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2.
BMC Med Educ ; 22(1): 304, 2022 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-35449040

RESUMO

BACKGROUND: Lesbian, gay, bisexual, transgender, queer, non-binary, intersex, and/or asexual (LGBTQ+) individuals continue to suffer worse health outcomes compared to the general population. Data on LGBTQ+ individuals in medicine, particularly in medical training, remain sparse. National studies of LGBTQ+ students in MD/PhD and DO/PhD training programs have not been reported. METHODS: Trainees pursuing MD, DO, MD/PhD, and DO/PhD degrees at 32 nationally representative institutions completed a 70-item survey about their future career and anticipated challenges using an online survey tool from September 2012 to December 2014. There were 4,433 respondents to the survey. Of those, 2,837 completed the gender identity questions and 2,849 completed the sexual orientation questions. Completion of these questions was required for inclusion. Survey results were analyzed to examine differences between LGBTQ+ and non-LGBTQ+ medical and dual degree trainees. RESULTS: LGBTQ+ students were underrepresented among MD/PhD and DO/PhD trainees (8.70%) compared to the US population, though their representation was higher than among MD and DO trainees (5.20%). LGBTQ+ dual degree trainees endorsed the greatest interest in pursuing careers involving academic medicine, with varying career focuses including research, clinical duties, education, and advocacy. LGBTQ+ dual degree trainees prioritized opportunities in patient care, work-life balance, and research as the most important factors for their career selection. Importantly, a higher percentage of LGBTQ+ dual degree trainees (15.50%) identified sexual harassment as a past barrier to career advancement compared to their non-LGBTQ+ peers (8.27%). LGBTQ+ dual degree trainees were more likely to report having a mentor who advocated for them. CONCLUSIONS: LGBTQ+ physician scientist trainees remain under-represented and under-studied. It is vital that medical institutions devote more time and resources towards identifying and addressing the unique needs of this group in training. Training programs should be aware of the current and prior challenges faced by their LGBTQ+ dual degree trainees, work to overcome the unique barriers they face, highlight the strengths and unique perspectives they bring, and foster their professional growth and goals during and beyond their training.


Assuntos
Pesquisa Biomédica , Minorias Sexuais e de Gênero , Pesquisa Biomédica/educação , Escolha da Profissão , Feminino , Identidade de Gênero , Humanos , Intenção , Masculino
3.
Immunohorizons ; 5(6): 466-476, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34398806

RESUMO

Lasting immunity will be critical for overcoming COVID-19. However, the factors associated with the development of high titers of anti-SARS-CoV-2 Abs and how long those Abs persist remain incompletely defined. In particular, an understanding of the relationship between COVID-19 symptoms and anti-SARS-CoV-2 Abs is limited. To address these unknowns, we quantified serum anti-SARS- CoV-2 Abs in clinically diverse COVID-19 convalescent human subjects 5 wk (n = 113) and 3 mo (n = 79) after symptom resolution with three methods: a novel multiplex assay to quantify IgG against four SARS-CoV-2 Ags, a new SARS-CoV-2 receptor binding domain-angiotensin converting enzyme 2 inhibition assay, and a SARS-CoV-2 neutralizing assay. We then identified clinical and demographic factors, including never-before-assessed COVID-19 symptoms, that consistently correlate with high anti-SARS-CoV-2 Ab levels. We detected anti-SARS-CoV-2 Abs in 98% of COVID-19 convalescent subjects 5 wk after symptom resolution, and Ab levels did not decline at 3 mo. Greater disease severity, older age, male sex, higher body mass index, and higher Charlson Comorbidity Index score correlated with increased anti-SARS-CoV-2 Ab levels. Moreover, we report for the first time (to our knowledge) that COVID-19 symptoms, most consistently fever, body aches, and low appetite, correlate with higher anti-SARS-CoV-2 Ab levels. Our results provide robust and new insights into the development and persistence of anti-SARS-CoV-2 Abs.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Imunoglobulina G/imunologia , SARS-CoV-2/imunologia , Adulto , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , COVID-19/epidemiologia , COVID-19/virologia , Estudos de Coortes , Feminino , Hospitalização/estatística & dados numéricos , Humanos , Imunoglobulina G/sangue , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Pandemias , SARS-CoV-2/fisiologia , Índice de Gravidade de Doença , Fatores de Tempo
4.
PLoS Biol ; 19(6): e3001265, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34143766

RESUMO

The search for potential antibody-based diagnostics, vaccines, and therapeutics for pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has focused almost exclusively on the spike (S) and nucleocapsid (N) proteins. Coronavirus membrane (M), ORF3a, and ORF8 proteins are humoral immunogens in other coronaviruses (CoVs) but remain largely uninvestigated for SARS-CoV-2. Here, we use ultradense peptide microarray mapping to show that SARS-CoV-2 infection induces robust antibody responses to epitopes throughout the SARS-CoV-2 proteome, particularly in M, in which 1 epitope achieved excellent diagnostic accuracy. We map 79 B cell epitopes throughout the SARS-CoV-2 proteome and demonstrate that antibodies that develop in response to SARS-CoV-2 infection bind homologous peptide sequences in the 6 other known human CoVs. We also confirm reactivity against 4 of our top-ranking epitopes by enzyme-linked immunosorbent assay (ELISA). Illness severity correlated with increased reactivity to 9 SARS-CoV-2 epitopes in S, M, N, and ORF3a in our population. Our results demonstrate previously unknown, highly reactive B cell epitopes throughout the full proteome of SARS-CoV-2 and other CoV proteins.


Assuntos
Anticorpos Antivirais/imunologia , COVID-19/imunologia , SARS-CoV-2/imunologia , Proteínas Virais/imunologia , Anticorpos Antivirais/sangue , COVID-19/patologia , Coronavirus/imunologia , Reações Cruzadas , Epitopos de Linfócito B , Humanos , Epitopos Imunodominantes , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Proteoma/imunologia , Índice de Gravidade de Doença
5.
medRxiv ; 2021 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-33655260

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) control in the United States remains hampered, in part, by testing limitations. We evaluated a simple, outdoor, mobile, colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay workflow where self-collected saliva is tested for SARS-CoV-2 RNA. From July 16 to November 19, 2020, 4,704 surveillance samples were collected from volunteers and tested for SARS-CoV-2 at 5 sites. A total of 21 samples tested positive for SARS-CoV-2 by RT-LAMP; 12 were confirmed positive by subsequent quantitative reverse-transcription polymerase chain reaction (qRT-PCR) testing, while 8 were negative for SARS-CoV-2 RNA, and 1 could not be confirmed because the donor did not consent to further molecular testing. We estimated the RT-LAMP assay's false-negative rate from July 16 to September 17, 2020 by pooling residual heat-inactivated saliva that was unambiguously negative by RT-LAMP into groups of 6 or less and testing for SARS-CoV-2 RNA by qRT-PCR. We observed a 98.8% concordance between the RT-LAMP and qRT-PCR assays, with only 5 of 421 RT-LAMP negative pools (2,493 samples) testing positive in the more sensitive qRT-PCR assay. Overall, we demonstrate a rapid testing method that can be implemented outside the traditional laboratory setting by individuals with basic molecular biology skills and can effectively identify asymptomatic individuals who would not typically meet the criteria for symptom-based testing modalities.

6.
medRxiv ; 2021 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-33442707

RESUMO

Lasting immunity will be critical for overcoming the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, factors that drive the development of high titers of anti-SARS-CoV-2 antibodies and how long those antibodies persist remain unclear. Our objective was to comprehensively evaluate anti-SARS-CoV-2 antibodies in a clinically diverse COVID-19 convalescent cohort at defined time points to determine if anti-SARS-CoV-2 antibodies persist and to identify clinical and demographic factors that correlate with high titers. Using a novel multiplex assay to quantify IgG against four SARS-CoV-2 antigens, a receptor binding domain-angiotensin converting enzyme 2 inhibition assay, and a SARS-CoV-2 neutralization assay, we found that 98% of COVID-19 convalescent subjects had anti-SARS-CoV-2 antibodies five weeks after symptom resolution (n=113). Further, antibody levels did not decline three months after symptom resolution (n=79). As expected, greater disease severity, older age, male sex, obesity, and higher Charlson Comorbidity Index score correlated with increased anti-SARS-CoV-2 antibody levels. We demonstrated for the first time that COVID-19 symptoms, namely fever, abdominal pain, diarrhea and low appetite, correlated consistently with higher anti-SARS-CoV-2 antibody levels. Our results provide new insights into the development and persistence of anti-SARS-CoV-2 antibodies.

7.
J Womens Health (Larchmt) ; 30(1): 90-102, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32349608

RESUMO

Background: Female physician-scientists have led major advances in medicine broadly and particularly in women's health. Women remain underrepresented in dual MD-PhD degree programs that train many physician-scientists despite gender parity among medical and biomedical research students. Materials and Methods: To explore how the training environment might be experienced differently for male and female students in one MD-PhD program, the authors analyzed gender differences in annual symposium speakers with exact binomial tests, student participation as question-askers at a weekly seminar with logistic regression, and number of publications with quasi-Poisson generalized linear models. They compared male and female students' perceptions of gender-based discrimination using a survey, including qualitative analysis of free text responses. The program consisted of 71 total students in the 2017-2018 and 2018-2019 academic years. Female students comprised 42.0% (81/191) of program matriculants from 1997 to 2019. Results: Male and female students were equally likely to present at the annual program symposium, but faculty (p = 0.001) and keynote (p = 0.012) presenters were more likely to be male. Compared with male counterparts, female students asked fewer seminar questions (p < 0.005) and female speakers received more questions (p = 0.03). Female students perceived less support and differed from men in reasons for asking or not asking seminar questions. Free text responses described repeated small acts of discrimination toward women with cumulative impact. Positive program changes followed presentation of findings to program leaders and students. Conclusions: The authors identified several aspects of one MD-PhD program that could discourage career or training persistence of female students. Increasing awareness of these issues was temporally related to positive programmatic changes.


Assuntos
Pesquisa Biomédica , Médicas , Educação de Pós-Graduação em Medicina , Feminino , Humanos , Masculino , Sexismo , Estudantes
8.
bioRxiv ; 2021 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-33052349

RESUMO

The search for potential antibody-based diagnostics, vaccines, and therapeutics for pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has focused almost exclusively on the spike (S) and nucleocapsid (N) proteins. Coronavirus membrane (M), ORF3a, and ORF8 proteins are humoral immunogens in other coronaviruses (CoVs) but remain largely uninvestigated for SARS-CoV-2. Here we use ultradense peptide microarray mapping to show that SARS-CoV-2 infection induces robust antibody responses to epitopes throughout the SARS-CoV-2 proteome, particularly in M, in which one epitope achieved excellent diagnostic accuracy. We map 79 B cell epitopes throughout the SARS-CoV-2 proteome and demonstrate that antibodies that develop in response to SARS-CoV-2 infection bind homologous peptide sequences in the six other known human CoVs. We also confirm reactivity against four of our top-ranking epitopes by enzyme-linked immunosorbent assay (ELISA). Illness severity correlated with increased reactivity to nine SARS-CoV-2 epitopes in S, M, N, and ORF3a in our population. Our results demonstrate previously unknown, highly reactive B cell epitopes throughout the full proteome of SARS-CoV-2 and other CoV proteins.

9.
J Biomol Tech ; 32(3): 137-147, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-35035293

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) control in the United States remains hampered, in part, by testing limitations. We evaluated a simple, outdoor, mobile, colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay workflow where self-collected saliva is tested for SARS-CoV-2 RNA. From July 16, 2020, to November 19, 2020, surveillance samples (n = 4704) were collected from volunteers and tested for SARS-CoV-2 at 5 sites. Twenty-one samples tested positive for SARS-CoV-2 by RT-LAMP; 12 were confirmed positive by subsequent quantitative reverse-transcription polymerase chain reaction (qRT-PCR) testing, whereas 8 tested negative for SARS-CoV-2 RNA, and 1 could not be confirmed because the donor did not consent to further molecular testing. We estimated the false-negative rate of the RT-LAMP assay only from July 16, 2020, to September 17, 2020 by pooling residual heat-inactivated saliva that was unambiguously negative by RT-LAMP into groups of 6 or fewer and testing for SARS-CoV-2 RNA by qRT-PCR. We observed a 98.8% concordance between the RT-LAMP and qRT-PCR assays, with only 5 of 421 RT-LAMP-negative pools (2493 total samples) testing positive in the more-sensitive qRT-PCR assay. Overall, we demonstrate a rapid testing method that can be implemented outside the traditional laboratory setting by individuals with basic molecular biology skills and that can effectively identify asymptomatic individuals who would not typically meet the criteria for symptom-based testing modalities.


Assuntos
COVID-19 , SARS-CoV-2 , Teste para COVID-19 , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/genética , Sensibilidade e Especificidade
10.
PLoS One ; 15(12): e0244882, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33382861

RESUMO

SARS-CoV-2 testing is crucial to controlling the spread of this virus, yet shortages of nucleic acid extraction supplies and other key reagents have hindered the response to COVID-19 in the US. Several groups have described loop-mediated isothermal amplification (LAMP) assays for SARS-CoV-2, including testing directly from nasopharyngeal swabs and eliminating the need for reagents in short supply. Frequent surveillance of individuals attending work or school is currently unavailable to most people but will likely be necessary to reduce the ~50% of transmission that occurs when individuals are nonsymptomatic. Here we describe a fluorescence-based RT-LAMP test using direct nasopharyngeal swab samples and show consistent detection in clinically confirmed primary samples with a limit of detection (LOD) of ~625 copies/µl, approximately 100-fold lower sensitivity than qRT-PCR. While less sensitive than extraction-based molecular methods, RT-LAMP without RNA extraction is fast and inexpensive. Here we also demonstrate that adding a lysis buffer directly into the RT-LAMP reaction improves the sensitivity of some samples by approximately 10-fold. Furthermore, purified RNA in this assay achieves a similar LOD to qRT-PCR. These results indicate that high-throughput RT-LAMP testing could augment qRT-PCR in SARS-CoV-2 surveillance programs, especially while the availability of qRT-PCR testing and RNA extraction reagents is constrained.


Assuntos
Teste de Ácido Nucleico para COVID-19 , COVID-19 , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/genética , SARS-CoV-2/genética , COVID-19/diagnóstico , COVID-19/genética , Primers do DNA/química , Primers do DNA/genética , Humanos , Limite de Detecção , Nasofaringe/virologia
11.
Nat Commun ; 11(1): 5558, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33144575

RESUMO

Evidence-based public health approaches that minimize the introduction and spread of new SARS-CoV-2 transmission clusters are urgently needed in the United States and other countries struggling with expanding epidemics. Here we analyze 247 full-genome SARS-CoV-2 sequences from two nearby communities in Wisconsin, USA, and find surprisingly distinct patterns of viral spread. Dane County had the 12th known introduction of SARS-CoV-2 in the United States, but this did not lead to descendant community spread. Instead, the Dane County outbreak was seeded by multiple later introductions, followed by limited community spread. In contrast, relatively few introductions in Milwaukee County led to extensive community spread. We present evidence for reduced viral spread in both counties following the statewide "Safer at Home" order, which went into effect 25 March 2020. Our results suggest patterns of SARS-CoV-2 transmission may vary substantially even in nearby communities. Understanding these local patterns will enable better targeting of public health interventions.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/transmissão , Genoma Viral/genética , Pneumonia Viral/epidemiologia , Pneumonia Viral/transmissão , COVID-19 , Infecções por Coronavirus/prevenção & controle , Geografia , Humanos , Programas de Rastreamento/métodos , Epidemiologia Molecular/métodos , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Distância Psicológica , Dispositivos de Proteção Respiratória , SARS-CoV-2 , Estados Unidos/epidemiologia , Wisconsin/epidemiologia
12.
Microorganisms ; 8(10)2020 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-33007921

RESUMO

From 2010 to 2015, 73 common marmosets (Callithrix jacchus) housed at the Wisconsin National Primate Research Center (WNPRC) were diagnosed postmortem with lymphocytic enterocolitis. We used unbiased deep-sequencing to screen the blood of deceased enterocolitis-positive marmosets for viruses. In five out of eight common marmosets with lymphocytic enterocolitis, we discovered a novel pegivirus not present in ten matched, clinically normal controls. The novel virus, which we named Southwest bike trail virus (SOBV), is most closely related (68% nucleotide identity) to a strain of simian pegivirus A isolated from a three-striped night monkey (Aotus trivirgatus). We screened 146 living WNPRC common marmosets for SOBV, finding an overall prevalence of 34% (50/146). Over four years, 85 of these 146 animals died or were euthanized. Histological examination revealed 27 SOBV-positive marmosets from this cohort had lymphocytic enterocolitis, compared to 42 SOBV-negative marmosets, indicating no association between SOBV and disease in this cohort (p = 0.0798). We also detected SOBV in two of 33 (6%) clinically normal marmosets screened during transfer from the New England Primate Research Center, suggesting SOBV could be exerting confounding influences on comparisons of common marmoset studies from multiple colonies.

13.
medRxiv ; 2020 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-32676620

RESUMO

Evidence-based public health approaches that minimize the introduction and spread of new SARS-CoV-2 transmission clusters are urgently needed in the United States and other countries struggling with expanding epidemics. Here we analyze 247 full-genome SARS-CoV-2 sequences from two nearby communities in Wisconsin, USA, and find surprisingly distinct patterns of viral spread. Dane County had the 12th known introduction of SARS-CoV-2 in the United States, but this did not lead to descendant community spread. Instead, the Dane County outbreak was seeded by multiple later introductions, followed by limited community spread. In contrast, relatively few introductions in Milwaukee County led to extensive community spread. We present evidence for reduced viral spread in both counties, and limited viral transmission between counties, following the statewide Safer-at-Home public health order, which went into effect 25 March 2020. Our results suggest that early containment efforts suppressed the spread of SARS-CoV-2 within Wisconsin.

15.
PLoS Negl Trop Dis ; 12(11): e0006903, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30481182

RESUMO

The specificity of the antibody response against Zika virus (ZIKV) is not well-characterized. This is due, in part, to the antigenic similarity between ZIKV and closely related dengue virus (DENV) serotypes. Since these and other similar viruses co-circulate, are spread by the same mosquito species, and can cause similar acute clinical syndromes, it is difficult to disentangle ZIKV-specific antibody responses from responses to closely-related arboviruses in humans. Here we use high-density peptide microarrays to profile anti-ZIKV antibody reactivity in pregnant and non-pregnant macaque monkeys with known exposure histories and compare these results to reactivity following DENV infection. We also compare cross-reactive binding of ZIKV-immune sera to the full proteomes of 28 arboviruses. We independently confirm a purported ZIKV-specific IgG antibody response targeting ZIKV nonstructural protein 2B (NS2B) that was recently reported in ZIKV-infected people and we show that antibody reactivity in pregnant animals can be detected as late as 127 days post-infection (dpi). However, we also show that these responses wane over time, sometimes rapidly, and in one case the response was elicited following DENV infection in a previously ZIKV-exposed animal. These results suggest epidemiologic studies assessing seroprevalence of ZIKV immunity using linear epitope-based strategies will remain challenging to interpret due to susceptibility to false positive results. However, the method used here demonstrates the potential for rapid profiling of proteome-wide antibody responses to a myriad of neglected diseases simultaneously and may be especially useful for distinguishing antibody reactivity among closely related pathogens.


Assuntos
Anticorpos Antivirais/imunologia , Complicações na Gravidez/imunologia , Proteínas não Estruturais Virais/imunologia , Infecção por Zika virus/imunologia , Zika virus/imunologia , Animais , Anticorpos Antivirais/sangue , Formação de Anticorpos , Reações Cruzadas , Mapeamento de Epitopos , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Feminino , Humanos , Macaca , Masculino , Gravidez , Complicações na Gravidez/sangue , Complicações na Gravidez/virologia , Estudos Soroepidemiológicos , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Zika virus/química , Zika virus/genética , Zika virus/isolamento & purificação , Infecção por Zika virus/sangue , Infecção por Zika virus/virologia
16.
Genome Announc ; 5(18)2017 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-28473378

RESUMO

The picornaviral genus Kunsagivirus has a single member, kunsagivirus A, which was discovered in migratory bird feces. We report here the discovery of a novel kunsagivirus in wild yellow baboon (Papio cynocephalus) blood. The genomic sequence of this virus indicates the probable need for the establishment of a second kunsagivirus species.

17.
J Org Chem ; 80(3): 1781-8, 2015 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-25562368

RESUMO

A series of isomeric 3-trimethylsilyl-1-arylcyclobutyl carbocations, 10 and 11, where the cross-ring 3-trimethylsilyl group has the potential to interact with the cationic center, have been generated under solvolytic conditions. When the cationic center can interact with the rear lobe of the carbon-silicon bond, rate enhancements become progressively larger as the substituent on the aryl group becomes more electron-withdrawing. When the potential interaction with the trimethylsilyl group is via a front lobe interaction, there is minimal rate enhancement over the range of substituents. Computational studies have also been carried out on these cations 10 and 11. Calculated trimethylsilyl stabilization energies progressively increase with electron-withdrawing character of the aryl groups when the trimethylsilyl interaction is via the rear lobe. By way of contrast, there are minimal changes in stabilization energies when the potential trimethylsilyl interaction is via the front lobe of the carbon-silicon bond. These computational studies, along with the solvolytic studies, point to a significant rear lobe 3-trimethylsilyl stabilization of arylcyclobutyl cations. They also argue against any front lobe stabilization of the isomeric arylcyclobutyl cations.

18.
J Org Chem ; 79(6): 2547-55, 2014 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-24548109

RESUMO

endo-2-Trimethylsilyl-anti-7-norbornyl triflate undergoes solvolysis reactions 1.8 × 10(4) faster than 7-norbornyl triflate in CD3CO2D and 1.3 × 10(5) times faster in CF3CH2OH. The exclusive substitution products with retained stereochemistry point to a significantly stabilized γ-trimethylsilyl carbocation intermediate. The endo-2-trimethylsilyl-7-norbornyl carbene gives a major rearrangement product where the trimethylsilyl-activated hydrogen migrates to the carbenic center. This rearrangement product implies stabilization of the carbene by the γ-trimethylsilyl group. Isodesmic computational studies (M062X/6-311+G**) indicate that the endo-2-trimethylsilyl-7-norbornyl cation is stabilized by 16.2 kcal/mol and that the endo-2-trimethylsilyl-7-norbornyl carbene is stabilized by a smaller factor of 1.8 kcal/mol. By way of contrast, anti-7-trimethylsilyl-endo-2-norbornyl mesylate undergoes solvolysis in CD3CO2D only 2.6 times faster than endo-2-norbornyl mesylate and 9.4 times faster in CF3CH2OH. The substitution products have only partially retained stereochemistry, and significant rearrangements are observed. The anti-7-trimethylsilyl-2-norbornyl carbene gives a rearrangement product via 1,3-hydrogen migration of the C6 hydrogen, which is completely analogous to the behavior of the unsubstituted 2-norbornyl carbene. Isodesmic calculations show that the anti-7-trimethylsilyl-2-norbornyl cation is stabilized by only 3.2 kcal/mol relative to the 2-norbornyl cation, and the corresponding anti-7-trimethylsilyl-2-norbornyl carbene is stabilized by a negligible 0.9 kcal/mol.

19.
J Antimicrob Chemother ; 67(10): 2388-95, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22740589

RESUMO

OBJECTIVES: To determine the stability/reversibility and mechanism of monensin adaptation in monensin-treated cattle isolates compared with reference bacterial isolates, exposed in vitro to high monensin concentrations. METHODS: We evaluated the potential for cattle-origin strains of Clostridium perfringens, Enterococcus faecium and Enterococcus faecalis exposed to monensin in vivo (in vivo monensin-exposed isolates) to maintain or achieve the ability to grow in the presence of high monensin concentrations (in vitro monensin-adapted isolates). Twenty-one consecutive subcultures of the in vitro monensin-adapted strains were performed, and monensin MICs were determined for the 3rd, 7th, 14th and 21st subcultures (subcultured isolates). SDS-PAGE and transmission electron microscopy (TEM) were used to determine protein expression and visualize extracellular morphology changes. RESULTS: Monensin-non-exposed isolates did not display monensin adaptation during in vitro monensin exposure. In contrast, in vivo monensin-exposed isolates displayed monensin adaptation enabling growth at 32× MIC. Upon consecutive subculturing, monensin MICs returned to baseline, or one dilution above, for the monensin-adapted strains. SDS-PAGE identified overexpression of a 14 kDa protein (C. perfringens) and a 20.5 kDa protein (E. faecium and E. faecalis) in the monensin-adapted isolates. TEM demonstrated that in vitro monensin-adapted strains had a significantly thicker cell wall or glycocalyx compared with in vivo monensin-exposed or subcultured isolates. CONCLUSIONS: In vivo monensin-exposed isolates of C. perfringens, E. faecium and E. faecalis have the ability to grow in the presence of high monensin concentrations in vitro. This is associated with an increased thickening of the cell wall or glycocalyx that is reversible upon serial passage, suggesting a phenotypically expressed, but not genetically stable, trait.


Assuntos
Antibacterianos/farmacologia , Clostridium perfringens/efeitos dos fármacos , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecium/efeitos dos fármacos , Inocuidade dos Alimentos , Monensin/farmacologia , Animais , Antibacterianos/metabolismo , Proteínas de Bactérias/análise , Bovinos , Doenças dos Bovinos/microbiologia , Parede Celular/ultraestrutura , Clostridium perfringens/isolamento & purificação , Clostridium perfringens/metabolismo , Clostridium perfringens/ultraestrutura , Eletroforese em Gel de Poliacrilamida , Enterococcus faecalis/isolamento & purificação , Enterococcus faecalis/metabolismo , Enterococcus faecalis/ultraestrutura , Enterococcus faecium/isolamento & purificação , Enterococcus faecium/metabolismo , Enterococcus faecium/ultraestrutura , Glicocálix/ultraestrutura , Infecções por Bactérias Gram-Positivas/microbiologia , Infecções por Bactérias Gram-Positivas/veterinária , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Transmissão , Monensin/metabolismo
20.
Vet Parasitol ; 168(3-4): 312-7, 2010 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-20045256

RESUMO

The efficacy of spinosad against adult fleas (Ctenocephalides felis) on dogs was evaluated in three controlled, blinded studies. One study was conducted to determine speed of kill on experimentally infested dogs. Two additional studies were designed to assess the efficacy of spinosad in preventing environmental contamination with flea eggs (USA study and EU study). An additional objective of the USA study was to assess the effects of skin and hair-coat debris from spinosad-treated dogs on eggs and larvae of C. felis. Dogs were randomly allocated to treatment with beef-flavored spinosad tablets, administered orally at a minimum dosage of 30mg/kg, or placebo. In the first study, speed of kill was determined by flea comb counts performed at 0.5, 1, 2, 4, 8, 12, 24 and 48h after spinosad treatment. Reductions in geometric mean flea counts for spinosad-treated dogs, compared to placebo were 53.7% at 0.5h, 64.2% at 1h, 85.8% at 2h and 100% at 4 through 48h post-treatment (p<0.05 at 1h and beyond). In the 2 flea egg production studies, dogs were treated (spinosad or placebo) once on day 0, infested with 600 fleas approximately 3h post-treatment and reinfested with approximately 600 fleas at intervals over 1 month. Flea eggs were collected starting at approximately 72h after each infestation. Eggs were examined for any effects of spinosad on egg viability. Efficacy of spinosad was also evaluated against environmental eggs and larvae exposed to canine hair-coat debris collected on days 3, 7, 14, 21, and 30. Spinosad was highly effective in reducing flea egg production (>99.8% across the entire study period) compared to control dogs in both egg collection studies. Insufficient numbers of eggs were recovered from spinosad-treated dogs to determine the viability of those eggs. There was no evidence of any effect on environmental flea stages, indicating that spinosad was not present in the skin debris of spinosad-treated dogs. The capability of spinosad to quickly kill adult fleas, and to greatly reduce egg production following challenge with high numbers of adult fleas is important in breaking the flea life cycle and preventing the introduction and establishment of new flea infestations in the household.


Assuntos
Doenças do Cão/tratamento farmacológico , Doenças do Cão/parasitologia , Ectoparasitoses/veterinária , Inseticidas , Macrolídeos , Sifonápteros , Administração Oral , Animais , Cães , Combinação de Medicamentos , Ectoparasitoses/tratamento farmacológico , Ectoparasitoses/parasitologia , Feminino , Masculino , Contagem de Ovos de Parasitas/veterinária , Distribuição Aleatória
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