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Merkel cell carcinoma (MCC) is a rare but aggressive neuroendocrine tumour of the skin with poor prognosis and rising global incidence. A recently published article in BMC Cancer, titled "Merkel cell carcinoma: a forty-year experience at the Peter MacCallum Cancer Centre" (Wang et al.), provides a contemporary analysis of locoregional disease outcomes in Australia which highlights the comparative effectiveness of radiotherapy for excisions with involved margins versus wide local excision. There is a persistent lack of clear, well-defined guidelines to manage MCC in Australia despite experiencing the highest rates globally. The advanced age at onset also provides inherent challenges for optimal management and often, a case-by-case approach is necessary based on patient preferences, baseline function and fitness for surgery. This paper responds to the recently published article by Wang et al. and will expand the discourse regarding management of localized MCC. Specifically, we will discuss the surgical excision approaches; alternative treatment options for MCC including radiotherapy, Mohs micrographic surgery and novel immunotherapy agents being investigated through several clinical trials.
Assuntos
Carcinoma de Célula de Merkel , Neoplasias Cutâneas , Carcinoma de Célula de Merkel/terapia , Carcinoma de Célula de Merkel/patologia , Humanos , Neoplasias Cutâneas/terapia , Neoplasias Cutâneas/patologia , Austrália/epidemiologiaRESUMO
BACKGROUND: Macrophages residing in milk are vital during intramammary infections. This study sought to develop a method enabling the investigation of macrophage responses to pathogens. Streptococcus uberis is the predominant cause of bovine mastitis UK-wide and its pathogenesis is unusual compared to other intramammary pathogens. Previous studies utilise macrophage cell lines, isolated bovine blood derived monocytes, or macrophages from raw milk through complex or inconsistent strategies such as fluorescence activated cell sorting (FACS), centrifugation and selective adherence, and CD14 antibody-microbeads. The centrifuge steps required in the initial stages often damage cells. Thus, the aim of this study was to develop a reliable, reproducible, and cost-effective method for isolating mammary macrophages from milk in a way that allows their culture, challenge with bacteria, and measurement of their response ex-vivo. RESULTS: This method achieves an average yield of 1.27 × 107 cells per litre of milk. Whole milk with somatic cell range of 45-65 cells/µL produced excellent yields, with efficient isolations accomplished with up to 150 cells/µL. This strategy uses milk diluted in PAE buffer to enable low-speed centrifugation steps followed by seeding on tissue-culture-treated plastic. Seeding 1,000,000 milk-extracted cells onto tissue culture plates was sufficient to obtain 50,000 macrophage. Isolated macrophage remained responsive to challenge, with the highest concentration of IL-1ß measured by ELISA at 20 h after challenge with S. uberis. In this model, the optimal multiplicity of infection was found to be 50:1 bacteria:macrophage. No difference in IL-1ß production was found between macrophages challenged with live or heat-killed S. uberis. Standardisation of the production of IL-1ß to that obtained following macrophage stimulation with LPS allowed for comparisons between preparations. CONCLUSIONS: A cost-effective method, utilising low-speed centrifugation followed by adherence to plastic, was established to isolate bovine mammary macrophages from raw milk. This method was shown to be appropriate for bacterial challenge, therefore providing a cost-effective, ex-vivo, and non-invasive model of macrophage-pathogen interactions. The optimal multiplicity of infection for S. uberis challenge was demonstrated and a method for standardisation against LPS described which removes sample variation. This robust method enables, reproducible and reliable interrogation of critical pathogen-host interactions which occur in the mammary gland.
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Doenças dos Bovinos , Mastite Bovina , Infecções Estreptocócicas , Feminino , Bovinos , Animais , Infecções Estreptocócicas/veterinária , Lipopolissacarídeos/metabolismo , Glândulas Mamárias Animais/metabolismo , Leite/microbiologia , Mastite Bovina/microbiologia , Macrófagos/metabolismo , Doenças dos Bovinos/metabolismoRESUMO
May Measurement Month (MMM) is a global and national blood pressure (BP) screening campaign initiated by the International Society of Hypertension to improve awareness of BP worldwide. This study reports on the findings of the MMM21 campaign in Australia. Adult participants (≥18 years) were screened through opportunistic sampling across Australia between 1 May and 30 November 2021. Trained volunteers recorded standardized BP measurements from community volunteer participants along with demographic data, lifestyle factors, comorbidities, and history of COVID-19 infection and vaccination. Hypertension was defined as systolic BP ≥ 140â mmHg and/or diastolic BP ≥ 90â mmHg and/or taking antihypertensive medication. Data were collated and analysed centrally using the current MMM protocol and presented after the imputation of missing BP readings. A total of 1307 participants were screened in 2021, comprising 652 (49.9%) females and 654 (50.0%) males with a mean age of 48 years (SD 20.1). Of all 1307 participants, 524 (40.1%) had hypertension. Of participants with hypertension, 65.4% were aware and 59.3% were on antihypertensive medication. Of 311 participants on antihypertensive medication, 54.7% had controlled BP. Of all 524 participants with hypertension, 32.5% had controlled BP. The current 2021 data may indicate some progress in creating BP awareness; however, consecutive Australian data obtained since 2017 demonstrated stagnating treatment, and control rates compared with global rates and those in other high-income countries. Concerted efforts from all stakeholders will be required to further improve BP awareness, treatment, and control rates in Australia.
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INTRODUCTION: Vaccine hesitancy represents a state of uncertainty before a decision about vaccination is made. It can lead to limited vaccine uptake despite adequate supply and an efficacious product. Western Australia (WA) presents a unique challenge with a population widely spread across metropolitan and rural sites and diverse opinions regarding vaccination. OBJECTIVE: To elicit and compare the common COVID-19 vaccine concerns in rural and metropolitan WA, and to identify proposed solutions to vaccine hesitancy. DESIGN: A voluntary online survey was distributed via social media over a 2-week period in August 2021 during Phase 2A of the National COVID-19 Vaccine Rollout Strategy. General practitioners and members of the public living in rural and metropolitan WA over the age of 18 were surveyed regarding COVID-19 vaccine concerns (blood clots; long- and short-term side effects; lack of testing; and other concerns) and suggested methods to address vaccine hesitancy. Data were analysed with a sequential mixed methods and thematic analysis approach. FINDINGS: There were 468 general population respondents to our survey, of whom 19.0% (n = 89) lived rurally. A majority (52.6% [n = 246]) of general respondents expressed concerns about COVID-19 vaccine safety. The commonest concerns were long- and short-term side effects, blood clots and inadequate testing. There was a positive correlation between rurality and vaccine concerns; an inverse relationship between rurality and vaccine uptake; and an inverse relationship between vaccine concerns and uptake. Improved media coverage was the commonest solution suggested to address COVID-19 vaccine hesitancy. DISCUSSION: A significant proportion of respondents had concerns about vaccine safety; concerns were more common in rural respondents. Rural communities may benefit from location-targeted media campaigns with a focus on breaking down barriers specific to these members of the population. Vaccine access is more challenging, and consistent messaging from trusted sources is of utmost importance to improve uptake. CONCLUSION: COVID-19 vaccine hesitancy is more common in rural populations. Targeted media-based education regarding vaccine safety may improve COVID-19 vaccine uptake.
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COVID-19 , Trombose , Humanos , Adulto , Pessoa de Meia-Idade , População Rural , Vacinas contra COVID-19 , Estudos Transversais , Austrália Ocidental , COVID-19/prevenção & controleRESUMO
BACKGROUND: Days alive and out of hospital (DAOH) is a composite, patient-centred outcome measure describing a patient's postoperative recovery, encompassing hospitalisation and mortality. DAOH is the number of days not in hospital over a defined postoperative period; patients who die have DAOH of zero. The Standardising Endpoints in Perioperative Medicine (StEP) group recommended DAOH as a perioperative outcome. However, DAOH has never been validated in patients undergoing emergency laparotomy. Here, we validate DAOH after emergency laparotomy and establish the optimal duration of observation. METHODS: Prospectively collected data of patients having emergency laparotomy in England (December 1, 2013-November 30, 2017) were linked to national hospital admission and mortality records for the year after surgery. We evaluated construct validity by assessing DAOH variation with known perioperative risk factors and predictive validity for 1 yr mortality using a multivariate Bayesian mixed-effects logistic regression. The optimal postoperative DAOH period (30 or 90 days) was judged on distributional and pragmatic properties. RESULTS: We analysed 78 921 records. The median 30-day DAOH (DAOH30) was 16 (inter-quartile range [IQR], 0-22) days and the median DAOH90 was 75 (46-82) days. DAOH was shorter in the presence of known perioperative risk factors. For patients surviving the first 30 postoperative days, shorter DAOH30 was associated with higher 1-yr mortality (odds ratio=0.94; 95% credible interval, 0.94-0.94). CONCLUSION: DAOH is a valid, patient-centred outcome after emergency laparotomy. We recommend its use in clinical trials, quality assurance, and quality improvement, measured at 30 days as mortality heavily skews DAOH measured at 90 days and beyond.
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Hospitalização/estatística & dados numéricos , Hospitais/provisão & distribuição , Laparotomia/estatística & dados numéricos , Avaliação de Resultados em Cuidados de Saúde/estatística & dados numéricos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Teorema de Bayes , Inglaterra , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Readmissão do Paciente/estatística & dados numéricos , Estudos Retrospectivos , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento , Adulto JovemRESUMO
Streptococcus uberis is a major causative agent of bovine mastitis worldwide, negatively affecting both milk production and animal welfare. Mammary infections result from environmental reservoirs, with cattle themselves required to propagate the infection cycle. Two longitudinal studies were performed to investigate the prevalence of Streptococcus uberis within feces and to evaluate factors which may affect gastrointestinal carriage. Bacterial detection was confirmed using a PCR-based method directed against sub0888 that detected S. uberis at an analytical sensitivity of 12 cfu/g of bovine feces. The first study sampled an entire herd at 8-wk intervals, over a 10-mo period and identified that maintenance of S. uberis within the dairy cow environment was due to a high proportion of animals shedding S. uberis and not due to a low number of "super-shedding" cows within the herd. Seasonality influenced detection rates, with detection levels significantly higher for housed cattle compared with those at pasture. Multilevel logistic regression was used to identify significant factors that affected S. uberis detection; these included parity, stage of lactation, and body condition score. An additional study involved screening a smaller cohort of cows housed over a 4-wk period and identified an increased probability of detection if cows were housed in loose straw yards, compared those in straw cubicles. This study highlighted several cow and management related factors that affect both detection of S. uberis and future infection risks.
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Doenças dos Bovinos , Mastite Bovina , Infecções Estreptocócicas , Animais , Bovinos , Fezes , Feminino , Mastite Bovina/epidemiologia , Leite , Prevalência , Infecções Estreptocócicas/epidemiologia , Infecções Estreptocócicas/veterinária , StreptococcusRESUMO
BACKGROUND: Utilising next generation sequencing to interrogate saturated bacterial mutant libraries provides unprecedented information for the assignment of genome-wide gene essentiality. Exposure of saturated mutant libraries to specific conditions and subsequent sequencing can be exploited to uncover gene essentiality relevant to the condition. Here we present a barcoded transposon directed insertion-site sequencing (TraDIS) system to define an essential gene list for Streptococcus equi subsp. equi, the causative agent of strangles in horses, for the first time. The gene essentiality data for this group C Streptococcus was compared to that of group A and B streptococci. RESULTS: Six barcoded variants of pGh9:ISS1 were designed and used to generate mutant libraries containing between 33,000-66,000 unique mutants. TraDIS was performed on DNA extracted from each library and data were analysed separately and as a combined master pool. Gene essentiality determined that 19.5% of the S. equi genome was essential. Gene essentialities were compared to those of group A and group B streptococci, identifying concordances of 90.2% and 89.4%, respectively and an overall concordance of 83.7% between the three species. CONCLUSIONS: The use of barcoded pGh9:ISS1 to generate mutant libraries provides a highly useful tool for the assignment of gene function in S. equi and other streptococci. The shared essential gene set of group A, B and C streptococci provides further evidence of the close genetic relationships between these important pathogenic bacteria. Therefore, the ABC of gene essentiality reported here provides a solid foundation towards reporting the functional genome of streptococci.
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Genes Bacterianos/genética , Sequenciamento de Nucleotídeos em Larga Escala , Streptococcus/genética , Genes Essenciais/genética , Genômica , MutaçãoRESUMO
BACKGROUND: The anionic-polyelectrolyte nature of the wall of Gram-positive bacteria has long been suspected to be involved in homeostasis of essential cations and bacterial growth. A better understanding of the coupling between the biophysics and the biology of the wall is essential to understand some key features at play in ion-homeostasis in this living system. METHODS: We consider the wall as a polyelectrolyte gel and balance the long-range electrostatic repulsion within this structure against the penalty entropy required to condense cations around wall polyelectrolytes. The resulting equations define how cations interact physically with the wall and the characteristic time required for a cation to leave the wall and enter into the bacterium to enable its usage for bacterial metabolism and growth. RESULTS: The model was challenged against experimental data regarding growth of Gram-positive bacteria in the presence of varying concentration of divalent ions. The model explains qualitatively and quantitatively how divalent cations interact with the wall as well as how the biophysical properties of the wall impact on bacterial growth (in particular the initiation of bacterial growth). CONCLUSION: The interplay between polymer biophysics and the biology of Gram positive bacteria is defined for the first time as a new set of variables that contribute to the kinetics of bacterial growth. GENERAL SIGNIFICANCE: Providing an understanding of how bacteria capture essential metal cations in way that does not follow usual binding laws has implications when considering the control of such organisms and their ability to survive and grow in extreme environments.
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Cátions Bivalentes/metabolismo , Parede Celular/metabolismo , Bactérias Gram-Positivas/metabolismo , Biofísica/métodos , Homeostase/fisiologia , Metais/metabolismo , Polieletrólitos/metabolismo , Polímeros/metabolismo , Eletricidade EstáticaRESUMO
BACKGROUND: Electroporation is a method of choice to transform living cells. The ability of electroporation to transfer small or large chemicals across the lipid bilayer membrane of eukaryotic cells or Gram-negative bacteria relies on the formation of transient pores across the membrane. To exist, these pores rely on an insulator (the bilayer membrane) and the presence of a potential difference on either side of the membrane mediated by an external electric field. In Gram-positive bacteria, however, the wall is not an insulator but pores can still form when an electric field is applied. Past works have shown that the electrostatic charge of teichoic acids, a major wall component; sensitizes the wall to pore formation when an external electric field is applied. These results suggest that teichoic acids mediate the formation of defects in the wall of Gram-positive bacteria. METHODS: We model the electrostatic repulsion between teichoic acids embedded in the bacterial wall composed of peptidoglycan when an electric field is applied. The repulsion between teichoic acids gives rise to a stress pressure that is able to rupture the wall when a threshold value has been reached. The size of such small defects can diverge leading to the formation of pores. RESULTS: It is demonstrated herein that for a bonding energy of about ~1-10 k(B)T between peptidoglycan monomers an intra-wall pressure of about ~5-120 k(B)T/nm(3) generates spherical defects of radius ~0.1-1 nm diverging in size to create pores. CONCLUSION: The electrostatic cavitation of the bacterial wall theory has the potential to highlight the role of teichoic acids in the formation pores, providing a new step in the understanding of electroporation in Gram-positive bacteria without requiring the use of an insulator.
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Eletroporação , Bactérias Gram-Positivas/fisiologia , Ácidos Teicoicos/metabolismo , Peptidoglicano/metabolismo , Eletricidade EstáticaRESUMO
Streptococcus uberis is frequently isolated from the mammary gland of dairy cattle. Infection with some strains can induce mild subclinical inflammation whilst others induce severe inflammation and clinical mastitis. We compared here the inflammatory response of primary cultures of bovine mammary epithelial cells (pbMEC) towards S. uberis strains collected from clinical or subclinical cases (seven strains each) of mastitis with the strong response elicited by Escherichia coli. Neither heat inactivated nor live S. uberis induced the expression of 10 key immune genes (including TNF, IL1B, IL6). The widely used virulent strain 0140J and the avirulent strain, EF20 elicited similar responses; as did mutants defective in capsule (hasA) or biofilm formation (sub0538 and sub0539). Streptococcus uberis failed to activate NF-κB in pbMEC or TLR2 in HEK293 cells, indicating that S. uberis particles did not induce any TLR-signaling in MEC. However, preparations of lipoteichoic acid (LTA) from two strains strongly induced immune gene expression and activated NF-κB in pbMEC, without the involvement of TLR2. The immune-stimulatory LTA must be arranged in the intact S. uberis such that it is unrecognizable by the relevant pathogen receptors of the MEC. The absence of immune recognition is specific for MEC, since the same S. uberis preparations strongly induced immune gene expression and NF-κB activity in the murine macrophage model cell RAW264.7. Hence, the sluggish immune response of MEC and not of professional immune cells to this pathogen may aid establishment of the often encountered belated and subclinical phenotype of S. uberis mastitis.
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Células Epiteliais/fisiologia , Macrófagos/fisiologia , Glândulas Mamárias Animais/microbiologia , Mastite Bovina/microbiologia , Infecções Estreptocócicas/veterinária , Streptococcus/classificação , Animais , Bovinos , Doenças dos Bovinos , Linhagem Celular , Feminino , Glândulas Mamárias Animais/citologia , Camundongos , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/microbiologiaRESUMO
Australia is known to have had the highest per-capita asbestos consumption level of any nation, reaching a peak in the 1970s. Although crocidolite was effectively banned in the late 1960s, and amosite use ceased in the mid 1980s, a complete asbestos ban was not implemented until 2003. This resulted in an epidemic of asbestos-related disease, which has only now reached its peak. Between 1982 and 2011, 13,036 individuals were newly diagnosed with malignant mesothelioma, with 690 diagnosed in 2011. A further 778 cases were identified between 1945 and 1981 from retrospective searches and the first 2 years of the Australian Mesothelioma Program. The age-standardized malignant mesothelioma incidence rate has leveled off in the last 10 years (2.8 per 100,000 in 2011). There has been a marked increase over time in the age-specific incidence rates for individuals aged 75 years or older. Data from the current Australian Mesothelioma Registry on asbestos exposure history in Australia is available for 449 subjects diagnosed between July 1, 2010, and April 1, 2015. This asbestos exposure history data show that 60% (n = 268) of cases had probable or possible occupational asbestos exposure, with trade-based jobs being the most frequent sources of occupational asbestos exposure. In addition, out of the 449 cases, 377 were recorded as having probable or possible nonoccupational asbestos exposure. Continuous vigilance toward changes over time in the settings in which people are exposed to asbestos and in the descriptive epidemiology of malignant mesothelioma is recommended to enable a comprehensive understanding of the current and future impact of asbestos-related diseases in Australia.
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Amianto/toxicidade , Exposição Ambiental , Neoplasias Pulmonares/epidemiologia , Mesotelioma/epidemiologia , Fatores Etários , Austrália/epidemiologia , Geografia , Humanos , Incidência , Neoplasias Pulmonares/induzido quimicamente , Mesotelioma/induzido quimicamente , Mesotelioma Maligno , Exposição Ocupacional , Fatores SexuaisRESUMO
BACKGROUND: Australia is known to have had one of the highest per-capita asbestos consumption rates, yet there are few contemporary reports on malignant mesothelioma trends. METHODS: Data on 10â 930 people with malignant pleural mesothelioma (MPM) and 640 people with malignant peritoneal mesothelioma diagnosed in Australia during 1982-2009 were analysed. Observed incidence rate trends were quantified. Incidence rates were projected up to 2030 using observed incident cases during 1982-2012. The relative per-decade change in excess mortality during 1999-2009 was estimated. RESULTS: During 1982-2009, acceleration in MPM age-standardised incidence rates were highest for women and those aged 75â years and above, with average annual percentage changes of +4.9 (95% CI 3.6 to 6.2) and +7.2 (95% CI 5.4 to 9.0), respectively. Age-standardised incidence rates for men with MPM aged 0-64â years decelerated rapidly during 2003-2009, an average annual percentage change of -5.1% (95% CI -7.6% to -2.5%). Overall, male age-specific MPM incidence rates in the age group of 65-74â year during 2010-2030 are projected to decline with rates projected to increase for older men and women with MPM. There was a statistically significant 16% relative reduction in the excess mortality rate (EMR) up to 5 years postdiagnosis for people diagnosed with malignant pleural and peritoneal mesothelioma combined in 2009 compared with those diagnosed in 1999, an EMR ratio of 0.84 (95% CI 0.77 to 0.92). CONCLUSIONS: Australia's malignant mesothelioma incidence rates appear to have reached maximum levels but with differences over time by age, gender and tumour location. Improvements over time in survival provide a glimpse of hope for this almost invariably fatal disease.
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Amianto/efeitos adversos , Carcinógenos , Exposição Ambiental , Neoplasias Pulmonares/epidemiologia , Mesotelioma/epidemiologia , Neoplasias Peritoneais/epidemiologia , Neoplasias Pleurais/epidemiologia , Adolescente , Adulto , Idoso , Austrália/epidemiologia , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Humanos , Incidência , Lactente , Recém-Nascido , Pulmão/patologia , Neoplasias Pulmonares/mortalidade , Masculino , Mesotelioma/mortalidade , Mesotelioma Maligno , Pessoa de Meia-Idade , Neoplasias Peritoneais/mortalidade , Peritônio/patologia , Neoplasias Pleurais/mortalidade , Adulto JovemRESUMO
BACKGROUND: Streptococcus uberis, a Gram-positive, catalase-negative member of the family Streptococcaceae is an important environmental pathogen responsible for a significant proportion of subclinical and clinical bovine intramammary infections. Currently, the genome of only a single reference strain (0140J) has been described. Here we present a comparative analysis of complete draft genome sequences of an additional twelve S. uberis strains. RESULTS: Pan and core genome analysis revealed the core genome common to all strains to be 1,550 genes in 1,509 orthologous clusters, complemented by 115-246 accessory genes present in one or more S. uberis strains but absent in the reference strain 0140J. Most of the previously predicted virulent genes were present in the core genome of all 13 strains but gene gain/loss was observed between the isolates in CDS associated with clustered regularly interspaced short palindromic repeats (CRISPRs), prophage and bacteriocin production. Experimental challenge experiments confirmed strain EF20 as non-virulent; only able to infect in a transient manner that did not result in clinical mastitis. Comparison of the genome sequence of EF20 with the validated virulent strain 0140J identified genes associated with virulence, however these did not relate clearly with clinical/non-clinical status of infection. CONCLUSION: The gain/loss of mobile genetic elements such as CRISPRs and prophage are a potential driving force for evolutionary change. This first "whole-genome" comparison of strains isolated from clinical vs non-clinical intramammary infections including the type virulent vs non-virulent strains did not identify simple gene gain/loss rules that readily explain, or be confidently associated with, differences in virulence. This suggests that a more complex dynamic determines infection potential and clinical outcome not simply gene content.
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Genoma Bacteriano , Streptococcus/genética , Virulência/genética , Animais , Bacteriocinas/metabolismo , Sequência de Bases , Bovinos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Hibridização Genômica Comparativa , Feminino , Mastite Bovina/genética , Mastite Bovina/microbiologia , Mastite Bovina/patologia , Leite/microbiologia , Dados de Sequência Molecular , Filogenia , Polimorfismo de Nucleotídeo Único , Alinhamento de Sequência , Análise de Sequência de DNA , Streptococcus/classificação , Streptococcus/patogenicidadeRESUMO
Re-engineered riboswitches that no longer respond to cellular metabolites, but that instead can be controlled by synthetic molecules, are potentially useful gene regulatory tools for use in synthetic biology and biotechnology fields. Previously, extensive genetic selection and screening approaches were employed to re-engineer a natural adenine riboswitch to create orthogonal ON-switches, enabling translational control of target gene expression in response to synthetic ligands. Here, we describe how a rational targeted approach was used to re-engineer the PreQ1 riboswitch from Bacillus subtilis into an orthogonal OFF-switch. In this case, the evaluation of just six synthetic compounds with seven riboswitch mutants led to the identification of an orthogonal riboswitch-ligand pairing that effectively repressed the transcription of selected genes in B. subtilis. The streamlining of the re-engineering approach, and its extension to a second class of riboswitches, provides a methodological platform for the creation of new orthogonal regulatory components for biotechnological applications including gene functional analysis and antimicrobial target validation and screening.
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Bacillus subtilis/genética , Regulação Bacteriana da Expressão Gênica , Engenharia Genética , Pirimidinonas/metabolismo , Pirróis/metabolismo , Riboswitch , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Bacillus subtilis/química , Bacillus subtilis/metabolismo , Sequência de Bases , Inativação Gênica , Mutagênese , Pirimidinonas/química , Pirróis/química , Biologia Sintética , Ativação TranscricionalRESUMO
The enzyme aryl/alkenyl malonate decarboxylase (AMDase) catalyses the enantioselective decarboxylative protonation (EDP) of a range of disubstituted malonic acids to give homochiral carboxylic acids that are valuable synthetic intermediates. AMDase exhibits a number of advantages over the non-enzymatic EDP methods developed to date including higher enantioselectivity and more environmentally benign reaction conditions. In this report, AMDase and engineered variants have been used to produce a range of enantioenriched heteroaromatic α-hydroxycarboxylic acids, including pharmaceutical precursors, from readily accessible α-hydroxymalonates. The enzymatic method described here represents an improvement upon existing synthetic chemistry methods that have been used to produce similar compounds. The relationship between the structural features of these new substrates and the kinetics associated with their enzymatic decarboxylation is explored, which offers further insight into the mechanism of AMDase.
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Carboxiliases/metabolismo , Ácidos Carboxílicos/química , Malonatos/química , Catálise , Descarboxilação , Estrutura Molecular , EstereoisomerismoRESUMO
Streptococcus uberis is a globally endemic and poorly controlled cause of bovine mastitis impacting the sustainability of the modern dairy industry. A core genome was derived from 579 newly sequenced S. uberis isolates, along with 305 publicly available genome sequences of S. uberis isolated from 11 countries around the world and used to develop a core genome multi-locus sequence typing (cgMLST) scheme. The S. uberis core genome comprised 1475 genes, and these were used to identify 1447 curated loci that were indexed into the cgMLST scheme. This was able to type 1012 of 1037 (>97ââ%) isolates used and differentiated the associated sequences into 932 discrete core genome sequence types (cgSTs). Analysis of the phylogenetic relationships of cgSTs revealed no clear clustering of isolates based on metadata such as disease status or year of isolation. Geographical clustering of cgSTs was limited to identification of a UK-centric clade, but cgSTs from UK isolates were also dispersed with those originating from other geographical regions across the entire phylogenetic topology. The cgMLST scheme offers a new tool for the detailed analysis of this globally important pathogen of dairy cattle. Initial analysis has re-emphasized and exemplified the genetically diverse nature of the global population of this opportunistic pathogen.
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Streptococcus , Animais , Bovinos , Feminino , Tipagem de Sequências Multilocus , Filogenia , Streptococcus/genética , Análise por ConglomeradosRESUMO
The regulation and control of gene expression in response to differing environmental stimuli is crucial for successful pathogen adaptation and persistence. The regulatory gene vru of Streptococcus uberis encodes a stand-alone response regulator with similarity to the Mga of group A Streptococcus. Mga controls expression of a number of important virulence determinants. Experimental intramammary challenge of dairy cattle with a mutant of S. uberis carrying an inactivating lesion in vru showed reduced ability to colonize the mammary gland and an inability to induce clinical signs of mastitis compared with the wild-type strain. Analysis of transcriptional differences of gene expression in the mutant, determined by microarray analysis, identified a number of coding sequences with altered expression in the absence of Vru. These consisted of known and putative virulence determinants, including Lbp (Sub0145), SclB (Sub1095), PauA (Sub1785) and hasA (Sub1696).
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Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Mastite Bovina/microbiologia , Infecções Estreptocócicas/veterinária , Streptococcus/metabolismo , Streptococcus/patogenicidade , Fatores de Virulência/metabolismo , Animais , Proteínas de Bactérias/genética , Bovinos , Feminino , Infecções Estreptocócicas/microbiologia , Streptococcus/genética , Virulência , Fatores de Virulência/genéticaRESUMO
Bovine mastitis remains the most common and costly disease of dairy cattle worldwide. A complementary control measure to herd hygiene and vaccine development would be to selectively breed cattle with greater resistance to mammary infection. Toll-like receptor 1 (TLR1) has an integral role for the initiation and regulation of the immune response to microbial pathogens, and has been linked to numerous inflammatory diseases. The objective of this study was to investigate whether single nucleotide polymorphisms (SNPs) within the bovine TLR1 gene (boTLR1) are associated with clinical mastitis (CM).Selected boTLR1 SNPs were analysed within a Holstein Friesian herd. Significant associations were found for the tagging SNP -79 T > G and the 3'UTR SNP +2463 C > T. We observed favourable linkage of reduced CM with increased milk fat and protein, indicating selection for these markers would not be detrimental to milk quality. Furthermore, we present evidence that some of these boTLR1 SNPs underpin functional variation in bovine TLR1. Animals with the GG genotype (from the tag SNP -79 T > G) had significantly lower boTLR1 expression in milk somatic cells when compared with TT or TG animals. In addition, stimulation of leucocytes from GG animals with the TLR1-ligand Pam3csk4 resulted in significantly lower levels of CXCL8 mRNA and protein.SNPs in boTLR1 were significantly associated with CM. In addition we have identified a bovine population with impaired boTLR1 expression and function. This may have additional implications for animal health and warrants further investigation to determine the suitability of identified SNPs as markers for disease susceptibility.
Assuntos
Mastite Bovina/genética , Leite/química , Leite/metabolismo , Polimorfismo de Nucleotídeo Único , Receptor 1 Toll-Like/genética , Animais , Bovinos , Contagem de Células/veterinária , Feminino , Estudos de Associação Genética/veterinária , Marcadores Genéticos , Lactação , Mastite Bovina/imunologia , Mastite Bovina/microbiologia , Reação em Cadeia da Polimerase/veterinária , Receptor 1 Toll-Like/metabolismoRESUMO
OBJECTIVE: To assess outcomes following Holmium Laser Enucleation of Prostate (HoLEP) in men with prostate volumes ≥150cc and compare this to men with prostate volumes ≤150 cc. PATIENTS & METHODS: We analyzed our prospective database of consecutive patients undergoing HoLEP in a single tertiary public hospital between October 2016 and January 2019. We excluded patients with clinically significant prostate cancer or neurogenic bladders. Preoperative prostate volume was measured on MRI or ultrasonography. Perioperative variables and functional outcomes were recorded. RESULTS: Of 304 HoLEPs performed, we included 97 patients with prostate volume of ≥150 cc and 186 patients with prostate volume <150 cc. Comparing both cohorts (≥150 cc vs <150 cc): mean age was 71.5 vs 68.3 years, prostate volume 195 cc vs 93 cc, preoperative Qmax 9.6mL/s vs 10mL/s, American Urology Association Symptom Score (IPSS) 21 points vs 20.5 points; mean PSA 13.2µg/L vs 8.8µg/L; laser duration 86 vs 59 minutes; morcellation duration 29 vs 14 minutes; enucleated weight was 124 g vs 60 g. One patient (1%) from the ≥150 cc cohort required a surgical procedure for stress urinary incontinence, and none from the <150 cc cohort, but this did not achieve statistical significance (Pâ¯=â¯.12). There were no statistically significant differences in postoperative Qmax (32.3 vs 26.4 mL/s; Pâ¯=â¯.12), IPSS (5.9 points vs 7.3points; Pâ¯=â¯.23), mean PSA (3.9 µg/L vs 2.2 µg/L; Pâ¯=â¯.60), stricture incidence (1% vs 2.7%; Pâ¯=â¯.63), or significant stress urinary incontinence (4.1% vs 0.5%; Pâ¯=â¯.08). CONCLUSION: Our large series demonstrates that HoLEP is safe and effective in patients with massive prostates (≥150 cc), with similar outcomes compared to patients with prostates <150 cc.