Clinical Approach to Patients with COVID-19 and Unrecognized Obstructive Sleep Apnea.

Purpose: We conducted a retrospective case series of seven male COVID-19 patients with respiratory failure and suspected OSA based on clinical features to evaluate the effects of undiagnosed obstructive sleep apnea (OSA) on COVID-19 outcomes and the response to a continuous positive airway pressure (CPAP) treatment. Cardiorespiratory polygraphy (CRP) and a continuous positive airway pressure treatment were used for diagnosis and management. They confirmed severe obstructive sleep apnea in all patients (apnea/hypopnea index > 30) and improved overnight oxygenation and symptoms at the 1-month follow-up. Conclusions: Undiagnosed obstructive sleep apnea may negatively impact COVID-19 outcomes by exacerbating respiratory failure. Recognition and treatment with continuous positive airway pressure can optimize the management of such patients.

Modified Titanium Surface with Nano Amorphous Calcium Phosphate@Chitosan Oligolactate as Ion Loading Platform with Multifunctional Properties for Potential Biomedical Application.

Titanium (Ti) is widely used in medical and dental implants. Calcium phosphate (CPs) coatings enhance Ti implants' osteoinductive properties, and additives further improve these coatings. Recently, a nano amorphous calcium phosphate (nACP) coating decorated with chitosan oligolactate (ChOL) and selenium (Se) showed immunomodulatory effects. This study investigates the surface morphology, composition, bioactivity, mechanical properties, and Se-release mechanism of the nACP@ChOL-Se hybrid coating on Ti substrates. Amorphous calcium phosphate (ACP) was synthesized, and the nACP@ChOL-Se hybrid coating was deposited on Ti substrates using in situ anaphoretic deposition. Physico-chemical characterization was used to analyze the surface of the coating (scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier Transform Infrared Spectroscopy). The distribution of Se within the coating was examined with energy-dispersive X-ray spectroscopy (EDS). Bioactivity was evaluated in simulated body fluid (SBF), and adhesion was tested using a scratch test method. In vitro testing determined the release mechanism of Se. SEM images illustrated the surface morphology, while AFM provided a detailed analysis of surface roughness. XRD analysis revealed structural and phase composition, and EDS confirmed Se distribution within the coating. The coating exhibited bioactivity in SBF and showed good adhesion according to the scratch test. In vitro testing uncovered the release mechanism of Se from the coating. This study successfully characterized the surface morphology, composition, bioactivity, and Se-release mechanism of the nACP@ChOL-Se hybrid coating on Ti substrates, offering insights for developing immunomodulatory coatings for medical and dental applications.

EGFR mutation testing from pleural effusions of non-small cell lung cancer patients at the institute for oncology and radiology of Serbia.

BACKGROUND: The use tumor-derived cell-free DNA extracted from body fluids is being evaluated for genetic testing in lung cancer. The aim of this study was to explore the feasibility and utility of implementation of EGFR molecular testing from pleural effusions in non-small cell lung cancer in the clinical diagnostics workflow. PATIENTS AND METHODS: This study included patients diagnosed with primary lung adenocarcinoma in the period July 2016 to June 2023. EGFR mutation testing was performed by qPCR (Cobas®) and dPCR. Testing was performed from 211 plasma samples when tissue was unavailable at diagnosis, and from 301 plasma samples and 18 pleural effusions at progression on first/second generation of EGFR TKIs. Descriptive methods of statistical analysis were used to summarize the sample data. Fisher's exact test, McNemar's test, Cohen's kappa tests were used for statistical analyses. Two-sided p-values <0.05 were considered statistically significant. RESULTS: A significantly higher detection rate of the T790M mutation in pleural effusion was obtained compared to blood (50% and 20%, p=0.047). When comparing the detection success rate of the resistant T790M mutation in pleural effusion and blood, a statistically significant difference was obtained in favor of pleural effusion (50% vs. 21.87%, p=0.01). CONCLUSIONS: Superior performance of pleural effusions compared to blood plasma was shown both in the analysis of success rate and in the detection of the resistant T790M mutation, at progression on EGFR TKIs. Pleural effusion should be considered in this setting whenever available, especially in countries with limited health resources.

Ghosts of the past: Elemental composition, biosynthesis reactions and thermodynamic properties of Zeta P.2, Eta B.1.525, Theta P.3, Kappa B.1.617.1, Iota B.1.526, Lambda C.37 and Mu B.1.621 variants of SARS-CoV-2.

From the perspectives of molecular biology, genetics and biothermodynamics, SARS-CoV-2 is the among the best characterized viruses. Research on SARS-CoV-2 has shed a new light onto driving forces and molecular mechanisms of viral evolution. This paper reports results on empirical formulas, biosynthesis reactions and thermodynamic properties of biosynthesis (multiplication) for the Zeta P.2, Eta B.1.525, Theta P.3, Kappa B.1.617.1, Iota B.1.526, Lambda C.37 and Mu B.1.621 variants of SARS-CoV-2. Thermodynamic analysis has shown that the physical driving forces for evolution of SARS-CoV-2 are Gibbs energy of biosynthesis and Gibbs energy of binding. The driving forces have led SARS-CoV-2 through the evolution process from the original Hu-1 to the newest variants in accordance with the expectations of the evolution theory.

Immunomodulatory Effects Mediated by Nano Amorphous Calcium Phosphate/Chitosan Oligosaccharide Lactate Coatings Decorated with Selenium on Titanium Implants.

The aim of this work is in situ anodization/anaphoretic deposition of a nano amorphous calcium phosphate (ACP)/chitosan oligosaccharide lactate (ChOL) multifunctional hybrid coating decorated with selenium (Se) on a titanium substrate and in vivo investigation of its immunomodulatory and anti-inflammatory effect. Investigating phenomena at the implant-tissue interface of interest for controlled inflammation and immunomodulation was also the aim of the research. In our earlier research, we designed coatings based on ACP and ChOL on titanium with anticorrosive, antibacterial and biocompatible properties, while in the presented results we show that selenium addition makes this coating an immunomodulator. The immunomodulatory effect of the novel hybrid coating is characterized by the examination of the functional aspects in the tissue around the implant (in vivo): proinflammatory cytokines' gene expression, M1 (iNOS) and M2 (Arg1) macrophages, fibrous capsule formation (TGF-ß) and vascularization (VEGF). The EDS, FTIR and XRD analyses prove the formation of a ACP/ChOL/Se multifunctional hybrid coating on Ti and the presence of Se. A higher M2/M1 macrophage ratio in the ACP/ChOL/Se-coated implants compared to pure titanium implants (a higher level of Arg1 expression) is noted at all time points examined (after 7, 14 and 28 days). Lower inflammation measured by gene expression of proinflammatory cytokines IL-1ß and TNF, lower expression of TGF-ß in the surrounding tissue and higher IL-6 expression (solely at day 7 post-implantation) is noted in presence of the ACP/ChOL/Se-coated implants.

Tumor selective Ru(III) Schiff bases complexes with strong in vitro activity toward cisplatin-resistant MDA-MB-231 breast cancer cells.

Novel ruthenium(III) complexes of general formula Na[RuCl2(L1-3-N,O)2] where L(1-3) denote deprotonated Schiff bases (HL1-HL3) derived from 5-substituted salicyladehyde and alkylamine (propyl- or butylamine) were prepared and characterized based on elemental analysis, mass spectra, infrared, electron spin/paramagnetic resonance (ESR/EPR) spectroscopy, and cyclovoltammetric study. Optimization of five isomers of complex C1 was done by DFT calculation. The interaction of C1-C3 complexes with DNA (Deoxyribonucleic acid) and BSA (Bovine serum albumin) was investigated by electron spectroscopy and fluorescence quenching. The cytotoxic activity of C1-C3 was investigated in a panel of four human cancer cell lines (K562, A549, EA.hy926, MDA-MB-231) and one human non-tumor cell line (MRC-5). Complexes displayed an apparent cytoselective profile, with IC50 values in the low micromolar range from 1.6 ± 0.3 to 23.0 ± 0.1 µM. Cisplatin-resistant triple-negative breast cancer cells MDA-MB-231 displayed the highest sensitivity to complexes, with Ru(III) compound containing two chlorides and two deprotonated N-propyl-5-chloro-salicylidenimine (hereinafter C1) as the most potent (IC50 = 1.6 µM), and approximately ten times more active than cisplatin (IC50 = 21.9 µM). MDA-MB-231 cells treated for 24 h with C1 presented with apoptotic morphology, as seen by acridine orange/ethidium bromide staining, while 48 h of treatment induced DNA fragmentation, and necrotic changes in cells, as seen by flow cytometry analysis. Drug-accumulation study by inductively coupled plasma mass spectrometry (ICP-MS) demonstrated markedly higher intracellular accumulation of C1 compared with cisplatin.

Arterial blood gases' analysis in elite breath-hold divers at extreme depths.

PURPOSE: To showcase results of arterial blood gases' analysis in elite breath-hold divers sampled at depths where their total lung capacities are below their residual lung volume on surface. METHODS: Three male elite breath-hold divers performed body plethysmographies to determine their lung volumes. Two dives were performed, one on normal inhalation to 60 m of depth and the second on complete exhalation to 10 m of depth. Blood samples were taken on five occasions; before the first dive, at 60 and 10 m of depth and immediately after resurfacing after both dives. RESULTS: Arterial blood gases' analysis at 60 m of depth showed an increase in partial pressures of oxygen and carbon dioxide, a consequent decrease in pH and an increase in concentration of HCO3-. After resurfacing, in two divers, values mostly returned to normal; hypoxemia was observed in one diver. At 10 m of depth, all values showed similar variation, and hypoxemia was observed in the same diver but at depth. Upon resurfacing, all values returned to normal. CONCLUSION: This is the first study performed at depths where the total lung capacities of participants are below their residual lung volumes at the surface. Partial pressure of carbon dioxide increases at depth to higher than normal values causing pH to decrease thus exceeding the buffering potential of the blood. In addition, previous assumptions that maximum depth in breath-hold divers is where total lung capacity is reduced to their residual volume proved wrong as our group of divers had no symptoms after resurfacing.

Anodizing/Anaphoretic Electrodeposition of Nano-Calcium Phosphate/Chitosan Lactate Multifunctional Coatings on Titanium with Advanced Corrosion Resistance, Bioactivity, and Antibacterial Properties.

The aim of this work was to investigate corrosion resistivity, bioactivity, and antibacterial activity of novel nano-amorphous calcium phosphate (ACP) potentially multifunctional composite coatings with and without chitosan oligosaccharide lactate (ChOL), ACP + ChOL/TiO2 and ACP/TiO2 ACP + ChOL/TiO2, respectively, on the titanium substrate. The coatings were obtained by new single-step in situ anodization of the substrate to generate TiO2 and the anaphoretic electrodeposition process of ACP and ChOL. The obtained coatings were around 300 ± 15 µm thick and consisted of two phases, namely, TiO2 and hybrid composite phases. Both ACP/TiO2 and ACP + ChOL/TiO2 have improved corrosion stability, whereas the ACP + ChOL/TiO2 coating showed better corrosion stability. It was shown that at the very start of the deposition process, the formation of the ChOL/TiO2 layer takes place predominantly, which is followed by the inclusion of ChOL into ACP with simultaneous growth of TiO2. This deposition mechanism resulted in the formation of strongly bonded uniform stable coating with high corrosion resistance. In vitro bioactivity was investigated by immersion of the samples in simulated body fluid (SBF). There is in-bone-like apatite formation on both ACP/TiO2 and ACP + ChOL/TiO2 surfaces upon immersion into SBF, which was proven by X-ray diffraction and Fourier transform infrared spectroscopy. While ACP/TiO2 shows no antibacterial activity, ACP + ChOL/TiO2 samples exhibited three- to fourfold decreases in the number of Staphylococcus aureus and Pseudomonas aeruginosa, respectively, after 420 min. The probable mechanism is binding ChOL with the bacterial cell wall, inhibiting its growth, altering the permeability of the cell membrane, and leading to bacterial death.

A Fluorescence-Based Assay for Measuring Glucose Uptake in Living Melanoma Cells.

Melanoma cells have high glycolytic capacity. Glucose uptake is a key rate-limiting step in glucose utilization. Here we describe a simple protocol for measuring direct glucose uptake in living melanoma cells by flow cytometry.

Exploring the real-world effect of the SARS-CoV-2 pandemic on the molecular diagnostics for cancer patients and high-risk individuals.

Background: The SARS-CoV-2 pandemic introduced a global distraction effect in cancer patients' care. The aim of this study was to explore the effect of the pandemic on the largest molecular diagnostics center for cancer patients and high-risk individuals in Serbia.Research design and methods: EGFR, KRAS/NRAS, BRAF, and BRCA1/2 mutation testing were performed by qPCR and NGS. NGS was used for panel testing of hereditary breast/ovarian cancer and cancers associated with Lynch syndrome. The analytical output during the state of emergency (SoE) was compared to the period before and after the outbreak using one-way ANOVA. Statistical significance was set at p < 0.05.Results: A 38% reduction in the number of analysis was detected during the SoE. After the SoE, a 19% reduction was noted compared to SoE and 50% compared to the period before the SoE (p = 0.038). Three of the 48 scheduled appointments for pretest genetic counseling were carried out during the SoE, but the number of NGS tests increased by 50%.Conclusions: The SARS-CoV-2 pandemic had a profound negative effect on the diagnostic output of our centralized molecular diagnostics center. The only positive effect was shortening of waiting lists for hereditary cancer patients and high-risk individuals.

Synthesis, chemical characterization, PARP inhibition, DNA binding and cellular uptake of novel ruthenium(II)-arene complexes bearing benzamide derivatives in human breast cancer cells.

Inhibitors of poly(ADP-ribose) polymerase-1 (PARP-1) showed remarkable clinical efficacy in BRCA-mutated tumors. Based on the rational drug design, derivatives of PARP inhibitor 3-aminobenzamide (3-AB), 2-amino-4-methylbenzamide (L1) and 3-amino-N-methylbenzamide (L2), were coordinated to the ruthenium(II) ion, to form potential drugs affecting DNA and inhibiting PARP enzyme. The four conjugated complexes of formula: C1 [(ƞ6-toluene)Ru(L1)Cl]PF6, C2 [(ƞ6-p-cymene)Ru(L1)Cl]PF6, C3 [(ƞ6-toluene)Ru(L2)Cl2] and C4 [(ƞ6-p-cymene)Ru(L2)Cl2], have been synthesized and characterized. Colorimetric 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT) assay showed the highest antiproliferative activity of C1 in HCC1937, MDA-MB-231, and MCF-7 breast cancer cells. Efficiency of inhibition of PARP-1 enzymatic activity in vitro decreased in order: C2 > C4 > 3-AB>C1 > C3. ICP-MS study of intracellular accumulation and distribution in BRCA1-mutated HCC1937 revealed that C1-C4 entered cells within 24 h. The complex C1 showed the highest intracellular accumulation, nuclear-targeting properties, and exhibited the highest DNA binding (39.2 ±â€¯0.6 pg of Ru per µg of DNA) that resulted in the cell cycle arrest in the S phase.

EGFR mutation testing from liquid biopsy of non-small cell lung cancer at the Institute for Oncology and Radiology of Serbia.

PURPOSE: Resistance to tyrosine kinase inhibitors (TKIs) in lung cancer often occurs, so mutation testing from liquid biopsy is the method of choice as a minimally invasive approach that quickly provides information for additional therapeutic options. The purpose of this study was to assess the success rate and usefulness of EGFR testing from liquid biopsy at the Institute for Oncology and Radiology of Serbia (IORS). METHODS: EGFR mutation testing was performed by real-time qPCR in 4750 tumor samples using the Cobas® EGFR Mutation Test v2. EGFR testing from 104 liquid biopsy samples was used to track the resistance on first-line EGFR-TKIs as well as for initial testing of 124 patients without tissue biopsies. RESULTS: Liquid biopsy samples were tested in cases with inadequate material for DNA isolation or without tissue biopsy at diagnosis. Nine mutated samples were detected (7.3 %) with a 99.2 % testing success rate. Testing liquid biopsy samples of patients who progressed on EGFR-TKIs showed an accordance rate of 67% with driver mutations, and 49% of mutated patients had the T790M mutation which rendered them eligible for third-generation EGFR-TKIs. An additional 5 patients tested EGFR wild type from plasma after progression were rebiopsied and 3 of them had the T790M mutation. CONCLUSIONS: EGFR mutation testing from liquid biopsy has been successfully implemented in Serbia and has proven invaluable for detecting molecular resistance mechanisms to EGFR-TKIs and as an alternative sample source for patients with scarce biopsy material or without any at all.

Telmisartan induces melanoma cell apoptosis and synergizes with vemurafenib in vitro by altering cell bioenergetics.

OBJECTIVE: Despite recent advancements in targeted therapy and immunotherapies, prognosis for metastatic melanoma patients remains extremely poor. Development of resistance to previously effective treatments presents a serious challenge and new approaches for melanoma treatment are urgently needed. The objective of this study was to examine the effects of telmisartan, an AGTR1 inhibitor and a partial agonist of PPARγ, on melanoma cells as a potential agent for repurposing in melanoma treatment. METHODS: Expression of AGTR1 and PPARγ mRNA in melanoma patient tumor samples was examined in publicly available datasets and confirmed in melanoma cell lines by qRT-PCR. A panel of melanoma cell lines was tested in viability, apoptosis and metabolic assays in presence of telmisartan by flow cytometry and immunocytochemistry. A cytotoxic effect of combinations of telmisartan and targeted therapy vemurafenib was examined using the Chou-Talalay combination index method. RESULTS: Both AGTR1 and PPARγ mRNA were expressed in melanoma patient tumor samples and decreased compared to the expression in the healthy skin. In vitro, we found that telmisartan decreased melanoma cell viability by inducing cell apoptosis. Increased glucose uptake, but not utilization, in the presence of telmisartan caused the fission of mitochondria and release of reactive oxygen species. Telmisartan altered the cell bioenergetics, thereby synergizing with vemurafenib in vitro, and even sensitized vemurafenib-resistant cells to the treatment. CONCLUSIONS: Given that the effective doses of telmisartan examined in our study can be administered to patients and that telmisartan is a widely used and safe antihypertensive drug, our findings provide the scientific rationale for testing its efficacy in treatment of melanoma progression.

New organoruthenium compounds with pyrido[2',3':5,6]pyrazino[2,3-f][1, 10]phenanthroline: synthesis, characterization, cytotoxicity, and investigation of mechanism of action.

Three new ruthenium(II)-arene complexes with pyrido[2',3':5,6]pyrazino[2,3-f][1, 10]phenanthroline (ppf) of general formula: C1 ([(ƞ6-benzene)Ru(ppf)Cl]PF6, C2 ([(ƞ6-toluene)Ru(ppf)Cl]PF6) and C3 ([(ƞ6-p-cymene)Ru(ppf)Cl]PF6) have been synthesized. The structures of complexes were determined by elemental analysis, IR, ESI-MS, as well as with 1H and 13C NMR spectroscopy. Cytotoxic activity has been evaluated in three different human neoplastic cell lines (A549, A375, LS 174T) and in one human non-tumor cell line (MRC-5), by the MTT assay. Complexes C1-C3 showed IC50 values in the micromolar range below 100 µM. Complex C3, carrying ƞ6-p-cymene as the arene ligand, exhibited cytoselective activity toward human malignant melanoma A375 cells (IC50 = 15.8 ± 2.7 µM), and has been selected for further analyses of its biological effects. Drug-accumulation study performed in the A375 cells disclosed that C3 possess lower ability of entering the cells compared to cisplatin and distributes approximately equally in the cytosol and membrane/organelle fraction of cells. Investigations in the 3D model of A375 cells, disclosed different effects of the complex C3 and cisplatin on growth of multicellular tumor spheroids (MCTSs). While the size of cisplatin-treated MCTSs decreased with time, MCTSs treated with C3 continued to growth. Differences in structural organization and biological activity of this type of ruthenium(II)-arene complexes versus cisplatin in A375 malignant melanoma cells pointed out their different modes of action, and necessity for further biological studies and optimizations for potential applications.

Propofol mediates signal transducer and activator of transcription 3 activation and crosstalk with phosphoinositide 3-kinase/AKT.

We previously demonstrated that propofol, an intravenous anesthetic with anti-oxidative properties, activated the phosphoinositide 3-kinase (PI3K)/AKT pathway to increase the expression of B cell lymphoma (Bcl)-2 and, therefore the anti-apoptotic potential on cardiomyocytes. Here, we wanted to determine if propofol can also activate the Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 3 pathway, another branch of cardioprotective signaling. The cellular response of nuclear factor kappa B (NFκB) and STAT3 was also evaluated. Cardiac H9c2 cells were treated by propofol alone or in combination with pretreatment by inhibitors for JAK2/STAT3 or PI3K/AKT pathway. STAT3 and AKT phosphorylation, and STAT3 translocation were measured by western blotting and immunofluorescence staining, respectively. Propofol treatment significantly increased STAT3 phosphorylation at both tyrosine 705 and serine 727 residues. Sustained early phosphorylation of STAT3 was observed with 25~75 µM propofol at 10 and 30 min. Nuclear translocation of STAT3 was seen at 4 h after treatment with 50 µM propofol. In cultured H9c2 cells, we further demonstrated that propofol-induced STAT3 phosphorylation was reduced by pretreatment with PI3K/AKT pathway inhibitors wortmannin or API-2. Conversely, pretreatment with JAK2/STAT3 pathway inhibitor AG490 or stattic inhibited propofol-induced AKT phosphorylation. In addition, propofol induced NFκB p65 subunit perinuclear translocation. Inhibition or knockdown of STAT3 was associated with increased levels of the NFκB p65 subunit. Our results suggest that propofol induces an adaptive response by dual activation and crosstalk of cytoprotective PI3K/AKT and JAK2/STAT3 pathways. Rationale to apply propofol clinically as a preemptive cardioprotectant during cardiac surgery is supported by our findings.