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The cerebrovascular system provides crucial functions that maintain metabolic and homeostatic states of the brain. Despite its integral role of supporting cerebral viability, the topological organization of these networks remains largely uncharacterized. This void in our knowledge surmises entirely from current technological limitations that prevent the capturing of data through the entire depth of the brain. We report high-resolution reconstruction and analysis of the complete vascular network of the entire brain at the capillary level in adult female and male mice using a vascular corrosion cast procedure. Vascular network analysis of the whole brain revealed sex-related differences of vessel hierarchy. In addition, region-specific network analysis demonstrated different patterns of angioarchitecture between brain subregions and sex. Furthermore, our group is the first to provide a three-dimensional analysis of the angioarchitecture and network organization in a single reconstructed tomographic data set that encompasses all hierarchy of vessels in the brain of the adult mouse.
Assuntos
Encéfalo/irrigação sanguínea , Imageamento Tridimensional/métodos , Neuroimagem/métodos , Microtomografia por Raio-X/métodos , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Recent investigations have shown that deep eutectic solvents provide a suitable environment for self-organisation of biomolecules, in particular phospholipids and proteins. However, the solvation of complex lyophilic moieties by deep eutectic solvents still remains unclear. Here we explore the behaviour of zwitterionic surfactants in choline chloride:glycerol eutectic mixture. Dodecyl-2-(trimethylammonio)ethylphosphate and N-alkyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (alkyl = dodecyl, tetradecyl) surfactants were investigated by means of surface tension, X-ray reflectivity and small-angle neutron scattering. These surfactants were found to remain surface active and form globular micelles in deep eutectic solvents. Still, the surface behaviour of these species was found to differ depending on the headgroup and tail structure. The morphology of the micelles also slightly varies between surfactants, demonstrating differences in the packing of individual monomers. The characteristics of mixtures of the dodecyl surfactants is also reported, showing a deviation from ideal mixing associated with attractive interactions between sulfobetaine and phosphocholine headgroups. Such non-ideality results in variation of the surface behaviour and self-assembly of these surfactant mixtures. The results presented here will potentially lead to the development of new alternatives for drug-delivery, protein solubilisation and biosensing through a better fundamental understanding of the behaviour of zwitterionic surfactants in deep eutectic solvents.
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Cryoinjury is a consequence of cryopreservation and may have a negative impact on sperm quality regarding motility, morphology, and viability. This study was designed to identify potential proteomic changes in human sperm cells throughout the cryopreservation process. Comparisons made within this study included the detection of the sperm proteomic changes induced by incubation of the sperm cells with a protein-free cryoprotectant (with and without CryoSperm), and the proteomic changes induced by freezing, thawing, and subsequent after-thawing incubation at two different temperatures (0 °C vs. 23 °C). Tandem Mass Tag (TMT) peptide labeling coupled with LC-MS/MS was used for protein quantification. LC-MS/MS resulted in the identification of 769 quantifiable proteins. The abundance of 105 proteins was altered upon CryoSperm incubation. Freezing and thawing also induced substantial protein changes. However, fewer changes were observed when semen was thawed and then maintained after-thawing at approximately 0 °C than when it was maintained after-thawing at 23 °C, with 60 and 99 differential proteins detected, respectively, as compared to unfrozen semen incubated in CryoSperm. Collectively, these differences indicate that substantial changes occur in the sperm proteome at every stage of the cryopreservation process which may ultimately impair the sperm fertilizing capability. This is the first study to compare protein levels in fresh and cryopreserved semen using the TMT technology coupled to LC-MS/MS.
Assuntos
Criopreservação/métodos , Preservação do Sêmen/métodos , Espermatozoides/metabolismo , Adulto , Crioprotetores , Fertilização/fisiologia , Humanos , Masculino , Proteômica , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/citologia , Espectrometria de Massas em TandemRESUMO
INTRODUCTION: The selection of a sperm with good genomic integrity is an important consideration for improving intracytoplasmic sperm injection (ICSI) outcome. Current convention selects sperm by vigour and morphology, but preliminary evidence suggests selection based on hyaluronic acid binding may be beneficial. The aim of the Hyaluronic Acid Binding Sperm Selection (HABSelect) trial is to determine the efficacy of hyaluronic acid (HA)-selection of sperm versus conventionally selected sperm prior to ICSI on live birth rate (LBR). The mechanistic aim is to assess whether and how the chromatin state of HA-selected sperm corresponds with clinical outcomes-clinical pregnancy rate (CPR), LBR and pregnancy loss (PL). METHODS AND ANALYSIS: Couples attending UK Centres will be approached, eligibility screening performed and informed consent sought. Randomisation will occur within 24â hours prior to ICSI treatment. Participants will be randomly allocated 1:1 to the intervention arm (physiological intracytoplasmic sperm injection, PICSI) versus the control arm using conventional methods (ICSI). The primary clinical outcome is LBR ≥37â weeks' gestation with the mechanistic study determining LBR's relationship with sperm DNA integrity. Secondary outcomes will determine this for CPR and PL. Only embryologists performing the procedure will be aware of the treatment allocation. Steps will be taken to militate against biases arising from embryologists being non-blinded. Randomisation will use a minimisation algorithm to balance for key prognostic variables. The trial is powered to detect a 5% difference (24-29%: p=0.05) in LBR ≥37â weeks' gestation. Selected residual sperm samples will be tested by one or more assays of DNA integrity. ETHICS AND DISSEMINATION: HABSelect is a UK NIHR-EME funded study (reg no 11/14/34; IRAS REF. 13/YH/0162). The trial was designed in partnership with patient and public involvement to help maximise patient benefits. Trial findings will be reported as per CONSORT guidelines and will be made available in lay language via the trial web site (http://www.habselect.org.uk/). TRIAL REGISTRATION NUMBER: ISRCTN99214271; Pre-results.
Assuntos
Coeficiente de Natalidade , Ácido Hialurônico , Resultado da Gravidez , Injeções de Esperma Intracitoplásmicas/métodos , Espermatozoides , Aborto Espontâneo , Adolescente , Adulto , Cromatina , Protocolos Clínicos , DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Gravidez , Taxa de Gravidez , Projetos de Pesquisa , Adulto JovemRESUMO
Industry transitions away from traditional photosystem II inhibiting (PSII) herbicides towards an 'alternative' herbicide suite are now widely advocated as a key component of improved environmental outcomes for Australia's Great Barrier Reef and improved environmental stewardship on the part of the Queensland sugar industry. A systematic desktop risk analysis found that based on current farming practices, traditional PSII herbicides can pose significant environmental risks. Several of the 'alternatives' that can directly fill a specific pre-emergent ('soil residual') weed control function similar to regulated PSII herbicides also, however, presented a similar environmental risk profile, regardless of farming systems and bio-climatic zones being considered. Several alternatives with a pre-emergent residual function as well as alternative post-emergent (contact or 'knockdown') herbicides were, predicted to pose lower environmental risks than the regulated PSII herbicides to most trophic levels, although environmental risks could still be present. While several herbicides may well be viable alternatives in terms of weed control, they can still present equal or possibly higher risks to the environment. Imposing additional regulations (or even de-registrations) on particular herbicides could result in marginal, and possibly perverse environmental impacts in the long term, if usage shifts to alternative herbicides with similar risk profiles. Regardless of any regulatory efforts, improved environmental sustainability outcomes in pesticide practices within the Great Barrier Reef catchment area will hinge primarily on the continuing adoption of integrated, strategic pest management systems and technologies applied to both traditional and 'alternative' herbicides. One of the emerging policy challenges is ensuring the requisite technical and extension support for cane growers to ensure effective adoption of rapidly evolving farming system technologies, in a very dynamic and scrutinised herbicide management environment.
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Agricultura/métodos , Recifes de Corais , Herbicidas/análise , Poluentes Químicos da Água/análise , Poluição Química da Água/prevenção & controle , Austrália , Poluição Química da Água/estatística & dados numéricosRESUMO
Heritable bacteria have been highlighted as important components of vector biology, acting as required symbionts with an anabolic role, altering competence for disease transmission, and affecting patterns of gene flow by altering cross compatibility. In this paper, we tested eight U.K. species of Culicoides (Diptera: Ceratopogonidae) midge for the presence of five genera of endosymbiotic bacteria: Cardinium (Bacteroidales: Bacteroidaceae); Wolbachia (Rickettsiales: Rickettsiaceae); Spiroplasma (Entomoplasmatales: Spiroplasmataceae); Arsenophonus (Enterobacteriales: Enterobacteriaceae), and Rickettsia (Rickettsiales: Rickettsiaceae). Cardinium spp. were detected in both sexes of Culicoides pulicaris and Culicoides punctatus, two known vectors of bluetongue virus. Cardinium spp. were not detected in any other species, including the Culicoides obsoletus group, the main vector of bluetongue and Schmallenberg viruses in northern Europe. The other endosymbionts were not detected in any Culicoides species. The Cardinium strain detected in U.K. Culicoides species is very closely related to the Candidatus Cardinium hertigii group C, previously identified in Culicoides species in Asia. Further, we infer that the symbiont is not a sex ratio distorter and shows geographic variation in prevalence within a species. Despite its detection in several species of Culicoides that vector arboviruses worldwide, the absence of Cardinium in the C. obsoletus group suggests that infections of these symbionts may not be necessary to the arboviral vector competence of biting midges.
Assuntos
Bacteroidaceae/genética , Bacteroidaceae/isolamento & purificação , Vírus Bluetongue/fisiologia , Ceratopogonidae/microbiologia , Insetos Vetores/microbiologia , Insetos Vetores/virologia , Animais , Feminino , Masculino , Filogenia , Especificidade da Espécie , SimbioseRESUMO
The Gene Ontology (GO) Consortium (GOC, http://www.geneontology.org) is a community-based bioinformatics resource that classifies gene product function through the use of structured, controlled vocabularies. Over the past year, the GOC has implemented several processes to increase the quantity, quality and specificity of GO annotations. First, the number of manual, literature-based annotations has grown at an increasing rate. Second, as a result of a new 'phylogenetic annotation' process, manually reviewed, homology-based annotations are becoming available for a broad range of species. Third, the quality of GO annotations has been improved through a streamlined process for, and automated quality checks of, GO annotations deposited by different annotation groups. Fourth, the consistency and correctness of the ontology itself has increased by using automated reasoning tools. Finally, the GO has been expanded not only to cover new areas of biology through focused interaction with experts, but also to capture greater specificity in all areas of the ontology using tools for adding new combinatorial terms. The GOC works closely with other ontology developers to support integrated use of terminologies. The GOC supports its user community through the use of e-mail lists, social media and web-based resources.
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Bases de Dados Genéticas , Genes , Anotação de Sequência Molecular , Vocabulário Controlado , Internet , FilogeniaRESUMO
Sperm DNA damage has a negative impact on pregnancy rates following assisted reproduction treatment (ART). The aim of the present study was to examine the relationship between sperm DNA fragmentation and live-birth rates after IVF and intracytoplasmic sperm injection (ICSI). The alkaline Comet assay was employed to measure sperm DNA fragmentation in native semen and in spermatozoa following density-gradient centrifugation in semen samples from 203 couples undergoing IVF and 136 couples undergoing ICSI. Men were divided into groups according to sperm DNA damage. Following IVF, couples with <25% sperm DNA fragmentation had a live-birth rate of 33%; in contrast, couples with >50% sperm DNA fragmentation had a much lower live-birth rate of 13%. Following ICSI, no significant differences in sperm DNA damage were found between any groups of patients. Sperm DNA damage was also associated with low live-birth rates following IVF in both men and couples with idiopathic infertility: 39% of couples and 41% of men with idiopathic infertility have high sperm DNA damage. Sperm DNA damage assessed by the Comet assay has a close inverse relationship with live-birth rates after IVF. Sperm DNA damage has a negative impact on assisted reproduction treatment outcome, in particular, on pregnancy rates. The aim of the present study was to examine the relationship between sperm DNA fragmentation and live-birth rates after IVF and intracytoplasmic sperm injection (ICSI). The alkaline Comet assay was employed to measure sperm DNA fragmentation in native semen and in spermatozoa following density-gradient centrifugation in semen samples from 203 couples undergoing IVF and 136 couples undergoing ICSI. Men were divided into groups according to sperm DNA damage and treatment outcome. Following IVF, couples with <25% sperm DNA fragmentation had a live birth rate of 33%. In contrast, couples with >50% sperm DNA fragmentation had a much lower live-birth rate of 13% following IVF. Following ICSI, there were no significant differences in levels of sperm DNA damage between any groups of patients. Sperm DNA damage was also associated with the very low live-birth rates following IVF in both men and couples with idiopathic infertility: 39% of couples and 41% of men have high level of sperm DNA damage. Sperm DNA damage assessed by the Comet assay has a close inverse relationship with live-birth rates after IVF.
Assuntos
Fragmentação do DNA , Fertilização in vitro , Taxa de Gravidez , Espermatozoides/fisiologia , Adulto , Ensaio Cometa , Feminino , Humanos , Nascido Vivo , Masculino , Gravidez , Reprodutibilidade dos Testes , Análise do SêmenRESUMO
STUDY QUESTION: Is there a need for a specific guide addressing studies of seminal quality? SUMMARY ANSWER: The proposed guidelines for the appraisal of SEMinal QUAlity studies (SEMQUA) reflect the need for improvement in methodology and research on semen quality. WHAT IS KNOWN ALREADY: From an examination of other instruments used to assess the quality of diagnostic studies, there was no guideline on studies of seminal quality. STUDY DESIGN, SIZE AND DURATION: Through systematic bibliographic search, potential items were identified and grouped into four blocks: participants, analytical methods, statistical methods and results. PARTICIPANTS/MATERIALS, SETTING AND METHODS: Our findings were presented to a panel of experts who were asked to identify opportunities for improvement. Then, a checklist was designed containing the questions generated by the items that summarize the essential points that need to be considered for the successful outcome of a SEMQUA. MAIN RESULTS AND THE ROLE OF CHANCE: Eighteen items were identified, from which 19 questions, grouped into four blocks, were generated to constitute the final checklist. An explanation for the inclusion of each item was provided and some examples found in the bibliographic search were cited. LIMITATIONS AND REASONS FOR CAUTION: We consider that not all items are equally applicable to all study designs, and so the hypothetical results are not comparable. For that reason, a score would not be fair to critically appraise a study. This checklist is presented as an instrument for appraising SEMQUAs and therefore remains open to constructive criticism. It will be further developed in the future, in parallel with the continuing evolution of SEMQUAs. WIDER IMPLICATIONS OF THE FINDINGS: The final configuration of the SEMQUA is in the form of a checklist, and includes the items generally considered to be essential for the proper development of a SEMQUA. The final checklist produced has various areas of application; for example, it would be useful for designing and constructing a SEMQUA, for reviewing a paper on the question, for educational purposes or as an instrument for appraising the quality of research articles in this field. STUDY FUNDING/COMPETING INTEREST(S): None.
Assuntos
Guias de Prática Clínica como Assunto , Análise do Sêmen/normas , Pesquisa Biomédica/tendências , Lista de Checagem , Europa (Continente) , Humanos , Infertilidade Masculina/diagnóstico , Masculino , Revisão da Pesquisa por Pares/métodos , Sociedades Científicas , Instituições Filantrópicas de SaúdeRESUMO
Recent societal acceptance of cannabinoids as recreational and therapeutic drugs has posed a potential hazard to male reproductive health. Mammals have a highly sophisticated endogenous cannabinoid (ECS) system that regulates male (and female) reproduction and exo-cannabinoids may influence it adversely. Therefore it is imperative to determine their effects on male reproduction so that men can make informed choices as to their use. Here, an animal model was used to administer HU210, a synthetic analogue of Δ9-tetrahydrocannabinol (THC) and potent cannabinoid receptor (CB) agonist to determine its effects on reproductive organ weights, spermatogenesis, testicular histology and sperm motility. Its effects on the physiological endocannabinoid system were also investigated. Spermatogenesis was markedly impaired with reductions in total sperm count after 2 weeks of exposure. Spermatogenic efficiency was depleted, and Sertoli cell number decreased as exposure time increased with seminiferous tubules showing germ cell depletion developing into atrophy in some cases. Sperm motility was also adversely affected with marked reductions from 2 weeks on. HU210 also acted on the sperm's endocannabinoid system. Long-term use of exo-cannabinoids has adverse effects on both spermatogenesis and sperm function. These findings highlight the urgent need for studies evaluating the fertility potential of male recreational drug users. HU210, a selective agonist for CB1 and CB2 cannabinoid receptors impairs spermatogenesis and sperm motility and deregulates the endocannabinoid system.
Assuntos
Agonistas de Receptores de Canabinoides/toxicidade , Dronabinol/análogos & derivados , Espermatogênese/efeitos dos fármacos , Animais , Dronabinol/toxicidade , Endocanabinoides/fisiologia , Masculino , Tamanho do Órgão/efeitos dos fármacos , Ratos , Células de Sertoli , Motilidade dos Espermatozoides/efeitos dos fármacosRESUMO
The Great Barrier Reef (GBR) is a World Heritage Area and contains extensive areas of coral reef, seagrass meadows and fisheries resources. From adjacent catchments, numerous rivers discharge pollutants from agricultural, urban, mining and industrial activity. Pollutant sources have been identified and include suspended sediment from erosion in cattle grazing areas; nitrate from fertiliser application on crop lands; and herbicides from various land uses. The fate and effects of these pollutants in the receiving marine environment are relatively well understood. The Australian and Queensland Governments responded to the concerns of pollution of the GBR from catchment runoff with a plan to address this issue in 2003 (Reef Plan; updated 2009), incentive-based voluntary management initiatives in 2007 (Reef Rescue) and a State regulatory approach in 2009, the Reef Protection Package. This paper reviews new research relevant to the catchment to GBR continuum and evaluates the appropriateness of current management responses.
Assuntos
Recifes de Corais , Poluentes da Água/análise , Poluição da Água/estatística & dados numéricos , Agricultura/estatística & dados numéricos , Austrália , Conservação dos Recursos Naturais/legislação & jurisprudência , Conservação dos Recursos Naturais/métodos , Monitoramento Ambiental , Política Ambiental , Sedimentos Geológicos/química , Água do Mar/química , Poluição da Água/legislação & jurisprudência , Poluição da Água/prevenção & controleRESUMO
Coral cores were collected along an environmental and water quality gradient through the Whitsunday Island group, Great Barrier Reef (Australia), for trace element and stable isotope analysis. The primary aim of the study was to examine if this gradient could be detected in coral records and, if so, whether the gradient has changed over time with changing land use in the adjacent river catchments. Y/Ca was the trace element ratio which varied spatially across the gradient, with concentrations progressively decreasing away from the river mouths. The Ba/Ca and Y/Ca ratios were the only indicators of change in the gradient through time, increasing shortly after European settlement. The Mn/Ca ratio responded to local disturbance related to the construction of tourism infrastructure. Nitrogen isotope ratios showed no apparent trend over time. This study highlights the importance of site selection when using coral records to record regional environmental signals.
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Antozoários/química , Recifes de Corais , Monitoramento Ambiental/métodos , Poluentes Químicos da Água/análise , Animais , Austrália , Rios/química , Oligoelementos/análise , Movimentos da Água , Poluição Química da Água/estatística & dados numéricosRESUMO
Anaesthetists in the Defence Medical Services (DMS) are currently dealing with casualties who have an increased prevalence of injuries due to blast, fragmentation and gunshot wounds. Despite guidelines already existing for unanticipated difficult tracheal intubation these have been designed for a civilian population and might not be relevant for the anticipated difficult airway experienced in the deployed field hospital. In order to establish an overview of current practice, three methods of investigation were undertaken; a literature review, a survey of DMS Anaesthetists and a search of the UKJoint Theatre Trauma Database. Results are discussed in terms of anatomical site, bleeding in the airway, facial distortion, patient positioning and an anaesthetic approach. There are certain key principles that should be considered in all cases and these are considered. Potential pitfalls are discussed and our initial proposed guidelines for use in the deployed field hospital are presented.
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Manuseio das Vias Aéreas/métodos , Traumatismos Faciais/cirurgia , Lesões do Pescoço/cirurgia , Ferimentos Penetrantes/cirurgia , Anestesia/métodos , Lesões das Artérias Carótidas/cirurgia , Humanos , Boca/lesões , Guias de Prática Clínica como AssuntoRESUMO
A great deal of circumstantial evidence has linked DNA damage in human spermatozoa with adverse reproductive outcomes including reduced fertility and high rates of miscarriage. Although oxidative stress is thought to make a significant contribution to DNA damage in the male germ line, the factors responsible for creating this stress have not been elucidated. One group of compounds that are thought to be active in this context are the estrogens, either generated as a result of the endogenous metabolism of androgens within the male reproductive tract or gaining access to the latter as a consequence of environmental exposure. In this study, a wide variety of estrogenic compounds were assessed for their direct effects on human spermatozoa in vitro. DNA integrity was assessed using the Comet and TUNEL assays, lesion frequencies were quantified by QPCR using targets within the mitochondrial and nuclear (beta-globin) genomes, DNA adducts were characterized by mass spectrometry and redox activity was monitored using dihydroethidium (DHE) as the probe. Of the estrogenic and estrogen analogue compounds evaluated, catechol estrogens, quercetin, diethylstilbestrol and pyrocatechol stimulated intense redox activity while genistein was only active at the highest doses tested. Other estrogens and estrogen analogues, such as 17beta-estradiol, nonylphenol, bisphenol A and 2,3-dihydroxynaphthalene were inactive. Estrogen-induced redox activity was associated with a dramatic loss of motility and, in the case of 2-hydroxyestradiol, the induction of significant DNA fragmentation. Mass spectrometry also indicated that catechol estrogens were capable of forming dimers that can cross-link the densely packed DNA strands in sperm chromatin, impairing nuclear decondensation. These results highlight the potential importance of estrogenic compounds in creating oxidative stress and DNA damage in the male germ line and suggest that further exploration of these compounds in the aetiology of male infertility is warranted.
Assuntos
Adutos de DNA/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Estrogênios/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Ensaio Cometa , Reagentes de Ligações Cruzadas , Etídio/análogos & derivados , Etídio/metabolismo , Humanos , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Oxirredução , Reação em Cadeia da Polimerase , Espécies Reativas de Oxigênio/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/fisiologiaRESUMO
The effects of diabetes mellitus on male reproductive health have not been clearly defined. A previous publication from this group reported significantly higher levels of nuclear DNA fragmentation and mitochondrial DNA deletions in spermatozoa from men with type 1 diabetes. This study compared semen profiles, sperm DNA fragmentation and levels of oxidative DNA modification in spermatozoa of diabetic and non-diabetic men. Semen samples from 12 non-diabetic, fertile men and 11 type 1 diabetics were obtained and subjected to conventional light microscopic semen analysis. Nuclear DNA fragmentation was assessed using an alkaline Comet assay and concentrations of 7,8-dihydro-8-oxo-2-deoxyguanosine (8-OHdG), an oxidative adduct of the purine guanosine, were assessed by high-performance liquid chromatography. Conventional semen profiles were similar in both groups, whilst spermatozoa from type 1 diabetics showed significantly higher levels of DNA fragmentation (44% versus 27%; P < 0.05) and concentrations of 8-OHdG (3.6 versus 2.0 molecules of 8-OHdG per 10(5) molecules of deoxyguanosine; P < 0.05). Furthermore, a positive correlation was observed between DNA fragmentation and concentrations of 8-OHdG per 10(5) molecules of deoxyguanosine (rs = 0.7, P < 0.05). The genomic damage evident in spermatozoa of type 1 diabetics may have important implications for their fertility and the outcome of pregnancies fathered by these individuals.
Assuntos
Fragmentação do DNA , Desoxiguanosina/análogos & derivados , Diabetes Mellitus Tipo 1/metabolismo , Estresse Oxidativo/fisiologia , Espermatozoides/metabolismo , 8-Hidroxi-2'-Desoxiguanosina , Estudos de Casos e Controles , Desoxiguanosina/metabolismo , Humanos , Masculino , Sêmen/citologiaRESUMO
We have previously shown that ExoU, a type III secreted cytotoxin of Pseudomonas aeruginosa, causes acute cytotoxicity towards corneal epithelial cells in vitro, and contributes to corneal disease pathology and ocular colonization in vivo. Subsequently, we reported that ExoU represses phagocyte infiltration of infected corneas in vivo. ExoU has patatin-like phospholipase activity that is required for cytotoxic activity in vitro (mammalian cell injury and death) and for disease in a murine model of pneumonia. We hypothesized that the phospholipase activity was required for ExoU-mediated corneal disease and ocular colonization. Using the murine scarification model, corneal disease pathology was examined after inoculation with approximately 10(6)cfu of a P. aeruginosa effector mutant (PA103DeltaexoUexoT::Tc) complemented with either exoU (pUCPexoU), phospholipase-inactive exoU (pUCPexoUD344A) or a plasmid control (pUCP18). Eyes were photographed and disease severity scored at 24 and 48h post-infection. Viable bacteria colonizing infected eyes were quantified at 6 and 48h. Complementation with exoU caused significantly more pathology (increased disease severity scores) and enabled bacteria to better colonize (by approximately 1000-fold) at 48h as compared to phospholipase-inactive exoU which did not differ from plasmid control. Surprisingly, exoU did not contribute to early (6h) colonization. In-vitro assays confirmed that the phospholipase domain of exoU was required for cytotoxicity towards human corneal epithelial cells. Taken together these data show that the phospholipase activity of the P. aeruginosa cytotoxin, ExoU, plays a role in the pathogenesis of corneal infection via mechanism(s) occurring after initial colonization of a susceptible cornea.
Assuntos
Proteínas de Bactérias/genética , Ceratite/microbiologia , Fosfolipases/metabolismo , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/patogenicidade , Animais , Proteínas de Bactérias/fisiologia , Domínio Catalítico/genética , Células Cultivadas , Infecções Oculares Bacterianas/microbiologia , Infecções Oculares Bacterianas/patologia , Humanos , Ceratite/patologia , Leucocidinas/genética , Leucocidinas/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Índice de Gravidade de Doença , Fatores de Tempo , Virulência/genéticaRESUMO
BACKGROUND: Diabetes mellitus (DM) is increasing in men of reproductive age. Despite this, the prevalence of diabetes in men attending fertility clinics is largely unknown. Furthermore, studies examining the effects of DM on sperm fertility potential have been limited to conventional semen analysis. METHODS: Conventional semen analysis (semen volume, sperm count, motility and morphology) was performed for 27 diabetic (mean age 34+/-2 years) and 29 non-diabetic subjects (control group, men undergoing routine infertility investigations, mean age 33+/-1 years). Nuclear DNA (nDNA) fragmentation was assessed using the alkaline Comet assay and mitochondrial DNA (mtDNA) deletions by Long-PCR. RESULTS: Other than a small, but significant, reduction in semen volume in diabetic men (2.6 versus 3.3 ml; P<0.05), conventional semen parameters did not differ significantly from control subjects. Diabetic subjects had significantly higher mean nDNA fragmentation (53 versus 32%; P<0.0001) and median number of mtDNA deletions (4 versus 3; P<0.05) compared with control subjects. CONCLUSIONS: Diabetes is associated with increased sperm nuclear and mtDNA damage that may impair the reproductive capability of these men.
Assuntos
Complicações do Diabetes , Diabetes Mellitus Tipo 1/diagnóstico , Infertilidade Masculina/diagnóstico , Adolescente , Adulto , Ensaio Cometa , Fragmentação do DNA , DNA Mitocondrial/metabolismo , Diabetes Mellitus Tipo 1/complicações , Eletroforese em Gel de Ágar , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Contagem de Espermatozoides , Espermatozoides/metabolismoRESUMO
BACKGROUND: Each year 40,000 men have a vasectomy in the UK whilst another 2400 request a reversal to begin a second family. Sperm can now be obtained by testicular biopsy and subsequently used in assisted conception with ICSI. The study aims were to compare sperm yields of men post-vasectomy or with obstructive azoospermia (OA) of unknown aetiology with yields of fertile men and to assess any alteration in the clinical pregnancy rates after ICSI. METHODS: Testicular tissue was obtained by Trucut needle from men who had undergone a vasectomy >5 years previously or had OA from other causes and from fertile men during vasectomy. Seminiferous tubules were milked to measure sperm yields. Numbers of Sertoli cells and spermatids and thickness of the seminiferous tubule walls were assessed using quantitative computerized analysis. RESULTS AND CONCLUSIONS: Sperm yields/g testis were significantly decreased in men post-vasectomy and in men with OA, relative to fertile men. Significant reductions were also observed in early (40%) and mature (29%) spermatid numbers and an increase of 31% was seen in the seminiferous tubule wall (basal membrane and collagen thickness) of vasectomized men compared with fertile men. Clinical pregnancy rates in couples who had had a vasectomy were also significantly reduced.
Assuntos
Injeções de Esperma Intracitoplásmicas/métodos , Espermatogênese , Espermatozoides/patologia , Testículo/patologia , Vasectomia , Adulto , Biópsia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Oligospermia/terapia , Gravidez , Taxa de Gravidez , Túbulos Seminíferos/patologia , Células de Sertoli/patologia , Contagem de Espermatozoides , Espermátides/patologia , VasovasostomiaRESUMO
DNA damage in the male germ line has been associated with poor semen quality, low fertilization rates, impaired preimplantation development, increased abortion and an elevated incidence of disease in the offspring, including childhood cancer. The causes of this DNA damage are still uncertain but the major candidates are oxidative stress and aberrant apoptosis. The weight of evidence currently favours the former and, in keeping with this conclusion, positive results have been reported for antioxidant therapy both in vivo and in vitro. Resolving the causes of DNA damage in the male germ line will be essential if we are to prevent the generation of genetically damaged human embryos, particularly in the context of assisted conception therapy.