Rothmund-Thomson syndrome, a disorder far from solved

Rothmund-Thomson syndrome (RTS) is a rare autosomal recessive disorder characterized by a range of clinical symptoms, including poikiloderma, juvenile cataracts, short stature, sparse hair, eyebrows/eyelashes, nail dysplasia, and skeletal abnormalities. While classically associated with mutations in the RECQL4 gene, which encodes a DNA helicase involved in DNA replication and repair, three additional genes have been recently identified in RTS: ANAPC1, encoding a subunit of the APC/C complex; DNA2, which encodes a nuclease/helicase involved in DNA repair; and CRIPT, encoding a poorly characterized protein implicated in excitatory synapse formation and splicing. Here, we review the clinical spectrum of RTS patients, analyze the genetic basis of the disease, and discuss molecular functions of the affected genes, drawing some novel genotype-phenotype correlations and proposing avenues for future studies into this enigmatic disorder

ATLANTIC EPIPHYTES: a data set of vascular and non-vascular epiphyte plants and lichens from the Atlantic Forest

Epiphytes are hyper-diverse and one of the frequently undervalued life forms in plant surveys and biodiversity inventories. Epiphytes of the Atlantic Forest, one of the most endangered ecosystems in the world, have high endemism and radiated recently in the Pliocene. We aimed to (1) compile an extensive Atlantic Forest data set on vascular, non-vascular plants (including hemiepiphytes), and lichen epiphyte species occurrence and abundance; (2) describe the epiphyte distribution in the Atlantic Forest, in order to indicate future sampling efforts. Our work presents the first epiphyte data set with information on abundance and occurrence of epiphyte phorophyte species. All data compiled here come from three main sources provided by the authors: published sources (comprising peer-reviewed articles, books, and theses), unpublished data, and herbarium data. We compiled a data set composed of 2,095 species, from 89,270 holo/hemiepiphyte records, in the Atlantic Forest of Brazil, Argentina, Paraguay, and Uruguay, recorded from 1824 to early 2018. Most of the records were from qualitative data (occurrence only, 88%), well distributed throughout the Atlantic Forest. For quantitative records, the most common sampling method was individual trees (71%), followed by plot sampling (19%), and transect sampling (10%). Angiosperms (81%) were the most frequently registered group, and Bromeliaceae and Orchidaceae were the families with the greatest number of records (27,272 and 21,945, respectively). Ferns and Lycophytes presented fewer records than Angiosperms, and Polypodiaceae were the most recorded family, and more concentrated in the Southern and Southeastern regions. Data on non-vascular plants and lichens were scarce, with a few disjunct records concentrated in the Northeastern region of the Atlantic Forest. For all non-vascular plant records, Lejeuneaceae, a family of liverworts, was the most recorded family. We hope that our effort to organize scattered epiphyte data help advance the knowledge of epiphyte ecology, as well as our understanding of macroecological and biogeographical patterns in the Atlantic Forest. No copyright restrictions are associated with the data set. Please cite this Ecology Data Paper if the data are used in publication and teaching events. © 2019 The Authors. Ecology © 2019 The Ecological Society of Americ

Participation of DNA polymerase iota in the absence of Pol eta in translesion synthesis of human cells UV-irradiated and characterization of cells from patients mutated in PCNA

A luz ultravioleta (UV), quando incide sobre as células humanas, é capaz de lesionar o DNA, gerando fotoprodutos conhecidos como dímeros de pirimidina, podendo resultar tanto em instabilidade gênica quanto em morte celular. Estas lesões, quando detectadas pela célula recrutam proteínas responsáveis pelo reparo por excisão de nucleotídeos (do inglês, NER). Caso não sejam reparadas até o momento da replicação, estas lesões bloqueiam a polimerase replicativa, sinalizando para um mecanismo de tolerância conhecido como síntese translesão (do inglês, TLS). Nesse processo, polimerases específicas, chamadas polimerases de TLS, são recrutadas para o local e realizam o bypass da lesão, permitindo à célula continuar a replicação. Dentre as polimerases de TLS descritas, a Pol&#951 (Pol eta) é a mais estudada, devido ao fato de a sua ausência levar a uma síndrome conhecida como xeroderma pigmentosum variante (XP-V), em que os pacientes apresentam hipersensibilidade à luz UV e aumento da incidência de tumores em regiões expostas ao sol. Apesar de diversos estudos, ainda não se sabe como a TLS dos dímeros ocorre nestes pacientes, deixando em aberto uma questão que implica, diretamente, na alta frequência de tumores de pele em pessoas XP-V. Sabe-se que a Pol&#953 (Pol iota), outra polimerase, está presente em foci de replicação, juntamente com a Pol&#951, porém sua função na TLS ainda não está definida. Nesse contexto, o objetivo principal do presente projeto é investigar o papel de Pol&#953 no bypass de lesões UV em células humanas. Para isso, utilizou-se linhagens celulares deficientes em NER (XP-C) silenciadas para Pol&#953 e/ou Pol&#951, previamente estabelecidas em nosso laboratório. Estas linhagens foram expostas a diferentes doses de luz UVC para a análise de viabilidade celular, morte celular (sub-G1 e ativação de caspase-3), progressão no ciclo, capacidade de migração e duplicação, quantificação da sinalização do dano, bloqueio da forquilha de replicação pelos dímeros de pirimidina e quantificação de quebras no DNA. Os resultados presentes nesta tese mostraram que a perda apenas de Pol&#953 não afeta a linhagem na maioria das metodologias. Entretanto, após a retirada de Pol&#953 de células que já não possuíam Pol&#951, observou-se uma redução acentuada da resistência da linhagem à luz UV, com morte elevada (24, 48 e 72 h após o tratamento) e bloqueio em fase G1/S do ciclo celular, porém sem aumento na sinalização do dano. Além disso, esta linhagem apresentou problemas na duplicação e migração celular após a irradiação com UVC e parada acentuada de forquilhas de replicação. Por fim, a quantificação de quebras 24 h após a irradiação evidenciou que a hipersensibilidade desta linhagem não é devido ao aumento da fragmentação do DNA, e sim uma incapacidade da célula em lidar com o dano quando ambas as polimerases de TLS estão ausentes. Baseado nestes resultados, concluiu-se que Pol&#953 atua de fato como backup na ausência de Pol&#951 na TLS de fotoprodutos. Também estudamos células mutadas em PCNA. Dados preliminares mostraram que esta mutação torna a linhagem mais sensível a lesões do tipo quebra-dupla e termosensibilidade, sendo interessante para o estudo da replicação e reparo de DNA.When the ultraviolet (UV) light reaches human cells, it damages the DNA, generating photoproducts known as pyrimidine dimers, which can result in both gene instability and cell death. These lesions, when detected by the cell, recruit proteins responsible for nucleotide excision repair (NER). If not repaired properly by the time of replication, these lesions block the replicative polymerase, signaling to a tolerance mechanism known as translesion synthesis (TLS). In this process, specific specific polymerases, called TLS polymerases, are recruited to the site to bypass the lesion, allowing the cell to continue replication. Among the described TLS polymerases, Pol&#951 (Pol eta) is the most studied, since its absence leads to a syndrome known as xeroderma pigmentosum variant (XP-V), in which patients have hypersensitivity to UV light and an increased incidence of tumors in regions exposed to the sunlight. Despite several studies, it is still not known how TLS occurs in the tolerance of photoproducts in these patients, leaving open a question that directly implies in the high frequency of skin tumors in XP-V people. It is known that Pol&#953 (Pol iota), another polymerase, is present in replication foci, along with Pol&#951, but its role in TLS is not yet defined. In this context, the main objective of the present project is to investigate the role of Pol&#953 in the bypass of UV-lesions in human cells. For this, NER-deficient cell lines (XP-C) silenced for Pol&#953 and/or Pol&#951, previously established in our laboratory, were used. These lineages were exposed to different doses of UVC light for the analysis of cell viability, cell death (sub-G1 and caspase-3 activation), cycle progression, migration and duplication capacities, quantification of signaling damage, blockage of the replication fork by pyrimidine dimers, and quantifying DNA breaks. The results presented in this thesis showed that the loss of Pol&#953 alone does not affect the lineage in most of the processes. However, after removal of Pol&#953 from cells that lack Pol&#951, an evident reduction in the resistance of the strain to UV light was observed, with high death (24, 48 and 72 h after treatment) and G1/S cell cycle arrest, but no increase in damage signaling. In addition, this cell lineage presented problems in duplication and migration after UVC irradiation and severe replication fork blockage, higher than the other cell lineages. Finally, the quantification of breaks 24 h after irradiation showed that the hypersensitivity of this cells is not due to an increase in DNA fragmentation, but an inability of the cell to deal with the damage when both TLS polymerases are absent. Based on these results, it was concluded that Pol&#953 is indeed the backup of Pol&#951 in the TLS of photoproducts. We also studied PCNA-mutated patient cells. Preliminary data showed that the mutation makes the cell line more sensitive to double stranded breaks lesions and presents thermosensitivity, which is an interesting finding in the study of DNA replication and repair

Translesion synthesis mechanisms depend on the nature of DNA damage in UV-irradiated human cells

Ultraviolet-induced 6-4 photoproducts (6-4PP) and cyclobutane pyrimidine dimers (CPD) can be tolerated by translesion DNA polymerases (TLS Pols) at stalled replication forks or by gap-filling. Here, we investigated the involvement of Polη, Rev1 and Rev3L (Polζ catalytic subunit) in the specific bypass of 6-4PP and CPD in repair-deficient XP-C human cells. We combined DNA fiber assay and novel methodologies for detection and quantification of single-stranded DNA (ssDNA) gaps on ongoing replication forks and postreplication repair (PRR) tracts in the human genome. We demonstrated that Rev3L, but not Rev1, is required for postreplicative gap-filling, while Polη and Rev1 are responsible for TLS at stalled replication forks. Moreover, specific photolyases were employed to show that in XP-C cells, CPD arrest replication forks, while 6-4PP are responsible for the generation of ssDNA gaps and PRR tracts. On the other hand, in the absence of Polη or Rev1, both types of lesion block replication forks progression. Altogether, the data directly show that, in the human genome, Polη and Rev1 bypass CPD and 6-4PP at replication forks, while only 6-4PP are also tolerated by a Polζ-dependent gap-filling mechanism, independent of S phase

Chloroquine - induced glioma cells death is associated with mitochondrial membrane potential loss, but not oxidative stress.

Chloroquine (CQ), a quinolone derivative widely used to treat and prevent malaria, has been shown to exert a potent adjuvant effect when combined with conventional glioblastoma therapy. Despite inducing lysosome destabilization and activating p53 in human glioma cells, the mechanisms under lying cell death induced by this drug are poorly under stood. Here, we analyzed inatime – anddose – dependent manner, the effects of CQ up on mitochondria integrity, autophagy regulation and redox processes in four human glioma cell lines that differin their resistance to this drug. NAC – containing media protected cells against CQ-induced loss of mitochondrial membrane potential (MMP), autophagyic vacuoles (LC3II) accumulation and loss of cell viability induced by CQ. However, we noticed that part of this protection was due to media acidification in NAC preparations, alerting for problems in experimental procedures using NAC. The results indicate that although CQ induces accumulation of LC3II, mitochondria, and oxidative stress, neither of these events is clearly correlated to cell death induced by this drug. The only event elicited in all cell lines at equitoxic doses of CQ was the loss of MMP, indicating that mitochondrial stability is important for cells resistance to this drug. Finally, the data indicate that higher steady-state MMP values can predict cell resistance to CQ treatment

Mycoplasma hominis Causes DNA Damage and Cell Death in Primary Human Keratinocytes

Mycoplasma hominis can be isolated from the human urogenital tract. However, its interaction with the host remains poorly understood. In this study, we aimed to assess the effects of M. hominis infection on primary human keratinocytes (PHKs). Cells were quantified at different phases of the cell cycle. Proteins involved in cell cycle regulation and apoptosis progression were evaluated. The expression of genes encoding proteins that are associated with the DNA damage response and Toll-like receptor pathways was evaluated, and the cytokines involved in inflammatory responses were quantified. A greater number of keratinocytes were observed in the Sub-G0/G1 phase after infection with M. hominis. In the viable keratinocytes, infection resulted in G2/M-phase arrest; GADD45A expression was increased, as was the expression of proteins such as p53, p27, and p21 and others involved in apoptosis regulation and oxidative stress. In infected PHKs, the expression of genes associated with the Toll-like receptor pathways showed a change, and the production of IFN-γ, interleukin (IL) 1β, IL-18, IL-6, and tumour necrosis factor alpha increased. The infection of PHKs by M. hominis causes cellular damage that can affect the cell cycle by activating the response pathways to cellular damage, oxidative stress, and Toll-like receptors. Overall, this response culminated in the reduction of cell proliferation/viability in vitro

NEOTROPICAL CARNIVORES: a data set on carnivore distribution in the Neotropics

Mammalian carnivores are considered a key group in maintaining ecological health and can indicate potential ecological integrity in landscapes where they occur. Carnivores also hold high conservation value and their habitat requirements can guide management and conservation plans. The order Carnivora has 84 species from 8 families in the Neotropical region: Canidae; Felidae; Mephitidae; Mustelidae; Otariidae; Phocidae; Procyonidae; and Ursidae. Herein, we include published and unpublished data on native terrestrial Neotropical carnivores (Canidae; Felidae; Mephitidae; Mustelidae; Procyonidae; and Ursidae). NEOTROPICAL CARNIVORES is a publicly available data set that includes 99,605 data entries from 35,511 unique georeferenced coordinates. Detection/non-detection and quantitative data were obtained from 1818 to 2018 by researchers, governmental agencies, non-governmental organizations, and private consultants. Data were collected using several methods including camera trapping, museum collections, roadkill, line transect, and opportunistic records. Literature (peer-reviewed and grey literature) from Portuguese, Spanish and English were incorporated in this compilation. Most of the data set consists of detection data entries (n = 79,343; 79.7%) but also includes non-detection data (n = 20,262; 20.3%). Of those, 43.3% also include count data (n = 43,151). The information available in NEOTROPICAL CARNIVORES will contribute to macroecological, ecological, and conservation questions in multiple spatio-temporal perspectives. As carnivores play key roles in trophic interactions, a better understanding of their distribution and habitat requirements are essential to establish conservation management plans and safeguard the future ecological health of Neotropical ecosystems. Our data paper, combined with other large-scale data sets, has great potential to clarify species distribution and related ecological processes within the Neotropics. There are no copyright restrictions and no restriction for using data from this data paper, as long as the data paper is cited as the source of the information used. We also request that users inform us of how they intend to use the data