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

Detection of bovine leukocyte adhesion deficiency, deficiency of uridine monophosphate synthase, and complex vertebral malformation in holstein cattle

This research aimed to evaluate the prevalence of the most common lethal diseases in the Lithuanian Holstein cattle population. Two hundred non-related (based on the documentation of origin) cattle (cows and heifers) were included in the study. DNA extraction from blood leukocytes was performed using the chloroform salt method. The cattle were tested for three inherited bovine disorders: bovine leukocyte adhesion deficiency (BLAD), deficiency of uridine monophosphate synthase (DUMPS), complex vertebral malformation (CVM). The PCR-RFLP test method was used to determine the polymorphism of the CD18 gene, which is responsible for BLAD inherited disorder development. A recessive allele with point mutation A→G (383), causing BLAD, was found in the Lithuanian cattle population with 0.0025 frequency. CVM disease is determined by the missense mutation, which has been found in the SLC35A3 gene. The study was performed using a sequencing method. A recessive allele with point mutation G→T (538), causing CVM, was found in the Lithuanian cattle population with 0.005 frequency. The PCR-RFLP test method was used to determine the polymorphism of the UMPS gene, responsible for DUMPS inherited disorder development. A recessive allele with point mutation C→T (1213), causing DUMPS, was not found in the Lithuanian Holstein cattle population. Because intensive selection programmes were performed over the previous decade, the number of heritable lethal diseases carriers has significantly decreased

The Association of Matrix Metalloproteinases Polymorphisms and interleukins in Advanced Age-Related Macular Degeneration

Age-related macular degeneration is the leading cause of permanent, irreversible, central blindness in patients over the age of 50 years [1]. Genetic influence, cumulative exposure to oxidative stress, and immune-inflammatory mediated processes may be considered as possible risk factors for development of AMD [2]. Purpose: To assess the impact of MMP1-1607 1G/2G (rs1799750), MMP7-181 A/G (rs11568818) SNP and systemic cytokins IL-1β, IL-6 levels on the development of exudative age-related macular degeneration (eAMD). Methodology: The study group comprised 282 patients with eAMD, and the control group enrolled 379 randomly selected persons. The genotyping of MMP1 (rs1799750) and MMP7-181 (rs11568818) was performed by using the PCR-RFLP method. To determine IL-1β and IL-6 serum level, the immunoenzymatic method with monoclonal antibodies coated plates was performed. Results: Only MMP-1 rs1799750 1G/2G genotype was more frequently found in the development of eAMD, it was associated with a 4.3-fold increased risk for eAMD under the codominant model and a 4.9-fold increased risk for eAMD under the overdominant model and the effect was more pronounced at the age of less than 65 years. IL-1β concentration was significantly higher in eAMD patients compared with control group subjects for MMP1 rs1799750 1G/1G genotype and MMP7 rs11568818 A/G genotype. Conclusions: Only MMP-1 rs1799750 1G/2G genotype was found to increase the risk of eAMD, and the effect was more pronounced at the age of less than 65 years. IL-1β concentration was significantly higher in eAMD patients compared with control group subjects for MMP1 rs1799750 1G/1G genotype and MMP7 rs11568818 A/G genotype

Pathomorphological Manifestations and the Course of the Cervical Cancer Disease Determined by Variations in the <i>TLR4</i> Gene

Cervical cancer (CC) is often associated with human papillomavirus (HPV). Chronic inflammation has been described as one of the triggers of cancer. The immune system fights diseases, including cancer. The genetic polymorphism of pathogen recognition receptors potentially influences the infectious process, development, and disease progression. Many candidate genes SNPs have been contradictory demonstrated to be associated with cervical cancer by association studies, GWAS. TLR4 gene activation can promote antitumor immunity. It can also result in immunosuppression and tumor growth. Our study aimed to investigate eight selected polymorphisms of the TLR4 gene (rs10759932, rs1927906, rs11536898, rs11536865, rs10983755, rs4986790, rs4986791, rs11536897) and to determine the impact of polymorphisms in genotypes and alleles on the pathomorphological characteristics and progression in a group of 172 cervical cancer subjects with stage I–IV. Genotyping was performed by RT-PCR assay. We detected that the CA genotype and A allele of rs11536898 were significantly more frequent in patients with metastases (p = 0.026; p = 0.008). The multivariate logistic regression analysis confirmed this link to be significant. The effect of rs10759932 and rs11536898 on progression-free survival (PFS) and overall survival (OS) has been identified as important. In univariate and multivariate Cox analyses, AA genotype of rs11536898 was a negative prognostic factor for PFS (p = 0.024; p = 0.057, respectively) and OS (p = 0.008; p = 0.042, respectively). Rs11536898 C allele predisposed for longer PFS (univariate and multivariate: p = 0.025; p = 0.048, respectively) and for better OS (univariate and multivariate: p = 0.010; p = 0.043). The worse prognostic factor of rs10759932 in a univariate and multivariate Cox analysis for survival was CC genotype: shorter PFS (p = 0.032) and increased risk of death (p = 0.048; p = 0.015, respectively). The T allele of rs10759932 increased longer PFS (univariate and multivariate: p = 0.048; p = 0.019, respectively) and longer OS (univariate and multivariate: p = 0.037; p = 0.009, respectively). Our study suggests that SNPs rs10759932 and rs11536898 may have the potential to be markers contributing to the assessment of the cervical cancer prognosis. Further studies, preferably with larger groups of different ethnic backgrounds, are needed to confirm the results of the current study

Systemic Optimization of Gene Electrotransfer Protocol Using Hard-to-Transfect UT-7 Cell Line as a Model

Non-adherent cells are difficult to transfect with chemical-mediated delivery methods. Electroporation is an attractive strategy to transfer the molecules of interest into suspension cells. Care must be taken with the viability of the transfected cells since parameters, which increase cell membrane permeability, subsequently increase transfection efficiency, leading to higher cell death indices. We intended to evaluate the distribution of hard-to-transfect UT-7 cells among different subpopulations: transfected/viable, untransfected/viable, transfected/dead, and untransfected/dead populations, for a better understanding of the relation between gene electrotransfer efficacy and cell death. The following electroporation parameters were tested: pulse strength, duration, plasmid DNA concentration, and ZnSO4 as DNase inhibitor. BTX T820 square-wave generator was used, and 48 h after electroporation, cells were observed for viability and fluorescence analysis. Increasing pulse strength correlated directly with an increased ratio of pEGFP-positive cells and inversely with cell viability. The best results, representing 21% pEGFP positive/viable cells, were obtained after EP with 1 HV 1400 V/cm pulse of 250 µs duration using 200 µg/mL plasmid concentration. Results demonstrated that plasmid concentration played the most significant role in pEGFP electrotransfer into UT-7 cells. These results can represent a relevant improvement of gene electrotransfer to obtain genetically modified suspension cells for further downstream experiments

The influence of TLR4 gene polymorphisms on milk quality and composition of Lithuanian Holstein cows

This study investigated bovine TRL4 gene c.9421C>T, c.2021C>T and c.-10C>T polymorphisms and their relationship with somatic cell count and indicators of milk composition. Blood samples were collected from 152 Lithuanian Holstein dairy cows. The method of polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) was used to genotype TLR4 gene polymorphisms. The data concerning somatic cell count and milk composition indicators (fat, protein, lactose) were analysed. The influence of genes and statistical significance of differences between different genotypes was evaluated by the one-factor dispersion analysis (ANOVA). Results showed that c.9421C>T was significantly associated with somatic cell count in milk. Also a significant association between the genotypes of c.9421C>T and somatic cell count was found (PT and c.-10C>T have no significant effect on mastitis resistance and milk composition. Analysis of the combined genotypes TC/CC of c.2021C>T and c.9421C>T allowed us to determine the possible association of SNPs with the somatic cell count and lactose content. The study showed a significant association between the somatic cell count (SCC) and TLR4 polymorphism c.9421C>T in Lithuanian Holstein cows. SCS of cows with a TT genotype was significantly lower and indicated the association of TT genotype with resistance to mastitis in c.9421C>T and allele T might be the beneficial allele for mastitis resistance

DDIT4 Downregulation by siRNA Approach Increases the Activity of Proteins Regulating Fatty Acid Metabolism upon Aspirin Treatment in Human Breast Cancer Cells

Repositioning of aspirin for a more effective breast cancer (BC) treatment requires identification of predictive biomarkers. However, the molecular mechanism underlying the anticancer activity of aspirin remains fully undefined. Cancer cells enhance de novo fatty acid (FA) synthesis and FA oxidation to maintain a malignant phenotype, and the mechanistic target of rapamycin (mTORC1) is required for lipogenesis. We, therefore, aimed to test if the expression of mTORC1 suppressor DNA damage-inducible transcript (DDIT4) affects the activity of main enzymes in FA metabolism after aspirin treatment. MCF-7 and MDA-MB-468 human BC cell lines were transfected with siRNA to downregulate DDIT4. The expression of carnitine palmitoyltransferase 1 A (CPT1A) and serine 79-phosphorylated acetyl-CoA carboxylase 1 (ACC1) were analyzed by Western Blotting. Aspirin enhanced ACC1 phosphorylation by two-fold in MCF-7 cells and had no effect in MDA-MB-468 cells. Aspirin did not change the expression of CPT1A in either cell line. We have recently reported DDIT4 itself to be upregulated by aspirin. DDIT4 knockdown resulted in 1.5-fold decreased ACC1 phosphorylation (dephosphorylation activates the enzyme), 2-fold increased CPT1A expression in MCF-7 cells, and 2.8-fold reduced phosphorylation of ACC1 following aspirin exposure in MDA-MB-468 cells. Thus, DDIT4 downregulation raised the activity of main lipid metabolism enzymes upon aspirin exposure which is an undesired effect as FA synthesis and oxidation are linked to malignant phenotype. This finding may be clinically relevant as DDIT4 expression has been shown to vary in breast tumors. Our findings justify further, more extensive investigation of the role of DDIT4 in aspirin’s effect on fatty acid metabolism in BC cells

Associations of MDM2 and MDM4 Polymorphisms with Early-Stage Breast Cancer

Breast cancer is one of the most common cancers worldwide. Single nucleotide polymorphisms (SNPs) in MDM2 and MDM4 have been associated with various cancers. However, the influence on clinical characteristics of breast cancer has not been sufficiently investigated yet. Thus, this study aimed to investigate the relationship between SNPs in MDM2 (rs2279744, rs937283, rs937282) and MDM4 (rs1380576, rs4245739) and I–II stage breast cancer. For analysis, the genomic DNA was extracted from 100 unrelated women peripheral blood. Polymorphisms were analyzed with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. The study showed that MDM2 rs937283 and rs937282 were significantly associated with estrogen receptor status and human epidermal growth factor receptor 2 (HER2) status. SNPs rs1380576 and rs4245739, located in MDM4, were significantly associated with status of estrogen and progesterone receptors. Our findings suggest that rs937283 AG, rs937282 CG, rs1380576 CC, and rs4245739 AA genotypes were linked to hormonal receptor positive breast cancer and may be useful genetic markers for disease assessment

VEGF-B, VEGF-A, FLT-1, KDR, ERBB2, EGFR, GRB2, RAC1, CDH1 and HYAL-1 Genes Expression Analysis in Canine Mammary Gland Tumors and the Association with Tumor ClinicoPathological Parameters and Dog Breed Assessment

Canine mammary gland tumors (CMTs) are one of the most prevalent cancers in dogs and a good model for human breast cancer (BC), however gene expression analysis of CMTs is scarce. Although divergence of genes expression has been found in BC of different human races, no such research of different dog’s breeds has been done. The purpose of this study was to investigate expression of the VEGF-B, VEGF-A, FLT-1, KDR, ERBB2, EGFR, GRB2, RAC1, CDH1 and HYAL-1 genes of canine mammary carcinomas, compare the expression levels with clinicopathological parameters and analyze expression disparities between different breeds. Carcinomas and adjacent tissues were collected from female dogs to perform routine histopathology, immunohistochemistry (IHC) and quantitative real-time polymerase chain reaction (qRT-PCR). We found that VEGF-B and EGFR genes were overexpressed in the mammary gland carcinomas compared to adjacent tissue. VEGF-B gene expression had associations with different parameters (tumor size, grade, and absence of metastasis). Furthermore, differences in VEGF-B, FLT1, ERBB2, GRB2, RAC1, CDH1 and HYAL-1 genes expression have been found in different breed dogs (German Shepherd, Yorkshire Terrier) and mixed-breed dogs indicating that a dog’s breed could determine a molecular difference, outcome of cancer and should be accounted as a confounding factor in the future gene expression research