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After sample processing for RNA and DNA analysis, the leftover protein pellets are usually discarded due to the limited efficiency of pellet reconstitution/solubilisation. As the pelleted proteins are tightly packed, they are most often solubilised using chaotropic agents (e.g., guanidine hydrochloride or urea), detergents (e.g., SDS), salts (NaCl) or basic buffer (Tris). The aim of this study was to define and optimise the procedure for the efficient extraction of proteins from human peripheral blood mononuclear cells (PBMCs), obtained by a single blood draw and lysed in TRIzol reagent, by varying experimental conditions in terms of protein precipitation solvent (isopropanol or acetone), washing (with or without guanidine hydrochloride) and solubilisation solution (containing SDS, NaCl, urea and/or Tris). We evaluated the efficacy of the final, optimised protocol to solubilise both small cytoplasmic and larger transmembrane proteins, and the compatibility with methods employed for the subsequent analysis of protein posttranslational modifications, such as glycosylation. The optimised protocol for the extraction and isolation of post-TRIzol leftover proteins from PBMCs can be defined as follows: protein precipitation from the organic phase with ice-cold acetone, pellet washing with absolute ethanol and solubilisation in 1 % SDS, employing 20 min heating at 50ºC and vortexing.

The knowledge on the general population health is important for creating public policies and organization of medical services. However, personal data are often limited, and mathematical models are employed to achieve a general overview. Cluster analysis was used in this study to assess general trends in population health based on laboratory data. Metabolic indicators were chosen to test the model and define population cohorts. Data on blood analysis of 33,049 persons, namely the concentrations of glucose, total cholesterol and triglycerides, were collected in a public health laboratory and used to define metabolic cohorts employing computational data clustering (CLARA method). The population was shown to be distributed in 3 clusters: persons with hypercholesterolemia with or without changes in the concentration of triglycerides or glucose, persons with reference or close to reference concentrations of all three analytes and persons with predominantly elevated all three parameters. Clustering of biochemical data, thus, is a useful statistical tool in defining population groups in respect to certain health aspect.

The binding of a popular food supplement and well-known antioxidant, dihydro-alpha-lipoic acid (DHLA) to human serum albumin (HSA) was characterised. The binding was monitored by several spectroscopic methods together with the molecular docking approach. HSA was able to bind DHLA with moderate affinity, 1.00±0.05×104 M-1. Spectroscopic data demonstrated that the preferential binding site for DHLA on HSA is IIA (Sudlow I). Both experimental and molecular docking analysis identified electrostatic (salt bridges) and hydrogen bonds as the key interactions involved in DHLA binding to HSA. Molecular docking confirmed that the Sudlow I site could accommodate DHLA and that the ligand is bound to the protein in a specific conformation. The molecular dynamic simulation showed that the formed complex is stable. Binding of DHLA does not affect the structure of the protein, but it thermally stabilises HSA. Bound DHLA had no effect on the susceptibility of HSA to trypsin digestion. Since DHLA is a commonly used food supplement, knowledge of its pharmacokinetics and pharmacodynamic properties in an organism is very important. This study further expands it by providing a detailed analysis of its interaction with HSA, the primary drug transporter in the circulation.

A simple and reliable method for the determination of the concentration and function of alpha-2-macroglobulin (α2M) by zymography was developed. The method is based on the covalent binding of α2M and trypsin followed by non-reducing PAGE and zymography with gelatine incorporated in the electrophoretic gel. The results showed that α2M binds trypsin in a concentration-dependent manner exhibiting a linear relation. The sensitivity of the method is 125 nM and the intra-assay coefficient of variation 4.2 %. Freezing of α2M induces its partial denaturation, which could be seen as the reduction in the amount of functional molecule and its reactivity with trypsin. The reported method enables measurement of α2M taking into consideration both its quantity and function, stressing the importance of the determination of the amount of physiologically active molecules and not just their presence in the sample. The method was further confirmed using α2M from patients with end-stage renal disease who are known to be under increased oxidative stress and inflammation, which are expected to modify the structure of proteins. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. 173042]

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The purpose of this paper is to introduce a model for measuring the efficiency in managing peer-review of scientific manuscripts by editors. The approach employed is based on the assumption that the editorial aim is to manage publication with high efficiency, employing the least amount of editorial resources. Efficiency is defined in this research as a measure based on 7 variables. An on-line survey was constructed and editors of journals originating from Serbia regularly publishing articles in the field of chemistry were invited to participate. An evaluation of the model is given based on responses from 24 journals and 50 editors. With this investigation we aimed to contribute to our understanding of the peer-review process and, possibly, offer a tool to improve the “efficiency” in journal editing. The proposed protocol may be adapted by other journals in order to assess the managing potential of editors.

Background: Insulin and insulin-like growth factor (IGF) activities are disturbed during critical illness. Time-course changes in the concentrations of insulin, IGF-I and IGFbinding proteins (IGFBPs) were monitored in this study and their correlation with interleukin (IL)-6 was assessed in patients subjected to total gastrectomy and specific nutritional regime. Methods: Patients were fed post-operatively according to the following scheme: parenteral nutrition on day 1, enteral nutrition combined with parenteral form from day 2 to 7, peroral nutrition from day 8 and full oral nutrition from day 14. Blood samples were taken periodically and the levels of IL-6, insulin, IGF-I and IGFBP-1 to -4 were determined. Results: On day 1 post-operatively, the concentration of IL6 reached its maximum and decreased afterwards. The concentration of insulin increased until day 3 and then started to fall. The concentration of IGF-I, already low preoperatively, continued to decrease. The concentration of IGFBP-1 peaked on day 1 post-operatively, whereas the concentration of IGFBP-3 decreased on that day. The concentration of IL-6 correlated positively with the concentration of IGFBP-1 and negatively with IGFBP-3. On day 14, the concentrations of IL-6, insulin and IGFBP-1 returned to or were close to their basal levels, whereas the concentrations of IGF-I and IGFBP-3 remained reduced. Conclusions: A 14-day post-operative recovery, which included specific nutritional support, was suitable to restore insulin concentration and re-establish IGFBP-1 regulation primarily by nutrition. Very low IGF-I level on day 14 after surgery and IGFBP-3 concentration still lower than before surgery indicated that the catabolic condition was not compensated.

A survey was conducted among editors of journals publishing in the field of chemistry, chemical technology and related topics in Serbia, aiming to collect information on their experience, problems and difficulties during peer-review process. Editors from 22 journals out of 27 which regularly published during 2015 replied. General data on journals were collected from responses obtained from editors-in-chief, whereas all editors (including sub-editors and section editors) participated in a questionnaire concerning peer-review procedure. Additionally, they were asked to evaluate quality of reports and attitude of reviewers, discuss present situation and suggest measures to improve peer-review process. Greatest problems encountered by editors in peer-review process can be summarized as follows: low rate of acceptance to review, low quality of reports, sometimes due to reviewer’s bias or his/her inability to properly understand review process. A method used to search for reviewers does not substantially influence quality of reports. Editors agree that introduction of On-Line process and creation of precise instructions for reviewers, education of potential reviewers, as well as social, public and professional recognition and appreciation of reviewers’ work, are the most important measures to improve quality of peer-review process and, consecutively, quality of published articles and journals.

Journal of the Serbian Chemical Society (JSCS) is an official journal of the Serbian Chemical Society and this year we are celebrating 85 years of its publishing and the 80th volume. After so many years of publishing, the idea of the Editorial board of the JSCS was to investigate what do authors, reviewers and Sub-editors think about the journal and whether their evaluation and suggestions can help to improve it. Questionnaires were sent to the three investigated groups as an e-mail link. Responses were analyzed and only the most general and the most important data are presented in this article. Grades, comments and suggestions have shown that most of the contributors are satisfied with the present handling and publishing policy of the JSCS, but certain technical aspects should be improved. After thorough inspection of the data, the Editorial Board decided to introduce fully automatic on-line system, to speed-up the peer review process, to improve Instructions to authors and Reviewer’s report form. All these novelties started from March 2015.

Background: Postoperative sepsis alters the growth hormone (GH)/insulin-like growth factor (IGF) axis leading to reduction in IGF-I and IGF-binding protein (IGFBP) 3 and elevation of GH-independent IGFBPs. The aim of this work was to investigate the differences in ligand binding and immunoreactivity of the circulating IGFBPs between surviving and non-surviving septic patients that underwent colorectal surgery. Methods: Ligand binding was detected only for IGFBP-3 and IGFBP-2 and, consecutively, immunoreactivity was assayed for these IGFBPs. Results: In survivors both ligand- and immunoblotting revea led two IGFBP-3 isoforms (40 and 45 kDa). In non-survivors ligand-blotting hardly detected the IGFBP-3 doublet, intact immunoreactive IGFBP-3 was not detected and the major species was the 29 kDa fragment. Immunoblotting with an anti-IGFBP-2 antibody indicated an increase in the amount of intact and fragmented IGFBP-2 along the progression of illness in the non-survivors. The amount of intact IGFBP-2 in the survivors did not differ during the treatment. The amount of immunoreactive IGFBP-2 corresponded to its ligand-binding activity. Conclusions: According to the results reported here, ligand-reactivity and immunoreactivity of IGFBP-3 should be taken into account as variables that affect the GH/IGF axis in critically ill patients. The degree of their variation is not directly correlated to the total amount of IGFBP-3 determined by the immunoassay, but they may be related to patients’ metabolic potential to combat a disease.

Diabetes mellitus is a complex disease that leads to alterations in the glycosylation of proteins. Insulin-like growth factor and insulin receptors are involved in the regulation of fetal and placental growth and development. In this work the N-glycans of these receptors, originating from placentas obtained from pregnancies complicated by pregestational insulin dependent diabetes mellitus, were studied. Diabetic mothers were under regular insulin therapy. Solubilised membrane samples from healthy and diabetic placentas were analysed using lectin-affinity chromatography. N-glycans bound to insulin-like growth factor and insulin receptors were studied in terms of their interaction with eleven aga roseimmobilised lectins: wheat germ agglutinin, succinyla ted wheat germ agglutinin, Ricinus communis agglutinin I, Sambucus nigra agglutinin, Erythrina cristagalli lectin, Ulex europaeus agglutinin, Lens culinaris agglutinin, Canavalia en siformis lectin, Phaseolus vulgaris erythro- and leukoagglutinin and Maackia amurensis agglutinin. A very similar type of N-glycans and content of the terminal saccharide residues were found in both groups of placentas. The results of this work suggest that the tight glycemic control may prevent alterations in the glycosylation of insulin-like growth factor and insulin receptors, thus maintaining physiological homeostasis during pregnancy and fetal growth.

Insulin-like growth factors (IGFs) play an important role in the regulation of cell growth, differentiation and metabolism. The amount of free, biologically active IGFs is regulated by the IGF-binding proteins (IGFBPs). IGFBP-3 is the most abundant binding protein and it is known to interact with other circulating proteins, including transferrin (Tf). In order to elucidate the possible role of IGF/IGFBP-3 in the iron metabolism, it is necessary to isolate IGFBP-3/Tf complexes. Several affinity-based techniques were employed. Results have shown that only double immunoprecipitation method with anti-Tf and anti-IGFBP-3 antibodies selectively separated complexes from other molecular forms, such as monomers, oligomers or fragments of IGFBP-3 and Tf. Isolated complexes can now be used to investigate the relationship between IGF/IGFBP-3 and iron, both in structural and metabolic tеrms.

The objective of this study was to examine the effect of dietary energy supplementation on hormones that are considered to be the main signals of a shift in energy balance around parturition. Sixty dry cows, 15 days before calving, were chosen and divided into two eaqual groups: control and experimental (GLY). Both groups were fed a standard ration balanced in accordance to the stage of the productivereproductive cycle. Additionally, each cow in the GLY group was given glycerol based dietary energy supplementation (250 mL daily during the dry and 300 mL daily during the lactation period), which provided additional 9.30 MJ NEL during the dry and 13.95 MJ NEL during the early lactation period. Milk production was measured on days 30 and 60 of lactation and milk production was significantly higher in GLY compared to control group at day 60 of lactation (p

Insulin-like growth factor-binding protein 1 (IGFBP-1) is a member of a family of six homologous proteins that regulate the action of the insulin-like growth factors. IGFBP-1 is a 25 kDa protein that beside its native form, may exist in several phosphoforms (30 kDa), which are predominant in the circulation of humans. Phosphorylation of IGFBP-1 is a post-translational modification that has a great influence on the IGF-I action. IGFBP-1 forms multimers and complexes with α2-macroglobulin (α2M). Polymerisation of IGFBP-1 was also reported. In order to analyse and separate these IGFBP-1 molecular species, affinity chromatography methods were used in this study. The results demonstrated that most of the IGFBP-1 circulates in complexes with α2M, which can be isolated by affinity chromatography using immobilised anti-α2M antibodies. IGFBP- 1/α2M complexes may be differentiated from IGFBP-1 dimer and multimers using lectin-affinity chromatography, since the latter do not interact with lectins. It seems that the complexes contain not only monomeric IGFBP-1, but also its multimers. Dimer and multimers are stable under reducing conditions, suggesting covalent linkage between units. Free IGFBP-1 monomer can be separated from multimers using Con A-affinity chromatography. The concentration of free IGFBP-1 is relatively low in the circulation.

The IGF system of human placenta consists of insulin-like growth factors (IGF)-I and -II, their receptors (IGF-1R and IGF-2R), and binding proteins (IGFBP-1 to -6). Due to many structural and metabolic similarities with insulin, the IGF system cannot be examined separately from insulin and its receptor (IR). In this study gel filtration was used to detect solubilized membrane proteins of the placenta obtained from healthy mothers and mothers with IDDM. In order to detect placental membrane proteins that bind IGF molecules (and insulin), the solubilized membranes were incubated with each of the three 125I-labelled ligands: 125I-IGF-I, 125I-IGF-II and 125I-insulin prior to gel filtration chromatography. The biochemical evidence of the presence of receptors for insulin and IGFs, as well as that of IGFBP-1 were obtained by immunoblotting. Herein we demonstrated that, considering IGF and insulin receptor content, the placental tissue obtained from mothers with IDDM was not different from that obtained from healthy mothers. However, the concentration of IGFBP-1 differed between the examined placentas. IDDM in mothers caused an increase in the amount of IGFBP-1 in their placentas and, consequently, the amount of the labelled ligand bound to it. The redistribution of IGFs between the receptors and IGFBP-1 may be involved in regulatory mechanisms in the placenta of mothers with IDDM.

Fetal growth in utero depends on the proper development and function of the placenta. Insulin-like growth factors (IGFs) are critically involved in placental development. During pregnancy, an IGF-binding protein, IGFBP-1, which is produced by maternal decidua, plays an important role in the control of the bioavailability of IGFs. It has recently been proposed that cleavage of decidual IGFBP-1 by matrix metalloproteases is a novel mechanism in the control of placental development. The presence of IGFBP-1 in solubilized placental cell membranes, i.e. its association with the membranes, was detected in an earlier work. Herein, it is shown that IGFBP-1 from the solubilized membranes forms dimers, as well as high molecular mass complexes. IGFBP-1 dimers preferably contain the non-phosphorylated form of IGFBP-1. The high molecular mass forms are polymers of IGFBP-1 or its complexes with other membrane proteins. Dimerization of IGFBP-1, together with its association with the placental cell membrane, could serve as an additional mechanism of the regulation of IGF availability to the type 1 IGF receptors.

In order to study whether the carbohydrate moieties of the human placental IGF-I receptor (IGF1R), IGF-II receptor (IGF2R) and insulin receptors (IRs) play a role in ligand binding, solubilised cell membrane preparations were incubated with 125I-labelled IGF-I, IGF-II and insulin in the presence of lectins with different sugar specificities. Three incubation procedures were tested: ligand-first, co-incubation and lectin-first incubation. Wheat germ agglutinin (WGA), concanavalin A (Con A) and phytohaemagglutinin (PHA) altered the binding of IGF-I and insulin to their high-affinity receptors in a lectin specific and dose-dependent manner, whereas these lectins did not affect the interaction of IGF-II with its receptor(s). Moreover, the same lectins either inhibited or enhanced IGF-I and insulin binding, depending on the incubation scheme. These results also suggest that IR-A and IR-B from human placenta might be differently glycosylated.

The life of an organism depends on its capacity to maintain equilibrium Disease induces acute adaptive responses specific to the stimulus and the organism achieves stability through change. These changes are associated with increased growth hormone resistance and activity of the hypothalamic-pituitary- adrenal axis, altered insulin response and cytokine synthesis and, closely related to all these events, alteration of the complex system interrelating insulin-like growth factors (IGFs), IGF-binding proteins (IGFBPs) and their receptors. This review aims to provide an overview of the results obtained in our laboratory from studies of the IGF system in patients with metabolic disturbances caused by infection, hepatobiliary and gastrointestinal diseases, open or laparoscopic surgery and postoperative sepsis.

The biological activities of IGF-I and –II are mediated mainly by the type 1 IGF receptor (IGF 1R) and controlled by their interaction with soluble proteins, the IGF binding proteins (IGFBPs). Although there is a growing body of evidence that some IGFBPs may be cell surface-bound, published data concerning cell association of IGFBP-1 are scarce and none of them concern placental cells. The cell membranes used in this study were isolated from term human placenta. Detergent-solubilized membranes were shown to contain two types of IGF binding structures that were separated by gel filtration on a Sephadex G-100 column. Proteins in the first peak were eluted at V0(Mr>100 kD) and they bound IGF-I with greater specificity and affinity than IGF-II and insulin. Most likely, they represented the IGF 1R. Small proteins (Mr≈45 kD) were eluted with the membrane proteins in the second maximum. They were able to bind IGF-I and IGF-II, but not insulin. The identity of these proteins was shown to be IGFBP-1 on the basis of their reaction with specific anti-IGFBP-1 antibodies. To the best of our knowledge, the existence of IGFBP-1 associated with human placental cell membranes has not been reported in the literature before. Colocalisation of IGFBP-1 with IGF 1R in cell membranes could provide efficient modulation of IGF 1R receptor-ligand interactions.

Mosses are biomonitors that accumulate large amounts of various pollutants including radionuclides. In this work we investigated the possibility of 137Cs extraction from mosses, as well as the significance of species specificity on the efficiency of 137Cs desorption. Salt and acid solutions were used as extraction media. It was shown that a 5% solution of both ammonium oxalate and phosphoric acid was able to desorb 81.8% of 137Cs from Homalothecium sericeum, which was 39.9%more than desorption from water. At the same time, most of the desorbed 137Cs+ was incorporated in crystals that precipitated from the solution. An interspecies difference in respect to 137Cs+ desorption was noticed.

A few years after the iodine content of salt in Serbia was increased from 7 to 15 mg/kg NaCI, iodine, thyroxine (T4) and triiodothyronine (T3) concentrations were measured in thyroid tissue obtained at autopsy from 21 human neonates who died within 30 days after birth. The thyroidal iodine as well as T4 and T3 content per gland in­creased progressively with gestational age of human neonates (r = 0.73, 0.70 and 0.67 respectively, p < 0.001). In seven newborns (gestational age 36 to 41 weeks) the mean values for total iodine, T4 and T3 per gland were 109.1 μg, 52.2 μg and 4.4 μg respectively. The results of iodine and iodothyroninc content found in neonatal thyroid gland, particularly at the end of gestation and a few days of postnatal life, indicates that the iodine supply was satisfactory.

Recent findings highlighted the potential of long non-coding RNAs (lncRNAs) as novel indicators of gestational diabetes mellitus (GDM), as they demonstrate altered expression in metabolic disorders, oxidative stress (OS) and inflammation (IFM). The aim of this study was to evaluate the diagnostic potential and prognostic significance of the OS/IFM-related lncRNAs H19, MALAT1 and MEG3 in GDM and their correlations with redox status-related parameters. The relative quantification of selected lncRNAs from peripheral blood mononuclear cells (PBMCs) of GDM patients and controls (n = 50 each) was performed by qPCR. The expression levels were tested for correlations with metal ion concentrations, NRF2 expression, activities of glutathione reductase (GR), superoxide dismutase (SOD), catalase (CAT), serum thiol content, protein carbonyl level and thiobarbituric acid reactive substances. MALAT1 and H19 were significantly downregulated in GDM patients (p = 0.0095 and p = 0.012, respectively). A correlation was observed between H19 expression and zinc levels in both GDM patients and controls. MALAT1 expression positively correlated with NFE2L2 levels in GDM patients (p = 0.026), while H19 exhibited a positive correlation with GR activity in controls (p = 0.018) and an inverse correlation with SOD activity (p = 0.048). Our data show the disturbance of OS/IFM-lncRNAs in GDM pathogenesis and illustrate the biomarker potential of the analyzed lncRNAs, as well as of certain redox status parameters. [ABSTRACT FROM AUTHOR]