Mismatch Tolerance Research Articles

Passive all-optical synchronization for polarization-maintaining mode-locked fiber lasers.

We have proposed and implemented for the first time to our knowledge a passive and all-optical pulse synchronization for polarization-maintaining fiber lasers. Specifically, the synchronization system was comprised of two independent Yb-doped and Er-doped mode-locked fiber lasers in a master-slave configuration. Master pulses were injected into the slave laser cavity consisting of a nonlinear amplifying loop mirror, which provided an effective fast intensity modulator due to the periodic introduction of nonreciprocal phase difference. As a result, robust and tight timing synchronization was achieved with a cavity mismatch tolerance of 800 µm and a relative timing jitter of 26 fs within 1-MHz bandwidth. In combination with the all-polarization-maintaining structure of fiber lasers, long-term stable operation was demonstrated over 12 hours without the need for temperature stabilization and vibration isolation. The implemented synchronous laser system could find immediate applications such as pump-probe microscopy, two-color spectroscopy and nonlinear frequency mixing.

A Market Center Based Clearing and Settlement of Pure Reactive Power Market in Deregulated Power System

DC Power Flow Model is a constant matrix, a non-iterative model which is built into DC Optimal Power Flow Model for market clearing and settlement of real power market. DC Optimal Power Flow Method is fast when compared to nonlinear Optimal Power Flow and the accuracy level is acceptable for security analysis. It is widely used for active power flow analysis in the deregulated power system. As of now, there are few papers available in DC Optimal Power Flow Model for reactive power flow analysis. This paper proposes a new Optimal Q Flow (OQF) method for market clearing and settlement of pure reactive power market. A lumped linear model is proposed for power balance equality constraint for fast convergence. In this method, a unique reference for delivery/withdrawal point for reactive power called Market Center is proposed to share the total transmission loss equitably between the GENCO and DISCO participants in a transparent manner for pure Q Market using incremental loss factor method. This model uses a slack bus independent loss factors measured with respect to “Market Center”. The objective function is to minimize the cost of the reactive power payable to GENCOs with respect to the market center. The compensation received by GENCO participants and Locational Marginal Price to be paid by DISCOs participants are derived for N bus. The OQF method is tested on a Radial Five Bus System, Ward and Hale 6 Bus System and IEEE 30 Bus System.The bus voltage magnitude computed using the Iterative Q Flow method is accurate and obtained for a mismatch tolerance of 0.1 MVAr. In the proposed method it is found that the total bus loss is reduced considerably thereby minimizing the cost of the objective function.

Mismatch tolerance during homologous recombination in mammalian cells

We investigated the homology dependency of recombination in thymidine kinase (tk)-deficient mouse fibroblasts. Cells were transfected with DNA constructs harboring a herpes tk gene (the “recipient”) rendered non-functional by an oligonucleotide containing the recognition site for endonuclease I-SceI. Constructs also contained a “donor” tk sequence that could restore function to the recipient gene through spontaneous gene conversion or via repair of a double-strand break (DSB) at the I-SceI site. Recombination events were recoverable by selection for tk-positive clones. Three different donors were used containing 16, 25, or 33 mismatches relative to the recipient. The mismatches were clustered, forming an interval of “homeology” relative to the recipient sequences. We show that when homeologous sequences were surrounded by high homology, mismatches were frequently included in gene conversion events. Notably, conversion tracts from spontaneous recombination included either all or none of the mismatches, suggesting that recombination must begin and end in high homology. This requirement was relaxed for events that occurred near an induced DSB, as a significant number of these latter conversion tracts had one end positioned within homeology. Knock-down of mismatch repair showed that incorporation of mismatches into gene conversion tracts can involve repair of mismatched heteroduplex intermediates, indicating that mismatch repair does not necessarily impede homeologous genetic exchange. Our results illustrate (1) genetic exchange between homeologous sequences in a mammalian genome is enabled by nearby homology, (2) proximity to a DSB impacts the homology requirements for where genetic exchange may begin and end, and (3) mismatch correction and previously documented anti-recombination activity are separable functions of the mismatch repair machinery in mammalian cells.

Multiplexed precision genome editing with trackable genomic barcodes in yeast.

Our understanding of how genotype controls phenotype is limited by the scale at which we can precisely alter the genome and assess phenotypic consequences of each perturbation. Here we describe a CRISPR/Cas9-based method for multiplexed accurate genome editing with short, trackable, integrated cellular barcodes (MAGESTIC) in S. cerevisiae. MAGESTIC uses array-synthesized guide-donor oligos for plasmid-based high-throughput editing and features genomic barcode integration to prevent plasmid barcode loss and to enable robust phenotyping. We demonstrate that editing efficiency can be increased >5-fold by recruiting donor DNA to the site of breaks using the LexA-Fkh1p fusion protein. We performed saturation editing of the essential gene SEC14 and identified amino acids critical for chemical inhibition of lipid signaling. We also constructed thousands of natural genetic variants, characterized guide mismatch tolerance at the genome-scale, and ascertained that cryptic Pol III termination elements substantially reduce guide efficacy. MAGESTIC will be broadly useful to uncover the genetic basis of phenotypes in yeast.

Validating auditory spatial awareness with virtual reality and vice-versa

“Immersive” technologies such as virtual (VR) and augmented reality (AR) stand to transform sensory and perceptual science, clinical assessments, and habilitation of spatial awareness. This talk explores some of the general challenges and opportunities for VR- and AR-enabled research, illustrated by specific studies in the area of spatial hearing. In one study, freefield localization and discrimination measures were compared across conditions which used VR to show, hide, or alter the visible locations of loudspeakers from trial to trial. The approach is well suited to understanding potential biases and cuing effects in real-world settings. A second study used headphone presentation to understand contextual effects on the relative weighting of binaural timing and intensity cues. Previous studies have used adjustment and lateralization to “trade” time and level cues in a sound-booth setting, but none have attempted to measure how listeners weight cues in realistic multisensory scenes or in realistic temporal contexts. Finally, a third study used simple VR “games” to measure implicit awareness of reverberation in multi-target scenes. Listeners’ tolerance of reverberant mismatch can be used to assess and habilitate auditory spatial awareness and provides a testbed for future applications of auditory augmented reality.

Analysis, Design, and Implementation of Passivity-Based Control for Multilevel Railway Power Conditioner

In recent years, railway power conditioner (RPC) has been used to improve the power quality of a traction power system. In engineering application of RPC, mismatches between parameters used in controller and actual values are inevitable, which will increase the difficulty of current controller design and deteriorate compensation performance. In this paper, a passivity-based control (PBC) system is studied for multilevel RPC to enhance its tolerance for mismatches. According to the topology of multilevel RPC, equivalent electrical and mathematical models are developed. To employ PBC, Euler-Lagrange system model of RPC is established by Park (αβ/dq) transformation, and the passivity of RPC and stability of PBC are proved. On this basis, the robustness of PBC is analyzed and criteria for damping selection are derived. Finally, simulation and experiments have been carried out to verify the structure and control method in the paper.

Partial DNA-guided Cas9 enables genome editing with reduced off-target activity.

CRISPR-Cas9 is a versatile RNA-guided genome editing tool. Here we demonstrate that partial replacement of RNA nucleotides with DNA nucleotides in CRISPR RNA (crRNA) enables efficient gene editing in human cells. This strategy of partial DNA replacement retains on-target activity when used with both crRNA and sgRNA, as well as with multiple guide sequences. Partial DNA replacement also works for crRNA of Cpf1, another CRISPR system. We find that partial DNA replacement in the guide sequence significantly reduces off-target genome editing through focused analysis of off-target cleavage, measurement of mismatch tolerance and genome-wide profiling of off-target sites. Using the structure of the Cas9-sgRNA complex as a guide, the majority of the 3' end of crRNA can be replaced with DNA nucleotide, and the 5 - and 3'-DNA-replaced crRNA enables efficient genome editing. Cas9 guided by a DNA-RNA chimera may provide a generalized strategy to reduce both the cost and the off-target genome editing in human cells.

Reduced structural flexibility for an exonuclease deficient DNA polymerase III mutant.

DNA synthesis, carried out by DNA polymerases, requires balancing speed and accuracy for faithful replication of the genome. High fidelity DNA polymerases contain a 3′–5′ exonuclease domain that can remove misincorporated nucleotides on the 3′ end of the primer strand, a process called proofreading. The E. coli replicative polymerase, DNA polymerase III, has spatially separated (~55 Å apart) polymerase and exonuclease subunits. Here, we report on the dynamics of E. coli DNA polymerase III proofreading in the presence of its processivity factor, the β2-sliding clamp, at varying base pair termini using single-molecule FRET. We find that the binding kinetics do not depend on the base identity at the termini, indicating a tolerance for DNA mismatches. Further, our single-molecule data and MD simulations show two previously unobserved features: (1) DNA Polymerase III is a highly dynamic protein that adopts multiple conformational states while bound to DNA with matched or mismatched ends, and (2) an exonuclease-deficient DNA polymerase III has reduced conformational flexibility. Overall, our single-molecule experiments provide high time-resolution insight into a mechanism that ensures high fidelity DNA replication to maintain genome integrity.

High power continuous wave second harmonic generation in a phase matching insensitive MgO doped LiNbO_3 waveguide

This paper reports an efficient method of using a chirped grating to reduce the phase-matching sensitivity and increase the output power of continuous wave second harmonic generation in a MgO doped LiNbO3 waveguide. It was found that using a chirped grating with decreasing periods from 6.96 μm to 6.95 μm significantly improved the phase mismatch tolerance and increased the second harmonic generation output power from 0.64 W (using uniform grating) to 1.13 W. This design was shown to lead to a phase matching insensitive waveguide. This indicates that there is a possibility of obtaining higher output power with the MgO doped LiNbO3 waveguide.

Glenohumeral Mismatch in Anatomic Total Shoulder Arthroplasty.

* The optimal amount of glenohumeral radial mismatch in anatomic shoulder arthroplasty remains undefined, but the consensus among multiple studies suggests between 4 and 8 mm.* Current implant designs offer mismatch of the radii of curvature between the humeral head and the glenoid ranging from 1 to 38 mm.* Clinically, conforming glenoids demonstrate greater loosening and lucency scores compared with nonconforming glenoid components; however, survivorship advantages have not been proven.* There remains a range of radial mismatch tolerances that improve glenohumeral kinematics and wear patterns, which appear to be greater when the glenoid is loaded centrally and is supported in neutral glenoid version.

Profiling the Mismatch Tolerance of Argonaute 2 through Deep Sequencing of Sliced Polymorphic Viral RNAs.

Low allelic and clonal variability among endogenous RNAi targets has focused mismatch tolerance studies to RNAi-active guide strands. However, the inherent genomic instability of RNA viruses such as hepatitis C virus (HCV) gives rise to quasi-species mutants within discrete clones: this facilitates mismatch tolerance studies from a target perspective. We recently quantified the slicing imprecision of Argonaute 2 using small interfering RNA (siRNA) analogs of the DNA-directed RNAi drug TT-034 and next-generation sequencing of 5′ RNA ligase-mediated rapid amplification of cDNA ends (RACE-SEQ). Here, we present an open-source, customizable, and computationally light RACE-SEQ bioinformatic pipeline, describing adaptations that semiquantitatively report the impact of RNAi hybridization site mismatches from the target perspective. The analysis shows that Argonaute 2 has a substitution-specific, 3- to 5-log activity window between fully complementary targets and targets with mismatches across positions 10–11. It further focuses the endonucleotic Slicer imprecision around positions 13–17, demonstrating its dependence on guide strand central region complementarity, and potentiation by even a single mismatch. We further propose pharmacogenomics value in testing endogenous targets using recombinant replicon systems and RACE-SEQ to report the pharmacodynamics of sequence-specific oligonucleotide therapeutics against all possible polymorphisms in a population, in a minimally biased, patient-free manner.

DNA recognition by an RNA-guided bacterial Argonaute.

Argonaute (Ago) proteins are widespread in prokaryotes and eukaryotes and share a four-domain architecture capable of RNA- or DNA-guided nucleic acid recognition. Previous studies identified a prokaryotic Argonaute protein from the eubacterium Marinitoga piezophila (MpAgo), which binds preferentially to 5′-hydroxylated guide RNAs and cleaves single-stranded RNA (ssRNA) and DNA (ssDNA) targets. Here we present a 3.2 Å resolution crystal structure of MpAgo bound to a 21-nucleotide RNA guide and a complementary 21-nucleotide ssDNA substrate. Comparison of this ternary complex to other target-bound Argonaute structures reveals a unique orientation of the N-terminal domain, resulting in a straight helical axis of the entire RNA-DNA heteroduplex through the central cleft of the protein. Additionally, mismatches introduced into the heteroduplex reduce MpAgo cleavage efficiency with a symmetric profile centered around the middle of the helix. This pattern differs from the canonical mismatch tolerance of other Argonautes, which display decreased cleavage efficiency for substrates bearing sequence mismatches to the 5′ region of the guide strand. This structural analysis of MpAgo bound to a hybrid helix advances our understanding of the diversity of target recognition mechanisms by Argonaute proteins.

The Musical Impact of Multicultural London English (MLE) Speech Rhythm

There is evidence that an emerging variety of English spoken by young Londoners—Multicultural London English (MLE)—has a more even syllable rhythm than Southern British English (SBE). Given findings that native language rhythm influences the production of musical rhythms and text setting, we investigated possible musical consequences of this development. We hypothesized that the lower vocalic durational variability in MLE and (putatively) less salient stress distinctions would go along with a preference by MLE speakers for lower melodic durational variability and a higher tolerance for stress mismatches (the non-coincidence of stress/beat strong-weak patterns) compared to SBE speakers. An analysis of two popchart song corpora by MLE and SBE artists confirmed that durational variability was lower in the MLE songs, and that there were more stress mismatches. In a follow-up experiment, MLE and SBE participants read four short English sentences and then rated text settings in pairs of specially constructed song fragments with and without stress mismatches. MLE participants’ speech showed the expected lower variability in vocalic duration and syllabic prominence compared to SBE participants’ speech, while their text setting ratings showed a greater tolerance of stress mismatches.

Increased hot forgeability of 2024Al/Al18B4O33w whisker composites at high strain rates

Whisker reinforced aluminum alloy composites are attractive for structural applications in automotive and aerospace applications. However, their forgeability is limited by their poor ductility at room temperature. The effects of temperatures (350–520°C) and strain rates (0.01–50s−1) on the resistance to microcracking and on the closure of voids are investigated for 2024Al/Al18B4O33w whisker composite as well as unreinforced AA2024 alloy. Under elevated temperature and high strain rate (>450°C, >1.0s−1), there are less cracks and no axial splitting on the surface of 2024Al/Al18B4O33w composite. Test results show lower flow stresses and incipient melting at higher temperatures enable better tolerance of whisker-matrix strain mismatch at the interface during hot compression resulting in increased stabilization of flow stresses. While higher strain rates contribute to increased strain rate hardening, whisker rotation and void closure. It is interesting to note that the unreinforced AA2024 alloy experiences flow localization, surface cracking and plastic instability at temperatures higher than 450°C but the presence of Al18B4O33 in the composite seems to better resist these failure mechanisms resulting in higher forgeability.

A New DC Power Flow Model for Q Flow Analysis for use in Reactive Power Market

DC Power Flow method is widely used for active power flow analysis in deregulated power system. DC Power Flow model is a constant matrix, non-iterative model which is built into DC Optimal Power Flow model for market clearing and settlement of real power market. As of now, there are few papers available in DC Power Flow model for reactive power flow analysis .Hence, this paper introduces a pure Q market and proposes a new DC Q flow model (DCQF) which can be built into a DC Optimal Q flow model for market clearing and settlement of pure Q market. In addition to the DCQF method, to obtain accurate result satisfying a specified mismatch tolerance, an Iterative QF method is also proposed whose results match exactly with the solution obtained using FDPF method. The DCQF method and Iterative QF method are tested on Ward and Hale 6-Bus System, IEEE-30 Bus System and Indian Utility 119-Bus System. The bus voltage magnitude computed using the proposed DCQF method is compared with the accurate solution obtained using Iterative QF method (proposed)/Fast Decoupled Power Flow method for a mismatch tolerance of 0.1MVAr. The maximum error value obtained is acceptable even for security analysis. The proposed DCQF and Iterative QF method are faster when compared to FDPF method.

Allele-specific locus binding and genome editing by CRISPR at the p16INK4a locus.

The clustered regularly interspaced short palindromic repeats (CRISPR) system has been adopted for a wide range of biological applications including genome editing. In some cases, dissection of genome functions requires allele-specific genome editing, but the use of CRISPR for this purpose has not been studied in detail. In this study, using the p16INK4a gene in HCT116 as a model locus, we investigated whether chromatin states, such as CpG methylation, or a single-nucleotide gap form in a target site can be exploited for allele-specific locus binding and genome editing by CRISPR in vivo. First, we showed that allele-specific locus binding and genome editing could be achieved by targeting allele-specific CpG-methylated regions, which was successful for one, but not all guide RNAs. In this regard, molecular basis underlying the success remains elusive at this stage. Next, we demonstrated that an allele-specific single-nucleotide gap form could be employed for allele-specific locus binding and genome editing by CRISPR, although it was important to avoid CRISPR tolerance of a single nucleotide mismatch brought about by mismatched base skipping. Our results provide information that might be useful for applications of CRISPR in studies of allele-specific functions in the genomes.

Repositionnement par le système ExacTrac® pour l’irradiation sans cadre invasif de métastases cérébrales en conditions stéréotaxiques : étude descriptive de cinq centres français

PurposeThis study aimed to analyse the positioning protocols with the ExacTrac® system, associated with a dedicated linear accelerator such as Novalis®, for stereotactic treatment of brain metastases in several French centres. Materials and methodsA survey, including three questions about the prescription of irradiation and twenty-one questions about how the ExacTrac® system is used, was sent to nine French centres owning a dedicated Novalis® accelerator. Five centres have accepted to participate in the study. ResultsAll centres checked the positioning before each treatment's bow, with residual mismatch tolerances of 0.5 to 0.7mm for the translations and 0.5 to 1° for the rotations. All centres except one also realised orthogonal planar images of classic incidences to help operators ensure proper isocentre positioning. Prescribed doses were 20Gy in one fraction, 30Gy and 33Gy in three fractions or 34Gy in four fractions, mainly depending on the size of the lesion. Finally, a physician validated the images at the treatment station before starting the irradiation. ConclusionsThe practices of the different centres concerning the positioning protocols were rather homogeneous, in agreement with the literature data on ExacTrac® system's accuracy, as well as proposed fractionations. The systematic medical validation at the treatment station may, however, be questioned because of the waiting time between the doctor's call and validation itself and because of its usefulness; indeed, corrections by the radiation oncologist are very rare and in some centres, non-existent.

A Passive Filter Building Block for Input or Output Current Ripple Cancellation in a Power Converter

This paper analyzes a coupled inductor based $LL$ – $LC$ network as a building block to achieve input or output current ripple cancellation in power converters. The proposed current ripple cancellation technique works on the principle of inductance matching. It also provides reduced current stress on the switching devices of the converter compared with the other current ripple cancellation networks discussed in the literature. The current ripple cancellation technique is verified in achieving input and output current ripple minimization in different converters. The effect of inductance mismatch, inductor and capacitor tolerances, and circuit parasitic is also studied. Some of the published research works are demonstrated to be a special case of the proposed idea. The effectiveness of the proposed method is validated by demonstrating the zero-ripple input current using a 31 V dc to 60 V dc, 240 W boost converter prototype and a 31 V dc to 115 V (rms) ac, 320 W current-fed-switched inverter prototype, and the zero-ripple output current using a 62 V to 42 V dc, 450 W buck converter prototype.

Distinct patterns of Cas9 mismatch tolerance in vitro and in vivo.

Cas9, a CRISPR-associated RNA-guided nuclease, has been rapidly adopted as a tool for biochemical and genetic manipulation of DNA. Although complexes between Cas9 and guide RNAs (gRNAs) offer remarkable specificity and versatility for genome manipulation, mis-targeted events occur. To extend the understanding of gRNA::target homology requirements, we compared mutational tolerance for a set of Cas9::gRNA complexes in vitro and in vivo (in Saccharomyces cerevisiae). A variety of gRNAs were tested with variant libraries based on four different targets (with varying GC content and sequence features). In each case, we challenged a mixture of matched and mismatched targets, evaluating cleavage activity on a wide variety of potential target sequences in parallel through high-throughput sequencing of the products retained after cleavage. These experiments evidenced notable and consistent differences between in vitro and S. cerevisiae (in vivo) Cas9 cleavage specificity profiles including (i) a greater tolerance for mismatches in vitro and (ii) a greater specificity increase in vivo with truncation of the gRNA homology regions.

Interactions of two large antiviral polyamides with the long control region of HPV16.

PA1 and PA25 are large hairpin polyamides that are effective in nearly eliminating HPV16 episomes (DNA) in cell culture, and PA25 has broad spectrum activity against three cancer-causing forms of HPV (Edwards, T. G., Koeller, K. J., Slomczynska, U., Fok, K., Helmus, M., Bashkin, J. K., Fisher, C., Antiviral Res. 91 (2011) 177-186). Described here are the interactions of these PAs with sequences in the long control region (LCR) of HPV16 (7348-122). Using an FeEDTA conjugate of PA1 (designed to recognize 5'-W2GW7-3'; W=A or T), 34 affinity cleavage (AC) patterns were detected for this fragment. These sites can be rationalized with sequences featuring perfect, single, double, triple and quadruple mismatches. Quantitative DNase I footprinting analysis indicates that perfect sites bind PA1 with Kds between 0.7 and 2.2nM. Kds for single, double, triple and quadruple mismatch sites range from 1-3nM-20nM. Using AC and EDTA conjugates, we report that unlike smaller 8-ring hairpin PAs, introduction of a chiral turn in this large polyamide has no effect on binding orientation (forward vs. reverse). Despite its design to recognize 5'-W2GW5GW4-3' via two Im residues, a motif not represented in this HPV sequence, a PA25-EDTA conjugate yielded 31 affinity cleavage sites on the region. Low nM Kds for PA25 without EDTA indicates a high tolerance for triple and quadruple mismatches. While there is extensive coverage of the sequence examined, AC cleavage patterns for the two PAs show discrete binding events and do not overlap significantly. This indicates that within the context of A/T rich sequences, these PAs do not recognize a simple shared sequence-related feature of the DNA. These insights continue to inform the complex nature of large hairpin PA-DNA interactions and antiviral behavior.