SAA1-dependent reprogramming of adipocytes by tumor cells is associated with triple negative breast cancer aggressiveness.

Triple negative breast cancers (TNBC) are characterized by a poor prognosis and a lack of targeted treatments. Their progression depends on tumor cell intrinsic factors, the tumor microenvironment and host characteristics. Although adipocytes, the primary stromal cells of the breast, have been determined to be plastic in physiology and cancer, the tumor-derived molecular mediators of tumor-adipocyte crosstalk have not been identified yet. In this study, we report that the crosstalk between TNBC cells and adipocytes in vitro beyond adipocyte dedifferentiation, induces a unique transcriptional profile that is characterized by inflammation and pathways that are related to interaction with the tumor microenvironment. Accordingly, increased cancer stem-like features and recruitment of pro-tumorigenic immune cells are induced by this crosstalk through CXCL5 and IL-8 production. We identified serum amyloid A1 (SAA1) as a regulator of the adipocyte reprogramming through CD36 and P2XR7 signaling. In human TNBC, SAA1 expression was associated with cancer-associated adipocyte infiltration, inflammation, stimulated lipolysis, stem-like properties, and a distinct tumor immune microenvironment. Our findings constitute evidence that the interaction between tumor cells and adipocytes through the release of SAA1 is relevant to the aggressiveness of TNBC.

Impact of a teat disinfectant based on Lactococcus cremoris on the cow milk proteome.

BACKGROUND: Dairy cow milking practices require cleaning and disinfection of the teat skin before and after milking to ensure the safety and quality of milk and prevent intramammary infections. Antimicrobial proteins of natural origin can be valuable alternatives to traditional disinfectants. In a recent field trial, we demonstrated that a teat dip based on a nisin A-producing Lactococcus cremoris (L) had comparable efficacy to conventional iodophor dip (C) in preventing dairy cow mastitis. Here, we present the differential shotgun proteomics investigation of the milk collected during the trial. METHODS: Four groups of quarter milk samples with low (LSCC) and high somatic cell count (HSCC) collected at the beginning (T0) and end (TF) of the trial were analyzed for a total of 28 LSCC (14 LSCC T0 and 14 LSCC TF) and 12 HSCC (6 HSCC T0 and 6 HSCC TF) samples. Milk proteins were digested into peptides, separated by nanoHPLC, and analyzed by tandem mass spectrometry (LC-MS/MS) on an Orbitrap Fusion Tribrid mass spectrometer. The proteins were identified with MaxQuant and interaction networks of the differential proteins were investigated with STRING. The proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD045030. RESULTS: In healthy milk (LSCC), we detected 90 and 80 differential proteins at T0 and TF, respectively. At TF, the Lactococcus group showed higher levels of antimicrobial proteins. In mastitis milk (HSCC), we detected 88 and 106 differential proteins at T0 and TF, respectively. In the Lactococcus group, 14 proteins with antimicrobial and immune defense functions were enriched at TF vs. 4 proteins at T0. Cathelicidins were among the most relevant enriched proteins. Western immunoblotting validation confirmed the differential abundance. CONCLUSIONS: At T0, the proteomic differences observed in healthy milk of the two groups were most likely dependent on physiological variation. On the other hand, antimicrobial and immune defense functions were higher in the milk of cows with mammary gland inflammation of the Lactococcus-treated group. Among other factors, the immunostimulatory action of nisin A might be considered as a contributor.

Environmental Temperature Variation Affects Brain Lipid Composition in Adult Zebrafish (Danio rerio).

This study delves deeper into the impact of environmental temperature variations on the nervous system in teleost fish. Previous research has demonstrated that exposing adult zebrafish (Danio rerio) to 18 °C and 34 °C for 4 or 21 days induces behavioural changes compared to fish kept at a control temperature of 26 °C, suggesting alterations in the nervous system. Subsequent studies revealed that these temperature conditions also modify brain protein expression, indicating potential neurotoxic effects. The primary aim of this work was to investigate the effects of prolonged exposure (21 days) to 18 °C or 34 °C on the brain lipidomes of adult zebrafish compared to a control temperature. Analysis of the brain lipidome highlighted significant alteration in the relative abundances of specific lipid molecules at 18 °C and 34 °C, confirming distinct effects induced by both tested temperatures. Exposure to 18 °C resulted in an increase in levels of phospholipids, such as phosphatidylethanolamine, alongside a general reduction in levels of sphingolipids, including sphingomyelin. Conversely, exposure to 34 °C produced more pronounced effects, with increases in levels of phosphatidylethanolamine and those of various sphingolipids such as ceramide, gangliosides, and sphingomyelin, alongside a reduction in levels of ether phospholipids, including lysophosphatidylethanolamine ether, phosphatidylethanolamine ether, and phosphatidylglycerol ether, as well as levels of glycolipids like monogalactosyldiacylglycerol. These results, when integrated with existing proteomic and behavioural data, offer new insights into the effects of thermal variations on the nervous system in teleost fish. Specifically, our proteomic and lipidomic findings suggest that elevated temperatures may disrupt mitochondrial function, increase neuronal susceptibility to oxidative stress and cytotoxicity, alter axonal myelination, impair nerve impulse transmission, hinder synapse function and neurotransmitter release, and potentially lead to increased neuronal death. These findings are particularly relevant in the fields of cell biology, neurobiology, and ecotoxicology, especially in the context of global warming.

A Prepandemic Nutrition-Sensitive Social Protection Program Has Sustained Benefits for Food Security and Diet Diversity in Myanmar during a Severe Economic Crisis.

BACKGROUND: One-third of preschool children in Myanmar were stunted in 2015-2016, and three-quarters of children 6-23 mo had inadequate diet diversity. In response, a large-scale nutrition-sensitive social protection program was implemented over 2016-2019. In 2020, however, Myanmar's economy was hit hard by the COVID-19 pandemic and harder still by a military takeover in 2021. OBJECTIVE: The objective of this study was to examine whether former beneficiaries of this program experienced better food security, food consumption, and diet diversity outcomes in the wake of major economic shocks. METHODS: In a previous cluster-randomized controlled trial conducted over 2016-2019, pregnant women and their children aged <2 y were randomly assigned to either: 1) CASH; 2) CASH + social and behavioral change communication (SBCC); or 3) a control group. Subsamples of these former participants were then resurveyed 10 times from June 2020 to December 2021 during Myanmar's protracted economic crisis. Randomized treatment exposure was used in a regression analysis to test for postprogram impacts on Food Insecurity Experience Scale indicators, household food consumption, and maternal and child diet diversity. We also examined the impacts on household income as a secondary outcome and potential impact pathway. RESULTS: Both intervention arms reported lower food insecurity, more frequent consumption of nutritious foods, and more diverse maternal and child diets compared with households in the control group. However, the improved dietary outcomes were larger for mothers and children exposed to CASH+SBCC compared with CASH, as was their monthly household income. CONCLUSIONS: The findings suggest that a program combining cash transfers with nutrition-related education can yield sustained benefits 1-2 y after the program was completed. This strengthens the evidence to support the expansion and scale-up of nutrition-sensitive social welfare programs to redress chronic malnutrition and enhance nutritional resilience in the face of a severe economic crisis.

The local health impacts of natural resource booms.

This paper uses novel micro-data on natural resources and administrative health data in Brazil to study how economic booms in minerals affect health at birth. By implementing a reduced-form estimation of shift-share research designs, the identification strategy relies on the exogeneity of global commodity prices to municipality-specific health outcomes. I find that, following changes in international prices, municipalities with historically more endowments have a higher number of premature births and births with low Appearance, Pulse, Grimace, Activity, Respiration scores. The impacts are primarily driven by metallic minerals. Instead, industrial minerals do not appear to have any effect on birth outcomes. Even though booms in metallic minerals generate benefits through resource windfalls-by increasing wealth and generating economic opportunities-the investigation of mechanisms reveals that they also result in costs-due to pollution-which seem to prevail. Hence, some metallic minerals remain a curse more than a blessing.

The Neurotoxic Effect of Environmental Temperature Variation in Adult Zebrafish (Danio rerio).

Neurotoxicity consists of the altered functionality of the nervous system caused by exposure to chemical agents or altered chemical-physical parameters. The neurotoxic effect can be evaluated from the molecular to the behavioural level. The zebrafish Danio rerio is a model organism used in many research fields, including ecotoxicology and neurotoxicology. Recent studies by our research group have demonstrated that the exposure of adult zebrafish to low (18 °C) or high (34 °C) temperatures alters their brain proteome and fish behaviour compared to control (26 °C). These results showed that thermal variation alters the functionality of the nervous system, suggesting a temperature-induced neurotoxic effect. To demonstrate that temperature variation can be counted among the factors that generate neurotoxicity, eight different protein datasets, previously published by our research group, were subjected to new analyses using an integrated proteomic approach by means of the Ingenuity Pathway Analysis (IPA) software (Release December 2022). The datasets consist of brain proteome analyses of wild type adult zebrafish kept at three different temperatures (18 °C, 26 °C, and 34 °C) for 4 days (acute) or 21 days (chronic treatment), and of BDNF+/- and BDNF-/- zebrafish kept at 26 °C or 34 °C for 21 days. The results (a) demonstrate that thermal alterations generate an effect that can be defined as neurotoxic (p value ≤ 0.05, activation Z score ≤ -2 or ≥2), (b) identify 16 proteins that can be used as hallmarks of the neurotoxic processes common to all the treatments applied and (c) provide three protein panels (p value ≤ 0.05) related to 18 °C, 34 °C, and BDNF depletion that can be linked to anxiety-like or boldness behaviour upon these treatments.

Molecular response of Sargassum vulgare to acidification at volcanic CO2 vents: Insights from proteomic and metabolite analyses.

Ocean acidification is impacting marine life all over the world. Understanding how species can cope with the changes in seawater carbonate chemistry represents a challenging issue. We addressed this topic using underwater CO2 vents that naturally acidify some marine areas off the island of Ischia. In the most acidified area of the vents, having a mean pH value of 6.7, comparable to far-future predicted acidification scenarios (by 2300), the biomass is dominated by the brown alga Sargassum vulgare. The novelty of the present study is the characterization of the S. vulgare proteome together with metabolite analyses to identify the key proteins, metabolites, and pathways affected by ocean acidification. A total of 367 and 387 proteins were identified in populations grown at pH that approximates the current global average (8.1) and acidified sites, respectively. Analysis of their relative abundance revealed that 304 proteins are present in samples from both sites: 111 proteins are either higher or exclusively present under acidified conditions, whereas 120 proteins are either lower or present only under control conditions. Functionally, under acidification, a decrease in proteins related to translation and post-translational processes and an increase of proteins involved in photosynthesis, glycolysis, oxidation-reduction processes, and protein folding were observed. In addition, small-molecule metabolism was affected, leading to a decrease of some fatty acids and antioxidant compounds under acidification. Overall, the results obtained by proteins and metabolites analyses, integrated with previous transcriptomic, physiological, and biochemical studies, allowed us to delineate the molecular strategies adopted by S. vulgare to grow in future acidified environments, including an increase of proteins involved in energetic metabolism, oxidation-reduction processes, and protein folding at the expense of proteins involved in translation and post-translational processes.

Achieving malaria testing and treatment targets for children under five in Mozambique: a cost-effectiveness analysis.

BACKGROUND: The entire population of Mozambique is at risk for malaria, which remains one of the leading causes of death. The 2017-2022 National Malaria Strategic Plan focuses on reducing malaria morbidity and mortality in high- and low-transmission areas. This study aimed to estimate the costs and health benefits of six variations of the World Health Organization's "test-and-treat" strategy among children under five. METHODS: A decision tree model was developed that estimates the costs and health outcomes for children under five. Data on probabilities, costs, weights for disability-adjusted life years (DALYs), and quality-adjusted life years (QALYs) were based on peer-reviewed, grey literature, and primary data analysis of the 2018 Malaria Indicator Survey. Six scenarios were compared to the status quo and calculated the incremental cost-effectiveness ratio (ICER) in terms of cost per QALY gained, DALY averted, and life saved. Deterministic and probabilistic sensitivity analyses were conducted to understand the effect of parameter uncertainty on the findings. RESULTS: In the base case, reaching the target of 100% testing with rapid diagnostic tests (RDTs; Scenario 1) is more cost-effective than improving the testing rate alone by 10% (Scenario 2). Achieving a 100% (Scenario 3) or a 10% increase in treatment rate (Scenario 4) have ICERs that are lower than Scenarios 1 and 2. Both Scenarios 5 and 6, which represent combinations of Scenarios 1-4, have lower ICERs than their constituent strategies on their own, which suggests that improvements in treatment are more cost-effective than improvements in testing alone. These results held when DALYs averted or lives saved were used as health outcomes. Deterministic and probabilistic sensitivity analyses revealed that the cost-effectiveness of Scenarios 1-6 are subject sensitive to parameter uncertainty, though Scenarios 4 and 5 are the optimal choice when DALYs averted or QALYs gained were used as the measure of health outcomes across all cost-effectiveness thresholds. CONCLUSIONS: Improving testing rates alone among children at risk for malaria has the potential to improve health but may not be the most efficient use of limited resources. Instead, small or large improvements in treatment, whether alone or in conjunction with improvements in testing, are the most cost-effective strategies for children under five in Mozambique.

Antimicrobial D-amino acid oxidase-derived peptides specify gut microbiota.

The flavoenzyme D-amino acid oxidase (DAAO) is deputed to the degradation of D-enantiomers of amino acids. DAAO plays various relevant physiological roles in different organisms and tissues. Thus, it has been recently suggested that the goblet cells of the mucosal epithelia secrete into the lumen of intestine, a processed and active form of DAAO that uses the intestinal D-amino acids to generate hydrogen peroxide (H2O2), an immune messenger that helps fighting gut pathogens, and by doing so controls the homeostasis of gut microbiota. Here, we show that the DAAO form lacking the 1-16 amino acid residues (the putative secretion signal) is unstable and inactive, and that DAAO is present in the epithelial layer and the mucosa of mouse gut, where it is largely proteolyzed. In silico predicted DAAO-derived antimicrobial peptides show activity against various Gram-positive and Gram-negative bacteria but not on Lactobacilli species, which represent the commensal microbiota. Peptidomic analysis reveals the presence of such peptides in the mucosal fraction. Collectively, we identify a novel mechanism for gut microbiota selection implying DAAO-derived antimicrobial peptides which are generated by intestinal proteases and that are secreted in the gut lumen. In conclusion, we herein report an additional, ancillary role for mammalian DAAO, unrelated to its enzymatic activity.

The role of Savings and Internal Lending Communities (SILCs) in improving community-level household wealth, financial preparedness for birth, and utilization of reproductive health services in rural Zambia: a secondary analysis.

BACKGROUND: Savings and Internal Lending Communities (SILCs) are a type of informal microfinance mechanism widely adapted in Zambia. The benefits of SILCs paired with other interventions have been studied in many countries. However, limited studies have examined SILCs in the context of maternal health. This study examined the association between having access to SILCs and: 1) household wealth, 2) financial preparedness for birth, and 3) utilization of various reproductive health services (RHSs). METHODS: Secondary analysis was conducted on baseline and endline household survey data collected as part of a Maternity Waiting Home (MWH) intervention trial in 20 rural communities across seven districts of Zambia. Data from 4711 women who gave birth in the previous year (baseline: 2381 endline: 2330) were analyzed. The data were stratified into three community groups (CGs): CG1) communities with neither MWH nor SILC, CG2) communities with only MWH, and CG3) communities with both MWH and SILC. To capture the community level changes with the exposure to SILCs, different women were randomly selected from each of the communities for baseline and endline data, rather than same women being surveyed two times. Interaction effect of CG and timepoint on the outcome variables - household wealth, saving for birth, antenatal care visits, postnatal care visits, MWH utilization, health facility based delivery, and skilled provider assisted delivery - were examined. RESULTS: Interaction effect of CGs and timepoint were significantly associated only with MWH utilization, health facility delivery, and skilled provider delivery. Compared to women from CG3, women from CG1 had lower odds of utilizing MWHs and delivering at health facility at endline. Additionally, women from CG1 and women from CG2 had lower odds of delivering with a skilled provider compared to women from CG3. CONCLUSION: Access to SILCs was associated with increased MWH use and health facility delivery when MWHs were available. Furthermore, access to SILCs was associated with increased skilled provider delivery regardless of the availability of MWH. Future studies should explore the roles of SILCs in improving the continuity of reproductive health services. TRIAL REGISTRATION: NCT02620436.

A Wide-Proteome Analysis to Identify Molecular Pathways Involved in Kidney Response to High-Fat Diet in Mice.

The etiopathogenesis of obesity-related chronic kidney disease (CKD) is still scarcely understood. To this aim, we assessed the effect of high-fat diet (HF) on molecular pathways leading to organ damage, steatosis, and fibrosis. Six-week-old male C57BL/6N mice were fed HF diet or normal chow for 20 weeks. Kidneys were collected for genomic, proteomic, histological studies, and lipid quantification. The main findings were as follows: (1) HF diet activated specific pathways leading to fibrosis and increased fatty acid metabolism; (2) HF diet promoted a metabolic shift of lipid metabolism from peroxisomes to mitochondria; (3) no signs of lipid accumulation and/or fibrosis were observed, histologically; (4) the early signs of kidney damage seemed to be related to changes in membrane protein expression; (5) the proto-oncogene MYC was one of the upstream transcriptional regulators of changes occurring in protein expression. These results demonstrated the potential usefulness of specific selected molecules as early markers of renal injury in HF, while histomorphological changes become visible later in obesity-related CDK. The integration of these information with data from biological fluids could help the identification of biomarkers useful for the early detection and prevention of tissue damage in clinical practice.

Brain Proteome and Behavioural Analysis in Wild Type, BDNF+/- and BDNF-/- Adult Zebrafish (Danio rerio) Exposed to Two Different Temperatures.

Experimental evidence suggests that environmental stress conditions can alter the expression of BDNF and that the expression of this neurotrophin influences behavioural responses in mammalian models. It has been recently demonstrated that exposure to 34 °C for 21 days alters the brain proteome and behaviour in zebrafish. The aim of this work was to investigate the role of BDNF in the nervous system of adult zebrafish under control and heat treatment conditions. For this purpose, zebrafish from three different genotypes (wild type, heterozygous BDNF+/- and knock out BDNF-/-) were kept for 21 days at 26 °C or 34 °C and then euthanized for brain molecular analyses or subjected to behavioural tests (Y-maze test, novel tank test, light and dark test, social preference test, mirror biting test) for assessing behavioural aspects such as boldness, anxiety, social preference, aggressive behaviour, interest for the novel environment and exploration. qRT-PCR analysis showed the reduction of gene expression of BDNF and its receptors after heat treatment in wild type zebrafish. Moreover, proteomic analysis and behavioural tests showed genotype- and temperature-dependent effects on brain proteome and behavioural responding. Overall, the absent expression of BDNF in KO alters (1) the brain proteome by reducing the expression of proteins involved in synapse functioning and neurotransmitter-mediated transduction; (2) the behaviour, which can be interpreted as bolder and less anxious and (3) the cellular and behavioural response to thermal treatment.

Rational Design of a User-Friendly Aptamer/Peptide-Based Device for the Detection of Staphylococcus aureus.

The urgent need to develop a detection system for Staphylococcus aureus, one of the most common causes of infection, is prompting research towards novel approaches and devices, with a particular focus on point-of-care analysis. Biosensors are promising systems to achieve this aim. We coupled the selectivity and affinity of aptamers, short nucleic acids sequences able to recognize specific epitopes on bacterial surface, immobilized at high density on a nanostructured zirconium dioxide surface, with the rational design of specifically interacting fluorescent peptides to assemble an easy-to-use detection device. We show that the displacement of fluorescent peptides upon the competitive binding of S. aureus to immobilized aptamers can be detected and quantified through fluorescence loss. This approach could be also applied to the detection of other bacterial species once aptamers interacting with specific antigens will be identified, allowing the development of a platform for easy detection of a pathogen without requiring access to a healthcare environment.

High-Resolution Mass Spectrometry-Based Approaches for the Detection and Quantification of Peptidase Activity in Plasma.

Proteomic technologies have identified 234 peptidases in plasma but little quantitative information about the proteolytic activity has been uncovered. In this study, the substrate profile of plasma proteases was evaluated using two nano-LC-ESI-MS/MS methods. Multiplex substrate profiling by mass spectrometry (MSP-MS) quantifies plasma protease activity in vitro using a global and unbiased library of synthetic peptide reporter substrates, and shotgun peptidomics quantifies protein degradation products that have been generated in vivo by proteases. The two approaches gave complementary results since they both highlight key peptidase activities in plasma including amino- and carboxypeptidases with different substrate specificity profiles. These assays provide a significant advantage over traditional approaches, such as fluorogenic peptide reporter substrates, because they can detect active plasma proteases in a global and unbiased manner, in comparison to detecting select proteases using specific reporter substrates. We discovered that plasma proteins are cleaved by endoproteases and these peptide products are subsequently degraded by amino- and carboxypeptidases. The exopeptidases are more active and stable in plasma and therefore were found to be the most active proteases in the in vitro assay. The protocols presented here set the groundwork for studies to evaluate changes in plasma proteolytic activity in shock.

In vitro-digested milk proteins: Evaluation of angiotensin-1-converting enzyme inhibitory and antioxidant activities, peptidomic profile, and mucin gene expression in HT29-MTX cells.

Over the past decades, several studies investigated the health-promoting functions of milk peptides. However, to date many hurdles still exist regarding the widespread use of milk-derived bioactive peptides, as they may be degraded during gastrointestinal digestion. Thus, the aim of our study was to in vitro digest intact whey protein isolate (WPI) and casein proteins (CNP), mimicking in vivo digestion, to investigate their bioactive effects and to identify the potential peptides involved. Whey protein isolate and CNP were digested using a pepsin-pancreatin protocol and ultra-filtered (3-kDa cutoff membrane). A permeate (<3 kDa) and a retentate (>3 kDa) were obtained. Soy protein was included as a control (CTR). Angiotensin-1-converting enzyme inhibitory (ACE1-I) and antioxidant activity (AOX) were assessed and compared with those observed in undigested proteins and CTR. Furthermore, the permeate was characterized by nano-liquid chromatography electrospray ionization tandem mass spectrometry (LC-nano ESI MS/MS) using a shotgun peptidomic approach, and retentate was further digested with trypsin and analyzed by MS using a shotgun proteomic approach to identify potentially bioactive peptides. Further, the effects of WPI, CNP, and CTR retentate on cell metabolic activity and on mucus production (MUC5AC and MUC2 gene expression) were assessed in intestinal goblet HT29-MTX-E12 cells. Results showed that WPI permeate induced a significant ACE1-I inhibitory effect [49.2 ± 0.64% (SEM)] compared with undigested WPI, CNP permeate, and retentate or CTR permeate (10.40 ± 1.07%). A significant increase in AOX (1.58 ± 0.04 and 1.61 ± 0.02 µmol of trolox AOX equivalents per mg of protein, respectively) upon digestion was found in WPI. Potentially bioactive peptides associated with ACE1-I and antihypertensive effects were identified in WPI permeate and CNP retentate. At specific concentrations, WPI, CNP, and CTR retentate were able to stimulate metabolic activity in HT29-MTX-E12 cells. Expression of MUC5AC was increased by CNP retentate and unaltered by WPI retentate; MUC2 expression was significantly increased by 0.33 mg/g of CNP and reduced by 1.33 mg/g of CNP. Our results confirm that milk proteins may be rich sources of bioactive compounds, with the greatest beneficial potential of CNP at the intestinal goblet cell level.

Improving rational use of ACTs through diagnosis-dependent subsidies: Evidence from a cluster-randomized controlled trial in western Kenya.

BACKGROUND: More than half of artemisinin combination therapies (ACTs) consumed globally are dispensed in the retail sector, where diagnostic testing is uncommon, leading to overconsumption and poor targeting. In many malaria-endemic countries, ACTs sold over the counter are available at heavily subsidized prices, further contributing to their misuse. Inappropriate use of ACTs can have serious implications for the spread of drug resistance and leads to poor outcomes for nonmalaria patients treated with incorrect drugs. We evaluated the public health impact of an innovative strategy that targets ACT subsidies to confirmed malaria cases by coupling free diagnostic testing with a diagnosis-dependent ACT subsidy. METHODS AND FINDINGS: We conducted a cluster-randomized controlled trial in 32 community clusters in western Kenya (population approximately 160,000). Eligible clusters had retail outlets selling ACTs and existing community health worker (CHW) programs and were randomly assigned 1:1 to control and intervention arms. In intervention areas, CHWs were available in their villages to perform malaria rapid diagnostic tests (RDTs) on demand for any individual >1 year of age experiencing a malaria-like illness. Malaria RDT-positive individuals received a voucher for a discount on a quality-assured ACT, redeemable at a participating retail medicine outlet. In control areas, CHWs offered a standard package of health education, prevention, and referral services. We conducted 4 population-based surveys-at baseline, 6 months, 12 months, and 18 months-of a random sample of households with fever in the last 4 weeks to evaluate predefined, individual-level outcomes. The primary outcome was uptake of malaria diagnostic testing at 12 months. The main secondary outcome was rational ACT use, defined as the proportion of ACTs used by test-positive individuals. Analyses followed the intention-to-treat principle using generalized estimating equations (GEEs) to account for clustering with prespecified adjustment for gender, age, education, and wealth. All descriptive statistics and regressions were weighted to account for sampling design. Between July 2015 and May 2017, 32,404 participants were tested for malaria, and 10,870 vouchers were issued. A total of 7,416 randomly selected participants with recent fever from all 32 clusters were surveyed. The majority of recent fevers were in children under 18 years (62.9%, n = 4,653). The gender of enrolled participants was balanced in children (49.8%, n = 2,318 boys versus 50.2%, n = 2,335 girls), but more adult women were enrolled than men (78.0%, n = 2,139 versus 22.0%, n = 604). At baseline, 67.6% (n = 1,362) of participants took an ACT for their illness, and 40.3% (n = 810) of all participants took an ACT purchased from a retail outlet. At 12 months, 50.5% (n = 454) in the intervention arm and 43.4% (n = 389) in the control arm had a malaria diagnostic test for their recent fever (adjusted risk difference [RD] = 9 percentage points [pp]; 95% CI 2-15 pp; p = 0.015; adjusted risk ratio [RR] = 1.20; 95% CI 1.05-1.38; p = 0.015). By 18 months, the ARR had increased to 1.25 (95% CI 1.09-1.44; p = 0.005). Rational use of ACTs in the intervention area increased from 41.7% (n = 279) at baseline to 59.6% (n = 403) and was 40% higher in the intervention arm at 18 months (ARR 1.40; 95% CI 1.19-1.64; p < 0.001). While intervention effects increased between 12 and 18 months, we were not able to estimate longer-term impact of the intervention and could not independently evaluate the effects of the free testing and the voucher on uptake of testing. CONCLUSIONS: Diagnosis-dependent ACT subsidies and community-based interventions that include the private sector can have an important impact on diagnostic testing and population-wide rational use of ACTs. Targeting of the ACT subsidy itself to those with a positive malaria diagnostic test may also improve sustainability and reduce the cost of retail-sector ACT subsidies. TRIAL REGISTRATION: ClinicalTrials.gov NCT02461628.

Structural and functional insights into the basic globulin 7S of soybean seeds by using trypsin as a molecular probe.

The basic 7S globulin (Bg7S) is one of the major globulins of soybean seeds. Despite its dual subunit composition and oligomeric assembly, Bg7S has a compact 3D structure (PDB: 3AUP) which is stabilized by a network of inter- and intra-chain disulphide bridges. Bg7S shares several structural elements with a number of homologous proteins from other seeds, whose function is still uncertain. In this work, Bg7S native conformation was probed by using the proteolytic enzyme trypsin. In spite of the presence of many arginine and lysine residues, the protein resulted extremely recalcitrant to in vitro enzymatic cleavage. Indeed, only two scissile bonds located near the C- and N-termini of the large and small subunits, respectively, were cleaved. The partially cleaved products were stable even at prolonged incubation times. Although the generated small peptide fragments were not covalently bound to the remnant of the main chains, they were held in place, as assessed by denaturing and non-denaturing chromatographic approaches. Moreover, both the already observed pH-dependent association/dissociation behaviour of the protein and its insulin binding capacity were preserved both at neutral and acidic pH values. These results are in line with the growing view that the degradation of seed proteins, either storage and non-storage, may be a controlled process related to specific functionalities.

Toward the Standardization of Mitochondrial Proteomics: The Italian Mitochondrial Human Proteome Project Initiative.

The Mitochondrial Human Proteome Project aims at understanding the function of the mitochondrial proteome and its crosstalk with the proteome of other organelles. Being able to choose a suitable and validated enrichment protocol of functional mitochondria, based on the specific needs of the downstream proteomics analysis, would greatly help the researchers in the field. Mitochondrial fractions from ten model cell lines were prepared using three enrichment protocols and analyzed on seven different LC-MS/MS platforms. All data were processed using neXtProt as reference database. The data are available for the Human Proteome Project purposes through the ProteomeXchange Consortium with the identifier PXD007053. The processed data sets were analyzed using a suite of R routines to perform a statistical analysis and to retrieve subcellular and submitochondrial localizations. Although the overall number of identified total and mitochondrial proteins was not significantly dependent on the enrichment protocol, specific line to line differences were observed. Moreover, the protein lists were mapped to a network representing the functional mitochondrial proteome, encompassing mitochondrial proteins and their first interactors. More than 80% of the identified proteins resulted in nodes of this network but with a different ability in coisolating mitochondria-associated structures for each enrichment protocol/cell line pair.

Hydrogen peroxide-mediated induction of SOD1 gene transcription is independent from Nrf2 in a cellular model of neurodegeneration.

BACKGROUND: It is still unclear whether oxidative stress (OS) is a disease consequence or is directly involved in the etiology of neurodegenerative disorders (NDs) onset and/or progression; however, many of these conditions are associated with increased levels of oxidation markers and damaged cell components. Previously we demonstrated the accumulation of reactive oxygen species (ROS) and increased SOD1 gene expression in H2O2-treated SH-SY5Y cells, recapitulating pathological features of Amyotrophic Lateral Sclerosis (ALS). Since we observed a post-transcriptional regulation of SOD1 gene in this cellular model, we investigated the transcriptional regulation of SOD1 mRNA under oxidative stress (OS). RESULTS: In response to H2O2 treatment, PolII increased its association to SOD1 promoter. Electrophoretic mobility shift assays and mass spectrometry analyses on SOD1 promoter highlighted the formation of a transcriptional complex bound to the ARE sequences. Western Blotting experiments showed that in our in vitro model, H2O2 exposure increases Nrf2 expression in the nuclear fraction while immunoprecipitation confirmed its phosphorylation and release from Keap1 inhibition. However, H2O2 treatment did not modify Nrf2 binding on SOD1 promoter, which seems to be regulated by different transcription factors (TFs). CONCLUSIONS: Although our data suggest that SOD1 is transcriptionally regulated in response to OS, Nrf2 does not appear to associate with SOD1 promoter in this cellular model of neurodegeneration. Our results open new perspectives in the comprehension of two key antioxidant pathways involved in neurodegenerative disorders.

Household beliefs about malaria testing and treatment in Western Kenya: the role of health worker adherence to malaria test results.

BACKGROUND: Although use of malaria diagnostic tests has increased in recent years, health workers often prescribe anti-malarial drugs to individuals who test negative for malaria. This study investigates how health worker adherence to malaria case management guidelines influences individuals' beliefs about whether their illness was malaria, and their confidence in the effectiveness of artemisinin-based combination therapy (ACT). METHODS: A survey was conducted with 2065 households in Western Kenya about a household member's treatment actions for a recent febrile illness. The survey also elicited the individual's (or their caregiver's) beliefs about the illness and about malaria testing and treatment. Logistic regressions were used to test the association between these beliefs and whether the health worker adhered to malaria testing and treatment guidelines. RESULTS: Of the 1070 individuals who visited a formal health facility during their illness, 82% were tested for malaria. ACT rates for malaria-positive and negative individuals were 89 and 49%, respectively. Overall, 65% of individuals/caregivers believed that the illness was "very likely" malaria. Individuals/caregivers had higher odds of saying that the illness was "very likely" malaria when the individual was treated with ACT, and this was the case both among individuals not tested for malaria [adjusted odds ratio (AOR) 3.42, 95% confidence interval (CI) [1.65 7.10], P = 0.001] and among individuals tested for malaria, regardless of their test result. In addition, 72% of ACT-takers said the drug was "very likely" effective in treating malaria. However, malaria-negative individuals who were treated with ACT had lower odds of saying that the drugs were "very likely" effective than ACT-takers who were not tested or who tested positive for malaria (AOR 0.29, 95% CI [0.13 0.63], P = 0.002). CONCLUSION: Individuals/caregivers were more likely to believe that the illness was malaria when the patient was treated with ACT, regardless of their test result. Moreover, malaria-negative individuals treated with ACT had lower confidence in the drug than other individuals who took ACT. These results suggest that ensuring health worker adherence to malaria case management guidelines will not only improve ACT targeting, but may also increase patient/caregivers' confidence in malaria testing and treatment.