively, for Dent disease and Lowe syndrome together with the possible use of alternative initiation codons might be related to their clinical expression, that is, Lowe syndrome or Dent-2 disease.     

Effects of serum-free culture at the air-liquid interface in a human tissue-engineered skin substitute

Previous studies have reported that well-defined culture conditions can improve keratinocytes terminal differentiation and reproducibility. The aim of our study was to compare skin substitutes cultured in a complete medium with those cultured in a serum-free medium at the air-liquid interface to optimize the self-assembly method. Skin substitutes, cultured in a serum-free medium over 7, 14, and 21 days, were compared with others cultured in a complete medium (5% serum) over the complete culture period. Masson's Trichrome staining showed that the substitutes cultured in a serum-free medium generated a well-developed and differentiated epidermis. Immunolabeling analyses between the substitutes cultured without serum and those cultured in complete serum showed similar expression of epidermal differentiation markers, dermo-epidermal junction, and dermal extracellular matrix components. On the basis of our Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) results, the skin substitutes cultured in serum-free condition over 21 days of culture at the air-liquid interface showed lower frequencies of the CH(2) symmetric mode of vibrations, which means a better lipid organization of the stratum corneum. No significant difference in hydrocortisone penetration was observed between serum-free medium substitutes and the controls. Results demonstrate that the absence of serum does not compromise the characteristics of the skin substitutes observed in this study.     

Genetic sequence variations and <Emphasis Type="Italic">ADPRT</Emphasis> haplotype analysis in French Canadian families with high risk of breast cancer

The poly(ADP-ribose) polymerase (PARP/ADPRT) protein family catalyzes the synthesis of cellular poly(ADP-ribose) following DNA damage and is involved in genomic integrity by regulating cellular responses to DNA damage and apoptosis. Moreover, ADPRT inhibition contributes to a protective effect against cancer development. These findings render ADPRT an attractive candidate susceptibility gene for breast cancer, and thus the goal of this study was to evaluate the possible involvement of ADPRT sequence variations in breast cancer susceptibility. The complete sequence of the 23 exons and flanking intronic sequences of the ADPRT gene was analyzed in 54 affected individuals from distinct high-risk non-BRCA1/2 French Canadian families. No deleterious truncating mutation was identified in the coding region. However, 34 sequence variations were identified, among which seven are coding variants and seven are novel changes. All coding variants and intronic changes located in the vicinity of the coding variants identified in the case series were also analyzed in a cohort of 73 unrelated healthy French Canadian individuals. Interestingly, one missense variant (Pro377Ser) was observed in three different breast cancer cases but was not present among unaffected individuals. We have conducted here an exhaustive detailed mutation and haplotype tagging analysis of the ADPRT gene with regard to breast cancer, providing useful data for other large-scale association studies. Additional studies in other cohorts and other populations are however needed to further evaluate the implication of the Pro377Ser missense variant with regard to breast cancer susceptibility. Other members of INHERIT BRCAs involved in clinical aspects of the study are listed in the Appendix. J. Simard holds a Canada Research Chair in Oncogenetics.