Feline herpesvirus 1 (FHV-1) enters the cell by receptor-mediated endocytosis.

Feline herpesvirus type 1 (FHV-1) is an enveloped dsDNA virus belonging to the Herpesviridae family and is considered one of the two primary viral etiological factors of feline upper respiratory tract disease. In this study, we investigated the entry of FHV-1 into host cells using two models: the AK-D cell line and primary feline skin fibroblasts (FSFs). We employed confocal microscopy, siRNA silencing, and selective inhibitors of various entry pathways. Our observations revealed that the virus enters cells via pH and dynamin-dependent endocytosis, as the infection was significantly inhibited by NH4Cl, bafilomycin A1, dynasore, and mitmab. Additionally, genistein, nystatin, and filipin treatments, siRNA knock-down of caveolin-1, as well as FHV-1 and caveolin-1 colocalization suggest the involvement of caveolin-mediated endocytosis during the entry process. siRNA knock-down of clathrin heavy chain and analysis of virus particle colocalization with clathrin indicated that clathrin-mediated endocytosis also takes part in the primary cells. This is the first study to systematically examine FHV-1 entry into host cells, and for the first time, we describe FHV-1 replication in AK-D and FSFs. IMPORTANCE Feline herpesvirus 1 (FHV-1) is one of the most prevalent viruses in cats, causing feline viral rhinotracheitis, which is responsible for over half of viral upper respiratory diseases in cats and can lead to ocular lesions resulting in loss of sight. Although the available vaccine reduces the severity of the disease, it does not prevent infection or limit virus shedding. Despite the clinical relevance, the entry mechanisms of FHV-1 have not been thoroughly studied. Considering the limitations of commonly used models based on immortalized cells, we sought to verify our findings using primary feline skin fibroblasts, the natural target for infection in cats.

Sirtuin 1 as a potential molecular target of resveratrol in selected diseases / Sirtuina 1 jako potencjalny cel molekularny resweratrolu w wybranych chorobach.

Cancer, type 2 diabetes, cardiovascular and neurological diseases are disorders commonly classified as diseases that have a significant impact on the length and quality of human life. Sirtuins play an important role in their pathogenesis and complications. Numerous studies indicate that modulation of the expression of these proteins can slow down the processes of aging and cell death, prevent inflammation, and regulate metabolic processes, and consequently modify the progression of the disease. One of the best-known sirtuins is sirtuin 1, whose strongest natural activator is resveratrol. The development of alternative therapies involving natural compounds such as resveratrol is highly desirable due to the significantly lower number of side effects compared to conventional therapies. Therefore, this review summarizes the possible benefits of resveratrol as a sirtuin 1 activator in the prevention and treatment of human diseases based on the results of the studies conducted so far.

The SARS-CoV-2 ORF10 is not essential in vitro or in vivo in humans.

SARS-CoV-2 genome annotation revealed the presence of 10 open reading frames (ORFs), of which the last one (ORF10) is positioned downstream of the N gene. It is a hypothetical gene, which was speculated to encode a 38 aa protein. This hypothetical protein does not share sequence similarity with any other known protein and cannot be associated with a function. While the role of this ORF10 was proposed, there is growing evidence showing that the ORF10 is not a coding region. Here, we identified SARS-CoV-2 variants in which the ORF10 gene was prematurely terminated. The disease was not attenuated, and the transmissibility between humans was maintained. Also, in vitro, the strains replicated similarly to the related viruses with the intact ORF10. Altogether, based on clinical observation and laboratory analyses, it appears that the ORF10 protein is not essential in humans. This observation further proves that the ORF10 should not be treated as the protein-coding gene, and the genome annotations should be amended.

The Role of Metalloproteinases and Their Tissue Inhibitors on Ocular Diseases: Focusing on Potential Mechanisms.

Eye diseases are associated with visual impairment, reduced quality of life, and may even lead to vision loss. The efficacy of available treatment of eye diseases is not satisfactory. The unique environment of the eye related to anatomical and physiological barriers and constraints limits the bioavailability of existing agents. In turn, complex ethiopathogenesis of ocular disorders that used drugs generally are non-disease specific and do not act causally. Therefore, there is a need for the development of a new therapeutic and preventive approach. It seems that matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) have a significant role in the development and progression of eye diseases and could be used in the therapy of these disorders as pharmacological targets. MMPs and TIMPs play an important role in the angiogenesis, epithelial-mesenchymal transition, cell invasion, and migration, which occur in ocular diseases. In this review, we aim to describe the participation of MMPs and TIMPs in the eye diseases, such as age-related macular degeneration, cataract, diabetic retinopathy, dry eye syndrome, glaucoma, and ocular cancers, posterior capsule opacification focusing on potential mechanisms.

Replication of Severe Acute Respiratory Syndrome Coronavirus 2 in Human Respiratory Epithelium.

Currently, there are four seasonal coronaviruses associated with relatively mild respiratory tract disease in humans. However, there is also a plethora of animal coronaviruses which have the potential to cross the species border. This regularly results in the emergence of new viruses in humans. In 2002, severe acute respiratory syndrome coronavirus (SARS-CoV) emerged and rapidly disappeared in May 2003. In 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) was identified as a possible threat to humans, but its pandemic potential so far is minimal, as human-to-human transmission is ineffective. The end of 2019 brought us information about severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emergence, and the virus rapidly spread in 2020, causing an unprecedented pandemic. At present, studies on the virus are carried out using a surrogate system based on the immortalized simian Vero E6 cell line. This model is convenient for diagnostics, but it has serious limitations and does not allow for understanding of the biology and evolution of the virus. Here, we show that fully differentiated human airway epithelium cultures constitute an excellent model to study infection with the novel human coronavirus SARS-CoV-2. We observed efficient replication of the virus in the tissue, with maximal replication at 2 days postinfection. The virus replicated in ciliated cells and was released apically.IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged by the end of 2019 and rapidly spread in 2020. At present, it is of utmost importance to understand the biology of the virus, rapidly assess the treatment potential of existing drugs, and develop new active compounds. While some animal models for such studies are under development, most of the research is carried out in Vero E6 cells. Here, we propose fully differentiated human airway epithelium cultures as a model for studies on SARS-CoV-2.

Entry of Human Coronavirus NL63 into the Cell.

The first steps of human coronavirus NL63 (HCoV-NL63) infection were previously described. The virus binds to target cells by use of heparan sulfate proteoglycans and interacts with the ACE2 protein. Subsequent events, including virus internalization and trafficking, remain to be elucidated. In this study, we mapped the process of HCoV-NL63 entry into the LLC-Mk2 cell line and ex vivo three-dimensional (3D) tracheobronchial tissue. Using a variety of techniques, we have shown that HCoV-NL63 virions require endocytosis for successful entry into the LLC-MK2 cells, and interaction between the virus and the ACE2 molecule triggers recruitment of clathrin. Subsequent vesicle scission by dynamin results in virus internalization, and the newly formed vesicle passes the actin cortex, which requires active cytoskeleton rearrangement. Finally, acidification of the endosomal microenvironment is required for successful fusion and release of the viral genome into the cytoplasm. For 3D tracheobronchial tissue cultures, we also observed that the virus enters the cell by clathrin-mediated endocytosis, but we obtained results suggesting that this pathway may be bypassed.IMPORTANCE Available data on coronavirus entry frequently originate from studies employing immortalized cell lines or undifferentiated cells. Here, using the most advanced 3D tissue culture system mimicking the epithelium of conductive airways, we systematically mapped HCoV-NL63 entry into susceptible cells. The data obtained allow for a better understanding of the infection process and may support development of novel treatment strategies.

Zika virus: mapping and reprogramming the entry.

BACKGROUND: The flaviviridae family comprises single-stranded RNA viruses that enter cells via clathrin-mediated pH-dependent endocytosis. Although the initial events of the virus entry have been already identified, data regarding intracellular virus trafficking and delivery to the replication site are limited. The purpose of this study was to map the transport route of Zika virus and to identify the fusion site within the endosomal compartment. METHODS: Tracking of viral particles in the cell was carried out with confocal microscopy. Immunostaining of two structural proteins of Zika virus enabled precise mapping of the route of the ribonucleocapsid and the envelope and, consequently, mapping the fusion site in the endosomal compartment. The results were verified using RNAi silencing and chemical inhibitors. RESULTS: After endocytic internalization, Zika virus is trafficked through the endosomal compartment to fuse in late endosomes. Inhibition of endosome acidification using bafilomycin A1 hampers the infection, as the fusion is inhibited; instead, the virus is transported to late compartments where it undergoes proteolytic degradation. The degradation products are ejected from the cell via slow recycling vesicles. Surprisingly, NH4Cl, which is also believed to block endosome acidification, shows a very different mode of action. In the presence of this basic compound, the endocytic hub is reprogrammed. Zika virus-containing vesicles never reach the late stage, but are rapidly trafficked to the plasma membrane via a fast recycling pathway after the clathrin-mediated endocytosis. Further, we also noted that, similarly as other members of the flaviviridae family, Zika virus undergoes furin- or furin-like-dependent activation during late steps of infection, while serine or cysteine proteases are not required for Zika virus maturation or entry. CONCLUSIONS: Zika virus fusion occurs in late endosomes and is pH-dependent. These results broaden our understanding of Zika virus intracellular trafficking and may in future allow for development of novel treatment strategies. Further, we identified a novel mode of action for agents commonly used in studies of virus entry. Schematic representation of differences in ZIKV trafficking in the presence of Baf A1 and NH4Cl.

Canine respiratory coronavirus employs caveolin-1-mediated pathway for internalization to HRT-18G cells.

Canine respiratory coronavirus (CRCoV), identified in 2003, is a member of the Coronaviridae family. The virus is a betacoronavirus and a close relative of human coronavirus OC43 and bovine coronavirus. Here, we examined entry of CRCoV into human rectal tumor cells (HRT-18G cell line) by analyzing co-localization of single virus particles with cellular markers in the presence or absence of chemical inhibitors of pathways potentially involved in virus entry. We also targeted these pathways using siRNA. The results show that the virus hijacks caveolin-dependent endocytosis to enter cells via endocytic internalization.

Binary Mixtures of Selected Bisphenols in the Environment: Their Toxicity in Relationship to Individual Constituents.

Bisphenol A (BPA) is one of the most popular and commonly used plasticizer in the industry. Over the past decade, new chemicals that belong to the bisphenol group have increasingly been used in industrial applications as alternatives to BPA. Nevertheless, information on the combined effects of bisphenol (BP) analogues is insufficient. Therefore, our current study aimed to find the biological response modulations induced by the binary mixtures of BP compounds. We determined the toxicity levels in Microtox and XenoScreen YES/YAS assays for several BP analogs alone, and for their binary mixtures. The results obtained constituted the database for chemometric intelligent data analysis to evaluate the possible interactions occurring in the mixtures. Several chemometric/biophysical models have been used (concentration addition-CA, independent action-IA and polynomial regression calculations) to realize this aim. The best fitting was found for the IA model and even in this description strong evidence for synergistic behaviors (modes of action) of some bisphenol analogue mixtures was demonstrated. Bisphenols A, S, F and FL were proven to be of significant endocrine threat (with respect to XenoScreen YES/YAS assay); thus, their presence in mixtures (including presence in tissues of living organisms) should be most strictly monitored and reported.

The Impact of Dietary Polyphenols on COX-2 Expression in Colorectal Cancer.

Polyphenols are natural compounds with high structural diversity whose common occurrence in plants renders them intrinsic dietary components. They are known to be secondary metabolites characterized by a wide spectrum of biological activities, and a growing body of evidence indicates they have anti-inflammatory potential. It is well known that inflammation plays a key role in many chronic diseases such as circulatory diseases, pulmonary diseases, autoimmune diseases, diabetes, cancer, and neurodegenerative diseases. Polyphenols influence the inflammatory process by controlling and inhibiting pro-inflammatory cytokines such as IL-1ß, IL-6, IL-8, and TNF-α, and cyclooxygenase-2 (COX-2) enzyme involved in the metabolism of arachidonic acid. Furthermore, polyphenols exhibit anti-inflammatory activity on many levels via NF-κB inhibition, and MAPK, iNOS, and growth factors regulation. This paper reviews the current state of knowledge concerning the potential of various dietary polyphenols to inhibit the effects of COX-2 in colon cancer, by examining the available evidence regarding the efficacy and safety of these compounds obtained from in vitro and animal studies.

Influence of polyphenol extract from evening primrose (Oenothera paradoxa) seeds on human prostate and breast cancer cell lines.

There is growing interest in plant polyphenols which exhibit pleiotropic biological activities, including anti-inflammatory, antioxidant, and anticancer effects. The objective of our study was to evaluate the influence of an evening primrose extract (EPE) from defatted seeds on viability and invasiveness of three human cell lines: PNT1A (normal prostate cells), DU145 (prostate cancer cells) and MDA-MB-231 (breast cancer cells). The results revealed that after 72 h of incubation the tested extract reduced the viability of DU 145 and MDA-MB-231 with IC50 equal to 14.5 µg/mL for both cell lines. In contrast, EPE did not inhibit the viability of normal prostate cells. Furthermore, EPE reduced PNT1A and MDA-MB-231 cell invasiveness; at the concentration of 21.75 µg/mL the suppression of invasion reached 92% and 47%, respectively (versus control). Additionally, zymographic analysis revealed that after 48 h of incubation EPE inhibited metalloproteinase-2 (MMP-2) and metalloproteinase-9 (MMP-9) activities in a dose-dependent manner. For PNT1A the activities of MMP-2 and MMP-9 decreased 4- and 2-fold, respectively, at EPE concentration of 29 µg/mL. In the case of MDA-MB-231 and DU 145 the decrease in MMP-9 activity at EPE concentration of 29 µg/mL was 5.5-fold and almost 1.9-fold, respectively. In conclusion, this study suggests that EPE may exhibit antimigratory, anti-invasive and antimetastatic potential towards prostate and breast cancer cell lines.

Synergistic interactions between anticancer chemotherapeutics and phenolic compounds and anticancer synergy between polyphenols.

Chemoprevention has recently gained a new dimension due to the possibility of studying the mechanisms of action of chemopreventive agents at the molecular level. Many compounds have been proved to inhibit early stages of carcinogenesis in experimental models. These compounds include both recognized drugs (such as tamoxifen and nonsteroidal anti-inflammatory drugs) and natural constituents of edible and therapeutic plants, particularly polyphenols. Phenolics are characterized by high structural diversity and, consequently, a very broad spectrum of biological activities. They are increasingly looked upon as a valuable alternative or a support for synthetic drugs, as evidenced by a growing number of clinical trials regarding the use of phenolic compounds and polyphenol-rich extracts in chemoprevention and therapy. In the present work, we discuss the effectiveness of natural polyphenols as cancer preventive and therapeutic agents resulting from their synergy with synthetic or semisynthetic anticancer drugs as well as with other phenolic compounds of plant origin.

Bisphenol A analogues and metabolic syndrome in women with polycystic ovary syndrome.

Bisphenols (BPs) have become a chemical group of special interest due to their ability to interfere with the endocrine system and their ubiquitous presence in the environment. As some of them possess mild estrogenic and anti-androgenic effects, they might be associated with the diagnosis of polycystic ovary syndrome (PCOS). Acting on multiple tissues, BPs exposure may lead to metabolic derangements characteristic for metabolic syndrome (MetS). Therefore, the aim of this study was to determine the potential relationship between exposure to some BPA analogues and features of the MetS in women with PCOS. Serum BPE, BPC, BPG, BPM, BPP, BPZ, BPFL, and BPBP concentrations did not differ significantly between the PCOS (n = 135) and the control subjects (n = 104). However, women whose serum BPM and BPP concentrations were in the highest tertile were more likely to be diagnosed with PCOS (adjusted OR; [95%CI] 0.43; [0.20; 0.89], P < 0.001 and 0.56; [0.27; 0.96], P = 0.049, consequently). Serum concentrations of BPs were not associated with the MetS diagnosis in the PCOS group. There was a negative correlation between the concentrations of serum BPBP and total serum cholesterol (r = - 0.153; P = 0.019), BPE and serum testosterone (r = - 0.160; P = 0.014) as well as BPC and HDL-cholesterol (r = - 0.138; P = 0.036). There was a positive correlation between the concentrations of BPP and serum triglycerides (r = 0.138; P = 0.036). Our results point to the potential association between exposure to BPM, BPP, and the diagnosis of PCOS, along with the impact of BPBP, BPE, BPC, and BPP on the metabolic features of the MetS.

Isolation, structural characterization and biological activity evaluation of melanoidins from thermally processed cocoa beans, carob kibbles and acorns as potential cytotoxic agents.

The aim of this study was to determine the chemical structure and biological activity of melanoidin fractions derived from cocoa beans, carob kibbles, and acorns roasted at different temperature-time conditions. The results showed that plant origin and roasting conditions had significant effects on the chemical composition, structural features, and morphology of melanoidins. All tested melanoidins exhibited significant antioxidant properties in three in vitro assays. In addition, they show significant in vitro anti-inflammatory activity by reducing lipoxygenase. The results from MTT assay showed that the all studied melanoidins had a cytotoxic effect against SW-480 cells in a dose- and time-dependent manner. Furthermore, the most pronounced activity was observed for acorn melanoidins. This is a unique finding, as the specific cytotoxic effect has not been reported for cocoa, carob and acorn melanoidins, and opens up a great opportunity to develop a potential novel cytotoxic agent against deadly colon cancer in the future.

Procyanidins from evening primrose (Oenothera paradoxa) defatted seeds inhibit invasiveness of breast cancer cells and modulate the expression of selected genes involved in angiogenesis, metastasis, and apoptosis.

There is a growing interest in plant polyphenols (including flavanols) that exhibit pleiotropic biological activities such as antiinflammatory, antioxidant, and anticancer effects. Here, we report for the first time the inhibition of MDA-MB-231 breast cancer cell viability and invasiveness by an evening primrose flavanol preparation (EPFP). We observed a decrease in MDA-MB-231 viability of 50% vs. a control after 72 h of incubation with EPFP at a concentration of 58 µM gallic acid equivalents (GAE) and an inhibition of their invasiveness of 65% vs. a control at 75 µM GAE after 48 h of incubation. EPFP caused a 10-fold reduction in matrix metalloproteinase-9 (MMP-9) activity at 100 µM GAE. Furthermore, through modulation of mRNA expression, EPFP reduced the expression levels of the following proteins: antiapoptotic Bcl-2, angiogenic vascular endothelial growth factor (VEGF), and 2 transcription factors (c-Jun, c-Fos). Moreover, analysis by flow cytometry revealed that EPFP induced apoptosis in MDA-MB-231 cells. In conclusion, our data shows that EPFP inhibits cell viability by increasing apoptosis and decreases cell invasiveness by decreasing angiogenesis.

Flavanols from Japanese quince (Chaenomeles japonica) fruit inhibit human prostate and breast cancer cell line invasiveness and cause favorable changes in Bax/Bcl-2 mRNA ratio.

Polyphenols are natural compounds of high structural diversity which translates into a very wide spectrum of biological activities, including chemoprevention. Here we report that a Japanese quince fruit flavanol preparation (JQFFP) caused favorable changes in Bax/Bcl-2 mRNA ratio, which rendered normal and cancer cells more resistant and more sensitive, respectively, to apoptosis. DU145 human prostate cancer cells were characterized by the most advantageous Bax/Bcl-2 ratio. The growth and invasiveness of MDA-MB-231 human breast cancer cells were strongly suppressed by JQFFP, which was accompanied with a decrease in MMP-9 activity and stimulation of TIMP-1 expression. Importantly, JQFFP did not decrease normal human prostate PNT1A cell number, whereas Bax/Bcl-2 ratio decreased which implies increased resistance to apoptosis. In conclusion, JQFFP exhibited a potent antiproliferative effect against cancer cells, inhibited their invasiveness, and decreased expression level of several genes involved in apoptosis, angiogenesis, and metastasis.

Interaction between polyamidoamine (PAMAM) dendrimers and bovine insulin.

OBJECTIVE: In this study the mechanism of interactions between polyamidoamine (PAMAM) dendrimers and bovine insulin was examined. The insulin is a 51 amino acid peptide-hormone involved in the homeostasis of blood glucose levels. This molecule consists of two chains - A and B - linked by two disulphide bridges. As insulin contains four tyrosine residues it was possible to evaluate dendrimers effect on protein conformation by measuring changes in the fluorescence spectra of insulin after addition of dendrimers or classical quenchers. METHODS: PAMAM dendrimers are based on ethylenediamine core and branched units which are built from methyl acrylate and ethylenediamine. PAMAM dendrimers with different surface groups (-COOH, -NH2, -OH) were used. Their size is comparable to insulin. RESULTS: The experiments show that interactions exist between PAMAM dendrimers and insulin. It was found that these interactions depend on a kind of dendrimer surface groups. CONCLUSIONS: It is very likely that interactions are of electrostatic nature and cause insulin conformational changes.

Lactoferrin improves symptoms of dextran sulfate sodium-induced colitis in mice through modulation of cellular senescence.

The multifaceted effects of lactoferrin (LF) on the digestive and immune systems make it an attractive therapeutic option in inflammatory bowel diseases. In this study, we aimed to explore the anti-inflammatory effects of LF in colitis, particularly in relation to cellular senescence. We hypothesize that LF has the potential to modulate the senescence process. The effects of LF on senescence were tested in vitro using HCT116 and SW480 cell lines, and in vivo, the dextran sulfate sodium-induced mouse model of colitis. LF (500 mg/kg) alleviated symptoms of colitis in mice with a significant decrease in colon damage (P < .0001 vs. control) and microscopic (P < .05 vs. control) scores. Cellular senescence markers p16 and p21 were significantly upregulated in the mouse colon during inflammation (both P < .01 vs. control), and LF at 500 mg/kg decreased these markers (both P < .05 vs. dextran sulfate sodium-treated mice). In vitro, LF significantly affected the expression of p16 and p21 (P < .05-P < .0001 vs. control), senescence associated secretory phenotype (P < .01-P < .0001 vs. control), and telomere-specific proteins: telomeric repeat binding factor 1 and 2 (P < .05-P < .0001 vs. control) in a concentration-dependent manner. LF modulates the expression of cellular senescence markers and shows hallmarks of senolytic and pro-senescent activity, depending on dose. Further studies are needed to fully understand the anti-inflammatory effect of LF in the context of senescence and safe utilization in patients with inflammatory bowel diseases.

SARS-CoV-2 infects an in vitro model of the human developing pancreas through endocytosis.

Recent studies showed that SARS-CoV-2 can infect adult human pancreas and trigger pancreatic damage. Here, using human fetal pancreas samples and 3D differentiation of human pluripotent cells into pancreatic endocrine cells, we determined that SARS-CoV-2 receptors ACE2, TMPRSS2, and NRP1 are expressed in precursors of insulin-producing pancreatic ß-cells, rendering them permissive to SARS-CoV-2 infection. We also show that SARS-CoV-2 enters and undergoes efficient replication in human multipotent pancreatic and endocrine progenitors in vitro. Moreover, we investigated mechanisms by which SARS-CoV-2 enters pancreatic cells, and found that ACE2 mediates the entry, while NRP1 and TMPRSS2 do not. Surprisingly, we found that in pancreatic progenitors, SARS-CoV-2 enters cells via cathepsin-dependent endocytosis, which is a different route than in respiratory tract. Therefore, pancreatic spheroids might serve as a model to study candidate drugs for endocytosis-mediated viral entry inhibition and to investigate whether SARS-CoV-2 infection may affect pancreas development, possibly causing lifelong health consequences.

Acriflavine, a clinically approved drug, inhibits SARS-CoV-2 and other betacoronaviruses.

The COVID-19 pandemic caused by SARS-CoV-2 has been socially and economically devastating. Despite an unprecedented research effort and available vaccines, effective therapeutics are still missing to limit severe disease and mortality. Using high-throughput screening, we identify acriflavine (ACF) as a potent papain-like protease (PLpro) inhibitor. NMR titrations and a co-crystal structure confirm that acriflavine blocks the PLpro catalytic pocket in an unexpected binding mode. We show that the drug inhibits viral replication at nanomolar concentration in cellular models, in vivo in mice and ex vivo in human airway epithelia, with broad range activity against SARS-CoV-2 and other betacoronaviruses. Considering that acriflavine is an inexpensive drug approved in some countries, it may be immediately tested in clinical trials and play an important role during the current pandemic and future outbreaks.