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Background: The prevalence of prediabetes is increasing in the global population and its metabolic derangements may expose to a higher risk to develop type 2 diabetes (T2D) and its cardiovascular burden. Lifestyle modifications might have considerable benefits on ameliorating metabolic status. Alternative biomarkers, such as circulating miR-21, has been recently discovered associated with dysglycemia. Here we evaluated, in a longitudinal cohort of dysglycemic population the relation between the circulating miR-21/ROS/HNE levels and the habit-intervention (HI) after 1 year of follow-up., Methods: 1506 subjects from DIAPASON study were screened based on the Findrisc score. Of them, 531 subjects with Findrisc ≥ 9 were selected for dysglycemia (ADA criteria) and tested for circulating miR-21, ROS and HNE levels, as damaging-axis. 207 subjects with dysglycemia were re-evaluated after 1-year of habit intervention (HI). Repeated measures tests were used to evaluate changes from baseline to 1-year of follow-up. The associations between glycemic parameters and miR-21/ROS/HNE were implemented by linear regression and logistic regression models., Results: After HI, we observed a significant reduction of miR-21/ROS/HNE axis in dysglycemic subjects, concomitantly with ameliorating of metabolic parameters, including insulin resistance, BMI, microalbuminuria, reactive hyperemia index and skin fluorescence. Significant positive interaction was observed between miR-21 axis with glycaemic parameters after HI. Lower miR-21 levels after HI, strongly associated with a reduction of glycemic damaging-axis, in particular, within-subjects with values of 2hPG < 200 mg/dL., Conclusions: Our findings demonstrated that HI influenced the epigenetic changes related to miR-21 axis, and sustain the concept of reversibility from dysglycemia. These data support the usefulness of novel biological approaches for monitoring glycemia as well as provide a screening tool for preventive programmes., (© 2022. The Author(s).)

Objectives: Familial hypercholesterolemia (FH) is the most common monogenic lipid disorder associated with premature coronary heart disease. Early cholesterol-lowering therapy could effectively reduce cardiovascular disease morbidity and mortality in these patients. However, the majority of people with FH are undiagnosed, also due to low awareness and knowledge of FH in general practice, despite the high number of contacts GPs have with most of their patients which allows a systematic and effective approach to the detection of this condition. Here, we present a simple method to improve detection and to enhance awareness of FH in primary care using GP electronic health records., Methods: We used electronic data from the Co.S. Consortium, involving more than 600 Italian affiliated GPs. Electronic data include demographic information, laboratory test results, recorded history of vascular disease and prescription of an HMG-CoA reductase inhibitor class medication. We performed a partial assessment of the Dutch Lipid Clinic Network (DLCN) score using those data that were recorded or available. We also sought to determine the prevalence of possible FH based on age-specific LDL-cholesterol thresholds employed by the diagnostic criteria of MEDPED and the non-age adjusted cut-off point (LDL-C ≥190 mg/dL)., Results: Data on LDL-C were available for 162,864 subjects. Mean LDL-C levels (SD) were 124.3 (33.6) mg/dL for non-treated subjects and 106.4 (38.5) mg/dL for statin-treated subjects. The cut-off of LDL-C ≥190 mg/dL yielded a prevalence of 2.9% among non-treated subjects and of 3.5% among statin-treated patients. Using the cut-off of ≥250 mg/dL, the prevalence was 0.1% among non-treated subjects and 0.3% among statin-treated patients. Using the cut-off ≥330 mg/dL (suggesting a probable diagnosis of FH according to the DLCN score) the prevalence was 0.01% and 0.02%. According to the stratification proposed by MEDPED criteria for the general population, the age-specific LDL-cholesterol thresholds identified 0.7% among non-treated subjects and 18.5% among statin-treated patients., Conclusion: The diagnosis of FH is possible in general medicine and should be an integral part of the GP's activity., (Copyright © 2017. Published by Elsevier B.V.)

Electrospray Ionization and collision induced dissociation tandem mass spectrometry are usually employed to obtain compound identification through a mass spectra match. Different algorithms have been developed for this purpose (for example the nist match algorithm). These approaches compare the tandem mass spectra of the unknown analyte with the tandem mass spectra spectra of known compounds inserted in a database. The compounds are usually identified on the basis of spectral match value associated with a probability of recognition. However, this approach is not usually applied to multiple reaction monitoring transition spectra achieved by means of triple quadrupole apparatus, mainly due to the lack of a transition spectra database. The Surface Activated Chemical Ionization-Electrospray-NIST Bayesian model database search (SANIST) platform has been recently developed for new potential metabolite biomarker discovery, to confirm their identity and to use them for clinical and diagnostic applications. Here, we present an improved version of the SANIST platform that extends its application to forensic, pharmaceutical, and food analysis studies, where the compound identification rules are strict. The European Union (EU) has set directives for compound identification (EU directive 2002/657/EC). We have applied the SANIST method to identification of 11-nor-9-carboxytetrahydro-cannabinol in urine samples (an example of a forensic application), circulating levels of the immunosuppressive drug tacrolimus in blood (an example of a pharmaceutical application) and glyphosate in fruit juice (an example of a food analysis application) that meet the EU directive requirements. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

A simple device effective for matrix-assisted laser desorption/ionization (MALDI) sample preparation, based on the spraying of matrix/sample solution through a stainless steel sieve (sieve-based device, SBD), has been employed for the preparation of MALDI samples of peptides, polysaccharides and high molecular weight proteins. The instrumental parameters, mainly the sprayer voltage, solution and auxiliary gas flows, and distances between sprayer-sieve and sieve-MALDI sample holder have been optimized. The MALDI spectra obtained by laser irradiation of the microspots so obtained, exhibit resolution and sensitivity higher than those achieved by the commonly employed dried droplets (DD) method. Worth noting is the fact that, in contrast to what is observed in the DD method, identical spectra are obtained by irradiation of different areas of the MALDI sample, showing that by using the SBD the matrix/analyte ratio is constant over the entire surface., (Copyright 2009 John Wiley & Sons, Ltd.)

The 2-iminothiolane reaction with protein amino groups adds a spacer arm ending with a thiol group, which can be further treated with molecules carrying a maleimido ring. This approach is currently used for the preparation of a candidate 'blood substitute' in which human Hb (haemoglobin) is conjugated with long chains of PEG [poly(ethylene glycol)]. To identify the thiolation sites by MS, we have carried out the reaction using deoxyHb bound to inositol hexaphosphate to protect some of the residues crucial for function and NEM (N-ethylmaleimide) to block and stabilize the thiol groups prior to enzymatic digestion by trypsin and pepsin. Under the conditions for the attachment of 5-8 PEG chains per tetramer, the thiolated residues were Lys7, Lys11, Lys16, Lys56 and Lys139 and, with lower accessibility, Lys90, Lys99 and Lys60 of the a-chain and Lys8, Lys17, Lys59, Lys61 and Lys66 and, with lower accessibility, Lys65, Lys95 and Lys144 of the b-chain. The a-amino groups of a- and b-chains were not modified and the reaction of the Cysb93 residues with NEM was minor or absent. After the modification with thiolane and NEM of up to five to eight lysine residues per tetramer, the products retained a large proportion of the properties of native Hb, such as low oxygen affinity, co-operativity, effect of the modulators and stability to autoxidation. Under identical anaerobic conditions, the conjugation of the thiolated Hb tetramer with five or six chains of the maleimido derivative of 6 kDa PEG yielded products with diminished co-operativity, Hill coefficient h=1.3-1.5, still retaining a significant proportion of the effects of the modulators of oxygen affinity and stability to autoxidation. Co-operativity was apparently independent of the topological distribution of the PEGylated sites as obtained by treating partly the thiolated protein with NEM prior to PEGylation [poly(ethylene glycol)ation].

We have applied the ligation detection reaction (LDR) combined with a universal array approach to the detection and quantitation of the polymerase chain reaction (PCR) amplified cry1A(b) gene from Bt-176 transgenic maize. We demonstrated excellent specificity and high sensitivity. Down to 0.5 fmol (nearly 60 pg) of PCR amplified transgenic material was unequivocally detected with excellent linearity within the 0.1-2.0% range with respect to wild-type maize. We suggest the feasibility of extending the LDR/universal array format to detect in parallel several transgenic sequences that are being developed for food applications.

Background: PCR amplification of bacterial 16S rRNA genes provides the most comprehensive and flexible means of sampling bacterial communities. Sequence analysis of these cloned fragments can provide a qualitative and quantitative insight of the microbial population under scrutiny although this approach is not suited to large-scale screenings. Other methods, such as denaturing gradient gel electrophoresis, heteroduplex or terminal restriction fragment analysis are rapid and therefore amenable to field-scale experiments. A very recent addition to these analytical tools is represented by microarray technology., Results: Here we present our results using a Universal DNA Microarray approach as an analytical tool for bacterial discrimination. The proposed procedure is based on the properties of the DNA ligation reaction and requires the design of two probes specific for each target sequence. One oligo carries a fluorescent label and the other a unique sequence (cZipCode or complementary ZipCode) which identifies a ligation product. Ligated fragments, obtained in presence of a proper template (a PCR amplified fragment of the 16s rRNA gene) contain either the fluorescent label or the unique sequence and therefore are addressed to the location on the microarray where the ZipCode sequence has been spotted. Such an array is therefore "Universal" being unrelated to a specific molecular analysis. Here we present the design of probes specific for some groups of bacteria and their application to bacterial diagnostics., Conclusions: The combined use of selective probes, ligation reaction and the Universal Array approach yielded an analytical procedure with a good power of discrimination among bacteria.

The Bohr protons released by oxygen exposure of the unliganded subunits of intermediates (alpha +CN-beta) (alpha +CN-beta) and (alpha beta +CN-) (alpha beta +CN-) were obtained by titrations of concentrated solutions of these species. The Bohr protons released by oxygen exposure of the other intermediates were obtained from titrations of equilibrium mixtures of two parental species, (alpha beta) (alpha beta), (alpha +CN-beta) (alpha +CN-beta), (alpha beta +CN-) (alpha beta +CN-), and (alpha +CN-beta +CN-) (alpha +CN-beta +CN-), in which the concentration of the hybrid intermediate was determined by cryogenic electrophoretic techniques. The Bohr effect of the intermediates was calculated by subtracting the Bohr protons released by oxygen exposure of the intermediates from the total Bohr protons of deoxyhemoglobin at the same pH. The Bohr effects of intermediates (alpha +CN-beta) (alpha beta) and (alpha beta +CN-) (alpha beta) were similar and vanished at pH 8 where the total Bohr effect of deoxyhemoglobin is still significant. This suggests that the Bohr effect in these intermediates is tertiary in the quaternary T structure. The curve of the Bohr effect of intermediate (alpha +CN-beta +CN-) (alpha beta), which was close to the curve obtained by adding the Bohr effects of the two monoliganded intermediates at acidic and physiological pH values, was significantly different from the curve obtained by adding the Bohr effects of one liganded subunit of intermediate (alpha +CN-beta) (alpha +CN-beta) and one liganded subunit of intermediate (alpha beta +CN-) (alpha beta +CN-).(ABSTRACT TRUNCATED AT 250 WORDS)

The intermediates in the oxidation of deoxyhemoglobin by ferricyanide in 0.1 M KCl, at 20 degrees C and three pH values, were studied by cryogenic techniques. Data analysis was carried out according to a simple four rate constant model, ignoring the functional heterogeneity of the subunits, to simulate the time courses of the oxidation reaction, as studied by the stopped-flow technique [Antonini et al., (1965) Biochemistry 4, 345], which show anticooperativity at neutral pH and cooperativity at alkaline pH. Data analysis according to a 12 rate constant model indicated that the rate of oxidation of the beta subunit in the first oxidation reaction was 4 times faster than the rate of oxidation of the alpha subunit at pH 6.2 and 12 times faster at pH 8.5. The reactions involving the alpha subunit were noncooperative except for the last oxidation step at acid and neutral pH, but were cooperative at alkaline pH. The reactions involving the beta subunit were partly noncooperative and partly anticooperative. These complex mechanistic patterns suggest that a simple two-state model requiring the concerted transition of the tertiary structures of the subunits from the T to the R conformation is not adequate to interpret the oxidation reaction and that tertiary structures contribute, positively and negatively, to cooperativity. A structural hypothesis is suggested to explain the difference in the reactivities of the alpha and beta subunits.

The populations of the intermediates in concentrated solutions of hemoglobin A0 equilibrated at various PCO values, pH 7.0, 0.1 M KCl, and 20 degrees C, have been determined using cryogenic methods. Data on CO saturations and distributions of intermediates were analysed in terms of the free energies of dimer-tetramer assembly of the intermediates (G.K. Ackers and F.R. Smith, Annu. Rev. Biophys. Chem. 16 (1987) 583). The cooperative free energy value of the singly ligated species was approximately one-half the total cooperative energy. The cooperative free energy value of the doubly ligated species was not significantly different from that of carboxyhemoglobin. Because of experimental error, the observed difference in concentrations among the populations of the doubly ligated species cannot be taken as indicative of their functional heterogeneity. Additional studies on some NO intermediates have emphasized that (alpha 1 beta 1)(alpha 2 beta 2)X, a key intermediate in the formulation of the 'third-state' hypothesis in the deoxy/cyanomethemoglobin system, has a free energy value for dimer-tetramer assembly which is critically dependent on the nature of the ligand X as suggested by Ackers and Smith (reference as cited above).

The application of a new technique based on the differential measurement of pH between two solutions to determine ethanol concentration in whole blood is reported. The ethanol is determined by measuring the change in pH following its enzymatic oxidation to acetaldehyde. The procedure correlates with the head space gas chromatographic method in the 0-108 mmol/l (0-5 g/l) ethanol whole blood concentration range according to the equation y = 1.344 + 1.013x (r = 0.997).

Human globin chain separation, a key procedure in the study of hemoglobinopathies, is routinely performed by chromatography on carboxymethylcellulose. This method, though relatively easy and highly reliable, is expensive and time consuming. A new procedure, based on isoelectric focusing, is presented which allows the simultaneous separation of globin chains from multiple samples (at least 20 per gel slab). The method is rapid, inexpensive and can be easily carried out in clinical laboratories, and its high sensitivity allows the identification of radioactive bands even with minute amounts of labelled material. A new phenomenon, called the 'Nonidet P-40 effect', which greatly enhances the separation between gamma and beta chains by binding to these two chains and shifting their pI values in opposite directions, is described.

The hemoglobinemia S is a genetic defect due to a replacement of a single amino acid in the beta-chain of the human hemoglobin, leading to the most characteristic case of primary blood hyperviscosity. The difference in beta-chain of normal HbA compared to that of HbS is represented by the replacement of glutamic acid, normally found in the 6th position, with valine. The sickling of HbS-containing erythrocytes is due to the polymerization of deoxygenated HbS tetramers with formation of linear structures and to the parallel distribution of these fibers in the red blood cell. The full deoxygenation of HbS blood induces the characteristic morphological changes of red blood cell and increases the blood viscosity from 40 to 120% if compared to the viscosity of the same oxygenated blood. Any change in the viscosity of normal blood following deoxygenation was observed. Almost all the clinical symptoms found in patients carrying HbS can be directly or indirectly correlated to the increased blood viscosity following deoxygenation.

The log P50 of normal human blood at 37 degrees C, PCO2 = 0, 21, 42, AND 57 MM Hg in the absence and in the presence of 2,3-DPG and ATP, has been determined in the pH range 7.0 to 7.6. Similar data have been obtained for human hemoglobin isotonic solutions at different protein concentrations in the presence of various amounts of each of the cofactors which are known to affect hemoglobin oxygen affinity in blood. It has been found that the addition of KC1, organic phosphates, magnesium ions, and CO2 confers to a 32% human hemoglobin solution the same oxygen affinity (over the entire physiological pH range) of whole blood. Thus there is no room for significant effects caused by some other unidentified molecules or ions.

We report here the development of a new method that allows continuous determination of the oxygen dissociation curve for microsamples (600 mul) of whole blood under conditions of pH, pCO2, methemoglobin concentration, and 2,3-diphosphoglycerate content closely approaching those found in the circulatory system. The method consists of gradually oxygenating a blood sample by adding H2O2 in the presence of catalase (EC 1.11.1.6), to produce the reaction H2O2 leads to H2O + 1/2 O2. Because the total oxygen content of blood can be derived from the known rate of H202 addition and the pO2 is determined in the liquid phase by an oxygen electrode, the two functions (total O2 content) and (% oxygen saturation) vs. pO2 are simple to calculate. pCO2 and pH are controlled by adding base simultaneously with the gradual oxygenation of blood. The method described thus avoids the direct measurement of oxygen saturation of whole blood.