Generation of rescued Japanese encephalitis virus genotype 1 from infectious full-size clone using reverse genetics.

Japanese encephalitis virus (JEV) is a pathogen responsible for high mortality and morbidity rates among children with encephalitis. Since JEV genotype 1 (GI) is the most prevalent strain in South Korea these days, corresponding research and vaccine development is urgently required. Molecular genetic studies on JEV vaccines can be boosted by obtaining genetically stable full-length infectious JEV complementary DNA (cDNA) clones. Furthermore, the significance of the reverse genetics system in facilitating molecular biological analyses of JEV properties has been demonstrated. This study constructed a recombinant JEV-GI strain using a reverse genetics system based on a Korean wild-type GI isolate (K05GS). RNA extracted from JEV-GI was used to synthesize cDNA, a recombinant full-length JEV clone, pTRE-JEVGI, was generated from the DNA fragment, and the virus was rescued. We performed in vitro and in vivo experiments to analyze the rescued JEV-GI virus. The rescued JEV-GI exhibited similar characteristics to wild-type JEV. These results suggest that our reverse genetics system can generate full-length infectious clones that can be used to analyze molecular biological factors that influence viral properties and immunogenicity. Additionally, it may be useful as a heterologous gene expression vector and help develop new strains for JEV vaccines.

Launch of the first canine mobile blood donation center in Asia: development, outcomes, and influence of an animal bloodmobile.

Introduction: A mobile blood donation station allows a maximum number of donors to donate blood at any location. In veterinary medicine, no previous studies have reported the use of bloodmobiles for blood donation in animals. We assessed Asia's first canine mobile blood donation center, which was trialed using a modified vehicle in South Korea. Methods: A vehicle was modified into a canine bloodmobile with two sections: the front as a laboratory and the back as a blood collection room with necessary equipment. To recruit companion dogs nationwide, the campaign was advertised on television and promoted via social media. Applications of the dogs meeting the following criteria were accepted: in general good health, between 2-8 years old, body weight above 25kg, vaccinated, regularly on heartworm and ectoparasite prophylactics. Pre-donation procedures included medical screening and informed consent, followed by blood collection in a routine fashion. Post-donation, dogs were monitored for complications and owners completed a post-donation survey. Results: Of the 750 applicants, 48 donor dogs were selected for investigation. Ten failed to donate blood owing to the following issues: behavioral problems (2/48), positive results on vector-borne disease screening tests (5/48), in-tubing clot formation (2/48), and absence on the relevant appointment date (1/48). Blood collection took approximately 12 minutes, and the entire procedure lasted an average of 1.5 hours per donor. The prevalence rates of dog erythrocyte antigen 1-negative and 1-positive blood were 32.6% and 67.4%, respectively. There were no donation-related complications, except for one dog that had contact dermatitis induced by clipper irritation. The post-donation survey completed by 46 owners revealed that most were satisfied with the campaign. The convenience of the mobile blood drive (93.5%) was a key factor contributing to high owner satisfaction and willingness to participate in future campaigns (95.7%), in line with findings from prior veterinary and human blood donation motivation research. Discussion: The bloodmobile effectively increased engagement in canine blood donation by enhancing accessibility. To optimize canine mobile blood drives, procuring larger vehicles and enhancing infrastructure for future campaigns would be beneficial. In conclusion, this study showed that Asia's first canine bloodmobile was successful in terms of improving the convenience, accessibility, and efficacy of canine blood donation. Although the concept is still unfamiliar to the public, active promotion of canine blood donation can help ensure a robust blood donation culture in the veterinary field.

Safety evaluation of a buffer used in the lyophilization of canine platelets: type I hypersensitivity reaction in dogs caused by bovine serum albumin.

Introduction: The present study was designed to evaluate the safety of substances generally used in the preparation of lyophilized platelet products (LPPs) because the possibility of an immune response to bovine serum albumin (BSA) was considered high when using previously described technology. Methods: An intradermal skin test, followed by a drug provocation test, was conducted to observe adverse events and identify the substances responsible for an immune response. Five male beagles (2 years old) weighing 12-14 kg were used. The dogs were clinically healthy and had no history of medication use. An intradermal skin test was conducted with each substance [i.e., 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, sodium chloride, potassium chloride, sodium bicarbonate, theophylline, trehalose, and BSA] used in the conventional freeze-dry method. Results: In the intradermal skin test, three dogs tested positive at the BSA injection site and showed clinical signs after the intradermal injection, including nausea and vomiting. For the drug provocation test, all dogs received two intravenous injections of an LPP buffer solution. The initial injection was devoid of BSA, whereas the subsequent injection contained BSA. The three dogs that had reacted to BSA in the intradermal skin test exhibited adverse events such as lethargy, vomiting, and nausea immediately after intravenous injection of the LPP buffer containing BSA. All dogs recovered uneventfully after symptomatic treatment in both tests. Discussion: The high incidence and severity of type I hypersensitivity reactions observed in this study suggested that BSA is unsuitable as a component of canine LPP.

The Effect of BI 730357 (Retinoic Acid-Related Orphan Receptor Gamma t Antagonist, Bevurogant) on the Pharmacokinetics of a Transporter Probe Cocktail, Including Digoxin, Furosemide, Metformin, and Rosuvastatin: An Open-Label, Non-randomized, 2-Period Fixed-Sequence Trial in Healthy Subjects.

Evaluating Drug-Drug Interactions (DDIs) for new investigational compounds requires several trials evaluating different drugs with different transporter specificities. By using a cocktail of drugs with different transporter specificities, a single trial could evaluate the pharmacokinetics (PKs) of each cocktail drug simultaneously, reducing the number of clinical DDI trials required for clinical development. We aimed to investigate the effect of steady-state Boehringer Ingelheim (BI) 730357 (bevurogant) on the PKs of a validated and optimized 4-component transporter cocktail. This open-label, non-randomized, 2-period fixed-sequence phase I trial compared transporter cocktail (0.25 mg digoxin/1 mg furosemide/10 mg metformin hydrochloride/10 mg rosuvastatin) with and without BI 730357 in healthy subjects aged 18-55 years with body mass index 18.5-29.9 kg/m2 . During reference treatment/period 1, transporter cocktail was administered 90 minutes after breakfast. After a washout period, during test treatment/period 2, BI 730357 was dosed twice daily for 13 days, with transporter cocktail administered on day 1. The primary endpoints were the area under the concentration-time curve of the analyte in plasma over the time interval from 0 extrapolated to infinity (AUC0-∞ ) and the maximum measured concentration of the analyte in plasma (Cmax ), and the secondary endpoint was the area under the concentration-time curve of the analyte in plasma over the time interval from 0 to the last quantifiable data point (AUC0-tz ). Steady-state BI 730357 increased digoxin (+48% to +94%), minimally affected metformin (-2% to -9%), furosemide (+12% to +18%), and rosuvastatin (+19% to +39%) exposure. Therefore, no clinically relevant inhibition of transporters OCT2/MATE-1/MATE-2K, OAT1/OAT3, OATP1B1/OATP1B3 was observed. Potential inhibition of breast cancer resistance protein noted as PK parameters of coproporphyrin I/III (OATP1B1/OATP1B3 biomarkers) remained within bioequivalence boundaries while rosuvastatin PK parameters (AUC0-∞ /Cmax /AUC0-tz ) exceeded the bioequivalence boundary. BI 730357 was safe and well tolerated. This trial confirms the usefulness and tolerability of the transporter cocktail consisting of digoxin, furosemide, metformin, and rosuvastatin in assessing drug-transporter interactions in vivo.

Synthesis and Anti-Inflammatory Activity of N(2)-Arylindazol-3(2H)-One Derivatives: Copper-Promoted Direct N-Arylation via Chan-Evans-Lam Coupling.

Inflammatory-related diseases are becoming increasingly prevalent, leading to a growing focus on the development of anti-inflammatory agents, with a particular emphasis on creating novel structural compounds. In this study, we present a highly efficient synthetic method for direct N-arylation to produce a variety of N(2)-arylindazol-3(2H)-ones 3, which exhibit anti-inflammatory activity. The Chan-Evans-Lam (CEL) coupling of N(1)-benzyl-indazol-3-(2H)-ones 1 with arylboronic acids 2 in the presence of a copper complex provided the corresponding N(2)-arylindazol-3(2H)-ones 3 in good-to-excellent yields, as identified with NMR, MS, and X-ray crystallography techniques. The cell viability and anti-inflammatory effects of the synthesized compounds (3 and 5) were briefly assessed using the MTT method and Griess assay. Among them, compounds 5 exhibited significant anti-inflammatory effects with negligible cell toxicity.

Pine Needle Extract Applicable to Topical Treatment for the Prevention of Human Papillomavirus Infection.

Most cervical cancers are associated with high-risk human papillomavirus (HPV) infection. Currently, cervical cancer treatment entails surgical removal of the lesion, but treatment of infection and preventing tissue damage are issues that still remain to be addressed. Herbal medicine and biological studies have focused on developing antiviral drugs from natural sources. In this study, we analyzed the potential antiviral effects of Pinus densiflora Sieb. et Zucc. leaf extracts against HPV. The pine needle extracts from each organic solvent were analyzed for antiviral activity. The methylene chloride fraction (PN-MC) showed the highest activity against HPV pseudovirus (PV). The PN-MC extract was more effective before, rather than after treatment, and therefore represents a prophylactic intervention. Mice were pre-treated with PN-MC via genital application or oral administration, followed by a genital or subcutaneous challenge with HPV PV, respectively. The HPV challenge results showed that mice treated via genital application exhibited complete protection against HPV. In conclusion, PN-MC represents a potential topical virucide for HPV infection.

Corallincola spongiicola sp. nov., isolated from sponge.

A gram-negative, motile, strictly aerobic, and rod-shaped bacterium designated 176GS2-150T was isolated from the sponge Hymeniacidon sinapium. The taxonomic position of the novel isolate was confirmed using the polyphasic approach. Strain 176GS2-150T grew well at 25 °C on marine agar. Based on its 16S rRNA gene sequence, we showed that strain 176GS2-150T belongs to the family Psychromonadaceae and class Gammaproteobacteria and is related to Corallincola platygyrae JLT2006T (96.84% sequence similarity). The G + C content of the genomic DNA was 49.0 mol%. The assembled draft genome of strain 176GS2-150T was 4.2 Mbp and consisted of 14 contigs. The major respiratory quinone was Q-8, and the major fatty acids were summed feature 3 (comprising C16 :1ω6c and/or C16:1ω7c), summed feature 8 (comprising C18 :1ω7c and/or C18:1ω6c), C17:0 iso, C16:0, and C15:0 iso. The polar lipids were phosphatidylglycerol, phosphatidylethanolamine, 3 unidentified phospholipids, and 1 unidentified polar lipid. On the basis of the genotypic and phenotypic characteristics, strain 176GS2-150T can be placed as a new species within the genus Corallincola; the name Corallincola spongiicola sp. nov. has been proposed, with type strain 176GS2-150T (= KACC 19890T = LMG 31317T).

Two-Dimensional Liquid Chromatography Analysis of Polystyrene/Polybutadiene Block Copolymers.

A detailed characterization of a commercial polystyrene/polybutadiene block copolymer material (Styrolux) was carried out using two-dimensional liquid chromatography (2D-LC). The Styrolux is prepared by statistical linking reaction of two different polystyrene- block-polybutadienyl anion precursors with a multivalent linking agent. Therefore, it is a mixture of a number of branched block copolymers different in molecular weight, composition, and chain architecture. While individual LC analysis, including size exclusion chromatography, interaction chromatography, or liquid chromatography at critical condition, is not good enough to resolve all the polymer species, 2D-LC separations coupling two chromatography methods were able to resolve all polymer species present in the sample; at least 13 block copolymer species and a homopolystyrene blended. Four different 2D-LC analyses combining a different pair of two LC methods provide their characteristic separation results. The separation characteristics of the 2D-LC separations are compared to elucidate the elution characteristics of the block copolymer species.

Lysobacter spongiae sp. nov., isolated from spongin.

A Gram-negative, motile, aerobic and rod-shaped bacterial strain designated 119BY6-57T was isolated from spongin. The taxonomic position of the novel isolate was confirmed using the polyphasic approach. Strain 119BY6-57T grew well at 25-30°C on marine agar. On the basis of 16S rRNA gene sequence similarity, strain 119BY6-57T belongs to the family Xanthomonadaceae and is related to Lysobacter aestuarii S2-CT (99.8% sequence similarity), L. maris KMU-14T (97.5%), and L. daejeonensis GH1-9T (97.3%). Lower sequence similarities (97.0%) were found with all of the other recognized members of the genus Lysobacter. The G + C content of the genomic DNA was 69.9 mol%. The major respiratory quinone was Q-8 and the major fatty acids were C16:0 iso, C15:0 iso, summed feature 9 (comprising C17:1 iso ω9c and/or C16:0 10-methyl), summed feature 3 (comprising C16:1ω7c and/or C16:1ω6c), and C11:0 iso 3-OH. The polar lipids were phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, three unidentified phospholipids, and an unidentified polar lipid. DNADNA relatedness values between strain 119BY6-57T and its closest phylogenetically neighbors were below 48.0 ± 2.1%. Based on genotypic and phenotypic characteristics, it is concluded that strain 119BY6-57T is a new member within the genus Lysobacter, for which the name Lysobacter spongiae sp. nov. is proposed. The type strain is 119BY6-57T (= KACC 19276T = LMG 30077T).