System-wide analysis of RNA and protein subcellular localization dynamics.

Although the subcellular dynamics of RNA and proteins are key determinants of cell homeostasis, their characterization is still challenging. Here we present an integrative framework to simultaneously interrogate the dynamics of the transcriptome and proteome at subcellular resolution by combining two methods: localization of RNA (LoRNA) and a streamlined density-based localization of proteins by isotope tagging (dLOPIT) to map RNA and protein to organelles (nucleus, endoplasmic reticulum and mitochondria) and membraneless compartments (cytosol, nucleolus and cytosolic granules). Interrogating all RNA subcellular locations at once enables system-wide quantification of the proportional distribution of RNA. We obtain a cell-wide overview of localization dynamics for 31,839 transcripts and 5,314 proteins during the unfolded protein response, revealing that endoplasmic reticulum-localized transcripts are more efficiently recruited to cytosolic granules than cytosolic RNAs, and that the translation initiation factor eIF3d is key to sustaining cytoskeletal function. Overall, we provide the most comprehensive overview so far of RNA and protein subcellular localization dynamics.

Proteomic analysis in primary T cells reveals IL-7 alters T cell receptor thresholding via CYTIP/cytohesin/LFA-1 localisation and activation.

The ability of the cellular immune system to discriminate self from foreign antigens depends on the appropriate calibration of the T cell receptor (TCR) signalling threshold. The lymphocyte homeostatic cytokine interleukin 7 (IL-7) is known to affect TCR thresholding, but the molecular mechanism is not fully elucidated. A better understanding of this process is highly relevant in the context of autoimmune disease therapy and cancer immunotherapy. We sought to characterise the early signalling events attributable to IL-7 priming; in particular, the altered phosphorylation of signal transduction proteins and their molecular localisation to the TCR. By integrating high-resolution proximity- phospho-proteomic and imaging approaches using primary T cells, rather than engineered cell lines or an in vitro expanded T cell population, we uncovered transduction events previously not linked to IL-7. We show that IL-7 leads to dephosphorylation of cytohesin interacting protein (CYTIP) at a hitherto undescribed phosphorylation site (pThr280) and alters the co-localisation of cytohesin-1 with the TCR and LFA-1 integrin. These results show that IL-7, acting via CYTIP and cytohesin-1, may impact TCR activation thresholds by enhancing the co-clustering of TCR and LFA-1 integrin.

Changes in the Oligodendrocyte Progenitor Cell Proteome with Ageing.

Following central nervous system (CNS) demyelination, adult oligodendrocyte progenitor cells (OPCs) can differentiate into new myelin-forming oligodendrocytes in a regenerative process called remyelination. Although remyelination is very efficient in young adults, its efficiency declines progressively with ageing. Here we performed proteomic analysis of OPCs freshly isolated from the brains of neonate, young and aged female rats. Approximately 50% of the proteins are expressed at different levels in OPCs from neonates compared with their adult counterparts. The amount of myelin-associated proteins, and proteins associated with oxidative phosphorylation, inflammatory responses and actin cytoskeletal organization increased with age, whereas cholesterol-biosynthesis, transcription factors and cell cycle proteins decreased. Our experiments provide the first ageing OPC proteome, revealing the distinct features of OPCs at different ages. These studies provide new insights into why remyelination efficiency declines with ageing and potential roles for aged OPCs in other neurodegenerative diseases.

Reductions in sugar sales from soft drinks in the UK from 2015 to 2018.

BACKGROUND: The consumption of free sugars in the UK is more than double the guideline intake for adults and close to triple for children, with soft drinks representing a significant proportion. The aim of this study was to assess how individual soft drink companies and consumers have responded to calls to reduce sugar consumption, including the soft drink industry levy (SDIL), between 2015 and 2018. METHODS: This was an annual cross-sectional study using nutrient composition data of 7377 products collected online, paired with volume sales data for 195 brands offered by 57 companies. The main outcome measures were sales volume, sugar content and volume of sugars sold by company and category, expressed in total and per capita per day terms. RESULTS: Between 2015 and 2018, the volume of sugars sold per capita per day from soft drinks declined by 30%, equivalent to a reduction of 4.6 g per capita per day. The sales-weighted mean sugar content of soft drinks fell from 4.4 g/100 ml in 2015 to 2.9 g/100 ml in 2018. The total volume sales of soft drinks that are subject to the SDIL (i.e. contain more than 5 g/100 ml of sugar) fell by 50%, while volume sales of low- and zero-sugar (< 5 g/100 ml) drinks rose by 40%. CONCLUSION: Action by the soft drinks industry to reduce sugar in products and change their product portfolios, coupled with changes in consumer purchasing, has led to a significant reduction in the total volume and per capita sales of sugars sold in soft drinks in the UK. The rate of change accelerated between 2017 and 2018, which also implies that the implementation of the SDIL acted as an extra incentive for companies to reformulate above and beyond what was already being done as part of voluntary commitments to reformulation, or changes in sales driven by consumer preferences.

Nutritional quality of packaged foods targeted at children in Brazil: which ones should be eligible to bear nutrient claims?

OBJECTIVES: This study aimed to assess the nutritional quality of food products marketed at children, with and without nutrient claims, using two different approaches. METHODS: Analyses were performed based on a data set with food composition and labelling data from every packaged food marketed at children sold in a major Brazilian supermarket (n=535). Foods were classified as 'healthier' and 'less healthy' according to the UK/Ofcom nutrient profile model and to the NOVA classification based on the level of food processing. Pearson's χ2 test was used to compare proportions between models. Agreement was assessed using Cohen's κ-statistic (P<0.05). RESULTS: The NOVA model was stricter than the UK/Ofcom model, classifying more products as 'less healthy' (91.4%) compared with the nutrient profile-based model (75.0%; P<0.001). Agreement between models was 79.4% (k=0.30), because 72.9% (n=390) of products were categorised as 'less healthy' by both models, and 6.5% (n=35) as 'healthier'. Half of the food products marketed at children from the database (270; 50.5%) bore nutrient claims. From these products with nutrient claims, 95.9% (92.8-98.0) were classified as 'less healthy' by the NOVA model, whereas this percentage was 74.1% (68.4-79.2) according to the UK/Ofcom model (P<0.05). CONCLUSIONS: The high number of foods with low nutritional quality being marketed at children via product packaging and nutrient claims should be of concern to policy makers wanting to improve children's diets and to tackle childhood obesity. The implementation of nutritional quality criteria to ensure that foods targeted at children should be eligible to bear nutrient claims on their labels could avoid a situation where claims mask the overall nutritional status of a food.

Quantitative proteomic and phosphoproteomic analysis of Trypanosoma cruzi amastigogenesis.

Chagas disease is a tropical neglected disease endemic in Latin America caused by the protozoan Trypanosoma cruzi. The parasite has four major life stages: epimastigote, metacyclic trypomastigote, bloodstream trypomastigote, and amastigote. The differentiation from infective trypomastigotes into replicative amastigotes, called amastigogenesis, takes place in vivo inside mammalian host cells after a period of incubation in an acidic phagolysosome. This differentiation process can be mimicked in vitro by incubating tissue-culture-derived trypomastigotes in acidic DMEM. Here we used this well-established differentiation protocol to perform a comprehensive quantitative proteomic and phosphoproteomic analysis of T. cruzi amastigogenesis. Samples from fully differentiated forms and two biologically relevant intermediate time points were Lys-C/trypsin digested, iTRAQ-labeled, and multiplexed. Subsequently, phosphopeptides were enriched using a TiO2 matrix. Non-phosphorylated peptides were fractionated via hydrophilic interaction liquid chromatography prior to LC-MS/MS analysis. LC-MS/MS and bioinformatics procedures were used for protein and phosphopeptide quantitation, identification, and phosphorylation site assignment. We were able to identify regulated proteins and pathways involved in coordinating amastigogenesis. We also observed that a significant proportion of the regulated proteins were membrane proteins. Modulated phosphorylation events coordinated by protein kinases and phosphatases that are part of the signaling cascade induced by incubation in acidic medium were also evinced. To our knowledge, this work is the most comprehensive quantitative proteomics study of T. cruzi amastigogenesis, and these data will serve as a trustworthy basis for future studies, and possibly for new potential drug targets.

Cell surface proteome analysis of human-hosted Trypanosoma cruzi life stages.

Chagas' disease is a neglected infectious illness, caused by the protozoan Trypanosoma cruzi. It remains a challenging health issue in Latin America, where it is endemic, and so far there is no immunoprophylatic vaccine or satisfactory chemotherapic treatment for its chronic stage. The present work addressed the analysis of the plasma membrane (PM) subproteome from T. cruzi human-hosted life stages, trypomastigote and axenic amastigote, by two complementary PM protein enrichment techniques followed by identification using an LC-MS/MS approach. The results revealed an extensive repertoire of proteins in the PM subproteomes, including enzymes that might be suitable candidates for drug intervention. The comparison of the cell surface proteome among the life forms revealed some potentially stage-specific enzymes, although the majority was shared by both stages. Bioinformatic analysis showed that the vast majority of the identified proteins are membrane-derived and/or possess predicted transmembrane domains. They are mainly involved in host cell infection, protein adhesion, cell signaling, and the modulation of mammalian host immune response. Several virulence factors and proteins potentially capable of acting at a number of metabolic pathways of the host and also to regulate cell differentiation of the parasite itself were also found.

Physicochemical and functional properties of short-chain fatty acid starch modified with different acyl groups and levels of modification.

Rice and quinoa starches are modified with short-chain fatty acids (SCFA) with different SCFA acyl chain lengths and levels of modification. This work is aimed to investigate the impact of modifying rice and quinoa starches with short-chain fatty acids (SCFAs) on various physicochemical properties, including particle size, protein and amylose content, thermal behavior, pasting characteristics, and in vitro digestibility. Both native and SCFA-starches showed comparable particle sizes, with rice starches ranging from 1.58 to 2.22 µm and quinoa starches from 5.18 to 5.72 µm. SCFA modification led to lower protein content in both rice (0.218-0.255 %) and quinoa starches (0.537-0.619 %) compared to their native counterparts. Esterification led to the reduction of gelatinization and pasting temperatures as well as the hardness of the paste of SCFA-starches were reduced while paste clarity increased. The highest level of modification in SCFA-starch was associated with the highest amount of resistant starch fraction. Principal component analysis revealed that modification levels exerted a greater influence on starch properties than the types of SCFA used (acetyl, propionyl, and butyryl). These findings is importance in considering the degree of substitution or level of modification when tailoring starch properties through SCFA modification, with implications for various applications in food applications.

Comprehensive proteomic analysis of Trypanosoma cruzi epimastigote cell surface proteins by two complementary methods.

Trypanosoma cruzi is a protozoan that causes Chagas' disease, a neglected infectious illness that affects millions of people, mostly in Latin America. Here, the cell surface subproteome of the T. cruzi epimastigote life form was characterized. In order to prepare samples enriched in epimastigote plasma membrane protein, two distinct methodologies were optimized and evaluated. The first methodology was based on cell surface trypsinization (Shave) of intact living cells while the second approach used biotinylation of cell surface proteins followed by streptavidin affinity chromatography isolation of the labeled proteins. Both T. cruzi subproteomes were analyzed by LC-MS/MS. The results showed that the methodologies offered comprehensive and complementary information about the parasite's plasma membrane subproteome.

The impact of badminton lessons on health and wellness of young adults with intellectual disabilities: a pilot study.

Background: Physical activity has been proposed as a context to foster the healthy development of individuals and reduce the risk of many chronic problems. This study evaluates the impact of badminton lessons on health and wellness in young adults with intellectual disabilities (ID). Methods: Eighteen participants with ID (14 males and 4 females, aged 19-26) and with little or no experience in badminton were assigned to an exercise group and a control group. The curriculum selected was Shuttle Time Starter Lessons. The exercise group practiced for 50 min each session, twice a week for 10 lessons with peers, while the control group maintained a regular life schedule. Physiological measures, motor performance, Special Olympics Individual Badminton Skills Assessment; and psychological measures were conducted before and after the program. A Wilcoxon signed-rank test was conducted to compare pre- and post-tests in each group. Results: The significantly reduced resting heart rate, longer walking distances in the 6-minute walk test, and better performance in badminton skills were evident in the exercise group. Further, a significantly increased left frontal alpha asymmetry was seen in the exercise group with participants expressing positive effects after the inclusive badminton program. Finally, resting EEG frontal asymmetry seemed to be reflective of emotion in persons with ID. Conclusions: Shuttle Time Badminton Lessons could be feasible for adults with ID. School teachers and coaches may adapt it to improve health and wellness and acquire badminton skills in adults with ID. In addition, the inclusive environment can motivate their participation.

Drosophila nicotinic acetylcholine receptor subunits and their native interactions with insecticidal peptide toxins.

Drosophila nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that represent a target for insecticides. Peptide neurotoxins are known to block nAChRs by binding to their target subunits, however, a better understanding of this mechanism is needed for effective insecticide design. To facilitate the analysis of nAChRs we used a CRISPR/Cas9 strategy to generate null alleles for all ten nAChR subunit genes in a common genetic background. We studied interactions of nAChR subunits with peptide neurotoxins by larval injections and styrene maleic acid lipid particles (SMALPs) pull-down assays. For the null alleles, we determined the effects of α-Bungarotoxin (α-Btx) and ω-Hexatoxin-Hv1a (Hv1a) administration, identifying potential receptor subunits implicated in the binding of these toxins. We employed pull-down assays to confirm α-Btx interactions with the Drosophila α5 (Dα5), Dα6, Dα7 subunits. Finally, we report the localisation of fluorescent tagged endogenous Dα6 during Drosophila CNS development. Taken together, this study elucidates native Drosophila nAChR subunit interactions with insecticidal peptide toxins and provides a resource for the in vivo analysis of insect nAChRs.

CFTR Rescue by Lumacaftor (VX-809) Induces an Extensive Reorganization of Mitochondria in the Cystic Fibrosis Bronchial Epithelium.

BACKGROUND: Cystic Fibrosis (CF) is a genetic disorder affecting around 1 in every 3000 newborns. In the most common mutation, F508del, the defective anion channel, CFTR, is prevented from reaching the plasma membrane (PM) by the quality check control of the cell. Little is known about how CFTR pharmacological rescue impacts the cell proteome. METHODS: We used high-resolution mass spectrometry, differential ultracentrifugation, machine learning and bioinformatics to investigate both changes in the expression and localization of the human bronchial epithelium CF model (F508del-CFTR CFBE41o-) proteome following treatment with VX-809 (Lumacaftor), a drug able to improve the trafficking of CFTR. RESULTS: The data suggested no stark changes in protein expression, yet subtle localization changes of proteins of the mitochondria and peroxisomes were detected. We then used high-content confocal microscopy to further investigate the morphological and compositional changes of peroxisomes and mitochondria under these conditions, as well as in patient-derived primary cells. We profiled several thousand proteins and we determined the subcellular localization data for around 5000 of them using the LOPIT-DC spatial proteomics protocol. CONCLUSIONS: We observed that treatment with VX-809 induces extensive structural and functional remodelling of mitochondria and peroxisomes that resemble the phenotype of healthy cells. Our data suggest additional rescue mechanisms of VX-809 beyond the correction of aberrant folding of F508del-CFTR and subsequent trafficking to the PM.

Insight into the salivary transcriptome and proteome of Dipetalogaster maxima.

Dipetalogaster maxima is a blood-sucking Hemiptera that inhabits sylvatic areas in Mexico. It usually takes its blood meal from lizards, but following human population growth, it invaded suburban areas, feeding also on humans and domestic animals. Hematophagous insect salivary glands produce potent pharmacologic compounds that counteract host hemostasis, including anticlotting, antiplatelet, and vasodilatory molecules. To obtain further insight into the salivary biochemical and pharmacologic complexity of this insect, a cDNA library from its salivary glands was randomly sequenced. Salivary proteins were also submitted to one- and two-dimensional gel electrophoresis (1DE and 2DE) followed by mass spectrometry analysis. We present the analysis of a set of 2728 cDNA sequences, 1375 of which coded for proteins of a putative secretory nature. The saliva 2DE proteome displayed approximately 150 spots. The mass spectrometry analysis revealed mainly lipocalins, pallidipins, antigen 5-like proteins, and apyrases. The redundancy of sequence identification of saliva-secreted proteins suggests that proteins are present in multiple isoforms or derive from gene duplications.

Resveratrol loaded Pickering emulsions stabilized by OSA modified rice starch granules.

Resveratrol is a photosensitive, bioactive molecule which has received increasing research interest during the past decade for its antioxidant properties. However, it has low solubility in water or common triglyceride oils. Resveratrol solubilization in oil can only be achieved in essential oils, such as flavour oils, but the stability of emulsions produced with this type of oils is low as they are prone to creaming phenomena and Oswald ripening. In this study, resveratrol was first dissolved in orange oil which was mixed into a medium-chain triglyceride (Miglyol) at different ratios and used as the internal phase of oil-in-water emulsions (O/W). The emulsions were stabilized by octenyl succinic anhydride (OSA) modified rice starch granules using two different ratios of starch particle:oil to study the influence of interfacial coverage on the final emulsion droplet size and emulsion stability. The results of this study indicated that stable Pickering emulsions could be prepared using OSA-modified rice starch granules even at partial coverage conditions. Emulsions prepared at an oil fraction of 0.5 using 30% v/v mixture of orange oil in Miglyol as the dispersed phase seemed to be an appropriate resveratrol carrier system, obtaining encapsulation efficiency values close to 90% which results in emulsions with a resveratrol concentration of 8.45 mg/L. Hence, the emulsions prepared are suitable for food fortification applications.

A new conceptual model of pesticide transfers from agricultural land to surface waters with a specific focus on metaldehyde.

Pesticide losses from agricultural land to water can result in the environmental deterioration of receiving systems. Mathematical models can make important contributions to risk assessments and catchment management. However, some mechanistic models have high parameter requirements which can make them difficult to apply in data poor areas. In addition, uncertainties in pesticide properties and applications are difficult to account for using models with long run-times. Alternative, simpler, conceptual models are easier to apply and can still be used as a framework for process interpretation. Here, we present a new conceptual model of pesticide behaviour in surface water catchments, based on continuous water balance calculations. Pesticide losses to surface waters are calculated based on the displacement of a limited fraction of the soil pore water during storm events occurring after application. The model was used to describe the behaviour of metaldehyde in a small (2.2 km2) under-drained catchment in Eastern England. Metaldehyde is a molluscicide which has been regularly detected at high concentrations in many drinking water supply catchments. Measured peak concentrations in stream water (to about 9 µg L-1) occurred in the first few storm events after application in mid-August. In each event, there was a quasi-exponential decrease in concentration during hydrograph recession. Peak concentrations decreased in successive events - responding to rainfall but reflecting an effective exhaustion in soil supply due to degradation and dissipation. Uncertain pesticide applications to the catchment were estimated using land cover analysis of satellite data, combined with a Poisson distribution to describe the timing of application. Model performance for both the hydrograph (after calibration of the water balance) and the chemograph was good and could be improved via some minor adjustments in assumptions which yield general insights into the drivers for pesticide transport. The use of remote sensing offers some promising opportunities for estimating catchment-scale pesticide applications and associated losses.

Synthesis of controlled size starch nanoparticles (SNPs).

Starch nanoparticles (SNPs) are a promising choice for the strategic development of new renewable and biodegradable nanomaterials for novel biomedical and pharmaceutical applications when loaded with antibiotics or with anticancer agents as target drug delivery systems. The final properties of the SNPs are strongly influenced by the synthesis method and conditions being a controlled and monodispersed size crucial for these applications. The aim of this work was to synthesize controlled size SNPs through nanoprecipitation and microemulsion methods by modifying main operating parameters regarding the effect of amylose and amylopectin ratio in maize starches. SNPs were characterized by size and shape. SNPs from 59 to 118 nm were obtained by the nanoprecipitation method, registering the higer values when surfactant was added to the aqueous phase. Microemulsion method led to 35-147 nm sizes observing a higher particle formation capacity. The composition of the maize used influenced the final particle size and shape.

Food labour, consumption hierarchies, and diet decision-making in Sri Lankan households: a qualitative study.

BACKGROUND: Sri Lanka faces the double burden of over- and undernutrition. To tackle this dual challenge, double duty interventions that improve the quality of the Sri Lankan diet in line with national dietary guidelines have been suggested. The success of these interventions depends upon an understanding of the context-specific factors that impact their uptake within the population. The purpose of this study was threefold: explore household responsibility for food-related labour; understand food decision-making influences; and investigate consumption hierarchies that might impact the distribution of intervention benefits. METHODS: We conducted face-to-face semi-structured interviews with 93 Sri Lankan adults residing in urban Colombo (n = 56), and urban and rural sectors in Kalutara (n = 29) and Trincomalee (n = 8). Interview data were analysed thematically. RESULTS: Findings from this study suggest that women in Sri Lanka continue to shoulder the burden of food-related labour disproportionately to men but that this responsibility is not always a proxy for dietary decision-making power. While men are often absent from the kitchen, their role in food purchasing and payment is prominent in many households. Despite these observed gender differences in food labour and provisioning, "traditional" age- and gender-based consumption hierarchies with negative nutrition consequences for women and children are not common, indicating that Sri Lankan 'table culture' may be changing. CONCLUSION: Dietary interventions with the aim of influencing day-to-day practice should be developed with an awareness of who is responsible for, who is able to perform, and who influences targeted behaviours.

Characterization and stability of short-chain fatty acids modified starch Pickering emulsions.

Acetylated, propionylated and butyrylated rice and quinoa starches at different levels of modification and starch concentrations, were used to stabilize oil-in-water starch Pickering emulsions at 10% oil fraction. Short-chain fatty acid modified starch Pickering emulsions (SPEs) were characterized after emulsification and after 50 days of storage. The particle size distribution, microstructure, emulsion index, and stability were evaluated. An increase in starch concentration led to a decrease of emulsion droplet sizes. Quinoa starch has shown the capability of stabilizing Pickering emulsions in both the native and modified forms. The emulsifying capacity of SPEs was improved by increasing the chain length of SCFA. Modified quinoa starch with higher chain lengths (i.e. propionylated and butyrylated), at higher levels of modification, showed higher emulsion index (>71%) and stability over the entire 50 days storage. At optimized formulation, SCFA-starch particles have the potential in stabilizing emulsions for functional foods, pharmaceutical formulations, or industrial food applications.

Quantifying regeneration in patients following peripheral nerve injury.

Healthy nerve function provides humans with the control of movement; sensation (such as pain, touch and temperature) and the quality of skin, hair and nails. Injury to this complex system creates a deficit in function, which is slow to recover, and rarely, if ever, returns to what patients consider to be normal. Despite promising results in pre-clinical animal experimentation effective translation is challenged by a current inability to quantify nerve regeneration in human subjects and relate this to measurable and responsible clinical outcomes. In animal models, muscle and nerve tissue samples can be harvested following experimental intervention. This allows direct quantification of muscle mass and quality and quantity of regeneration of axons; such an approach is not applicable in human medicine as it would ensure a significant functional deficit. Nevertheless a greater understanding of this process would allow the relationship that exists between neural and neuromuscular regeneration and functional outcome to be more clearly understood. This article presents a combined commentary of current practice from a specialist clinical unit and research team with regard to laboratory and clinical quantification of nerve regeneration. We highlight how electrophysiological diagnostic methods (which are used with significant recognised limitations in the assessment of clinical medicine) can potentially be used with more validity to interpret and assess the processes of neural regeneration in the clinical context, thus throwing light on the factors at play in translating lab advances into the clinic.