Bacterial Ligase D preternary-precatalytic complex performs efficient abasic sites processing at double strand breaks during nonhomologous end joining

Abasic (AP) sites, the most common DNA lesions are frequently associated with double strand breaks (DSBs) and can pose a block to the final ligation. In many prokaryotes, nonhomologous end joining (NHEJ) repair of DSBs relies on a two-component machinery constituted by the ring-shaped DNA-binding Ku that recruits the multicatalytic protein Ligase D (LigD) to the ends. By using its polymerization and ligase activities, LigD fills the gaps that arise after realignment of the ends and seals the resulting nicks. Here, we show the presence of a robust AP lyase activity in the polymerization domain of Bacillus subtilis LigD (BsuLigD) that cleaves AP sites preferentially when they are proximal to recessive 5'-ends. Such a reaction depends on both, metal ions and the formation of a Watson¿Crick base pair between the incoming nucleotide and the templating one opposite the AP site. Only after processing the AP site, and in the presence of the Ku protein, BsuLigD catalyzes both, the in-trans addition of the nucleotide to the 3'-end of an incoming primer and the ligation of both ends. These results imply that formation of a preternary-precatalytic complex ensures the coupling of AP sites cleavage to the end-joining reaction by the bacterial LigDMinistry of Science, Innovation and Universities [BFU2017-83900-P AEI/FEDER UE to M.V.]; institutional grants from Fundacion Ramón Areces and Banco ´ de Santander to the Centro de Biología Molecular Severo Ocho

Análisis bioquímico de las proteínas de reparación del DNA Ku y Ligasa D de Bacillus subtilis

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 19-12-2016Esta tesis tiene embargado el acceso al texto completo hasta el 19-06-2018Bacillus subtilis en una bacteria Gram-positiva formadora de esporas provista del sistema de reparación de roturas de las dos cadenas del DNA mediante la Unión de Extremos No Homólogos (NHEJ). Este sistema está constituido por la proteína de unión a DNA Ku (BsuKu) que se une a las roturas y recluta a la Ligasa D dependiente de ATP (BsuLigD) que posteriormente cataliza el relleno de los pequeños gaps que pudieran surgir durante el alineamiento de los extremos y el sellado de los nicks resultantes, contribuyendo a la estabilidad genómica durante la fase estacionaria y la germinación de las esporas. Hasta el momento, las únicas funciones descritas para la proteína Ku procariota eran la unión a los extremos del DNA procedentes de roturas y el reclutamiento de la LigD para su posterior procesamiento y reparación. En esta Tesis Doctoral, se ha identificado la presencia de una actividad AP/5´-dRP liasa en BsuKu, capaz de reconocer y procesar sitios abásicos (AP) tanto en ssDNA, dsDNAs con extremos 5´-protuberantes y romos, así como en moléculas de DNA sin extremos. Esta nueva actividad de BsuKu, en coordinación con las actividades de polimerización y ligasa de BsuLigD, hacen posible la unión eficiente de fragmentos de DNA portadores de sitios AP próximos a extremos 5´-protuberantes. Además, hemos demostrado la presencia de la actividad AP-liasa también en la proteína Ku de la bacteria Gram-negativa Pseudomonas aeruginosa, hecho que nos permite expandir nuestros resultados a otras proteínas Ku bacterianas. Por otra parte, hemos identificado la presencia de una actividad dRP-liasa localizada en el dominio ligasa N-terminal de la proteína BsuLigD. Esta actividad, junto con las de polimerización y ligación, permite la reparación eficiente de un DNA portador de un sitio AP en una reacción reconstituida in vitro de Reparación por Escisión de Bases (BER). El requerimiento de los pasos de polimerización, eliminación del grupo 5´-dRP y ligación final, junto con el hecho de que BsuLigD y Nfo (la AP-endonucleasa de la espora), confieren a las esporas resistencia a la desecación, sugiere que BsuLigD podría estar participando activamente en una potencial vía BER en la espora. Hemos demostrado que la actividad dRP-liasa no es específica de BsuLigD, sino que se encuentra también en la de P. aeruginosa, permitiéndonos extrapolar nuestras observaciones al resto de LigDs bacterianas. Asimismo, se ha determinado que BsuLigD tiene una actividad AP-liasa clásica en sustratos de DNA con extremos 5´-recesivos. Esta actividad requiere la presencia de metal, así como la unión (aunque no la incorporación) en el centro activo de polimerización del nucleótido correcto que habría de reemplazar el sitio AP tras su eliminación, sugiriendo la necesidad de la formación de un complejo preternario estable para que se lleve a cabo el procesamiento del sitio AP a la espera de la posterior reacción de ligación a un primer. Por último, se ha mostrado la capacidad de BsuLigD de incorporar ribonucleótidos frente a lesiones en el molde de moléculas de DNA, tales como 8-oxodG, timidin-glicol y 5-hidroxi desoxicitidina, y la posterior ligación del producto elongado a un extremo 5´-P. Estos resultados podrían implicar a esta proteína no solo en las rutas NHEJ y BER, sino también en la ruta de tolerancia al daño en el DNA conocida como Síntesis a Través de Lesión.Bacillus subtilis is a Gram-positive sporulating bacterium endowed with a double-strand break Non- Homologous End-Joining (NHEJ) repair system. This system is constituted by the DNA-binding protein Ku (BsuKu) that binds to the broken ends and recruits the ATP-dependent DNA ligase D (BsuLigD) that further catalyses both, filling of the short gaps that could arise after synapsis, and ligation of the resulting nicks, contributing to genome stability during the stationary phase and the spore germination. So far, the only described functions for the prokaryotic Ku were binding to the broken ends of DNA and recruitment of LigD. In this Thesis, we have identified the presence of an AP/5´-dRP lyase activity in BsuKu. This protein can recognize and process AP sites present in ssDNA, in both, 5´-protruding and blunt ends, as well as in DNA molecules without ends. This newly identified activity in BsuKu, in coordination with BsuLigD’s polymerization and ligation activities, makes possible the efficient joining of 5´-protruding DNA ends with near terminal AP sites. We have shown that the homolog protein from the distantly related Gram-negative bacterium Pseudomonas aeruginosa also exhibits this activity, a fact that allows us to infer its general presence in the bacterial Ku proteins. Additionally, we have identified the presence of a 5´-dRP-lyase activity located at the N-terminal ligase domain of BsuLigD. This activity, along with its polymerization and ligation activities, allows efficient repairing of AP-containing DNA in an in vitro reconstituted Base Excision Repair (BER) reaction. The requirement of a polymerization, a 5´-dRP removal and a final sealing step in BER, together with the joint participation of BsuLigD with the spore specific AP-endonuclease Nfo in conferring spore resistance to desiccation, suggests that BsuLigD could actively participate in this pathway. We have demonstrated that this 5´-dRP-lyase is also present in P. aeruginosa LigD, allowing us to expand our results to other bacterial LigDs. Likewise, we have determined that BsuLigD has a classical AP-lyase activity that acts on 5´-recessive DNA ends. In this case the cleavage at the AP site requires the presence of metal ions, as well as the binding of the correct nucleotide that would replace the AP site at the polymerization active site, a fact that suggests the formation of a stable preternary complex required to process the AP site before the subsequent ligation to an incoming primer strand. Finally, it is shown the ability of BsuLigD to incorporate ribonucleotides opposite lesions in the template strand, such as 8-oxodG, thymidine-glycol and 5-hydroxy deoxycytidine, ligating the extended product to a 5´-P end. These results could involve BsuLigD not only in NHEJ and BER, but also in the DNA damage tolerance pathway Translesion Synthesis

Evaluation of the LIAISON XL Zika Capture IgM II for the Diagnosis of Zika Virus Infections

The aim of this study is to evaluate the performance characteristics of the LIAISON XL Zika Capture IgM II. For this purpose we tested 128 samples obtained from recent infections caused by the Zika (ZIKV; 74 samples), dengue (DENV; 10 samples), chikungunya (CHIK V; 11 samples), rubella (RUBV; 10 samples) and measles (MeV; 10 samples) viruses, as well as human parvovirus B19 (HPVB19; 13 samples). The results of the assay under evaluation are compared with those obtained from an indirect immunofluorescence (IIF) assay, and the discrepancies are resolved by considering other laboratory results (PCR and a plaque-reduction neutralization test). The LIAISON showed excellent sensitivity (100%). The specificity (91.25%) was hampered by some false-positive results in recent dengue virus, chikungunya virus, measles virus and human parvovirus B19 infections. The method evaluated is adequate, but the low specificity makes it necessary to consider the clinical and epidemiological contexts of patients, as well as other laboratory results.Funding: This work has been financed by a contract between the Instituto de Salud Carlos III and Diasorin Iberia(MVP280/18).S

Imported Human West Nile Virus Lineage 2 Infection in Spain: Neurological and Gastrointestinal Complications

We report the first human case of West Nile virus (WNV) lineage 2 infection imported to Spain by a traveler returning from Romania. Serum, cerebrospinal fluid and urine samples were analyzed and West Nile virus infection was identified by PCR and serological tests. The patient developed fever, diarrhea and neurological symptoms, accompanied by mild pancreatitis, described previously in very few cases as a complication of WNV infection and by alithiasic cholecystitis. Viral RNA was detected in urine until 30 days after the onset of symptoms and neutralizing antibodies were detected at very low titers. The phylogenetic analysis in a fragment of the NS5 gene of the virus showed a homology with sequences from WNV lineage 2 belonging to the monophyletic Central/Southern European group.This work was partially funded by the Instituto de Salud Carlos III Projects “PI14CIII/00014” and “PI19CIII_00014”.S

Comparative Evaluation of Indirect Immunofluorescence and NS-1-Based ELISA to Determine Zika Virus-Specific IgM

Differential diagnosis of the Zika virus (ZIKV) is hampered by cross-reactivity with other flaviviruses, mainly dengue viruses. The aim of this study was to compare two commercial methods for detecting ZIKV immunoglobulin M (IgM), an indirect immunofluorescence (IIF) and an enzyme immunoassay (ELISA), using the non-structural (NS) 1 protein as an antigen, both from EuroImmun, Germany. In total, 255 serum samples were analyzed, 203 of which showed laboratory markers of ZIKV infections (PCR-positive in serum and/or in urine and/or positive or indeterminate specific IgM). When tested with IIF, 163 samples were IgM-positive, while 13 samples were indeterminate and 78 were negative. When IIF-positive samples were tested using ELISA, we found 61 positive results, 14 indeterminate results, and 88 negative results. Among the indeterminate cases tested with IIF, ELISA analysis found two positive, two indeterminate, and nine negative results. Finally, 74 of the 78 IIF-negative samples proved also to be negative using ELISA. For the calculations, all indeterminate results were considered to be positive. The agreement, sensitivity, and specificity between ELISA and IIF were 60.2%, 44.9%, and 94.9%, respectively. Overall, 101 samples showed discrepant results; these samples were finally classified on the basis of other ZIKV diagnostic approaches (PCR-positive in serum and/or in urine, IgG determinations using IIF or ELISA, and ZIKV Plaque Reduction Neutralization test-positive), when available. A final classification of 228 samples was possible; 126 of them were positive and 102 were negative. The corresponding values of agreement, sensitivity, and specificity of IIF were 86.0%, 96.8%, and 72.5%, respectively. The corresponding figures for ELISA were 81.1%, 65.9%, and 100%, respectively. The ELISA and IIF methods are both adequate approaches for detecting ZIKV-specific IgM. However, considering their respective weaknesses (low sensitivity in ELISA and low specificity in IIF), serological results must be considered jointly with other laboratory results.The study was partially financed by a contract between the ISCIII and Euroimmun Diagnostics España SLU (MVP216/17), and by ISCIII, project RD16CIII/0003/0003, “Red de Enfermedades Tropicales”, Subprogram RETICS Plan Estatal de I+D+I 2013-2016, and co-funded by FEDER “Una manera de hacer Europa” and project PI16CIII/00037.S

Screening for Zika virus infection in 1057 potentially exposed pregnant women, Catalonia (northeastern Spain)

Dear editor: A recent editorial in Travel Medicine and Infectious Diseases highlighted the lack of studies about Zika virus (ZIKV) in pregnancy and its implications in many countries [1]. Zika virus infection can induce congenital defects in the newborn such as microcephaly and miscarriage when mothers are infected during pregnancy [2]. However, relevant questions remain to be completely understood, such as the risk of infection for pregnant women and of subsequent congenital defects, and the ratio between symptomatic and asymptomatic ZIKV infections in the general population and in pregnant women. Here, we describe the results of a ZIKV screening of pregnant women in Catalonia, northeastern Spain. Testing for ZIKV was recommended for all pregnant women with history of travel to ZIKV endemic areas during pregnancy or in the 8 weeks before conception [3]. Symptomatic patients were screened by serological methods from day four after the onset of symptoms and by molecular methods within the first week (serum) and two weeks (urine) after illness. Asymptomatic patients were tested by serological methods. Seroneutralization assay for ZIKV was perfomed in samples positive for antibodies. Commercial diagnostic assays were used (RT-PCR, Altona Diagnostics and IIFT, Euroimmun). Neutralization titers ≥1/32 were considered indicative of the presence of ZIKV neutralizing antibodies. Follow up at two designated reference obstetrical departments for early detection of microcephaly or other malformations was offered to pregnant women with laboratory evidence of ZIKV infection. When available, amniotic fluid and placental tissue samples were tested for ZIKV by RT-PCR in cases of microcephaly or miscarriage, respectively

Zika virus infection: a new public health emergency with great media impact

[ES] La infección por virus Zika (VZ) está afectando intensamente a los países latinoamericanos y se ha convertido en una nueva epidemia mediática. Su posible asociación con microcefalia y síndrome de Guillain-Barré motivó que la Organización Mundial de la Salud (OMS) declarase el 1 de febrero de 2016 que esta epidemia constituye una emergencia de salud pública de importancia internacional. Los datos epidemiológicos muestran una incidencia creciente en países como Brasil y Colombia, y que la epidemia sigue expandiéndose por muchos otros países. Desde enero de 2007 hasta el 27 de abril de 2016, la OMS ha detectado transmisión autóctona en 55 países (en 42 de ellos ha sido el primer brote de Zika), y 1198 microcefalias y otros trastornos neurológicos en Brasil. Así mismo, durante 2015 y 2016, 13 países detectaron un incremento de los casos de síndrome de Guillain-Barré y de confirmación de VZ asociado a este. En relación a las microcefalias y otras graves alteraciones cerebrales en recién nacidos de madres afectadas por VZ, las investigaciones ya evidencian una relación causal. Clínicamente muchos casos son asintomáticos y el diagnóstico ofrece dificultades con otras arbovirosis. El control de vectores en España es prioritario, dada la existencia de Aedes albopictus (mosquito tigre). También se recomienda el diagnóstico precoz, evitar viajes a zonas endémicas, mantener relaciones sexuales protegidas y procurar que la prioridad política, que puede evitar que esta epidemia se convierta en una enfermedad endémica de alta prevalencia, no nos haga olvidar otros problemas de salud. [EN] Infection with Zika virus (ZV) has become a new epidemic, with great impact on the media, and is having a strong effect in Latin American countries. Its possible association with microcephaly and Guillain-Barré syndrome prompted the World Health Organization (WHO) to declare on 1 February 2016 that this epidemic is a public health emergency of international concern. Epidemiological data show an increasing incidence in countries like Brazil and Colombia, and that the epidemic is still expanding in many other countries. Between January 2007 and 27 April 2016, the WHO detected transmission in 55 countries (in 42 of these, this was the first outbreak of Zika) and 1,198 microcephalies and other neurological disorders in Brazil. Also, during 2015-2016, 13 countries detected an increase in Guillain-Barré syndrome and/or confirmation of ZV associated with Guillain-Barré syndrome. Research has already demonstrated a causal relationship between microcephaly and other serious brain disorders in newborns and ZV infection in the mother. Clinically, many cases are asymptomatic and it can be difficult to distinguish this diagnosis from that of other arboviruses. Vector control in Spain is a priority because of the presence of the Aedes albopictus (tiger mosquito). Early diagnosis is recommended, as is avoiding travel to endemic areas and unprotected sex, and ensuring that the high political profile, which can prevent this epidemic from becoming a high prevalence endemic disease, does not cause us to forget about other health problems.S

Chikungunya virus infections among travellers returning to Spain, 2008 to 2014

Since the first documented autochthonous transmission of chikungunya virus in the Caribbean island of Saint Martin in 2013, the infection has been reported within the Caribbean region as well as North, Central and South America. The risk of autochthonous transmission of chikungunya virus becoming established in Spain may be elevated due to the large numbers of travellers returning to Spain from countries affected by the 2013 epidemic in the Caribbean and South America, as well as the existence of the Aedes albopictus vector in certain parts of Spain. We retrospectively analysed the laboratory diagnostic database of the National Centre for Microbiology, Institute of Health Carlos III (CNM-ISCIII) from 2008 to 2014. During the study period, 264 confirmed cases, of 1,371 suspected cases, were diagnosed at the CNM-ISCIII. In 2014 alone, there were 234 confirmed cases. The highest number of confirmed cases were reported from the Dominican Republic (n = 136), Venezuela (n = 30) and Haiti (n = 11). Six cases were viraemic in areas of Spain where the vector is present. This report highlights the need for integrated active case and vector surveillance in Spain and other parts of Europe where chikungunya virus may be introduced by returning travellers

First Report of Sylvatic DENV-2-Associated Dengue Hemorrhagic Fever in West Africa

Dengue virus (DENV) circulates in human and sylvatic cycles. Sylvatic strains are both ecologically and evolutionarily distinct from endemic viruses. Although sylvatic dengue cycles occur in West African countries and Malaysia, only a few cases of mild human disease caused by sylvatic strains and one single case of dengue hemorrhagic fever in Malaysia have been reported. Here we report a case of dengue hemorrhagic fever (DHF) with thrombocytopenia (13000/µl), a raised hematocrit (32% above baseline) and mucosal bleeding in a 27-year-old male returning to Spain in November 2009 after visiting his home country Guinea Bissau. Sylvatic DENV-2 West African lineage was isolated from blood and sera. This is the first case of DHF associated with sylvatic DENV-2 in Africa and the second case worldwide of DHF caused by a sylvatic strain

Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to <90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], >300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of <15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P<0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P<0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years