Ribo-polymerase chain reaction--a facile method for the preparation of chimeric RNA/DNA applied to DNA sequencing

We describe ribo-polymerase chain reaction (PCR), a method for the preparation of chimeric RNA/DNA. The RNA/DNA chimeric nucleic acids are generated directly from genomic DNA starting templates with two locus-specific primers, three nucleotides in their deoxy form and the fourth in its ribo form, a DNA polymerase capable of incorporating ribo bases, a suitable buffer, and thermal cycling. We have applied ribo-PCR to resequence DNA by directly fragmenting the RNA/DNA chimeras with alkali and analyzing the fragments by mass spectrometry (MS). Mass fingerprint is used to identify deviations from the reference sequence. This method readily detects homozygous sequence deviations as well as heterozygous positions directly from genomic DNA samples. With the high-throughput capability of MS, this facile method is well suited for screening DNA sequences of limited regions of the genome in a large number of individuals. It can also be used to sequence multiple distant genomic loci in a single reaction. This novel ribo-PCR resequencing protocol was applied to different genomic loci involving nitric oxide synthase 1 (NOS1) and H19 in 30 individuals and SLCO1B1 in 95 individuals.     

Primary B-CLL Resistance to NK Cell Cytotoxicity can be Overcome In Vitro and In Vivo by Priming NK Cells and Monoclonal Antibody Therapy

Despite recent advances with monoclonal antibody therapy, chronic lymphocytic leukemia (CLL) remains incurable. Natural killer (NK) cells are potent antitumoral effectors, particularly against hematological malignancies. Defective recognition of B-CLL leukemic cells by NK cells has been previously described. Here, we deciphered the mechanisms that hamper NK cell-mediated clearance of B-CLL and evaluated the potential of NK cells as therapeutic tools for treatment of CLL. First of all, leukemic B cells resemble to normal B cells with a weak expression of ligands for NK receptors. Conversely, NK cells from B-CLL patients were functionally and phenotypically competent, despite a decrease of expression of the activating receptor NKp30. Consequently, resting allogeneic NK cells were unable to kill leukemic B cells in vitro. These data suggest that patients' NK cells cannot initiate a proper immune reaction due to a lack of leukemic cell recognition. We next set up a xenotransplantation mouse model to study NK-CLL cell interactions. Together with our in vitro studies, in vivo data revealed that activation of NK cells is required in order to control B-CLL and that activated NK cells synergize to enhance rituximab effect on tumor load. This study points out the requirements for immune system manipulation for treatment of B-CLL in combination with monoclonal antibody therapy.     

MLPA and sequence analysis of DPY19L2 reveals point mutations causing globozoospermia

STUDY QUESTION: Do DPY19L2 heterozygous deletions and point mutations account for some cases of globozoospermia? SUMMARY ANSWER: Two DPY19L2 heterozygous deletions and three point mutations were identified, thus further confirming that genetic alterations of the DPY19L2 gene are the main cause of globozoospermia and indicating that DPY19L2 molecular diagnostics should not be stopped in the absence of a homozygous gene deletion. WHAT IS KNOWN ALREADY: Globozoospermia is a rare phenotype of primary male infertility characterized by the production of a majority of round-headed spermatozoa without acrosome. We demonstrated previously that most cases in man were caused by a recurrent homozygous deletion of the totality of the DPY19L2 gene, preventing sperm head elongation and acrosome formation. In mammals, DPY19L2 has three paralogs of yet unknown function and one highly homologous pseudogene showing >95% sequence identity with DPY19L2. Specific amplification and sequencing of DPY19L2 have so far been hampered by the presence of this pseudogene which has greatly complicated specific amplification and sequencing. STUDY DESIGN, SIZE, DURATION: In this cohort study, 34 patients presenting with globozoospermia were recruited during routine infertility treatment in infertility centers in France and Tunisia between January 2008 and December 2011. The molecular variants identified in patients were screened in 200 individuals from the general population to exclude frequent non-pathological polymorphisms. PARTICIPANTS/MATERIALS, SETTING, METHODS: We developed a Multiplex Ligation-dependent Probe Amplification test to detect the presence of heterozygous deletions and identified the conditions to specifically amplify and sequence the 22 exons and intronic boundaries of the DPY19L2 gene. The pathogenicity of the identified mutations and their action on the protein were evaluated in silico. MAIN RESULTS AND THE ROLE OF CHANCE: There were 23 patients who were homozygous for the DPY19L2 deletion (67.6%). Only eight of the eleven non-homozygously deleted patients could be sequenced due to poor DNA quality of three patients. Two patients were compound heterozygous carrying one DPY19L2 deleted allele associated respectively with a nonsense (p.Q342*) and a missense mutation (p.R290H). One patient was homozygous for p.M358K, another missense mutation affecting a highly conserved amino acid. Due to the localization of this mutation and the physicochemical properties of the substituted amino acids, we believe that this variant is likely to disrupt one of the protein transmembrane domains and destabilize the protein. Overall, 84% of the fully analysed patients (n = 31) had a molecular alteration of DPY19L2. There was no clear phenotypic difference between the homozygous deleted individual, patients carrying a point mutation and undiagnosed patients. LIMITATIONS, REASONS FOR CAUTION: Globally poor fertilization rates are observed after intracytoplasmic sperm injection of round spermatozoa. Further work is needed to assess whether DPY19L2 mutated patients present a better or worse prognostic than the non-diagnosed patients. Evaluation of the potential benefit of treatment with a calcium ionophore, described to improve fertilization, should be evaluated in these two groups. WIDER IMPLICATIONS OF THE FINDINGS: In previous work, deletions of DPY19L2 had only been identified in North African patients. Here we have identified DPY19L2 deletions and point mutations in European patients, indicating that globozoospemia caused by a molecular defect of DPY19L2 can be expected in individuals from any ethnic background. STUDY FUNDING/COMPETING INTEREST(S): None of the authors have any competing interest. This work is part of the project 'Identification and Characterization of Genes Involved in Infertility (ICG2I)' funded by the program GENOPAT 2009 from the French Research Agency (ANR).     

RCL2, a potential formalin substitute for tissue fixation in routine pathological specimens

Masir N, Ghoddoosi M, Mansor S, Abdul‐Rahman F, Florence C S, Mohamed‐Ismail N A, Tamby M‐R & Md‐Latar N H
(2012) Histopathology  60, 804–815 RCL2, a potential formalin substitute for tissue fixation in routine pathological specimens Aims: To investigate RCL2 as a fixative for tissue fixation in routine histopathological examination and to assess tissue suitability for ancillary investigations. Methods and results: Forty‐nine samples from 36 fresh specimens were cut into three equal pieces and fixed in RCL2 diluted in 100% ethanol, RCL2 in 95% ethanol, or neutral buffered formalin as control. Suitability for microtomy, quality of histomorphology, histochemistry, immunohistochemistry, fluorescent and silver in‐situ hybridization analysis and extracted genomic DNA were assessed. Microtomy was straightforward in most tissue blocks, but there was difficulty in cutting in approximately a quarter of samples, which required careful handling by an experienced technician. There were no significant differences in tissue morphology between RCL2‐ and formalin‐fixed tissues (P = 0.08). Generally, the quality of histochemical staining, immunohistochemistry and in‐situ hybridization were comparable to that of formalin‐fixed tissues. Inconsistent immunoreactivity was noted, however, with antibodies against pan‐cytokeratin and progesterone receptor. Genomic DNA concentration was higher in RCL2‐fixed tissues. Using RCL2 diluted in 95% ethanol did not affect fixation quality. Conclusion: RCL2 is a potential formalin substitute suitable as a fixative for use in routine histopathological examination; however, difficulty in microtomy and occasional discrepancies in immunohistochemical reactivity require further optimization of the methodology.     

Epidemiology of and diagnostic strategies for toxoplasmosis

The apicomplexan parasite Toxoplasma gondii was discovered a little over 100 years ago, but knowledge of its biological life cycle and its medical importance has grown in the last 40 years. This obligate intracellular parasite was identified early as a pathogen responsible for congenital infection, but its clinical expression and the importance of reactivations of infections in immunocompromised patients were recognized later, in the era of organ transplantation and HIV infection. Recent knowledge of host cell-parasite interactions and of parasite virulence has brought new insights into the comprehension of the pathophysiology of infection. In this review, we focus on epidemiological and diagnostic aspects, putting them in perspective with current knowledge of parasite genotypes. In particular, we provide critical information on diagnostic methods according to the patient's background and discuss the implementation of screening tools for congenital toxoplasmosis according to health policies.     

A study of magnesium deficiency in human and experimental pulmonary hypertension

Pulmonary hypertension (PH) is defined as an increase in mean pulmonary arterial pressure above 25?mmHg. Pulmonary vasoconstriction, cellular proliferation, inflammation, and oxidative stress are involved in the pathophysiology of PH. Since hypomagnesemia was reported to promote endothelial cell dysfunction leading to inflammation and oxidative stress, we investigated the potential involvement of magnesium (Mg) deficiency in experimental and human PH. Our results indicate that Mg deficiency has no impact on hypoxia-induced PH development or severity, and that no reduction in Mg plasma concentration was observed in patients with severe pulmonary arterial hypertension. Thus, hypomagnesemia does not appear to play a role in the pathophysiology of experimental and human pulmonary hypertension.     

Les eaux embouteillées

Bottled waters, natural mineral water and spring water, meet the specific criteria defined by the regulation. Natural mineral water is characterized by its underground origin, the stability of its composition and its “original purity”. Water is an essential nutrient whose primary function is to hydrate. However, some natural mineral water can contribute to meet the recommended daily allowances (RDA) of minerals such as calcium and magnesium. These minerals from water are well absorbed and as consequence can contribute to meeting until 64 and 44% of daily intake in calcium and magnesium respectively. This recommendation is particularly relevant for children and adolescents, pregnant and lactating women, menopausal women and the elderly whom daily needs are increased. Concerning sulphates, the studies conducted to date do not allow establishing an upper limit of consumption via drinking water as they do not conclude on adverse effects in human being. In conclusion, as part of a balanced diet, natural mineral waters represent a natural source of minerals not negligible all the more they may have beneficial effects on health.     

Echographic measurement of skin thickness in sites suitable for intradermal vaccine injection in infants and children

Whereas the knowledge of skin thickness is essential to determine microneedle length and ensure proper administration of and better responses to intradermal vaccines, very few figures are available, especially in infants and children. Using ultrasound echography, we investigated skin thickness in 384 children aged 4-7, 12-18, and 54-66 months at potential body sites for intradermal vaccine delivery: deltoid, suprascapular, upper back, and lumbar area. The mean epidermis plus dermis thickness was significantly higher at the suprascapular than at the deltoid site (1.29 mm vs. 1.22 mm) and remained relatively unchanged whatever the BMI, age, sex, and skin phototype. In the 43 children aged 54-66 months, the mean skin thickness was significantly higher in the upper than in the lumbar area (1.39 mm vs. 1.31 mm). In this study setting, the heterogeneity in skin thickness cannot be considered sufficient to indicate various microneedle lengths for various ages or injection sites.     

Abnormal brain activation during working memory in children with prenatal exposure to drugs of abuse: the effects of methamphetamine, alcohol, and polydrug exposure

Structural and metabolic abnormalities in fronto-striatal structures have been reported in children with prenatal methamphetamine (MA) exposure. The current study was designed to quantify functional alterations to the fronto-striatal circuit in children with prenatal MA exposure using functional magnetic resonance imaging (fMRI). Because many women who use MA during pregnancy also use alcohol, a known teratogen, we examined 50 children (age range 7-15), 19 with prenatal MA exposure, 15 of whom had concomitant prenatal alcohol exposure (the MAA group), 13 with heavy prenatal alcohol but no MA exposure (ALC group), and 18 unexposed controls (CON group). We hypothesized that MA exposed children would demonstrate abnormal brain activation during a visuospatial working memory (WM) "N-Back" task. As predicted, the MAA group showed less activation than the CON group in many brain areas, including the striatum and frontal lobe in the left hemisphere. The ALC group showed less activation than the MAA group in several regions, including the right striatum. We found an inverse correlation between performance and activity in the striatum in both the CON and MAA groups. However, this relationship was significant in the caudate of the CON group but not the MAA group, and in the putamen of the MAA group but not the CON group. These findings suggest that structural damage in the fronto-striatal circuit after prenatal MA exposure leads to decreased recruitment of this circuit during a WM challenge, and raise the possibility that a rewiring of cortico-striatal networks may occur in children with prenatal MA exposure.