Proteomic and glycomic analyses of the potential protective role of zeolite on an osteoporotic rat model

Bone homeostasis is secured by a combined action of bone forming osteoblasts and bone resorbing osteoclasts. When this balance is impaired, osteopenia is induced, which in time develops into osteoporosis. Antibody glycosylation influences osteoclast differentiation. It is known that immunoglobulin G (IgG) immune complexes positively stimulate osteoclast differentiation, resulting in subsequent inflammatory bone loss. In order to further investigate this process, a method was developed to isolate IgG from serum and to investigate the changes in IgG glycosylation patterns in osteoporotic rats treated with clinoptilolite, a natural zeolite with great detoxification and ion exchange properties. An experimental rat model was set up, and following groups of animals were analysed: healthy control, sham control, ovariectomized control (OVX), OVX supplemented with synthetic zeolite, OVX supplemented with natural clinoptilolite and OVX supplemented with micro activate clinoptilolite. Using affinity chromatography by use of monolithic supports with immobilized immunoglobulin binding ligands as a robust tool for isolation of immunoglobulins, IgG can be purified from rat sera. Compared to proteins A and G, recombinant protein L binds by far the largest number of isoforms of all immunoglobulins. For this reason, this ligand immobilized on a monolithic column has been used in this work. To procure a highly enriched IgG preparation, the fraction with proteins eluted from the column was analysed on a 1D polyacrylamide gel, after which in-gel tryptic digestion of this protein was performed. The resulting peptides were successfully identified by MALDI-TOF/TOF mass spectrometry as parts of IgG heavy and light chains. In the next step, the IgG glycan structure was analysed by use of the same technique, after the glycans have been removed from the protein using deglycosylation enzyme PNGase F. Next to the IgG analysis, liver proteomes of healthy, sham operated and ovariectomized (OVX) rats treated with zeolites were fractionated according to hydrophobicity and each fraction was separately analysed by SDS PAGE. Finally, liver cryo slices were examined by Synchrotron radiation. Present results give us the evidence that the developed high throughput protocols for analysis of glycosylation of rat immunoglobulins, namely IgG, IgA and IgM, as well as the protocol for quantitative proteomic investigations of rat liver proteome, give us the fundament for further investigations by use of a larger number of experimental animals. We suggest that clinoptilolite positively affects bone status in osteoporotic rats as a consequence of signalling changes in the body, particularly those initiated by the liver and the systemic spread of IgG molecules.Rad ne sadrži sažetak na drugom jeziku

Proteomic and glycomic analyses of the potential protective role of zeolite on an osteoporotic rat model

Bone homeostasis is secured by a combined action of bone forming osteoblasts and bone resorbing osteoclasts. When this balance is impaired, osteopenia is induced, which in time develops into osteoporosis. Antibody glycosylation influences osteoclast differentiation. It is known that immunoglobulin G (IgG) immune complexes positively stimulate osteoclast differentiation, resulting in subsequent inflammatory bone loss. In order to further investigate this process, a method was developed to isolate IgG from serum and to investigate the changes in IgG glycosylation patterns in osteoporotic rats treated with clinoptilolite, a natural zeolite with great detoxification and ion exchange properties. An experimental rat model was set up, and following groups of animals were analysed: healthy control, sham control, ovariectomized control (OVX), OVX supplemented with synthetic zeolite, OVX supplemented with natural clinoptilolite and OVX supplemented with micro activate clinoptilolite. Using affinity chromatography by use of monolithic supports with immobilized immunoglobulin binding ligands as a robust tool for isolation of immunoglobulins, IgG can be purified from rat sera. Compared to proteins A and G, recombinant protein L binds by far the largest number of isoforms of all immunoglobulins. For this reason, this ligand immobilized on a monolithic column has been used in this work. To procure a highly enriched IgG preparation, the fraction with proteins eluted from the column was analysed on a 1D polyacrylamide gel, after which in-gel tryptic digestion of this protein was performed. The resulting peptides were successfully identified by MALDI-TOF/TOF mass spectrometry as parts of IgG heavy and light chains. In the next step, the IgG glycan structure was analysed by use of the same technique, after the glycans have been removed from the protein using deglycosylation enzyme PNGase F. Next to the IgG analysis, liver proteomes of healthy, sham operated and ovariectomized (OVX) rats treated with zeolites were fractionated according to hydrophobicity and each fraction was separately analysed by SDS PAGE. Finally, liver cryo slices were examined by Synchrotron radiation. Present results give us the evidence that the developed high throughput protocols for analysis of glycosylation of rat immunoglobulins, namely IgG, IgA and IgM, as well as the protocol for quantitative proteomic investigations of rat liver proteome, give us the fundament for further investigations by use of a larger number of experimental animals. We suggest that clinoptilolite positively affects bone status in osteoporotic rats as a consequence of signalling changes in the body, particularly those initiated by the liver and the systemic spread of IgG molecules.Rad ne sadrži sažetak na drugom jeziku

Proteomic and glycomic analyses of the potential protective role of zeolite on an osteoporotic rat model

Bone homeostasis is secured by a combined action of bone forming osteoblasts and bone resorbing osteoclasts. When this balance is impaired, osteopenia is induced, which in time develops into osteoporosis. Antibody glycosylation influences osteoclast differentiation. It is known that immunoglobulin G (IgG) immune complexes positively stimulate osteoclast differentiation, resulting in subsequent inflammatory bone loss. In order to further investigate this process, a method was developed to isolate IgG from serum and to investigate the changes in IgG glycosylation patterns in osteoporotic rats treated with clinoptilolite, a natural zeolite with great detoxification and ion exchange properties. An experimental rat model was set up, and following groups of animals were analysed: healthy control, sham control, ovariectomized control (OVX), OVX supplemented with synthetic zeolite, OVX supplemented with natural clinoptilolite and OVX supplemented with micro activate clinoptilolite. Using affinity chromatography by use of monolithic supports with immobilized immunoglobulin binding ligands as a robust tool for isolation of immunoglobulins, IgG can be purified from rat sera. Compared to proteins A and G, recombinant protein L binds by far the largest number of isoforms of all immunoglobulins. For this reason, this ligand immobilized on a monolithic column has been used in this work. To procure a highly enriched IgG preparation, the fraction with proteins eluted from the column was analysed on a 1D polyacrylamide gel, after which in-gel tryptic digestion of this protein was performed. The resulting peptides were successfully identified by MALDI-TOF/TOF mass spectrometry as parts of IgG heavy and light chains. In the next step, the IgG glycan structure was analysed by use of the same technique, after the glycans have been removed from the protein using deglycosylation enzyme PNGase F. Next to the IgG analysis, liver proteomes of healthy, sham operated and ovariectomized (OVX) rats treated with zeolites were fractionated according to hydrophobicity and each fraction was separately analysed by SDS PAGE. Finally, liver cryo slices were examined by Synchrotron radiation. Present results give us the evidence that the developed high throughput protocols for analysis of glycosylation of rat immunoglobulins, namely IgG, IgA and IgM, as well as the protocol for quantitative proteomic investigations of rat liver proteome, give us the fundament for further investigations by use of a larger number of experimental animals. We suggest that clinoptilolite positively affects bone status in osteoporotic rats as a consequence of signalling changes in the body, particularly those initiated by the liver and the systemic spread of IgG molecules.Rad ne sadrži sažetak na drugom jeziku

Ultrastructural alterations in peripheral blood lymphocytes in type 2 diabetes patients

Tip 2 dijabetes melitus (T2DM), trenutno jedna od najvećih globalnih epidemija, predstavlja heterogenu grupu poremećaja koja se karakteriše različitim nivoom insulinske rezistencije, poremećenom sekrecijom insulina i hiperglikemijom. Kao početna terapija T2DM koristi se metformin, dok se u kasnijem toku bolesti u terapiji koristi insulin. Nedavni nalazi autoantitela na β ćelije pankreasa i autoreaktivnih T limfocita u T2DM dovode do pretpostavke da u patogenezi T2DM postoji autoimunska komponenta. Savremena istraživanja ukazuju na značaj autofagije u patogenezi T2DM, ali je njena uloga kontroverzna. Rezultati nekih studija ukazuju na povećanu aktivnost autofagije u mononuklearnim ćelijama periferne krvi, dok su u drugim studijama detektovali njenu sniženu aktivnost u ovim ćelijama u dijabetesu. Autofagija je lizozomalni put degradacije intracelularnih komponenti u fiziološkim uslovima i u ćelijskom stresu, i ima ključnu ulogu intracelularnom metabolizmu. Promene u autofagičnom procesu mogu nastati kao odgovor na hiperglikemiju kod bolesnika sa T2DM. Novija elektronsko mikroskopska istraživanja su pokazala da u T2DM dolazi do morfoloških promena u izgledu nukleusa u vidu nepravilnosti oblika i binukelacije nukleusa. Pretpostavka je da su nepravilnosti oblika nukleusa u normalnim humanim limfocitima rezultat aktivacije T limfocita. Cilj: Cilj ovog istraživanja bio je da se metodama ultrastrukturne morfometrije kvantifikuje broj autofagičnih struktura u limfocitima periferne krvi T2DM bolesnika na elektronskom mikroskopu; da se koreliraju dobijeni rezultati sa ekspresijom proteina uključenih u proces autofagije; da se uradi ultrastrukturna analiza aktivacije limfocita i oštećenja subcelularnih struktura kod bolesnika sa T2DM; fraktalna analiza ultrastrukturnog izgleda nukleusa i citoplazme limfocita sa T2DM; i korelacija statusa autofagije sa aktivacijom i oštećenjem subcelularnih struktura u limfocitima bolesnika sa T2DM, kao i sa rezultatima dobijenim fraktalnom analizom nukleusa i citoplazme...Type 2 diabetes mellitus (T2DM), currently one of the largest global epidemics, is a heterogeneous group of disorders characterized by varying levels of insulin resistance, impaired insulin secretion, and hyperglycemia. Initial therapy for T2DM is metformin, but with the progression of disease, insulin therapy is used. Recent findings of autoantibodies against pancreatic β-cell and autoreactive T cell in T2DM lead to the assumption that there is an autoimmune component in the pathogenesis of T2DM. Recent researches points out the importance of autophagy in the pathogenesis of T2DM, but its role is controversial. Some studies indicate an increased autophagy level in peripheral blood mononuclear cells, while others showed decreased level of autophagy in these cells in diabetes. Autophagy is a lysosomal pathway of degradation of intracellular components in physiological conditions and in cellular stress, and plays a key role in intracellular metabolism. Changes in the autophagy may arise in response to hyperglycemia in patients with T2DM. Recent electron microscopy studies have shown that cells in T2DM have morphological changes in the appearance of the nuclei in the form of shape irregularities and binucleation. It has been proposed that nuclear contour irregularity in normal human lymphocytes, could result from T cell activation. Aim: The aim of this study was to quantify the number of autophagic structures in the peripheral blood lymphocytes of the T2DM patients on an electron microscope, using the methods of ultrastructural morphometry; and to correlate the results obtained with the expression of proteins involved in the autophagy; to perform ultrastructural analysis of lymphocyte activation and subcellular structure damage in T2DM patients; fractal analysis of the ultrastructural appearance of T2DM lymphocyte nuclei and cytoplasm; to correlate autophagy level with activation and damage of subcellular structures in the lymphocytes of T2DM patients, as well as with the results obtained from the fractal analysis of the nuclei and cytoplasm..

Double-hit primary unilateral adrenal lymphoma with good outcome

Introduction. Primary adrenal non-Hodgkin’s lymphoma (NHL) is a rare neoplasm with poor prognosis. On the other side, double-hit lymphomas with BCL2 and MYC translocation are characterized by advanced disease stage, extranodal and central nervous system involvements at presentation or disease progression. Case report. We reported a 73-year-old male patient with double-hit primary adrenal lymphoma and preserved adrenal function, showing a favorable clinical course. Computed tomography of abdomen showed a 9 7 cm mass of the left adrenal gland. Laparatomy with left adrenalectomy was done and histological examination revealed diagnosis of a diffuse large B-cell NHL (DLBCL), non-GCB subtype. The patient was treated with 6 cycles of R-CHOP chemotherapy with reduced doses of doxorubicin because of the decreased left verticle ejection fraction. The patient was followed up regularly for 20 months with no evidence of tumor recurrence despite the inherently poor prognostic profile and double-hit phenotype of the disease. Conclusion. R-CHOP chemotherapy in combination with adrenalectomy can be an effective first-line regimen for primary adrenal DLBCL, despite the inherently poor prognostic profile (non-GCB subtype, bulky disease, elevated lactate dehydrogenase and double-hit phenotype of the disease)

Adsorption isotherms of removing copper from aqueous solutions by low cost adsorbent-lignite

Serbian lignite from ’’Kolubara’’ deposit was used as a low cost adsorbent for removal of copper ions (Cu2+) from aqueous solutions. Lignite was subjected to the elementary and technical analysis as well as BET and FTIR analysis to provide complete characterization. Basic comparison between lignite and activated carbon was also done. Adsorption efficiency was studied as function of initial metal concentration and amount of adsorbent. Three different amounts of lignite were used (30, 45, and 60 g). Initial solutions with three different concentrations of copper (50, 200, and 330 mg/l) were prepared. Results proved that investigated lignite is very efficient sorbent material, especially in case of low copper concentration where the usual methods are either economically unrewarding or technically complicated. The effect of adsorbent amount on adsorption kinetics is visible but not crucial

Right ventricular-pulmonary artery coupling in the early stages of infarction could optimize patient risk stratification over five years

Background: Right ventricular (RV) dysfunction is recognized as a cardinal prognostic marker in heart failure (HF) patients. Myocardial infarction (MI) is often followed by unrecognized RV dysfunction, which can be associated with worse outcome. It is recently shown that the ratio between TAPSE and PASP (RV/PA) may depict cardiopulmonary hemodynamics better than the two parameters alone. Aim: To evaluate the interactions between left ventricular (LV) and RV function in early phase of MI and to assess the prognostic significance of RV/PA coupling in patients with first MI during 5 years follow up. Methods: The prospective study included 144 patients with the first MI treated with the primary percutaneous coronary intervention (p PCI) who underwent Doppler echocardiography within 2±1 days of MI. LV function analysis included: LV ejection fraction (EF), ratio between early diastolic velocity and tissue annular velocity (E/e) and global longitudinal strain (GLS). RV function and RV-PA interaction was expressed as ratio between TAPSE and PASP. During the five-year follow-up, major cardiovascular events and especially hospitalization for HF were analyzed. Results: Progressive RV/PA uncoupling was associated with higher degree of LV impairment and dysfunction (EF p<0.001, E/e p=0.002, GLS p<0.001) and severity of mitral regurgitation (p=0.013). Lower baseline RV/PA coupling significantly reflects the frequency of hospitalizations for HF in the population of patients with first MI during five-year follow-up (0.62 v.s.0.51, p=0.021). After multivariate adjustment RV/PA remained an independent predictor of all major cardiac events (MACE) after five years (OR 14.0 [1.5–130.8], p=0.019). Conclusion: A lower baseline RV-PA coupling, reflecting a higher degree of LV-induced pulmonary hypertension and secondary RV-dysfunction, is associated with decline of LV function in early phase of MI, and is independently associated with worse prognosis after five years. The value of RV-PA ratio as an prognstic marker warrants further investigation.ESC Congress 2023 25–28 August 2023 Amsterdam, Netherland

Kinetics of copper adsorption from wastewater by lignite

Kolubara lignite was used as a natural and low cost adsorption material for removal of copper (Cu2+) from the aqueous solutions. As a method, batch adsorption procedure was applied. Results show that domestic lignite is good adsorbent material since about 90 % of copper cations were removed in 5 minutes of contact time from the solutions of low copper concentration (50 mg/l) regardless the adsorbent amount, while the same effect of adsorption was achieved in 60 minutes in case of solutions with high concentrations of copper (330 mg/l). Optimum removal of copper ions was achieved at pH = 5.0. The study proved that tested lignite is very efficient adsorbent material. This behavior can be explained by FTIR spectrum despite a small specific surface area of lignite. Many bands are attributed to the functional groups involved in chemisorption and ionic exchange, basic mechanisms of copper adsorption