A vector system is described that combines reliable, very low level, regulated protein expression in human cells with two affinity purification tags (Sequential Peptide Affinity, or SPA, system). By avoiding overproduction of the target protein, this system allows for the efficient purification of natural protein complexes and their identification by mass spectrometry. We also present an adaptation of the SPA system for the efficient purification and identification of protein complexes in E. coli and, potentially, other bacteria.

ObjectiveThe aim of the study was to determine the serum levels of CRP, IL-6 and TNF-α in elderly diabetic patients with depressive syndrome alone or with coexisting mild cognitive impairment (MCI).Methods276 diabetics elders were screened for depressive symptoms (using Geriatric Depression Scale: GDS-30) and MCI (using the Montreal Cognitive Assessment: MoCA score). Data of HbA1c, blood lipids and inflammatory markers levels were collected.ResultsIn all groups of patients levels of CRP, IL-6 and TNF-α were significantly higher as compared to controls. The highest level of inflammatory markers was detected in group with depressive mood and coexisting MCI, however IL-6 level didn’t significantly differ as compared to MCI group. We founded correlations between all inflammatory markers in group of patients with depressive mood and in group of subjects with depressive symptoms and coexisting MCI. GDS-30 score was correlated with levels of inflammatory markers in group with depressive mood, and with levels of CRP and TNF-α in group with depressive mood and coexisting MCI. In the group with depressive mood and coexisting MCI we founded that MoCA score was negatively correlated with CRP and TNF-α levels; and HbA1c level was positively correlated with all inflammatory markers. The univariate logistic regression models revealed that variables which increased the likelihood of having been diagnosed with MCI in depressed patients were: higher levels of HbA1c, CRP, IL-6 and TNF-α, previous CVD or stroke, increased number of co-morbidities and microvascular complications, older age, less years of formal education. The multivariable model showed that previous CVD, higher HbA1c and IL-6 levels are significant factors.ConclusionsWe demonstrated that the presence of depressive syndrome is associated with higher levels of inflammatory markers in elderly patients with diabetes. The presence of MCI in these depressed subjects has additive effect on levels of inflammatory mediators.

The aim of the study was to determine the serum levels of adiponectin, leptin and IL-1 β in elderly diabetic patients with and without mild cognitive impairment (MCI) and to examine the associations of these markers with clinical and cognitive parameters. A biochemical evaluation was performed of 62 seniors with type 2 diabetes (T2DM) and MCI, and 132 seniors with T2DM but without MCI (controls). Serum leptin and IL-1 β levels were higher and adiponectin concentration was lower in MCI patients than controls. In MCI subjects, adiponectin level was negatively correlated with leptin, IL-1 β levels and BMI. Leptin concentration was correlated with IL-1 β level. Univariate logistic regression models revealed that the factors which increased the likelihood of diagnosis of MCI in elderly patients with T2DM were higher levels of HbA1c, leptin, IL-1 β and triglycerides, as well as lower levels of adiponectin and HDL cholesterol. Similarly, previous CVD, hypertension, hyperlipidemia, retinopathy, nephropathy, hypoglycemia, longer duration of diabetes, increased number of co-morbidities, older age, fewer years of formal education were found to be associated with MCI. The multivariable model indicated fewer years of formal education, previous CVD, hypertension, increased number of co-morbidities, higher HbA1c and IL-1 β levels and lower adiponectin level. Elderly diabetic patients with MCI have higher levels of leptin and IL-1 β and lower levels of adiponectin. Further prospective studies are needed to determine the role of these markers in the progression to dementia.

This paper describes a new technique for noninvasive diagnostic analysis of metabolic regulation and the microcirculation. Flow Mediated Skin Fluorescence (FMSF) is based on monitoring the intensity of NADH fluorescence emitted from skin tissue on the forearm. The principles of the technique are discussed, in particular, concerning experimental procedures and the definition and interpretation of the measured parameters. The unique features and potential avenues for development of the FMSF technique are also outlined.

ObjectiveThe aim of the study was to evaluate serum levels of advanced glycation end products (AGEs), receptor for advanced glycation end products (RAGE), and C-reactive protein (CRP) in elderly patients with type 2 diabetes mellitus with and without mild cognitive impairment (MCI) and to determine the predictors (including AGEs, RAGE, and CRP levels) of having MCI in elderly patients with type 2 diabetes.MethodsTwo hundred seventy-six diabetics elders were screened for MCI (using the Montreal Cognitive Assessment: MoCA score). Data of biochemical parameters and biomarkers were collected.ResultsSerum AGEs, RAGE, and CRP levels were significantly increased in MCI patients compared to controls. In group of patients with MCI, serum RAGE level was positively correlated with AGEs level and with CRP level. RAGE, AGEs, and CRP concentrations were positively correlated with HbA1c levels and negatively correlated with MoCA score. The univariate logistic regression models revealed that variables, which increased the likelihood of diagnosis of MCI in elderly patients with type 2 diabetes were higher levels of HbA1c, RAGE, AGEs, CRP, TG, lower level of HDL cholesterol, previous CVD, HA, or use of HA drugs, hyperlipidemia, retinopathy, nephropathy, increased number of co-morbidities, older age, and less years of formal education. HA or use of HA drugs, previous CVD, higher level of RAGE and CRP, older age and less years of formal education are the factors increasing the likelihood of having MCI in elderly patients with type 2 diabetes in multivariable model.ConclusionIn summary, serum AGEs, RAGE, and CRP are increased in the circulation of MCI elderly diabetic patients compared to controls. A larger population-based prospective study needs to be performed to further confirm the relationship between AGEs, RAGE, and the cognitive decline or progress to dementia.

1H Nuclear magnetic resonance (NMR) relaxometry was exploited to investigate the dynamics of solid proteins. The relaxation experiments were performed at 37 °C over a broad frequency range, from approximately 10 kHz to 40 MHz. Two relaxation contributions to the overall 1H spin–lattice relaxation were revealed; they were associated with 1H–1H and 1H–14N magnetic dipole–dipole interactions, respectively. The 1H–1H relaxation contribution was interpreted in terms of three dynamical processes occurring on timescales of 10−6 s, 10−7 s, and 10−8 s, respectively. The 1H–14N relaxation contribution shows quadrupole relaxation enhancement effects. A thorough analysis of the data was performed revealing similarities in the protein dynamics, despite their different structures. Among several parameters characterizing the protein dynamics and structure (e.g., electric field gradient tensor at the position of 14N nuclei), the orientation of the 1H–14N dipole–dipole axis, with respect to the principal axis system of the electric field gradient, was determined, showing that, for lysozyme, it was considerably different than for the other proteins. Moreover, the validity range of a closed form expression describing the 1H–14N relaxation contribution was determined by a comparison with a general approach based on the stochastic Liouville equation.

PurposeSignificant progress has been made in the technological and physical aspects of dose delivery and distribution in proton therapy. However, mode of cell killing induced by protons is less understood in comparison with X-rays. The purpose of this study is to see if there is any difference in the mode of cell-killing, induced by protons and X-rays in an ex vivo human peripheral blood lymphocyte (HPBL) model.Materials and methodsHPBL were irradiated with 60 MeV proton beam or 250-kVp X-rays in the dose range of 0.3–4.0 Gy. Frequency of apoptotic and necrotic cells was determined by the Fluorescein (FITC)-Annexin V labelling procedure, 1 and 4 h after irradiation. Chip-based DNA Ladder Assay was used to confirm radiation-induced apoptosis and necrosis. Chip-based DNA Ladder Assay was used to confirm radiation-induced apoptosis.ResultsEx vivo irradiation of HPBL with proton beams of 60 MeV or 250 kVp X-rays resulted in apoptotic as well as necrotic modes of cell-killing, which were evident at both 1 and 4 h after irradiation in the whole dose and time range. Generally, our results indicated that protons cause relatively higher yields of cell death that appears to be necrosis compared to X-rays. The analysis also demonstrates that radiation type and dose play a critical role in mode of cell-killing.ConclusionObtained results suggest that X-rays and protons induce cell-killing by different modes. Such differences in cell-killing modes may have implications on the potential of a given therapeutic modality to cause immune modulation via programmed cell death (X-rays) or necrotic cell death (proton therapy). These studies point towards exploring for gene expression biomarkers related necrosis or apoptosis to predict immune response after proton therapy.

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