RESUMO
Importance: Implemented in 18 regions, Comprehensive Primary Care Plus (CPC+) was the largest US primary care delivery model ever tested. Understanding its association with health outcomes is critical in designing future transformation models. Objective: To test whether CPC+ was associated with lower health care spending and utilization and improved quality of care. Design, Setting, and Participants: Difference-in-differences regression models compared changes in outcomes between the year before CPC+ and 5 intervention years for Medicare fee-for-service beneficiaries attributed to CPC+ and comparison practices. Participants included 1373 track 1 (1â¯549â¯585 beneficiaries) and 1515 track 2 (5â¯347â¯499 beneficiaries) primary care practices that applied to start CPC+ in 2017 and met minimum care delivery and other eligibility requirements. Comparison groups included 5243 track 1 (5â¯347â¯499 beneficiaries) and 3783 track 2 (4â¯507â¯499 beneficiaries) practices, matched, and weighted to have similar beneficiary-, practice-, and market-level characteristics as CPC+ practices. Interventions: Two-track design involving enhanced (higher for track 2) and alternative payments (track 2 only), care delivery requirements (greater for track 2), data feedback, learning, and health information technology support. Main Outcomes and Measures: The prespecified primary outcome was annualized Medicare Part A and B expenditures per beneficiary per month (PBPM). Secondary outcomes included expenditure categories, utilization (eg, hospitalizations), and claims-based quality-of-care process and outcome measures (eg, recommended tests for patients with diabetes and unplanned readmissions). Results: Among the CPC+ patients, 5% were Black, 3% were Hispanic, 87% were White, and 5% were of other races (including Asian/Other Pacific Islander and American Indian); 85% of CPC+ patients were older than 65 years and 58% were female. CPC+ was associated with no discernible changes in the total expenditures (track 1: $1.1 PBPM [90% CI, -$4.3 to $6.6], P = .74; track 2: $1.3 [90% CI, -$5 to $7.7], P = .73), and with increases in expenditures including enhanced payments (track 1: $13 [90% CI, $7 to $18], P < .001; track 2: $24 [90% CI, $18 to $31], P < .001). Among secondary outcomes, CPC+ was associated with decreases in emergency department visits starting in year 1, and in acute hospitalizations and acute inpatient expenditures in later years. Associations were more favorable for practices also participating in the Medicare Shared Savings Program and independent practices. CPC+ was not associated with meaningful changes in claims-based quality-of-care measures. Conclusions and Relevance: Although the timing of the associations of CPC+ with reduced utilization and acute inpatient expenditures was consistent with the theory of change and early focus on episodic care management of CPC+, CPC+ was not associated with a reduction in total expenditures over 5 years. Positive interaction between CPC+ and the Shared Savings Program suggests transformation models might be more successful when provider cost-reduction incentives are aligned across specialties. Further adaptations and testing of primary care transformation models, as well as consideration of the larger context in which they operate, are needed.
Assuntos
Gastos em Saúde , Medicare , Idoso , Humanos , Feminino , Estados Unidos , Masculino , Atenção à Saúde , Assistência Integral à Saúde , Planos de Pagamento por Serviço Prestado , Atenção Primária à Saúde/organização & administraçãoRESUMO
AbstractIntergenerational effects, also known as parental effects in which the offspring phenotype is influenced by the parental phenotype, can occur in response to factors that occur not only in early but also in late parental life. However, little is known about how these parental life stage-specific environments interact with each other and with the offspring environment to influence offspring phenotypes, particularly in organisms that realize distinct niches across ontogeny. We examined the effects of parental larval starvation and adult reproductive environment on offspring traits under matching or mismatching offspring larval starvation conditions using the holometabolous, haplodiploid insect Athalia rosae (turnip sawfly). We show that parental larval starvation had trait-dependent intergenerational effects on both life history and consumption traits of offspring larvae, partly in interaction with offspring conditions, while there was no significant effect of parental adult reproductive environment. In addition, while offspring larval starvation led to numerous gene- and pathway-level expression differences, parental larval starvation impacted far fewer genes and only the ribosomal pathway. Our findings reveal that parental starvation evokes complex intergenerational effects on offspring life history traits, consumption patterns, and gene expression, although the effects are less pronounced than those of offspring starvation.
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Características de História de Vida , Transcriptoma , Animais , Insetos/genética , Larva/genética , ReproduçãoRESUMO
Timely and effective antibiotic treatment is vital for sepsis, with increasing incidence of antimicrobial-resistant bacteremia driving interest in rapid phenotypic susceptibility testing. To enable the widespread adoption needed to make an impact, antibiotic susceptibility testing (AST) systems need to be accurate, enable rapid intervention, have a broad antimicrobial menu and be easy to use and affordable. We evaluated the Specific Reveal (Specific Diagnostics, San Jose, CA) rapid AST system on positive blood cultures with Gram-negative organisms in a relatively resistant population in a large urban hospital to assess its potential for routine clinical use. One hundred four randomly selected positive blood cultures (Virtuo; bioMérieux) were Gram stained, diluted 1:1,000 in Pluronic water, inoculated into 96-well antibiotic plates, sealed with the Reveal sensor panel, and placed in the Reveal instrument for incubation and reading. The MIC and susceptible/intermediate/resistant category was determined and compared to results from Vitek 2 (bioMérieux) for the 17 antimicrobials available and to Sensititre (Thermo Fisher) for 24 antimicrobials. Performance was also assessed with contrived blood cultures with 33 highly resistant strains. Reveal was in 98.0% essential agreement (EA) and 96.3% categorical agreement (CA) with Sensititre, with just 1.3% very major error (VME) and 97.0%/96.2%/1.3% EA/CA/VME versus Vitek 2. Reveal results for contrived highly resistant strains were equivalent, with EA/CA/VME of 97.7%/95.2%/1.0% with CDC/FDA Antibiotic Resistance Isolate Bank references. Average time to result (TTR) for Reveal was 4.6 h. Sample preparation was relatively low skill and averaged 3 min. We conclude that the Reveal system enables accurate and rapid susceptibility testing of Gram-negative blood cultures.
Assuntos
Bacteriemia , Hemocultura , Antibacterianos/farmacologia , Bacteriemia/diagnóstico , Hemocultura/métodos , Bactérias Gram-Negativas , Hospitais Urbanos , Humanos , Testes de Sensibilidade MicrobianaRESUMO
Chemical defense is a widespread anti-predator strategy exhibited by organisms, with individuals either synthesizing or extrinsically acquiring defensive chemicals. In some species, such defences can also be transferred among conspecifics. Here, we tested the effects of pharmacophagy on the defense capability of the turnip sawfly, Athalia rosae, which can acquire neo-clerodane diterpenoids (clerodanoids) via pharmacophagy when having access to the plant Ajuga reptans. We show that clerodanoid access mediates protection against predation by mantids for the sawflies, both in a no-choice feeding assay and a microcosm setup. Even indirect access to clerodanoids, via nibbling on conspecifics that had access to the plant, resulted in protection against predation albeit to a lower degree than direct access. Furthermore, sawflies that had no direct access to clerodanoids were consumed less frequently by mantids when they were grouped with conspecifics that had direct access. Most, but not all, of such initially undefended sawflies could acquire clerodanoids from conspecifics that had direct access to the plant, although in low quantities. Together our results demonstrate that clerodanoids serve as a chemical defense that can also be transferred by interactions among conspecifics. Moreover, the presence of chemically defended individuals in a group can confer protection onto conspecifics that had no direct access to clerodanoids.
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Brassicaceae , Himenópteros , Animais , Humanos , Larva , Plantas , Comportamento PredatórioRESUMO
BACKGROUND: The Centers for Medicare & Medicaid Services launched the 4-year Comprehensive Primary Care Initiative (CPC Classic) in 2012 and its 5-year successor, CPC Plus (CPC+), in 2017 to test whether improving primary care delivery in five areas-and providing practices with financial and technical support-reduced spending and improved quality. This is the first study to examine long-term effects of a primary care practice transformation model. OBJECTIVE: To test whether long-term primary care transformation-the 4-year CPC Classic and the first 2 years of its successor, CPC+-reduced hospitalizations, emergency department (ED) visits, and spending over 6 years. DESIGN: We used a difference-in-differences analysis to compare outcomes for beneficiaries attributed to CPC Classic practices with outcomes for beneficiaries attributed to comparison practices during the year before and 6 years after CPC Classic began. PARTICIPANTS: The study involved 565,674 Medicare fee-for-service beneficiaries attributed to 502 CPC Classic practices and 1,165,284 beneficiaries attributed to 908 comparison practices, with similar beneficiary-, practice-, and market-level characteristics as the CPC Classic practices. INTERVENTIONS: The interventions required primary care practices to improve 5 care areas and supported their transformation with substantially enhanced payment, data feedback, and learning support and, for CPC+, added health information technology support. MAIN MEASURES: Hospitalizations (all-cause), ED visits (outpatient and total), and Medicare Part A and B expenditures. KEY RESULTS: Relative to comparison practices, beneficiaries in intervention practices experienced slower growth in hospitalizations-3.1% less in year 5 and 3.5% less in year 6 (P < 0.01) and roughly 2% (P < 0.1) slower growth each year in total ED visits during years 3 through 6. Medicare Part A and B expenditures (excluding care management fees) did not change appreciably. CONCLUSIONS: The emergence of favorable effects on hospitalizations in years 5 and 6 suggests primary care transformation takes time to translate into lower hospitalizations. Longer tests of models are needed.
Assuntos
Gastos em Saúde , Medicare , Idoso , Assistência Integral à Saúde , Planos de Pagamento por Serviço Prestado , Humanos , Atenção Primária à Saúde , Estados UnidosRESUMO
Sex allocation theory in simultaneous hermaphrodites predicts that optimal sex allocation is influenced by local sperm competition, which occurs when related sperm compete to fertilize a given set of eggs. Different factors, including the mating strategy and the ability to self-fertilize, are predicted to affect local sperm competition and hence the optimal SA. Moreover, since the local sperm competition experienced by an individual can vary temporally and spatially, this can favour the evolution of sex allocation plasticity. Here, using seven species of the free-living flatworm genus Macrostomum, we document interspecific variation in sex allocation, but neither their mating strategy nor their ability to self-fertilize significantly predicted sex allocation among these species. Since we also found interspecific variation in sex allocation plasticity, we further estimated standardized effect sizes for plasticity in response to (i) the presence of mating partners (i.e. in isolation vs. with partners) and (ii) the strength of local sperm competition (i.e. in small vs. large groups). We found that self-fertilization predicted sex allocation plasticity with respect to the presence of mating partners, with plasticity being lower for self-fertilizing species. Finally, we showed that interspecific variation in sex allocation is higher than intraspecific variation due to sex allocation plasticity. Our study suggests that both sex allocation and sex allocation plasticity are evolutionarily labile, with self-fertilization predicting the latter in Macrostomum.
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Platelmintos , Animais , Fertilização , Masculino , Reprodução/fisiologia , Autofertilização , Espermatozoides/fisiologiaRESUMO
OBJECTIVE: To explore the differentials of postpartum contraceptive adoption between rural and urban poor after adjusting for utilization of MCH services and other selected socioeconomic and demographic covariates. METHODS: The data for this study is taken from the 4th round of NFHS survey conducted in India during 2015-16. The analysis is limited to 125,340 currently married women whose menses had returned at the time of survey. Discrete time complementary log-log multilevel model was applied. RESULTS: The results clearly indicate that women from rural areas had a lower chance of early initiation of modern spacing methods after having recent birth as compare to that of Urban Poor and Urban non-poor areas. The contributions of several socioeconomic and demographic characteristics that were important for family planning practice were also highlighted in this study. CONCLUSION: There is an urgent need of designing an intervention that will result in effective delivery of services to achieve the greatest impact. Policy planners must focus on targeted interventions for family planning use in the postpartum period than simply focusing on family planning.
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Serviços de Planejamento Familiar , Educação Sexual , Feminino , Humanos , População Urbana , População Rural , Período Pós-PartoRESUMO
Nitric oxide (NO) is a toxic gas, which is dangerous for human health and causes many respiratory infections, poisoning, and lung damage. In this work, we have successfully grown ZnO nanorod film on annealed ZnO seed layer in different ambient temperatures, and the morphology of the nanorods sensing layer that affects the gas sensing response to nitric oxide (NO) gas were investigated. To acknowledge the effect of annealing treatment, the devices were fabricated with annealed seed layers in air and argon ambient at 300 °C and 500 °C for 1 h. To simulate a vertical device structure, a silver nanowire electrode covered in ZnO nanorod film was placed onto the hydrothermal grown ZnO nanorod film. We found that annealing treatment changes the seed layer's grain size and defect concentration and is responsible for this phenomenon. The I-V and gas sensing characteristics were dependent on the oxygen defects concentration and porosity of nanorods to react with the target gas. The resulting as-deposited ZnO seed layer shows better sensing response than that annealed in an air and argon environment due to the nanorod morphology and variation in oxygen defect concentration. At room temperature, the devices show good sensing response to NO concentration of 10 ppb and up to 100 ppb. Shortly, these results can be beneficial in the NO breath detection for patients with chronic inflammatory airway disease, such as asthma.
Assuntos
Nanotubos , Óxido de Zinco , Eletrodos , Humanos , Óxido Nítrico , PrataRESUMO
BACKGROUND: Maternal deaths among young women (15-24 years) shares 38% of total maternal mortality in India. Utilizing maternal health care services can reduce a substantial proportion of maternal mortality. However, there is a paucity of studies focusing on young women in this context. This paper, therefore, aimed to examine the trends and determinants of full antenatal care (ANC) and skilled birth attendance (SBA) utilization among young married women in India. METHODS: The study analysed data from the four rounds of National Family Health Surveys conducted in India during the years 1992-93, 1998-99, 2005-06 and 2015-16. Young married women aged 15-24 years with at least one live birth in the 3 years preceding the survey were considered for analysis in each survey round. We used descriptive statistics to assess the prevalence and trends in full ANC and SBA use. Pooled multivariate logistic regression was conducted to identify the demographic and socioeconomic determinants of the selected maternity care services. The significance level for all analyses was set at p ≤ 0.05. RESULTS: The use of full ANC among young mothers increased from 27 to 46% in India, and from 9 to 28% in EAG (Empowered Action Group) states during 1992-2016. SBA utilization was 88 and 83% during 2015-16 by showing an increment of 20 and 50% since 1992 in India and EAG states, respectively. Findings from multivariate analysis revealed a significant difference in the use of selected maternal health care services by maternal age, residence, education, birth order and wealth quintile. Additionally, Muslim women, women belonging to scheduled caste (SC)/ scheduled tribe (ST) social group, and women unexposed to mass media were less likely to utilize both the maternal health care services. Concerning the time effect, the odds of the utilization of full ANC and SBA among young women was found to increase over time. CONCLUSIONS: In India coverage of full ANC among young mothers remained unacceptably low, with a wide and persistent gap in utilization between EAG and non-EAG states since 1992. Targeted health policies should be designed to address low coverage of ANC and SBA among underprivileged young mothers and increased efforts should be made to ensure effective implementation of ongoing programs, especially in EAG states.
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Serviços de Saúde Materna/estatística & dados numéricos , Mães , Aceitação pelo Paciente de Cuidados de Saúde/estatística & dados numéricos , Cuidado Pré-Natal/estatística & dados numéricos , Adolescente , Escolaridade , Feminino , Humanos , Índia , Casamento , Serviços de Saúde Materna/tendências , Gravidez , Cuidado Pré-Natal/tendências , Fatores Socioeconômicos , Adulto JovemRESUMO
PURPOSE: Comprehensive Primary Care Plus (CPC+) is the largest test of primary care payment and delivery reform. This program aims to strengthen primary care via enhanced and alternative payment, data feedback, learning, and health information technology support for practice transformation for more than 3,000 practices. We analyzed participation rates and how CPC+ practices differ from other primary care practices in CPC+ regions. METHODS: We assembled a unique data set describing all US primary care practices and compared primary care practices in CPC+ regions, CPC+ applicants, and CPC+ participants. Among CPC+ participants, we compared across 2 model tracks. RESULTS: Of the primary care practices in CPC+ regions, 22% applied for CPC+ and 15% participated. Practices that applied to CPC+ were diverse, but they were generally larger, more sophisticated electronic health record users, more likely to be owned by a hospital or health system, more likely to have experience with transformation efforts, and more likely to be in urban areas than practices that did not apply. Applicants also generally served slightly healthier and more advantaged Medicare fee-for-service beneficiaries. Differences between practices that applied but did not join CPC+ and CPC+ participants were smaller yet systematic. CONCLUSIONS: Participants in CPC+ are diverse but not representative of all primary care practices, underscoring the need to further engage practices that are small, independent, in rural areas, and lack experience with practice and payment transformation models, as well as the need to extrapolate evaluation results carefully.
Assuntos
Assistência Integral à Saúde/organização & administração , Inovação Organizacional , Adulto , Tomada de Decisões , Planos de Pagamento por Serviço Prestado , Feminino , Acessibilidade aos Serviços de Saúde , Humanos , Masculino , Medicare , Relações Médico-Paciente , Desenvolvimento de Programas , Estados UnidosRESUMO
BACKGROUND: Indian women are more prone to first birth at a relatively younger age after marriage. Also, we do not have sufficient literature available that focuses on contraceptive use before first birth. The analysis of the present study was done using data from the fourth round of National Family Health Survey (2015-16), India. The objectives of the present study were to measure the levels and trends of contraceptive use before first birth among Indian ever married women, aged 15-34 years. METHODS: The study includes 279,896 ever married women aged 15-34 years at the time of the NFHS-4 survey. To identify the socio-demographic determinants governing the pioneering study behavior, multivariable techniques have been used in the analysis. The statistical significance of the relationship between socio-demographic factors and contraceptive use prior to first birth was tested using a chi-squared test for association. Hosmer Lemeshow statistics and Nagelkerke R square have been used to check how well the logistic regression model fits the data. Map of India showing different zonal classification is made using the ArcGIS software version 10.3. RESULT: The trends of contraceptive usage show a decline in use before first birth and the various socio-demographic factors affecting the use of contraceptive before first birth are religion, caste, education, wealth index, media exposure, age at marriage and the zonal classifications. CONCLUSION: The noticeable result in this study is the comparative decline in contraceptive use by women in India before first birth in NFHS-4 with respect to previous NFHS done in India. The likelihood of using contraception before first birth is significantly affected by factors like place of residence, religion, caste, current age of women, age at marriage, education level of women, wealth index, media exposure and zonal classification.
Assuntos
Fatores Etários , Ordem de Nascimento/psicologia , Comportamento Contraceptivo/tendências , Anticoncepção/tendências , Casamento/psicologia , Adolescente , Adulto , Anticoncepção/psicologia , Comportamento Contraceptivo/psicologia , Estudos Transversais , Países em Desenvolvimento , Feminino , Humanos , Índia , Modelos Logísticos , Casamento/estatística & dados numéricos , Gravidez , Religião , Classe Social , Fatores Socioeconômicos , Adulto JovemRESUMO
We successfully demonstrated a simple and low-cost nitric oxide (NO) gas sensor to deliver parts-per-billion (ppb) regime detection at room temperature operation. A vertical-channel ZnO nanorods resistor is fabricated by putting silver nanowire electrode onto the hydrothermal ZnO nanorods film. With suitable process condition, the nanorods film exhibits a uniform morphology to enable the formation of gas-permeable nanowire top electrode while also the nanorods provide good surface-to-volume ratio to deliver strong reaction with NO gas. A detection limit to 10 ppb NO is observed while the sensing dynamic range from 10 ppb to 100 ppb is obtained. The sensor is reversible and the real-time sensing response is within 30 s. The results benefit the NO breath detection for patients with chronic inflammatory airway disease, such as asthma.
RESUMO
Measuring changes in protein or organelle abundance in the cell is an essential, but challenging aspect of cell biology. Frequently-used methods for determining organelle abundance typically rely on detection of a very few marker proteins, so are unsatisfactory. In silico estimates of protein abundances from publicly available protein spectra can provide useful standard abundance values but contain only data from tissue proteomes, and are not coupled to organelle localization data. A new protein abundance score, the normalized protein abundance scale (NPAS), expands on the number of scored proteins and the scoring accuracy of lower-abundance proteins in Arabidopsis. NPAS was combined with subcellular protein localization data, facilitating quantitative estimations of organelle abundance during routine experimental procedures. A suite of targeted proteomics markers for subcellular compartment markers was developed, enabling independent verification of in silico estimates for relative organelle abundance. Estimation of relative organelle abundance was found to be reproducible and consistent over a range of tissues and growth conditions. In silico abundance estimations and localization data have been combined into an online tool, multiple marker abundance profiling, available in the SUBA4 toolbox (http://suba.live).
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteoma , Proteômica , Biomarcadores/metabolismo , Organelas/metabolismo , Transporte ProteicoRESUMO
Mitochondria play important roles in multiple aspects of viral tumorigenesis. Mitochondrial genomes contribute to the host's genetic background. After viruses enter the cell, they modulate mitochondrial function and thus alter bioenergetics and retrograde signaling pathways. At the same time, mitochondria also regulate and mediate viral oncogenesis. In this context, oncogenesis by oncoviruses like Hepatitis B virus (HBV), Hepatitis C virus (HCV), Human papilloma virus (HPV), Human Immunodeficiency virus (HIV) and Epstein-Barr virus (EBV) will be discussed.
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Mitocôndrias/metabolismo , Neoplasias/patologia , Neoplasias/virologia , Retroviridae/metabolismo , Animais , Carcinogênese , Transformação Celular Neoplásica , Genoma Mitocondrial , HIV , Hepacivirus , Vírus da Hepatite B , Herpesvirus Humano 4 , Humanos , Invasividade Neoplásica , Metástase Neoplásica , Papillomaviridae , Transdução de SinaisAssuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Herpesvirus Humano 6/isolamento & purificação , Linfo-Histiocitose Hemofagocítica/patologia , Osteopetrose/patologia , Infecções por Roseolovirus/patologia , Imunodeficiência Combinada Severa/patologia , Humanos , Recém-Nascido , Linfo-Histiocitose Hemofagocítica/complicações , Linfo-Histiocitose Hemofagocítica/tratamento farmacológico , Masculino , Osteopetrose/complicações , Osteopetrose/tratamento farmacológico , Prognóstico , Infecções por Roseolovirus/complicações , Infecções por Roseolovirus/tratamento farmacológico , Imunodeficiência Combinada Severa/complicações , Imunodeficiência Combinada Severa/tratamento farmacológicoRESUMO
Unicornuate uterus with rudimentary horn occurs due to failure of complete development and partial fusion of one of the Müllerian ducts. Pregnancy in a non-communicating rudimentary horn is extremely rare, with a reported incidence of 1 in 76 000-150 000 pregnancies, and usually terminates in rupture during the first or second trimester. Clinical diagnosis of rudimentary horn pregnancy in a woman with history of normal vaginal delivery in prior gestations is difficult. The role of sonography, and more recently, magnetic resonance imaging, in the presurgical diagnosis of rudimentary horn pregnancy is established. We present a case of magnetic resonance imaging diagnosis of 20-week pregnancy in the unruptured non-communicating rudimentary horn in a patient with previous history of two full-term normal vaginal deliveries. The novelty of the case lies in the fact that there was associated torsion of the gravid rudimentary horn and ipsilateral ovary, which has not been reported previously.
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Doenças Ovarianas/diagnóstico por imagem , Anormalidade Torcional/diagnóstico por imagem , Anormalidades Urogenitais/diagnóstico por imagem , Útero/anormalidades , Adulto , Feminino , Humanos , Imageamento por Ressonância Magnética , Gravidez , Segundo Trimestre da Gravidez , Útero/diagnóstico por imagemRESUMO
In Ethiopia many women do not practice appropriate complementary feeding (CF). The Health Belief Model (HBM) asserts that change in behavior is determined after consideration of severity, benefit, and barriers to change. This study examined the effectiveness of 3 months of HBM-based education compared to the traditional (didactic) method on CF practices of mothers, with no education as control, using three randomized groups. One hundred sixty-six mother-infant (6-18 months) pairs were recruited. At baseline and after intervention, knowledge, perceptions, and practices about CF and related areas were determined. It was only diet diversity that increased significantly in the HBM group (from 3.05±0.94 food groups to 3.79±0.82, p<.05) while the other two groups had no change. Improvements in food groups were most noticeable as legumes & nuts (from 35.6% use to 83.9% in HBM group). Thus, nutrition education about diet diversity improvement needs to be conducted promotes behavior change.
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Dieta , Comportamento Alimentar , Educação em Saúde/métodos , Conhecimentos, Atitudes e Prática em Saúde , Fenômenos Fisiológicos da Nutrição do Lactente , Modelos Educacionais , Mães , Adulto , Aleitamento Materno , Cultura , Etiópia , Fabaceae , Feminino , Humanos , Lactente , Masculino , Estado Nutricional , Nozes , DesmameRESUMO
The type VI secretion system (T6SS) has emerged as a critical virulence factor for the group of closely related Burkholderia spp. that includes Burkholderia pseudomallei, B. mallei, and B. thailandensis. While the genomes of these bacteria, referred to as the Bptm group, appear to encode several T6SSs, we and others have shown that one of these, type VI secretion system 5 (T6SS-5), is required for virulence in mammalian infection models. Despite its pivotal role in the pathogenesis of the Bptm group, the effector repertoire of T6SS-5 has remained elusive. Here we used quantitative mass spectrometry to compare the secretome of wild-type B. thailandensis to that of a mutant harboring a nonfunctional T6SS-5. This analysis identified VgrG-5 as a novel secreted protein whose export depends on T6SS-5 function. Bioinformatics analysis revealed that VgrG-5 is a specialized VgrG protein that harbors a C-terminal domain (CTD) conserved among Bptm group species. We found that a vgrG-5 ΔCTD mutant is avirulent in mice and is unable to stimulate the fusion of host cells, a hallmark of the Bptm group previously shown to require T6SS-5 function. The singularity of VgrG-5 as a detected T6SS-5 substrate, taken together with the essentiality of its CTD for virulence, suggests that the protein is critical for the effector activity of T6SS-5. Intriguingly, we show that unlike the bacterial-cell-targeting T6SSs characterized so far, T6SS-5 localizes to the bacterial cell pole. We propose a model whereby the CTD of VgrG-5-, propelled by T6SS-5-, plays a key role in inducing membrane fusion, either by the recruitment of other factors or by direct participation.
Assuntos
Sistemas de Secreção Bacterianos/fisiologia , Burkholderia/patogenicidade , Células Gigantes/fisiologia , Animais , Western Blotting , Burkholderia/metabolismo , Células Cultivadas , Células Gigantes/metabolismo , Interações Hospedeiro-Parasita/fisiologia , Macrófagos/metabolismo , Espectrometria de Massas , Fusão de Membrana/fisiologia , Camundongos , Microscopia de Fluorescência , Virulência/genética , Virulência/fisiologia , Fatores de Virulência/metabolismoRESUMO
Transformation of engineered Escherichia coli into a robust microbial factory is contingent on precise control of metabolism. Yet, the throughput of omics technologies used to characterize cell components has lagged far behind our ability to engineer novel strains. To expand the utility of quantitative proteomics for metabolic engineering, we validated and optimized targeted proteomics methods for over 400 proteins from more than 20 major pathways in E. coli metabolism. Complementing these methods, we constructed a series of synthetic genes to produce concatenated peptides (QconCAT) for absolute quantification of the proteins and made them available through the Addgene plasmid repository (www.addgene.org). To facilitate high sample throughput, we developed a fast, analytical-flow chromatography method using a 5.5-min gradient (10 min total run time). Overall this toolkit provides an invaluable resource for metabolic engineering by increasing sample throughput, minimizing development time and providing peptide standards for absolute quantification of E. coli proteins.
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Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Perfilação da Expressão Gênica/métodos , Ensaios de Triagem em Larga Escala/métodos , Peptídeos/metabolismo , Engenharia de Proteínas/métodos , Peptídeos/genética , Mapeamento de Interação de Proteínas/métodos , Proteômica/métodosRESUMO
The interplay of the three-dimensional (3D) distribution of various subcellular components and their interactions are expected to control overall cellular morphology in biology. In this study, we aimed to determine whether the pleomorphy observed at the whole-cell level is being reflected by the components constituting the cells by focusing on the 3D distribution of pixel intensities at the single-cell level of the whole (cell) and its parts (the seven subcellular components of the cells-self-assemblies of smaller units). We rigorously acquired and analyzed the image data of RAW264.7 cells at the single-cell level. We report asymmetries in the spatial distribution of pixel intensities at the whole-cell and subcellular component levels along with the occurrence of alterations when pleomorphism is reduced by synchronization of the cell cycle. From our repertoire of seven subcellular components, we report ER, mitochondria, and tubulin to be independent of whole-cell apico-basal heterogeneity of optical density while nuclear, plasma membrane, lysosomal, and actin fluorescence distributions are found to contribute to the apico-basal polarity of the whole cell. While doing so, we have also developed an image analysis algorithm utilizing 2D segmentation to analyze the single cells in 3D using confocal microscopy, a technique that allows us to analyze cellular states in their native hydrated state.