Emergence and spread of SARS-CoV-2 lineage B.1.620 with variant of concern-like mutations and deletions.

Distinct SARS-CoV-2 lineages, discovered through various genomic surveillance initiatives, have emerged during the pandemic following unprecedented reductions in worldwide human mobility. We here describe a SARS-CoV-2 lineage - designated B.1.620 - discovered in Lithuania and carrying many mutations and deletions in the spike protein shared with widespread variants of concern (VOCs), including E484K, S477N and deletions HV69Δ, Y144Δ, and LLA241/243Δ. As well as documenting the suite of mutations this lineage carries, we also describe its potential to be resistant to neutralising antibodies, accompanying travel histories for a subset of European cases, evidence of local B.1.620 transmission in Europe with a focus on Lithuania, and significance of its prevalence in Central Africa owing to recent genome sequencing efforts there. We make a case for its likely Central African origin using advanced phylogeographic inference methodologies incorporating recorded travel histories of infected travellers

Alterations of hsa-miR-24-3p and hsa-miR-34a-5p gene expression in blood plasma of the patients with stable angina pectoris

Dovydas Gečys. Alterations of hsa-miR-24-3p and hsa-miR-34a-5p gene expression in blood plasma of the patients with stable angina pectoris Supervisor: Ph.D Vacis Tatarūnas The aim of this study was to investigate the expression of hsa-miR-24-3p and hsa-miR-34a-5p genes in blood plasma of the patients with stable angina pectors and to evaluate the impact of these molecules, CYP4F2 rs2108622, rs1558139, rs3093135, rs20749102 genetic variants and non-genetic factors on CYP4F2 enzyme levels. Objectives: 1. To determine and to compare the expression levels of hsa-miR-24-3p and hsa-miR-34a-5p genes, and CYP4F2 enzyme concentration in blood plasma of the patients with stable angina pectoris patients and in healthy subjects. 2. To evaluate the impact of CYP4F2 rs2108622, rs1558139, rs3093135, rs20749102 genetic variants, gender, age and diabetes mellitus on the expression levels of hsa-miR-24-3p and hsa-miR-34a-5p genes in blood plasma. 3. To evaluate the impact of CYP4F2 rs2108622, rs1558139, rs3093135, rs20749102 genetic variants, gender, age and diabetes mellitus on CYP4F2 enzyme concentration in blood plasma. 4. To evaluate the impact of drugs on hsa-miR-24-3p, hsa-miR-34a-5p, and ώ-hydroxilase levels in blood plasma of patients with stable angina pectoris. Totally 34 patients with stable angina pectoris (SA) and 15 control subjects were examined. Total RNA was extracted from blood plasma using commercial kit. The CYP4F2 enzyme levels were analyzed using commercial kit. Gene expression analysis and genotyping were performed using real-time polymerase chain reaction (RT-PCR) method. Results. The expression of hsa-miR-24-3p and hsa-miR-34a-5p genes was upregulated by 5,5 (p=0,0001) and 4,3-fold (p=0,002), respectively, in the patients with SA as compared to healthy subjects. The concentration of CYP4F2 enzyme was 2 fold lower (p=0,0001) in blood plasma of the patients compared to healthy individuals. Hsa-mir-24-3p expression levels were inversely proportional to CYP4F2 levels (Spearman correlation coeficient r=-0,346, p=0,016). Male patients had 1,6 times lower CYP4F2 concentration as compared to female patients (p=0,04). CYP4F2 rs2108622 CC carriers had 2 fold lower enzyme concentration in blood plasma as compared to CT carriers (p=0,04). CYP4F2 rs1558139 AA carriers had 4-fold (p=0,04) and 3-fold (p=0,01) lower enzyme concentrations as compared to AG and GG variant carriers. No significant associations were found among sex, age, DM, CYP4F2 genetic variants and micro-RNA levels. SA patients, users of atorvastatin and ACE inhibitors, had 2,9 (p=0,02) and 2,1 fold (p=0,05), respectively, higher hsa-miR-24-3p gene expression in plasma as compared to non-users. Hsa-miR-34a-5p levels were higher by 5,8 fold (p=0,09) in blood plasma of the patients users of ACE inhibitors. Conclusions. Hsa-miR-24-3p and hsa-miR-34a-5p expression was higher in blood plasma of the patients with SA. CYP4F2 levels were lower in the patients. Variants of CYP4F2 and non-genetic factors had no significant effect on hsa-miR-24-3p and hsa-miR-34a-5p gene expression levels. SA males had lower CYP4F2 concentration than females. CYP4F2 rs2108622 CC carriers had lower enzyme concentration as compared to the carriers of CT variant. The carriers of CYP4F2 rs1558139 AA variant, had lower CYP4F2 enzyme concentration as compared to AG and GG variant carriers. Users of atorvastatin and ACE inhibitors had higher hsa-miR-24a-3p gene expression in blood plasma. Other drugs had no significant effect on CYP4F2 concentration in blood plasma

Adipose Tissue-Derived Stem Cell Extracellular Vesicles Suppress Glioblastoma Proliferation, Invasiveness and Angiogenesis

Extracellular vesicles (EVs) are attractive anticancer drug delivery candidates as they confer several fundamental properties, such as low immunogenicity and the ability to cross biological barriers. Mesenchymal stem cells (MSCs) are convenient producers for high EV yields, and patient-derived adipose tissue MSC-EVs could serve as personalised carriers. However, MSC-EV applications raise critical concerns as their natural cargo can affect tumour progression in both inducing and suppressing ways. In this study, we investigated the effect of adipose tissue-derived mesenchymal stem cell EVs (ASC-EVs) on several glioblastoma (GBM) cell lines to define their applicability for anticancer therapies. ASC-EVs were isolated from a cell-conditioned medium and characterised by size and specific markers. The internalisation of fluorescently labelled ASC-EVs by human GBM cells HROG36, U87 MG, and T98G was evaluated by fluorescent microscopy. Changes in GBM cell proliferation after ASC-EV application were determined by the metabolic PrestoBlue assay. Expression alterations in genes responsible for cell adhesion, proliferation, migration, and angiogenesis were evaluated by quantitative real-time PCR. ASC-EV effects on tumour invasiveness and neoangiogenesis in ovo were analysed on the chicken embryo chorioallantoic membrane model (CAM). ASC-EV treatment reduced GBM proliferation in vitro and significantly downregulated invasiveness-related genes ITGα5 (in T98G and HROG63) and ITGβ3 (in HROG36) and the vascularisation-inducing gene KDR (in all GBM lines). Additionally, an approximate 65% reduction in the GBM invasion rate was observed in CAM after ASC-EV treatment. Our study indicates that ASC-EVs possess antitumour properties, reducing GBM cell proliferation and invasiveness, and can be applied as anticancer therapeutics and medicine carriers

Long Noncoding RNAs CARMN, LUCAT1, SMILR, and MALAT1 in Thoracic Aortic Aneurysm: Validation of Biomarkers in Clinical Samples

Background and Objectives. Thoracic aortic aneurysm (TAA) is a silent disease characterised by aortic wall expansion and vascular smooth muscle cell (VSMC) dedifferentiation from contractile to synthetic phenotype. Long noncoding RNAs (lncRNAs) involved in VSMC phenotypic regulation could be considered as potential diagnostic indicators and therapeutic targets of TAA. In vitro studies show that lncRNAs CARMN, LUCAT1, MALAT1, and SMILR are associated with the VSMC phenotypic state. Our aim was to test if these lncRNAs are dysregulated during TAA formation in clinical patient samples. Materials and Methods. Relative expression of lncRNAs CARMN, LUCAT1, SMILR, and MALAT1 was tested in clinical aortic tissue and blood plasma samples from TAA and non-TAA patients using the qRT-PCR method. The Mann–Whitney U test was used to compare ΔCt values between the study groups. ROC curve analysis was performed to evaluate the diagnostic value of plasma lncRNAs. Results. We found significantly reduced CARMN (p = 0:033) and LUCAT1 (p = 0:009) expression in aortic tissue samples from TAA patients. Relative expression of MALAT1 (p = 0:117) and SMILR (p = 0:610) did not differ in aortic tissue between the TAA and non-TAA groups. Expression of both LUCAT1 and SMILR was significantly decreased in TAA patients’ blood plasma compared to controls (p = 0:018 and p = 0:032, respectively). However, only LUCAT1 showed the ability to discriminate aneurysmal disease in patients’ blood plasma (AUC = 0:654, 95%CI = 0:534‐0:775, p = 0:018). Conclusions. We have shown that the expression of lncRNAs CARMN and LUCAT1 is reduced in dilated aortic tissue and that the LUCAT1 and SMILR expression is lower in the blood plasma of TAA patients. Decreased LUCAT1 expression in TAA patients’ blood plasma may have diagnostic potential in discriminating patients with TAA

Internalisation of RGD-Engineered Extracellular Vesicles by Glioblastoma Cells

Glioblastoma multiforme (GBM) is the most aggressive CNS tumour with no efficient treatment, partly due to the retention of anticancer drugs by the blood–brain barrier (BBB) and their insufficient concentration in tumour cells. Extracellular vesicles (EVs) are attractive drug carriers because of their biocompatibility and ability to cross the BBB. Additional efficiency can be achieved by adding GBM-cell-specific ligands. GBM cells overexpress integrins; thus, one of the most straightforward targeting strategies is to modify EVs with integrin-recognising molecules. This study investigated the therapeutic potential of genetically engineered EVs with elevated membrane levels of the integrin-binding peptide RGD (RGD-EVs) against GBM cells in vitro. For RGD-EV production, stable RGD-HEK 293FT cells were generated by using a pcDNA4/TO-Lamp2b-iRGD-HA expression vector and performing antibiotic-based selection. RGD-EVs were isolated from RGD-HEK 293FT-cell-conditioned medium and characterised by size (Zetasizer), specific markers (ELISA) and RGD expression (Western Blot). Internalisation by human GBM cells HROG36 and U87 MG and BJ-5ta human fibroblasts was assessed by fluorescent EV RNA labelling. The effect of doxorubicin-loaded RGD-EVs on GBM cells was evaluated by the metabolic PrestoBlue viability assay; functional GAPDH gene knockdown by RGD-EV-encapsulated siRNA was determined by RT-qPCR. RGD-EVs had 40% higher accumulation in GBM cells (but not in fibroblasts) and induced significantly stronger toxicity by loaded doxorubicin and GAPDH silencing by loaded siRNA compared to unmodified EVs. Thus, RGD modification substantially increases the specific delivery capacity of HEK 293FT-derived EVs to GBM cells

Effects of Combined Treatment with Sodium Dichloroacetate and Sodium Valproate on the Genes in Inflammation- and Immune-Related Pathways in T Lymphocytes from Patients with SARS-CoV-2 Infection with Pneumonia: Sex-Related Differences

The study presents data on the anti-inflammatory effects of a combination of sodium dichloroacetate and sodium valproate (DCA–VPA) on the expression of inflammation- and immune response-related genes in T lymphocytes of SARS-CoV-2 patients. The study aimed to assess the effects of DCA–VPA on the genes of cytokine activity, chemokine-mediated signaling, neutrophil chemotaxis, lymphocyte chemotaxis, T-cell chemotaxis, and regulation of T-cell proliferation pathways. The study included 21 patients with SARS-CoV-2 infection and pneumonia: 9 male patients with a mean age of 68.44 ± 15.32 years and 12 female patients with a mean age of 65.42 ± 15.74 years. They were hospitalized between December 2022 and March 2023. At the time of testing, over 90% of sequences analyzed in Lithuania were found to be of the omicron variant of SARS-CoV-2. The T lymphocytes from patients were treated with 5 mmol DCA and 2 mmol VPA for 24 h in vitro. The effect of the DCA–VPA treatment on gene expression in T lymphocytes was analyzed via gene sequencing. The study shows that DCA–VPA has significant anti-inflammatory effects and apparent sex-related differences. The effect is more potent in T cells from male patients with SARS-CoV-2 infection and pneumonia than in females

Differential Mitochondrial, Oxidative Stress and Inflammatory Responses to SARS-CoV-2 Spike Protein Receptor Binding Domain in Human Lung Microvascular, Coronary Artery Endothelial and Bronchial Epithelial Cells

Recent evidence indicates that the SARS-CoV-2 spike protein affects mitochondria with a cell type-dependent outcome. We elucidate the effect of the SARS-CoV-2 receptor binding domain (RBD) on the mitochondrial network and cristae morphology, oxygen consumption, mitoROS production, and inflammatory cytokine expression in cultured human lung microvascular (HLMVECs), coronary artery endothelial (HCAECs), and bronchial epithelial cells (HBECs). Live Mito Orange staining, STED microscopy, and Fiji MiNa analysis were used for mitochondrial cristae and network morphometry; an Agilent XFp analyser for mitochondrial/glycolytic activity; MitoSOX fluorescence for mitochondrial ROS; and qRT-PCR plus Luminex for cytokines. HLMVEC exposure to SARS-CoV-2 RBD resulted in the fragmentation of the mitochondrial network, mitochondrial swelling, increased cristae area, reduced cristae density, and suppressed mitochondrial oxygen consumption and glycolysis. No significant mitochondrial morphology or oxygen consumption changes were observed in HCAECs and HBECs. SARS-CoV-2 RBD induced mitoROS-mediated expression of cytokines GM-CSF and IL-1β in all three investigated cell types, along with IL-8 expression in both endothelial cell types. The findings suggest mitochondrial ROS control SARS-CoV-2 RBD-induced inflammation in HLMVECs, HCAECs, and HBECs, with the mitochondria of HLMVECs being more sensitive to SARS-CoV-2 RBD

SARS-CoV-2 infection, sex-related differences, and a possible personalized treatment approach with valproic acid: a review

Sex differences identified in the COVID-19 pandemic are necessary to study. It is essential to investigate the efficacy of the drugs in clinical trials for the treatment of COVID-19, and to analyse the sex-related beneficial and adverse effects. The histone deacetylase inhibitor valproic acid (VPA) is a potential drug that could be adapted to prevent the progression and complications of SARS-CoV-2 infection. VPA has a history of research in the treatment of various viral infections. This article reviews the preclinical data, showing that the pharmacological impact of VPA may apply to COVID-19 pathogenetic mechanisms. VPA inhibits SARS-CoV-2 virus entry, suppresses the pro-inflammatory immune cell and cytokine response to infection, and reduces inflammatory tissue and organ damage by mechanisms that may appear to be sex-related. The antithrombotic, antiplatelet, anti-inflammatory, immunomodulatory, glucose- and testosterone-lowering in blood serum effects of VPA suggest that the drug could be promising for therapy of COVID-19. Sex-related differences in the efficacy of VPA treatment may be significant in developing a personalised treatment strategy for COVID-19

Virus Mimetic Poly (I:C)-Primed Airway Exosome-like Particles Enter Brain and Induce Inflammatory Cytokines and Mitochondrial Reactive Oxygen Species in Microglia

Viral infections induce extracellular vesicles (EVs) containing viral material and inflammatory factors. Exosomes can easily cross the blood-brain barrier during respiratory tract infection and transmit the inflammatory signal to the brain; however, such a hypothesis has no experimental evidence. The study investigated whether exosome-like vesicles (ELVs) from virus mimetic poly (I:C)-primed airway cells enter the brain and interact with brain immune cells microglia. Airway cells were isolated from Wistar rats and BALB/c mice; microglial cell cultures—from Wistar rats. ELVs from poly (I:C)-stimulated airway cell culture medium were isolated by precipitation, visualised by transmission electron microscopy, and evaluated by nanoparticle analyser; exosomal markers CD81 and CD9 were determined by ELISA. For in vitro and in vivo tracking, particles were loaded with Alexa Fluor 555-labelled RNA. Intracellular reactive oxygen species (ROS) were evaluated by DCFDA fluorescence and mitochondrial superoxide—by MitoSOX. ELVs from poly (I:C)-primed airway cells entered the brain within an hour after intranasal introduction, were internalised by microglia and induced intracellular and intramitochondrial ROS production. There was no ROS increase in microglial cells was after treatment with ELVs from airway cells untreated with poly (I:C). In addition, poly (I:C)-primed airway cells induced inflammatory cytokine expression in the brain. The data indicate that ELVs secreted by virus-primed airway cells might enter the brain, cause the activation of microglial cells and neuroinflammation

Emergence and spread of SARS-CoV-2 lineage B.1.620 with variant of concern-like mutations and deletions

Distinct SARS-CoV-2 lineages, discovered through various genomic surveillance initiatives, have emerged during the pandemic following unprecedented reductions in worldwide human mobility. We here describe a SARS-CoV-2 lineage - designated B.1.620 - discovered in Lithuania and carrying many mutations and deletions in the spike protein shared with widespread variants of concern (VOCs), including E484K, S477N and deletions HV69Δ, Y144Δ, and LLA241/243Δ. As well as documenting the suite of mutations this lineage carries, we also describe its potential to be resistant to neutralising antibodies, accompanying travel histories for a subset of European cases, evidence of local B.1.620 transmission in Europe with a focus on Lithuania, and significance of its prevalence in Central Africa owing to recent genome sequencing efforts there. We make a case for its likely Central African origin using advanced phylogeographic inference methodologies incorporating recorded travel histories of infected travellers