16 research outputs found
Structural and functional changes of fibrinogen in patients on peritoneal dialysis
Cilj disertacije je da se kod bolesnika u završnom stadijumu bubrežne slabosti lečenih peritoneumskom dijalizom (PD-om) analiziraju strukturne i funkcionalne promene fibrinogena i fibrina, i da se ispita povezanost uočenih promena sa nastankom neželjenih događaja kardiovaskularnog sistema (KVS-a), nakon perioda praćenja od 18 meseci. U ispitivanju je učestvovala grupa od 52 bolesnika i grupa od 32 zdrave kontrole, uporediva prema polu i starosti. Fibrinogen je izolovan iz plazme isoljavanjem amonijum-sulfatom, i analiziran spektroskopskim, imuno- i lektin-afinitetnim metodama. Fibrinogeni bolesnika na PD-u imaju značajno viši sadržaj karbonila od fibrinogena zdravih kontrola, a Aα i Bβ lanci su podložniji oksidaciji od γ lanaca. Vreme i brzina koagulacije su slični u obe ispitivane grupe, kao i vrednosti za maksimalnu apsorbancu (UV VIS spektri, na 350 nm) i emisioni maksimum ispitivanih fibrinogena (fluorescentni spektri, na 344 nm). Od 16 korišćenih lektina, kod 10 je odnos signal/šum >10, dok je za dva lektina <3. Fibrinogeni ispitanika iz obe grupe su reagovali sa istim lektinima, i sadrže: a) biantenarne N-glikane sa α2,6-Sia; b) umetnuti GlcNAc i/ ili α1,6-Fuc, i c) α1,3-Fuc. Primećena je mala zastupljenost multiantenarnih i paucimanoznih i/ili visokomanoznih N-glikana, i O-glikana tipa T i Tn antigena sa ili bez Sia. Kao prediktor ultrafiltracione slabosti se izdvojio sadržaj fukozilovanih glikana koje vezuje AAL (sa vrednošću AUC od 72,4%). Povišen sadržaj (pauci)manoznih struktura (koje vezuje GNL) nađen je kod bolesnika koji su preminuli nakon 18 meseci od početka studije. Fibrinogen izolovan iz plazme bolesnika lečenih PD-om ima značajno višu koncentraciju karbonila, izmenjenu sekundarnu i tercijarnu strukturu i izmenjen sadržaj glikana. Stepen fukozilacije ima visok potencijal da bude prediktivni faktor malfunkcije peritoneumske membrane, dok sadržaj paucimanoznih/visokomanoznih struktura korelira sa neželjenim kardiovaskularnim događajima i letalnim ishodom.The aim of the thesis is to analyze the structural and functional changes of fibrinogen and fibrin in patients with end - stage renal disease treated with peritoneal dialysis (PD), and to examine the relationship between the observed changes and the occurrence of adverse cardiovascular (CVD) events after a follow - up period of 18 months. The study involved 52 patients and 32 healthy volunteers, comparable by gender and age. Fibrinogen was isolated from plasma by ammonium sulfate isolation, and analyzed by spectroscopic, immuno- and lectin-affinity methods.
The fibrinogens of patients on PD have a significantly higher carbonyl content than the fibrinogens of healthy volunteers, and the Aα and Bβ chains are more susceptible to oxidation than the γ chains. The time and rate of coagulation are similar in both study groups, as well as the values for maximum absorbance (UV VIS spectra, at 350 nm) and emission maximum of analyzed fibrinogens (fluorescent spectra, at 344 nm). Of the 16 lectins used, 10 had a signal-to-noise ratio of > 10, while two lectins had <3. Fibrinogens of both groups reacted with the same lectins, and contain: a) biantenary N-glycans with α2,6-Sia; b) insert GlcNAc and / or α1,6-Fuc, and c) α1,3-Fuc. A small presence of multiantennar and paucimannosidic and/or high-mannose N-glycans, and O-glycans of type T and Tn antigens with or without Sia was observed. The content of fucosylated N-glycans (AAL binding) emerged as a predictor of ultrafiltration strength, (with an AUC value of 72.4%), while an elevated content of (pauci)mannosidic glycans (GNL singal) was found in patients died 18 months after the start of the study.
Fibrinogen isolated from the plasma of patients treated with PD has a significantly higher carbonyl concentration, altered secondary and tertiary structure, and altered glycan content. The degree of fucosylation has a high potential to be a predictive factor of peritoneal membrane dysfunction, while the content of paucimannosidic/highly mannose structures correlates with adverse cardiovascular events and lethal outcome
Mijalgični encefalomijelitis - enigma na raskršću medicine
Myalgic encephalomyelitis is a complex, multisystem disease with chronic course significantly affecting patients' quality of life. Physical and mental exertion intolerability, muscle pain, and sleep problems are the main features accompanied often with cognitive inefficacy and vegetative symptoms. Prevalence is 7-3000 per 100,000 adults. It is estimated that 90% of the patients are misdiagnosed. Pathogenesis is still only speculative but current research points to disturbances in the immunological system, inflammatory pathways, autonomic and central nervous system, muscle and mitochondria, as well as alterations of gut microbiota and gut permeability. The onset is typically acute, following an infectious disease. Exertional intolerance lasting for more than six months is an important diagnostic factor. The core features must be moderate to severe and present at least 50% of the time. Diagnostic criteria should be fulfilled and differential diagnosis should be made to exclude other potential pathological conditions or to diagnose comorbidities. Brain magnetic resonance imaging morphometry has shown gray matter atrophy in occipital lobes bilaterally, right angular gyrus, and the posterior division of the left parahippocampal gyrus, consistent with memory problems and potentially with impairment of visual processing. Treatment is still symptomatic and of partial benefit. Symptomatic treatment can include medications for controlling pain and sleep problems, graded exercise and cognitive behavioral therapy. Larger controlled trials are needed to shed more light on this challenging condition.Mijalgični encefalomijelitis je složeno, multisistemsko oboljenje sa hroničnim tokom koje značajno utiče na kvalitet života bolesnika. Slaba tolerancija na fizički i mentalni napor, bolovi u mišićima i problemi sa spavanjem glavne su odlike i često su praćene kognitivnom neefikasnošću i vegetativnim simptomima. Prevalencija je 7-3000 na 100.000 odraslih. Procenjuje se da je 90% ovih bolesnika pogrešno dijagnostikovano. Patogeneza je i dalje samo spekulativna, ali trenutna istraživanja ukazuju na poremećaje u imunološkom sistemu, inflamatornom odgovoru, autonomnom i centralnom nervnom sistemu, mišićima i mitohondrijama, kao i promene mikrobiote i propustljivosti creva. Početak bolesti je tipično akutan i prati infektivnu bolest. Netolerancija napora koja traje duže od šest meseci važan je dijagnostički kriterijum. Osnovne karakteristike moraju biti umerene do teške i prisutne najmanje 50% vremena. Isključivanje drugih mogućih patoloških stanja ili komorbiditetnih dijagnoza zahteva zadovoljavanje dijagnostičkih kriterijuma i diferencijalno dijagnostičko sagledavanje. Morfometrijska snimanja mozga magnetnom rezonancom pokazala su atrofiju sive mase u okcipitalnim režnjevima bilateralno, desnom angularnom girusu i posteriornom levom parahipokampalnom girusu, što može dovesti do problema sa pamćenjem i oštećenja vizuelne obrade informacija. Lečenje je i dalje simptomatsko i samo delimično uspešno. Simptomatski tretman može da uključuje lekove za kontrolu bola i problema sa spavanjem, doziranu fizičku aktivnost i kognitivno-bihevioralnu terapiju. Potrebne su veće studije da bi se razjasnilo ovo medicinsko stanje
Uticaj brzine rezanja i dubine reza na geometriju prednje linije reza pri obradi abrazivnim vodenim mlazom
Abrasive water jet machining is an unconventional machining process, which is increasingly being used. It is suitable for cutting complex contoured elements, in all types of materials. The major disadvantages of this machining technique are ununiform quality of the machined surface at different depths of cut, lagging of the cut front of the machined surface from the vertical plane and the phenomenon of striation patterns on the cut surface. The striation patterns on machined surface are the trail of the abrasive water jet through the material, and as such, they represents the cut front. Striation may result in different flaws occurring at cutting closed contours and may cause irregularities at cutting angles. The objective of this paper was to determine traverse speed and depth of cut effect on the cut front geometry i.e. on the striation geometry. The experiments were carried out on 6 mm and 10 mm thick samples of aluminum alloy AA 6060 (EN AW-6060; ISO AlMgSi). Samples were machined with several traverse speeds. The lagging of the cut front, at different depths of cut, was measured and analyzed. The obtained results enabled us to define the influence of traverse speed on striation geometry.Publishe
Quantitation of the active alpha-2-macroglobulin by trypsin protease zymography
Alpha-2-macroglobulin (α2M) is a homotetrameric blood glycoprotein having molecular
mass of 720 kDa which acts as a general protease inhibitor 1. So far, the methods to
estimate the quantity of α2M and its activity were separate procedures. The quantity is
usually measured by immunochemical assays and the anti-protease activity of α2M by
measuring the activity of trypsin bound to α2M using chromogenic substrate BAPNA 2. A
simple and reliable method for determination of the concentration and function of α2M by
zymography was developed. This 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 have shown that α2M binds trypsin in a linear,
concentration-dependent manner. The sensitivity of the method is 125 nM with an intraassay
coefficient of variation 4.2 %. Freezing of α2M induces its partial denaturation,
which can be seen as the reduction in the amount of functional molecule and its reactivity
with trypsin. The method was further tested using α2M from patients with an end-stage
renal disease who are known to be under an increased oxidative stress and inflammation,
which are expected to modify the structure of proteins. Using α2M from these patients,
lower affinity of α2M towards trypsin was detected when compaired to α2M isolated from
healthy persons. The reported zymographic method enables measurement of α2M taking
into consideration both its quantity and function, stressing the importance of determination
of the amount of physiologically active molecules and not just their total amount present in
the sample. Monitoring of the relation quantity/activity becomes very important when the
sample originates from an individual exposed to a stress or with a disease accompanied by
post-translational modifications of proteins such as diabetes, renal disease or cancer 3.
Presented method also enables determination of α2M in the presence of different modifying
chemical substances
Analiza opijata u urinu nakon konzumacije proizvoda sa makom
This study presents the case of opiate analysis in urine of two female participants, aged between 20 and 25 who consumed commonly available bakery products with poppy seeds. One of the participants consumed strudel, the product with larger amount of poppy seeds, while the other consumed braid pastry. Urine samples of both participants (n=8) were collected 5, 12, 24, 30, 36, 48, 60 and 72 h after the consummation. Immunochromatographic screening test (cuttof 300 ng/mL) and LC-MS analysis were preformed on the presence of opiates. The screening test for opiates was positive for a very long period of time, 48 h after the consumption of braid pastry and even 72 h after the consumption of strudel. LC-MS analysis verified the results of screening test and the presence of the main opioide alcaloides, morphine and codeine, was confirmed in all positive samples. Determined morphine level was higher than the level of codeine in all samples. The concentration of morphine (0.25 mg/L) was higher in urine of the participant who consumed strudel, compared to the concentration of morphine (0.06 mg/L) after the consumption of braid pastry. The results of this research show that opiates can be found in urine after the consumption of products which contain poppy seed. Morphine and codeine can be detected even during the same time period as in the samples of users of psychoactive substances (3 days and longer). According to the results, retention time of opiates in urine is longer after the consumption of products with higher amounts of poppy. This finding should be taken into account in routine testing of employees in the workplace, members of the military, students in schools, as well as athletes.U ovom radu je prikazan slučaj dve osobe ženskog pola starosti između 20 i 25 godina, koje su konzumirale široko dostupne pekarske proizvode sa makom, sa ciljem da se izvrši analiza opijata u urinu. Jedna osoba je konzumirala štrudlu a druga pletenicu, s tim da je štrudla vizuelno sadržala veću količinu maka. Uzorci urina od obe osobe (po 8 uzoraka) su sakupljeni nakon 5, 12, 24, 30, 36, 48, 60 i 72 sata od konzumacije. Urađena je imunohromatografska i LC/MS analiza na prisustvo opijata. Rezultati imunohromatografske analize su pokazali da je test na opijate (cuttof 300 ng/mL) bio pozitivan u veoma dugom vremenskom periodu, 48 sati nakon konzumacije pletenice i čak 72 sata nakon konzumacije štrudle. LC-MS analizom su potvrđeni rezultati dobijeni preliminarnim testom i u svim pozitivnim uzorcima je dokazano prisustvo glavnih opijatnih alkaloida, morfina i kodeina. Koncentracija morfina je bila veća od koncentracije kodeina u svim uzorcima. Uočena je veća koncentracija morfina (0,25 mg/L) u urinu osobe koja je konzumirala štrudlu u odnosu na koncentraciju morfina (0,06 mg/L) nakon konzumacije pletenice. Rezultati ovog rada potvrđuju da se opijati nalaze u urinu nakon konzumacije proizvoda sa makom i da ih je moguće detektovati čak tokom istog vremenskog perioda kao i kod korisnika psihoaktivnih supstanci, tri dana, i više. Takođe, prema dobijenim rezultatima vreme zadržavanja opijata u urinu je duže nakon konzumacije proizvoda sa većom količinom maka. Navedene rezultate treba imati u vidu prilikom rutinskih testiranja zaposlenih na radnom mestu, pripadnika vojske, učenika u školama i sportista
Prediction of Mortality in Patients on Peritoneal Dialysis Based on the Fibrinogen Mannosylation
As we already reported, fibrinogen fucosylation emerged as a prognostic marker of peritoneal
membrane function in end-stage renal disease (ESRD) patients on peritoneal dialysis. After a
follow-up period of 18 months, we estimated the ability of employed lectins, as well as other biochemical
parameters, to serve as mortality predictors in these patients. Following a univariate Cox
regression analysis, ferritin, urea clearance, residual diuresis, hyperglycemia, and an increase in the
signal intensity obtained with Galanthus nivalis lectin (GNL) emerged as potential mortality predictors,
but additional multivariate Cox regression analysis pointed only to glucose concentration
and GNL as mortality predictors. Higher signal intensity obtained with GNL in patients that died
suggested the importance of paucimannosidic/highly mannosidic N-glycan structures on fibrinogen
as factors that are related to unwanted cardiovascular events and all-cause mortality and can possibly
be seen as a prediction tool. Altered glycan structures composed of mannose residues are expected
to affect the reactivity of mannosylated glycoproteins with mannose-binding lectin and possibly
the entire cascade of events linked to this lectin. Since patients with ESRD are prone to cardiovascular
complications and the formation of atherosclerotic plaques, one can hypothesize that fibrinogen
with increasingly exposed mannose residues may contribute to the unwanted events
Proteomic profiling of anti-transferrin pull-down in patients with underlying oxidative stress
Human serum tansferrin (hsTf) is a major circulatory protein crucial for the
transport/metabolism of Fe3+ ions. By sequestering and delivering ferric ions to target
tissues/cells hsTf maintains redox homeostasis. Oxidative stress (OS), one of the hallmarks
of (patho)physiological conditions, alters protein structure and function. The main role of
hsTf hinges on specific interaction with cellular Tf receptor (TfR) while other interactions
contribute to diverse functions. The aim of this study was to profile interacting partners of
hsTf in the samples of serum coming from patients diagnosed with a wide range of
pathological conditions with underlying OS status. Anti-hsTf pull-down samples were
analysed using mass spectrometry. Data went through analysis by appropriate
bioinformatic tools. Results reveal differences in expression of hsTf interacting proteins in
sample groups of patients suffering from kidney insufficiency subjected to dialysis
treatment (peritoneal-PD or hemo-HD) also with patients with gestational diabetes
compared to respective healthy sample groups. Colorectal cancer stage T3 versus T2 stage
shows an inverse distribution of expression profiles in comparison to healthy samples.
Most prominent differences are seen in hsTf interacting partners involved in the
complement and coagulation cascades and cholesterol metabolic pathways, suggesting a
multifaceted role of hsTf in these processes throughout the course of the disease
Glycosylation and Characterization of Human Transferrin in an End-Stage Kidney Disease
Chronic kidney disease (CKD) is a global health concern affecting approximately one billion individuals worldwide. End-stage kidney disease (ESKD), the most severe form of CKD, is
often accompanied by anemia. Peritoneal dialysis (PD), a common treatment for ESKD, utilizes the
peritoneum for solute transfer but is associated with complications including protein loss, including
transferrin (Tf) a key protein involved in iron transport. This study investigated Tf characteristics in
ESKD patients compared to healthy individuals using lectin microarray, spectroscopic techniques and immunocytochemical analysis to assess Tf interaction with transferrin receptors (TfRs). ESKD patients exhibited altered Tf glycosylation patterns, evidenced by significant changes in lectin reactivity compared to healthy controls. However, structural analyses revealed no significant differences in the Tf secondary or tertiary structures between the two groups. A functional analysis demonstrated comparable Tf-TfR interaction in both PD and healthy samples. Despite significant alterations in Tf glycosylation, structural integrity and Tf-TfR interaction remained preserved in PD patients. These findings suggest that while glycosylation changes may influence iron metabolism, they do not impair Tf function. The study highlights the importance of a glucose-free dialysis solutions in managing anemia exacerbation in PD patients with poorly controlled anemia, potentially offering a targeted therapeutic approach to improve patient outcomes
Structural and functional changes of fibrinogen in patients on peritoneal dialysis
Cilj disertacije je da se kod bolesnika u završnom stadijumu bubrežne slabosti lečenih peritoneumskom dijalizom (PD-om) analiziraju strukturne i funkcionalne promene fibrinogena i fibrina, i da se ispita povezanost uočenih promena sa nastankom neželjenih događaja kardiovaskularnog sistema (KVS-a), nakon perioda praćenja od 18 meseci. U ispitivanju je učestvovala grupa od 52 bolesnika i grupa od 32 zdrave kontrole, uporediva prema polu i starosti. Fibrinogen je izolovan iz plazme isoljavanjem amonijum-sulfatom, i analiziran spektroskopskim, imuno- i lektin-afinitetnim metodama. Fibrinogeni bolesnika na PD-u imaju značajno viši sadržaj karbonila od fibrinogena zdravih kontrola, a Aα i Bβ lanci su podložniji oksidaciji od γ lanaca. Vreme i brzina koagulacije su slični u obe ispitivane grupe, kao i vrednosti za maksimalnu apsorbancu (UV VIS spektri, na 350 nm) i emisioni maksimum ispitivanih fibrinogena (fluorescentni spektri, na 344 nm). Od 16 korišćenih lektina, kod 10 je odnos signal/šum >10, dok je za dva lektina <3. Fibrinogeni ispitanika iz obe grupe su reagovali sa istim lektinima, i sadrže: a) biantenarne N-glikane sa α2,6-Sia; b) umetnuti GlcNAc i/ ili α1,6-Fuc, i c) α1,3-Fuc. Primećena je mala zastupljenost multiantenarnih i paucimanoznih i/ili visokomanoznih N-glikana, i O-glikana tipa T i Tn antigena sa ili bez Sia. Kao prediktor ultrafiltracione slabosti se izdvojio sadržaj fukozilovanih glikana koje vezuje AAL (sa vrednošću AUC od 72,4%). Povišen sadržaj (pauci)manoznih struktura (koje vezuje GNL) nađen je kod bolesnika koji su preminuli nakon 18 meseci od početka studije. Fibrinogen izolovan iz plazme bolesnika lečenih PD-om ima značajno višu koncentraciju karbonila, izmenjenu sekundarnu i tercijarnu strukturu i izmenjen sadržaj glikana. Stepen fukozilacije ima visok potencijal da bude prediktivni faktor malfunkcije peritoneumske membrane, dok sadržaj paucimanoznih/visokomanoznih struktura korelira sa neželjenim kardiovaskularnim događajima i letalnim ishodom.The aim of the thesis is to analyze the structural and functional changes of fibrinogen and fibrin in patients with end - stage renal disease treated with peritoneal dialysis (PD), and to examine the relationship between the observed changes and the occurrence of adverse cardiovascular (CVD) events after a follow - up period of 18 months. The study involved 52 patients and 32 healthy volunteers, comparable by gender and age. Fibrinogen was isolated from plasma by ammonium sulfate isolation, and analyzed by spectroscopic, immuno- and lectin-affinity methods.
The fibrinogens of patients on PD have a significantly higher carbonyl content than the fibrinogens of healthy volunteers, and the Aα and Bβ chains are more susceptible to oxidation than the γ chains. The time and rate of coagulation are similar in both study groups, as well as the values for maximum absorbance (UV VIS spectra, at 350 nm) and emission maximum of analyzed fibrinogens (fluorescent spectra, at 344 nm). Of the 16 lectins used, 10 had a signal-to-noise ratio of > 10, while two lectins had <3. Fibrinogens of both groups reacted with the same lectins, and contain: a) biantenary N-glycans with α2,6-Sia; b) insert GlcNAc and / or α1,6-Fuc, and c) α1,3-Fuc. A small presence of multiantennar and paucimannosidic and/or high-mannose N-glycans, and O-glycans of type T and Tn antigens with or without Sia was observed. The content of fucosylated N-glycans (AAL binding) emerged as a predictor of ultrafiltration strength, (with an AUC value of 72.4%), while an elevated content of (pauci)mannosidic glycans (GNL singal) was found in patients died 18 months after the start of the study.
Fibrinogen isolated from the plasma of patients treated with PD has a significantly higher carbonyl concentration, altered secondary and tertiary structure, and altered glycan content. The degree of fucosylation has a high potential to be a predictive factor of peritoneal membrane dysfunction, while the content of paucimannosidic/highly mannose structures correlates with adverse cardiovascular events and lethal outcome
Health risk assessment of toxic metals and toxigenic fungi in commercial herbal tea samples from Belgrade, Serbia
This study aimed to: (i) determine the content of toxic metals and toxigenic fungi in 14 commercial herbal tea samples from the market of Belgrade, Serbia; and (ii) assess their risk to human health. After the microwave digestion (HNO3/H2O2, 7:1,v/v), toxic metal content was determined by graphite furnace atomic absorption spectroscopy (GF-AAS). The health risk as a result of Cd, Pb, As, Mn, Ni, and Cr exposure via herbal tea intake was assessed through estimated daily intake (EDI), target hazard quotient (THQ), and hazard index (HI). Fungi isolated from the tested herbal tea samples were identified on the basis of colony morphology and microscopic characteristics of reproductive structures. Toxic metals concentration in herbal tea samples for Cd, Pb, As, Mn, Ni and Cr ranged 0.04-0.93, 0.09-2.54, 0.03-0.77, 1.82-651.04, 0.97-9.01, 0.49-3.47 mg/kg of dried plant material, respectively. Values of THQ for investigated toxic metals and HI were below 1, indicating the absence of human health risk. All 14 samples of the tested herbal teas were contaminated with fungi, while total count of cultivable fungi in each sample did not exceed national standards (105 CFU/g). Presence of 23 morphologically different isolates suggested moderate fungal diversity of the samples, while the highest isolation frequency (IF) of 100% was documented for Penicillium spp. and Aspergillus spp. The obtained risk assessment results suggest that all the tested samples are safe for human health regarding the toxic metal and fungal content and provide an important basis for understanding the potential risks of toxic metals and toxigenic fungi intake via commercially available herbal teas