The role of mismatch repair in the prevention of base pair mutations in Saccharomyces cerevisiae

In most organisms, the mismatch repair (MMR) system plays an important role in substantially lowering mutation rates and blocking recombination between nonidentical sequences. In Saccharomyces cerevisiae, the products of three genes homologous to Escherichia coli mutS—MSH2, MSH3, and MSH6—function in MMR by recognizing mispaired bases. To determine the effect of MMR on single-base pair mismatches, we have measured reversion rates of specific point mutations in the CYC1 gene in both wild-type and MMR-deficient strains. The reversion rates of all of the point mutations are similar in wild-type cells. However, we find that in the absence of MSH2 or MSH6, but not MSH3, reversion rates of some mutations are increased by up to 60,000-fold, whereas reversion rates of other mutations are essentially unchanged. When cells are grown anaerobically, the reversion rates in MMR-deficient strains are decreased by as much as a factor of 60. We suggest that the high reversion rates observed in these MMR-deficient strains are caused by misincorporations opposite oxidatively damaged bases and that MMR normally prevents these mutations. We further suggest that recognition of mispairs opposite damaged bases may be a more important role for MMR in yeast than correction of errors opposite normal bases.

ATM-dependent expression of the insulin-like growth factor-I receptor in a pathway regulating radiation response

The ATM gene is mutated in the syndrome of ataxia telangiectasia (AT), associated with neurologic dysfunction, growth abnormalities, and extreme radiosensitivity. Insulin-like growth factor-I receptor (IGF-IR) is a cell surface receptor with tyrosine kinase activity that can mediate mitogenesis, cell transformation, and inhibition of apoptosis. We report here that AT cells express low levels of IGF-IR and show decreased IGF-IR promoter activity compared with wild-type cells. Complementation of AT cells with the ATM cDNA results in increased IGF-IR promoter activity and elevated IGF-IR levels, whereas expression in wild-type cells of a dominant negative fragment of ATM specifically reduces IGF-IR expression, results consistent with a role for ATM in regulating IGF-IR expression at the level of transcription. When expression of IGF-IR cDNA is forced in AT cells via a heterologous viral promoter, near normal radioresistance is conferred on the cells. Conversely, in ATM cells complemented with the ATM cDNA, specific inhibition of the IGF-IR pathway prevents correction of the radiosensitivity. Taken together, these results establish a fundamental link between ATM function and IGF-IR expression and suggest that reduced expression of IGF-IR contributes to the radiosensitivity of AT cells. In addition, because IGF-I plays a major role in human growth and metabolism and serves as a survival and differentiation factor for developing neuronal tissue, these results may provide a basis for understanding other aspects of the AT syndrome, including the growth abnormalities, insulin resistance, and neurodegeneration.

Somatic mosaicism in Fanconi anemia: Evidence of genotypic reversion in lymphohematopoietic stem cells

Somatic mosaicism has been observed previously in the lymphocyte population of patients with Fanconi anemia (FA). To identify the cellular origin of the genotypic reversion, we examined each lymphohematopoietic and stromal cell lineage in an FA patient with a 2815–2816ins19 mutation in FANCA and known lymphocyte somatic mosaicism. DNA extracted from individually plucked peripheral blood T cell colonies and marrow colony-forming unit granulocyte–macrophage and burst-forming unit erythroid cells revealed absence of the maternal FANCA exon 29 mutation in 74.0%, 80.3%, and 86.2% of colonies, respectively. These data, together with the absence of the FANCA exon 29 mutation in Epstein–Barr virus-transformed B cells and its presence in fibroblasts, indicate that genotypic reversion, most likely because of back mutation, originated in a lymphohematopoietic stem cell and not solely in a lymphocyte population. Contrary to a predicted increase in marrow cellularity resulting from reversion in a hematopoietic stem cell, pancytopenia was progressive. Additional evaluations revealed a partial deletion of 11q in 3 of 20 bone marrow metaphase cells. By using interphase fluorescence in situ hybridization with an MLL gene probe mapped to band 11q23 to identify colony-forming unit granulocyte–macrophage and burst-forming unit erythroid cells with the 11q deletion, the abnormal clone was exclusive to colonies with the FANCA exon 29 mutation. Thus, we demonstrate the spontaneous genotypic reversion in a lymphohematopoietic stem cell. The subsequent development of a clonal cytogenetic abnormality in nonrevertant cells suggests that ex vivo correction of hematopoietic stem cells by gene transfer may not be sufficient for providing life-long stable hematopoiesis in patients with FA.

Formation of Holliday junctions by regression of nascent DNA in intermediates containing stalled replication forks: RecG stimulates regression even when the DNA is negatively supercoiled

Replication forks formed at bacterial origins often encounter template roadblocks in the form of DNA adducts and frozen protein–DNA complexes, leading to replication-fork stalling and inactivation. Subsequent correction of the corrupting template lesion and origin-independent assembly of a new replisome therefore are required for survival of the bacterium. A number of models for replication-fork restart under these conditions posit that nascent strand regression at the stalled fork generates a Holliday junction that is a substrate for subsequent processing by recombination and repair enzymes. We show here that early replication intermediates containing replication forks stalled in vitro by the accumulation of excess positive supercoils could be cleaved by the Holliday junction resolvases RusA and RuvC. Cleavage by RusA was inhibited by the presence of RuvA and was stimulated by RecG, confirming the presence of Holliday junctions in the replication intermediate and supporting the previous proposal that RecG could catalyze nascent strand regression at stalled replication forks. Furthermore, RecG promoted Holliday junction formation when replication intermediates in which the replisome had been inactivated were negatively supercoiled, suggesting that under intracellular conditions, the action of RecG, or helicases with similar activities, is necessary for the catalysis of nascent strand regression.

The precursor of PsaD assembles into the photosystem I complex in two steps.

The present study addresses the assembly in the chloroplast thylakoid membranes of PsaD, a peripheral membrane protein of the photosystem I complex. Located on the stromal side of the thylakoids, PsaD was found to assemble in vitro into the membranes in its precursor (pre-PsaD) and also in its mature (PsaD) form. Newly assembled unprocessed pre-PsaD was resistant to NaBr and alkaline wash. Yet it was sensitive to proteolytic digestion. In contradistinction, when the assembled precursor was processed, the resulting mature PsaD was resistant to proteases to the same extent as endogenous [correction of endogeneous] PsaD. The accumulation of protease-resistant PsaD in the thylakoids correlated with the increase of mature-PsaD in the membranes. This protection of mature PsaD from proteolysis could not be observed when PsaD was in a soluble form-i.e. not assembled within the thylakoids. The data suggest that pre-PsaD assembles to the membranes and only in a second step processing takes place. The observation that the assembly of pre-PsaD is affected by salts to a much lesser extent than that of mature-PsaD supports a two-step assembly of pre-PsaD.

Cardioprotective effects of 70-kDa heat shock protein in transgenic mice.

Heat shock proteins are proposed to limit injury resulting from diverse environmental stresses, but direct metabolic evidence for such a cytoprotective function in vertebrates has been largely limited to studies of cultured cells. We generated lines of transgenic mice to express human 70-kDa heat shock protein constitutively in the myocardium. Hearts isolated from these animals demonstrated enhanced recovery of high energy phosphate stores and correction of metabolic acidosis following brief periods of global ischemia sufficient to induce sustained abnormalities of these variables in hearts from nontransgenic littermates. These data demonstrate a direct cardioprotective effect of 70-kDa heat shock protein to enhance postischemic recovery of the intact heart.

Toward the therapeutic editing of mutated RNA sequences.

If RNA editing could be rationally directed to mutated RNA sequences, genetic diseases caused by certain base substitutions could be treated. Here we use a synthetic complementary RNA oligonucleotide to direct the correction of a premature stop codon mutation in dystrophin RNA. The complementary RNA oligonucleotide was hybridized to a premature stop codon and the hybrid was treated with nuclear extracts containing the cellular enzyme double-stranded RNA adenosine deaminase. When the treated RNAs were translated in vitro, a dramatic increase in expression of a downstream luciferase coding region was observed. The cDNA sequence data are consistent with deamination of the adenosine in the UAG stop codon to inosine by double-stranded RNA adenosine deaminase. Injection of oligonucleotide-mRNA hybrids into Xenopus embryos also resulted in an increase in luciferase expression. These experiments demonstrate the principle of therapeutic RNA editing.

Phenotype correction in retinal pigment epithelium in murine mucopolysaccharidosis VII by adenovirus-mediated gene transfer.

We have studied the use of adenovirus-mediated gene transfer to reverse the pathologic changes of lysosomal storage disease caused by beta-glucuronidase deficiency in the eyes of mice with mucopolysaccharidosis VII. A recombinant adenovirus carrying the human beta-glucuronidase cDNA coding region under the control of a non-tissue-specific promoter was injected intravitreally or subretinally into the eyes of mice with mucopolysaccharidosis VII. At 1-3 weeks after injection, the treated and control eyes were examined histochemically for beta-glucuronidase expression and histologically for phenotypic correction of the lysosomal storage defect. Enzymatic expression was detected 1-3 weeks after injection. Storage vacuoles in the retinal pigment epithelium (RPE) were still present 1 week after gene transfer but were reduced to undetectable levels by 3 weeks in both intravitreally and subretinally injected eyes. There was minimal evidence of ocular pathology associated with the viral injection. These data indicate that adenovirus-mediated gene transfer to the eye may provide for adjunctive therapy for lysosomal storage diseases affecting the RPE in conjunction with enzyme replacement and/or gene therapies for correction of systemic disease manifestations. The data also support the view that recombinant adenovirus may be useful as a gene therapy vector for retinal degenerations that result from a primary genetic defect in the RPE cells.

Visual and optical performance and quality of life after implantation of posterior chamber phakic intraocular lens

Purpose To evaluate visual, optical, and quality of life (QoL) outcomes and intercorrelations after bilateral implantation of posterior chamber phakic intraocular lenses. Methods Twenty eyes with high to moderate myopia of 10 patients that underwent PRL implantation (Phakic Refractive Lens, Carl Zeiss Meditec AG) were examined. Refraction, visual acuity, photopic and low mesopic contrast sensitivity (CS) with and without glare, ocular aberrations, as well as QoL outcomes (National Eye Institute Refractive Error Quality of Life Instrument-42, NEI RQL-42) were evaluated at 12 months postoperatively. Results Significant improvement in uncorrected (UDVA) and best-corrected distance (CDVA) visual acuities were found postoperatively (p < 0.01), with significant reduction in spherical equivalent (p < 0.01). Low mesopic CS without glare was significantly better than measurements with glare for 1.5, 3, and 6 cycles/degree (p < 0.01). No significant correlations between higher order root mean square (RMS) with CDVA (r = −0.26, p = 0.27) and CS (r ≤ 0.45, p ≥ 0.05) were found. Postoperative binocular photopic CS for 12 cycles/degree and 18 cycles/degree correlated significantly with several RQL-42 scales. Glare index correlated significantly with CS measures and scotopic pupil size (r = −0.551, p = 0.04), but not with higher order RMS (r = −0.02, p = 0.94). Postoperative higher order RMS, postoperative primary coma and postoperative spherical aberration was significant higher for 5-mm pupil diameter (p < 0.01) compared with controls. Conclusions Correction of moderate to high myopia by means of PRL implantation had a positive impact on CS and QoL. The aberrometric increase induced by the surgery does not seem to limit CS and QoL. However, perception of glare is still a relevant disturbance in some cases possibly related to the limitation of the optical zone of the PRL.

Analysis of internal astigmatism and higher order aberrations in eyes implanted with a new diffractive multifocal toric intraocular lens

Background To evaluate the intraocular lens (IOL) position by analyzing the postoperative axis of internal astigmatism as well as the higher-order aberration (HOA) profile after cataract surgery following the implantation of a diffractive multifocal toric IOL. Methods Prospective study including 51 eyes with corneal astigmatism of 1.25D or higher of 29 patients with ages ranging between 20 and 61 years old. All cases underwent uneventful cataract surgery with implantation of the AT LISA 909 M toric IOL (Zeiss). Visual, refractive and corneal topograpy changes were evaluated during a 12-month follow-up. In addition, the axis of internal astigmatism as well as ocular, corneal, and internal HOA (5-mm pupil) were evaluated postoperatively by means of an integrated aberrometer (OPD Scan II, Nidek). Results A significant improvement in uncorrected distance and near visual acuities (p < 0.01) was found, which was consistent with a significant correction of manifest astigmatism (p < 0.01). No significant changes were observed in corneal astigmatism (p = 0.32). With regard to IOL alignment, the difference between the axes of postoperative internal and preoperative corneal astigmatisms was close to perpendicularity (12 months, 87.16° ± 7.14), without significant changes during the first 6 months (p ≥ 0.46). Small but significant changes were detected afterwards (p = 0.01). Additionally, this angular difference correlated with the postoperative magnitude of manifest cylinder (r = 0.31, p = 0.03). Minimal contribution of intraocular optics to the global magnitude of HOA was observed. Conclusions The diffractive multifocal toric IOL evaluated is able to provide a predictable astigmatic correction with apparent excellent levels of optical quality during the first year after implantation.

Pilot study of hyperopic LASIK using the solid-state laser technology

Background To evaluate and report the visual, refractive, and aberrometric outcomes of LASIK for the correction of low to moderate hyperopia in a pilot group using a commercially available solid-state laser. Methods Prospective pilot study including 11 consecutive eyes with low to moderate hyperopia of six patients undergoing LASIK surgery using the Pulzar Z1 solid-state laser (CustomVis Laser Pty Ltd., currently CV Laser). Visual, refractive, and aberrometric changes were evaluated. Potential complications were evaluated as well. Mean follow-up time was 6.6 months (range, 3 to 11 months). Results A significant improvement in LogMAR uncorrected distance visual acuity (UDVA) was observed postoperatively (p = 0.01). No significant change was detected in LogMAR corrected distance visual acuity (CDVA) (p = 0.21). Postoperative LogMAR UDVA was 0.1 (about 20/25) or better in ten eyes (90.9 %). Mean overall efficacy and safety indices were 1.03 and 1.12. Postoperatively, no losses of lines of CDVA were observed. Postoperative spherical equivalent was within ±1.00 D in ten eyes (90.9 %). With regard to aberrations, no statistically significant changes were found in higher order and primary coma RMS postoperatively (p ≥ 0.21), and only minimal but statistically significant negativization of primary spherical aberration (p = 0.02) was observed. No severe complications were observed. Conclusion LASIK surgery using the solid-state laser technology seems to be a useful procedure for the correction of low to moderate hyperopia, with minimal induction of higher order aberrations.

Algorithm for Correcting the Keratometric Error in the Estimation of the Corneal Power in Eyes With Previous Myopic Laser Refractive Surgery

Purpose: To calculate theoretically the errors in the estimation of corneal power when using the keratometric index (nk) in eyes that underwent laser refractive surgery for the correction of myopia and to define and validate clinically an algorithm for minimizing such errors. Methods: Differences between corneal power estimation by using the classical nk and by using the Gaussian equation in eyes that underwent laser myopic refractive surgery were simulated and evaluated theoretically. Additionally, an adjusted keratometric index (nkadj) model dependent on r1c was developed for minimizing these differences. The model was validated clinically by retrospectively using the data from 32 myopic eyes [range, −1.00 to −6.00 diopters (D)] that had undergone laser in situ keratomileusis using a solid-state laser platform. The agreement between Gaussian (PGaussc) and adjusted keratometric (Pkadj) corneal powers in such eyes was evaluated. Results: It was found that overestimations of corneal power up to 3.5 D were possible for nk = 1.3375 according to our simulations. The nk value to avoid the keratometric error ranged between 1.2984 and 1.3297. The following nkadj models were obtained: nkadj= −0.0064286r1c + 1.37688 (Gullstrand eye model) and nkadj = −0.0063804r1c + 1.37806 (Le Grand). The mean difference between Pkadj and PGaussc was 0.00 D, with limits of agreement of −0.45 and +0.46 D. This difference correlated significantly with the posterior corneal radius (r = −0.94, P < 0.01). Conclusions: The use of a single nk for estimating the corneal power in eyes that underwent a laser myopic refractive surgery can lead to significant errors. These errors can be minimized by using a variable nk dependent on r1c.

Influence of the Difference Between Corneal and Refractive Astigmatism on LASIK Outcomes Using Solid-State Technology

Purpose: To evaluate the influence of the difference between preoperative corneal and refractive astigmatism [ocular residual astigmatism (ORA)] on outcomes obtained after laser in situ keratomileusis (LASIK) surgery for correction of myopic astigmatism using the solid-state laser technology. Methods: One hundred one consecutive eyes with myopia or myopic astigmatism of 55 patients undergoing LASIK surgery using the Pulzar Z1 solid-state laser (CustomVis Laser Pty Ltd, currently CV Laser) were included. Visual and refractive changes at 6 months postoperatively and changes in ORA and anterior corneal astigmatism and posterior corneal astigmatism (PCA) were analyzed. Results: Postoperatively, uncorrected distance visual acuity improved significantly (P < 0.01). Likewise, refractive cylinder magnitude and spherical equivalent were reduced significantly (P < 0.01). In contrast, no significant changes were observed in ORA magnitude (P = 0.81) and anterior corneal astigmatism (P = 0.12). The mean overall efficacy and safety indices were 0.96 and 1.01, respectively. These indices were not correlated with preoperative ORA (r = −0.15, P = 0.15). Furthermore, a significant correlation was found between ORA (r = 0.81, P < 0.01) and PCA postoperatively, but not preoperatively (r = 0.12, P = 0.25). Likewise, a significant correlation of ORA with manifest refraction was only found postoperatively (r = −0.38, P < 0.01). Conclusions: The magnitude of ORA does not seem to be a predictive factor of efficacy and safety of myopic LASIK using a solid-state laser platform. The higher relevance of PCA after surgery in some cases may explain the presence of unexpected astigmatic residual refractive errors.

Fixed Mydriatic Pupil Associated with an Intraocular Pressure Rise as a Complication of the Implant of a Phakic Refractive Lens (PRL)

We describe a case report of a patient that was implanted with a posterior chamber phakic intraocular lens (Phakic Refractive Lens, PRL) for the correction of moderate myopia and who developed postoperatively a fixed mydriasis compatible with an Urrets-Zavalia Syndrome (UZS). Specifically, a sudden acute increase of IOP in the left eye was observed in the immediate postoperative period. After IOP stabilization, the refractive result was good, but a fixed and mydriatic pupil appeared. This condition led the patient to experience visual discomfort, halos, and glare associated with high levels of higher-order aberrations in spite of the good visual result. A tinted-contact lens was fitted in order to minimize those symptoms. The UZS should be considered as a possible complication after implantation of posterior chamber phakic intraocular lenses.

Construction of Structures I in Moodle: a new teaching methodology in the Degree of Building Engineer

The subject of Construction of Structures I studies, from a constructive point of view and taking into account current legislation, reinforced concrete structures used in buildings, through the acquisition of knowledge and construction criteria required in the profession of a Technical Architect. The contents acquired in this course are essential for further professional development of technicians and are closely related to many of the subjects taught in the same or other courses of the Degree in Technical Architecture at the University of Alicante. The aim of this paper is to present, analyze and discuss the development of a new methodology proposed in the mentioned subject, as it supposed an important change in the traditional way of teaching Construction and Structures I. In order to incorporate new teaching tools in 2013-2014, the course has been implemented by using a Moodle software tool to promote blended learning with online exercises. Our Moodle community allows collaborative work within an open-source platform where teachers and students share a new and personalized learning environment. Students are easily used to the interface and the platform, value the constant connection with teachers or other fellows and completely agree with the possibility of making questions or share documents 24 hours a day. The proposed methodology consists of lectures and practical classes. In the lectures, the basics of each topic are discussed; class attendance, daily study and conducting scheduled exercises are indispensable. Practical classes allow to consolidate the knowledge gained in theory classes by solving professional exercises and actual construction problems related to structures, that shall be compulsorily delivered online. So, after the correction of the teacher and the subsequent feedback of students, practical exercises ensure lifelong learning of the student, who can download any kind of material at any time (constructive details, practical exercises and even corrected exams). Regarding the general evaluation system, goals achievement is assessed on an ongoing basis (65% of the final mark) along the course through written and graphic evidences in person and online, as well as a individual development of a workbook. In all cases, the acquisition of skills, the ability to synthesize, the capacity of logical and critical thinking are assessed. The other 35 % of the mark is evaluated by a complementary graphic exam. Participation in the computing platform is essential and the student is required to do and present, at least 90% of the practices proposed. Those who do not comply with the practices in each specific date could not be assessed continuously and may only choose the final exam. In conclusion, the subject of Construction of Structures I is essential in the development of the regulated profession of Technical Architect as they are considered, among other professional profiles, as specialists in construction of building structures. The use of a new communication platform and online teaching allows the acquisition of knowledge and constructive approaches in a continuous way, with a more direct and personal monitoring by the teacher that has been highly appreciated by almost 100% of the students. Ultimately, it is important to say that the use of Moodle in this subject is a very interesting tool, which was really well welcome by students in one of the densest and important subjects of the Degree of Technical Architecture.