Effect of multiple transverse modes in self-mixing sensors based on vertical-cavity surface-emitting lasers

We investigate the effect of coexisting transverse modes on the operation of self-mixing sensors based on vertical-cavity surface-emitting lasers (VCSELs). The effect of multiple transverse modes on the measurement of displacement and distance were examined by simulation and in laboratory experiment. The simulation model shows that the periodic change in the shape and magnitude of the self-mixing signal with modulation current can be properly explained by the different frequency-modulation coefficients of the respective transverse modes in VCSELs. The simulation results are in excellent agreement with measurements performed on single-mode and multimode VCSELs and on self-mixing sensors based on these VCSELs.

Bellerophon: a program to detect chimeric sequences in multiple sequence alignments

Bellerophon is a program for detecting chimeric sequences in multiple sequence datasets by an adaption of partial treeing analysis. Bellerophon was specifically developed to detect 16S rRNA gene chimeras in PCR-clone libraries of environmental samples but can be applied to other nucleotide sequence alignments.

Using a combination of MLPA kits to detect chromosomal imbalances in patients with multiple congenital anomalies and mental retardation is a valuable choice for developing countries

Conventional karyotyping detects anomalies in 3-15% of patients with multiple congenital anomalies and mental retardation (MCA/MR). Whole-genome array screening (WGAS) has been consistently suggested as the first choice diagnostic test for this group of patients, but it is very costly for large-scale use in developing countries. We evaluated the use of a combination of Multiplex Ligation-dependent Probe Amplification (MLPA) kits to increase the detection rate of chromosomal abnormalities in MCA/MR patients. We screened 261 MCA/MR patients with two subtelomeric and one microdeletion kits. This would theoretically detect up to 70% of all submicroscopic abnormalities. Additionally we scored the de Vries score for 209 patients in an effort to find a suitable cut-off for MLPA screening. Our results reveal that chromosomal abnormalities were present in 87 (33.3%) patients, but only 57 (21.8%) were considered causative. Karyotyping detected 15 abnormalities (6.9%), while MLPA identified 54 (20.7%). Our combined MLPA screening raised the total detection number of pathogenic imbalances more than three times when compared to conventional karyotyping. We also show that using the de Vries score as a cutoff for this screening would only be suitable under financial restrictions. A decision analytic model was constructed with three possible strategies: karyotype, karyotype + MLPA and karyotype + WGAS. Karyotype + MLPA strategy detected anomalies in 19.8% of cases which account for 76.45% of the expected yield for karyotype + WGAS. Incremental Cost Effectiveness Ratio (ICER) of MLPA is three times lower than that of WGAS, which means that, for the same costs, we have three additional diagnoses with MLPA but only one with WGAS. We list all causative alterations found, including rare findings, such as reciprocal duplications of regions deleted in Sotos and Williams-Beuren syndromes. We also describe imbalances that were considered polymorphisms or rare variants, such as the new SNP that confounded the analysis of the 22q13.3 deletion syndrome. (C) 2011 Elsevier Masson SAS. All rights reserved.

A Duplex Allele-Specific Amplification PCR to Detect SMN1 Deletion

Spinal muscular atrophy (SMA), the leading genetic cause of death in childhood, is an autosomal recessive neuromuscular disorder characterized by progressive muscle weakness, associated with deletions of the survival motor neuron (SMN) gene identified and mapped to chromosome 5q13. SMN is present in two highly homologous copies (SMN1 and SMN2). In the general population, normal individuals (noncarriers) have at least one telomeric (SMN1) copy, and 5% of them have no copies of SMN2. Approximately 95% of SMA patients carry homologous deletions of SMN1 exon(s) 7 (and 8). SMN1 and SMN2 exons 7 and 8 differ only by 1 bp each, and SMA diagnosis might be performed by single-strand conformational polymorphism, PCR amplification followed by restriction fragment length polymorphism (RFLP), multiple ligation-dependent probe amplification, or realtime PCR of SMNs exons 7 and 8. We developed a simpler and cost-effective method to detect SMN1 exon 7 deletion based on allele-specific amplification PCR.

Morphological investigation of Toxoplasma gondii in vivo by a multiple beam interference microscope

A recently developed technique, namely multiple beam interference microscopy, has been applied to investigate the morphology of the parasite Toxoplasma gondii for the first time. The interference pattern obtained from the multiple internal reflection of a T. gondii, sandwiched between a glass plate and a cover plate, was focused on the objective of a conventional microscope. Because of the enhance contrast, several details of sub cellular structure and separating compartments are clearly visible. Details reveal the presence of a nucleus, lipid body, dense granule, rhoptry and amylopectin. The wall thickness of the membrane of the lipid body and the amylopectin is of the order of 0.02 µm and can be clearly distinguished with the help of the present technique. The same parasite has also been examined with the help of atomic force microscopy, and because of its thick membrane, the inner structural details were not observed at all. Sub cellular details of T. gondii observed with the present technique have been reported earlier only by low amplification transmission electron microscopy and not by any optical microscopic technique.

Preliminary results of random amplification of polymorphic DNA among Triatominae of the phyllosoma complex (Hemiptera, Reduviidae)

In Mexico, Triatoma longipennis (Usinger), Triatoma picturata (Usinger), and Triatoma pallidipennis (Stal), primary Chagas disease vector species of the phyllosoma complex, were analyzed by randomly amplified polymorphic DNA (RAPD). Sixteen decametric primers resolved individual profiles not identical, but partially discriminative between species. Analysis based on pairwise presence/absence comparisons between the three species was performed using three primers and two outgroup species Triatoma infestans (Klug) and Triatoma barberi (Usinger). Fifty-three bands in total were scored, although only two bands were constant among the three phyllosoma complex species. Two other bands were constant only for T. longipennis and T. picturata together, and not present in T. pallidipennis. Neighbor Joining tree and the multiple correspondence analysis discriminated T. pallidipennis clearly from the other two species, although there was overlap between T. longipennis and T. picturata. The results indicate a close relationship between the studied species and support the hypothesis of their recent evolution. The suitability of RAPD to discern populations within the species is discussed.

Genetic variability of Aedes aegypti in the Americas using a mitochondrial gene: evidence of multiple introductions

To analyze the genetic relatedness and phylogeographic structure of Aedes aegypti, we collected samples from 36 localities throughout the Americas (Brazil, Peru, Venezuela, Guatemala, US), three from Africa (Guinea, Senegal, Uganda), and three from Asia (Singapore, Cambodia, Tahiti). Amplification and sequencing of a fragment of the mitochondrial NADH dehydrogenase subunit 4 gene identified 20 distinct haplotypes, of which 14 are exclusive to the Americas, four to African/Asian countries, one is common to the Americas and Africa, and one to the Americas and Asia. Nested clade analysis (NCA), pairwise distribution, statistical parsimony, and maximum parsimony analyses were used to infer evolutionary and historic processes, and to estimate phylogenetic relationships among haplotypes. Two clusters were found in all the analyses. Haplotypes clustered in the two clades were separated by eight mutational steps. Phylogeographic structure detected by the NCA was consistent with distant colonization within one clade and fragmentation followed by range expansion via long distance dispersal in the other. Three percent of nucleotide divergence between these two clades is suggestive of a gene pool division that may support the hypothesis of occurrence of two subspecies of Ae. aegypti in the Americas.

Non-invasive prenatal diagnosis of multiple endocrine neoplasia type 2A using COLD-PCR combined with HRM genotyping analysis from maternal serum.

The multiple endocrine neoplasia type 2A (MEN2A) is a monogenic disorder characterized by an autosomal dominant pattern of inheritance which is characterized by high risk of medullary thyroid carcinoma in all mutation carriers. Although this disorder is classified as a rare disease, the patients affected have a low life quality and a very expensive and continuous treatment. At present, MEN2A is diagnosed by gene sequencing after birth, thus trying to start an early treatment and by reduction of morbidity and mortality. We first evaluated the presence of MEN2A mutation (C634Y) in serum of 25 patients, previously diagnosed by sequencing in peripheral blood leucocytes, using HRM genotyping analysis. In a second step, we used a COLD-PCR approach followed by HRM genotyping analysis for non-invasive prenatal diagnosis of a pregnant woman carrying a fetus with a C634Y mutation. HRM analysis revealed differences in melting curve shapes that correlated with patients diagnosed for MEN2A by gene sequencing analysis with 100% accuracy. Moreover, the pregnant woman carrying the fetus with the C634Y mutation revealed a melting curve shape in agreement with the positive controls in the COLD-PCR study. The mutation was confirmed by sequencing of the COLD-PCR amplification product. In conclusion, we have established a HRM analysis in serum samples as a new primary diagnosis method suitable for the detection of C634Y mutations in MEN2A patients. Simultaneously, we have applied the increase of sensitivity of COLD-PCR assay approach combined with HRM analysis for the non-invasive prenatal diagnosis of C634Y fetal mutations using pregnant women serum.

Violence and displacement. Evidence from the Spanish civil war (1936-1939)

This paper explores the relationship between violence and displacement during civil war focusing on two different forms of population movements (i.e. incoming and outgoing), and two different forms of violence (i.e. direct and indirect). The paper explores the relationship between displacement and violence at the local level in the context of a civil war fought conventionally using fine-grained data from 1,062 municipalities of the region of Catalonia during the Spanish Civil War (1936-1939). First, the paper suggests that exogenous and endogenous to the war factors combine to generate patterns of resettlement. Second, the evidence indicates that, in acivil war context, refugee flows and violence are interrelated in multiple ways: the arrival of internal refugees in a locality promotes the perpetration of direct violence against civilians; this, in turn, triggers the departure of people from the locality when the other group approaches. Third, indirect violence (i.e. bombings) shows to be the most significant factor accounting for external displacement at the local level, suggesting that bombing can serve as a strong signal for civilians of the type of armed group they are facing. Finally, the Spanish case suggests that the demographic changes provoked by displacement, combined with the lethality of the conflict, are likely to have long-term political consequences.

Influence of the LILRA3 Deletion on Multiple Sclerosis Risk: Original Data and Meta-Analysis.

BACKGROUND Multiple sclerosis (MS) is a neurodegenerative, autoimmune disease of the central nervous system. Genome-wide association studies (GWAS) have identified over hundred polymorphisms with modest individual effects in MS susceptibility and they have confirmed the main individual effect of the Major Histocompatibility Complex. Additional risk loci with immunologically relevant genes were found significantly overrepresented. Nonetheless, it is accepted that most of the genetic architecture underlying susceptibility to the disease remains to be defined. Candidate association studies of the leukocyte immunoglobulin-like receptor LILRA3 gene in MS have been repeatedly reported with inconsistent results. OBJECTIVES In an attempt to shed some light on these controversial findings, a combined analysis was performed including the previously published datasets and three newly genotyped cohorts. Both wild-type and deleted LILRA3 alleles were discriminated in a single-tube PCR amplification and the resulting products were visualized by their different electrophoretic mobilities. RESULTS AND CONCLUSION Overall, this meta-analysis involved 3200 MS patients and 3069 matched healthy controls and it did not evidence significant association of the LILRA3 deletion [carriers of LILRA3 deletion: p = 0.25, OR (95% CI) = 1.07 (0.95-1.19)], even after stratification by gender and the HLA-DRB1*15:01 risk allele.

High-resolution 3D whole-heart coronary MRA: a study on the combination of data acquisition in multiple breath-holds and 1D residual respiratory motion compensation.

OBJECT: To study a scan protocol for coronary magnetic resonance angiography based on multiple breath-holds featuring 1D motion compensation and to compare the resulting image quality to a navigator-gated free-breathing acquisition. Image reconstruction was performed using L1 regularized iterative SENSE. MATERIALS AND METHODS: The effects of respiratory motion on the Cartesian sampling scheme were minimized by performing data acquisition in multiple breath-holds. During the scan, repetitive readouts through a k-space center were used to detect and correct the respiratory displacement of the heart by exploiting the self-navigation principle in image reconstruction. In vivo experiments were performed in nine healthy volunteers and the resulting image quality was compared to a navigator-gated reference in terms of vessel length and sharpness. RESULTS: Acquisition in breath-hold is an effective method to reduce the scan time by more than 30 % compared to the navigator-gated reference. Although an equivalent mean image quality with respect to the reference was achieved with the proposed method, the 1D motion compensation did not work equally well in all cases. CONCLUSION: In general, the image quality scaled with the robustness of the motion compensation. Nevertheless, the featured setup provides a positive basis for future extension with more advanced motion compensation methods.

Simultaneous identification of multiple mammalian species from mixed forensic samples based on mtDNA control region length polymorphism

Molecular species identification in mixed or contaminated biological material has always been problematic. We developed a simple and accurate method for mammal DNA identification in mixtures, based on interspecific mitochondrial DNA control region length polymorphism. Contrary to other published methods dealing with species mixtures, our protocol requires a single universal primer pair and amplification step, and is not based on a pre-defined panel of species. This protocol has been routinely employed by our laboratory for species identification in dozens of human and animal forensic caseworks. Six representative forensic caseworks involving the specific identification of mixed animal samples are reported in this paper, in order to demonstrate the applicability and usefulness of the method.

Holographic optical tweezers combined with back-focal-plane displacement detection

A major problem with holographic optical tweezers (HOTs) is their incompatibility with laser-based position detection methods, such as back-focal-plane interferometry (BFPI). The alternatives generally used with HOTs, like high-speed video tracking, do not offer the same spatial and temporal bandwidths. This has limited the use of this technique in precise quantitative experiments. In this paper, we present an optical trap design that combines digital holography and back-focal-plane displacement detection. We show that, with a particularly simple setup, it is possible to generate a set of multiple holographic traps and an additional static non-holographic trap with orthogonal polarizations and that they can be, therefore, easily separated for measuring positions and forces with the high positional and temporal resolutions of laser-based detection. We prove that measurements from both polarizations contain less than 1% crosstalk and that traps in our setup are harmonic within the typical range. We further tested the instrument in a DNA stretching experiment and we discuss an interesting property of this configuration: the small drift of the differential signal between traps.

Continuous quantification of HER2 expression by microfluidic precision immunofluorescence estimates HER2 gene amplification in breast cancer.

Chromogenic immunohistochemistry (IHC) is omnipresent in cancer diagnosis, but has also been criticized for its technical limit in quantifying the level of protein expression on tissue sections, thus potentially masking clinically relevant data. Shifting from qualitative to quantitative, immunofluorescence (IF) has recently gained attention, yet the question of how precisely IF can quantify antigen expression remains unanswered, regarding in particular its technical limitations and applicability to multiple markers. Here we introduce microfluidic precision IF, which accurately quantifies the target expression level in a continuous scale based on microfluidic IF staining of standard tissue sections and low-complexity automated image analysis. We show that the level of HER2 protein expression, as continuously quantified using microfluidic precision IF in 25 breast cancer cases, including several cases with equivocal IHC result, can predict the number of HER2 gene copies as assessed by fluorescence in situ hybridization (FISH). Finally, we demonstrate that the working principle of this technology is not restricted to HER2 but can be extended to other biomarkers. We anticipate that our method has the potential of providing automated, fast and high-quality quantitative in situ biomarker data using low-cost immunofluorescence assays, as increasingly required in the era of individually tailored cancer therapy.

Formation and differentiation of multiple mesenchymal lineages during lung development is regulated by {beta}-catenin signaling.

BACKGROUND: The role of ss-catenin signaling in mesodermal lineage formation and differentiation has been elusive. METHODOLOGY: To define the role of ss-catenin signaling in these processes, we used a Dermo1(Twist2)(Cre/+) line to target a floxed beta-catenin allele, throughout the embryonic mesenchyme. Strikingly, the Dermo1(Cre/+); beta-catenin(f/-) conditional Knock Out embryos largely phenocopy Pitx1(-/-)/Pitx2(-/-) double knockout embryos, suggesting that ss-catenin signaling in the mesenchyme depends mostly on the PITX family of transcription factors. We have dissected this relationship further in the developing lungs and find that mesenchymal deletion of beta-catenin differentially affects two major mesenchymal lineages. The amplification but not differentiation of Fgf10-expressing parabronchial smooth muscle progenitor cells is drastically reduced. In the angioblast-endothelial lineage, however, only differentiation into mature endothelial cells is impaired. CONCLUSION: Taken together these findings reveal a hierarchy of gene activity involving ss-catenin and PITX, as important regulators of mesenchymal cell proliferation and differentiation.