The effects of mismatches on hybridization in DNA microarrays: determination of nearest neighbor parameters

Quantifying interactions in DNA microarrays is of central importance for a better understanding of their functioning. Hybridization thermodynamics for nucleic acid strands in aqueous solution can be described by the so-called nearest-neighbor model, which estimates the hybridization free energy of a given sequence as a sum of dinucleotide terms. Compared with its solution counterparts, hybridization in DNA microarrays may be hindered due to the presence of a solid surface and of a high density of DNA strands. We present here a study aimed at the determination of hybridization free energies in DNA microarrays. Experiments are performed on custom Agilent slides. The solution contains a single oligonucleotide. The microarray contains spots with a perfect matching complementary sequence and other spots with one or two mismatches: in total 1006 different probe spots, each replicated 15 times per microarray. The free energy parameters are directly fitted from microarray data. The experiments demonstrate a clear correlation between hybridization free energies in the microarray and in solution. The experiments are fully consistent with the Langmuir model at low intensities, but show a clear deviation at intermediate (non-saturating) intensities. These results provide new interesting insights for the quantification of molecular interactions in DNA microarrays.Comment: 31 pages, 5 figure

Nonequilibrium effects in DNA microarrays: a multiplatform study

It has recently been shown that in some DNA microarrays the time needed to reach thermal equilibrium may largely exceed the typical experimental time, which is about 15h in standard protocols (Hooyberghs et al. Phys. Rev. E 81, 012901 (2010)). In this paper we discuss how this breakdown of thermodynamic equilibrium could be detected in microarray experiments without resorting to real time hybridization data, which are difficult to implement in standard experimental conditions. The method is based on the analysis of the distribution of fluorescence intensities I from different spots for probes carrying base mismatches. In thermal equilibrium and at sufficiently low concentrations, log I is expected to be linearly related to the hybridization free energy $\Delta G$ with a slope equal to $1/RT_{exp}$, where $T_{exp}$ is the experimental temperature and R is the gas constant. The breakdown of equilibrium results in the deviation from this law. A model for hybridization kinetics explaining the observed experimental behavior is discussed, the so-called 3-state model. It predicts that deviations from equilibrium yield a proportionality of $\log I$ to $\Delta G/RT_{eff}$. Here, $T_{eff}$ is an effective temperature, higher than the experimental one. This behavior is indeed observed in some experiments on Agilent arrays. We analyze experimental data from two other microarray platforms and discuss, on the basis of the results, the attainment of equilibrium in these cases. Interestingly, the same 3-state model predicts a (dynamical) saturation of the signal at values below the expected one at equilibrium.Comment: 27 pages, 9 figures, 1 tabl

Efficacy of celecoxib in treating symptoms of viral pharyngitis: A double-blind, randomized study of celecoxib versus diclofenac

This study compared the efficacy and safety of the cyclooxygenase-2 specific inhibitor celecoxib with the conventional non-steroidal anti-inflammatory drug diclofenac in the symptomatic treatment of viral pharyngitis. Adult patients from 27 study centers in Latin America were treated with oral doses of celecoxib 200 mg once daily or 200 mg twice daily, or diclofenac 75 mg twice daily for 5 days in a double-blind, randomized study. The primary efficacy assessment was 'Throat Pain on Swallowing' on day 3. In addition, secondary quality-of-life assessments were performed on days 3 and 5. All adverse events and treatment-emergent signs and symptoms were recorded. Data from 313 patients were evaluable for efficacy (105 celecoxib 200 mg once daily, 107 celecoxib 200 mg twice daily, 101 diclofenac 75 mg twice daily). The upper 95% confidence limits for the visual analog scale of 'Throat Pain on Swallowing' on day 3 for celecoxib 200 mg once daily relative to diclofenac 75 mg twice daily, and celecoxib 200 mg twice daily relative to diclofenac 75 mg twice daily were 9.26 and 7.83, respectively. All secondary efficacy and quality-of-life measures were clinically similar for the three treatment groups, and no statistically significant differences were detected. The incidences of treatment-emergent adverse events and withdrawals due to adverse events were similar for all groups, but numerically higher among patients taking diclofenac than celecoxib. More patients in the diclofenac group reported gastrointestinal complaints (7.3%) compared with those in the celecoxib groups (4.3% in the celecoxib 200 mg once-daily group and 3.4% in the celecoxib 200 mg twice-daily group). In conclusion, 5 days of treatment with celecoxib 200 mg once daily is as effective as diclofenac 75 mg twice daily in the symptomatic treatment of viral pharyngitis. Celecoxib 200 mg once daily is also as effective as celecoxib 200 mg twice daily in this condition

Efficacy of ChAdOx1 nCoV-19 (AZD1222) vaccine against SARS-CoV-2 lineages circulating in Brazil

Several COVID-19 vaccines have shown good efficacy in clinical trials, but there remains uncertainty about the efficacy of vaccines against different variants. Here, we investigate the efficacy of ChAdOx1 nCoV-19 (AZD1222) against symptomatic COVID-19 in a post-hoc exploratory analysis of a Phase 3 randomised trial in Brazil (trial registration ISRCTN89951424). Nose and throat swabs were tested by PCR in symptomatic participants. Sequencing and genotyping of swabs were performed to determine the lineages of SARS-CoV-2 circulating during the study. Protection against any symptomatic COVID-19 caused by the Zeta (P.2) variant was assessed in 153 cases with vaccine efficacy (VE) of 69% (95% CI 55, 78). 49 cases of B.1.1.28 occurred and VE was 73% (46, 86). The Gamma (P.1) variant arose later in the trial and fewer cases (N = 18) were available for analysis. VE was 64% (−2, 87). ChAdOx1 nCoV-19 provided 95% protection (95% CI 61%, 99%) against hospitalisation due to COVID-19. In summary, we report that ChAdOx1 nCoV-19 protects against emerging variants in Brazil despite the presence of the spike protein mutation E484K

Genes for hereditary sensory and autonomic neuropathies: a genotype–phenotype correlation

Hereditary sensory and autonomic neuropathies (HSAN) are clinically and genetically heterogeneous disorders characterized by axonal atrophy and degeneration, exclusively or predominantly affecting the sensory and autonomic neurons. So far, disease-associated mutations have been identified in seven genes: two genes for autosomal dominant (SPTLC1 and RAB7) and five genes for autosomal recessive forms of HSAN (WNK1/HSN2, NTRK1, NGFB, CCT5 and IKBKAP). We performed a systematic mutation screening of the coding sequences of six of these genes on a cohort of 100 familial and isolated patients diagnosed with HSAN. In addition, we screened the functional candidate gene NGFR (p75/NTR) encoding the nerve growth factor receptor. We identified disease-causing mutations in SPTLC1, RAB7, WNK1/HSN2 and NTRK1 in 19 patients, of which three mutations have not previously been reported. The phenotypes associated with mutations in NTRK1 and WNK1/HSN2 typically consisted of congenital insensitivity to pain and anhidrosis, and early-onset ulcero-mutilating sensory neuropathy, respectively. RAB7 mutations were only found in patients with a Charcot-Marie-Tooth type 2B (CMT2B) phenotype, an axonal sensory-motor neuropathy with pronounced ulcero-mutilations. In SPTLC1, we detected a novel mutation (S331F) corresponding to a previously unknown severe and early-onset HSAN phenotype. No mutations were found in NGFB, CCT5 and NGFR. Overall disease-associated mutations were found in 19% of the studied patient group, suggesting that additional genes are associated with HSAN. Our genotype–phenotype correlation study broadens the spectrum of HSAN and provides additional insights for molecular and clinical diagnosis

Linkage and association studies identify a novel locus for Alzheimer disease at 7q36 in a Dutch population-based sample

We obtained conclusive linkage of Alzheimer disease (AD) with a candidate region of 19.7 cM at 7q36 in an extended multiplex family, family 1270, ascertained in a population-based study of early-onset AD in the northern Netherlands. Single-nucleotide polymorphism and haplotype association analyses of a Dutch patient-control sample further supported the linkage at 7q36. In addition, we identified a shared haplotype at 7q36 between family 1270 and three of six multiplex AD-affected families from the same geographical region, which is indicative of a founder effect and defines a priority region of 9.3 cM. Mutation analysis of coding exons of 29 candidate genes identified one linked synonymous mutation, g.38030G-->C in exon 10, that affected codon 626 of the PAX transactivation domain interacting protein gene (PAXIP1). It remains to be determined whether PAXIP1 has a functional role in the expression of AD in family 1270 or whether another mutation at this locus explains the observed linkage and sharing. Together, our linkage data from the informative family 1270 and the association data in the population-based early-onset AD patient-control sample strongly support the identification of a novel AD locus at 7q36 and re-emphasize the genetic heterogeneity of AD

Correlation in chicken between the marker LEI0258 alleles and Major Histocompatibility Complex sequences

<p>Abstract</p> <p>Background</p> <p>The LEI0258 marker is located within the B region of the chicken Major Histocompatibility Complex (MHC), and is surprisingly well associated with serology. Therefore, the correlation between the LEI0258 alleles and the MHC class I and the class II alleles at the level of sequences is worth investigating in chickens. Here we describe to which extent the LEI0258 alleles are associated with alleles of classical class I genes and non-classical class II genes, in reference animals as well as local breeds with unknown MHC haplotypes.</p> <p>Methods</p> <p>For the class I region, in an exploratory project, we studied 10 animals from 3 breeds: Rhode Island Red, White Leghorn and Fayoumi chickens, by cloning and sequencing <it>B-F1</it> and <it>B-F2</it> cDNA from exon 1 to 3’UTR. For the class II region, we reconstructed haplotypes of the 8.8 kb genomic region encompassing three non-classical class II genes: <it>B-DMA</it>, <it>B-DMB1</it> and <it>B-DMB2</it>, for 146 animals from more than 50 breeds including wild species of jungle fowls.</p> <p>Results</p> <p>Overall we found that the LEI0258 marker genotypes gave good indications of the MHC haplotypes, and a very good predictions (>0.95) of the heterozygosity of an animal at the MHC locus.</p> <p>Conclusions</p> <p>Our results show that the LEI0258 alleles are strongly associated with haplotypes of classical class I genes and non-classical class II genes, unravelling the reasons why this marker is becoming the reference marker for MHC genotyping in chickens.</p

Decoding of Superimposed Traces Produced by Direct Sequencing of Heterozygous Indels

Direct Sanger sequencing of a diploid template containing a heterozygous insertion or deletion results in a difficult-to-interpret mixed trace formed by two allelic traces superimposed onto each other. Existing computational methods for deconvolution of such traces require knowledge of a reference sequence or the availability of both direct and reverse mixed sequences of the same template. We describe a simple yet accurate method, which uses dynamic programming optimization to predict superimposed allelic sequences solely from a string of letters representing peaks within an individual mixed trace. We used the method to decode 104 human traces (mean length 294 bp) containing heterozygous indels 5 to 30 bp with a mean of 99.1% bases per allelic sequence reconstructed correctly and unambiguously. Simulations with artificial sequences have demonstrated that the method yields accurate reconstructions when (1) the allelic sequences forming the mixed trace are sufficiently similar, (2) the analyzed fragment is significantly longer than the indel, and (3) multiple indels, if present, are well-spaced. Because these conditions occur in most encountered DNA sequences, the method is widely applicable. It is available as a free Web application Indelligent at http://ctap.inhs.uiuc.edu/dmitriev/indel.asp

Novel computational methods for increasing PCR primer design effectiveness in directed sequencing

<p>Abstract</p> <p>Background</p> <p>Polymerase chain reaction (PCR) is used in directed sequencing for the discovery of novel polymorphisms. As the first step in PCR directed sequencing, effective PCR primer design is crucial for obtaining high-quality sequence data for target regions. Since current computational primer design tools are not fully tuned with stable underlying laboratory protocols, researchers may still be forced to iteratively optimize protocols for failed amplifications after the primers have been ordered. Furthermore, potentially identifiable factors which contribute to PCR failures have yet to be elucidated. This inefficient approach to primer design is further intensified in a high-throughput laboratory, where hundreds of genes may be targeted in one experiment.</p> <p>Results</p> <p>We have developed a fully integrated computational PCR primer design pipeline that plays a key role in our high-throughput directed sequencing pipeline. Investigators may specify target regions defined through a rich set of descriptors, such as Ensembl accessions and arbitrary genomic coordinates. Primer pairs are then selected computationally to produce a minimal amplicon set capable of tiling across the specified target regions. As part of the tiling process, primer pairs are computationally screened to meet the criteria for success with one of two PCR amplification protocols. In the process of improving our sequencing success rate, which currently exceeds 95% for exons, we have discovered novel and accurate computational methods capable of identifying primers that may lead to PCR failures. We reveal the laboratory protocols and their associated, empirically determined computational parameters, as well as describe the novel computational methods which may benefit others in future primer design research.</p> <p>Conclusion</p> <p>The high-throughput PCR primer design pipeline has been very successful in providing the basis for high-quality directed sequencing results and for minimizing costs associated with labor and reprocessing. The modular architecture of the primer design software has made it possible to readily integrate additional primer critique tests based on iterative feedback from the laboratory. As a result, the primer design software, coupled with the laboratory protocols, serves as a powerful tool for low and high-throughput primer design to enable successful directed sequencing.</p

A Variable Stiffness Actuator Module With Favorable Mass Distribution for a Bio-inspired Biped Robot

Achieving human-like locomotion with humanoid platforms often requires the use of variable stiffness actuators (VSAs) in multi-degree-of-freedom robotic joints. VSAs possess 2 motors for the control of both stiffness and equilibrium position. Hence, they add mass and mechanical complexity to the design of humanoids. Mass distribution of the legs is an important design parameter, because it can have detrimental effects on the cost of transport. This work presents a novel VSA module, designed to be implemented in a bio-inspired humanoid robot, Binocchio, that houses all components on the same side of the actuated joint. This feature allowed to place the actuator's mass to more proximal locations with respect to the actuated joint instead of concentrating it at the joint level, creating a more favorable mass distribution in the humanoid. Besides, it also facilitated it's usage in joints with centralized multi-degree of freedom (DoF) joints instead of cascading single DoF modules. The design of the VSA module is presented, including it's integration in the multi-DoFs joints of Binocchio. Experiments validated the static characteristics of the VSA module to accurately estimate the output torque and stiffness. The dynamic responses of the driving and stiffening mechanisms are shown. Finally, experiments show the ability of the actuation system to replicate the envisioned human-like kinematic, torque and stiffness profiles for Binocchio