Clinical manifestations of SARS-CoV-2 infection range from mild to critically severe. The aim of the study was to highlight the immunological events associated with the severity of SARS-CoV-2 ...infection, with an emphasis on cells of innate immunity. Thirty COVID-19 patients with mild/moderate symptoms and 27 patients with severe/critically severe symptoms were recruited from the Clinical Center of Kragujevac during April 2020. Flow cytometric analysis was performed to reveal phenotypic and functional alterations of peripheral blood cells and to correlate them with the severity of the disease. In severe cases, the number of T and B lymphocytes, dendritic cells, NK cells, and HLA-DR-expressing cells was drastically decreased. In the monocyte population proportion between certain subsets was disturbed and cells coexpressing markers of M1 and M2 monocytes were found in intermediate and non-classical subsets. In mild cases decline in lymphocyte number was less pronounced and innate immunity was preserved as indicated by an increased number of myeloid and activated dendritic cells, NK cells that expressed activation marker at the same level as in control and by low expression of M2 marker in monocyte population. In patients with severe disease, both innate and adoptive immunity are devastated, while in patients with mild symptoms decline in lymphocyte number is lesser, and the innate immunity is preserved.
•The changes in tissue histomorphology and DNA quality in six different healthy tissues (brain, heart, lung, liver, spleen and kidney) exempted during autopsy of healthy individuals and storage at ...−20 °C and −150 °C three month were investigated.•Samples stored at −20 °C sustained the less degree of histomorphological damage compared to samples stored at −150 °C.•The highest DNA concentration was obtained from the liver tissue that was stored at −20 °C.•The integrity of molecule DNA was better on samples stored at −20 °C than at −150 °C.•Freezing and storing tissues at −20 °C has a number of advantages over freezing and storing tissues at −150 °C.
The aim of this study was to compare the changes in tissue histomorphology and DNA quality in six different healthy tissues (brain, heart, lung, liver, spleen and kidney) exempted during autopsy of healthy individuals and storage at −20 °C and −150 °C three month. Tissue samples were obtained, divided by tissue and temperature group, and for each sample, tissue histomorphology and DNA (isolated from all tissues in duplicated − 72 samples of DNA) quality were analysed. Morphology of tissue samples was studied using H&E staining. DNA was isolated using the phenol–chloroform-isoamyl alcohol method. To assess the concentration and purity of the DNA samples, we used a spectrophotometer to measure absorbance at wavelengths of 280 nm and 260 nm. The fragments of human telomerase reverse transcriptase (hTERT) gene were amplified from the DNA using PCR reaction and then visualised using the 2 % agarose gel. Samples stored at −150 °C sustained the highest degree of histomorphological damage, while samples stored at −20 °C were less degraded, compared to control. The liver samples stored at −20 °C had a mean DNA concentration (1030.4 ± 51.5 ng/μl) higher than the samples of liver tissue stored at −150 °C (497.4 ± 167.1 ng/μl) (p < 0.001). Other tissues did not have statistically significantly different DNA concentration at both temperatures. Liver samples at −20 °C had degraded DNA, showed as the absence of hTERT gene in most of samples. Other tissue samples in both temperature groups had unfragmented DNA. Storing tissue samples at −20 °C is not inferior in terms of DNA yield and integrity, and possibly superior for tissue histomorphology, comparing with samples stored at −150⁰C.
Tissue formalin fixation and paraffin embedding (FFPE) is a standard method for long-term preservation and morphological and molecular analysis. The aim of this study was to analyze the effect of ...storage time on the integrity of DNA isolated from three different healthy FFPE tissues. DNA was isolated from FFPE heart, liver and brain tissues obtained from autopsy and archived from 1988 to 2017 using two different methods of DNA isolation: phenol–chloroform-isoamyl alcohol (PCI) and PureLink Genomic DNA Kit. The quantification and purity of DNA was measured spectrophotometrically at 260 nm and 280 nm. The quality of isolated DNA was evaluated by PCR amplification of GPD1 (150 bp), ACTB (262 bp) and RPL4 (407 bp) genes. The histomorphological characteristics of FFPE tissues were not significantly changed during 30 years of storage. Higher yield (272.9 ± 10.3 µg) and purity (A260/280 = 2.05) of DNA was obtained using the PCI method for DNA isolation from FFPE liver tissue. The PCI extraction method showed reproducible and consistent results in PCR amplification of all of three examined genes. The GPD1 gene can be amplified up to 30 years, the ACTB gene in the same samples up to 26 years and the RPL4 gene up to 6 years of storage in FFPE blocks. Although the best yield and purity of isolated DNA (using both isolation methods) was obtained from FFPE liver tissue, the DNA with the most preserved integrity was obtained from brain tissue archived up to 30 years. This is the first report using long-term archived healthy FFPE tissues (up to 30 years) that shows that the DNA isolated from these tissues is of preserved integrity and can be used in molecular autopsy.
The aim of this study was to investigate effects of irradiations with the therapeutic proton and carbon ion beams in two non-small cell lung cancers, CRL5876 adenocarcinoma and HTB177 large cell lung ...carcinoma. The DNA damage response dynamics, cell cycle regulation, and cell death pathway activation were followed. Viability of both cell lines was lower after carbon ions compared to the therapeutic proton irradiations. HTB177 cells showed higher recovery than CRL5876 cells seven days following the treatments, but the survival rates of both cell lines were lower after exposure to carbon ions with respect to therapeutic protons. When analyzing cell cycle distribution of both CRL5876 and HTB177 cells, it was noticed that therapeutic protons predominantly induced G1 arrest, while the cells after carbon ions were arrested in G2/M phase. The results illustrated that differences in the levels of phosphorylated H2AX, a double-strand break marker, exist after therapeutic proton and carbon ion irradiations. We also observed dose- and time-dependent increase in the p53 and p21 levels after applied irradiations. Carbon ions caused larger increase in the quantity of p53 and p21 compared to therapeutic protons. These results suggested that various repair mechanisms were induced in the treated cells. Considering the fact that we have not observed any distinct change in the Bax/Bcl-2 ratio following irradiations, it seemed that different types of cell death were involved in the response to the two types of irradiations that were applied.
Acetylshikonin (AcSh), as a red colored pigment found in roots of the plants from family Boraginaceae, showed excellent cytotoxic activity. Due to its hydrophobic nature, and thus poor ...bioavailability, the aim of this study was to prepare acetylshikonin/β-cyclodextrin (AcSh/β-CD) inclusion complex by using coprecipitation method, characterize obtained system by using UV/VIS, IR and 1H NMR spectroscopy, and determine cytotoxic activity. Phase solubility test indicated formation of AL-type binary system (substrate/ligand ratio was 1:1 M/M), with stability constant Ks of 306.01 M−1. Formation of noncovalent bonds between inner layer of the hole of β-CD and AcSh was observed using spectroscopic methods. Notable changes in chemical shifts of two protons (−0.020 ppm) from naphthoquinone moiety (C6-H and C7-H), as well as protons from hydroxyl groups (−0.013 and −0.009, respectively) attached to C5 and C8 carbons from naphthoquinone part indicate that the molecule of AcSh enters the β-CD cavity from the aromatic side. Cytotoxic activity against HCT-116 and MDA-MB-231 cell lines was measured by MTT test and clonogenic assay. Mechanisms of action of free AcSh and inclusion complex were assessed by flow cytometry. In comparison to free AcSh, AcSh/β-CD showed stronger short-term effect on HCT-116 cells and superior long-term effect on both cell lines. Inclusion complex induced more pronounced cell cycle arrest and autophagy inhibition, and induced increase in accumulation of intracellular ROS more effectively than free AcSh. In conclusion, AcSh/β-CD binary system showed better performances regarding cytotoxic activity against tested tumor cell lines.
Background/Aim. In road traffic accidents, chest injuries are a critical factor since death usually occurs as a result of injuries to the heart and lungs, flail chest, pneumothorax, exsanguination, ...etc. The aim of this study was to analyze the most vulnerable subjects in road accidents, as well as the most frequent and most significant types of chest injuries sustained by different categories of subjects, and to examine the relevance of certain injuries or factors to outliving the injuries. Methods. The autopsy, the retrospective, and the cross-sectional study were performed at the Clinical Centre of Kragujevac, Department of Forensic Medicine and Toxicology. The study included all participants in road traffic accidents on the territory of Kragujevac and the surrounding area who died from the injuries sustained from the accidents or due to complications of the injuries during the period from 2001 to 2016. The subjects were divided into the following groups: pedestrians, motor vehicle drivers, front-seat passengers, back-seat passengers, bicyclists, motorcyclists, and tractor drivers. The occurrence of injuries in three regions of the body (chest, head, and abdomen) was analyzed in all the subjects. Results. The study included 525 subjects who died due to the injuries sustained in traffic accidents, which makes up to 38.4% of the total number of 1,366 autopsy cases covered by the study period. The average age of the subjects was 52 ? 19 years. The study sample consisted of 391 (74.5%) men and 134 (25.5%) women. The most vulnerable subjects were pedestrians (220, i.e. 41.9%), followed by motor vehicle drivers ( 98, i .e. 1 8.7%), front-seat passengers (79, i.e. 15%), motorcyclists (39, i.e. 7.4%), bicyclists (38, i.e. 7.2%), back-seat passengers (29, i.e. 5.5%) and tractor drivers (22, i.e. 4.2%). Chest injuries were identified in 408 subjects (77.7%), while the most frequent type of in-jury was rib fracture, observed in two-thirds of the sample. Out of the total number, 291 (55.4%) subjects died at the scene of the accident or on their way to the hospital, while 234 (44.6%) of them outlived injuries for a certain period of time. Drivers exhibited the highest risk of dying at the scene, while bicyclists outlived their injuries more frequently. Conclusion. Chest injuries are very common in subjects who died at the scene of the road traffic accident.
Four new ligands and their palladium(II) complexes of general formula R2-S,S-eddtyr (L1–L4) and PdCl2(R2-S,S-eddtyr) (C1–C4) (R=ethyl, n-propyl, n-butyl and n-pentyl; ...S,S-eddtyr·2HCl=ethylenediamine-N,N′-di-(2,2′-di(4-hydroxy-benzyl))-acetic acid dihydrochloride have been synthesized and characterized by microanalysis, infrared, 1H and 13C NMR spectroscopy. Cytotoxicity for ligands and complexes on two different cell lines (human breast cancer, MDA-MB-231 and human lung cancer, A549 cell lines) and human chronic lymphocytic leukemia cells (CLL) was investigated using MTT (3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) assay.
The complexes have been obtained by reaction of potassium-tetrachloridopalladate(II) with corresponding esters of ethylenediamine-N,N′-di-(2,2′-di(4-hydroxy-benzyl))-acetate dihydrochloride dihydrate. We investigated in vitro antitumor activity of these complexes and their ligands on two cell lines (human breast cancer, MDA-MB-231 and human lung cancer, A549 cell lines) and human chronic lymphocytic leukemia cells (CLL). Display omitted
•Four new ligands and their palladium(II) complexes were synthesized.•Characterized by microanalysis, infrared, 1H and 13C NMR spectroscopy•Cytotoxicity for ligands and complexes is investigated.
Three series of sulfonyl esters were synthesized in reactions of sulfonyl chlorides with three different phenolic chalcone analogues (dehydrozingerone (4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one), (
...E
)-1-(4-hydroxy-3-methoxyphenyl)pent-1-en-3-one, and (
E
)-1-(4-hydroxy-3-methoxyphenyl)-5-methylhex-1-en-3-one). The structures of the new compounds were determined by IR, MS, and NMR methods. Screening of the new sulfonyl esters’ in vitro cytotoxic activities against human epithelial cervical carcinoma (HeLa) and normal human fibroblast (MRC-5) cell lines by the MTT method was performed. The five most active were selected and further tested on HeLa, MRC-5, and MCF-7 (breast carcinoma) cell lines. The examined compounds exhibit strong in vitro anticancer activities with moderate-to-high selectivity, inducing apoptotic cell death and cell cycle arrest in both HeLa and MCF-7 cell lines, but have little to no effect on the non-cancerous MRC-5 cell line.
This paper presents the synthesis and structural characterization of a series of new ruthenium(
ii
) complexes
1-7
, with the general formula
mer
-RuL
3
(
N
-
N
)ClCl, where L is ...2,2′:6′,2′′-terpyridine (tpy) or 4′-(4-chlorophenyl)-2,2′:6′,2′′-terpyridine (Cl-Ph-tpy) and
N
-
N
is
o
-benzoquinonediimine (
o
-bqdi), 2,3-naphthoquinonediimine (nqdi), 4,4′-dimethyl-2,2′-bipyridine (dmbpy) or 2,2′-bipyridine-4,4′-dicarboxylic acid (dcbpy). The kinetic results showed that the ligand substitution reactions of new Ru(
ii
)-polypyridyl complexes with biomolecules were affected by different substituents and the aromaticity of meridional tridentate and bidentate spectator ligands as well as by the nature of the entering nucleophile. The reactivity of the complexes increases in the order: dmbpy < dcbipy < nqdi <
o
-bqdi. In addition, quantum chemical calculations were performed to support the interpretation and discussion of the experimental data. Furthermore, combining ethidium bromide (EB) and Hoechst 33258 (2-(4-hydroxyphenyl)-5-5-(4-methylpiperazine-1-yl)benzimidazo-2-yl-benzimidazole) fluorescence assay results implied that
1-7
might interact with calf thymus DNA through partial intercalation and/or minor groove binding. The human serum albumin (HAS)-fluorescence binding studies involving the site markers, eosin Y, as a marker for site I of subdomain IIA, and ibuprofen, as a marker for site II of subdomain IIIA, showed that Ru(
ii
) compounds bind to both sites with moderately strong affinity (
K
b
= 10
4
-10
6
M
−1
). Moreover, these DNA/HSA experimental results were confirmed by molecular docking. Complexes
2
,
5
and
6
exerted good to strong and highly selective cytotoxic activity against breast adenocarcinoma (MDA-MB 231), colorectal carcinoma (HCT116) and cervix adenocarcinoma (HeLa). Depending on their structure and cell line, the complexes acted differently in terms of their influence on autophagy, the cell cycle and the engaged apoptotic pathway.
New ruthenium(
ii
)-tpy complexes strongly and selectively limited cancer cell growth and replication, and induced apoptosis, at least partly through damaging DNA or blockade of DNA synthesis.
•Autopsy findings reveal that the cause of death is a combination of hypothermia, postural asphyxia, carboxyhemoglobin, and benzodiazepine intoxication.•Chemical toxicological analyses support the ...conclusion that the victim's demise resulted from the synergistic effects of multiple factors.•The manner of death is determined to be a combination of accidental and suicidal, reflecting the victim's attempted suicide and subsequent exposure to low external temperatures.•The autopsy suggests that the fatal outcome is a consequence of the interplay between environmental conditions and the presence of toxic substances in the body.•The investigation underscores the complexity of the case, highlighting the importance of considering various factors contributing to the tragic event, including both intentional and unintentional elements.
Suicide is the intentional and voluntary act of destroying one's own life, while an accident is an unintended event that involuntarily causes injury to one's health or destruction of life.
We report a case of a 54-year-old male who was found in a forest in late November, approximately 500 m away from his car. He was positioned bent over a trunk of a tree with his head beneath the rest of his body. His pants were down to his knees, and there were soiled blades of grass and leaves on his body. Investigation of the case circumstances revealed that he had attempted suicide by carbon monoxide poisoning, using a hose connected to the exhaust pipe and running it through the window into the cabin. Window on the driver’s side was broken with glass particles on the driver's seat. Wrappers from “Rivotril” tablets, a generic benzodiazepine, were also found in the car.
Autopsy revealed the following: postmortem hypostasis was of a cherry red color and well pronounced on the upper part of the front of the body and face. Numerous bruises, contusions, and erosions were present all over the body. Frostbites were especially pronounced in the knees and elbows area. The synovial membranes were partially bloodstained and reddish in color. Opening the stomach revealed erosions of the gastric mucosa (Wischnewsky sign).
Chemical toxicological analysis detected presence of benzodiazepines and carboxyhemoglobin (25%).
Based on the autopsy findings, chemical toxicological analyses, and investigation of the case circumstances, it has been concluded that the death occurred due to the combined effects of hypothermia, postural asphyxia and carboxyhemoglobin and benzodiazepine intoxication. The manner of death in this case is a combination of accidental and suicidal, as the victim attempted suicide but ultimately died due to exposure to low external temperature.
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