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Optical methodologies in the analysis of erythrocyte deformability and heterogeneity
Get PDFEktacytometry, a diffraction-based method, measures the deformability of entire erythrocyte populations and does not provide information on the altered deformability of individual erythrocytes or affected subpopulation induced by constant oxidative stress, physical stress, metabolic depletion, and loss of ion gradients during their 120 days life span. We introduced an approach based on ektacytometry coupled to flow cytometry analysis to monitor the deformability and heterogeneity of subpopulations of erythrocytes. The effects of in vitro changes of osmotic gradient (from 155 mM to 93 mM phosphate buffer) and treatment by oxidative agent (0.5 mM and 0.75 mM terc-bytil hydroperoxide (TBPH)) on human erythrocyte isolated from healthy male donors, were tested using RheoScan D 300 (RheoMeditech. Inc., Korea) and BD FACSCalibur flow cytometer (Becton Dickinson, USA).
A decrease in erythrocyte deformability by changes in osmolality or treatment with TBPH
per se was demonstrated by ektacytometry. Nevertheless, this method could not analyze the
erythrocytes that underwent both treatments due to their lysis by the shear stress in the
device. The samples of an equal population of normal erythrocytes and erythrocytes
rigidified by 0.5 mM TBPH (slightly rigid) showed elongation indices in the physiological
range, i.e., the effect of the treatments was annulled. The same result was obtained by flow
cytometry. On the other hand, an altered population of oxidized cells by 0.5 mM TBPH was
detected in hypoosmotic 93 mM buffer based on their forward scatter (FSC) (Figure 1) and
side scatter (SSC) parameters. The subtle changes in the erythrocyte’s subpopulation
mechanobiology are essential to monitoring even in healthy people exposed to physical
or/and environmental stress and stored erythrocytes, but some additional techniques are
needed for the established optical-based approaches
Graphene quantum dots protect SH-SY5Y neuronal cells from SNP-induced apoptotic death
Get PDFIntroduction: We examined the molecular mechanisms of graphene quantum dot (GQD)- mediated
protection of SH-SY5Y human neuroblastoma cells from oxidative/nitrosative stress induced by iron-nitrosyl complex sodium nitroprusside (SNP).
Methods: GQD was produced by electrochemical oxidation of graphite and characterized by AFM, UVVIS
and FTIR spectroscopy. The antioxidant activity of GQD in cell-free conditions was assessed by DPPH, NBT and EPR analysis. The neuroprotective potential of GQD was determined by cell viability assays MTT, CV. Flow cytometry was used to assess markers of apoptosis and GQD scavenging of intracellular ROS/RNS as well. Cellular internalization of GQD was determined using TEM.
Results: GQD prevented SNP-induced apoptosis, caspase activation and mitochondrial depolarization in neuroblastoma cells. Although GQD diminished the NO levels in SNP-treated cells, NO scavengers displayed only a slight protection. GQD significantly protected SH-SY5Y cells from neurotoxicity of lightexhausted SNP, incapable of producing NO, implying that protective mechanism is independent of NO-scavenging. GQD reduced SNP-triggered increase in intracellular levels of ROS, particularly •OH, O2•− in cells and cell-free condition. Nonselective antioxidants, •OH scavengers and iron chelators, mimicked GQD cytoprotection, indicating that GQD protect cells by neutralizing •OH generated in the Fenton reaction.
Cellular GQD internalization was required for optimal protection since the removal of extracellular
GQD by extensive washing partly diminished their protective effect, suggesting that GQD exerted
neuroprotective effect intra- and extracellularly.
Conclusion: By demonstrating that GQD protect neuroblastoma cells from SNP-induced apoptosis by •OH/NO scavenging, our results suggest that GQD could be valuable candidates for treatment of neurodegenerative diseases associated with oxidative/nitrosative stress
Antimikrobna aktivnost jaja i voska poreklom od jaja krpelja vrsta Ixodes ricinus i Dermacentor reticulatus
Get PDFEksperimentalna terapija malarije – novi vidici
Get PDFWith an estimated 247 million cases annually and 619.000 deaths (in 2021) malaria remains a major
disease of the developing world and globally the most important parasitic disease. Because of
widespread resistance to available antimalarials including chloroquine (CQ) and its derivatives, new
drugs are urgently needed. Here we report on the antimalarial efficacy of new 4-aminoquinoline
derivatives, with modifications at the linker and at the quinoline nucleus.
In vitro screening was performed by the lactate dehydrogenase assay, based on measurement of the
plasmodial lactate dehydrogenase activity in both a CQ-sensitive (3D7) and a CQ-resistant (Dd2)
strain of Plasmodium falciparum, with a CQ as a control. In vivo antimalarial activity was investigated
in C57BL/6 mice infected with Plasmodium berghei ANKA strain by the modified Thompson test.
Compounds were first tested for toxicity. A total of 37 compounds were screened in vitro. Of the 22
that passed the first screening, 18 had IC50 values lower than CQ in the Dd2 strain while only one
was efficient in the 3D7 strain. However, even 15 compounds showed in vivo activity, significantly
(P<0.05) prolonging survival of treated vs. untreated mice. Among these, seven compounds afforded
the survival of 20–100% of treated mice up to Day 31, with or without the detection of parasites
in peripheral blood.
Most importantly, three of these, including ClAQ1, FClAQ1 and ClAQ8, afforded survival of 100% of
animals, the first two at 80 and 160 mg/kg/day and the last only at 160 mg/kg/day.Survival was associated with complete parasite clearance, as shown by both microscopy and qPCR.
Of note, continuous monitoring of parasitemia allowed the observation of a potentially important
phenomenon, that a number of compounds were able to confer resistance to cerebral malaria and
afford a switch to hyperparasitaemia to mice prone to the neurological syndrome.
By comparing the antimalarial activity of this group of novel compounds, we found that even minor
structural modifications substantially affect activity. The results of this extensive study are important,
as they may guide future work involving structural modifications of aminoquinolines, and as a contribution
to the knowledge in the field of malarial chemotherapy.Malarija ostaje globalno najznačajnija parazitska infekcija sa procenjenih 247 miliona slučajeva
i 619.000 smrtnih slučajeva godišnje (2021.). Zbog široko rasprostranjene rezistencije na dostupne
antimalarike, uključujući hlorokvin (CQ) i njegove derivate, hitno su potrebni novi lekovi.
U ovom istraživanju ispitana je potencijalna antimalarijska aktivnost 37 novosintetisanih aminohinolina
sa hemijskim modifikacijama na aminohinolinskom jezgru i bočnom lancu. In vitro skrining aktivnosti
jedinjenja vršen je kolorimetrijskim esejom laktat dehidrogenaze na dva soja Plasmodium falciparum,
osetljivim (3D7) i rezistentnim (Dd2) na CQ, uz CQ kao pozitivnu kontrolu. Aktivnost u in
vivo sistemu je ispitana na ženkama miševa soja C57Bl/6 inficiranim ANKA sojem Plasmodium berghei
primenom modifikovanog Thompson-ovog testa. Ispitivanju aktivnosti jedinjenja prethodila je
faza kliničkog praćenja zdravih životinja terapiranih eksperimentalnim jedinjenjima. Od 37 jedinjenja
ispitanih u fazi in vitro skrininga, 22 koja su inhibirala ≥50% rast bar jednog od dva soja P. falciparum
odabrana su za titraciju do IC50 vrednosti.
Prema soju rezistentnom na CQ, 18 jedinjenja se pokazalo aktivnijim od CQ, dok je među njima samo
jedno jedinjenje bilo aktivnije i prema osetljivom soju. Čak 15 jedinjenja ispitanih u in vivo sistemu
značajno je produžilo život inficiranim životinjama u odnosu na kontrolnu grupu (P < 0.05).
Među njima, sedam jedinjenja je omogućilo preživljavanje 20–100% tretiranih miševa do dana 31, sa
ili bez nalaza parazita u perifernoj krvi. Posebno treba istaći tri jedinjenja koja su dovela do izlečenja
svih tretiranih životinja, ClAQ1 i FClAQ1 (80 i 160 mg/kg/dan) i ClAQ8 (160 mg/kg/dan).Preživljavanje je bilo praćeno i kompletnim klirensom parazita što je dokazano mikroskopskim pregledom
razmaza kao i qPCR analizom krvi i tkiva jetre preživelih životinja. Važno je pomenuti da
je kontinuirano praćenje parazitemije svih tretiranih miševa omogućilo da se zapazi potencijalno
značajan fenomen.
Naime, neka jedinjenja su omogućila da miševi postanu otporni na razvoj cerebralne malarije
i uzrokovala da miševi skloni razvoju neurološkog sindroma tolerišu preživljavanje sa izuzetno velikim
brojem parazita. Poređenjem antimalarijske aktivnosti novosintetisanih aminohinolina uočeno je da
i male strukturne promene u velikoj meri menjaju aktivnost. Rezultati ovog opsežnog istraživanja
su od značaja za buduća istraživanja strukturne modifikacije aminohinolina i doprinose proširenju
znanja u oblasti hemioterapije malarije
Easily Applicable Predictive Score for Differential Diagnosis of Prefibrotic Primary Myelofibrosis from Essential Thrombocythemia
Get PDFEssential thrombocythemia (ET) and prefibrotic primary myelofibrosis (prePMF) initially have a similar phenotypic presentation with thrombocytosis. The aim of our study was to determine significant clinical-laboratory parameters at presentation to differentiate prePMF from ET as well as to develop and validate a predictive diagnostic prePMF model. This retrospective study included 464 patients divided into ET (289 pts) and prePMF (175 pts) groups. The model was built using data from a development cohort (229 pts; 143 ET, 86 prePMF), which was then tested in an internal validation cohort (235 pts; 146 ET, 89 prePMF). The most important prePMF predictors in the multivariate logistic model were age ≥ 60 years (RR = 2.2), splenomegaly (RR = 13.2), and increased lactat-dehidrogenase (RR = 2.8). Risk scores were assigned according to derived relative risk (RR) for age ≥ 60 years (1 point), splenomegaly (2 points), and increased lactat-dehidrogenase (1 point). Positive predictive value (PPV) for pre-PMF diagnosis with a score of ≥points was 69.8%, while for a score of ≥3 it was 88.2%. Diagnostic performance had similar values in the validation cohort. In MPN patients with thrombocytosis at presentation, the application of the new model enables differentiation of pre-PMF from ET, which is clinically relevant considering that these diseases have different prognoses and treatments
Understanding the Male Perspective: Evaluating Quality of Life and Psychological Distress in Serbian Men Undergoing Infertility Treatment
Get PDFThe experience of an infertility diagnosis and treatment imposes a profound burden on affected individuals, encompassing not only physical and medical aspects but also a plethora of psychological, social, and emotional factors. By employing a multimodal assessment featuring validated self-report questionnaires, physical measurements, and clinical records, the present study aimed to explore the quality of life and psycho-emotional distress of men undergoing infertility treatment in Serbia, thereby addressing the dearth of research on the underrepresented male perspective in this domain. Findings revealed diverse semen abnormalities among participants (n = 96, average age 37.69 ± 5.72), with significant associations between longer treatment durations and reduced sperm motility. The observed rates of men surpassing predetermined DASS-42 questionnaire thresholds for depression, anxiety, and stress in the analyzed cohort were 13.54%, 11.46%, and 22.92%, respectively. Summary scores in conceptual areas comprised in the SF-36 questionnaire ranged from 49.00 ± 6.25 for the mental health dimension to 90.16 ± 17.75 obtained in the physical functioning subscale. Patients with a longer treatment duration demonstrated lower scores in the role emotional domain, indicative of a less favorable emotional state. Expectedly, inverse correlations were found between the SF-36 mental health score and DASS-42 subscales. By addressing the existing knowledge gap and highlighting the unique needs of infertile men, the finding of this study may contribute to a more inclusive and holistic approach to infertility research and management
Factors Associated with Toxoplasma gondii Seroprevalence in Pregnant Women: A Cross-Sectional Study in Belgrade, Serbia
Get PDFToxoplasmosis, caused by the cosmopolitan protozoan Toxoplasma gondii, has particular implications during pregnancy due to the possible transmission of infection to the fetus. Very few studies have assessed seroprevalence and the risk factors for toxoplasmosis in healthy pregnant women. The aim of this study was to examine the seroprevalence of T. gondii infection in healthy pregnant women and to identify the associated risk factors for toxoplasmosis. The cross-sectional study involved 300 healthy pregnant women who came to the Institute for Blood Transfusion in Belgrade between November 2018 and February 2019 for routine blood group and Rh factor testing before delivery, who were also tested using serological screening for the presence of specific antibodies. Positives were further examined using enzyme immunoassay. Of the total sera of participants analyzed, 38 were positive for specific IgG, resulting in a seroprevalence rate of 12.7% (95% Confidence Interval (CI) 9.1–17.0%). All pregnant women presented negative anti-T. gondii IgM antibodies. The multivariate logistic regression analysis revealed that living in a house with a garden was independently associated with the risk of T. gondii infections, while eating chicken meat was connected with a lower risk compared to eating other types of meat with an odds ratio (OR) of 2.5 (95% CI 1.21–5.02) and an OR of 0.3 (95% CI 0.09–0.83), respectively. Although the prevalence of anti-T. gondii IgG antibodies is relatively low, it is essential to maintain and adapt evidence-based preventive measures for toxoplasmosis continually
Comparative assessment of erythrocyte sphingolipid levels as potential cardiovascular health markers in women from Libya and Serbia: a small-scale study
Get PDFAim: Cardiovascular diseases (CVDs) represent the major cause of morbidity and mortality worldwide including Libya, where they account for 43% of all deaths. Sphingolipids are involved in the pathology of numerous diseases including cardiovascular diseases and are proposed as potential biomarkers of cardiovascular health that could be more effective compared to traditional clinical biomarkers. The aim of this study was to determine the sphingolipid content in the erythrocyte membrane of Libyan migrant and Serbian resident women. In addition, to examine if sphingolipid levels could be used as a novel indicator of cardiovascular risk, we evaluated possible correlations with some well-established biomarkers of cardiovascular health. Materials and Methods: A total of 13 Libyan and 15 Serbian healthy women participated in the study. The high-performance version thin-layer chromatography (HPTLC) using the image analysis tool JustTLC was applied for quantification of erythrocytes’ sphingolipids. Results: Lower mean values of erythrocytes’ sphingolipids and cholesterol concentrations were found in the group of Libyan emigrants compared to Serbian resident women. Besides, in this group of apparently healthy women (n = 28), the sphingolipid content of erythrocytes was inversely related to the Omega-3 index (r =-0.492, p = 0.008) and directly linked to vitamin D status (r = 0.433, p = 0.021) and membrane cholesterol levels (r = 0.474, p = 0.011). Conclusion: The erythrocytes’ sphingolipid levels should be measured/assessed as an additional biomarker of CV health, by applying a simple and routine method. Still, further investigation in a larger population-specific context is warranted
Disturbed Plasma Lipidomic Profiles in Females with Diffuse Large B-Cell Lymphoma: A Pilot Study
Get PDFLipidome dysregulation is a hallmark of cancer and inflammation. The global plasma lipidome and sub-lipidome of inflammatory pathways have not been reported in diffuse large B-cell lymphoma (DLBCL). In a pilot study of plasma lipid variation in female DLBCL patients and BMI-matched disease-free controls, we performed targeted lipidomics using LC-MRM to quantify lipid mediators of inflammation and immunity, and those known or hypothesised to be involved in cancer progression: sphingolipids, resolvin D1, arachidonic acid (AA)-derived oxylipins, such as hydroxyeicosatetraenoic acids (HETEs) and dihydroxyeicosatrienoic acids, along with their membrane structural precursors. We report on the role of the eicosanoids in the separation of DLBCL from controls, along with lysophosphatidylinositol LPI 20:4, implying notable changes in lipid metabolic and/or signalling pathways, particularly pertaining to AA lipoxygenase pathway and glycerophospholipid remodelling in the cell membrane. We suggest here the set of S1P, SM 36:1, SM 34:1 and PI 34:1 as DLBCL lipid signatures which could serve as a basis for the prospective validation in larger DLBCL cohorts. Additionally, untargeted lipidomics indicates a substantial change in the overall lipid metabolism in DLBCL. The plasma lipid profiling of DLBCL patients helps to better understand the specific lipid dysregulations and pathways in this cancer
Antimikrobna antitela u fiziološkim i patološkim stanjima
Get PDFU ovom izlaganju biće predstavljeni naši dosadašnji rezultati na temu antimikrobnih antitela kod profesionalnih sportista, odraslih osoba koje se ne bave profesionalno sportom; promene nivoa antibakterijskih antitela pri starenju; poređenje specifičnosti serumskih i salivarnih IgA antitela kod mladih odraslih osoba, kao i nivoi ukupnih i antibakterijskih antitela u Covid-19 virusnoj infekciji i u sepsi. Nivoi i titri antimikrobnih antitela različitih klasa i potklasa određivani su ELISA testom iz uzoraka seruma, plazme ili salive. Ispitivano je vezivanje za izolovane antigene mikroorganizama (lipopolisaharid, peptidoglikan i zimozan) i za cele mikroorganizme, različitih vrsta i sojeva. Vezivanje prečišćenog salivarnog IgA za mikroorganizme praćeno je i protočnom citometrijom. Od rezultata izdvajamo da izlaganje fizičkom naporu visokog intenziteta dovodi do promena na nivou antibakterijskih antitela, i to u pravcu sveobuhvatnijeg prepoznavanja lipopolisaharida1. Suplementacija profesionalnih sportista određenim probioticima dovodi do održanja nivoa ukupnih salivarnih i serumskih IgA antitela, kao i IgG antitela specifičnih prema Enterococcus faecalis, koja su u ovoj populaciji pokazala sezonsku varijaciju. Pri starenju dolazi do smanjenja nivoa antipneumokoknih antitela i to zavisno od pola, pa su stariji mukarci naročito pogođeni ovim smanjenjem3. Salivarna IgA antitela razlikuju se po specifičnosti od serumskih IgA antitela i pružaju nespecifičnu zaštitu. Poređenje pacijenata sa sepsom, hospitalizovanih Covid-19 pacijenta i starosnih kontrola pokazalo je brojne razlike u nivoima kako ukupnih, tako i antibakterijskih antitela. Nivoi antibakterijskih antitela kod pacijentata koji nisu preživeli bili su niži nego kod onih koji su preživeli, što potvrđuje značaj ovih anititela za preživljavanje sepse