Quantitative phosphoproteome analysis of lysophosphatidic acid induced chemotaxis applying dual-step (18)O labeling coupled with immobilized metal-ion affinity chromatography

Reversible protein phosphorylation is a central cellular regulatory mechanism in modulating protein activity and propagating signals within cellular pathways and networks. Development of more effective methods for the simultaneous identification of phosphorylation sites and quantification of temporal changes in protein phosphorylation could provide important insights into molecular signaling mechanisms in various cellular processes. Here we present an integrated quantitative phosphoproteomics approach and its application for comparative analysis of Cos-7 cells in response to lysophosphatidic acid (LPA) gradient stimulation. The approach combines trypsin-catalyzed (16)O/ (18)O labeling plus (16)O/ (18)O-methanol esterification for quantitation, a macro-immobilized metal-ion affinity chromatography trap for phosphopeptide enrichment, and LC-MS/MS analysis. LC separation and MS/MS are followed by neutral loss-dependent MS/MS/MS for phosphopeptide identification using a linear ion trap (LTQ)-FT mass spectrometer. A variety of phosphorylated proteins were identified and quantified including receptors, kinases, proteins associated with small GTPases, and cytoskeleton proteins. A number of hypothetical proteins were also identified as differentially expressed followed by LPA stimulation, and we have shown evidence of pseudopodia subcellular localization of one of these candidate proteins. These results demonstrate the efficiency of this quantitative phosphoproteomics approach and its application for rapid discovery of phosphorylation events associated with LPA gradient sensing and cell chemotaxis.     

Late‐life time‐restricted feeding and exercise differentially alter healthspan in obesity

Aging and obesity increase multimorbidity and disability risk, and determining interventions for reversing healthspan decline is a critical public health priority. Exercise and time‐restricted feeding (TRF) benefit multiple health parameters when initiated in early life, but their efficacy and safety when initiated at older ages are uncertain. Here, we tested the effects of exercise versus TRF in diet‐induced obese, aged mice from 20 to 24 months of age. We characterized healthspan across key domains: body composition, physical, metabolic, and cardiovascular function, activity of daily living (ADL) behavior, and pathology. We demonstrate that both exercise and TRF improved aspects of body composition. Exercise uniquely benefited physical function, and TRF uniquely benefited metabolism, ADL behavior, and circulating indicators of liver pathology. No adverse outcomes were observed in exercised mice, but in contrast, lean mass and cardiovascular maladaptations were observed following TRF. Through a composite index of benefits and risks, we conclude the net healthspan benefits afforded by exercise are more favorable than those of TRF. Extrapolating to obese older adults, exercise is a safe and effective option for healthspan improvement, but additional comprehensive studies are warranted before recommending TRF.     

Compressed sensing approach for wrist vein biometrics

The work describes features of the compressed sensing (CS) approach utilized for development of a wearable system for wrist vein recognition with single‐pixel detection; we consider this system useful for biometrics authentication purposes. The CS approach implies use of a spatial light modulation (SLM) which, in our case, can be performed differently—with a liquid crystal display or diffusely scattering medium. We show that compressed sensing combined with above‐mentioned means of SLM allows us to avoid using an optical system—a limiting factor for wearable devices. The trade‐off between the 2 different SLM approaches regarding issues of practical implementation of CS approach for wrist vein recognition purposes is discussed. A possible solution of a misalignment problem—a typical issue for imaging systems based upon 2D arrays of photodiodes—is also proposed. Proposed design of the wearable device for wrist vein recognition is based upon single‐pixel detection.      

The development of modified human Hsp70 (HSPA1A) and its production in the milk of transgenic mice

The production of major human heat shock protein Hsp70 (HSPA1A) in a eukaryotic expression system is needed for testing and possible medical applications. In this study, transgenic mice were produced containing wild-type human Hsp70 allele in the vector providing expression in the milk. The results indicated that human Hsp70 was readily expressed in the transgenic animals but did not apparently preserve its intact structure and, hence, it was not possible to purify the protein using conventional isolation techniques. It was suggested that the protein underwent glycosylation in the process of expression, and this quite common modification for proteins expressed in the milk complicated its isolation. To check this possibility, we mutated all presumptive sites of glycosylation and tested the properties of the resulting modified Hsp70 expressed in E. coli. The investigation demonstrated that the modified protein exhibited all beneficial properties of the wild-type Hsp70 and was even superior to the latter for a few parameters. Based on these results, a transgenic mouse strain was obtained which expressed the modified Hsp70 in milk and which was easy to isolate using ATP columns. Therefore, the developed construct can be explored in various bioreactors for reliable manufacture of high quality, uniform, and reproducible human Hsp70 for possible medical applications including neurodegenerative diseases and cancer.     

3-(N-arylsulfamoyl)benzamides, inhibitors of human sirtuin type 2 (SIRT2)

Inhibition of sirtuin 2 (SIRT2) is known to be protective against the toxicity of disease proteins in Parkinson's and Huntington's models of neurodegeneration. Previously, we developed SIRT2 inhibitors based on the 3-(N-arylsulfamoyl)benzamide scaffold, including3-(N-(4-bromophenyl)sulfamoyl)-N-(4-bromophenyl)benzamide(C2-8, 1a), which demonstrated neuroprotective effects in a Huntington's mouse model, but had low potency of SIRT2 inhibition. Here we report that N-methylation of 1a greatly increases its potency and results in excellent selectivity for SIRT2 over SIRT1 and SIRT3 isoforms. Structure-activity relationships observed for 1a analogs and docking simulation data suggest that the para-substituted amido moiety of these compounds could occupy two potential hydrophobic binding pockets in SIRT2. These results provide a direction for the design of potent drug-like SIRT2 inhibitors.     

Biosensors to measure inositol 1,4,5-trisphosphate concentration in living cells with spatiotemporal resolution

Phosphoinositides participate in many signaling cascades via phospholipase C stimulation, which hydrolyzes phosphatidylinositol 4,5-bisphosphate, producing second messengers diacylglycerol and inositol 1,4,5-trisphosphate (InsP3). Destructive chemical approaches required to measure [InsP3] limit spatiotemporal understanding of subcellular InsP3 signaling. We constructed novel fluorescence resonance energy transfer-based InsP3 biosensors called FIRE (fluorescent InsP3-responsive element) by fusing plasmids encoding the InsP3-binding domain of InsP3 receptors (types 1-3) between cyan fluorescent protein and yellow fluorescent protein sequences. FIRE was expressed and characterized in COS-1 cells, cultured neonatal cardiac myocytes, and incorporated into an adenoviral vector for expression in adult cardiac ventricular myocytes. FIRE-1 exhibits an approximately 11% increase in the fluorescence ratio (F530/F480) at saturating [InsP3] (apparent K(d) = 31.3 +/- 6.7 nm InsP3). In COS-1 cells, neonatal rat cardiac myocytes and adult cat ventricular myocytes FIRE-1 exhibited comparable dynamic range and a 10% increase in donor (cyan fluorescent protein) fluorescence upon bleach of yellow fluorescent protein, indicative of fluorescence resonance energy transfer. In FIRE-1 expressing ventricular myocytes endothelin-1, phenylephrine, and angiotensin II all produced rapid and spatially resolved increases in [InsP3] using confocal microscopy (with free [InsP3] rising to approximately 30 nm). Local entry of intracellular InsP3 via membrane rupture by a patch pipette (containing InsP3)in myocytes expressing FIRE-1 allowed detailed spatiotemporal monitoring of intracellular InsP3 diffusion. Both endothelin-1-induced and direct InsP3 application (via pipette rupture) revealed that InsP3 diffusion into the nucleus occurs with a delay and blunted rise of [InsP3] versus cytosolic [InsP3]. These new biosensors allow studying InsP3 dynamics at high temporal and spatial resolution that will be powerful in under-standing InsP3 signaling in intact cells.     

Thermal stress defense in freshwater amphipods from contrasting habitats with emphasis on small heat shock proteins (sHSPs)

This study comparatively evaluated small heat shock proteins (sHSP) (related to α-crystallin) and antioxidant enzymes such as peroxidase (POD), catalase (CAT), glutathione S-transferase (GST) as anti-thermal stress response in two contrasting freshwater amphipods, the stenoecious Baikalean endemic Eulimnogammarus cyaneus and the Palearctic Gammarus lacustris.The thermal stress modulated the activity of antioxidant enzymes as well as the sHSP synthesis in both species. In both species, only the declining POD activity showed a clear dependency on exposure time.The most expressed response to elevated temperatures has been the activation of sHSP synthesis, with clear differences in the patterns: in G. lacustris, sHSP concentrations peaked after 12 h with a subsequent decline, while they increased steadily in E. cyaneus. Hence, the stenoecious species did not acclimate to the thermal stress within the given exposure time as the euryecious did.     

Late ontogeny growth acceleration and size form transformations in Transbaikalian Arctic charr, <Emphasis Type="Italic">Salvelinus alpinus</Emphasis> complex: evidence from fin ray cross section growth layers

In some polymorphic populations of Arctic charr in Transbaikalia, an individual can transform from a smaller to larger size form during their lifetime as a result of accelerated growth that follows a period of slow growth and reproduction as a small size form. Alternating periods of slow and fast growth are reflected in growth layer patterns visible in fin ray cross sections. Stained microtome fin ray cross sections were used to reveal the incidence of transformations from one form to another. Data were collected from 14 northern Transbaikalian lakes containing two or three sympatric Arctic charr forms (‘dwarf’, ‘small’, and ‘large’) exhibiting varying levels of morphological separation. Individuals recruited from the dwarf or small form were found in varying proportions among the small and/or large form in 12 lakes. Small or large form charr that grew without noticeable acceleration to the adult size typical of the form or experienced accelerated growth as juveniles prior to maturation were also observed. There were no transformations between sympatric forms that differed in the length and number of gill rakers and in some other meristic characters. Results indicate that in the region under study, transformations of sympatric Arctic charr size forms are a widespread, but not a ubiquitous, phenomenon. Such transformations reflect the plasticity of the developmental channels of the forms. In the course of intra-lacustrine form divergence and genetic differentiation, the frequency of the observed transformations decreases to zero.