NASCENT DNA PROTEOMICS ANALYSIS UNCOVERS DNA REPLICATION DYNAMICS IN THE HUMAN PATHOGEN TRYPANOSOMA BRUCEI

DNA is the substrate of many cellular processes including DNA replication, transcription and chromatin remodeling. These processes are coordinated to maintain genome integrity and ensure accurate duplication of genetic and epigenetic information. Genome-wide studies have provided evidence of the relationship between transcription and DNA replication timing. A global analysis of DNA replication initiation in T. brucei showed that TbORC1 (subunit of the origin recognition complex, ORC) binding sites are located at the boundaries of transcription units. Although recent studies in T. brucei indicate functional links among DNA replication and transcription, the underlying mechanisms remain unknown. In this study, we adapted an unbiased technology for the identification of replication fork proteins called iPOND (isolation of proteins on nascent DNA) to T. brucei, its first application to a parasite system.The iPOND approach relies on labeling newly replicated DNA with the thymidine analog EdU (5-ethynyl-2′-deoxyuridine), which contains an alkyne functional group that enables the cycloaddition of a biotin azide. This click chemistry reaction yields a stable covalent linkage, facilitating streptavidin capture of cross-linked biotinylated DNA-protein complexes. First, we described how we adapted the iPOND protocol in T. brucei cells to generate a suitable sample for mass spectrometry analysis (Chapter 2). We performed MS label-free quantification to determine which proteins are enriched in an active replication fork in T. brucei (Chapter 3). We identified 410 proteins, including key DNA replication factors and proteins associated with transcription, chromatin organization, DNA repair and mRNA splicing. Around 25% of the proteins identified were of unknown function that might have the potential to be essential trypanosome-specific replication proteins. Additionally, we characterized two proteins from our iPOND-derived protein list (Chapter 3). These are a protein annotated as a Replication Factor C subunit (Tb927.10.7990), and a protein of unknown function (Tb927.3.5370). Both revealed nuclear localization. Tb927.10.7990 proved to be essential since its silencing caused a growth defect and impaired DNA replication. However,Tb927.3.5370 appeared to be a dispensable gene. We propose nucleosomes are assembled close to the replication fork followed by RNA pol II recruitment, transcription, and co-transcriptional RNA splicing. Further studies are needed to determine how these processes are linked and co-regulated, and how rapidly they are initiated during DNA replication

Influencia de la maduración del fruto de Arbutus xalapensis Kunth sobre la germinación de semillas y embriones cigóticos

En este trabajo se determinó el efecto del estado de maduración del fruto en la germinación in vitro de semillas y embriones cigóticos deArbutus xalapensisKunt. Se colectaron frutos de 10 árboles en cada uno de los dos sitios de estudio y se clasifi caron según su tamaño y peso dentro de tres grupos caracterizados por el color del fruto: 1 a 6 frutos verde oscuro (FVO), 7 y 8 frutos verde-amarillo (FVA), y 9 y 10 frutos naranja-rojizo (FNR). De cada estado se seleccionaron 50 semillas y se colocaron en medio MS para evaluar su germinación. El experimento se repitió dos veces y los resultados obtenidos fueron sometidos a un análisis de comparación de medias de Tukey el cual indicó que los tres grupos de frutos presentan características de peso fresco y diámetro diferentes (P = 0.05), con un promedio de 6.3 g y de 0.21 mm para FVO, 9.5 g y 0.46 mm para FVA y 10.8 g y 0.70 mm para FNR. El análisis de microscopía estereoscópica demostró que todos los estados de maduración presentan semillas y embriones; los estados 1 al 4 presentan embriones no desarrollados, y en los estados 5 al 10 los frutos contienen un mayor número de semillas con embriones desarrollados y con una mayor germinación (7.6 a 8.8 semillas/unidad experimental). Se propone el uso de semillas provenientes de frutos de los estados de maduración 5 al 10 para la germinación adecuada de semillas de madroño, mientras que para los embriones cigóticos es necesario realizar estudios de prueba de medios de cultivo para su germinación

Investigating the impact of UV-C/H2O2 and sunlight/H2O2 on the removal of antibiotics, antibiotic resistance determinants and toxicity present in urban wastewater

This work aimed at exploring the impact of UV-C/H2O2 and sunlight/H2O2 processes, applied at pilot scale, on removing: (i) ciprofloxacin and sulfamethoxazole, (ii) cultivable Escherichia coli and Pseudomonas aeruginosa grown in the presence and absence of sub-minimal inhibitory concentrations of ciprofloxacin and sulfamethoxazole and (iii) the genes 16S rRNA and selected antibiotic resistance genes (ARGs) (i.e., sul1, blaCTX-M, qnrS, tetM, etc.) from urban wastewater. The major antibiotic transformation products (TPs) formed, were elucidated and the chronic toxicity of the whole effluent mixture against Vibrio fischeri was evaluated. The capability of the processes, in terms of the elimination of the antibiotics present in urban wastewater, varied among the two light sources used: both antibiotics were fully removed during UV-C/Η2Ο2, whereas only ciprofloxacin was removed during the sunlight/H2O2. The photo-transformation of the antibiotics led to the identification of 21 and 18 TPs of ciprofloxacin and sulfamethoxazole, respectively, while all of them retained their core moiety, responsible for the antibacterial activity. All the UV-C/H2O2-treated samples were found to be toxic, whereas the luminescence of V. fischeri was not inhibited when tested in the sunlight/H2O2-treated samples. During both processes, E. coli, P. aeruginosa and the colonies of these species still viable in the presence of antibiotics, were successfully inactivated to values below the detection limit. However, sunlight/H2O2 has not achieved complete disinfection, as regrowth of E. coli and P. aeruginosa colonies was observed after 48 h of storage of the treated effluent. Finally, none of the technologies tested was able to completely remove the target ARGs, confirming their inability to prevent the spread of resistance determinants to the environment.info:eu-repo/semantics/acceptedVersio

Differential effects of the second SARS-CoV-2 mRNA vaccine dose on T cell immunity in naive and COVID-19 recovered individuals

The rapid development of mRNA-based vaccines against the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) led to the design of accelerated vaccination schedules that have been extremely effective in naive individuals. While a two-dose immunization regimen with the BNT162b2 vaccine has been demonstrated to provide a 95% efficacy in naive individuals, the effects of the second vaccine dose in individuals who have previously recovered from natural SARS-CoV-2 infection has not been investigated in detail. In this study, we characterize SARS-CoV-2 spike-specific humoral and cellular immunity in naive and previously infected individuals during and after two doses of BNT162b2 vaccination. Our results demonstrate that, while the second dose increases both the humoral and cellular immunity in naive individuals, COVID-19 recovered individuals reach their peak of immunity after the first dose. These results suggests that a second dose, according to the current standard regimen of vaccination, may be not necessary in individuals previously infected with SARS-CoV-2.Funding: Research reported in this publication was supported in part by the National Cancer Institute of the NIH (5R01HD102614-02; R01CA249204 and R01CA248984) and an ISMMS seed fund to E.G. The authors gratefully acknowledge use of the services and facilities of the Tisch Cancer Institute supported by a NCI Cancer Center Support Grant (P30 CA196521). M.S. was supported by a NCI training grant (T32CA078207). This work was supported by an ISMMS seed fund to J.O.; Instituto de Salud Carlos III (COV20-00668) to R.C.R.; the Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation (COVID-19 research call COV20/00181) co-financed by the European Development Regional Fund ‘‘A way to achieve Europe’’ to E.P.; the Instituto de Salud Carlos III, Spain (COV20/00170); the Government of Cantabria, Spain (2020UIC22-PUB-0019) to M.L.H.; the Instituto de Salud Carlos III (PI16CIII/00012) to P.P.; the Fondo Social Europeo e Iniciativa de Empleo Juvenil YEI (Grant PEJ2018-004557-A) to M.P.E.; and by REDInREN 016/009/009 ISCIII. This project has received funding from the European Union Horizon 2020 research and innovation programs VACCELERATE and INsTRuCT under grant agreements 101037867 and 860003

Differential effects of the second SARS-CoV-2 mRNA vaccine dose on T cell immunity in naive and COVID-19 recovered individuals

The rapid development of mRNA-based vaccines against the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) led to the design of accelerated vaccination schedules that have been extremely effective in naive individuals. While a two-dose immunization regimen with the BNT162b2 vaccine has been demonstrated to provide a 95% efficacy in naive individuals, the effects of the second vaccine dose in individuals who have previously recovered from natural SARS-CoV-2 infection has not been investigated in detail. In this study, we characterize SARS-CoV-2 spike-specific humoral and cellular immunity in naive and previously infected individuals during and after two doses of BNT162b2 vaccination. Our results demonstrate that, while the second dose increases both the humoral and cellular immunity in naive individuals, COVID-19 recovered individuals reach their peak of immunity after the first dose. These results suggests that a second dose, according to the current standard regimen of vaccination, may be not necessary in individuals previously infected with SARS-CoV-2.Research reported in this publication was supported in part by the National Cancer Institute of the NIH (5R01HD102614-02; R01CA249204 and R01CA248984) and an ISMMS seed fund to E.G. The authors gratefully acknowledge use of the services and facilities of the Tisch Cancer Institute supported by a NCI Cancer Center Support Grant (P30 CA196521). M.S. was supported by a NCI training grant (T32CA078207). This work was supported by an ISMMS seed fund to J.O.; Instituto de Salud Carlos III (COV20-00668) to R.C.R.; the Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation (COVID-19 research call COV20/00181) co-financed by the European Development Regional Fund “A way to achieve Europe” to E.P.; the Instituto de Salud Carlos III, Spain (COV20/00170); the Government of Cantabria, Spain (2020UIC22-PUB-0019) to M.L.H.; the Instituto de Salud Carlos III (PI16CIII/00012) to P.P.; the Fondo Social Europeo e Iniciativa de Empleo Juvenil YEI (Grant PEJ2018-004557-A) to M.P.E.; and by REDInREN 016/009/009 ISCIII. This project has received funding from the European Union Horizon 2020 research and innovation programs VACCELERATE and INsTRuCT under grant agreements 101037867 and 860003.S

Trans* Pregnancy and Lactation: A Literature Review from a Nursing Perspective

Pregnancy and lactationinvolve two aspects that are socially and culturally associated with women. However, there are a few biological differences between male and female breast tissue. Lactation and pregnancy are viable processes that do not depend on sex. Even for the latter,it is only necessary to have an organ capable of gestation. Ways to favor mammogenesis and lactogenesis in trans* women have been established. There are protocols to promote lactation in trans* women, usually used for adoptive mothers or those whose children have been born through gestational surrogacy. Chestfeeding a baby could be the cause of feelings as diverse as gender dysphoria in the case of trans* men, and euphoria and affirmation of femininity in trans* women. This study involves a review of the available scientific literature addressing medical aspects related to pregnancy and lactation in trans* individuals, giving special attention to nursing care during perinatal care. There are scarce studies addressing care and specificallynursing care in trans* pregnancy and lactation. Our study indicates the factors that can be modified and the recommendations for optimizing the care provided to these individuals in order to promote and maintain the lactation period in search of improvement and satisfaction with the whole proces

Nuclear DNA Replication in Trypanosomatids:There Are No Easy Methods for Solving Difficult Problems

In trypanosomatids, etiological agents of devastating diseases, replication is robust and finely controlled to maintain genome stability and function in stressful environments. However, these parasites encode several replication protein components and complexes that show potentially variant composition compared with model eukaryotes. This review focuses on the advances made in recent years regarding the differences and peculiarities of the replication machinery in trypanosomatids, including how such divergence might affect DNA replication dynamics and the replication stress response. Comparing the DNA replication machinery and processes of parasites and their hosts may provide a foundation for the identification of targets that can be used in the development of chemotherapies to assist in the eradication of diseases caused by these pathogens

Real-Time PCR in HIV/Trypanosoma cruzi Coinfection with and without Chagas Disease Reactivation: Association with HIV Viral Load and CD4+ Level

Chagas disease is endemic in Latin America and is caused by the flagellate protozoan T. cruzi. The acute phase is asymptomatic in the majority of the cases and rarely causes inflammation of the heart or the central nervous system. Most infected patients progress to a chronic phase, characterized by cardiac or digestive involvement when not asymptomatic. However, when patients are also exposed to an immunosuppressant (such as chemotherapy), neoplasia, or other infections such as HIV, T. cruzi infection may develop into a severe disease (Chagas disease reactivation) involving the heart and central nervous system. The current microscopic methods for diagnosing Chagas disease reactivation are not sensitive enough to prevent the high rate of death observed in these cases. Therefore, we propose a quantitative method to monitor blood levels of the parasite, which will allow therapy to be administered as early as possible, even if the patient has not yet presented symptoms