ABSTRACT
2,2'-Bis(4-dimethylaminophenyl)- and 2,2'-dicyclohexyl-1,1',3,3'-tetramethyl-2,2',3,3'-tetrahydro-2,2'-bibenzo[d]imidazole ((N-DMBI)2 and (Cyc-DMBI)2) are quite strong reductants with effective potentials of ca. -2 V vs ferrocenium/ferrocene, yet are relatively stable to air due to the coupling of redox and bond-breaking processes. Here, we examine their use in accomplishing electron transfer-induced bond-cleavage reactions, specifically dehalogenations. The dimers reduce halides that have reduction potentials less cathodic than ca. -2 V vs ferrocenium/ferrocene, especially under UV photoexcitation (using a 365 nm LED). In the case of benzyl halides, the products are bibenzyl derivatives, whereas aryl halides are reduced to the corresponding arenes. The potentials of the halides that can be reduced in this way, quantum-chemical calculations, and steady-state and transient absorption spectroscopy suggest that UV irradiation accelerates the reactions via cleavage of the dimers to the corresponding radical monomers.
ABSTRACT
A simple metal-free method for the synthesis of quinazolinones from commercially available 2-nitrobenzyl alcohols and tetrahydroisoquinolines is developed. The reaction conditions were tolerant of an array of functionalities such as halogen, tertiary amine, protected alcohol, and ester groups. Under nearly identical conditions, quinazolinethiones were obtained in the presence of elemental sulfur and suitable mediators.
Subject(s)
Tetrahydroisoquinolines , Metals , QuinazolinonesABSTRACT
Driven by a resurgence of interest in electrode-driven synthetic methods, this paper covers recent activity in the field of mediated electrochemical and photoelectrochemical bond activation, inclusive of C-H, C-C, C-N, and other C-heteroatom bonds.
Subject(s)
Carbon , Carbon/chemistry , CatalysisABSTRACT
Previously, rainbow trout fed deoxynivalenol (DON) or partially fed (pair-fed) for 4 weeks before and during experimental infection with Flavobacterium psychrophilum had significantly decreased mortality rates. Similar results were obtained in the present study after 12 days, but not after 6 days, feeding 5 ppm DON or pair-fed before infection. Furthermore, feeding 250 ppm chloroquine (CQ) also reduced mortality (p = .052) compared with controls and may have promise for treatment of some fish disease. Parallel groups of fish were maintained on the respective treatments for 15 days, with an additional group that was fasted, but were not infected to monitor autophagy. Fish that were fasted or fed DON had significantly increased LC3II in the liver and fasted fish had significantly decreased LC3II in muscle compared with controls using western blot. There was no difference in LC3II signal in the spleen of any treatment group. Fish that were fasted or pair-fed had significant up-regulation of the Atg genes atg4, atg7, lc3, gabarap and atg12 in muscle using quantitative PCR. Less alteration of Atg expression was seen in liver. Fish treated with CQ had significantly increased expression of atg4, becn1, lc3 and atg12 in the liver. Fish fed DON for 15 days had few alterations of Atg genes in either the liver or muscle. It is still not clear if autophagy is responsible for the resistance of rainbow trout fed DON, CQ or pair-fed before F. psychrophilum infection.
Subject(s)
Fish Diseases , Flavobacteriaceae Infections , Oncorhynchus mykiss , Animals , Autophagy/genetics , Fish Diseases/microbiology , Flavobacteriaceae Infections/microbiology , Flavobacterium/physiology , Oncorhynchus mykiss/microbiologyABSTRACT
Pyrido-fused quinazolinones were synthesized via copper-catalyzed cascade C(sp2)-H amination and annulation of 2-aminoarylmethanols with isoquinolines or pyridines. The transformation proceeded readily in the presence of a commercially available CuCl2 catalyst with molecular oxygen as a green oxidant. Moreover, the dehydrogenative cross-coupling of 2-aminoarylmethanols with tetrahydroisoquinolines was explored, in which CuBr exhibited higher catalytic activity than CuCl2. Broad substrate scope with good tolerance of functionalities was observed under the optimized reaction conditions. The bioactive naturally occurring alkaloid rutaecarpine could be obtained by this strategy. The remarkable feature of this protocol is that complicated heterocyclic structures are readily achieved in a single synthetic step from easily accessible reactants and catalysts. This pathway to pyrido-fused quinazolinones would be complementary to existing protocols.
ABSTRACT
Magnesium is involved in a variety of physiological processes in marine animals and is known to be deleterious in both excess and deficiency. The effects of magnesium concentration ranging from 700 mg/L (low), 1344 mg/L (control), and 2000 mg/L (high) on size and pulse rate in upside-down jellyfish (Cassiopea andromeda) medusae were examined in two separate 28-day trials. Exposure to low magnesium resulted in significantly (p < .05) higher pulse rates and decreased bell diameter and also produced oral arm degradation. Exposure to high magnesium resulted in significantly (p < .05) lower pulse rates and decreased bell diameter as well as oral arm cupping. In both low and high magnesium, almost all specimens changed color from pale blue on Day 1, to brown by Day 28, suggesting a loss of zooxanthellae. The decrease in bell diameter and color change was more pronounced and occurred more rapidly in low magnesium. The results of both trials demonstrate the deleterious effects of high and low magnesium on C. andromeda and emphasize the importance of monitoring magnesium concentration to maintain healthy display animals in public aquaria.
Subject(s)
Magnesium , Scyphozoa , Animals , Animals, Zoo , Heart RateABSTRACT
BACKGROUND: Aquatic bird bornavirus 1 (ABBV-1) has been associated with neurological diseases in wild waterfowls. In Canada, presence of ABBV-1 was demonstrated by RT-qPCR and immunohistochemistry in tissues of waterfowls with history of neurological disease and inflammation of the central and peripheral nervous tissue, although causation has not been proven by pathogenesis experiments, yet. To date, in vitro characterization of ABBV-1 is limited to isolation in primary duck embryo fibroblasts. The objectives of this study were to describe isolation of ABBV-1 in primary duck embryonic fibroblasts (DEF), and characterize replication in DEF and three immortalized avian fibroblast cell lines (duck CCL-141, quail QT-35, chicken DF-1) in order to evaluate cellular permissivity and identify suitable cell lines for routine virus propagation. METHODS: The virus was sequenced, and phylogenetic analysis performed on a segment of the N gene coding region. Virus spread in cell cultures, viral RNA and protein production, and titres were evaluated at different passages using immunofluorescence, RT-qPCR, western blotting, and tissue culture dose 50% (TCID50) assay, respectively. RESULTS: The isolated ABBV-1 showed 97 and 99% identity to European ABBV-1 isolate AF-168 and North American ABBV-1 isolates 062-CQ and CG-N1489, and could infect and replicate in DEF, CCL-141, QT-35 and DF-1 cultures. Viral RNA was detected in all four cultures with highest levels observed in DEF and CCL-141, moderate in QT-35, and lowest in DF-1. N protein was detected in western blots from infected DEF, CCL-141 and QT-35 at moderate to high levels, but minimally in infected DF-1. Infectious titre was highest in DEF (between approximately 105 to 106 FFU / 106 cells). Regarding immortalized cell lines, CCL-141 showed the highest titre between approximately 104 to 105 FFU / 106 cells. DF-1 produced minimal infectious titre. CONCLUSIONS: This study confirms the presence of ABBV-1 among waterfowl in Canada and reported additional in vitro characterization of this virus in different avian cell lines. ABBV-1 replicated to highest titre in DEF, followed by CCL-141 and QT-35, and poorly in DF-1. Our results showed that CCL-141 can be used instead of DEF for routine ABBV-1 production, if a lower titre is an acceptable trade-off for the simplicity of using immortalized cell line over primary culture.
Subject(s)
Bornaviridae/isolation & purification , Bornaviridae/physiology , Fibroblasts/virology , Virus Replication , Animals , Bird Diseases/virology , Bornaviridae/classification , Canada , Cell Culture Techniques , Cell Line, Transformed , Chickens/virology , Ducks/virology , Phylogeny , Quail/virologyABSTRACT
Autophagy modulation influences the success of intracellular pathogens, and an understanding of the mechanisms involved might offer practical options to reduce the impact of infectious disease. Viral haemorrhagic septicaemia virus (VHSV) can cause high mortality and economic loss in some commercial fish species. VHSV IVb was used to infect a rainbow trout gill cell line, RTgill-W1, followed by the treatment of the cells with different autophagy-modulating reagents. LC3II protein using Western blot was significantly (p < .05) decreased for two days following VHSV infection, and immunofluorescence confirmed that LC3II-positive intracytoplasmic puncta were also decreased. Infection with VHSV resulted in significantly decreased expression of the autophagy-related (Atg) genes atg4, at12, atg13 and becn1 after one day using quantitative PCR. Both viral gene copy number and VHSV N protein were significantly decreased by treating the cells with autophagy-blocking (chloroquine) and autophagy-inhibiting reagents (deoxynivalenol and 3-methyladenine) after three days, while autophagy induction (restricted nutrition and rapamycin) had limited effect. Only treatment of RTgill-W1 with deoxynivalenol resulted in a significant increase in expression of type I interferon. Therefore, the suppression of autophagy initially occurs after VHSV IVb infection, but the modulation of autophagy can also inhibit VHSV IVb infection in RTgill-W1 after three days.
Subject(s)
Autophagy , Epithelial Cells/virology , Hemorrhagic Septicemia, Viral/pathology , Novirhabdovirus/pathogenicity , Oncorhynchus mykiss/virology , Animals , Cell Line , Epithelial Cells/drug effects , Gene Dosage , Gills/cytology , Novirhabdovirus/genetics , Nucleocapsid Proteins/geneticsABSTRACT
The present study helps clarify when the fish type I IFN groups/subgroups evolved, by examination of the IFN genes present in the Holostean spotted gar, Lepisosteus oculatus, in relation to the IFN genes present in the Chondrostea (sturgeon). It confirms that all three IFN groups (I-III), and group II subgroups, existed prior to the appearance of teleost fish. Preliminary expression analysis in a gar cell line (GARL) suggests these IFN genes will have a role in antiviral defence in Holostean fish, in that they are induced by poly(I:C). A refined model of IFN evolution within the actinopterygian fish is proposed.
Subject(s)
Evolution, Molecular , Fishes/genetics , Fishes/immunology , Interferons/genetics , Skates, Fish/genetics , Skates, Fish/immunology , Animals , Cell Line , Interferons/classification , Poly I-C/immunology , Poly I-C/pharmacologyABSTRACT
As global warming and environmental pollution modify aquatic environments, the thermal biology of fish could be affected by interactions between temperature and pollutants, such as selenium (Se). Therefore, selenomethionine (SeMet) was studied for effects on cell viability and on heat shock protein 70 (HSP70) levels in the rainbow trout intestinal epithelial cell, RTgutGC, at hypothermic (4⯰C), normothermic (14 and 18⯰C) and hyperthermic (26⯰C) temperatures. RTgutGC cultures remained viable for at least a week at all temperatures, although energy metabolism as measured with Alamar Blue (resazurin) was appreciably diminished at 4⯰C. Over a 7-day incubation, HSP 70 levels in cultures remained steady at 4⯰C, declined at 18⯰C, and increased slightly at 26⯰C. When 125⯵M SeMet was present, cultures remained viable and HSP70 levels were neither increased nor decreased relative to control cultures, regardless of the temperature. With 500 and 1000⯵M SeMet, cell viability was profoundly impaired after 7 days in cultures at 14, 18 and 26⯰C but was unchanged at 4⯰C. Overall the results suggest that only hypothermia modulated the response of rainbow trout cells to SeMet.
Subject(s)
Epithelial Cells/drug effects , Epithelial Cells/metabolism , HSP70 Heat-Shock Proteins/metabolism , Selenomethionine/toxicity , Animals , Cell Line , Cell Survival/drug effects , Energy Metabolism , Intestinal Mucosa/cytology , Oncorhynchus mykiss , TemperatureABSTRACT
In order to develop an in vitro system to study the cell biology of starvation in the fish intestine, rainbow trout intestinal epithelial cells were subjected to three kinds of nutrient deprivation and evaluated for 7 days. The RTgutGC cell line was grown into monolayers in Leibovitz's basal medium supplemented with fetal bovine serum (L15/FBS) and then subjected to deprivation of serum (L15); of serum, amino acids, and vitamin (L15/ex); and of all nutrients (L15/salts). After 7 days of nutrient deprivation, the cells remained attached to the plastic surface as monolayers but changes were seen in shape, with the cells becoming more polygonal, actin and α-tubulin cytoskeleton organization, and in tight junction protein-1 (ZO-1) localization. Two barrier functions, transepithelial electrical resistance (TEER) and Lucifer Yellow (LY) retention, were impaired by nutrient deprivation. In L15/FBS, cells rapidly healed a gap or wound in the monolayer. In L15 and L15/ex, some cells moved into the gap, but after 7 days, the wound remained unhealed, whereas in L15/salts, cells did not even migrate into the gap. Upon nutrient replenishment (L15/FBS) after 7 days in L15, L15/ex, or L15/salts, cells proliferated again and healed a wound. After 7 days of nutrient deprivation, monolayers were successfully passaged with trypsin and cells in L15/FBS grew to again form monolayers. Therefore, rainbow trout intestinal epithelial cells survived starvation, but barrier and wound healing functions were impaired.
Subject(s)
Epithelial Cells/physiology , Fish Diseases/physiopathology , Intestinal Mucosa/cytology , Malnutrition/veterinary , Oncorhynchus mykiss , Animals , Cell Line , Cells, Cultured , Malnutrition/physiopathologyABSTRACT
Heart diseases caused by viruses are major causes of Atlantic salmon aquaculture loss. Two Atlantic salmon cardiovascular cell lines, an endothelial cell line (ASHe) from the heart and a fibroblast cell line (BAASf) from the bulbus arteriosus, were evaluated for their response to four fish viruses, CSV, IPNV, VHSV IVa and VHSV IVb, and the innate immune agonist, double-stranded RNA mimic poly IC. All four viruses caused cytopathic effects in ASHe and BAASf. However, ASHe was more susceptible to all four viruses than BAASf. When comparing between the viruses, ASHe cells were found to be moderately susceptible to CSV and VHSV IVb, but highly susceptible to IPNV and VHSV IVa induced cell death. All four viruses were capable of propagating in the ASHe cell line, leading to increases in virus titre over time. In BAASf, CSV and IPNV produced more than one log increase in titre from initial infection, but VHSV IVb and IVa did not. When looking at the antiviral response of both cell lines, Mx proteins were induced in ASHe and BAASf by poly IC. All four viruses induced Mx proteins in BAASf, while only CSV and VHSV IVb induced Mx proteins in ASHe. IPNV and VHSV IVa suppressed Mx proteins expression in ASHe. Pretreatment of ASHe with poly IC to allow for Mx proteins accumulation protected the culture from subsequent infections with IPNV and VHSV IVa, resulting in delayed cell death, reduced virus titres and reduced viral proteins expression. These data suggest that endothelial cells potentially can serve as points of infections for viruses in the heart and that two of the four viruses, IPNV and VHSV IVa, have mechanisms to avoid or downregulate antiviral responses in ASHe cells. Furthermore, the high susceptibility of the ASHe cell line to IPNV and VHSV IVa can make it a useful tool for studying antiviral compounds against these viruses and for general detection of fish viruses.
Subject(s)
Fish Diseases/therapy , Heart Diseases/veterinary , RNA Virus Infections/veterinary , RNA Viruses/physiology , Salmo salar , Animals , Cell Line , Endothelial Cells , Female , Fibroblasts , Fish Diseases/virology , Fish Proteins/metabolism , Heart Diseases/therapy , Heart Diseases/virology , Myxovirus Resistance Proteins/metabolism , RNA Virus Infections/therapy , RNA Virus Infections/virology , RNA, Double-Stranded/pharmacologyABSTRACT
Spaghetti meat (SM) and woody breast (WB) are breast muscle myopathies of broiler chickens, characterized by separation of myofibers and by fibrosis, respectively. This study sought to investigate the transcriptomic profiles of breast muscles affected by SM and WB. Targeted sampling was conducted on a flock to obtain 10 WB, 10 SM, and 10 Normal Pectoralis major muscle samples from 37-day-old male chickens. Total RNA was extracted, cDNA was used for pair-end sequencing, and differentially expressed genes (DEGs) were determined by a false discovery rate of <0.1 and a >1.5-fold change. Principal component and heatmap cluster analyses showed that the SM and WB samples clustered together. No DEGs were observed between SM and WB fillets, while a total of 4018 and 2323 DEGs were found when comparing SM and WB, respectively, against Normal samples. In both the SM and WB samples, Gene Ontology terms associated with extracellular environment and immune response were enriched. The KEGG analysis showed enrichment of cytokine-cytokine receptor interaction and extracellular matrix-receptor interaction pathways in both myopathies. Although SM and WB are macroscopically different, the similar transcriptomic profiles suggest that these conditions may share a common pathogenesis. This is the first study to compare the transcriptomes of SM and WB, and it showed that, while both myopathies had profiles different from the normal breast muscle, SM and WB were similar, with comparable enriched metabolic pathways and processes despite presenting markedly different macroscopic features.
ABSTRACT
Aquatic bird bornavirus 1 (ABBV-1) has a high prevalence of infection in certain North American populations of Canada geese (Branta canadensis), suggesting a possible role of these birds as an ABBV-1 reservoir. The goal of this study was to evaluate the ability of Canada geese to become experimentally infected with ABBV-1, develop lesions, and transmit the virus to conspecifics. One-week-old Canada geese (n, 65) were inoculated with ABBV-1 through the intramuscular (IM) or cloacal (CL) routes, with the control group receiving carrier only. An additional 6 geese were added to each group to test horizontal transmission (sentinel birds). Geese were monitored daily, and selected birds were euthanized at 1, 8, and 15-weeks post infection (wpi) to assess virus replication in tissues and lesion development. At 15 wpi, over 70% of IM birds were infected, while the CL route yielded only 1 infected goose. Of the infected IM geese, 26% developed encephalitis and/or myelitis after 8 wpi. No clinical signs were observed, and no sentinel birds became infected in any group. Only 1 oropharyngeal swab (IM group) tested positive for ABBV-1 RNA, while the water from the enclosures was consistently negative for virus RNA. This study documents successful experimental infection of Canada geese with ABBV-1, with findings comparable to what is described in infection trials with other waterfowl species. However, minimal shedding and lack of environmental dispersal indicate that Canada geese have little potential to disseminate the virus among wild waterfowl, and that other species could be better suited to act as chronic ABBV-1 shedders in the wild.
Subject(s)
Bird Diseases , Bornaviridae , Animals , Geese , Bornaviridae/genetics , Ducks/genetics , RNA, Viral , Canada/epidemiologyABSTRACT
The rapid development and deployment of vaccines following the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been estimated to have saved millions of lives. Despite their immense success, there remains a need for next-generation vaccination approaches for SARS-CoV-2 and future emerging coronaviruses and other respiratory viruses. Here we utilized a Newcastle Disease virus (NDV) vectored vaccine expressing the ancestral SARS-CoV-2 spike protein in a pre-fusion stabilized chimeric conformation (NDV-PFS). When delivered intranasally, NDV-PFS protected both Syrian hamsters and K18 mice against Delta and Omicron SARS-CoV-2 variants of concern. Additionally, intranasal vaccination induced robust, durable protection that was extended to 6 months post-vaccination. Overall, our data provide evidence that NDV-vectored vaccines represent a viable next-generation mucosal vaccination approach.
ABSTRACT
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged following an outbreak of unexplained viral illness in China in late 2019. Since then, it has spread globally causing a pandemic that has resulted in millions of deaths and has had enormous economic and social consequences. The emergence of SARS-CoV-2 saw the rapid and widespread development of a number of vaccine candidates worldwide, and this never-before-seen pace of vaccine development led to several candidates progressing immediately through clinical trials. Many countries have now approved vaccines for emergency use, with large-scale vaccination programs ongoing. Despite these successes, there remains a need for ongoing pre-clinical and clinical development of vaccine candidates against SARS-CoV-2, as well as vaccines that can elicit strong mucosal immune responses. Here, we report on the efficacy of a Newcastle disease virus-vectored vaccine candidate expressing SARS-CoV-2 spike protein (NDV-FLS) administered to cynomolgus macaques. Macaques given two doses of the vaccine via respiratory immunization developed robust immune responses and had reduced viral RNA levels in nasal swabs and in the lower airway. Our data indicate that NDV-FLS administered mucosally provides significant protection against SARS-CoV-2 infection, resulting in reduced viral burden and disease manifestation, and should be considered as a viable candidate for clinical development.
ABSTRACT
Aquatic bird bornavirus 1 (ABBV-1) is a neurotropic virus that causes persistent infection in the nervous system of wild waterfowl. This study evaluated whether Pekin ducks, the most common waterfowl raised worldwide, are susceptible to ABBV-1 infection and associated disease. Groups of Pekin ducks were inoculated with ABBV-1 through the intracranial (IC; n, 32), intramuscular (IM; n, 30), and choanal (CH; n, 30) routes. Controls (CO; n, 29) received carrier only. At 1, 12, and 21 weeks postinfection (wpi), 7-14 birds were euthanized to assess virus distribution and lesions. Infection rates in the IC and IM groups were over 70%, while only 4 ducks in the CH group became infected. Neurological signs were observed in 8 ducks only, while over 25% of IC and IM birds had encephalitis and/or myelitis. Seroconversion was highest in the IC and IM groups, and mucosal ABBV-1 RNA shedding was most frequent in the IC group (53%). None of the fertile eggs laid during the experiment tested positive for ABBV-1 RNA. This study shows that Pekin ducks are permissive to ABBV-1 infection and partly susceptible to associated disease. While mucosal shedding may be an important route of transmission, congenital infection appears unlikely.
Subject(s)
Influenza in Birds , RNA Viruses , Animals , Ducks , RNA Viruses/genetics , RNAABSTRACT
Herein, we report a method to estimate the thermodynamic potentials of electrochemical reactions at different temperatures. We use a two-term Taylor series approximation of thermodynamic potential as a function of temperature, and we calculate the temperature sensitivity for a family of twenty seven known half reactions. We further analyze pairs of cathode and anode half-cells to pinpoint optimal voltage matches and discuss implications of changes in temperature on overall cell voltages. Using these observations, we look forward to increased interest in temperature and idealized half-reaction pairing as experimental choices for the optimization of electrochemical processes.