DNA nicks promote efficient and safe targeted gene correction

Targeted gene correction employs a site-specific DNA lesion to promote homologous recombination that eliminates mutation in a disease gene of interest. The double-strand break typically used to initiate correction can also result in genomic instability if deleterious repair occurs rather than gene correction, possibly compromising the safety of targeted gene correction. Here we show that single-strand breaks (nicks) and double-strand breaks both promote efficient gene correction. However, breaks promote high levels of inadvertent but heritable genomic alterations both locally and elsewhere in the genome, while nicks are accompanied by essentially no collateral local mutagenesis, and thus provide a safer approach to gene correction. Defining efficacy as the ratio of gene correction to local deletion, nicks initiate gene correction with 70-fold greater efficacy than do double-strand breaks (29.0±6.0% and 0.42±0.03%, respectively). Thus nicks initiate efficient gene correction, with limited local mutagenesis. These results have clear therapeutic implications, and should inform future design of meganucleases for targeted gene correction.     

时间序列修订对森林二氧化碳通量的影响

对长白山阔叶红松林2003年生长季的涡动相关实测时间序列进行了去倾修订与超声风速仪倾斜修订,并分析了不同修订方法对森林CO2通量计算值的影响．结果表明,基于未修订时间序列计算得到的森林CO2通量(Fcraw)被高估．线性与非线性去倾对Fcraw的修订量分别为1．6％、1．8％,两者差异很小．平面拟合坐标变换与流线坐标变换对Fcraw的修订量分别为3．7％、4．7％,两者差异较大．对线性去倾后的时间序列分别进行流线坐标变换与平面拟合坐标变换,二者对Fcraw的修订量分别为5．5％与4．6％．建议对时间序列进行线性去倾与平面拟合坐标变换综合修订．     

Comparing the performances of Diggle's tests of spatial randomness for small samples with and without edge-effect correction: application to ecological data

Diggle's tests of spatial randomness based on empirical distributions of interpoint distances can be performed with and without edge-effect correction. We present here numerical results illustrating that tests without the edge-effect correction proposed by Diggle (1979, Biometrics 35, 87-101) have a higher power for small sample sizes than those with correction. Ignoring the correction enables detection of departure from spatial randomness with smaller samples (down to 10 points vs. 30 points for the tests with correction). These results are confirmed by an example with ecological data consisting of maps of two species of trees in a West African savanna. Tree numbers per species per map were often less than 20. For one of the species, for which maps strongly suggest an aggregated pattern, tests without edge-effect correction enabled rejection of the null hypothesis on three plots out of five vs. on only one for the tests with correction.     

Increased SFHR gene correction efficiency with sense single-stranded DNA

BACKGROUND: The correction of a mutated gene by the small fragment homologous replacement (SFHR) method is a highly attractive approach for gene therapy. However, the current SFHR method with a heat-denatured double-stranded PCR fragment yielded a low correction efficiency. METHODS: Single-stranded (ss) DNA fragments were prepared from ss phagemid DNA and tested in a gene correction assay with an inactivated Hyg-EGFP fusion gene, as a model target. RESULTS: A 606-nt sense, ss DNA fragment dramatically (12-fold) improved the gene correction efficiency, although the antisense strand showed only minimal correction efficiency. CONCLUSIONS: These results suggest that the use of a sense, single-stranded DNA fragment is useful in the SFHR method for the correction of mutated genes.     

Improved gene correction efficiency with a tailed duplex DNA fragment

A 606-base single-stranded (ss) DNA fragment, prepared by restriction enzyme digestion of ss phagemid DNA, corrects a hygromycin resistance and enhanced green fluorescent protein (Hyg-EGFP) fusion gene more efficiently than a PCR fragment, which is the conventional type of DNA fragment used in gene correction. Here, a tailed duplex, obtained by annealing an oligonucleotide to the ss DNA fragment, was used in the correction. The tailed duplex may be a good substrate for the RAD51 protein, an important enzyme in homologous recombination, which could be the gene correction pathway. The annealing of the oligonucleotides enhanced the correction efficiency of the Hyg-EGFP gene, especially when annealed in the 3'-region of the ss DNA fragment. Both the length and backbone structure of the oligonucleotides affected the gene correction efficiency. This type of gene correction device was also effective for another target gene, the rpsL gene. The results obtained in this study indicate that tailed duplex DNA fragments are effective nucleic acids for gene correction.     

Instrumented forceps for measuring tensile forces in the rod of the VDS implant during correction of scoliosis.

Ventral derotation spondylodesis (VDS) is the standard in ventral scoliosis surgery. Especially in the thoracic spine, there are no alternatives to VDS with compression and derotation as its correction forces. However, pull-out of the end-vertebra screw during correction of scoliosis with the VDS implant is a common complication involving particularly the cranial end-vertebra screw in the thoracic region. This complication requires an extension of the fusion length or reduces at least the outcome of the correction. There are no in vivo data on correction forces in ventral scoliosis surgery. Thus the correction depends on the skill and experience of the surgeon. An instrumented forceps developed and built to measure forces in the longitudinal rod allows axial tensile forces to be determined in the longitudinal rod during surgery. The instrumented forceps has the advantage of reducing the risk of screw pull-out. Furthermore, viscoelastic behavior of the spine can be measured during ventral correction. In addition, knowledge of the correction forces improves our biomechanical understanding of the spine, especially during correction of scoliosis. Intraoperative force measurement is in no way detrimental to the patient.     

Multiplicative background correction for spotted microarrays to improve reproducibility

We propose a simple approach, the multiplicative background correction, to solve a perplexing problem in spotted microarray data analysis: correcting the foreground intensities for the background noise, especially for spots with genes that are weakly expressed or not at all. The conventional approach, the additive background correction, directly subtracts the background intensities from foreground intensities. When the foreground intensities marginally dominate the background intensities, the additive background correction provides unreliable estimates of the differential gene expression levels and usually presents M-A plots with fishtails or fans. Unreliable additive background correction makes it preferable to ignore the background noise, which may increase the number of false positives. Based on the more realistic multiplicative assumption instead of the conventional additive assumption, we propose to logarithmically transform the intensity readings before the background correction, with the logarithmic transformation symmetrizing the skewed intensity readings. This approach not only precludes the fishtails and fans in the M-A plots, but provides highly reproducible background-corrected intensities for both strongly and weakly expressed genes. The superiority of the multiplicative background correction to the additive one as well as the no background correction is justified by publicly available self-hybridization datasets.     

Targeted gene correction of hprt mutations by 45 base single-stranded oligonucleotides

Targeted correction of a single base in a gene of an eucaryotic cell by specific oligonucleotides is a yet controversial technique. Here, we introduce the correction of point mutations in the hypoxanthine-guanine-phosphoribosyl-transferase (HPRT) gene as an additional model system to test targeted gene correction. In human, Hprt mutations cause Lesch-Nyhan syndrome. Using hamster V79 cells, we generated three cell lines with one hprt point mutation each. These cell lines were treated with specific single-stranded 45 base phosphothioate modified oligonucleotides and selected by HAT medium. The surviving clones were investigated for the correction of the respective hprt mutation. Treatment with the oligonucleotides was successful in repairing all three hprt mutations (hprt cDNA position 74, C --> T; position 151, C --> T; and position 400, G --> A). The correction efficiency was very low but reproducible. We suggest that this system allows one to investigate targeted gene correction in dependence on the target sequence and the oligonucleotides used.     

Effects of target sequence and sense versus anti-sense strands on gene correction with single-stranded DNA fragments

The correction of an inactivated hygromycin resistance and enhanced green fluorescent protein (Hyg-EGFP) fusion gene by a several hundred-base single-stranded (ss) DNA fragment has been reported. In this study, the effectiveness of this type of gene correction was examined for various positions in the rpsL gene. Sense and anti-sense ssDNA fragments were prepared, and the gene correction efficiencies were determined by co-introduction of the target plasmid containing the gene with the ssDNA fragments. The gene correction efficiency varied (0.8-9.3%), depending on target positions and sense/anti-sense strands. Sense ssDNA fragments corrected the target gene with equal or higher efficiencies as compared to their anti-sense counterparts. The target positions corrected with high efficiency by the sense fragments also tended to be corrected efficiently by the anti-sense fragments. These results suggest that the sense ssDNA fragments are useful for the correction of mutated genes. The variation in the correction efficiency may depend on the sequence of the target position in double-stranded DNA.     

Presurgical finite element simulation of scoliosis correction

For surgical correction of scoliotic spinal deformity, internal fixation systems apply lateral and distractive corrective forces. In order to gain maximal correction, a finite-element analysis of the spinal deformity correction technique has been carried out preoperatively, after first employing the spinal deformity correction finite-element model to determine the in vivo spinal stiffness. The presurgical analysis also gives us an appreciation of how the parameters of deformity, stiffness and corrective forces jointly contribute to the value of the correction index. The paper presents the methodology and clinical application. It also summarizes the results for ten patients, whereby the efficacy of presurgical analysis is assessed by comparing the corrective index values by presurgical simulation with the surgical results for equivalent levels of corrective forces.     

The effect of the superior colliculus on the function of the contralateral lateral geniculate body in the cat

In acute experiments on rats it was shown that stimulation of the superior colliculus [correction of upper bimounding] leads to the formation in the contralateral lateral geniculate [correction of external geniculated] body of a colliculus-geniculate response. The nature of the changes in a considerable degree is determined by the fact, to which neurones of the lateral geniculate [correction of external geniculated] body, the effect of contralateral superior colliculus [correction of upper bimounding] is addressed.     

Integral correction of end-plate potentials: A useful alternative to peak amplitude correction

Validity of correction of peak amplitudes of end-plate potentials for non-linear summation of unit potentials may be affected by membrane capacitance and asynchrony of action of unit potentials. Compensatory adjustment for the opposing and interacting effects of these factors on the correction is difficult to calculate but may be circumvented by use of an integral correction procedure described here. This procedure involves correction of the voltage of the potential over its entire time course rather than at the peak only.     

Caméras hybrides TEMP-TDM et semi-quantification du I-FPCIT : évaluation de l’apport de la TDM

Objectives

Tomoscintigraphy of dopamine transporters with ¹²³I-FP-CIT is nowadays essential to visualise impairment of nigro-striatal system for the diagnosis of parkinsonism and for the differential diagnosis of dementia. With the development of hybrid cameras (SPECT-CT), the CT contribution in nuclear neurology needs to be assessed in diagnostic and semi-quantification performances. The main purpose of our study is to compare attenuation correction using CT to attenuation correction using the linear algorithm of Chang. SPECT-CT with parallel collimation results were also weighed against fan beam collimation and the contribution of partial volume effect correction was studied in secondary objective.

Materials and methods

We used a trilinear phantom to define spatial resolution and an anthropomorphic striatal phantom to quantify the activity in striatal cavities. We compared the impact of attenuation and scatter correction on spatial resolution and semi-quantification in striatum. We performed the partial volume effect correction on reconstructed images according to the method of Rousset.

Results

Attenuation correction by CT did not improve significantly spatial resolution compared to the algorithm of Chang. The semi-quantification of ¹²³I-FPCIT in striata was not significantly different according to the various CA, but was significantly improved with CT attenuation and scatter correction. Partial volume effect correction improved the quantification from 40 to 60% in the striatal structures, when the activity was superior in at least twice the background noise.

Conclusion

SPECT-CT hybrid cameras increase spatial resolution and improve semi-quantification of ¹²³I-FPCIT because of CT attenuation and scatter correction. Another use of CT is the possibility of calibrating anatomic segmentation of striata for partial volume effect correction. Partial volume effect correction improves quantification and is essential for early diagnosis of nigro-striatal disease.     

热带季节雨林林冠碳通量不同校正方法的比较分析

利用西双版纳热带季节雨林2003年3月1～9日的CO₂和水汽通量数据,比较了不同校正方法的差异.结果表明,对季节雨林林冠碳通量观测数据进行校正是必要的,但不同校正方法所起的作用有所差异;昼间WPL校正的作用最大,虚温校正的作用最小;t检验证明,经WPL修正后的碳通量值与基准值、无显著差异;聚类分析表明,进行自然风3次旋转校正有利于数值精度的提高.夜间是自然风旋转校正的贡献最大、虚温校正贡献最小;t检验表明,经过WPL修正和平面旋转校正及其组合得到的碳通量值与基准值均有显著差异;而经包含有自然风旋转校正处理后的碳通量值与基准值均无显著差异;聚类分析表明,经过自然风坐标旋转后,再进行WPL修正,有利于数值精度的提高.     

Correction of lower eyelid ptosis in the anophthalmic orbit: a long-term follow-up

We describe a 10-year review of 53 patients having had correction of lower eyelid ptosis using fascia lata sling suspension by the operation first described in 1973. The overall conclusion is that this continues to be a reliable procedure with a low complication rate. Four major changes relating to operative technique that create a better result are as follows: (1) the surgical correction must begin with a prosthesis that is ideal for the socket; (2) the fascial strip is narrower at 2 mm; (3) the lateral orbital rim burr hole is placed higher; and (4) the passage of the fascial strip is facilitated by the use of Wright's needle. The optimal sequence of operative procedures in the anophthalmic orbit syndrome is (1) correction of enophthalmos and superior sulcus depression, (2) correction of lower eyelid ptosis, and (3) correction of upper eyelid ptosis.     

Insertion and Deletion Mismatches Distant from the Target Position Improve Gene Correction with a Tailed Duplex

A 5′-tailed duplex (TD) DNA corrects a base-substitution mutation. In this study, the effects of insertion and deletion (indel) mismatches distant from the target position on the gene correction were examined. Three target plasmid DNAs with and without indel mismatches ～330 bases distant from the correction target position were prepared, and introduced into HeLa cells together with the TD. The indel mismatches improved the gene correction efficiency and specificity without sequence conversions at the indel mismatch site. These results suggested that the gene correction efficiency and specificity are increased when an appropriate second mismatch is introduced into the TD fragment.     

Membrane capacitance and correction of end-plate potentials for nonlinear summation of quantal units

The question of effect of muscle membrane capacitance on Martin's correction of end-plate potential amplitudes for nonlinear summation of unit potentials was examined. By addition of capacitance to the membrane circuit model which serves as the basis for the correction, it is demonstrated through use of the appropriate circuit equations that the correction is unaffected by capacitance.     

Biomechanical design and long-term stability of trees: Morphological and wood traits involved in the balance between weight increase and the gravitropic reaction

Studies on tree biomechanical design usually focus on stem stiffness, resistance to breakage or uprooting, and elastic stability. Here we consider another biomechanical constraint related to the interaction between growth and gravity. Because stems are slender structures and are never perfectly symmetric, the increase in tree mass always causes bending movements. Given the current mechanical design of trees, integration of these movements over time would ultimately lead to a weeping habit unless some gravitropic correction occurs. This correction is achieved by asymmetric internal forces induced during the maturation of new wood.The long-term stability of a growing stem therefore depends on how the gravitropic correction that is generated by diameter growth balances the disturbance due to increasing self weight. General mechanical formulations based on beam theory are proposed to model these phenomena. The rates of disturbance and correction associated with a growth increment are deduced and expressed as a function of elementary traits of stem morphology, cross-section anatomy and wood properties. Evaluation of these traits using previously published data shows that the balance between the correction and the disturbance strongly depends on the efficiency of the gravitropic correction, which depends on the asymmetry of wood maturation strain, eccentric growth, and gradients in wood stiffness. By combining disturbance and correction rates, the gravitropic performance indicates the dynamics of stem bending during growth. It depends on stem biomechanical traits and dimensions. By analyzing dimensional effects, we show that the necessity for gravitropic correction might constrain stem allometric growth in the long-term. This constraint is compared to the requirement for elastic stability, showing that gravitropic performance limits the increase in height of tilted stem and branches. The performance of this function may thus limit the slenderness and lean of stems, and therefore the ability of the tree to capture light in a heterogeneous environment.     

The correction of eye blink artefacts in the EEG: a comparison of two prominent methods

Background

The study investigated the residual impact of eyeblinks on the electroencephalogram (EEG) after application of different correction procedures, namely a regression method (eye movement correction procedure, EMCP) and a component based method (Independent Component Analysis, ICA).

Methodology/Principle Findings

Real and simulated data were investigated with respect to blink-related potentials and the residual mutual information of uncorrected vertical electrooculogram (EOG) and corrected EEG, which is a measure of residual EOG contribution to the EEG. The results reveal an occipital positivity that peaks at about 250ms after the maximum blink excursion following application of either correction procedure. This positivity was not observable in the simulated data. Mutual information of vertical EOG and EEG depended on the applied regression procedure. In addition, different correction results were obtained for real and simulated data. ICA yielded almost perfect correction in all conditions. However, under certain conditions EMCP yielded comparable results to the ICA approach.

Conclusion

In conclusion, for EMCP the quality of correction depended on the EMCP variant used and the structure of the data, whereas ICA always yielded almost perfect correction. However, its disadvantage is the much more complex data processing, and that it requires a suitable amount of data.