Complete assembly, annotation of virulence genes and CRISPR editing of the genome of Leishmania amazonensis PH8 strain.

We report the sequencing and assembly of the PH8 strain of Leishmania amazonensis one of the etiological agents of leishmaniasis. After combining data from long Pacbio reads, short Illumina reads and synteny with the Leishmania mexicana genome, the sequence of 34 chromosomes with 8317 annotated genes was generated. Multigene families encoding three virulence factors, A2, amastins and the GP63 metalloproteases, were identified and compared to their annotation in other Leishmania species. As they have been recently recognized as virulence factors essential for disease establishment and progression of the infection, we also identified 14 genes encoding proteins involved in parasite iron and heme metabolism and compared to genes from other Trypanosomatids. To follow these studies with a genetic approach to address the role of virulence factors, we tested two CRISPR-Cas9 protocols to generate L. amazonensis knockout cell lines, using the Miltefosine transporter gene as a proof of concept.

Impact of Uncertainty Parameter Distribution on Robust Decision Making Outcomes for Climate Change Adaptation under Deep Uncertainty.

Deep uncertainty in future climatic and economic conditions complicates developing infrastructure designed to last several generations, such as water reservoirs. In response, analysts have developed multiple robust decision frameworks to help identify investments and policies that can withstand a wide range of future states. Although these frameworks are adept at supporting decisions where uncertainty cannot be represented probabilistically, analysts necessarily choose probabilistic bounds and distributions for uncertain variables to support exploratory modeling. The implications of these assumptions on the analytical outcomes of robust decision frameworks are rarely evaluated, and little guidance exists in terms of how to select uncertain variable distributions. Here, we evaluate the impact of these choices by following the robust decision-making procedure, using four different assumptions about the probabilistic distribution of exogenous uncertainties in future climatic and economic states. We take a water reservoir system in Ethiopia as our case study, and sample climatic parameters from uniform, normal, extended uniform, and extended normal distributions; we similarly sample two economic parameters. We compute regret and satisficing robustness decision criteria for two performance measures, agricultural water demand coverage and net present value, and perform scenario discovery on the most robust reservoir alternative. We find lower robustness scores resulting from extended parameter distributions and demonstrate that parameter distributions can impact vulnerabilities identified through scenario discovery. Our results suggest that exploratory modeling within robust decision frameworks should sample from extended, uniform parameters distributions.

Retrospective Parameter Estimation and Forecast of Respiratory Syncytial Virus in the United States.

Recent studies have shown that systems combining mathematical modeling and Bayesian inference methods can be used to generate real-time forecasts of future infectious disease incidence. Here we develop such a system to study and forecast respiratory syncytial virus (RSV). RSV is the most common cause of acute lower respiratory infection and bronchiolitis. Advanced warning of the epidemic timing and volume of RSV patient surges has the potential to reduce well-documented delays of treatment in emergency departments. We use a susceptible-infectious-recovered (SIR) model in conjunction with an ensemble adjustment Kalman filter (EAKF) and ten years of regional U.S. specimen data provided by the Centers for Disease Control and Prevention. The data and EAKF are used to optimize the SIR model and i) estimate critical epidemiological parameters over the course of each outbreak and ii) generate retrospective forecasts. The basic reproductive number, R0, is estimated at 3.0 (standard deviation 0.6) across all seasons and locations. The peak magnitude of RSV outbreaks is forecast with nearly 70% accuracy (i.e. nearly 70% of forecasts within 25% of the actual peak), four weeks before the predicted peak. This work represents a first step in the development of a real-time RSV prediction system.

Cost Savings and Effectiveness of Long-Acting Reversible Contraception (LARC) on the Prevention of Pregnancy in Adolescents: A Systematic Review.

Adolescent pregnancy is a major public health issue with profound implications for health and socioeconomic factors. The use of long-acting reversible contraception (LARC) could be an interesting strategy to reduce the unintended pregnancy rate. However, the cost of LARC is still a barrier to widespread adoption. This study aimed to analyze the effectiveness and economic impact of LARC compared with non-LARC methods in preventing unintended pregnancy among adolescent girls. This systematic review was registered in PROSPERO (CRD42023387735) and conducted following the PRISMA guidelines. We included articles covering adolescents aged 10-19 years without language restrictions that evaluated the use of LARC compared with non-LARC in terms of effectiveness and the public health costs of unintended pregnancy. The search for articles included the databases MEDLINE/PubMed, Cochrane Library, Embase, and Lilacs, using the entry terms "Adolescent" and "Long-Acting Reversible Contraception." We evaluated the risk of bias and the certainty of the evidence for each outcome of interest. The search retrieved a total of 1,169 articles and, after the title and abstract, we identified 40 articles for full-text analysis. Out of the 40 studies evaluated, 4 articles met the eligibility criteria for cost evaluation, and 1 met the eligibility criteria for effectiveness as an outcome. In conclusion, LARC emerges as the most effective and cost-effective contraceptive method. The cost of utilizing LARC, especially the copper IUD, is significantly lower than the costs attributable to unintended pregnancies in adolescence.

Proteasome protease mediated regulation of cytokine induction and inflammation.

We have previously demonstrated that proteasome serves as a central regulator of inflammation and macrophage function. Until recently, proteasomes have generally been considered to play a relatively passive role in the regulation of cellular activity, i.e., any ubiquitinated protein was considered to be in discriminatively targeted for degradation by the proteasome. We have demonstrated, however, by using specific proteasome protease inhibitors and knockout mice lacking specific components of immunoproteasomes, that proteasomes (containing X, Y, and Z protease subunits) and immunoproteasomes (containing LMP7, LMP2, and LMP10 protease subunits) have well-defined functions in cytokine induction and inflammation based on their individual protease activities. We have also shown that LPS-TLR mediated signaling in the murine RAW 264.7 macrophage cell line results in the replacement of macrophage immunoproteasomal subunits. Such modifications serve as pivotal regulators of LPS-induced inflammation. Our findings support the relatively novel concept that defects in structure/function of proteasome protease subunits caused by genetic disorders, aging, diet, or drugs may well have the potential to contribute to modulation of proteasome activity. Of particular relevance, we have identified quercetin and resveratrol, significant constituents present in berries and in red wine respectively, as two novel proteasome inhibitors that have been previously implicated as disease-modifying natural products. We posit that natural proteasome inhibitors/activators can potentially be used as therapeutic response modifiers to prevent/treat diseases through pathways involving the ubiquitin-proteasome pathway (UP-pathway), which likely functions as a master regulator involved in control of overall inflammatory responses. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.

Comparison of automated methods for measurement of 25-hydroxyvitamin D.

BACKGROUND: The objective of the study was to compare the analytical performance of three automated immunoassays for the determination of serum 25-hydroxyvitamin D. METHODS: Quantitative determination of serum 25(OH)D were performed by "Vitamin D total" (n = 131) and "Vitamin D3(25-OH)" (n = 77) assays (Roche Diagnostics) on a Cobas e411 and by "25-OH Vitamin D" (n = 131) assay (Abbott Laboratories) on an Architect. The inter-assay precision was calculated and methods were compared by the Passing Bablok regression and Bland-Altman analysis. RESULTS: The "Vitamin D total" demonstrated stronger correlation (r = 0.863) and better agreement (bias = -7.89 nmol/L) with the "25-OH Vitamin D" than with the "Vitamin D3(25-OH)" (r = 0.716; bias = +18.6 nmol/L). The inter-assay precision (% CV) for the "Vitamin D total" and "25-OH Vitamin D" assays, were respectively 3.47 to 6.14 and 4.27 to 8.56. CONCLUSIONS: The results of this study demonstrate that Abbott "25-OH Vitamin D" and Roche "Vitamin D total" are rapid and precise methods for 25(OH)D serum measurement over a wide reportable range on automated immunoassay platforms. The Abbott assay exhibited better correlation and agreement with the new Roche assay than the "Vitamin D3(25-OH)" Roche assay. Between method differences observed in this study still indicates the need for standardization of 25(OH)D assays.

Postural control and functional strength in patients with type 2 diabetes mellitus with and without peripheral neuropathy.

OBJECTIVE: To assess the influence of diabetic neuropathy (DN) on balance and functional strength in patients with diabetes mellitus type 2 (DM2). DESIGN: Cross-sectional study. SETTING: Diabetes outpatient unit. PARTICIPANTS: Adults (N=62; age range, 40-65y): 32 with DM2 (19 subjects without DN and 13 with DN) and 30 without DM2 (control group). INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Upright balance, evaluated in 4 situations (fixed platform, unstable platform, with eyes open, with eyes closed), and functional strength, assessed with a five-times-sit-to-stand test, were analyzed using an electromagnetic system, with a sensor placed over C7 to allow maximum trunk displacements in the anterior-posterior and medial-lateral directions. The Berg Balance Scale and the Timed Up & Go test were also used. RESULTS: Subjects with DM2 had greater anterior-posterior displacement (P<.05) in the unstable platform with eyes closed condition compared with those without DM2, whereas no difference in medial-lateral displacement was observed between these groups. A difference in time was observed in the five-times-sit-to-stand test (P<.05), with subjects in the control group performing the tasks faster than either group of subjects with DM2. Additionally, subjects in the control group showed a higher score in the Berg Balance Scale and performed the Timed Up & Go test in less time compared with subjects in other groups. CONCLUSIONS: Subjects with DM2, with or without DN, showed deficits in postural control and functional strength compared with healthy individuals of the same age group.

Inhibition of nitric oxide and inflammatory cytokines in LPS-stimulated murine macrophages by resveratrol, a potent proteasome inhibitor.

BACKGROUND: Altered immune function during ageing results in increased production of nitric oxide (NO) and other inflammatory mediators. Recently, we have reported that NO production was inhibited by naturally-occurring proteasome inhibitors (quercetin, δ-tocotrienol, and riboflavin) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells, and thioglycolate-elicited peritoneal macrophages from C57BL/6 mice. In a continuous effort to find more potent, non-toxic, commercially available, naturally-occurring proteasome inhibitors that suppress inflammation, the present study was carried out to describe the inhibition of NF-κB activation and NO, TNF-α, IL-6, IL-1ß, and iNOS expression by trans-resveratrol, trans-pterostilbene, morin hydrate, and nicotinic acid in LPS-induced RAW 264.7 cells and thioglycolate-elicited peritoneal macrophages from C57BL/6 and BALB/c mice. RESULTS: The present results indicate that resveratrol, pterostilbene, and morin hydrate caused significant inhibition (>70% to 90%; P < 0.02) in the activities of chymotrypsin-like, trypsin-like, and post-acidic (post-glutamase) proteasome sites in RAW 264.7 cells at a dose of only 20 µM. These compounds also inhibited the production of NO by RAW-264.7 cells stimulated with LPS alone (>40%; P < 0.05), or LPS + interferon-γ (IFN-γ; >60%; P < 0.02). Furthermore, resveratrol, pterostilbene, morin hydrate, and quercetin suppressed secretion of TNF-α (>40%; P < 0.05) in LPS-stimulated RAW 264.7 cells, and suppressed NF-κB activation (22% to 45%; P < 0.05) in LPS-stimulated HEK293T cells. These compounds also significantly suppressed LPS-induced expression of TNF-α, IL-1ß, IL-6, and iNOS genes in RAW 264.7 cells, and also in thioglycolate-elicited peritoneal macrophages from C57BL/6 and BALB/c mice. CONCLUSIONS: The present results clearly demonstrate that resveratrol and pterostilbene are particularly potent proteasome inhibitors that suppress expression of genes, and production of inflammatory products in LPS-stimulated RAW 264.7 cells, and macrophages from C57BL/6 and BALB/c mice. Resveratrol and pterostilbene which are present in grapes, blueberries, and red wine, have been implicated as contributing factors to the lower incidence of cardiovascular disease in the French population, despite their relatively high dietary fat intake. Consequently, it appears likely that the beneficial nutritional effects of resveratrol and pterostilbene are due at least in part, to their ability to inhibit NF-κB activation by the proteasome, thereby suppressing activation of pro-inflammatory cytokines and iNOS genes, resulting in decreased secretion of TNF-α, IL-1ß, IL-6, and NO levels, in response to inflammatory stimuli. This is the first report demonstrating that resveratrol and pterostilbene act as proteasome inhibitors, thus providing a mechanism for their anti-inflammatory effects.

Suicide mortality among older adults in Brazil between 2000 and 2019 - estimates from the Global Burden of Disease Study 2019.

INTRODUCTION: Older adults present a higher risk of suicide, and Brazil is experiencing a fast population aging. To understand the impact of demographic transition, we compared Brazilian suicide mortality rates (MR) among adults (50+ years) with global rates, those from one high-income country, and those from one middle-income country. Looking for regional disparities, the MR was analyzed among older adults (60+ years) by Brazilian states. METHODS: This was an ecological study based on estimates from the Global Burden of Disease Study, from 2000 to 2019. Age-standardized MR and age-specific MR per 100,000 inhabitants were described, with 95% uncertainty intervals (UI). RESULTS: During the period, the annual estimates and the declining trend in mortality were higher in the world than in the studied countries. In 2019, global age-standardized MR was 9.39 (95% UI 8.48-10.29), compared to 5.68 (95% UI 5.40-6.19), 6.01 (95% UI 5.10-7.04), and 6.63 (95% UI 6.43-6.95) in Brazil, Mexico, and England, respectively. In Brazil, despite a significant decline in national rates, stability was observed in 15 states. An increase in aging was only found for men, who presented 3-4 times higher MR than women. The states' rates presented large differences: in 2019, the rates among men aged 60-64 years varied from 7.24 (95% UI 5.31; 9.85) to 26.32 (95% UI 20.21; 34.50). CONCLUSIONS: The smaller decline in suicide mortality among older Brazilian adults, the increasing risk with aging, and the higher mortality among men indicate the need for specific prevention policies. The variation within states suggests differences in the data quality or in socio-cultural and historical aspects, which requires further investigation.

Sodium butyrate-loaded nanoparticles coated with chitosan for the treatment of neovascularization in age-related macular degeneration: Ocular biocompatibility and antiangiogenic activity.

Sodium butyrate-loaded nanoparticles coated chitosan (NaBu-loaded nanoparticles/CS) were developed to treat the choroidal neovascularization in wet age-related macular degeneration (AMD). The nanoparticles were produced by double emulsification and solvent evaporation technique, optimized by experimental statistical design, characterized by analytical methods, investigated in terms of in vitro and in vivo ocular biocompatibility, and evaluated as an antiangiogenic system in vivo. The NaBu-loaded nanoparticles/CS were 311.1 ± 3.1 nm in diameter with a 0.208 ± 0.007 polydispersity index; had a +56.3 ± 2.6 mV zeta potential; showed a 92.3 % NaBu encapsulation efficiency; and sustained the drug release over 35 days. The NaBu-loaded nanoparticles/CS showed no toxicity to human retinal pigment epithelium cells (ARPE-19 cells); was not irritant to the chorioallantoic membrane (CAM); did not interfere in the integrity of the retinal layers of rat's eyes, as detected by the Optical Coherence Tomography and histopathology; and inhibited the angiogenesis in CAM assay. The NaBu-loaded nanoparticles/CS could be a therapeutic alternative to limit the neovascularization in AMD.

Nitazoxanide in Patients Hospitalized With COVID-19 Pneumonia: A Multicentre, Randomized, Double-Blind, Placebo-Controlled Trial.

Background: Nitazoxanide exerts antiviral activity in vitro and in vivo and anti-inflammatory effects, but its impact on patients hospitalized with COVID-19 pneumonia is uncertain. Methods: A multicentre, randomized, double-blind, placebo-controlled trial was conducted in 19 hospitals in Brazil. Hospitalized adult patients requiring supplemental oxygen, with COVID-19 symptoms and a chest computed tomography scan suggestive of viral pneumonia or positive RT-PCR test for COVID-19 were enrolled. Patients were randomized 1:1 to receive nitazoxanide (500 mg) or placebo, 3 times daily, for 5 days, and were followed for 14 days. The primary outcome was intensive care unit admission due to the need for invasive mechanical ventilation. Secondary outcomes included clinical improvement, hospital discharge, oxygen requirements, death, and adverse events within 14 days. Results: Of the 498 patients, 405 (202 in the nitazoxanide group and 203 in the placebo group) were included in the analyses. Admission to the intensive care unit did not differ between the groups (hazard ratio [95% confidence interval], 0.68 [0.38-1.20], p = 0.179); death rates also did not differ. Nitazoxanide improved the clinical outcome (2.75 [2.21-3.43], p < 0.0001), time to hospital discharge (1.37 [1.11-1.71], p = 0.005), and reduced oxygen requirements (0.77 [0.64-0.94], p = 0.011). C-reactive protein, D-dimer, and ferritin levels were lower in the nitazoxanide group than the placebo group on day 7. No serious adverse events were observed. Conclusions: Nitazoxanide, compared with placebo, did not prevent admission to the intensive care unit for patients hospitalized with COVID-19 pneumonia. Clinical Trial Registration: Brazilian Registry of Clinical Trials (REBEC) RBR88bs9x; ClinicalTrials.gov, NCT04561219.

δ-Tocotrienol and quercetin reduce serum levels of nitric oxide and lipid parameters in female chickens.

BACKGROUND: Chronic, low-grade inflammation provides a link between normal ageing and the pathogenesis of age-related diseases. A series of in vitro tests confirmed the strong anti-inflammatory activities of known inhibitors of NF-κB activation (δ-tocotrienol, quercetin, riboflavin, (-) Corey lactone, amiloride, and dexamethasone). δ-Tocotrienol also suppresses ß-hydroxy-ß-methylglutaryl coenzyme A (HMG-CoA) reductase activity (the rate-limiting step in de novo cholesterol synthesis), and concomitantly lowers serum total and LDL cholesterol levels. We evaluated these compounds in an avian model anticipating that a dietary additive combining δ-tocotrienol with quercetin, riboflavin, (-) Corey lactone, amiloride, or dexamethasone would yield greater reductions in serum levels of total cholesterol, LDL-cholesterol and inflammatory markers (tumor necrosis factor-α [TNF-α], and nitric oxide [NO]), than that attained with the individual compounds. RESULTS: The present results showed that supplementation of control diets with all compounds tested except riboflavin, (-) Corey lactone, and dexamethasone produced small but significant reductions in body weight gains as compared to control. (-) Corey lactone and riboflavin did not significantly impact body weight gains. Dexamethasone significantly and markedly reduced weight gain (>75%) compared to control. The serum levels of TNF-α and NO were decreased 61% - 84% (P < 0.001), and 14% - 67%, respectively, in chickens fed diets supplemented with δ-tocotrienol, quercetin, riboflavin, (-) Corey lactone, amiloride, or dexamethasone as compared to controls. Significant decreases in the levels of serum total and LDL-cholesterol were attained with δ-tocotrienol, quercetin, riboflavin and (-) Corey lactone (13% - 57%; P < 0.05), whereas, these levels were 2-fold higher in dexamethasone treated chickens as compared to controls. Parallel responses on hepatic lipid infiltration were confirmed by histological analyses. Treatments combining δ-tocotrienol with the other compounds yielded values that were lower than individual values attained with either δ-tocotrienol or the second compound. Exceptions were the significantly lower total and LDL cholesterol and triglyceride values attained with the δ-tocotrienol/(-) Corey lactone treatment and the significantly lower triglyceride value attained with the δ-tocotrienol/riboflavin treatment. δ-Tocotrienol attenuated the lipid-elevating impact of dexamethasone and potentiated the triglyceride lowering impact of riboflavin. Microarray analyses of liver samples identified 62 genes whose expressions were either up-regulated or down-regulated by all compounds suggesting common impact on serum TNF-α and NO levels. The microarray analyses further identified 41 genes whose expression was differentially impacted by the compounds shown to lower serum lipid levels and dexamethasone, associated with markedly elevated serum lipids. CONCLUSIONS: This is the first report describing the anti-inflammatory effects of δ-tocotrienol, quercetin, riboflavin, (-) Corey lactone, amiloride, and dexamethasone on serum TNF-δ and NO levels. Serum TNF-δ levels were decreased by >60% by each of the experimental compounds. Additionally, all the treatments except with dexamethasone, resulted in lower serum total cholesterol, LDL-cholesterol and triglyceride levels. The impact of above mentioned compounds on the factors evaluated herein was increased when combined with δ-tocotrienol.

Suppression of nitric oxide induction and pro-inflammatory cytokines by novel proteasome inhibitors in various experimental models.

BACKGROUND: Inflammation has been implicated in a variety of diseases associated with ageing, including cancer, cardiovascular, and neurologic diseases. We have recently established that the proteasome is a pivotal regulator of inflammation, which modulates the induction of inflammatory mediators such as TNF-α, IL-1, IL-6, and nitric oxide (NO) in response to a variety of stimuli. The present study was undertaken to identify non-toxic proteasome inhibitors with the expectation that these compounds could potentially suppress the production of inflammatory mediators in ageing humans, thereby decreasing the risk of developing ageing related diseases. We evaluated the capacity of various proteasome inhibitors to suppress TNF-α, NO and gene suppression of TNF-α, and iNOS mRNA, by LPS-stimulated macrophages from several sources. Further, we evaluated the mechanisms by which these agents suppress secretion of TNF-α, and NO production. Over the course of these studies, we measured the effects of various proteasome inhibitors on the RAW 264.7 cells, and peritoneal macrophages from four different strains of mice (C57BL/6, BALB/c, proteasome double subunits knockout LMP7/MECL-1-/-, and peroxisome proliferator-activated receptor-α,-/- (PPAR-α,-/-) knockout mice. We also directly measured the effect of these proteasome inhibitors on proteolytic activity of 20S rabbit muscle proteasomes. RESULTS: There was significant reduction of chymotrypsin-like activity of the 20S rabbit muscle proteasomes with dexamethasone (31%), mevinolin (19%), δ-tocotrienol (28%), riboflavin (34%), and quercetin (45%; P < 0.05). Moreover, quercetin, riboflavin, and δ-tocotrienol also inhibited chymotrypsin-like, trypsin-like and post-glutamase activities in RAW 264.7 whole cells. These compounds also inhibited LPS-stimulated NO production and TNF-α, secretion, blocked the degradation of P-IκB protein, and decreased activation of NF-κB, in RAW 264.7 cells. All proteasome inhibitors tested also significantly inhibited NO production (30% to 60% reduction) by LPS-induced thioglycolate-elicited peritoneal macrophages derived from all four strains of mice. All five compounds also suppressed LPS-induced TNF-α, secretion by macrophages from C57BL/6 and BALB/c mice. TNF-α, secretion, however, was not suppressed by any of the three proteasome inhibitors tested (δ-tocotrienol, riboflavin, and quercetin) with LPS-induced macrophages from LMP7/MECL-1-/- and PPAR-α,-/- knockout mice. Results of gene expression studies for TNF-α, and iNOS were generally consistent with results obtained for TNF-α, protein and NO production observed with four strains of mice. CONCLUSIONS: Results of the current study demonstrate that δ-tocotrienol, riboflavin, and quercetin inhibit NO production by LPS-stimulated macrophages of all four strains of mice, and TNF-α, secretion only by LPS-stimulated macrophages of C57BL/6 and BALB/c mice. The mechanism for this inhibition appears to be decreased proteolytic degradation of P-IκB protein by the inhibited proteasome, resulting in decreased translocation of activated NF-κB to the nucleus, and depressed transcription of gene expression of TNF-α, and iNOS. Further, these naturally-occurring proteasome inhibitors tested appear to be relatively potent inhibitors of multiple proteasome subunits in inflammatory proteasomes. Consequently, these agents could potentially suppress the production of inflammatory mediators in ageing humans, thereby decreasing the risk of developing a variety of ageing related diseases.

Inhibition of nitric oxide in LPS-stimulated macrophages of young and senescent mice by δ-tocotrienol and quercetin.

BACKGROUND: Changes in immune function believed to contribute to a variety of age-related diseases have been associated with increased production of nitric oxide (NO). We have recently reported that proteasome inhibitors (dexamethasone, mevinolin, quercetin, δ-tocotrienol, and riboflavin) can inhibit lipopolysaccharide (LPS)-induced NO production in vitro by RAW 264.7 cells and by thioglycolate-elicited peritoneal macrophages derived from four strains of mice (C57BL/6, BALB/c, LMP7/MECL-1(-/-) and PPAR-α(-/-) knockout mice). The present study was carried out in order to further explore the potential effects of diet supplementation with naturally-occurring inhibitors (δ-tocotrienol and quercetin) on LPS-stimulated production of NO, TNF-α, and other pro-inflammatory cytokines involved in the ageing process. Young (4-week-old) and senescent mice (42-week old) were fed control diet with or without quercetin (100 ppm), δ-tocotrienol (100 ppm), or dexamethasone (10 ppm; included as positive control for suppression of inflammation) for 4 weeks. At the end of feeding period, thioglycolate-elicited peritoneal macrophages were collected, stimulated with LPS, LPS plus interferon-ß (IFN-ß), or LPS plus interferon-γ (IFN-γ), and inflammatory responses assessed as measured by production of NO and TNF-α, mRNA reduction for TNF-α, and iNOS genes, and microarray analysis. RESULTS: Thioglycolate-elicited peritoneal macrophages prepared after four weeks of feeding, and then challenged with LPS (10 ng or 100 ng) resulted in increases of 55% and 73%, respectively in the production of NO of 46-week-old compared to 8-week-old mice fed control diet alone (respective control groups), without affecting the secretion of TNF-α among these two groups. However, macrophages obtained after feeding with quercetin, δ-tocotrienol, and dexamethasone significantly inhibited (30% to 60%; P < 0.02) the LPS-stimulated NO production, compared to respective control groups. There was a 2-fold increase in the production of NO, when LPS-stimulated macrophages of quercetin, δ-tocotrienol, or dexamethasone were also treated with IFN-ß or IFN-γ compared to respective control groups. We also demonstrated that NO levels and iNOS mRNA expression levels were significantly higher in LPS-stimulated macrophages from senescent (0.69 vs 0.41; P < 0.05), compared to young mice. In contrast, age did not appear to impact levels of TNF-α protein or mRNA expression levels (0.38 vs 0.35) in LPS-stimulated macrophages. The histological analyses of livers of control groups showed lesions of peliosis and microvesicular steatosis, and treated groups showed Councilman body, and small or large lymphoplasmacytic clusters. CONCLUSIONS: The present results demonstrated that quercetin and δ-tocotrienols inhibit the LPS-induced NO production in vivo. The microarray DNA analyses, followed by pathway analyses indicated that quercetin or δ-tocotrienol inhibit several LPS-induced expression of several ageing and pro-inflammatory genes (IL-1ß, IL-1α, IL-6, TNF-α, IL-12, iNOS, VCAM1, ICAM1, COX2, IL-1RA, TRAF1 and CD40). The NF-κB pathway regulates the production of NO and inhibits the pro-inflammatory cytokines involved in normal and ageing process. These ex vivo results confirmed the earlier in vitro findings. The present findings of inhibition of NO production by quercetin and δ-tocotrienol may be of clinical significance treating several inflammatory diseases, including ageing process.

Tocotrienols inhibit lipopolysaccharide-induced pro-inflammatory cytokines in macrophages of female mice.

BACKGROUND: Inflammation has been implicated in cardiovascular disease, and the important role of proteasomes in the development of inflammation and other macrophage functions has been demonstrated. Tocotrienols are potent hypocholesterolemic agents that inhibit ß-hydroxy-ß-methylglutaryl coenzyme A reductase activity, which is degraded via the ubiquitin-proteasome pathway. Our objective was to evaluate the effect of tocotrienols in reducing inflammation. Lipopolysaccharide (LPS) was used as a prototype for inflammation in murine RAW 264.7 cells and BALB/c female mice. RESULTS: The present results clearly demonstrate that α-, γ-, or δ-tocotrienol treatments inhibit the chymotrypsin-like activity of 20 S rabbit muscle proteasomes (> 50%; P < 0.05). Chymotrypsin, trypsin, and post-glutamase activities were decreased > 40% (P < 0.05) with low concentrations (< 80 µM), and then increased gradually with concentrations of (80--640 µM) in RAW 264.7 whole cells. Tocotrienols showed 9--33% (P < 0.05) inhibitions in TNF-α secretion in LPS-stimulated RAW 264.7 cells. Results of experiments carried out in BALB/c mice demonstrated that serum levels of TNF-α after LPS treatment were also reduced (20--48%; P < 0.05) by tocotrienols with doses of 1 and 10 µg/kg, and a corresponding rise in serum levels of corticosterone (19--41%; P < 0.05) and adrenocorticotropic hormone (81--145%; P < 0.02) was observed at higher concentrations (40 µM). Maximal inhibition of LPS-induced TNF-α was obtained with δ-tocotrienol (10 µg/kg). Low concentrations of δ-Tocotrienols (< 20 µM) blocked LPS-induced gene expression of TNF-α, IL-1ß, IL-6 and iNOS (> 40%), while higher concentrations (40 µM) increased gene expression of the latter in peritoneal macrophages (prepared from BALB/c mice) as compared to control group. CONCLUSIONS: These results represent a novel approach by using natural products, such as tocotrienols as proteasome modulators, which may lead to the development of new dietary supplements of tocotrienols for cardiovascular diseases, as well as others that are based on inflammation.

Mothers as facilitators for a parent group intervention for children with Congenital Zika Syndrome: Qualitative findings from a feasibility study in Brazil.

BACKGROUND: The Zika virus outbreak in Brazil (2015-2016) affected thousands of children who were born with Congenital Zika Syndrome (CZS). Families play an important role in their care of children with complex needs, yet their knowledge, experience and skills are rarely harnessed in existing interventions to best support these families. OBJECTIVE: This study explores the use of mothers as facilitators for a community-based group intervention for children with CZS and their caregivers in Brazil. METHODS: Four facilitators were trained to deliver the 10-week intervention called "Juntos". Two were mothers of a child with CZS ("expert mothers") and two were therapists (speech therapist and physiotherapist). The intervention was delivered to three groups, generally including 8-10 caregivers. Two researchers, who were psychologists, observed the groups and held focus group discussions at the end of each session. They undertook semi-structured interviews post intervention with a purposive sample of caregivers, and with the facilitators. Observation notes were collated and summarised. Transcripts were transcribed and thematically analysed using five elements to assess feasibility: acceptability, demand, implementation, practicality and adaptation. RESULTS: The use of expert mothers as facilitators was considered to be acceptable and there was demand for their role. Their experiential knowledge was viewed as important for sharing and learning, and supporting and encouraging the group. The intervention was delivered with fidelity by the expert mothers. The practicality of the intervention was facilitated by holding the group sessions in the community, providing transport costs to facilitators and participants, paying expert mothers and therapist facilitators equally and supporting the expert mothers through a mentorship programme. Equal payment with the therapist enabled the expert mothers to better facilitate the groups, through increased confidence in the value of their role. Adaptation of the intervention included development of video resources and mentoring guidelines. CONCLUSION: The use of expert mothers as facilitators of caregiver groups provides a unique approach to harness the knowledge, experience, and skills of families to provide care, and is likely to be feasible in similar contexts.

Conscientious Objection to Legal Abortion in Minas Gerais State.

OBJECTIVE: The aim of this study was to verify the existence of conscientious objection to comprehensive health care for the victim of sexual violence, as well as to understand the service structure of institutions authorized in the health care system for victims of sexual violence in the state of Minas Gerais. METHODS: This is a quantitative, cross-sectional, descriptive, and analytical field study aiming to collect data from institutions authorized to assist victims of sexual violence in the state. The instrument was handed in to the coordinators of these services. RESULTS: It was found that 11% have no physician in service and that 31% had no training for this type of care. It was revealed that 85% of these institutions have already encountered patients wishing to have a legal abortion, but 83% of them have not had their request granted. There was a 60% presence of conscientious objection by the entire medical team, the main reason being religious (57%). CONCLUSION: The assistance system is not prepared for comprehensive care for victims of sexual violence, especially in terms of legal abortions, with conscientious objection being the main obstacle. A functional referral and counter-referral system is needed to alleviate such a serious and evident problem. It is hoped that the research results will promote dialogues in the state that favor appropriate actions on legal abortion, and respect the medical professional, in case of conscientious objection.

OBJETIVO: O objetivo do estudo foi verificar a existência da objeção de consciência na atenção integral da saúde à vítima de violência sexual, bem como conhecer a estrutura de atendimento das instituições credenciadas na rede de atenção à vítima de violência sexual no Estado de Minas Gerais. MéTODOS: Trata-se de um estudo de campo de caráter quantitativo, transversal, descritivo e analítico, com proposta de coleta de dados das instituições credenciadas ao atendimento às vítimas de violência sexual no estado. O instrumento foi entregue às(aos) coordenadora(es) destes serviços. RESULTADOS: Verificou-se que 11% dos serviços não possuem médicos e 31% não fornecem treinamento para este tipo de atendimento. Foi revelado que 85% dessas instituições já encontraram pacientes que desejam fazer o aborto legal, mas 83% destas não tiveram seu pedido atendido. Houve 60% da presença de objeção de consciência por parte de toda a equipe médica, sendo o principal motivo religioso (57%). CONCLUSãO: O sistema de assistência no Estado não está preparado para o atendimento integral às vítimas de violência sexual, principalmente no quesito resolução do aborto legal, sendo a objeção de consciência o maior obstáculo. Se faz necessária uma rede de referência e contra referência funcionante para amenizar problema tão sério e evidente. Espera-se que o resultado da pesquisa crie espaços de diálogos dentro do estado que favoreçam ações adequadas sobre o aborto legal, e o profissional médico respeitado, se houver objeção de consciência.

Superensemble forecast of respiratory syncytial virus outbreaks at national, regional, and state levels in the United States.

Respiratory syncytial virus (RSV) infections peak during the winter months in the United States, yet the timing, intensity, and onset of these outbreaks vary each year. An RSV vaccine is on the cusp of being released; precise models and accurate forecasts of RSV epidemics may prove vital for planning where and when the vaccine should be deployed. Accurate forecasts with sufficient spatial and temporal resolution could also be used to support the prevention or treatment of RSV infections. Previously, we developed and validated an RSV forecast system at the regional scale in the United States. This model-inference system had considerable forecast skill, relative to the historical expectance, for outbreak peak intensity, total outbreak size, and onset, but only marginal skill for predicting the timing of the outbreak peak. Here, we use a superensemble approach to combine three forecasting methods for RSV prediction in the US at three different spatial resolutions: national, regional, and state. At the regional and state levels, we find a substantial improvement of forecast skill, relative to historical expectance, for peak intensity, timing, and onset outbreak up to two months in advance of the predicted outbreak peak. Moreover, due to the greater variability of RSV outbreaks at finer spatial scales, we find that improvement of forecast skill at the state level exceeds that at the regional and national levels. Such finer scale superensemble forecasts may be more relevant for effecting local-scale interventions, particularly in communities with a high burden of RSV infection.

Simulation of four respiratory viruses and inference of epidemiological parameters.

While influenza has been simulated extensively to better understand its behavior and predict future outbreaks, most other respiratory viruses have seldom been simulated. In this study, we provide an overview of four common respiratory viral infections: respiratory syncytial virus (RSV), respiratory adenovirus, rhinovirus and parainfluenza, present specimen data collected 2004-2014, and simulate outbreaks in 19 overlapping regions in the United States. Pairing a compartmental model and data assimilation methods, we infer key epidemiological parameters governing transmission: the basic reproductive number R 0 and length of infection D. RSV had been previously simulated, and our mean estimate of D and R 0 of 5.2 days and 2.8, respectively, are within published clinical and modeling estimates. Among the four virus groupings, mean estimates of R 0 range from 2.3 to 3.0, with a lower and upper quartile range of 2.0-2.8 and 2.6-3.2, respectively. As rapid PCR testing becomes more common, estimates of the observed virulence and duration of infection for these viruses could inform decision making by clinicians and officials for managing patient treatment and response.

Serum 25-hydroxyvitamin D levels in a healthy population from the North of Portugal.

Vitamin D status in human populations has become a matter of great concern, in the wake of a multitude of published works that document widespread vitamin D deficiency across Europe, even in countries with abundant sunlight. In Portugal there are no measures of 25-hydroxyvitamin D - 25(OH)D - levels in the general adult population. The purpose of this study was to measure 25(OH)D levels in a healthy population cohort and investigate the possible association with season and selected demographic and laboratory measurements. A cohort of 198 participants (18-67 years) living in the north of Portugal, Porto, conducted in July and August 2015 (summer time) and April 2016 (winter time) was studied to evaluate serum 25(OH)D levels. Sociodemographic characteristics (age, sex and body mass index) and season of the year were taken into account as possible 25(OH)D levels codeterminants. In the whole group, the mean level of serum 25(OH)D was 55.4±23.4 nmol/L, with 48% of the population presenting levels compatible with vitamin D deficiency (below 50 nmol/L). In the winter period, this value reaches 74%. No statistically significant differences were observed between genders (57.4±23.9 vs. 53.3±22.8 nmol/L, p=0.219) as well as no statistically significant correlation was found between age and 25(OH)D levels (p=0.349). As expected higher levels of 25(OH)D were observed in summer than in winter (68.2±21.5 vs. 42.2±16.9 nmol/L; p<0.0001). Serum 25(OH)D levels were significantly lower in obese compared to non-obese subjects (46.6±17.6 vs. 57.7±24.2 nmol/L, p=0.012). Vitamin D deficiency is prevalent in this area, affecting almost half of the population. Body mass index and season are predictors for lower 25-hydroxyvitamin D levels and vitamin D status. An effective strategy to prevent vitamin D deficiency and insufficiency should be envisaged and implemented in our population.