Search

Spherical gold nanoparticles (GNPs), whose unique properties regarding biomedical applications were broadly investigated, are an object of interest as nanocarriers in drug targeted delivery systems (DTDSs). The possibility of surface functionalization, especially in enabling longer half-life in the bloodstream and enhancing cellular uptake, provides an opportunity to overcome the limitations of popular anticancer drugs (such as cisplatin) that cause severe side effects due to their nonselective transportation. Herein, we present investigations of gold nanoparticle–cisplatin systems formation (regarding reaction kinetics and equilibrium) in which it was proved that the formation efficiency and stability strongly depend on the nanoparticle surface functionalization. In this study, the capillary electrophoresis hyphenated with inductively coupled plasma tandem mass spectrometry (CE-ICP-MS/MS) was used for the first time to monitor gold–drug nanoconjugates formation. The research included optimizing CE separation conditions and determining reaction kinetics using the CE-ICP-MS/MS developed method. To characterize nanocarriers and portray changes in their physicochemical properties induced by the surface’s processes, additional hydrodynamic size and ζ-potential by dynamic light scattering (DLS) measurements were carried out. The examinations of three types of functionalized GNPs (GNP-PEG-COOH, GNP-PEG-OCH3, and GNP-PEG-biotin) distinguished the essential differences in drug binding efficiency and nanoconjugate stability.

Even cyanobacteria from ecosystems of low biodiversity, such as the Baltic Sea, can constitute a rich source of bioactive metabolites. Potent toxins, enzyme inhibitors, and anticancer and antifungal agents were detected in both bloom-forming species and less commonly occurring cyanobacteria. In previous work on the Baltic Pseudanabaena galeata CCNP1313, the induction of apoptosis in the breast cancer cell line MCF-7 was documented. Here, the activity of the strain was further explored using human dermal fibroblasts, African green monkey kidney, cancer cell lines (T47D, HCT-8, and A549ACE2/TMPRSS2) and viruses (SARS-CoV-2, HCoV-OC43, and WNV). In the tests, extracts, chromatographic fractions, and the main components of the P. galeata CCNP1313 fractions were used. The LC-MS/MS analyses of the tested samples led to the detection of forty-five peptides. For fourteen of the new peptides, putative structures were proposed based on MS/MS spectra. Although the complex samples (i.e., extracts and chromatographic fractions) showed potent cytotoxic and antiviral activities, the effects of the isolated compounds were minor. The study confirmed the significance of P. galeata CCNP1313 as a source of metabolites with potent activity. It also illustrated the difficulties in assigning the observed biological effects to specific metabolites, especially when they are produced in minute amounts.

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 β-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

There are currently no cures for coronavirus infections, making the prevention of infections the only course open at the present time. The COVID-19 pandemic has been difficult to prevent, as the infection is spread by respiratory droplets and thus effective, scalable and safe preventive interventions are urgently needed. We hypothesise that preventing viral entry into mammalian nasal epithelial cells may be one way to limit the spread of COVID-19. Here we show that N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (GCPQ), a positively charged polymer that has been through an extensive Good Laboratory Practice toxicology screen, is able to reduce the infectivity of SARS-COV-2 in A549ACE2+ and Vero E6 cells with a log removal value of − 3 to − 4 at a concentration of 10–100 μg/ mL (p p 50/mL) showed a trend for nasal GCPQ (20 mg/kg) to inhibit viral load in the respiratory tract and brain, although the study was not powered to detect statistical significance. GCPQ’s electrostatic binding to the virus, preventing viral entry into the host cells, is the most likely mechanism of viral inhibition. Radiolabelled GCPQ studies in mice show that at a dose of 10 mg/kg, GCPQ has a long residence time in mouse nares, with 13.1% of the injected dose identified from SPECT/CT in the nares, 24 h after nasal dosing. With a no observed adverse effect level of 18 mg/kg in rats, following a 28-day repeat dose study, clinical testing of this polymer, as a COVID-19 prophylactic is warranted.

There are currently no cures for coronavirus infections, making the prevention of infections the only course open at the present time. The COVID-19 pandemic has been difficult to prevent, as the infection is spread by respiratory droplets and thus effective, scalable and safe preventive interventions are urgently needed. We hypothesise that preventing viral entry into mammalian nasal epithelial cells may be one way to limit the spread of COVID-19. Here we show that N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (GCPQ), a positively charged polymer that has been through an extensive Good Laboratory Practice toxicology screen, is able to reduce the infectivity of SARS-COV-2 in A549ACE2+ and Vero E6 cells with a log removal value of −3 to −4 at a concentration of 10 – 100 μg/ mL (p < 0.05 compared to untreated controls) and to limit infectivity in human airway epithelial cells at a concentration of 500 μg/ mL (p < 0.05 compared to untreated controls). GCPQ is currently being developed as a pharmaceutical excipient in nasal and ocular formulations. GCPQ’s electrostatic binding to the virus, preventing viral entry into the host cells, is the most likely mechanism of viral inhibition. Radiolabelled GCPQ studies in mice show that at a dose of 10 mg/ kg, GCPQ has a long residence time in mouse nares, with 13.1% of the injected dose identified from SPECT/CT in the nares, 24 hours after nasal dosing. With a no observed adverse effect level of 18 mg/ kg in rats, following a 28-day repeat dose study, clinical testing of this polymer, as a COVID-19 prophylactic is warranted.

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., Author summary Coronaviral genomes code for several proteins, with the large 1a/1ab being expressed directly from genomic (g)RNA. For the expression of other viral proteins, a set of subgenomic mRNAs is produced during replication. It includes mRNAs for structural (S-E-M-N) and accessory proteins. While the function of structural proteins is well described, the function of the latter ones is under debate. Some of them are required for replication, while others are dispensable in vitro but essential in vivo. Initially, 10 open reading frames (ORFs) were annotated in the SARS-CoV-2 genome, amongst which ORF10 is the most peculiar, as it does not share sequence homology with any known protein. Shortly after the genomic sequences became available, speculations on this protein's role in pathogenesis and innate immunity breaching started. Here, we identified two patients infected with SARS-CoV-2 variants with the ORF10 gene prematurely terminated. The disease was not attenuated, and the transmissibility was maintained. The in vitro study showed that the ORF10 is also not essential for replication. Consequently, ORF10 should not be treated as the protein-coding gene, and the genome annotations should be amended.

The protease kallikrein 13 cleaves the spike protein of the human coronavirus HKU1 to facilitate viral entry., Priming viral entry Unlike SARS-CoV-2, the human coronavirus HKU1 normally causes relatively mild respiratory tract infections; however, it shares with SARS-CoV-2 the mechanism of using its surface spike (S) protein to enter target cells. Because the host receptor for HCoV-HKU1 is unknown, efforts to study the virus in cell culture systems have proved difficult. Milewska et al. found that knockout of the protease kallikrein 13 (KLK13) in human airway epithelial cells blocked their infection by HCoV-HKU1, that overexpression of KLK13 in nonpermissive cells enabled their infection by the virus, and that KLK13 cleaved the viral S protein. Together, these findings suggest that KLK13 is a priming enzyme for viral entry and may help to establish cell lines that can facilitate further investigation of the mechanism of viral pathogenesis., Human coronavirus HKU1 (HCoV-HKU1) is associated with respiratory disease and is prevalent worldwide, but an in vitro model for viral replication is lacking. An interaction between the coronaviral spike (S) protein and its receptor is the primary determinant of tissue and host specificity; however, viral entry is a complex process requiring the concerted action of multiple cellular elements. Here, we found that the protease kallikrein 13 (KLK13) was required for the infection of human respiratory epithelial cells and was sufficient to mediate the entry of HCoV-HKU1 into nonpermissive RD cells. We also demonstrated the cleavage of the HCoV-HKU1 S protein by KLK13 in the S1/S2 region, suggesting that KLK13 is the priming enzyme for this virus. Together, these data suggest that protease distribution and specificity determine the tissue and cell specificity of the virus and may also regulate interspecies transmission.

Zika virus is a flavivirus discovered in 1947, but the association between Zika virus infection and brain disorders was not demonstrated until 2015 in Brazil. Infection mostly poses a threat to women during pregnancy, since it may cause microcephaly and other neurological dysfunctions in the developing fetus. However, infection is also associated with Guillain-Barré syndrome. The nonstructural NS3 protein is essential for virus replication because it helps to remodel the cellular microenvironment. Several reports show that this protease can process cellular substrates and thereby modify cellular pathways that are important for the virus. Herein, we explored some of the targets of NS3, but we could not confirm the biological relevance of its protease activity. Thus, although mass spectrometry is highly sensitive and useful in many instances, being also able to show directions, where cell/virus interaction occurs, we believe that biological validation of the observed results is essential.

The beginning of 2020 brought us information about the novel coronavirus emerging in China. Rapid research resulted in the characterization of the pathogen, which appeared to be a member of the SARS-like cluster, commonly seen in bats., Among seven coronaviruses that infect humans, three (severe acute respiratory syndrome coronavirus [SARS-CoV], Middle East respiratory syndrome coronavirus [MERS-CoV], and the newly identified severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) are associated with a severe, life-threatening respiratory infection and multiorgan failure. We previously proposed that the cationically modified chitosan N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC) is a potent inhibitor of human coronavirus NL63 (HCoV-NL63). Next, we demonstrated the broad-spectrum antiviral activity of the compound, as it inhibited all low-pathogenicity human coronaviruses (HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1). Here, using in vitro and ex vivo models of human airway epithelia, we show that HTCC effectively blocks MERS-CoV and SARS-CoV-2 infection. We also confirmed the mechanism of action for these two viruses, showing that the polymer blocks the virus entry into the host cell by interaction with the S protein. IMPORTANCE The beginning of 2020 brought us information about the novel coronavirus emerging in China. Rapid research resulted in the characterization of the pathogen, which appeared to be a member of the SARS-like cluster, commonly seen in bats. Despite the global and local efforts, the virus escaped the health care measures and rapidly spread in China and later globally, officially causing a pandemic and global crisis in March 2020. At present, different scenarios are being written to contain the virus, but the development of novel anticoronavirals for all highly pathogenic coronaviruses remains the major challenge. Here, we describe the antiviral activity of an HTCC compound, previously developed by us, which may be used as a potential inhibitor of currently circulating highly pathogenic coronaviruses—SARS-CoV-2 and MERS-CoV.

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., 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.

Ammonia borane (AB) based nanocomposites have been investigated with the aim of developing a promising solid-state hydrogen store that complies with the requirements of a modular polymer electrolyte membrane fuel cell (PEM FC) in a portable power pack system. AB-carbon nanocomposites (prepared via ball milling or solution-impregnation) demonstrate improved hydrogen release performance compared to AB itself in terms of onset temperature and hydrogen purity, while maintaining a gravimetric density of more than 5 wt. % H2. The most promising of these materials is an AB-AC (activated carbon) composite, synthesised via solution-impregnation with an optimal dehydrogenation temperature of 96 °C. When combined with an external nickel chloride filter downstream, no evolved gaseous by-products can be detected above 100 ppb. The feasibility of an AB-AC storage tank has been further endorsed by simulations in which the reaction rate and the hydrogen flux was found to be almost constant as the temperature front propagated from the bottom to the top of the tank after initiation.

The beginning of 2020 brought us information about the novel coronavirus emerging in China. Rapid research resulted in the characterization of the pathogen, which appeared to be a member of the SARS-like cluster, commonly seen in bats. Despite the global and local efforts, the virus escaped the healthcare measures and rapidly spread in China and later globally, officially causing a pandemic and global crisis in March 2020. At present, different scenarios are being written to contain the virus, but the development of novel anticoronavirals for all highly pathogenic coronaviruses remains the major challenge. Here, we describe the antiviral activity of previously developed by us HTCC compound (N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride), which may be used as potential inhibitor of currently circulating highly pathogenic coronaviruses – SARS-CoV-2 and MERS-CoV.

SARS-CoV-2 emerged by the end of 2019 to rapidly spread in 2020. At present, it is of utmost importance to understand the virus biology and to rapidly assess the 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 the Vero E6 cells. Here, we propose fully differentiated human airway epithelium cultures as a model for studies on the SARS-CoV-2. Further, we also provide basic characteristics of the system.

Human coronavirus HKU1 (HCoV-HKU1) is associated with respiratory disease and is prevalent worldwide, but in vitro model for virus replication is lacking. Interaction between the coronaviral spike (S) protein and its receptor is the major determinant of virus tissue and host specificity, but virus entry is a complex process requiring a concerted action of multiple cellular elements. Here, we show that KLK13 is required for the infection of the human respiratory epithelium and is sufficient to mediate the entry of HCoV-HKU1 to non-permissive RD cells. We also demonstrated HCoV-HKU1 S protein cleavage by KLK13 in the S1/S2 region, proving that KLK13 is the priming enzyme for this virus. Summarizing, we show for the first time that protease distribution and specificity predetermines the tissue and cell specificity of the virus and may also regulate interspecies transmission. It is also of importance that presented data may be relevant for the emerging coronaviruses, including SARS-CoV-2 and may help to understand the differences in their zoonotic potential.

Coronavirus entry encompasses the initial steps of infection, from virion attachment to genome release. Advances in fluorescent labeling of viral and cellular components and confocal imaging enable broad spectrum studies on this process. Here, we describe methods for visualization of coronavirus entry into immortalized cell lines and 3D tissue culture models.

Virology, as a branch of the life sciences, discovered mass spectrometry (MS) to be the pivotal tool around two decades ago. The technique unveiled the complex network of interactions between the living world of pro- and eukaryotes and viruses, which delivered "a piece of bad news wrapped in protein" as defined by Peter Medawar, Nobel Prize Laureate, in 1960. However, MS is constantly evolving, and novel approaches allow for a better understanding of interactions in this micro- and nanoworld. Currently, we can investigate the interplay between the virus and the cell by analyzing proteomes, interactomes, virus-cell interactions, and search for the compounds that build viral structures. In addition, by using MS, it is possible to look at the cell from the broader perspective and determine the role of viral infection on the scale of the organism, for example, monitoring the crosstalk between infected tissues and the immune system. In such a way, MS became one of the major tools for the modern virology, allowing us to see the infection in the context of the whole cell or the organism. © 2019 John Wiley & Sons Ltd. Mass Spec Rev.

Human coronavirus NL63 (HCoV-NL63) is a common respiratory virus that causes moderately severe infections. We have previously shown that the virus uses heparan sulfate proteoglycans (HSPGs) as the initial attachment factors, facilitating viral entry into the cell. In the present study, we show that the membrane protein (M) of HCoV-NL63 mediates this attachment. Using viruslike particles lacking the spike (S) protein, we demonstrate that binding to the cell is not S protein dependent. Furthermore, we mapped the M protein site responsible for the interaction with HSPG and confirmed its relevance using a viable virus. Importantly, in silico analysis of the region responsible for HSPG binding in different clinical isolates and the Amsterdam I strain did not exhibit any signs of cell culture adaptation. IMPORTANCE It is generally accepted that the coronaviral S protein is responsible for viral interaction with a cellular receptor. Here we show that the M protein is also an important player during early stages of HCoV-NL63 infection and that the concerted action of the two proteins (M and S) is a prerequisite for effective infection. We believe that this study broadens the understanding of HCoV-NL63 biology and may also alter the way in which we perceive the first steps of cell infection with the virus. The data presented here may also be important for future research into vaccine or drug development.

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. Electronic supplementary material The online version of this article (10.1186/s13567-018-0551-9) contains supplementary material, which is available to authorized users.

APOBEC3 family members are cytidine deaminases with roles in intrinsic responses to infection by retroviruses and retrotransposons, and in the control of other DNA viruses, such as herpesviruses, parvoviruses and hepatitis B virus. Although effects of APOBEC3 members on viral DNA have been demonstrated, it is not known whether they edit RNA genomes through cytidine deamination. Here, we investigated APOBEC3-mediated restriction of Coronaviridae. In experiments in vitro, three human APOBEC3 proteins (A3C, A3F and A3H) inhibited HCoV-NL63 infection and limited production of progeny virus, but did not cause hypermutation of the coronaviral genome. APOBEC3-mediated restriction was partially dependent on enzyme activity, and was reduced by the use of enzymatically inactive APOBEC3. Moreover, APOBEC3 proteins bound to the coronaviral nucleoprotein, and this interaction also affected viral replication. Although the precise molecular mechanism of deaminase-dependent inhibition of coronavirus replication remains elusive, our results further our understanding of APOBEC-mediated restriction of RNA virus infections.

Aspiration pneumonia is a life-threatening infectious disease often caused by oral anaerobic and periodontal pathogens such as Porphyromonas gingivalis. This organism produces proteolytic enzymes, known as gingipains, which manipulate innate immune responses and promote chronic inflammation. Here, we challenged mice with P. gingivalis W83 and examined the role of gingipains in bronchopneumonia, lung abscess formation, and inflammatory responses. Although gingipains were not required for P. gingivalis colonization and survival in the lungs, they were essential for manifestation of clinical symptoms and infection-related mortality. Pathologies caused by wild-type (WT) P. gingivalis W83, including hemorrhage, necrosis, and neutrophil infiltration, were absent from lungs infected with gingipain-null isogenic strains or WT bacteria preincubated with gingipain-specific inhibitors. Damage to lung tissue correlated with systemic inflammatory responses, as manifested by elevated levels of TNF, IL-6, IL-17, and C-reactive protein. These effects were unequivocally dependent on gingipain activity. Gingipain activity was also implicated in the observed increase in IL-17 in lung tissues. Furthermore, gingipains increased platelet counts in the blood and activated platelets in the lungs. Arginine-specific gingipains made a greater contribution to P. gingivalis-related morbidity and mortality than lysine-specific gingipains. Thus, inhibition of gingipain may be a useful adjunct treatment for P. gingivalis-mediated aspiration pneumonia.

Human coronavirus (hCoV) HKU1 is one of six hCoVs identified to date and the only one with an unidentified cellular receptor. hCoV-HKU1 encodes a hemagglutinin-esterase (HE) protein that is unique to the group a betacoronaviruses (group 2a). The function of HKU1-HE remains largely undetermined. In this study, we examined binding of the S1 domain of hCoV-HKU1 spike to a panel of cells and found that the S1 could specifically bind on the cell surface of a human rhabdomyosarcoma cell line, RD. Pretreatment of RD cells with neuraminidase (NA) and trypsin greatly reduced the binding, suggesting that the binding was mediated by sialic acids on glycoproteins. However, unlike other group 2a CoVs, e.g., hCoV-OC43, for which 9- O -acetylated sialic acid (9- O -Ac-Sia) serves as a receptor determinant, HKU1-S1 bound with neither 9- O -Ac-Sia-containing glycoprotein(s) nor rat and mouse erythrocytes. Nonetheless, the HKU1-HE was similar to OC43-HE, also possessed sialate- O -acetylesterase activity, and acted as a receptor-destroying enzyme (RDE) capable of eliminating the binding of HKU1-S1 to RD cells, whereas the O -acetylesterase-inactive HKU1-HE mutant lost this capacity. Using primary human ciliated airway epithelial (HAE) cell cultures, the only in vitro replication model for hCoV-HKU1 infection, we confirmed that pretreatment of HAE cells with HE but not the enzymatically inactive mutant blocked hCoV-HKU1 infection. These results demonstrate that hCoV-HKU1 exploits O -Ac-Sia as a cellular attachment receptor determinant to initiate the infection of host cells and that its HE protein possesses the corresponding sialate- O -acetylesterase RDE activity. IMPORTANCE Human coronaviruses (hCoV) are important human respiratory pathogens. Among the six hCoVs identified to date, only hCoV-HKU1 has no defined cellular receptor. It is also unclear whether hemagglutinin-esterase (HE) protein plays a role in viral entry. In this study, we found that, similarly to other members of the group 2a CoVs, sialic acid moieties on glycoproteins are critical receptor determinants for the hCoV-HKU1 infection. Interestingly, the virus seems to employ a type of sialic acid different from those employed by other group 2a CoVs. In addition, we determined that the HKU1-HE protein is an O -acetylesterase and acts as a receptor-destroying enzyme (RDE) for hCoV-HKU1. This is the first study to demonstrate that hCoV-HKU1 uses certain types of O -acetylated sialic acid residues on glycoproteins to initiate the infection of host cells and that the HKU1-HE protein possesses sialate- O -acetylesterase RDE activity.

West Nile virus (WNV) is a member of the flavivirus genus belonging to the Flaviviridae family. The viral serine protease NS2B/NS3 has been considered an attractive target for the development of anti-WNV agents. Although several NS2B/NS3 protease inhibitors have been described so far, most of them are reversible inhibitors. Herein, we present a series of α-aminoalkylphosphonate diphenyl esters and their peptidyl derivatives as potent inhibitors of the NS2B/NS3 protease. The most potent inhibitor identified was Cbz-Lys-Arg-(4-GuPhe)P(OPh)2 displaying Ki and k2/Ki values of 0.4 µM and 28 265 M−1s−1, respectively, with no significant inhibition of trypsin, cathepsin G, and HAT protease.

The Coronaviridae family clusters a number of large RNA viruses, which share several structural and functional features. However, members of this family recognize different cellular receptors and exploit different entry routes, what affects their species specificity and virulence. The aim of this study was to determine how human coronavirus OC43 enters the susceptible cell. Using confocal microscopy and molecular biology tools we visualized early events during infection. We found that the virus employs caveolin-1 dependent endocytosis for the entry and the scission of virus-containing vesicles from the cell surface is dynamin-dependent. Furthermore, the vesicle internalization process requires actin cytoskeleton rearrangements. With our research we strove to broaden the understanding of the infection process, which in future may be beneficial for the development of a potential therapeutics.

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.

The paper elaborates the influence of the non-equimolar and equimolar counterdiffusion on combustion of single coal char particles in the oxy-fuel combustion conditions. The phenomenon of the non-equimolar counterdiffusion is usually neglected and superseded by the equimolar counterdiffusion. Such a replacement can lead to results, e.g. to char burnout, particle temperature and species concentrations, which do not agree with the real process. The paper presents the numerical results of single coal char particle combustion in the oxy-fuel combustion conditions at which the effect of the equimolar and non-equimolar counterdiffusion has been taken into consideration. It has been assumed that the char carbon heterogeneously reacts with O2, CO2 and H2O forming CO or CO2 and H2 depending on the combustion conditions. The reactions in the gas phase have been neglected to achieve the state of pure diffusion. The combustion of the particle is described by the mass and energy conservation equations commonly used in Euler–Lagrange computations of pulverized coal combustion. Numerical simulations performed for various values of the particle diameter and reagent concentrations clearly show that the use of the equimolar counterdiffusion model always overpredicts the non-equimolar one. Since the molar fluxes of the equimolar counterdiffusion are not coupled each other, the mass transfer towards the particle burning is higher which gives particle temperature and reaction rates too high compared to the non-equimolar counterdiffusion. Occurring discrepancy further develops during combustion also for other quantities describing the process, i.e. char burnout and gas concentrations at the particle surface. A simple correction which is proposed consists in reducing the value of the equimolar mass transfer coefficient that decreases the mass transfer and consequently letting the equimolar counterdiffusion model to be effortlessly used and get results which well follow the non-equimolar model. Such a simplified treatment of complex non-equimolar counterdiffusion can be easily implemented into numerical codes and needs no numerical solution of coupled non-linear equations describing the non-equimolar counterdiffusion.

Oxy-fuel combustion is considered as a promising solution to reduce greenhouse-gases and pollutant emissions. The main advantage of oxy-fuel combustion over other technologies for pollution reduction from pulverized coal combustion is that it can be applied to the existing coal-fired power plants. However, switching from conventional to oxy-fired coal combustion brings significant challenges. One of the most important is change of pulverized coal ignition characteristics. This paper presents the results of experimental and numerical analysis of ignition phenomena under oxy-fuel conditions. The main focus of the presented paper is to evaluate the effectiveness of the mathematical devolatilisation sub-model, in predicting the ignition point of pulverized coal flames under oxy-firing conditions. Regarding this, the performance of several devolatilisation models, from simple to more complex ones, in predicting ignition point position have been investigated. Numerically determined values of the ignition point position, and ignition temperature for various O-2-N-2 and O-2-CO2 conditions were compared with experimental data from the laboratory ignition test facility. Obtained results pointed out that network devolatilisation models (CPD and FG) give more accurate results in comparison with standard devolatilisation models (single rate and two competing rates). The best performance is achieved using FG devolatilisation model. Thus, newly implemented FG model will be used for future numerical simulations of oxy-fuel pulverized coal combustion on 0.5 MW pilot plant facility. (C) 2011 Elsevier Ltd. All rights reserved. 8th European conference on coal research and its applications, Sep 06-10, 2010, Leeds, England

Human coronavirus NL63 was identified in 2004 in the Netherlands. Due to the high prevalence and world-wide distribution of this pathogen, it is essential to develop a sensitive and specific detection assay suitable for use in a routine diagnostic laboratory. Techniques based on PCR or real-time PCR are laborious and expensive. Detailed analysis of the HCoV-NL63 genome permitted the identification of a conserved nucleic acid sequential motif, which was sufficient for the design of a loop-mediated isothermal amplification (LAMP) assay. Evaluation of the method showed that the test is specific to HCoV-NL63 and that it does not cross-react with other respiratory viruses. The detection limit was found to be 1 copy of RNA template per reaction in cell culture supernatants and clinical specimens.

It is expected that pulverized coal combustion will continue to play a major role in electricity generation for the foreseeable future. Oxy-fuel coal combustion is actively being investigated, as alternative to conventional pulverized-coal combustion, due to its potential to easier carbon dioxide sequestration. This paper presents experimental and numerical analysis of ignition phenomena in oxy-fuel conditions. A modification of standard sequential coal combustion model is proposed. The new model is developed following the criteria for the particle ignition mechanism as the function of surrounding conditions. The implemented model was validated based on ignition point position obtained from the drop tube facility experiments in various O2–N2 and O2–CO2 conditions. The obtained numerical results showed a much better agreement with the experimental results when compared with the simulations performed with the default FLUENT sub-models for coal particle ignition/combustion, thus enabling a quantitative determination of pulverized coal flame ignition point position using numerical analysis.

Relatively recently discovered, human metapneumovirus (HMPV) is a human pathogen with worldwide prevalence, accounting for a substantial percentage of respiratory tract diseases. Concurrent viral and bacterial infections enable intricate mechanisms of cooperation between pathogens, which complicate the symptoms and outcome of the disease. Such bilateral interactions are based on the modulation of bacterial growth on epithelium pathologically altered during viral illness and the modulation of immune responses, as well as the enhancement of virus replication by bacterial virulence factors. This study showed that proteases produced byPorphyromonas gingivalis, a Gram-negative bacterium implicated in the development of periodontitis, named gingipains, facilitated HMPV replication in LLC-MK2 cells and may contribute to HMPV pathogenicity in patients with periodontitis. Gingipains at low nanomolar concentrations enabled HMPV replication and allowed virus propagationin vitro. In contrast to previously published data for influenza virus, however,Staphylococcus aureusproteases and human neutrophil elastase did not affect virus replication.

Safety aspects in modelling of batch and semibatch stirred tank reactors as well as a model based safety analysis have been considered. Applicability of two basic types of models – i.e. the perfectly mixed reactor model and the CFD model, both formulated for laboratory scale as well as pilot plant scale reactors – has been discussed. A formulation of the appropriate reactor model, which is adequate to the considered case study has been demonstrated and tested experimentally. Particular attention has been devoted to the formulation of robust CFD models employed to simulate a performance of the stirred tank reactors. It has been found that models for perfectly mixed reactors may have quite wide range of application, while the CFD models should be definitely used in case of fast reactions, high viscosity of the reacting mixture as well as of failure leading to stopping of the impeller. The CFD models are able to predict a dynamic behaviour of reactors at any circumstances, so they can play a significant role in safety analysis carried out for industrial scale reactors, for which experimental safety tests are expensive and dangerous.

Novel sulfonated derivatives of poly(allylamine hydrochloride) (NSPAHs) and N-sulfonated chitosan (NSCH) have been synthesized, and their activity against influenza A and B viruses has been studied and compared with that of a series of carrageenans, marine polysaccharides of well-documented anti-influenza activity. NSPAHs were found to be nontoxic and very soluble in water, in contrast to gel-forming and thus generally poorly soluble carrageenans. In vitro and ex vivo studies using susceptible cells (Madin-Darby canine kidney epithelial cells and fully differentiated human airway epithelial cultures) demonstrated the antiviral effectiveness of NSPAHs. The activity of NSPAHs was proportional to the molecular mass of the chain and the degree of substitution of amino groups with sulfonate groups. Mechanistic studies showed that the NSPAHs and carrageenans inhibit influenza A and B virus assembly in the cell.

In this work, we have analyzed the application of CFD (computational fluid dynamics) to simulate consequences of stirrer failures, which can lead to reactor thermal runaway. For a typical industrial batch reactor, several “CFD computational experiments” have been carried out to explore runaway and non-runaway situations. Then the results of the CFD simulations, supported by an on-line divergence criterion, have been used to detect a dangerous reactor behavior. It has been found out, that even for the reactor operated at potentially safe conditions a failure of the stirring system can lead to serious thermal runaways. A significance of a proper location of temperature probe for an early runaway detection has also been discussed. A modification of the applied divergence criterion to avoid false alarms has been proposed and tested.

This study deals with safety aspects of batch reactors for styrene suspension polymerization. Polymerization is known to be the most frequent cause of thermal runaway incidents in the industrial chemical processes. Two sorts of safety problems have been studied in this work. The first one is related to an influence of process parameters on the thermal behavior of the well-mixed polymerization reactor. With regard to this part of the work the modeling results have been presented in a form of the maximum reaction temperature maps, which allow for choosing the safe process conditions. The second one is related to a stirrer malfunction in the suspension polymerization reactor. As the computational fluid dynamics (CFD) simulations have shown, thermal runaway starts when the mixing problems occur even if an emergency cooling is applied.

The presented work is a part of studies carried out to develop a practical method to prevent runaway events in chemical reactors. The use of computational fluid dynamics (CFD) techniques is proposed here to indicate in advance the local hot-spots in the reaction mixture, so as to elaborate an efficient and sensitive method for early warning detection of runaway. The CFD method has been chosen to support, elaborated by Bosch et al. (Comput. Chem. Eng. 28 (2004) 527), the on-line runaway detection method in batch reactors, which is based on the divergence criterion. Series of CFD simulations have been executed for two exothermic reactions, being the esterification reaction of the 2-butanol and the propionic anhydrite catalysed by the sulphuric acid (homogeneous system) and the hydrolysis of the propionic anhydrite catalysed by the sulphuric acid (heterogeneous system). For both the considered reactions the results of calculations have been verified with the results of experimental measurements performed in a bench scale RC1 Mettler-Toledo reaction calorimeter. The elaborated conclusions can be further employed in the parametric sensitivity analysis (Comput. Chem. Eng. 28 (2004) 527; Am. Inst. Chem. Eng. J. 45 (1999) 2429) to indicate a number of temperature sensors and their location inside the reactor, which will help to apply efficiently the divergence criterion method.

Elevated total bilirubin (TB) and transaminases are frequently reported in patients with heart failure and are related to their worse prognosis. On the basis of hemodynamic data from previous studies, the investigators hypothesized that elevated bilirubin and transaminases are associated with different patterns of cardiac remodeling and dysfunction in patients with heart failure (i.e., elevated bilirubin with predominantly right-heart dysfunction and elevated transaminases with predominantly left-heart dysfunction). Therefore, the aim of this study was to evaluate prospectively echocardiographic correlates of elevated TB and transaminases on admission in patients with exacerbation of chronic heart failure.The following echocardiographic parameters were prospectively analyzed in 150 patients (mean age, 75 years; 59% men): right ventricular end-diastolic diameter, right atrial area, tricuspid regurgitation, right ventricular systolic pressure, tricuspid annular plane systolic excursion, tricuspid lateral annulus systolic velocity, estimated right atrial pressure, portal vein pulsatility index (PVPI), left ventricular end-diastolic diameter (LVEDD), left ventricular ejection fraction, and cardiac index.Elevated TB was found in 61 patients (41%) and elevated transaminases in 46 patients (31%). In univariate logistic regression analysis, right ventricular end-diastolic diameter, right atrial area, tricuspid regurgitation, estimated right atrial pressure, tricuspid annular plane systolic excursion, tricuspid lateral annulus systolic velocity, PVPI, left ventricular ejection fraction, and cardiac index were significant predictors of elevated TB (P .05 for all). LVEDD indexed to body surface area, right ventricular end-diastolic diameter, and systolic blood pressure on admission were significant predictors of elevated transaminases (P .05 for all). In a multivariate regression model, only PVPI remained a significant predictor of elevated TB and LVEDD indexed to body surface area of elevated transaminases. Sensitivity, specificity, and positive and negative predictive values of PVPI 0.5 in the prediction of elevated TB were 81%, 87%, 82%, and 87%, respectively.Several echocardiographic indices of right-heart dysfunction and low cardiac index are related to elevated TB, with an increased PVPI having the best predictive value. A weak statistically significant association was found between elevated transaminase levels and left ventricular end-diastolic diameter indexed to body surface area.

Human coronavirus NL63 (HCoV-NL63) is an alphacoronavirus that was first identified in 2004 in the nasopharyngeal aspirate from a 7-month-old patient with a respiratory tract infection. Previous studies showed that HCoV-NL63 and the genetically distant severe acute respiratory syndrome (SARS)-CoV employ the same receptor for host cell entry, angiotensin-converting enzyme 2 (ACE2), but it is largely unclear whether ACE2 interactions are sufficient to allow HCoV-NL63 binding to cells. The present study showed that directed expression of angiotensin-converting enzyme 2 (ACE2) on cells previously resistant to HCoV-NL63 renders them susceptible, showing that ACE2 protein acts as a functional receptor and that its expression is required for infection. However, comparative analysis showed that directed expression or selective scission of the ACE2 protein had no measurable effect on virus adhesion. In contrast, binding of HCoV-NL63 to heparan sulfates was required for viral attachment and infection of target cells, showing that these molecules serve as attachment receptors for HCoV-NL63. IMPORTANCE ACE2 protein was proposed as a receptor for HCoV-NL63 already in 2005, but an in-depth analysis of early events during virus infection had not been performed thus far. Here, we show that the ACE2 protein is required for viral entry but that it is not the primary binding site on the cell surface. Conducted research showed that heparan sulfate proteoglycans function as adhesion molecules, increasing the virus density on cell surface and possibly facilitating the interaction between HCoV-NL63 and its receptor. Obtained results show that the initial events during HCoV-NL63 infection are more complex than anticipated and that a newly described interaction may be essential for understanding the infection process and, possibly, also assist in drug design.

Porphyromonas gingivalis is a Gram-negative bacterium associated with the development of periodontitis. The evolutionary success of this pathogen results directly from the presence of numerous virulence factors, including peptidylarginine deiminase (PPAD), an enzyme that converts arginine to citrulline in proteins and peptides. Such posttranslational modification is thought to affect the function of many different signaling molecules. Taking into account the importance of tissue remodeling and repair mechanisms for periodontal homeostasis, which are orchestrated by ligands of the epidermal growth factor receptor (EGFR), we investigated the ability of PPAD to distort cross talk between the epithelium and the epidermal growth factor (EGF) signaling pathway. We found that EGF preincubation with purified recombinant PPAD, or a wild-type strain of P. gingivalis , but not with a PPAD-deficient isogenic mutant, efficiently hindered the ability of the growth factor to stimulate epidermal cell proliferation and migration. In addition, PPAD abrogated EGFR-EGF interaction-dependent stimulation of expression of suppressor of cytokine signaling 3 and interferon regulatory factor 1. Biochemical analysis clearly showed that the PPAD-exerted effects on EGF activities were solely due to deimination of the C-terminal arginine. Interestingly, citrullination of two internal Arg residues with human endogenous peptidylarginine deiminases did not alter EFG function, arguing that the C-terminal arginine is essential for EGF biological activity. Cumulatively, these data suggest that the PPAD-activity-abrogating EGF function in gingival pockets may at least partially contribute to tissue damage and delayed healing within P. gingivalis -infected periodontia.

Highlights ► Polymer HTCC and its hydrophobic derivative HM-HTCC can inhibit human coronavirus NL63 and murine hepatitis virus. ► HTCC binds the S protein and most likely hampers viral entry to susceptible cells., The human coronavirus NL63 is generally classified as a common cold pathogen, though the infection may also result in severe lower respiratory tract diseases, especially in children, patients with underlying disease, and elderly. It has been previously shown that HCoV-NL63 is also one of the most important causes of croup in children. In the current manuscript we developed a set of polymer-based compounds showing prominent anticoronaviral activity. Polymers have been recently considered as promising alternatives to small molecule inhibitors, due to their intrinsic antimicrobial properties and ability to serve as matrices for antimicrobial compounds. Most of the antimicrobial polymers show antibacterial properties, while those with antiviral activity are much less frequent. A cationically modified chitosan derivative, N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC), and hydrophobically-modified HTCC were shown to be potent inhibitors of HCoV-NL63 replication. Furthermore, both compounds showed prominent activity against murine hepatitis virus, suggesting broader anticoronaviral activity.