Astaxanthin: Past, Present, and Future.

Astaxanthin (AX), a lipid-soluble pigment belonging to the xanthophyll carotenoids family, has recently garnered significant attention due to its unique physical properties, biochemical attributes, and physiological effects. Originally recognized primarily for its role in imparting the characteristic red-pink color to various organisms, AX is currently experiencing a surge in interest and research. The growing body of literature in this field predominantly focuses on AXs distinctive bioactivities and properties. However, the potential of algae-derived AX as a solution to various global environmental and societal challenges that threaten life on our planet has not received extensive attention. Furthermore, the historical context and the role of AX in nature, as well as its significance in diverse cultures and traditional health practices, have not been comprehensively explored in previous works. This review article embarks on a comprehensive journey through the history leading up to the present, offering insights into the discovery of AX, its chemical and physical attributes, distribution in organisms, and biosynthesis. Additionally, it delves into the intricate realm of health benefits, biofunctional characteristics, and the current market status of AX. By encompassing these multifaceted aspects, this review aims to provide readers with a more profound understanding and a robust foundation for future scientific endeavors directed at addressing societal needs for sustainable nutritional and medicinal solutions. An updated summary of AXs health benefits, its present market status, and potential future applications are also included for a well-rounded perspective.

Astaxanthin as a Novel Mitochondrial Regulator: A New Aspect of Carotenoids, beyond Antioxidants.

Astaxanthin is a member of the carotenoid family that is found abundantly in marine organisms, and has been gaining attention in recent years due to its varied biological/physiological activities. It has been reported that astaxanthin functions both as a pigment, and as an antioxidant with superior free radical quenching capacity. We recently reported that astaxanthin modulated mitochondrial functions by a novel mechanism independent of its antioxidant function. In this paper, we review astaxanthin's well-known antioxidant activity, and expand on astaxanthin's lesser-known molecular targets, and its role in mitochondrial energy metabolism.

Optimizing the Design of Diatom Biosilica-Targeted Fusion Proteins in Biosensor Construction for Bacillus anthracis Detection.

In vivo functionalization of diatom biosilica frustules by genetic manipulation requires careful consideration of the overall structure and function of complex fusion proteins. Although we previously had transformed Thalassiosira pseudonana with constructs containing a single domain antibody (sdAb) raised against the Bacillus anthracis Sterne strain, which detected an epitope of the surface layer protein EA1 accessible in lysed spores, we initially were unsuccessful with constructs encoding a similar sdAb that detected an epitope of EA1 accessible in intact spores and vegetative cells. This discrepancy limited the usefulness of the system as an environmental biosensor for B. anthracis. We surmised that to create functional biosilica-localized biosensors with certain constructs, the biosilica targeting and protein trafficking functions of the biosilica-targeting peptide Sil3T8 had to be uncoupled. We found that retaining the ER trafficking sequence at the N-terminus and relocating the Sil3T8 targeting peptide to the C-terminus of the fusion protein resulted in successful detection of EA1 with both sdAbs. Homology modeling of antigen binding by the two sdAbs supported the hypothesis that the rescue of antigen binding in the previously dysfunctional sdAb was due to removal of steric hindrances between the antigen binding loops and the diatom biosilica for that particular sdAb.

Distance-Matched Tagging Sequence Optimizes Live-Cell Protein Labeling by a Biarsenical Fluorescent Reagent AsCy3_E.

Cell permeable biarsenical fluorescent dyes built around a cyanine scaffold (AsCy3) create the ability to monitor the structural dynamics of tagged proteins in living cells. To extend the capability of this photostable and bright biarsenical probe to site-specifically label cellular proteins, we have compared the ability of AsCy3 to label two different tagging sequences (i.e., CCKAEAACC and CCKAEAAKAEAAKCC), which were separately engineered onto enhanced green fluorescent proteins (EGFPs) and expressed in Escherichia coli. The cysteine pairs within the shorter protein tag (i.e., Cy3TAG) are designed to specifically match the 14.5 Å interarsenic atomic separation within AsCy3, whereas the longer protein tag (Cy3TAG+6) was identified using a peptide screening approach and reported to enhance the binding affinity and brightness. We report that AsCy3 binds both the tagged proteins with similar high affinities (Kd < 1 µM) under both in vivo labeling conditions and following isolation and labeling of the tagged EGFP protein. Greater experimental reproducibility and substantially larger AsCy3 labeling stoichiometries are observed under in vivo conditions using the shorter Cy3TAG in comparison to the Cy3TAG+6. These results suggest that the use of the distance-matched and conformationally restricted Cy3TAG avoids nonspecific protein interactions, thereby enabling routine measurements of protein localization and conformational dynamics in living cells.

Hydrogel Tethering Enhances Interdomain Stabilization of Single-Chain Antibodies.

Here, we identify the importance of molecular crowding agents in the functional stabilization of scFv antibodies. Antibodies were tethered through an engineered calmodulin (CaM)-binding peptide into a stimulus-responsive hydrogel composed of poly(ethylene glycol) (PEG)-functionalized CaM. Macromolecular crowding is modulated by transient heating, which decreases effective pore sizes. Using a fluorescent ligand bound to the scFv, frequency-domain fluorescence spectroscopy was used to assess the structural coupling between the VH and the VL domains and relationships with functional stabilization. There is minimal structural coupling between the VH and the VL domains in solution, as is apparent from the substantial rotational mobility for the bound ligand, that is suggestive of an independent mobility for the VH and the VL domains. In comparison, the hydrogel matrix acts to structurally couple the VH and the VL domains, resulting in a reduction in rotational mobility and a retention of ligand binding in the presence of 8.0 M urea. Under these same conditions, ligand binding is disrupted for scFv antibodies in solution. Increases in the stabilization of scFv antibodies in hydrogels is not simply the result of molecular crowding because decreases in pore size act to destabilize ligand binding. Rather, our results suggest that the functional stabilization of the scFv antibody within the PEG hydrogel matrix includes important factors involving protein solvation that stabilize interdomain interactions between the VH and the VL domains necessary for ligand binding.

Dynamic Stabilization of Expressed Proteins in Engineered Diatom Biosilica Matrices.

Self-assembly of recombinant proteins within the biosilica of living diatoms represents a means to construct functional materials in a reproducible and scalable manner that will enable applications that harness the inherent specificities of proteins to sense and respond to environmental cues. Here we describe the use of a silaffin-derived lysine-rich 39-amino-acid targeting sequence (Sil3T8) that directs a single chain fragment variable (scFv) antibody or an enhanced green fluorescent protein (EGFP) to assemble within the biosilica frustule, resulting in abundance of >200 000 proteins per frustule. Using either a fluorescent ligand bound to the scFv or the intrinsic fluorescence of EGFP, we monitored protein conformational dynamics, accessibility to external quenchers, binding affinity, and conformational stability. Like proteins in solution, proteins within isolated frustules undergo isotropic rotational motion, but with 2-fold increases in rotational correlation times that are indicative of weak macromolecular associations within the biosilica. Solvent accessibilities and high-affinity (pM) binding are comparable to those in solution. In contrast to solution conditions, scFv antibodies within the biosilica matrix retain their binding affinity in the presence of chaotropic agents (i.e., 8 M urea). Together, these results argue that dramatic increases in protein conformational stability within the biosilica matrices arise through molecular crowding, acting to retain native protein folds and associated functionality with the potential to allow the utility of engineered proteins under a range of harsh environmental conditions associated with environmental sensing and industrial catalytic transformations.

Antigen Binding and Site-Directed Labeling of Biosilica-Immobilized Fusion Proteins Expressed in Diatoms.

The diatom Thalassiosira pseudonana was genetically modified to express biosilica-targeted fusion proteins comprising either enhanced green fluorescent protein (EGFP) or single chain antibodies engineered with a tetracysteine tagging sequence. Of interest were the site-specific binding of (1) the fluorescent biarsenical probe AsCy3 and AsCy3e to the tetracysteine tagged fusion proteins and (2) high and low molecular mass antigens, the Bacillus anthracis surface layer protein EA1 or small molecule explosive trinitrotoluene (TNT), to biosilica-immobilized single chain antibodies. Analysis of biarsenical probe binding using fluorescence and structured illumination microscopy indicated differential colocalization with EGFP in nascent and mature biosilica, supporting the use of either EGFP or bound AsCy3 and AsCy3e in studying biosilica maturation. Large increases in the lifetime of a fluorescent analogue of TNT upon binding single chain antibodies provided a robust signal capable of discriminating binding to immobilized antibodies in the transformed frustule from nonspecific binding to the biosilica matrix. In conclusion, our results demonstrate an ability to engineer diatoms to create antibody-functionalized mesoporous silica able to selectively bind chemical and biological agents for the development of sensing platforms.

The proteolytic landscape of the yeast vacuole.

The vacuole in the yeast Saccharomyces cerevisiae plays a number of essential roles, and to provide some of these required functions the vacuole harbors at least seven distinct proteases. These proteases exhibit a range of activities and different classifications, and they follow unique paths to arrive at their ultimate, common destination in the cell. This review will first summarize the major functions of the yeast vacuole and delineate how proteins are targeted to this organelle. We will then describe the specific trafficking itineraries and activities of the characterized vacuolar proteases, and outline select features of a new member of this protease ensemble. Finally, we will entertain the question of why so many proteases evolved and reside in the vacuole, and what future research challenges exist in the field.

Tryptophan breakdown is increased in euthymic overweight individuals with bipolar disorder: a preliminary report.

OBJECTIVES: Individuals with bipolar disorder (BD) are disproportionately affected by symptoms of being overweight and metabolic syndrome when compared to the general population. The pertinence of this observation is underscored by observations that excess weight is associated with a more complex illness presentation, course, and outcome in BD. We present the first preliminary report of our BIPFAT study, which explored shared hypothesized pathophysiological pathways between being overweight and having BD. METHODS: We investigated the tryptophan-kynurenine metabolism pathway as a proxy of dysregulated inflammatory homeostasis in euthymic, overweight individuals with BD (n = 78) compared to healthy controls (n = 156). RESULTS: Both blood kynurenine concentrations and the kynurenine to tryptophan ratio [(Kyn:Trp); an estimate of tryptophan breakdown] were significantly higher in the total sample of euthymic patients with BD, with greater increases noted in both parameters in the subsample of overweight patients with BD. When compared to controls, peripheral neopterin concentrations were significantly lower. Within the BD group, there were also significant between-group differences in neopterin concentrations, with higher levels in those who were overweight and in subjects with BD in the later stages of illness compared to earlier stages. CONCLUSIONS: Increased tryptophan breakdown, as well as neopterin levels in BD, may be an indirect mediator of immune-mediated inflammation. In BD, this may account for the high prevalence of medical comorbidities and increased mortality. The observation of increased kynurenine levels and Kyn:Trp, and altered circulating neopterin levels provides indirect evidence of increased activity of tryptophan-degrading indoleamine 2,3-dioxygenase in euthymic individuals with BD, underscoring the role of inflammatory mediators as a causative and/or consequent factor. More robust abnormalities in the overweight subsample underscore the additional inflammatory burden of medical comorbidity and suggest a shared pathophysiology as well as a mechanism mediating BD and cardiovascular disease.

Characterization of an M28 metalloprotease family member residing in the yeast vacuole.

The systematic and complete characterization of the Saccharomyces cerevisiae genome and proteome has been stalled in some cases by misannotated genes. One such gene is YBR074W, which was initially annotated as two independent open reading frames (ORFs). We now report on Ybr074, a metalloprotease family member that was initially predicted to reside in the endoplasmic reticulum (ER). Therefore, we tested the hypothesis that Ybr074 may be an ER quality control protease. Instead, indirect immunofluorescence images indicate that Ybr074 is a vacuolar protein, and by employing protease protection assays, we demonstrate that a conserved M28 metalloprotease domain is oriented within the lumen. Involvement of Ybr074 in ER protein quality control was ruled out by examining the stabilities of several well-characterized substrates in strains lacking Ybr074. Finally, using a proteomic approach, we show that disrupting Ybr074 function affects the levels of select factors implicated in vacuolar trafficking and osmoregulation. Together, our data indicate that Ybr074 is the only multispanning vacuolar membrane protease found in the yeast Saccharomyces cerevisiae.

Sexual behavior, body image, and partnership in chronic illness: a comparison of Huntington's disease and multiple sclerosis.

Huntington's disease (HD) and multiple sclerosis (MS) are both chronic progressive illnesses posing a serious challenge to affected patients and families. Sexual dysfunction in HD as well as in MS is a very common problem, although it is unclear whether the dysfunction is caused by the chronic illness itself or by the sociopsychiatric burden related to the illness. Twenty-nine patients with HD and 27 patients with MS each participated in a semistructured interview and several standardized questionnaires concerning partnership, sexual function, and body image. The results display significant differences in both patient groups, displaying higher sexual desire and activity in HD patients, but MS patients also reported fewer sexual problems compared to the norming values. Conversely, the MS patients' relationships seemed to be stable despite subjectively perceived lower initiative on sexual activities. The results are discussed under the possible influences of the underlying organic changes and the psychosocial consequences of chronic progressive disorders.

A continuum method for determining membrane protein insertion energies and the problem of charged residues.

Continuum electrostatic approaches have been extremely successful at describing the charged nature of soluble proteins and how they interact with binding partners. However, it is unclear whether continuum methods can be used to quantitatively understand the energetics of membrane protein insertion and stability. Recent translation experiments suggest that the energy required to insert charged peptides into membranes is much smaller than predicted by present continuum theories. Atomistic simulations have pointed to bilayer inhomogeneity and membrane deformation around buried charged groups as two critical features that are neglected in simpler models. Here, we develop a fully continuum method that circumvents both of these shortcomings by using elasticity theory to determine the shape of the deformed membrane and then subsequently uses this shape to carry out continuum electrostatics calculations. Our method does an excellent job of quantitatively matching results from detailed molecular dynamics simulations at a tiny fraction of the computational cost. We expect that this method will be ideal for studying large membrane protein complexes.

Aromatic and cation-pi interactions enhance helix-helix association in a membrane environment.

The cation-pi interaction is an electrostatic attraction between a positive charge and the conjugated pi electrons of an aromatic ring. These interactions are well documented in soluble proteins and can be both structurally and functionally important. Catalyzed by observations in our laboratory that an Ala- and Ile-rich two-helix transmembrane segment tended to form SDS-resistant dimers upon the incorporation of suitably located Trp residues, here we have constructed a library of related constructs to study systematically the impact of aromatic-aromatic and cation-pi interactions on tertiary structure formation within an Escherichia coli membrane. Using the TOXCAT oligomerization assay with the hydrophobic segment AIAIAIIAZAXAIIAIAIAI, where Z = A, W, Y, or F and X = A, H, R, or K in all possible combinations of cation and/or aromatic pairings, to assess the TM-TM dependent expression of the chloramphenicol acetyltransferase reporter gene, we find that cation-pi interactions, particularly between Lys and Trp, Tyr, or Phe, as well as weakly polar interactions between pairs of aromatic residues, significantly enhance the strength of oligomerization of these hydrophobic helices, in some instances forming oligomers four times stronger than the high-affinity glycophorin A dimer. The contribution of these forces to the tertiary structure formation in designed transmembrane segments suggests that similar forces may also be a significant factor in the folding and stability of native membrane proteins.