In Vitro Effects of Combining Genistein with Aromatase Inhibitors: Concerns Regarding Its Consumption during Breast Cancer Treatment.

INTRODUCTION: The third-generation of aromatase inhibitors (AIs)-Exemestane (Exe), Letrozole (Let), and Anastrozole (Ana)-is the main therapeutic approach applied for estrogen receptor-positive (ER+) breast cancer (BC), the most common neoplasm in women worldwide. Despite their success, the development of resistance limits their efficacy. Genistein (G), a phytoestrogen present in soybean, has promising anticancer properties in ER+ BC cells, even when combined with anticancer drugs. Thus, the potential beneficial effects of combining G with AIs were investigated in sensitive (MCF7-aro) and resistant (LTEDaro) BC cells. METHODS: The effects on cell proliferation and expression of aromatase, ERα/ERß, and AR receptors were evaluated. RESULTS: Unlike the combination of G with Ana or Let, which negatively affects the Ais' therapeutic efficacy, G enhanced the anticancer properties of the steroidal AI Exe, increasing the antiproliferative effect and apoptosis relative to Exe. The hormone targets studied were not affected by this combination when compared with Exe. CONCLUSIONS: This is the first in vitro study that highlights the potential benefit of G as an adjuvant therapy with Exe, emphasizing, however, that soy derivatives widely used in the diet or applied as auxiliary medicines may increase the risk of adverse interactions with nonsteroidal AIs used in therapy.

An Exemestane Derivative, Oxymestane-D1, as a New Multi-Target Steroidal Aromatase Inhibitor for Estrogen Receptor-Positive (ER+) Breast Cancer: Effects on Sensitive and Resistant Cell Lines.

Around 70-85% of all breast cancer (BC) cases are estrogen receptor-positive (ER+). The third generation of aromatase inhibitors (AIs) is the first-line treatment option for these tumors. Despite their therapeutic success, they induce several side effects and resistance, which limits their efficacy. Thus, it is crucial to search for novel, safe and more effective anti-cancer molecules. Currently, multi-target drugs are emerging, as they present higher efficacy and lower toxicity in comparison to standard options. Considering this, this work aimed to investigate the anti-cancer properties and the multi-target potential of the compound 1α,2α-epoxy-6-methylenandrost-4-ene-3,17-dione (Oxy), also designated by Oxymestane-D1, a derivative of Exemestane, which we previously synthesized and demonstrated to be a potent AI. For this purpose, it was studied for its effects on the ER+ BC cell line that overexpresses aromatase, MCF-7aro cells, as well as on the AIs-resistant BC cell line, LTEDaro cells. Oxy reduces cell viability, impairs DNA synthesis and induces apoptosis in MCF-7aro cells. Moreover, its growth-inhibitory properties are inhibited in the presence of ERα, ERß and AR antagonists, suggesting a mechanism of action dependent on these receptors. In fact, Oxy decreased ERα expression and activation and induced AR overexpression with a pro-death effect. Complementary transactivation assays demonstrated that Oxy presents ER antagonist and AR agonist activities. In addition, Oxy also decreased the viability and caused apoptosis of LTEDaro cells. Therefore, this work highlights the discovery of a new and promising multi-target drug that, besides acting as an AI, appears to also act as an ERα antagonist and AR agonist. Thus, the multi-target action of Oxy may be a therapeutic advantage over the three AIs applied in clinic. Furthermore, this new multi-target compound has the ability to sensitize the AI-resistant BC cells, which represents another advantage over the endocrine therapy used in the clinic, since resistance is a major drawback in the clinic.

Understanding the bioaccumulation of pharmaceutical active compounds by clams Ruditapes decussatus exposed to a UWWTP discharge.

Twenty-four pharmaceutical active compounds (PhACs) were evaluated in the soft tissues of clams Ruditappes decussatus exposed along a 1.5-km dispersal gradient of the treated effluent from an urban wastewater treatment plant discharging in Ria Formosa, and compared with those in the marine waters and discharged effluents. The clams were exposed for 1 month, in June-July 2016, 2017 and 2018. PhACs were quantified by high performance liquid chromatography coupled to tandem mass spectrometry after the quick, easy, cheap, effective, rugged and safe (QuEChERS) method (clams) or solid-phase extraction (water samples). The most representative PhACs in the effluents and receiving waters (regardless of the tidal dilution effect) were diclofenac, carbamazepine and caffeine (on average ≤ 2 µg/L) and only caffeine exhibited significant inter-annual differences, with higher values in 2017. In turn, the most bioaccumulated PhACs in clams were caffeine (0.54-27 ng/g wet weight, significantly higher in 2016) and acetaminophen (0.37-3.7 ng/g wet weight, significant lower in 2016). A multivariate principal component analysis showed (i) PhAC bioaccumulation primarily depended on biotic factors (clams length and weight), (ii) PhAC physicochemical properties Log Kow, pKa and water solubility interplaying with water abiotic variables were more relevant for explaining data variability in water than the physical dilution/tidal mixing, (iii) this process, reflected by the salinity gradient, had a tertiary role in data variation, responsible for spatial discrimination of marine waters. This study provides a better understanding of PhACs bioaccumulation by clams Ruditapes decussatus in real environmental conditions, under the influence of urban treated effluent dispersal in Ria Formosa coastal lagoon, a major producer of bivalves, ultimately disentangling key factors of PhAC bioaccumulation.

Effects of PI3K inhibition in AI-resistant breast cancer cell lines: autophagy, apoptosis, and cell cycle progression.

INTRODUCTION: Breast cancer is the leading cause of cancer death in women. The aromatase inhibitors (AIs), Anastrozole (Ana), Letrozole (Let), and Exemestane (Exe) are a first-line treatment option for estrogen receptor-positive (ER+) breast tumors, in postmenopausal women. Nevertheless, the development of acquired resistance to this therapy is a major drawback. The involvement of PI3K in resistance, through activation of the PI3K/AKT/mTOR survival pathway or through a cytoprotective autophagic process, is widely described. MATERIALS AND METHODS: The involvement of autophagy in response to Ana and Let treatments and the effects of the combination of BYL-719, a PI3K inhibitor, with AIs were explored in AI-resistant breast cancer cell lines (LTEDaro, AnaR, LetR, and ExeR). RESULTS: We demonstrate that Ana and Let treatments do not promote autophagy in resistant breast cancer cells, contrary to Exe. Moreover, the combinations of BYL-719 with AIs decrease cell viability by different mechanisms by nonsteroidal vs. steroidal AIs. The combination of BYL-719 with Ana or Let induced cell cycle arrest while the combination with Exe promoted cell cycle arrest and apoptosis. In addition, BYL-719 decreased AnaR, LetR, and ExeR cell viability in a dose- and time-dependent manner, being more effective in the ExeR cell line. This decrease was further exacerbated by ICI 182,780. CONCLUSION: These results corroborate the lack of cross-resistance between AIs verified in the clinic, excluding autophagy as a mechanism of resistance to Ana or Let and supporting the ongoing clinical trials combining BYL-719 with AIs.

Cannabinoids in Breast Cancer: Differential Susceptibility According to Subtype.

Although cannabinoids have been used for centuries for diverse pathological conditions, recently, their clinical interest and application have emerged due to their diverse pharmacological properties. Indeed, it is well established that cannabinoids exert important actions on multiple sclerosis, epilepsy and pain relief. Regarding cancer, cannabinoids were first introduced to manage chemotherapy-related side effects, though several studies demonstrated that they could modulate the proliferation and death of different cancer cells, as well as angiogenesis, making them attractive agents for cancer treatment. In relation to breast cancer, it has been suggested that estrogen receptor-negative (ER-) cells are more sensitive to cannabinoids than estrogen receptor-positive (ER+) cells. In fact, most of the studies regarding their effects on breast tumors have been conducted on triple-negative breast cancer (TNBC). Nonetheless, the number of studies on human epidermal growth factor receptor 2-positive (HER2+) and ER+ breast tumors has been rising in recent years. However, besides the optimistic results obtained thus far, there is still a long way to go to fully understand the role of these molecules. This review intends to help clarify the clinical potential of cannabinoids for each breast cancer subtype.

Screening and Seasonal Behavior of Analgesics, Non-steroidal Anti-inflammatory Drugs, and Antibiotics in Two Urban Wastewater Treatment Plants.

Pharmaceutical active compounds (PhACs) belonging to analgesics, antibiotics, and non-steroidal anti-inflammatory drugs (NSAIDs) therapeutic classes were monitored in wastewater influents and effluents from two Portuguese urban wastewater treatment plants (UWWTPs) for 24 months. Both facilities were chosen due to their effluents are discharged in highly touristic and sensitive areas, Tagus river and Ria Formosa coastal lagoon, respectively. Target PhACs, acetaminophen, diclofenac, ibuprofen, naproxen, sulfadiazine, and sulfamethoxazole were measured using solid-phase extraction (SPE) coupled to liquid chromatography tandem mass spectrometry (LC-MS/MS). PhACs occurrence in the influents was higher than 98%, with acetaminophen presenting the highest concentrations, with values between 16.3 µg/L and 124 µg/L. In the effluents, distinct behavior was observed with diclofenac and sulfamethoxazole showing recalcitrant characteristics, whereas acetaminophen, ibuprofen, and naproxen showed removal efficiencies above 95%. Acetaminophen and ibuprofen amount in influents showed consistently higher concentration levels in autumn (in Beirolas and Faro Nw UWWTPs) and winter (only in Beirolas UWWTP) seasons. These seasonal trends were observed to a greater extent in Beirolas UWWTP than in Faro Nw UWWTP. This study enabled the comprehensive definition of a behavior pattern for these target contaminants, contributing to better characterization and build-up a library of PhACs occurrence. It also allowed a robust seasonal profiling of the target compounds due to the high number of samples analyzed by each season and a longer monitoring campaign, making the obtained results more significant.

Removal of veterinary antibiotics in constructed wetland microcosms - Response of bacterial communities.

This study aimed to evaluate the response of bacteria, in terms of microbial community structure, from constructed wetland (CW) microcosms exposed to two veterinary antibiotics, enrofloxacin (ENR) and ceftiofur (CEF), alone or in a mixture, identifying which bacterial groups were dominant in CWs substrate during livestock wastewater treatment. Wastewater, not-doped or doped with ENR and/or CEF (100 µg/L each), was treated during 18 one-week cycles. Systems showed removal percentages > 85% for the added antibiotics, showing also high removal percentages for nutrients and organic matter and confirming CWs systems were working properly. However, both time of exposure and presence of antibiotics influenced significantly CWs substrate bacterial community structure. Pyrosequencing results showed bacterial communities were dominated by phyla Proteobacteria (38-48%), Firmicutes (20-27%), Bacteroidetes (12-15%) and Actinobacteria (4-9%), and that their relative abundance was clearly affected by the presence of the antibiotics. Results suggest the applicability of CWs for the removal of veterinary antibiotics from livestock wastewaters and provide new knowledge about the bacteria within the system, which can potentially be involved in removal processes. This information could in the future be used to improve CWs removal rates of pharmaceuticals from livestock wastewaters.

A comparison between visual, intraoral scanner, and spectrophotometer shade matching: A clinical study.

STATEMENT OF PROBLEM: Visual shade matching is subjective and a cause of concern for clinicians. Different measurement devices have been developed to assist in tooth color selection and to achieve better esthetic results. However, consensus is lacking as to which method of tooth shade selection provides more predictable results. PURPOSE: The purpose of this clinical study was to compare the reliability of different visual and instrumental methods for dental shade matching. MATERIAL AND METHODS: Visual shade matching was performed by 3 experienced clinicians using 2 different shade guides (VITA Classical A1-D4 and VITA Toothguide 3D-MASTER with 29 tabs; VITA Zahnfabrik) with and without the aid of a light-correcting device (Smile Lite; Smile Line). An intraoral scanner (TRIOS; 3Shape A/S) and a spectrophotometer (VITA Easyshade Advance 4.0; VITA Zahnfabrik) were also used for color shade matching. The instrumental methods were repeated 3 times to determine repeatability. Shade-matching sessions for each method were performed under controlled lighting on the middle third of the maxillary right central incisor of 28 participants. The Fleiss' kappa statistical test was used to assess the reliability of each method. The weighted kappa statistical test was used to assess the agreement between the shades matched by different methods (α=.05). RESULTS: Instrumental methods were more accurate than visual methods. The best performance was found for the intraoral scanner configured for the 3D-MASTER scale (Fleiss' kappa value of .874) and for the spectrophotometer configured for the VITA Classical scale (Fleiss' kappa value of .805). The best visual shade-matching method was the VITA Classical scale associated with the light-correcting device (Fleiss' kappa value of .322). The Classical scale without the light-correcting device showed the poorest reliability (Fleiss' kappa value of .177) (P<.05). CONCLUSIONS: Instrumental methods for color shade matching were more reliable than the visual methods tested.

Investigation of Endophytic Bacterial Community in Supposedly Axenic Cultures of Pineapple and Orchids with Evidence on Abundant Intracellular Bacteria.

Asepsis, defined as the absence of microbial contamination, is one of the most important requirements of plant micropropagation. In long-term micropropagated cultures, there may occasionally occur scattered microorganism growth in the culture medium. These microorganisms are common plant components and are known as latent endophytes. Thus, the aim of this research was to investigate the presence of endophytic bacteria in asymptomatic pineapple and orchid microplants, which were cultivated in three laboratories for 1 year. Isolation and characterization of bacterial isolates, PCR-DGGE from total genomic DNA of microplants and ultrastructural analysis of leaves were performed. In the culture-dependent technique, it was only possible to obtain bacterial isolates from pineapple microplants. In this case, the bacteria genera identified in the isolation technique were Bacillus, Acinetobacter, and Methylobacterium. The scanning electron microscopy and transmission electron microscopy (SEM and TEM) analyses revealed the presence of endophytic bacteria in intracellular spaces in the leaves of pineapple and orchid microplants, independent of the laboratory or cultivation protocol. Our results strongly indicate that there are endophytic bacterial communities inhabiting the microplants before initiation of the in vitro culture and that some of these endophytes persist in their latent form and can also grow in the culture medium even after long-term micropropagation, thus discarding the concept of "truly axenic plants."

Dyschromatopsia in Multiple Sclerosis Patients: A Marker of Subclinical Involvement?

BACKGROUND: In multiple sclerosis (MS), even in the absence of a clinical episode of optic neuritis (ON), the optic nerve and retinal nerve fiber layer (RNFL) may be damaged leading to dyschromatopsia. Subclinical dyschromatopsia has been described in MS associated with lower motor and cognitive performances. OBJECTIVES: To set the prevalence of dyschromatopsia in eyes of MS patients without a history of ON, to compare its prevalence in patients with and without ON history, and to explore the association between dyschromatopsia and disease duration, average peripapillary RNFL thickness, macular volume, and cognitive and motor performances. METHODS: An observational cross-sectional study was conducted at multiple medical centers. Data were collected after single neurological and ophthalmological evaluations. Dyschromatopsia was defined by the presence of at least 1 error using Hardy-Rand-Rittler plates. RESULTS: In our population of 125 patients, 79 patients (63.2%) never had ON and 35 (28.8%) had unilateral ON. The prevalence of dyschromatopsia in eyes of patients without ON was 25.7%. Patients with dyschromatopsia had a statistically significant lower RNFL thickness (P = 0.004 and P = 0.040, right and left eyes, respectively) and worse performance in symbol digit modalities test (P = 0.012). No differences were found in macular volume or motor function tasks. CONCLUSIONS: Dyschromatopsia occurs frequently in MS patients. It may be associated with a worse disease status and possibly serve as a marker for the detection of subclinical disease progression since it was detected even in the absence of ON. It correlated with thinner peripapillary RNFL thickness and inferior cognitive performance.

Chlorination and oxidation of sulfonamides by free chlorine: Identification and behaviour of reaction products by UPLC-MS/MS.

Sulfonamides (SAs) are one class of the most widely used antibiotics around the world and have been frequently detected in municipal wastewater and surface water in recent years. Their transformation in waste water treatment plants (WWTP) and in water treatment plants (WTP), as well as, their fate and transport in the aquatic environment are of concern. The reaction of six sulfonamides (sulfamethoxazole, sulfapyridine, sulfamethazine, sulfamerazine, sulfathiazole and sulfadiazine) with free chlorine was investigated at a laboratory scale in order to identify the main chlorination by-products. A previously validated method, liquid chromatography/mass spectrometry, was used to analyse SAs and their chlorination by-products. At room temperature, pH 6-7, reaction times of up to 2 h and an initial concentration of 2 mg/L of free chlorine, the majority of SAs suffered degradation of around 65%, with the exception of sulfamethoxazole and sulfathiazole (20%). The main reaction of SAs with free chlorine occurred in the first minute.

Endophytic bacterial communities associated with two explant sources of Eucalyptus benthamii Maiden & Cambage.

Micropropagation has been applied in the recovery and rejuvenation of adult trees, which is achieved by various subcultures in the multiplication phase. This strategy has brought questions about the endophytic microbiota associated with these plants along its manipulation. Therefore, the aim of this study was to evaluate the composition of the endophytic bacterial communities associated with two explants sources [the canopy branches (CB) and the trunk base of the tree (TB)] under prolonged in vitro cultivation. In addition we analyzed the bacterial community dynamic along the subcultures in different micropropagation phases. Bacterial DNA was extracted from samples of mini-stumps (in vivo) from CB and TB and in micro-stumps produced by in vitro cultivations of these explants sources--both originated from one single matrix plant of Eucalyptus benthamii. In vitro establishment occurred in two dates and the evaluation of endophytic bacterial communities was made in vivo and in vitro samples (on 10th, 13th and 16th subcultures), when elongated shoots and roots were analyzed. Analysis was performed by PCR-DGGE based on the V6 region of ribosomal gene 16S rDNA. Bands profiles showed differences in communities between in vivo and in vitro samples, and also distinctions of communities assessed in the subcultures, elongated and rooted samples. Distinctions in the composition of endophytic bacterial communities were greater in CB micro-stumps. These results indicate a differential colonization of explants by endophytic bacteria, with predominance of common (ever-present) endophytes in TB samples and casual, here named opportunistic, in CB samples.

Partially Edentulous Arches: A 5-Year Survey of Patients Treated at the Fluminense Federal University Removable Prosthodontics Clinics in Brazil.

PURPOSE: This study investigated the number and Kennedy Classification of the edentulous arches in patients treated at the Removable Partial Denture (RPD) Clinics of the Fluminense Federal University School of Dentistry (FO-UFF) in Rio de Janeiro, Brazil, from 2005 to 2010. MATERIALS AND METHODS: A cross-sectional retrospective survey was conducted on patient record charts to identify gender, age, number, and location of the edentulous arches, and Kennedy Class type. RESULTS: One hundred and forty-six patients were analyzed for this study (96 [65.8%] women and 50 [34.2%] men). Two hundred and ninety-two arches were analyzed: 74 arches (25%) were found with intact dentitions, 18 (6.1%) were edentulous arches, and 200 (68.8%) were partially edentulous arches. Ninety-one patients (62.3%) needed oral rehabilitation with RPDs on one arch and 55 (37.7%) in both arches. One hundred and eight (53.2%) partially edentulous mandibles and 92 (46.8%) partially edentulous maxillae were found. Kennedy Class I was more frequent in the mandibular arch (58 patients; 29%) whereas Kennedy Class III was more frequent in the maxillary arch (40 patients; 20%). Patients aged between 51 and 60 years presented the highest percentage of partially edentulous arches (33.6%). CONCLUSION: Mandibular Kennedy Class I and maxillary Kennedy Class III presented the highest frequency in patients treated at the FO-UFF. These results are in agreement with previous studies that evaluated the different Kennedy classes in partially edentulous arches.

Trace element compositional changes in human milk during the first four months of lactation.

The aims of this paper were to evaluate changes in specific oligoelements in human milk during the first four months of lactation and to correlate such changes with total antioxidant status (TAS) and other parameters, such as the mother's age, primipara versus multipara, and supplement intake. Milk samples were collected from 31 lactating women following 1, 4, 8, 12 and 16 weeks after birth. Trace levels of 13 elements were measured by inductively coupled plasma-mass spectrometry (ICP-MS). The results obtained for the oligoelements exhibited a decrease in concentration from 7 days to 4 months of breast-feeding, with exceptions. Correlations were found between TAS and Co, V, Rb and Tl. Between primipara and multipara, differences were found for Ni and Rb. Regarding the mother's age, correlation was found for Rb and Ba (increased for mothers older than 30 years). Increased amounts of Rb, Mo and Tl at any lactation period appeared in women who took supplements.

Prevalence and determinants of insomnia symptoms among schoolteachers.

INTRODUCTION: Insomnia is the most common sleep disorder. In Portugal, teachers have a specific socioeconomic situation, caused by the distance between home and workplace, unstable job situation and students' behavioral problems. The aim of this study was to determine the prevalence of insomnia in a sample of Portuguese schoolteachers. PARTICIPANTS AND METHODS: In a cross-sectional study 604 teachers were assessed of seventeen public schools, from the districts of Aveiro and Viseu, Portugal. Data was collected through a self-administered questionnaire. Insomnia had been defined according to the fourth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) as the presence of one or more of the following symptoms: a) difficulty initiating sleep; b) difficulty maintaining sleep; c) early morning awakening and difficulty getting back to sleep; d) non-restorative sleep, that lasts for a period of 1 month. RESULTS: Prevalence of insomnia symptoms in the sample was 40.6%. Prevalence of the variables difficulty initiating sleep, difficulty maintaining sleep, early morning awakening and difficulty getting back to sleep and non-restorative sleep were 14.3%, 28.7%, 19.7% and 20.7%, respectively. Insomnia symptoms had been associated with marital status (divorced; OR=1.65; 95%CI, 0.78-3.48), years of teaching experience (10 to 20 years; OR=0.46; 95%CI, 0.28-0.75) and job satisfaction (OR=0.74; 95%CI, 0.53-1.0). CONCLUSION: Portuguese schoolteachers show a high prevalence of insomnia. Insomnia was associated with sociodemographic and occupational variables.

Influence of tumor microenvironment on the different breast cancer subtypes and applied therapies.

Despite the significant improvements made in breast cancer therapy during the last decades, this disease still has increasing incidence and mortality rates. Different targets involved in general processes, like cell proliferation and survival, have become alternative therapeutic options for this disease, with some of them already used in clinic, like the CDK4/6 inhibitors for luminal A tumors treatment. Nevertheless, there is a demand for novel therapeutic strategies focused not only on tumor cells, but also on their microenvironment. Tumor microenvironment (TME) is a very complex and dynamic system that, more than surrounding and supporting tumor cells, actively participates in tumor development and progression. During the last decades, it has become clear that the cellular and acellular components of TME differ between the various breast cancer subtypes and shape the differences regarding their severity and prognosis. The pivotal role of the TME in controlling tumor growth and influencing responses to therapy represents a potential source for novel targets and therapeutic strategies. In this review, we present a description of the multiple therapeutic options used for different breast cancer subtypes, as well as the influence that the TME may exert on the development of the disease and on the response to the distinct therapies, which in some cases may explain their failure by the occurrence of relapses and resistance. Furthermore, the ongoing studies focused on the use of TME components for developing potential cancer treatments are described.

Validation of an FT-IR microscopy method for the monitorization of microplastics in water for human consumption in Portugal: Lisbon case study.

The growing anthropogenic contamination of natural water by microplastics (MPs) confirms the urgent need to preserve this precious resource. MPs are part of the group of contaminants of emerging concern, and the occurrence studies in surface water and water for human consumption (WHC) are mandatory for environmental and human health risk assessment. This study aims to optimize and validate a Fourier transform infrared spectroscopy method coupled with optical microscopy (micro-FTIR) in transmission mode to monitor MPs in WHC. Water sample (250 mL; without sample pre-treatment) was filtered through 5 µm silicon filters. The infrared spectra identification was performed by OMNIC mathematical correlation, using various spectra libraries for polymers (including the in-house IR spectra library), a background reading on a clean silicon filter, and an aperture of 100 µm × 100 µm. The validated method showed good accuracy, with an average recovery for representative polymers of 91%, a relative standard deviation of 13%, and a reporting limit (RL) of 44 MPs/L. Sixty WHC samples from the Lisbon water supply system showed MPs ranging from 0 (< RL) to 934 MPs/L, with an average value of 309 MPs/L. The most representative polymers were polyethylene (PE, 76.8%), polyethylene terephthalate (PET, 6.9%), polypropylene (PP, 6%), polystyrene (PS, 4%), and polyamide (PA,4%). In terms of size, the microplastic particles had an average length and width of 76 µm and 39 µm, respectively.

Can primary health care mitigate the effects of economic crises on child health in Latin America? An integrated multicountry evaluation and forecasting analysis.

BACKGROUND: Latin American and Caribbean countries are dealing with the combined challenges of pandemic-induced socicoeconomic stress and increasing public debt, potentially leading to reductions in welfare and health-care services, including primary care. We aimed to evaluate the impact of primary health-care coverage on child mortality in Latin America over the past two decades and to forecast the potential effects of primary health-care mitigation during the current economic crisis. METHODS: This multicountry study integrated retrospective impact evaluations in Brazil, Colombia, Ecuador, and Mexico from 2000 to 2019 with forecasting models covering up to 2030. We estimated the impact of coverage of primary health care on mortality rates in children younger than 5 years (hereafter referred to as under-5 mortality) across different age groups and causes of death, adjusting for all relevant demographic, socioeconomic, and health-care factors, with fixed-effects multivariable negative binomial models in 5647 municipalities with an adequate quality of vital statistics. We also performed several sensitivity and triangulation analyses. We integrated previous longitudinal datasets with validated dynamic microsimulation models and projected trends in under-5 mortality rates under alternative policy response scenarios until 2030. FINDINGS: High primary health-care coverage was associated with substantial reductions in post-neonatal mortality rates (rate ratio [RR] 0·72, 95% CI 0·71-0·74), toddler (ie, aged between 1 year and <5 years) mortality rates (0·75, 0·73-0·76), and under-5 mortality rates (0·81, 0·80-0·82), preventing 305 890 (95% CI 251 826-360 517) deaths of children younger than 5 years over the period 2000-19. High primary health-care coverage was also associated with lower under-5 mortality rates from nutritional deficiencies (RR 0·55, 95% CI 0·52-0·58), anaemia (0·64, 0·57-0·72), vaccine-preventable and vaccine-sensitive conditions (0·70, 0·68-0·72), and infectious gastroenteritis (0·78, 0·73-0·84). Considering a scenario of moderate economic crisis, a mitigation response strategy implemented in the period 2020-30 that increases primary health-care coverage could reduce the under-5 mortality rate by up to 23% (RR 0·77, 95% CI 0·72-0·84) when compared with a fiscal austerity response, and this strategy would avoid 142 285 (95% CI 120 217-164 378) child deaths by 2030 in Brazil, Colombia, Ecuador, and Mexico. INTERPRETATION: The improvement in primary health-care coverage in Brazil, Colombia, Ecuador, and Mexico over the past two decades has substantially contributed to improving child survival. Expansion of primary health-care coverage should be considered an effective strategy to mitigate the health effects of the current economic crisis and to achieve Sustainable Development Goals related to child health. FUNDING: UK Medical Research Council. TRANSLATIONS: For the Spanish and Portuguese translations of the abstract see Supplementary Materials section.

Analyzing the safety of the parasiticide fungus Mucor circinelloides: first insights on its virulence profile and interactions with the avian gut microbial community.

Parasiticide fungi are considered an accurate, sustainable, and safe solution for the biocontrol of animal gastrointestinal (GI) parasites. This research provides an initial characterization of the virulence of the native parasiticide fungus Mucor circinelloides (FMV-FR1) and an assessment of its impact on birds' gut microbes. The genome of this fungus was sequenced to identify the genes coding for virulence factors. Also, this fungus was checked for the phenotypic expression of proteinase, lecithinase, DNase, gelatinase, hemolysin, and biofilm production. Finally, an in vivo trial was developed based on feeding M. circinelloides spores to laying hens and peacocks three times a week. Bird feces were collected for 3 months, with total genomic DNA being extracted and subjected to long-read 16S and 25S-28S sequencing. Genes coding for an iron permease (FTR1), iron receptors (FOB1 and FOB2), ADP-ribosylation factors (ARFs) (ARF2 and ARF6), and a GTPase (CDC42) were identified in this M. circinelloides genome. Also, this fungus was positive only for lecithinase activity. The field trial revealed a fecal microbiome dominated by Firmicutes and Proteobacteria in laying hens, and Firmicutes and Bacteroidetes in peacocks, whereas the fecal mycobiome of both bird species was mainly composed of Ascomycetes and Basidiomycetes fungi. Bacterial and fungal alpha-diversities did not differ between sampling time points after M. circinelloides administrations (P = 0.62 and P = 0.15, respectively). Although findings from this research suggest the lack of virulence of this M. circinelloides parasiticide isolate, more complementary in vitro and in vivo research is needed to conclude about the safety of its administration to birds, aiming at controlling their GI parasites.IMPORTANCEA previous study revealed that the native Mucor circinelloides isolate (FMV-FR1) can develop parasiticide activity toward coccidia oocysts, one of the most pathogenic GI parasites in birds. However, ensuring its safety for birds is of utmost importance, namely by studying its virulence profile and potential effect on commensal gut microbes. This initial study revealed that although this M. circinelloides isolate had genes coding for four types of virulence factors-iron permease, iron receptors, ADP-ribosylation factors, and GTPase-and only expressed phenotypically the enzyme lecithinase, the administration of its spores to laying hens and peacocks did not interfere with the abundances and diversities of their gut commensal bacteria and fungi. Although overall results suggest the lack of virulence of this M. circinelloides isolate, more complementary research is needed to conclude about the safety of its administration to birds in the scope of parasite biocontrol programs.

Assessing the efficacy of the ovicidal fungus Mucor circinelloides in reducing coccidia parasitism in peacocks.

The biological control of gastrointestinal (GI) parasites using predatory fungi has been recently proposed as an accurate and sustainable approach in birds. The current study aimed to assess for the first time the efficacy of using the native ovicidal fungus Mucor circinelloides (FMV-FR1) in reducing coccidia parasitism in peacocks. For this purpose, an in vivo trial was designed in the resident peacock collection (n = 58 birds) of the São Jorge Castle, at Lisbon, Portugal. These animals presented an initial severe infection by coccidia of the genus Eimeria (20106 ± 8034 oocysts per gram of feces, OPG), and thus received commercial feed enriched with a M. circinelloides suspension (1.01 × 108 spores/kg feed), thrice-weekly. Fresh feces were collected every 15 days to calculate the coccidia shedding, using the Mini-FLOTAC technique. The same bird flock served simultaneously as control (t0 days) and test groups (t15-t90 days). The average Eimeria sp. shedding in peacocks decreased up to 92% following fungal administrations, with significant reduction efficacies of 78% (p = 0.004) and 92% (p = 0.012) after 45 and 60 days, respectively. Results from this study suggest that the administration of M. circinelloides spores to birds is an accurate solution to reduce their coccidia parasitism.