RESUMO
Giant hydronephrosis (GH), characterized by the presence of more than 1 L of fluid in the renal collecting system, is a rare urological condition, particularly in adults. Obstruction of the pyeloureteral junction is the most common cause of GH. We report the case of a 51-year-old man who presented with dyspnea, edema of the lower limbs, and major abdominal distension. The patient was diagnosed with obstruction of the pyeloureteral junction, causing a left giant hydronephrotic kidney. After renal drainage of 27 L of urine, a laparoscopic nephrectomy was performed. GH usually manifests as asymptomatic abdominal distension or vague symptoms. However, very few published reports describe cases of GH initially presenting with respiratory and vascular manifestations.
Assuntos
Hidronefrose , Masculino , Adulto , Humanos , Pessoa de Meia-Idade , Hidronefrose/diagnóstico , Rim/diagnóstico por imagem , Pelve Renal , Tomografia Computadorizada por Raios X , Drenagem/efeitos adversosRESUMO
De novo germline mutations arise preferentially in male owing to fundamental differences between spermatogenesis and oogenesis. Post-meiotic chromatin remodeling in spermatids results in the elimination of most of the nucleosomal supercoiling and is characterized by transient DNA fragmentation. Using three alternative methods, DNA from sorted populations of mouse spermatids was used to confirm that double-strand breaks (DSB) are created in elongating spermatids and repaired at later steps. Specific capture of DSB was used for whole-genome mapping of DSB hotspots (breakome) for each population of differentiating spermatids. Hotspots are observed preferentially within introns and repeated sequences hence are more prevalent in the Y chromosome. When hotspots arise within genes, those involved in neurodevelopmental pathways become preferentially targeted reaching a high level of significance. Given the non-templated DNA repair in haploid spermatids, transient DSBs formation may, therefore, represent an important component of the male mutation bias and the etiology of neurological disorders, adding to the genetic variation provided by meiosis.
Assuntos
Montagem e Desmontagem da Cromatina/genética , Quebras de DNA de Cadeia Dupla , Fragmentação do DNA , Espermátides/metabolismo , Animais , Ensaio Cometa , DNA/genética , DNA/metabolismo , Reparo do DNA , Masculino , Meiose/genética , Camundongos Endogâmicos C57BL , Nucleossomos/genéticaRESUMO
Transient DNA breaks and evidence of DNA damage response have recently been reported during the chromatin remodeling process in haploid spermatids, creating a potential window of enhanced genetic instability. We used flow cytometry to achieve separation of differentiating spermatids into four highly purified populations using transgenic mice harboring 160 CAG repeats within exon 1 of the human Huntington disease gene (HTT). Trinucleotic repeat expansion was found to occur immediately following the chromatin remodeling steps, confirming the genetic instability of the process and pointing to the origin of paternal anticipation observed in some trinucleotidic repeats diseases.
Assuntos
Montagem e Desmontagem da Cromatina , Instabilidade Genômica , Espermátides/metabolismo , Repetições de Trinucleotídeos , Animais , Citometria de Fluxo , Masculino , Camundongos , Espermatogênese/genética , Expansão das Repetições de TrinucleotídeosRESUMO
BACKGROUND: COVID-19 severity is associated with its respiratory manifestations. Neutralising antibodies against SARS-CoV-2 administered systemically have shown clinical efficacy. However, immediate and direct delivery of neutralising antibodies via inhalation might provide additional respiratory clinical benefits. IBIO123 is a cocktail of three, fully human, neutralising monoclonal antibodies against SARS-CoV-2. We aimed to assess the safety and efficacy of inhaled IBIO123 in individuals with mild-to-moderate COVID-19. METHODS: This double-blind, dose-ascending, placebo-controlled, first-in-human, phase 1/2 trial recruited symptomatic and non-hospitalised participants with COVID-19 in South Africa and Brazil across 11 centres. Eligible participants were adult outpatients (aged ≥18 years; men and non-pregnant women) infected with COVID-19 (first PCR-confirmed within 72 h) and with mild-to-moderate symptoms, the onset of which had to be within 10 days of randomisation. Using permuted blocks of four, stratified by site, we randomly assigned participants (1:3) to receive single-dose placebo or IBIO123 (1 mg, 5 mg, or 10 mg) in phase 1, and single-dose placebo or IBIO123 (10 mg) in phase 2, in addition to local standard of care. Participants underwent serological testing to identify antibodies against SARS-CoV-2. Participants, investigators, and the study team were masked to treatment assignment. In phase 1, the primary outcome was the safety assessment in the safety population (ie, all participants who received an intervention). In phase 2, the primary outcome was the mean absolute change from baseline to day 5 in SARS-CoV-2 viral load measured by nasopharyngeal swabs analysed using a mixed model for repeated measures in the full analysis set (FAS; ie, participants with one analysable viral load value at baseline and at least one analysable viral load value at day 3 or day 5). Secondary clinical outcomes included safety from baseline to day 29, assessed by evaluating adverse events; the effect of IBIO123 on baseline COVID-19 symptoms resolution until day 6, with symptoms systemically evaluated by the investigators; and disease progression as measured by the COVID-19 WHO Clinical Progression Scale. For clinical endpoints in phase 2, we used a modified FAS (ie, participants who had at least one analysable viral load value over the course of the study, confirming that they were infected with SARS-CoV-2). This trial is now completed and is registered with ClinicalTrials.gov, NCT05298813. FINDINGS: Between Dec 4, 2021, and May 23, 2022, 24 participants were enrolled in phase 1. Between July 20, 2022, and Jan 4, 2023, 138 participants were enrolled in phase 2 and five were excluded because they did not meet the inclusion criteria. Participants were randomly assigned to receive IBIO123 (n=18) or placebo (n=6) in phase 1, and randomly assigned to receive IBIO123 (n=104) or placebo (n=34) in phase 2. In phase 2, the study was stopped before reaching the planned accrual because of a decline in COVID-19 incidence. In phase 1, no safety issues were observed. In phase 2, the difference in mean absolute change from baseline viral load to day 5 between participants in the IBIO123 group and participants in the placebo group was -0·29 log10 copies per mL (95% CI -1·32 to 0·75; p=0·45) in the FAS population and -0·49 log10 copies per mL (-1·56 to 0·58; p=0·20) in seropositive participants. In the modified FAS, 81 (69%) of 118 participants were at high risk of severe disease progression. The number of participants with resolution of respiratory symptoms at day 6 was 34 (42%) of 81 in the IBIO123 group versus five (17%) of 29 in the placebo group (p=0·017) in the modified FAS population and 19 (35%) of 55 versus three (14%) of 21 among participants at high risk (p=0·083). One participant died and one participant was hospitalised in the placebo group, whereas no deaths or hospitalisations were reported in the IBIO123 group. 39 (38%) of 104 participants in the IBIO123 group had adverse events, compared with 13 (38%) of 34 in the placebo group. INTERPRETATION: Inhalation of IBIO123 was safe. Despite the lack of significant reduction of viral load at day 5, treatment with IBIO123 resulted in a higher proportion of participants with complete resolution of respiratory symptoms at day 6. This study supports further clinical research on inhaled monoclonal antibodies in COVID-19 and respiratory diseases in general. FUNDING: Canadian Strategic Innovation Fund and Immune Biosolutions.
Assuntos
COVID-19 , Adolescente , Adulto , Feminino , Humanos , Masculino , Anticorpos Monoclonais , Anticorpos Neutralizantes , Canadá , Método Duplo-Cego , SARS-CoV-2RESUMO
At the sequence level, genetic diversity is provided by de novo transmittable mutations that may act as a substrate for natural selection. The gametogenesis process itself is considered more likely to induce endogenous mutations and a clear male bias has been demonstrated from recent next-generation sequencing analyses. As new experimental evidence accumulates, the post-meiotic events of the male gametogenesis (spermiogenesis) appear as an ideal context to induce de novo genetic polymorphism transmittable to the next generation. It may prove to be a major component of the observed male mutation bias. As spermatids undergo chromatin remodeling, transient endogenous DNA double-stranded breaks are produced and trigger a DNA damage response. In these haploid cells, one would expect that the non-templated, DNA end-joining repair processes may generate a repertoire of sequence alterations in every sperm cell potentially transmittable to the next generation. This may therefore represent a novel physiological mechanism contributing to genetic diversity and evolution.
Assuntos
Dano ao DNA/genética , Reparo do DNA/genética , Células Germinativas/citologia , Espermatogênese/genética , Espermatozoides/citologia , Variação Genética , Haploidia , Humanos , Masculino , Mutação , Polimorfismo Genético , Espermatozoides/metabolismoRESUMO
BACKGROUND The blind-ending branch of a bifid ureter is a rare congenital anomaly which is usually asymptomatic but can occasionally give rise to various symptoms, such as chronic abdominal pain. Diagnosis is most often confirmed radiologically, and treatment is usually conservative. Surgical resection of the blind ending of a bifid ureter should be considered in cases of persistent symptoms. CASE REPORT A female patient of 49 years of age presented with intermittent right lumbar pain, repetitive urinary infections and microscopic hematuria. We present here the diagnostic work-up of the case, leading to the identification of the existence of ureteral bifidity located at the lower third of the ureter and of a blind ending of the bifid ureter. Several regimens of various antibiotics failed to resolve the symptoms. It was decided to carry out a laparoscopic resection of the blind ending of the bifid ureter. We describe the practical procedures of the surgical operation and discuss briefly the embryological etiology and the physiopathology of the condition as well as the principal diagnostic modalities. Since the surgery, the patient has been symptom-free. CONCLUSIONS Despite being usually asymptomatic, the rare congenital anomaly of a bifid ureter with a blind ending can occasionally give rise to symptoms such as recurrent infections and persistent abdominal pain. Laparoscopic-based resection of the blind ending should be considered in such cases.
Assuntos
Laparoscopia , Ureter , Obstrução Ureteral , Infecções Urinárias , Dor Abdominal/etiologia , Dor Abdominal/cirurgia , Feminino , Humanos , Ureter/cirurgiaRESUMO
Within the sperm nucleus, the paternal genome remains functionally inert and protected following protamination. This is marked by a structural morphogenesis that is heralded by a striking reduction in nuclear volume. Despite these changes, both human and mouse spermatozoa maintain low levels of nucleosomes that appear non-randomly distributed throughout the genome. These regions may be necessary for organizing higher order genomic structure through interactions with the nuclear matrix. The promoters of this transcriptionally quiescent genome are differentially marked by modified histones that may poise downstream epigenetic effects. This notion is supported by increasing evidence that the embryo inherits these differing levels of chromatin organization. In concert with the suite of RNAs retained in the mature sperm, they may synergistically interact to direct early embryonic gene expression. Irrespective, these features reflect the transcriptional history of spermatogenic differentiation. As such, they may soon be utilized as clinical markers of male fertility. In this review, we explore and discuss how this may be orchestrated.
Assuntos
Núcleo Celular/metabolismo , Cromatina/metabolismo , Matriz Nuclear/metabolismo , RNA/metabolismo , Espermatozoides/metabolismo , Animais , Montagem e Desmontagem da Cromatina , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Masculino , Camundongos , Nucleossomos/metabolismoRESUMO
The observation that PrP is present in the cytosol of some neurons and non-neuronal cells and that the N-terminal signal peptide is slightly inefficient has brought speculations concerning a possible function of the protein in the cytosol. Here, we show that cells expressing a cytosolic form of PrP termed cyPrP display a large juxtanuclear cytoplasmic RNA organelle. Although cyPrP spontaneously forms aggresomes, we used several mutants to demonstrate that the assembly of this RNA organelle is independent from cyPrP aggregation. Components of the organelle fall into three classes: mRNAs; proteins, including the RNAseIII family polymerase Dicer, the decapping enzyme Dcp1a, the DEAD-box RNA helicase DDX6, and the small nuclear ribonucleoprotein-associated proteins SmB/B'/N; and non-coding RNAs, including rRNA 5S, tRNAs, U1 small nuclear RNA, and microRNAs. This composition is similar to RNA granules or chromatoid bodies from germ cells, or planarian stem cells and neurons, which are large ribonucleoprotein complexes predicted to function in RNA processing and posttranscriptional gene regulation. The domain of PrP encompassing residues 30 to 49 is essential for the formation of the RNA particle. Our findings confirm the intriguing relation between PrP and RNA in cells, and underscore an unexpected function for cytosolic PrP: assembling a large RNA processing center which we have termed PrP-RNP for PrP-induced ribonucleoprotein particle.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Regulação da Expressão Gênica , Príons/metabolismo , RNA/metabolismo , Ribonucleoproteínas/metabolismo , Transcrição Gênica , Animais , Linhagem Celular Tumoral , Células Germinativas/metabolismo , Camundongos , Neurônios/metabolismo , Poro Nuclear/metabolismo , Planárias/citologia , Planárias/metabolismo , Ligação Proteica , RNA Ribossômico 18S/metabolismo , RNA Ribossômico 28S/metabolismo , RNA Nuclear Pequeno/metabolismo , RNA de Transferência/metabolismo , Ribonucleoproteínas/química , Células-Tronco/metabolismo , Vimentina/metabolismoRESUMO
We present a case of a leiomyoma of the seminal vesicle that occurred in a 52-year-old man who presented with symptoms of bladder outlet obstruction. Prostate-specific antigen was within normal limit. Computed tomography scan and magnetic resonance imaging revealed a mass in the patient's right seminal vesicle. Laparoscopic excision of the seminal vesicle tumor was performed successfully. The patient was discharged from the hospital on the fourth postoperative day.
RESUMO
DNA double-strand breaks (DSBs) represent a major threat to the genetic integrity of the cell. Knowing both their genome-wide distribution and number is important for a better assessment of genotoxicity at a molecular level. Available methods may have underestimated the extent of DSBs as they are based on markers specific to those undergoing active repair or may not be adapted for the large diversity of naturally occurring DNA ends. We have established conditions for an efficient first step of DNA nick and gap repair (NGR) allowing specific determination of DSBs by end labeling with terminal transferase. We used DNA extracted from HeLa cells harboring an I-SceI cassette to induce a targeted nick or DSB and demonstrated by immunocapture of 3'-OH that a prior step of NGR allows specific determination of loci-specific or genome wide DSBs. This method can be applied to the global determination of DSBs using radioactive end labeling and can find several applications aimed at understanding the distribution and kinetics of DSBs formation and repair.
Assuntos
Mapeamento Cromossômico/métodos , DNA Nucleotidilexotransferase/genética , Reparo do DNA , DNA/genética , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Genoma Humano , Proteínas de Saccharomyces cerevisiae/genética , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Nucleotidilexotransferase/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Loci Gênicos , Células HeLa , Humanos , Cinética , Radioisótopos de Fósforo/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , TransfecçãoRESUMO
A strategy amenable to the genome-wide study of DNA damage and repair kinetics is described. The ultraviolet damage endonuclease (UVDE) generates 3'-OH ends at the two major UV induced DNA lesions, cyclobutane pyrimidine dimers (CPDs) and 6,4 pyrimidine-pyrimidone dimers (6,4 PPs), allowing for their capture after biotin end-labeling. qPCR amplification of biotinylated DNA enables parallel measuring of DNA damage in several loci, which can then be combined with high-throughput screening of cell survival to test genotoxic reagents. Alternatively, a library of captured sequences could be generated for a genome wide study of damage sites and large-scale assessment of repair kinetics in different regions of the genome, using next-generation sequencing. The assay is suitable to study any DNA lesion that can be converted into 3'-OH by UVDE, or other enzymes. Toward these goals, we compared UVDE with the classical T4 endonuclease V (T4V) assay. We showed that there is a linear correlation between UV dose, 3'-OH formation and capture by immunoprecipitation, together with its potential application for in vivo studies.
Assuntos
Dano ao DNA , Genoma Fúngico , Imunoprecipitação , Testes de Mutagenicidade , Dímeros de Pirimidina/análise , DNA Fúngico/química , Endodesoxirribonucleases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to the next generation. So far, molecular studies of this morphological transition have been hampered by the lack of a method allowing adequate separation of these important steps of spermatid differentiation for subsequent analyses. Earlier attempts at proper gating of these cells using flow cytometry may have been difficult because of a peculiar increase in DNA fluorescence in spermatids undergoing chromatin remodeling. Based on this observation, we provide details of a simple flow cytometry scheme, allowing reproducible purification of four populations of mouse spermatids fixed with ethanol, each representing a different state in the nuclear remodeling process. Population enrichment is confirmed using step-specific markers and morphological criterions. The purified spermatids can be used for genomic and proteomic analyses.
Assuntos
Citometria de Fluxo/métodos , Espermátides/classificação , Animais , Diferenciação Celular/fisiologia , DNA/análise , Masculino , Camundongos , Proteômica/métodos , Espermátides/química , Espermátides/citologia , EspermatogêneseRESUMO
Determination of cellular DNA damage has so far been limited to global assessment of genome integrity whereas nucleotide-level mapping has been restricted to specific loci by the use of specific primers. Therefore, only limited DNA sequences can be studied and novel regions of genomic instability can hardly be discovered. Using a well-characterized yeast model, we describe a straightforward strategy to map genome-wide DNA strand breaks without compromising nucleotide-level resolution. This technique, termed "damaged DNA immunoprecipitation" (dDIP), uses immunoprecipitation and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labeling (TUNEL) to capture DNA at break sites. When used in combination with microarray or next-generation sequencing technologies, dDIP will allow researchers to map genome-wide DNA strand breaks as well as other types of DNA damage and to establish a clear profiling of altered genes and/or intergenic sequences in various experimental conditions. This mapping technique could find several applications for instance in the study of aging, genotoxic drug screening, cancer, meiosis, radiation and oxidative DNA damage.
Assuntos
Mapeamento Cromossômico/métodos , Quebras de DNA , Saccharomyces cerevisiae/genética , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/metabolismo , Dano ao DNA/genética , Dano ao DNA/fisiologia , Genes Fúngicos Tipo Acasalamento/genética , Loci Gênicos , Marcação In Situ das Extremidades Cortadas , Modelos Biológicos , Organismos Geneticamente Modificados , Plasmídeos/genética , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
Wood preserved with chromated copper arsenate (CCA) and alkaline copper quaternary (ACQ) was mixed with artificial rainwater, to generate leachates containing As, Cr and Cu. Then, leachates were applied to two soils at rates of 13-169 mg As kg(-1) soil (dry weight basis), 12-151 mg Cr kg(-1) and 10-216 mg Cu kg(-1). Metal bioavailability was evaluated after 28 days using the earthworm Eisenia fetida (Savigny). Metal concentrations in earthworm tissue ranged from negligible to 80 mg As kg(-1) (dry weight basis), 89 mg Cr kg(-1) and 90 mg Cu kg(-1), which appeared to be non-lethal to E. fetida. There was less Cu available to earthworms in the Courval soil (pH 7.8) than the Châteauguay soil (pH 6.8), but earthworm growth and reproduction were not affected by exposure to Cu from ACQ-treated wood. In contrast, earthworms exposed to As, Cr and Cu from the CCA-treated wood gained weight more quickly in the Courval soil (1.3-21 mg g(-1) initial biomass days) than in the Châteauguay soil (0.2-7.8 mg g(-1) day(-1)), but fewer than 20% of the cocoons deposited by the faster-growing earthworms hatched by the end of the 56 days ecotoxicology test. It appeared that E. fetida can allocate more energy to growth than reproduction, delaying cocoon development and hatching in some situations. Further information is needed on the soil factors that may induce such behavior, as it can affect the interpretation of results from the earthworm ecotoxicology test.
Assuntos
Arseniatos/toxicidade , Oligoquetos/efeitos dos fármacos , Poluentes do Solo/toxicidade , Animais , Arseniatos/farmacocinética , Arsênio/análise , Arsênio/metabolismo , Arsênio/toxicidade , Disponibilidade Biológica , Cromo/análise , Cromo/metabolismo , Cromo/toxicidade , Cobre/análise , Cobre/metabolismo , Cobre/toxicidade , Oligoquetos/crescimento & desenvolvimento , Oligoquetos/fisiologia , Compostos Organometálicos/análise , Compostos Organometálicos/metabolismo , Compostos Organometálicos/toxicidade , Chuva , Reprodução/efeitos dos fármacos , Poluentes do Solo/análise , Poluentes do Solo/farmacocinética , MadeiraRESUMO
This paper reviews the possible origin of sperm DNA fragmentation and focuses on the nuclear events associated with spermiogenesis as a potential source of genetic instability and reduced fertilizing potential of the mature male gamete. Recent findings suggest a programmed DNA fragmentation and DNA damage response during the chromatin remodeling steps in spermatids. We also discuss the spermatid DNA repair mechanisms and the possible involvement of condensing proteins, such as transition proteins and protamines, in the process, as this DNA fragmentation is normally not found in late spermatids. We propose that alterations in the chromatin remodeling steps or DNA repair in elongating spermatids may lead to persistent DNA breaks. This vulnerable step of spermiogenesis may provide a clue to the etiology of sperm DNA fragmentation associated with infertility in humans. This vulnerability is further emphasized given the haploid character of spermatids that must resolve programmed double-stranded breaks by an error-prone DNA repair mechanism. Therefore, spermiogenesis has probably been overlooked as an important source of genetic instability.
Assuntos
Transtornos Cromossômicos , Reparo do DNA , Infertilidade Masculina/genética , Espermatogênese/genética , Espermatozoides/fisiologia , Animais , Apoptose , Instabilidade Cromossômica , Fragmentação do DNA , Humanos , Masculino , Idade PaternaRESUMO
A precise packaging of the paternal genome during spermiogenesis is essential for fertilization and embryogenesis. Most of the nucleosomal DNA supercoiling must be eliminated in elongating spermatids (ES), and transient DNA strand breaks are observed that facilitate the process. Topoisomerases have been considered as ideal candidates for the removal of DNA supercoiling, but their catalytic activity, in the context of such a major chromatin remodeling, entails genetic risks. Using immunofluorescence, we confirmed that topoisomerase II beta (TOP2B) is the type II topoisomerase present in ES between steps 9 and 13. Interestingly, the detection of TOP2B was found coincident with detection of tyrosyl-DNA phosphodiesterase 1 (TDP1), an enzyme known to resolve topoisomerase-mediated DNA damage. The presence of gamma-H2AX (also known as H2AFX) coincident with DNA strand breakage was also confirmed at these steps and indicates that a DNA damage response is triggered. Active DNA repair in ES was demonstrated using a fluorescent in situ DNA polymerase activity assay on squash preparations of staged tubules. In the context of haploid spermatids, any unresolved double-strand breaks, resulting from a failure in the rejoining process of TOP2B, must likely rely on the error-prone nonhomologous end joining, because homologous recombination cannot proceed in the absence of a sister chromatid. Because this process is part of the normal developmental program of the spermatids, dramatic consequences for the genomic integrity of the developing male gamete may arise should any alteration in the process occur.