RESUMO
The production of PER-like extended-spectrum ß-lactamases has recently been associated with reduced susceptibility to the last resort drugs aztreonam/avibactam and cefiderocol. PER-2 has been mainly confined to Argentina and neighboring countries. Until now, only three plasmids harboring blaPER-2 genes have been characterized but very little is known about the involvement of different plasmid groups in its dissemination. The diversity of genetic platforms associated with blaPER-2 genes from a collection of PER-producing Enterobacterales was analysed by describing the close environment and the plasmid backbones. Full sequences of 11 plasmids were obtained by short read (Illumina) and long read (Oxford Nanopore or PacBio) sequencing technologies. De novo assemblies, annotation and sequence analysis were performed by Unicycler, Prokka and BLAST. Plasmid analysis revealed that the blaPER-2 gene is encoded on plasmids of different incompatibility groups (A, C, FIB, HI1B, N2), indicating that this gene may have been disseminated through a variety of plasmids. Comparison with the few publicly available nucleotide sequences describing the blaPER-2 genetic environment, including those from the environmental species Pararheinheimera spp. (considered as the progenitor of blaPER genes), indicates a role of ISPa12 in blaPER-2 gene mobilization from the chromosome of Pararheinheimera spp. Also, the blaPER-2 gene was carried by a novel ISPa12-composite transposon, Tn7390. In addition, its association with ISKox2-like elements in the close genetic environment in all plasmids analysed suggests a role of these insertion sequence elements in further dissemination of blaPER-2 genes.
Assuntos
Antibacterianos , Chromatiaceae , Antibacterianos/farmacologia , beta-Lactamases/genética , beta-Lactamases/metabolismo , Plasmídeos/genética , Elementos de DNA Transponíveis/genética , Sequência de Bases , Chromatiaceae/genéticaRESUMO
Alzheimer's disease (AD) is characterized by the presence of neuropsychiatric or behavioral and psychological symptoms of dementia (BPSD). BPSD have been associated with the APOE_ε4 allele, which is also the major genetic AD risk factor. Although the involvement of some circadian genes and orexin receptors in sleep and behavioral disorders has been studied in some psychiatric pathologies, including AD, there are no studies considering gene-gene interactions. The associations of one variant in PER2, two in PER3, two in OX2R and two in APOE were evaluated in 31 AD patients and 31 cognitively healthy subjects. Genotyping was performed using real-time PCR and capillary electrophoresis from blood samples. The allelic-genotypic frequencies of variants were calculated for the sample study. We explored associations between allelic variants with BPSD in AD patients based on the NPI, PHQ-9 and sleeping disorders questionnaires. Our results showed that the APOE_ε4 allele is an AD risk variant (p = 0.03). The remaining genetic variants did not reveal significant differences between patients and controls. The PER3_rs228697 variant showed a nine-fold increased risk for circadian rhythm sleep-wake disorders in Mexican AD patients, and our gene-gene interaction analysis identified a novel interaction between PERIOD and APOE gene variants. These findings need to be further confirmed in larger samples.
Assuntos
Doença de Alzheimer , Humanos , Alelos , Doença de Alzheimer/diagnóstico , Apolipoproteína E4/genética , Apolipoproteínas E/genética , Frequência do Gene , Genótipo , Proteínas Circadianas Period/genéticaRESUMO
The molecular circadian clock is based on a transcriptional/translational feedback loop in which the stability and half-life of circadian proteins is of importance. Cysteine residues of proteins are subject to several redox reactions leading to S-thiolation and disulfide bond formation, altering protein stability and function. In this work, the ability of the circadian protein period 2 (PER2) to undergo oxidation of cysteine thiols was investigated in HEK-293T cells. PER2 includes accessible cysteines susceptible to oxidation by nitroso cysteine (CysNO), altering its stability by decreasing its monomer form and subsequently increasing PER2 homodimers and multimers. These changes were reversed by treatment with 2-mercaptoethanol and partially mimicked by hydrogen peroxide. These results suggest that cysteine oxidation can prompt PER2 homodimer and multimer formation in vitro, likely by S-nitrosation and disulphide bond formation. These kinds of post-translational modifications of PER2 could be part of the redox regulation of the molecular circadian clock.
Assuntos
Relógios Circadianos , Proteínas Circadianas Period , Ritmo Circadiano/fisiologia , Cisteína/metabolismo , Dimerização , Oxirredução , Proteínas Circadianas Period/química , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Proteínas/metabolismoRESUMO
The blaPER-2-harboring plasmid pCf587 (191,541 bp) belongs to lineage IncA/C1 and is closely related to pRA1. It contains a large resistance island including the blaPER-2 gene between two copies of ISKox2-like elements, the toxin-antitoxin module pemK-pemI, several other resistance genes inserted within a Tn2 transposon, a Tn21-like structure, and a class 1 integron. pCf587 belongs to sequence type 13 (ST13), a new plasmid multilocus sequence typing (pMLST) ST.
Assuntos
Citrobacter freundii/enzimologia , Plasmídeos/genética , Antibacterianos/farmacologia , Citrobacter freundii/efeitos dos fármacos , Citrobacter freundii/genética , Farmacorresistência Bacteriana Múltipla/genética , Integrons/genética , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Análise de Sequência de DNA , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
The objective of this study was to investigate the molecular mechanisms explaining the multidrug-resistant (MDR) phenotype found in a novel clinical Shewanella sp. strain (Shew256) recovered from a diabetic patient. Whole-genome shotgun sequencing was performed using Illumina MiSeq-I and Nextera XT DNA library. De novo assembly was performed with SPAdes. RAST Server was used to predict the open-reading frames and the predictions were confirmed using BLAST. Further genomic analysis was carried out using average nucleotide identity (ANI), ACT (Artemis), OrthoMCL, ARG-ANNOT, ISfinder, PHAST, tRNAscan-SE, plasmidSPAdes, PlasmidFinder and MAUVE. PCR and plasmid extraction were also performed. Genomic analysis revealed a total of 456 predicted genes unique to Shew256 compared with other Shewanella genomes. Moreover, the presence of different resistance genes, including blaPER-2, was found. A complex class 1 integron containing the ISCR1 gene, disrupted by two putative transposase genes, was identified. Furthermore, other resistance genes, a transposon containing aph(3'), insertion sequences, phages and non-coding RNAs were also found. In conclusion, evidence of acquisition of resistance genes and mobile elements that could explain the MDR phenotype were observed. This Shewanella sp. represents a prime example of how antibiotic resistance determinants can be acquired by uncommon pathogens.
Assuntos
Elementos de DNA Transponíveis/genética , Farmacorresistência Bacteriana Múltipla/genética , Genes Bacterianos/genética , Shewanella/enzimologia , Shewanella/genética , beta-Lactamases/genética , Idoso , Antibacterianos/farmacologia , DNA Bacteriano/genética , Transferência Genética Horizontal , Genoma Bacteriano , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Integrons , Masculino , Testes de Sensibilidade Microbiana , Fases de Leitura Aberta , Filogenia , Plasmídeos/genética , Fatores R/genética , RNA não Traduzido , Análise de Sequência de DNA , Shewanella/efeitos dos fármacos , Shewanella/isolamento & purificação , Sequenciamento Completo do GenomaRESUMO
The suprachiasmatic nucleus (SCN), which is the main circadian biological clock in mammals, is composed of multiple cells that function individually as independent oscillators to express the self-sustained mRNA and protein rhythms of the so-called clock genes. Knowledge regarding the presence and localization of the proteins and neuroactive substances of the SCN are essential for understanding this nucleus and for its successful manipulation. Although there have been advances in the investigation of the intrinsic organization of the SCN in rodents, little information is available in diurnal species, especially in primates. This study, which explores the pattern of expression and localization of PER2 protein in the SCN of capuchin monkey, evaluates aspects of the circadian system that are common to both primates and rodents. Here, we showed that PER2 protein immunoreactivity is higher during the light phase. Additionally, the complex organization of cells that express vasopressin, vasoactive intestinal polypeptide, neuron-specific nuclear protein, calbindin and calretinin in the SCN, as demonstrated by their immunoreactivity, reveals an intricate network that may be related to the similarities and differences reported between rodents and primates in the literature.
Assuntos
Expressão Gênica/fisiologia , Proteínas Circadianas Period/metabolismo , Núcleo Supraquiasmático/metabolismo , Animais , Arginina Vasopressina/metabolismo , Calbindina 2/metabolismo , Calbindinas/metabolismo , Cebus , Ritmo Circadiano/genética , Masculino , Proteínas do Tecido Nervoso/metabolismo , Proteínas Circadianas Period/genética , Estimulação Luminosa , RNA Mensageiro/metabolismo , Serotonina/metabolismo , Peptídeo Intestinal Vasoativo/metabolismoRESUMO
Vertebrates have a central clock and also several peripheral clocks. Light responses might result from the integration of light signals by these clocks. The dermal melanophores of Xenopus laevis have a photoreceptor molecule denominated melanopsin (OPN4x). The mechanisms of the circadian clock involve positive and negative feedback. We hypothesize that these dermal melanophores also present peripheral clock characteristics. Using quantitative PCR, we analyzed the pattern of temporal expression of Opn4x and the clock genes Per1, Per2, Bmal1, and Clock in these cells, subjected to a 14-h light:10-h dark (14L:10D) regime or constant darkness (DD). Also, in view of the physiological role of melatonin in the dermal melanophores of X. laevis, we determined whether melatonin modulates the expression of these clock genes. These genes show a time-dependent expression pattern when these cells are exposed to 14L:10D, which differs from the pattern observed under DD. Cells kept in DD for 5 days exhibited overall increased mRNA expression for Opn4x and Clock, and a lower expression for Per1, Per2, and Bmal1. When the cells were kept in DD for 5 days and treated with melatonin for 1 h, 24 h before extraction, the mRNA levels tended to decrease for Opn4x and Clock, did not change for Bmal1, and increased for Per1 and Per2 at different Zeitgeber times (ZT). Although these data are limited to one-day data collection, and therefore preliminary, we suggest that the dermal melanophores of X. laevis might have some characteristics of a peripheral clock, and that melatonin modulates, to a certain extent, melanopsin and clock gene expression.