A qualitative inquiry of food insecurity in Belize.

OBJECTIVE: To explore and provide contextual meaning around issues surrounding food insecurity, namely factors influencing food access, as one domain of food security. DESIGN: A community-based, qualitative inquiry using semi-structured face-to-face interviews was conducted as part of a larger sequential mixed-methods study. SETTING: Cayo District, Belize, May 2019-August 2019. PARTICIPANTS: Thirty English-speaking individuals (eight males, twenty-two females) between the ages of 18-70, with varying family composition residing within the Cayo District. RESULTS: Participants describe a complex interconnectedness between family- and individual-level barriers to food access. Specifically, family composition, income, education and employment influence individuals' ability to afford and access food for themselves or their families. Participants also cite challenges with transportation and distance to food sources and educational opportunities as barriers to accessing food. CONCLUSION: These findings provide insight around food security and food access barriers in a middle-income country and provide avenues for further study and potential interventions. Increased and sustained investment in primary and secondary education, including programmes to support enrollment, should be a priority to decreasing food insecurity. Attention to building public infrastructure may also ease burdens around accessing foods.

Proteolipidic composition of exosomes changes during reticulocyte maturation.

During the orchestrated process leading to mature erythrocytes, reticulocytes must synthesize large amounts of hemoglobin, while eliminating numerous cellular components. Exosomes are small secreted vesicles that play an important role in this process of specific elimination. To understand the mechanisms of proteolipidic sorting leading to their biogenesis, we have explored changes in the composition of exosomes released by reticulocytes during their differentiation, in parallel to their physical properties. By combining proteomic and lipidomic approaches, we found dramatic alterations in the composition of the exosomes retrieved over the course of a 7-day in vitro differentiation protocol. Our data support a previously proposed model, whereby in reticulocytes the biogenesis of exosomes involves several distinct mechanisms for the preferential recruitment of particular proteins and lipids and suggest that the respective prominence of those pathways changes over the course of the differentiation process.

Agonist-selective dynamic compartmentalization of human Mu opioid receptor as revealed by resolutive FRAP analysis.

Techniques for analyzing the membrane diffusion of molecules are the most promising methods for investigating the compartmentalization of G-protein-coupled receptors, particularly as relevant to receptor signaling processes. Here, we report fluorescence recovery after photobleaching (FRAP) measurements performed at variable spot radius for human mu opioid (hMOP) receptors on SH-SY5Y neuroblastoma cells in the presence of ligands. Although an antagonist did not affect the behavior of the receptors compared with the basal state, two different agonists, DAMGO and morphine, caused markedly different changes to receptor diffusion. Like receptors in the absence of ligand, receptors bound to morphine exhibited diffusion confined to joined semipermeable domains, but with smaller domain size and diffusion coefficient. This effect was inhibited by pertussis toxin, strongly suggesting that this dynamic behavior is associated with early steps of signaling. In the presence of DAMGO, half of the receptors displayed free long-range diffusion and the other half were confined to smaller isolated domains. Hypertonic sucrose buffer suppressed this effect, which we attribute to receptor entry into clathrin-coated pits. It is likely that the observation of distinct receptor dynamics in the presence of DAMGO and morphine involves the agonist-selective phosphorylation of the receptor.

Phthiocerol dimycocerosates of M. tuberculosis participate in macrophage invasion by inducing changes in the organization of plasma membrane lipids.

Phthiocerol dimycocerosates (DIM) are major virulence factors of Mycobacterium tuberculosis (Mtb), in particular during the early step of infection when bacilli encounter their host macrophages. However, their cellular and molecular mechanisms of action remain unknown. Using Mtb mutants deleted for genes involved in DIM biosynthesis, we demonstrated that DIM participate both in the receptor-dependent phagocytosis of Mtb and the prevention of phagosomal acidification. The effects of DIM required a state of the membrane fluidity as demonstrated by experiments conducted with cholesterol-depleting drugs that abolished the differences in phagocytosis efficiency and phagosome acidification observed between wild-type and mutant strains. The insertion of a new cholesterol-pyrene probe in living cells demonstrated that the polarity of the membrane hydrophobic core changed upon contact with Mtb whereas the lateral diffusion of cholesterol was unaffected. This effect was dependent on DIM and was consistent with the effect observed following DIM insertion in model membrane. Therefore, we propose that DIM control the invasion of macrophages by Mtb by targeting lipid organisation in the host membrane, thereby modifying its biophysical properties. The DIM-induced changes in lipid ordering favour the efficiency of receptor-mediated phagocytosis of Mtb and contribute to the control of phagosomal pH driving bacilli in a protective niche.

Membrane functional organisation and dynamic of mu-opioid receptors.

The activation and signalling activity of the membrane mu-opioid receptor (MOP-R) involve interactions among the receptor, G-proteins, effectors and many other membrane or cytosolic proteins. Decades of investigation have led to identification of the main biochemical processes, but the mechanisms governing the successive protein-protein interactions have yet to be established. We will need to unravel the dynamic membrane organisation of this complex and multifaceted molecular machinery if we are to understand these mechanisms. Here, we review and discuss advances in our understanding of the signalling mechanism of MOP-R resulting from biochemical or biophysical studies of the organisation of this receptor in the plasma membrane.

Membrane partitioning of various delta-opioid receptor forms before and after agonist activations: the effect of cholesterol.

Lipid rafts depicted as densely packed and thicker membrane microdomains, based on the dynamic clustering of cholesterol and sphingolipids, may help as platforms involved in a wide variety of cellular processes. The reasons why proteins segregate into rafts are yet to be clarified. The human delta opioid receptor (hDOR) reconstituted in a model system has been characterised after ligand binding by an elongation of its transmembrane part, inducing rearrangement of its lipid microenvironment [Alves, Salamon, Hruby, and Tollin (2005) Biochemistry 44, 9168-9178]. We used hDOR to understand better the correlation between its function and its membrane microdomain localisation. A fusion protein of hDOR with the Green Fluorescent Protein (DOR*) allows precise receptor membrane quantification. Here we report that (i) a fraction of the total receptor pool requires cholesterol for binding activity, (ii) G-proteins stabilize a high affinity state conformation which does not seem modulated by cholesterol. In relation to its distribution, and (iii) a fraction of DOR* is constitutively associated with detergent-resistant membranes (DRM) characterised by an enrichment in lipids and proteins raft markers. (iv) An increase in the quantity of DOR* was observed upon agonist addition. (v) This DRM relocation is prevented by uncoupling the receptor-G-protein interaction.

Changes of the membrane lipid organization characterized by means of a new cholesterol-pyrene probe.

We synthesized 3beta-hydroxy-pregn-5-ene-21-(1-methylpyrenyl)-20-methylidene (Py-met-chol), consisting of cholesterol steroid rings connected to a pyrene group via a linker without polar atoms. This compound has interesting spectroscopic properties when probing membranes: 1), The pyrene has hypochromic properties resulting from probe self-association processes in membranes. Using liposomes of various lipid compositions, we determined the association constants of the probe (K): K(DOPC) >> K(POPC) >> K(DMPC) > K(DMPC/15 mol % Chol) > K(DMPC/30 mol % Chol). This indicates a better probe solvation in saturated than in unsaturated lipids, and this effect is enhanced as the cholesterol concentration increases. 2), The pyrene fluorophore is characterized by monomer (I(1)-I(5)) and excimer (I(E)) emission bands. In model membranes, I(1)/I(3) and I(E)/I(3) ratios revealed a correlation between the polarity of the lipid core of the membrane and the amount of cholesterol. 3), Using this probe, we monitored the first steps of the signaling pathway of the mouse delta-opioid receptor, a G-protein-coupled receptor. The thickness of the membrane around this receptor is known to change after agonist binding. Fluorescence spectra of living Chinese hamster ovary cells overexpressing mouse delta-opioid receptor specifically revealed the agonist binding. These results indicate that Py-met-chol may be useful for screening ligands of this family of receptors.

Diffusion of the mu opioid receptor at the surface of human neuroblastoma SH-SY5Y cells is restricted to permeable domains.

Previous single-molecule studies have shown a long-term diffusion superimposed to a short-term confinement of the human mu opioid (hMOP) receptors at the surface of heterologous cells. However, additional ensemble average measurements are required to reach a complete understanding of the undergoing process. Here, we analyse, by fluorescence recovery after photobleaching measurements, the lateral diffusion of fully functional T7-EGFP-hMOP receptors in neuroblastoma SH-SY5Y cells naturally expressing a low level of the wild-type receptor. Experiments carried out at variable observation radii demonstrate the restriction of the receptors diffusion to sub-micrometer sized domains. Furthermore, consistently with the long-term single-molecule data, the domains are found permeable.

Modes of membrane interaction of a natural cysteine-rich peptide: viscotoxin A3.

Among the very homologous family of alpha- and beta-thionins, known for their antimicrobial activity, the viscotoxin subfamily differs from other members because it is cytotoxic against tumoral cells but weakly hemolytic. We studied the interactions between the most active of these toxins, viscotoxin A3 (VA3), and model membranes made of phosphatidylcholine and phosphatidylserine (PS), the major zwitterionic and acidic phospholipids found in eukaryotic cells. Monolayer studies showed that electrostatic forces are essential for the interaction and are mainly involved in modulating the embedding of the toxin in the PS head group region. This in turn induces membrane stiffening, as shown by fluorescence polarization assays with 1,6-diphenyl-1,3,5-hexatriene and its derivatives. Moreover, vesicle permeabilization analyses showed that there are two modes of interaction, which are directly related to the stiffening effect and depend on the amount of VA3 bound to the surface of the vesicles. We propose an interaction model in which the embedding of VA3 in the membrane induces membrane defects leading to the gradual release of encapsulated dye. When the surfaces of the vesicles are saturated with the viscotoxin, complete vesicle destabilization is induced which leads to bilayer disruption, all-or-none encapsulated dye release and rearrangement of the vesicles.

Highly ordered supra-molecular organization of the mycobacterial lipoarabinomannans in solution. Evidence of a relationship between supra-molecular organization and biological activity.

The complex mycobacterial mannosylated lipoarabinomannans (ManLAMs) are currently considered to be the major virulence factors of the pathogenic Mycobacterium tuberculosis. The recognition and the interaction of ManLAMs with immune system receptors have been shown to promote M.tuberculosis phagocytosis but also to down-regulate the bactericidal immune response of the host in favor of the survival of the pathogenic bacilli. To date these original biological activities were mainly associated to the presence of mannose residues capping the non-reducing ends of the ramified polysaccharide moiety of these complex lipoglycans. However, we demonstrated recently that the molecular recognition of ManLAM terminal mannose units by human pulmonary surfactant protein A (hSP-A) carbohydrate recognition domains depends on the presence of the lipid moiety of the ManLAMs as proposed by Sidobre et al. in 2002. Thus, we investigated the putative role of the ManLAM aglycon moiety. The data presented here, indicate that the hydrophobic aglycon part of ManLAM is associated to a characteristic concentration-dependent supra-molecular organization of these complex molecules. Furthermore, we observed that the deacylated ManLAMs or the lipid-free mannosylated arabinomannans, which do not exhibit characteristic ManLAM activities, do not display this supra-molecular organization. These observations strongly suggest that the ManLAMs immunomodulatory activities might be associated to their particular organization. Finally, the determination of the critical micellar concentration of ManLAMs obviously supports the notion that this supra-molecular organization may be responsible for the specific biological activities of these complex molecules.

Characterization of M-laurdan, a versatile probe to explore order in lipid membranes.

Microdomains corresponding to localized partition of lipids between ordered and less ordered environments are the subject of intensive investigations, because of their putative participation in modulating cellular responses. One popular approach in the field consists in labelling membranes with solvatochromic fluorescent probes such as laurdan and C-laurdan. In this report, we describe a high-yield procedure for the synthesis of laurdan, C-laurdan and two new fluorophores, called MoC-laurdan and M-laurdan, as well as their extensive photophysical characterization. We find that the latter probe, M-laurdan, is particularly suited to discriminate lipid phases independently of the chemical nature of the lipids, as measured by both fluorescence Generalized Polarization (GP) and anisotropy in large unilamellar vesicles made of various lipid compositions. In addition, staining of live cells with M-laurdan shows a good stability over time without any apparent toxicity, as well as a wider distribution in the various cell compartments than the other probes.

Functional membrane diffusion of G-protein coupled receptors.

G-protein-coupled receptor function involves interactions between the receptor, G-proteins and effectors in the cell plasma membrane. The main biochemical processes have been individually identified but the mechanisms governing the successive protein-protein interactions of this complex multi-molecular machinery have yet to be established. We discuss advances in understanding the functional dynamics of the receptor resulting from diffusion measurements, and in the context of the plasma membrane organization.

CD4 interacts constitutively with multiple CCR5 at the plasma membrane of living cells. A fluorescence recovery after photobleaching at variable radii approach.

The entry of human immunodeficiency virus into target cells requires successive interactions of the viral envelope glycoprotein gp120 with CD4 and the chemokine receptors CCR5 or CXCR4. We previously demonstrated, by Förster resonance energy transfer experiments, the constitutive association of CD4 and CCR5 at the surface of living cells. We therefore speculated that this interaction may correlate with compartmentalization of CD4 and CCR5 within the plasma membrane. Here, we characterize the lateral distribution, the dynamics, and the stoichiometry of these receptors in living cells stably expressing CD4 and/or CCR5 by means of fluorescence recovery after photobleaching at variable radii experiments. We found that (i) these receptors expressed alone are confined into 1-microm-sized domains, (ii) CD4-CCR5 associations occur outside and inside smaller domains, and (iii) these interactions involve multiple CCR5 molecules per CD4.

CD4 and CCR5 constitutively interact at the plasma membrane of living cells: a confocal fluorescence resonance energy transfer-based approach.

Human immunodeficiency virus entry into target cells requires sequential interactions of the viral glycoprotein envelope gp120 with CD4 and chemokine receptors CCR5 or CXCR4. CD4 interaction with the chemokine receptor is suggested to play a critical role in this process but to what extent such a mechanism takes place at the surface of target cells remains elusive. To address this issue, we used a confocal microspectrofluorimetric approach to monitor fluorescence resonance energy transfer at the cell plasma membrane between enhanced blue and green fluorescent proteins fused to CD4 and CCR5 receptors. We developed an efficient fluorescence resonance energy transfer analysis from experiments carried out on individual cells, revealing that receptors constitutively interact at the plasma membrane. Binding of R5-tropic HIV gp120 stabilizes these associations thus highlighting that ternary complexes between CD4, gp120, and CCR5 occur before the fusion process starts. Furthermore, the ability of CD4 truncated mutants and CCR5 ligands to prevent association of CD4 with CCR5 reveals that this interaction notably engages extracellular parts of receptors. Finally, we provide evidence that this interaction takes place outside raft domains of the plasma membrane.

Probing functionalized gold colloids for single particle tracking experiments.

Functionalized submicroscopic particles are currently used to label proteins or lipids at the surface of living cells for single particle tracking experiments. In many cases, it can be of crucial importance for the particle to be anchored to a single molecule. We have addressed this question for the labeling at the plasma membrane of NRK cells of the mu-opioid receptor bearing a T7 epitope at the N-terminus. Using biophysical methods we were able to prepare quasi-monovalent anti-T7 antibody conjugated gold colloids (40 nm diameter) leading to stable and specific binding to the receptor. The rational method, we report here, can be extended to design customized probes for the labeling of various tagged molecules.

Confined diffusion without fences of a g-protein-coupled receptor as revealed by single particle tracking.

Single particle tracking is a powerful tool for probing the organization and dynamics of the plasma membrane constituents. We used this technique to study the micro -opioid receptor belonging to the large family of the G-protein-coupled receptors involved with other partners in a signal transduction pathway. The specific labeling of the receptor coupled to a T7-tag at its N-terminus, stably expressed in fibroblastic cells, was achieved by colloidal gold coupled to a monoclonal anti T7-tag antibody. The lateral movements of the particles were followed by nanovideomicroscopy at 40 ms time resolution during 2 min with a spatial precision of 15 nm. The receptors were found to have either a slow or directed diffusion mode (10%) or a walking confined diffusion mode (90%) composed of a long-term random diffusion and a short-term confined diffusion, and corresponding to a diffusion confined within a domain that itself diffuses. The results indicate that the confinement is due to an effective harmonic potential generated by long-range attraction between the membrane proteins. A simple model for interacting membrane proteins diffusion is proposed that explains the variations with the domain size of the short-term and long-term diffusion coefficients.

Dynamic confinement of NK2 receptors in the plasma membrane. Improved FRAP analysis and biological relevance.

A functional fluorescent neurokinin NK2 receptor, EGFP-NK2, was previously used to follow, by fluorescence resonance energy transfer measurements in living cells, the binding of its fluorescently labeled agonist, bodipy-neurokinin A (NKA). Local agonist application suggested that the activation and desensitization of the NK2 receptors were compartmentalized at the level of the plasma membrane. In this study, fluorescence recovery after photobleaching experiments are carried out at variable observation radius (vrFRAP) to probe EGFP-NK2 receptor mobility and confinement. Experiments are carried out at 20 degrees C to maintain the number of receptors constant at the cell surface during recordings. In the absence of agonist, 35% EGFP-NK2 receptors diffuse within domains of 420 +/- 80 nm in radius with the remaining 65% of receptors able to diffuse with a long range lateral diffusion coefficient between the domains. When cells are incubated with a saturating concentration of NKA, 30% EGFP-NK2 receptors become immobilized in small domains characterized by a radius equal to 170 +/- 50 nm. Biochemical experiments show that the confinement of EGFP-NK2 receptor is not due to its association with rafts at any given time. Colocalization of the receptor with beta-arrestin and transferrin supports that the small domains, containing 30% of activated EGFP-NK2, correspond to clathrin-coated pre-pits. The similar amount of confined EGFP-NK2 receptors found before and after activation (30-35%) is discussed in term of putative transient interactions of the receptors with preexisting scaffolds of signaling molecules.

Comparative membrane interaction study of viscotoxins A3, A2 and B from mistletoe (Viscum album) and connections with their structures.

Viscotoxins A2 (VA2) and B (VB) are, together with viscotoxin A3 (VA3), among the most abundant viscotoxin isoforms that occur in mistletoe-derived medicines used in anti-cancer therapy. Although these isoforms have a high degree of amino-acid-sequence similarity, they are strikingly different from each other in their in vitro cytotoxic potency towards tumour cells. First, as VA3 is the only viscotoxin whose three-dimensional (3D) structure has been solved to date, we report the NMR determination of the 3D structures of VA2 and VB. Secondly, to account for the in vitro cytotoxicity discrepancy, we carried out a comparative study of the interaction of the three viscotoxins with model membranes. Although the overall 3D structure is highly conserved among the three isoforms, some discrete structural features and associated surface properties readily account for the different affinity and perturbation of model membranes. VA3 and VA2 interact in a similar way, but the weaker hydrophobic character of VA2 is thought to be mainly responsible for the apparent different affinity towards membranes. VB is much less active than the other two viscotoxins and does not insert into model membranes. This could be related to the occurrence of a single residue (Arg25) protruding outside the hydrophobic plane formed by the two amphipathic alpha-helices, through which viscotoxins are supposed to interact with plasma membranes.

Análise de tratamento anti-rábico humano pós-exposição em região da Grande São Paulo, Brasil / Analysis of human anti rabic post exposure treatment in an urban region of Southeastern Brazil

O tratamento de seres humanos expostos ao risco de infecção pelo vírus rábico ainda pode incluir a ocorrência de reações pós-vacinais indesejáveis, tanto de ordem local como geral. A análise sistemática dos informes epidemiológicos de pacientes submetidos a este tipo de tratamento poderá oferecer subsídios para a modificação desta situação. Foram analisados os registros de tratamento dessa zoonose visando à melhoria do seu controle. Métodos - Foram analisadas através do programa EPI Info as fichas de investigação epidemiológica da raiva de 8.758 habitantes do Município de Osasco, SP (Brasil), atendidos no período de 1984 a 1994. A vacina utilizada foi do tipo Fuenzalida & Palacios. Resultados - Constatou-se a existência de maior risco de exposição para os indivíduos do sexo masculino, com cinco a nove anos de idade. As agressões ocorreram com maior freqüência no domicílio da vítima e os cães foram os principais responsáveis. Dos cães e gatos envolvidos, respectivamente 51,0 por cento e 73,2 por cento não haviam sido imunizados mais freqüentes foram: cabeça (36,6 por cento) e membros superiores (35,1 por cento); quando a faixa etária ultrapassava os nove anos as áreas mais acometidas foram membros superiores (45,8 por cento) e membros inferiores (43,7 por cento). Dos pacientes analisados, 26,5 por cento já haviam recebido vacinação anti-rábica anterior e 90,7 por cento procurou a orientação médica em até cinco dias da agressão. Para 41,9 por cento foi prescrita unicamente a vacinação e para 0,05 por cento a soro-vacinação. Conclusões - Houve 11,7 por cento de abandonos a tratamentos e 51,3 por cento foram dispensados do mesmo em função da observação animal. Dos pacientes tratados com vacina ou soro-vacinação houve 0,25 por cento de acidentes pós-vacinais, dos quais 0,3 por cento to tipo neutrológico. Os meses de março, julho, agosto e setembro foram os de maior procura.