Multivalent interactions of the disordered regions of XLF and XRCC4 foster robust cellular NHEJ and drive the formation of ligation-boosting condensates in vitro.

In mammalian cells, DNA double-strand breaks are predominantly repaired by non-homologous end joining (NHEJ). During repair, the Ku70/80 heterodimer (Ku), XRCC4 in complex with DNA Ligase 4 (X4L4), and XLF form a flexible scaffold that holds the broken DNA ends together. Insights into the architectural organization of the NHEJ scaffold and its regulation by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) have recently been obtained by single-particle cryo-electron microscopy analysis. However, several regions, especially the C-terminal regions (CTRs) of the XRCC4 and XLF scaffolding proteins, have largely remained unresolved in experimental structures, which hampers the understanding of their functions. Here, we used magnetic resonance techniques and biochemical assays to comprehensively characterize the interactions and dynamics of the XRCC4 and XLF CTRs at atomic resolution. We show that the CTRs of XRCC4 and XLF are intrinsically disordered and form a network of multivalent heterotypic and homotypic interactions that promotes robust cellular NHEJ activity. Importantly, we demonstrate that the multivalent interactions of these CTRs led to the formation of XLF and X4L4 condensates in vitro which can recruit relevant effectors and critically stimulate DNA end ligation. Our work highlights the role of disordered regions in the mechanism and dynamics of NHEJ and lays the groundwork for the investigation of NHEJ protein disorder and its associated condensates inside cells with implications in cancer biology, immunology and the development of genome editing strategies.

Refractive Index Fiber Laser Sensor by Using a Fiber Ball Lens Interferometer with Adjustable Free Spectral Range.

In this work, a fiber laser refractometer based on a fiber ball lens (FBL) interferometer is proposed. The linear cavity erbium-doped fiber laser uses an FBL structure acting as a spectral filter and sensing element for determining the RI of a liquid medium surrounding the fiber. The optical interrogation of the sensor is the wavelength displacement of the generated laser line as a function of the RI variations. For the proposed FBL interferometric filter, the free spectral range of its wavelength-modulated reflection spectrum is adjusted to maximum in order to obtain RI measurements in a range of 1.3939 to 1.4237 RIU, from laser wavelength displacements in a range from 1532.72 to 1565.76 nm. The obtained results show that the wavelength of the generated laser line is a linear function of the RI variations on the medium surrounding the FBL with a sensitivity of 1130.28 nm/RIU. The reliability of the proposed fiber laser RI sensor is analytically and experimentally investigated.

The DNA binding domain and the C-terminal region of DNA Ligase IV specify its role in V(D)J recombination.

DNA Ligase IV is responsible for the repair of DNA double-strand breaks (DSB), including DSBs that are generated during V(D)J recombination. Like other DNA ligases, Ligase IV contains a catalytic core with three subdomains-the DNA binding (DBD), the nucleotidyltransferase (NTD), and the oligonucleotide/oligosaccharide-fold subdomain (OBD). Ligase IV also has a unique C-terminal region that includes two BRCT domains, a nuclear localization signal sequence and a stretch of amino acid that participate in its interaction with XRCC4. Out of the three mammalian ligases, Ligase IV is the only ligase that participates in and is required for V(D)J recombination. Identification of the minimal domains within DNA Ligase IV that contribute to V(D)J recombination has remained unresolved. The interaction of the Ligase IV DNA binding domain with Artemis, and the interaction of its C-terminal region with XRCC4, suggest that both of these regions that also interact with the Ku70/80 heterodimer are important and might be sufficient for mediating participation of DNA Ligase IV in V(D)J recombination. This hypothesis was investigated by generating chimeric ligase proteins by swapping domains, and testing their ability to rescue V(D)J recombination in Ligase IV-deficient cells. We demonstrate that a fusion protein containing Ligase I NTD and OBDs flanked by DNA Ligase IV DBD and C-terminal region is sufficient to support V(D)J recombination. This chimeric protein, which we named Ligase 37, complemented formation of coding and signal joints. Coding joints generated with Ligase 37 were shorter than those observed with wild type DNA Ligase IV. The shorter length was due to increased nucleotide deletions and decreased nucleotide insertions. Additionally, overexpression of Ligase 37 in a mouse pro-B cell line supported a shift towards shorter coding joints. Our findings demonstrate that the ability of DNA Ligase IV to participate in V(D)J recombination is in large part mediated by its DBD and C-terminal region.

TiOxNy Thin Film Sputtered on a Fiber Ball Lens as Saturable Absorber for Passive Q-Switched Generation of a Single-Tunable/Dual-Wavelength Er-Yb Double Clad Fiber Laser.

The use of titanium oxynitride (TiOxNy) thin films as a saturable absorber (SA) element for generation of passive Q-switched (PQS) laser pulses, from a linear cavity Er-Yb double-clad fiber (EYDCF) laser, is demonstrated. Additionally, the deposition of the material as a thin film covering a fiber micro-ball lens (MBL) structure is reported for the first time. The TiOxNy coating is deposited by a direct current (DC) magnetron-sputtering technique. The MBL is inserted within the laser cavity in a reflection configuration, alongside a reflecting mirror. As a result, the coated fiber MBL simultaneously acts as a SA element for PQS laser pulses generation and as an interference filter for wavelength selection and tuning of the generated laser line. Tunable single-laser emission in a wavelength range limited by dual-wavelength laser generation at 1541.96 and 1547.04 nm is obtained. PQS laser pulses with a repetition rate from 18.67 to 124.04 kHz, minimum pulse duration of 3.57 µs, maximum peak power of 0.359 W, and pulse energy of 1.28 µJ were obtained in a pump power range from 1 to 1.712 W.

Bilateral enucleation at birth modifies calcium spike amplitude, but not frequency, in neurons of the somatosensory thalamus and cortex: Implications for developmental cross-modal plasticity.

Bilateral eye enucleation at birth (BE) leads to an expansion of the primary somatosensory cortex (S1) in rat pups. Although increased growth of the somatosensory thalamo-cortical afferents (STCAs) in part explains S1 expansion, timing mechanisms governing S1 formation are also involved. In this work, we begin the search of a developmental clock by intending to document the existence of putative clock neurons in the somatosensory thalamus (VPM) and S1 based upon changes of spontaneous spike amplitude; a biophysical property sensitive to circadian regulation; the latter known to be shifted by enucleation. In addition, we also evaluated whether STCAs growth rate and segregation timing were modified, as parameters the clock might time. We found that spontaneous spike amplitude transiently, but significantly, increased or decreased in VPM and S1 neurons of BE rat pups, respectively, as compared to their control counterparts. The growth rate and segregation timing of STCAs was, however, unaffected by BE. These results support the existence of a developmental clock that ticks differently in the VPM and S1 after BE. This observation, together with the fact that STCAs growth rate and segregation timing is unchanged, suggests that S1 expansion in BE rats may in part be controlled at the cortical level.

Lossy Mode Resonance Generation on Sputtered Aluminum-Doped Zinc Oxide Thin Films Deposited on Multimode Optical Fiber Structures for Sensing Applications in the 1.55 µm Wavelength Range.

In this work, we demonstrated lossy mode resonance (LMR) generation in optical fiber structures based on multimode fibers coated with aluminum-doped zinc oxide (AZO) films. AZO thin films were deposited by using radio frequency magnetron sputtering. In order to exhibit the usefulness of the LMR effect for sensing applications in optical fiber based systems, the deposition conditions of the AZO film coatings were set to obtain the second LMR order within the 1.55 µm wavelength range. An optical transmission configuration setup was used to investigate the LMR effect on fiber structures based on the use of no-core and cladding-removed multimode fibers coated with AZO films synthesized from metallic sputtering targets with different proportions of Zn:Al, 92:8% and 98:2%, at atomic concentrations. The optical and electrical/chemical features of the AZO films were characterized with UV-vis and XPS spectroscopy, respectively. The optical response of the proposed sensing configuration to refractive index (RI) variations was experimentally demonstrated. For the best approach, the sensitivity of wavelength displacement to RI variations on the liquid surrounding media was found to be 1214.7 nm/RIU.

Biallelic mutations in DNA ligase 1 underlie a spectrum of immune deficiencies.

We report the molecular, cellular, and clinical features of 5 patients from 3 kindreds with biallelic mutations in the autosomal LIG1 gene encoding DNA ligase 1. The patients exhibited hypogammaglobulinemia, lymphopenia, increased proportions of circulating γδT cells, and erythrocyte macrocytosis. Clinical severity ranged from a mild antibody deficiency to a combined immunodeficiency requiring hematopoietic stem cell transplantation. Using engineered LIG1-deficient cell lines, we demonstrated chemical and radiation defects associated with the mutant alleles, which variably impaired the DNA repair pathway. We further showed that these LIG1 mutant alleles are amorphic or hypomorphic, and exhibited variably decreased enzymatic activities, which lead to premature release of unligated adenylated DNA. The variability of the LIG1 genotypes in the patients was consistent with that of their immunological and clinical phenotypes. These data suggest that different forms of autosomal recessive, partial DNA ligase 1 deficiency underlie an immunodeficiency of variable severity.

All-fiber laser with simultaneous Tm3+ passive Q-switched and Ho3+ gain-switched operation.

We experimentally demonstrate simultaneous Tm3+ passive Q-switched (PQS) and Ho3+ gain-switched laser operations at 1888.8 and 2021.2 nm, respectively, in a single-cavity all-fiber laser. The PQS operation of the Tm3+ laser is based on the use of a high-concentration holmium-doped fiber as a fiber saturable absorber. Then the Tm3+ laser emission is used as a pulsed pump source to achieve Ho3+ gain-switched pulses. A high birefringence fiber optical loop mirror used as a spectral filter allows the tuning of both Tm3+ and Ho3+ laser emissions.

All-Fiber Laser Curvature Sensor Using an In-Fiber Modal Interferometer Based on a Double Clad Fiber and a Multimode Fiber Structure.

An all-fiber curvature laser sensor by using a novel modal interference in-fiber structure is proposed and experimentally demonstrated. The in-fiber device, fabricated by fusion splicing of multimode fiber and double-clad fiber segments, is used as wavelength filter as well as the sensing element. By including a multimode fiber in an ordinary modal interference structure based on a double-clad fiber, the fringe visibility of the filter transmission spectrum is significantly increased. By using the modal interferometer as a curvature sensitive wavelength filter within a ring cavity erbium-doped fiber laser, the spectral quality factor Q is considerably increased. The results demonstrate the reliability of the proposed curvature laser sensor with advantages of robustness, ease of fabrication, low cost, repeatability on the fabrication process and simple operation.

Structure and Dynamics of an Intrinsically Disordered Protein Region That Partially Folds upon Binding by Chemical-Exchange NMR.

Many intrinsically disordered proteins (IDPs) and protein regions (IDRs) engage in transient, yet specific, interactions with a variety of protein partners. Often, if not always, interactions with a protein partner lead to partial folding of the IDR. Characterizing the conformational space of such complexes is challenging: in solution-state NMR, signals of the IDR in the interacting region become broad, weak, and often invisible, while X-ray crystallography only provides information on fully ordered regions. There is thus a need for a simple method to characterize both fully and partially ordered regions in the bound state of IDPs. Here, we introduce an approach based on monitoring chemical exchange by NMR to investigate the state of an IDR that folds upon binding through the observation of the free state of the protein. Structural constraints for the bound state are obtained from chemical shifts, and site-specific dynamics of the bound state are characterized by relaxation rates. The conformation of the interacting part of the IDR was determined and subsequently docked onto the structure of the folded partner. We apply the method to investigate the interaction between the disordered C-terminal region of Artemis and the DNA binding domain of Ligase IV. We show that we can accurately reproduce the structure of the core of the complex determined by X-ray crystallography and identify a broader interface. The method is widely applicable to the biophysical investigation of complexes of disordered proteins and folded proteins.

Proteinases during Early Development of the Pacific Whiteleg Shrimp Penaeus vannamei.

During shrimp larval development, changes occur in molecular components. Enzyme activity and mRNA expression of proteinases were assayed in Penaeus vannamei during larval development, which consists of 5 nauplius stages, 3 protozoeal stages, 3 mysis stages, and 12 postlarval stages. Trypsin activity reached a maximum at the beginning of postlarval stages 1 and 2, and significantly decreased in subsequent postlarval stages. Chymotrypsin activity increased at the third protozoeal stage, then significantly decreased in subsequent stages. Identification of proteinase by mass spectrometry and inhibitors allowed us to track their appearance in zymograms and to distinguish between isoenzymes. Chymotrypsin BI and BII had a distinguishing pattern of appearance during larval development, which could compensate for the reduction in trypsin activity. The mRNA content of isotrypsin 21, chymotrypsin 1, and zinc proteinase was differentially expressed in larvae. Zinc proteinase and chymotrypsin 1 mRNA were expressed at a basal content at the beginning of the protozoeal stages, increased by the end of the mysis stages and onward, while isotrypsin 21 mRNA had a peak at mysis stage 3. Transcript changes reflect transcriptional regulation of the proteinases tested. Proteinase mRNA in tissues, other than the digestive gland, suggests potentially different roles besides digestion during ontogeny.

Effectiveness of current disinfection procedures against biofilm on contaminated GI endoscopes.

BACKGROUND AND AIMS: Attention to patient safety has increased recently due to outbreaks of nosocomial infections associated with GI endoscopy. The aim of this study was to evaluate current cleaning and disinfection procedures of endoscope channels with high bioburden and biofilm analysis, including the use of resistant mycobacteria associated with postsurgical infections in Brazil. METHODS: Twenty-seven original endoscope channels were contaminated with organic soil containing 10(8) colony-forming units/mL of Pseudomonas aeruginosa, Staphylococcus aureus, or Mycobacterium abscessus subsp bolletii. Biofilms with the same microorganisms were developed on the inner surface of channels with the initial inoculum of 10(5) colony-forming units/mL. Channels were reprocessed following current protocol, and samples from cleaning and disinfection steps were analyzed by bioluminescence for adenosine triphosphate, cultures for viable microorganisms, and confocal microscopy. RESULTS: After contamination, adenosine triphosphate levels increased dramatically, and high bacterial growth was observed in all cultures. After cleaning, adenosine triphosphate levels decreased to values comparable to precontamination levels, and bacterial growth was demonstrated in 5 of 27 catheters, 2 with P aeruginosa and 3 with M abscessus. With regard to induced biofilm, a remarkable reduction occurred after cleaning, but significant microbial growth inhibition occurred only after disinfection. Nevertheless, viable microorganisms within the biofilm were still detected by confocal microscopy, more so with glutaraldehyde than with peracetic acid or O-phataladehyde. CONCLUSION: After the complete disinfection procedure, viable microorganisms could still be detected within the biofilm on endoscope channels. Prevention of biofilm development within endoscope channels should be a priority in disinfection procedures, particularly for ERCP and EUS.

Biochemical characterisation of chymotrypsin from the midgut gland of yellowleg shrimp, Penaeus californiensis.

Chymotrypsin from shrimp, Penaeus californiensis, was compared to Bos taurus chymotrypsin, and its structure-function relationship was studied. Catalytic efficiency toward synthetic substrate is lower, but it has a broad specificity and higher activity toward protein substrates, including collagen. It is active at pH 4-10 and fully active up to 50 °C for 2 h and at least nine days at room temperature. The activation peptide is twice as long as bovine chymotrypsinogen, has less disulfide bridges, and is a single polypeptide. Only one activation step is necessary from chymotrypsinogen to the mature enzyme. Postmortem implications in muscle softening and melanisation, resistance to temperature and pH and efficiency with proteinaceous substrates make chymotrypsin useful as a biotechnological tool in food processing. This makes shrimp processing wastes useful as a material for production of fine reagents.

Conocimiento de los pacientes sobre el consentimiento informado en un hospital general / Patients' knowledge about informed consent in a general hospital

Resumen Introducción: El consentimiento informado es un documento escrito firmado por el paciente o su representante legal en el que acepta, bajo debida información otorgada por el médico, los riesgos y beneficios esperados. Objetivo: Determinar el grado de conocimiento del consentimiento informado en los pacientes de un hospital general de zona. Método: Encuesta transversal. Se estudiaron 220 pacientes (tamaño muestral determinado) hospitalizados por tamaño muestral, seleccionados por aleatorización sistemática en los servicios de cirugía, pediatría y medicina interna. Se construyó y aplicó un instrumento válido y consistente con 13 reactivos (validado por dos anestesiólogos, un pediatra, un urgenciólogo y un intensivista en dos rondas, miembros del Comité de Bioética) que indagaban edad, género, escolaridad, autorización y tipo de atención médica, responsable legal, lectura completa del documento, causas de no lectura, información médica previa firma. Variable dependiente: conocimiento general (una pregunta con 5 reactivos, un punto para cada uno si se conocía qué indagaban las características del consentimiento), los resultados se clasificaron como sabe mucho 5 puntos, sabe poco 2 a 3 puntos, y no sabe 0 a 1 puntos y además del trato del personal hospitalario. Encuesta autoaplicada. Se incluyeron documentos con las firmas del paciente, del médico, dos testigos y procedimiento a autorizar. Se eliminaron encuestas incompletas. Las encuestas se cotejaron con el expediente. Se determinó la consistencia del instrumento y se compararon las respuestas cualitativas por chi cuadrada. Resultados: Se estudiaron 74 pacientes por servicio; 26% ignoraba el consentimiento informado y 63% tuvo poco conocimiento, aun así 62% lo firmó. Sólo 56% de los expedientes tuvieron consentimiento considerado válido. Conclusiones: La mayoría de los pacientes ignora a qué se refiere el consentimiento informado, pero aun así lo firman. El índice de consentimientos informados en los expedientes fue deficiente.

Abstract Introduction: The informed consent is a written document signed by the patient or by his legal representative, in which risks and benefits are understood and accepted, once the physician has informed them about medical procedures to be performed. Objective: To determine the level of knowledge about the informed consent in patients from a general hospital. Methods: Transversal survey. 220 hospitalized patients of surgery from pediatrics and internal medicine wards were studied, sampling was chosen randomly, systematically and by ward. A validated and consistent instrument was applied consisting of 13 questions (validated by two anesthesiologists, one pediatrician, one emergency physician and one intensive care physician in two rounds, which were members of the Bioethics Committee), that explored age, genre, education, authorization and type of medical care, the legal responsible, full reading of the document, reasons for not reading, and medical information before signature. The general knowledge was the dependent variable (explored through a question with five possible answers, each one worth one point, that analyzed the document characteristics, classifying results as "knows a lot" with five points, "knows little" two to three points and "doesn't know" with 0 to one point. The survey was self-applied. A document was valid when it included the signatures of the patient, the physician, and two witnesses, as well as the proposed procedure. Incomplete surveys were eliminated. The surveys were compared to the clinical file. The consistency was determined and the qualitative answers were compared through chi-square. Results: 74 patients by service; 26% had no knowledge about the informed consent, 63% had limited knowledge, even though 62% signed the document. Only 56% of the clinical files had a valid informed consent. Conclusion: the majority of patients ignore the informed consent, although they still sign it. The rate of informed consents present in clinical files was deficient.

Escasa lectura crítica de investigación entre los alumnos de pre grado de medicina / Little critical reading of research at an undergraduate medical internship

Introducción: La lectura crítica de la investigación está escasamente desarrollada entre los internos de pregrado y los programas institucionales la ignoran. Informes transversales lo confirman, así como intervenciones exitosas. Los estudios longitudinales son escasos. Objetivo: Evaluar el desarrollo y la persistencia de la lectura crítica de investigación en un internado de pregrado con énfasis en investigación (IPI). Material y métodos: Estudio de cohortes; cinco grupos con 77 alumnos, aleatorizados, de cinco universidades, con cinco profesores (diferentes años de docencia). Estrategia: Frecuencia semanal (90 min); lectura de artículo y resolución de guía, discusión plenaria (profesor y compañeros) y elaboración de protocolo. Se aplicó (inicio, 6 y 11 meses) un instrumento de 108 reactivos, equilibrado (interpretación, juicio y propuestas), validado, consistente, con seis resúmenes (encuesta, instrumento, casos y controles, ensayos controlados y aleatorizados [ECA], prueba diagnóstica y cohorte). Estadísticos: No paramétricos intragrupo e intergrupo y el azar. Resultados: No se observaron diferencias iniciales entre los promedios universitarios, medianas globales (13, rango 10 a 17), o sub componentes. Se observaron diferencias posteriores al curso en la calificación global a favor del G2 (mediana 23, rango: 22 a 39); todos los grupos avanzaron (p < 0.05). El avance se mantiene en la calificación global final (mediana 29, rango: 23 a 35). El azar disminuyó (60% a 8%) del inicio al final. No hubo avances en ECA y cohortes. Conclusiones: El aprendizaje de la investigación es fundamental en el pregrado y permite evaluar las publicaciones médicas. Un programa y actividades específicas que incluyan este aprendizaje, así como la guía por parte de los docentes con la implementación de estrategias participativas permitirán desarrollar esta habilidad que persistirá a mediano plazo

Introduction: Critical reading of research papers is poorly developed among undergraduate interns, and institutional programs ignore it. Cross-sectional reports confirm this, as well as successful interventions. Longitudinal studies are scarce. Objective: To evaluate the development and continuity of critical reading of research papers in undergraduate internship with emphasis on research. Material and methods: co-hort study; five groups with 77 students, randomized, from five universities, with five teachers (of varied teaching experience). Strategy: Weekly basis (90 min); reading of an article and guidance resolution, plenary discussion (teacher and classroom) and creation of a protocol. A balanced (interpretation, judg-ment and proposals), validated, consistent 108-item instrument was applied (baseline, 6-11 months) plus six abstracts (survey, instrument, cases and controls, randomized controlled trials [RCTs], diagnostic test and cohort). Statistics: Nonparametric, intra- and intergroup, randomized analysis. Results: No baseline differences between the grade-point averages, median global rating (13, range 10 to 17), or subcom-ponents were observed. Overall rating favored G2 (median 23, range 22-39) by the end of the course; all groups performed better (p < 0.05). Progress was maintained at the overall final rating (median 29, range: 23-35). Random performance decreased (60% to 8%) from beginning to end. No progress was observed in RCTs or cohorts. Conclusions: Learning about research is essential in undergraduate studies and allows medical literature to be assessed. A study program and specific activities including it, and a guide from teachers with participatory strategies, help develop this skill, which is maintained in the medium term.

DNA Ligase IV regulates XRCC4 nuclear localization.

DNA Ligase IV, along with its interacting partner XRCC4, are essential for repairing DNA double strand breaks by non-homologous end joining (NHEJ). Together, they complete the final ligation step resolving the DNA break. Ligase IV is regulated by XRCC4 and XLF. However, the mechanism(s) by which Ligase IV control the NHEJ reaction and other NHEJ factor(s) remains poorly characterized. Here, we show that a C-terminal region of Ligase IV (aa 620-800), which encompasses a NLS, the BRCT I, and the XRCC4 interacting region (XIR), is essential for nuclear localization of its co-factor XRCC4. In Ligase IV deficient cells, XRCC4 showed deregulated localization remaining in the cytosol even after induction of DNA double strand breaks. DNA Ligase IV was also required for efficient localization of XLF into the nucleus. Additionally, human fibroblasts that harbor hypomorphic mutations within the Ligase IV gene displayed decreased levels of XRCC4 protein, implicating that DNA Ligase IV is also regulating XRCC4 stability. Our results provide evidence for a role of DNA Ligase IV in controlling the cellular localization and protein levels of XRCC4.

Foreign nurse importation and the supply of native nurses.

The importation of foreign registered nurses has been used as a strategy to ease nursing shortages in the United States. The effectiveness of this policy depends critically on the long-run response of native nurses. We examine the effects of immigration of foreign-born registered nurses on the long-run employment and occupational choice of native nurses. Using a variety of empirical strategies that exploit the geographical distribution of immigrant nurses across US cities, we find evidence of large displacement effects - over a ten-year period, for every foreign nurse that migrates to a city, between 1 and 2 fewer native nurses are employed in the city. We find similar results using data on nursing board exam-takers at the state level - an increase in the flow of foreign nurses significantly reduces the number of natives sitting for licensure exams in more dependent states relative to less dependent states. Using data on self-reported workplace satisfaction among a sample of California nurses, we find suggestive evidence that part of the displacement effects could be driven by a decline in the perceived quality of the workplace environment.

Recombination-activating gene 1 (Rag1)-deficient mice with severe combined immunodeficiency treated with lentiviral gene therapy demonstrate autoimmune Omenn-like syndrome.

BACKGROUND: Recombination-activating gene 1 (RAG1) deficiency results in severe combined immunodeficiency (SCID) caused by a complete lack of T and B lymphocytes. If untreated, patients succumb to recurrent infections. OBJECTIVES: We sought to develop lentiviral gene therapy for RAG1-induced SCID and to test its safety. METHODS: Constructs containing the viral spleen-focus-forming virus (SF), ubiquitous promoters, or cell type-restricted promoters driving sequence-optimized RAG1 were compared for efficacy and safety in sublethally preconditioned Rag1(-/-) mice undergoing transplantation with transduced bone marrow progenitors. RESULTS: Peripheral blood CD3(+) T-cell reconstitution was achieved with SF, ubiquitous promoters, and cell type-restricted promoters but 3- to 18-fold lower than that seen in wild-type mice, and with a compromised CD4(+)/CD8(+) ratio. Mitogen-mediated T-cell responses and T cell-dependent and T cell-independent B-cell responses were not restored, and T-cell receptor patterns were skewed. Reconstitution of mature peripheral blood B cells was approximately 20-fold less for the SF vector than in wild-type mice and often not detectable with the other promoters, and plasma immunoglobulin levels were abnormal. Two months after transplantation, gene therapy-treated mice had rashes with cellular tissue infiltrates, activated peripheral blood CD44(+)CD69(+) T cells, high plasma IgE levels, antibodies against double-stranded DNA, and increased B cell-activating factor levels. Only rather high SF vector copy numbers could boost T- and B-cell reconstitution, but mRNA expression levels during T- and B-cell progenitor stages consistently remained less than wild-type levels. CONCLUSIONS: These results underline that further development is required for improved expression to successfully treat patients with RAG1-induced SCID while maintaining low vector copy numbers and minimizing potential risks, including autoimmune reactions resembling Omenn syndrome.