Muscle Daily Undulating Periodization for Strength and Body Composition: The Proposal of a New Model.

The traditional linear periodization model is designed for modifications to be performed over several weeks, whereas alterations in the undulating model are applied on a more frequent basis. The study investigated a novel periodization scheme, the muscle daily undulating periodization model (mDUP). Thirty-seven men were randomly assigned into 2 groups: (a) a group that performed 12 weeks of daily undulating periodization with fix overload (DUP-F) resistance training (n = 19) and (b) a group that performed 12-weeks of muscle daily undulating periodization with variation overload (mDUP) (n = 18). Body composition and strength assessments (muscular endurance and one repetition maximum [1 RM] for barbell bench press, 45º leg press, lat pull down, and standing arm curl) were completed before and after the program. Two-way MANOVA with repeated measures was used to compare groups with significance set at p<0.05. There were no differences between periodization programs for anthropometric variables (p > 0.05, η2p = 0.04), but improvement was noted over time (p < 0.001, η2p = 0.60). No differences were observed between periodization programs for strength (p > 0.05, η2p = 0.056), but strength increased over time (p < 0.001, η2p = 0.95). Similarly, no muscular endurance differences were seen between periodization programs (p > 0.05, η2p = 0.15), but measures increased over time (p < 0.001, η2p = 0.60). When it comes to body composition, muscle strength, and muscle endurance, the present study provides evidence that both periodization models displayed similar results, with more evident improvements in strength. Thus, it seems pertinent to consider this new periodization model plausible for RT practitioners in order to achieve new adaptations.

Enhanced receptor-clathrin interactions induced by N-glycan-mediated membrane micropatterning.

Glycan-protein interactions are emerging as important modulators of membrane protein organization and dynamics, regulating multiple cellular functions. In particular, it has been postulated that glycan-mediated interactions regulate surface residence time of glycoproteins and endocytosis. How this precisely occurs is poorly understood. Here we applied single-molecule-based approaches to directly visualize the impact of glycan-based interactions on the spatiotemporal organization and interaction with clathrin of the glycosylated pathogen recognition receptor dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN). We find that cell surface glycan-mediated interactions do not influence the nanoscale lateral organization of DC-SIGN but restrict the mobility of the receptor to distinct micrometer-size membrane regions. Remarkably, these regions are enriched in clathrin, thereby increasing the probability of DC-SIGN-clathrin interactions beyond random encountering. N-glycan removal or neutralization leads to larger membrane exploration and reduced interaction with clathrin, compromising clathrin-dependent internalization of virus-like particles by DC-SIGN. Therefore, our data reveal that cell surface glycan-mediated interactions add another organization layer to the cell membrane at the microscale and establish a novel mechanism of extracellular membrane organization based on the compartments of the membrane that a receptor is able to explore. Our work underscores the important and complex role of surface glycans regulating cell membrane organization and interaction with downstream partners.

Ceramide: a simple sphingolipid with unique biophysical properties.

Ceramides are involved in a variety of cellular processes and in disease. Their biological functions are thought to depend on ceramides' unique biophysical properties, which promote strong alterations of cell membrane properties and consequent triggering of signaling events. Over the last decades, efforts were made to understand the impact of ceramide on membrane biophysical features. Several studies, performed in a multitude of membrane models, address ceramides' specific interactions, the effect of their acyl chain structure and the influence of membrane lipid composition and properties on ceramide biophysical outcome. In this review, a rationale for the multiple and complex changes promoted by ceramide is provided, highlighting, on a comprehensive and critical manner, the interactions between ceramides and specific lipids and/or lipid phases. Focus is also given to the interplay between ceramide and cholesterol, particularly in lipid raft-mimicking mixtures, an issue of intense debate due to the urgent need to understand the biophysical impact of ceramide formation in models resembling the cell membrane. The implications of ceramide-induced biophysical changes on lipid-protein interactions and cell signaling are also discussed, together with the emerging evidence for the existence of ceramide-gel like domains in cellular membranes.

Biochemical and imaging methods to study receptor membrane organization and association with lipid rafts.

Lipid rafts, cell membrane domains with unique composition and properties, modulate the membrane distribution of receptors and signaling molecules facilitating the assembly of active signaling platforms. However, the underlying mechanisms that link signal transduction and lipid rafts are not fully understood, mainly because of the transient nature of these membrane assemblies. Several methods have been used to study the association of membrane receptors with lipid rafts. In the first part of this chapter, a description of how biochemical methods such as raft disruption by cholesterol depletion agents are useful in qualitatively establishing protein association with lipid rafts is presented. The second part of this chapter is dedicated to imaging techniques used to study membrane receptor organization and lipid rafts. We cover conventional approaches such as confocal microscopy to advanced imaging techniques such as homo-FRET microscopy and superresolution methods. For each technique described, their advantages and drawbacks are discussed.

Edelfosine and miltefosine effects on lipid raft properties: membrane biophysics in cell death by antitumor lipids.

Edelfosine (1-O-octadecyl-2-O-methyl-sn-glycero-phosphocholine) and miltefosine (hexadecylphosphocholine) are synthetic alkylphospholipids (ALPs) that are reported to selectively accumulate in tumor cell membranes, inducing Fas clustering and activation on lipid rafts, triggering apoptosis. However, the exact mechanism by which these lipids elicit these events is still not fully understood. Recent studies propose that their mode of action might be related with alterations of lipid rafts biophysical properties caused by these lipid drugs. To achieve a clear understanding of this mechanism, we studied the effects of pharmacologically relevant amounts of edelfosine and miltefosine in the properties of model and cellular membranes. The influence of these molecules on membrane order, lateral organization, and lipid rafts molar fraction and size were studied by steady-state and time-resolved fluorescence methods, Förster resonance energy transfer (FRET), confocal and fluorescence lifetime imaging microscopy (FLIM). We found that the global membrane and lipid rafts biophysical properties of both model and cellular membranes were not significantly affected by both the ALPs. Nonetheless, in model membranes, a mild increase in membrane fluidity induced by both alkyl lipids was detected, although this effect was more noticeable for edelfosine than miltefosine. This absence of drastic alterations shows for the first time that ALPs mode of action is unlikely to be directly linked to alterations of lipid rafts biophysical properties caused by these drugs. The biological implications of this result are discussed in the context of ALPs effects on lipid metabolism, mitochondria homeostasis modulation, and their relationship with tumor cell death.

The photophysics of a Rhodamine head labeled phospholipid in the identification and characterization of membrane lipid phases.

The organization of lipids and proteins into domains in cell membranes is currently an established subject within biomembrane research. Fluorescent probes have been used to detect and characterize these membrane lateral heterogeneities. However, a comprehensive understanding of the link between the probes' fluorescence features and membrane lateral organization can only be achieved if their photophysical properties are thoroughly defined. In this work, a systematic characterization of N-(lyssamine Rhodamine B sulfonyl)-1,2-dioleoyl-sn-3-phosphatidylehanolamine (Rhod-DOPE) absorption and fluorescence behavior in gel, liquid-ordered (l(o)) and liquid-disordered (l(d)) model membranes was performed. In agreement with a previous study, it was found that Rhod-DOPE fluorescence lifetimes present a strong sensitivity to lipid phases, becoming significantly shorter in l(o) membranes as the probe membrane concentration increases. The sensitivity of Rhod-DOPE absorption and fluorescence properties to the membrane phase was further explored. In particular, the fluorescence lifetime sensitivity was shown to be a consequence of the enhanced Rhod-DOPE fluorescence dynamic self-quenching, due to the formation of probe-rich membrane domains in these condensed phases that cannot be considered as typical probe aggregates, as excitonic interaction is not observed. The highly efficient dynamic self-quenching was shown to be specific to l(o) phases, pointing to an important effect of membrane dipole potential in this process. Altogether, this work establishes how to use Rhod-DOPE fluorescence properties in the study of membrane lipid lateral heterogeneities, in particular cholesterol-enriched lipid rafts.

Organization and dynamics of Fas transmembrane domain in raft membranes and modulation by ceramide.

To comprehend the molecular processes that lead to the Fas death receptor clustering in lipid rafts, a 21-mer peptide corresponding to its single transmembrane domain (TMD) was reconstituted into mammalian raft model membranes composed of an unsaturated glycerophospholipid, sphingomyelin, and cholesterol. The peptide membrane lateral organization and dynamics, and its influence on membrane properties, were studied by steady-state and time-resolved fluorescence techniques and by attenuated total reflection Fourier transformed infrared spectroscopy. Our results show that Fas TMD is preferentially localized in liquid-disordered membrane regions and undergoes a strong reorganization as the membrane composition is changed toward the liquid-ordered phase. This results from the strong hydrophobic mismatch between the length of the peptide hydrophobic stretch and the hydrophobic thickness of liquid-ordered membranes. The stability of nonclustered Fas TMD in liquid-disordered domains suggests that its sequence may have a protective function against nonligand-induced Fas clustering in lipid rafts. It has been reported that ceramide induces Fas oligomerization in lipid rafts. Here, it is shown that neither Fas TMD membrane organization nor its conformation is affected by ceramide. These results are discussed within the framework of Fas membrane signaling events.

Cholesterol-rich fluid membranes solubilize ceramide domains: implications for the structure and dynamics of mammalian intracellular and plasma membranes.

A uniquely sensitive method for ceramide domain detection allowed us to study in detail cholesterol-ceramide interactions in lipid bilayers with low (physiological) ceramide concentrations, ranging from low or no cholesterol (a situation similar to intracellular membranes, such as endoplasmic reticulum) to high cholesterol (similar to mammalian plasma membrane). Diverse fluorescence spectroscopy and microscopy experiments were conducted showing that for low cholesterol amounts ceramide segregates into gel domains that disappear upon increasing cholesterol levels. This was observed in different raft (sphingomyelin/cholesterol-containing) and non-raft (sphingomyelin-absent) membranes, i.e. mimicking different types of cell membranes. Cholesterol-ceramide interactions have been described mainly as raft sphingomyelin-dependent. Here sphingomyelin independence is demonstrated. In addition, ceramide-rich domains re-appear when either cholesterol is converted by cholesterol oxidase to cholestenone or the temperature is decreased. Ceramide is more soluble in cholesterol-rich fluid membranes than in cholesterol-poor ones, thereby increasing the chemical potential of cholesterol. Ceramide solubility depends on the average gel-fluid transition temperature of the remaining membrane lipids. The inability of cholestenone-rich membranes to dissolve ceramide gel domains shows that the cholesterol ordering and packing properties are fundamental to the mixing process. We also show that the solubility of cholesterol in ceramide domains is low. The results are rationalized by a ternary phospholipid/ceramide/cholesterol phase diagram, providing the framework for the better understanding of biochemical phenomena modulated by cholesterol-ceramide interactions such as cholesterol oxidase activity, lipoprotein metabolism, and lipid targeting in cancer therapy. It also suggests that the lipid compositions of different organelles are such that ceramide gel domains are not formed unless a stress or pathological situation occurs.

Formation of ceramide/sphingomyelin gel domains in the presence of an unsaturated phospholipid: a quantitative multiprobe approach.

To better understand how ceramide modulates the biophysical properties of the membrane, the interactions between palmitoyl-ceramide (PCer) and palmitoyl-sphingomyelin (PSM) were studied in the presence of the fluid phospholipid palmitoyl-oleoyl-phosphatidylcholine (POPC) in membrane model systems. The use of two fluorescent membrane probes distinctly sensitive to lipid phases allowed a thorough biophysical characterization of the ternary system. In these mixtures, PCer recruits POPC and PSM in the fluid phase to form extremely ordered and compact gel domains. Gel domain formation by low PCer mol fraction (up to 12 mol %) is enhanced by physiological PSM levels (approximately 20-30 mol % total lipid). For higher PSM content, a three-phase situation, consisting of fluid (POPC-rich)/gel (PSM-rich)/gel (PCer-rich) coexistence, is clearly shown. To determine the fraction of each phase a quantitative method was developed. This allowed establishing the complete ternary phase diagram, which helps to predict PCer-rich gel domain formation and explains its enhancement through PSM/PCer interactions.

Ceramide-domain formation and collapse in lipid rafts: membrane reorganization by an apoptotic lipid.

The effect of physiologically relevant ceramide concentrations (< or = 4 mol %) in raft model membranes with a lipid composition resembling that of cell membranes, i.e., composed of different molar ratios of an unsaturated glycerophospholipid, sphingomyelin, and cholesterol (Chol) along a liquid-disordered-liquid-ordered tie line was explored. The application of a fluorescence multiprobe and multiparameter approach, together with multiple fluorescence resonance energy transfer (FRET) pairs, in the well-characterized palmitoyl-oleoyl-phosphocholine (POPC)/palmitoyl-sphingomyelin (PSM)/Chol ternary mixture, revealed that low palmitoyl-ceramide (PCer) concentrations strongly changed both the biophysical properties and lipid lateral organization of the ternary mixtures in the low-to-intermediate Chol/PSM-, small raft size range (<25 mol % Chol). For these mixtures, PCer recruited up to three PSM molecules for the formation of very small ( approximately 4 nm) and highly ordered gel domains, which became surrounded by rafts (liquid-ordered phase) when Chol/PSM content increased. However, the size of these rafts did not change, showing that PCer did not induce the formation of large platforms or the coalescence of small rafts. In the high Chol/PSM-, large raft domains range (>33 mol % Chol), Chol completely abolished the effect of PCer by competing for PSM association. Lipid rafts govern the biophysical properties and lateral organization in these last mixtures.