Antiviral Lipid Nanocarrier Loaded with Remdesivir Effective Against SARS-CoV-2 in vitro Model.

Introduction: The ongoing SARS-CoV-2 pandemic has affected public health, the economy, and society. This study reported a nanotechnology-based strategy to enhance the antiviral efficacy of the antiviral agent remdesivir (RDS). Results: We developed a nanosized spherical RDS-NLC in which the RDS was encapsulated in an amorphous form. The RDS-NLC significantly potentiated the antiviral efficacy of RDS against SARS-CoV-2 and its variants (alpha, beta, and delta). Our study revealed that NLC technology improved the antiviral effect of RDS against SARS-CoV-2 by enhancing the cellular uptake of RDS and reducing SARS-CoV-2 entry in cells. These improvements resulted in a 211% increase in the bioavailability of RDS. Conclusion: Thus, the application of NLC against SARS-CoV-2 may be a beneficial strategy to improve the antiviral effects of antiviral agents.

Development of a long-acting tablet with ticagrelor high-loaded nanostructured lipid carriers.

Ticagrelor (TCG), an antiplatelet agent, has low solubility and permeability; thus, there are many trials to apply the pharmaceutical technology for the enhancement of TCG solubility and permeability. Herein, we have developed the TCG high-loaded nanostructured lipid carrier (HL-NLC) and solidified the HL-NLC to develop the oral tablet. The HL-NLC was successfully fabricated and optimized with a particle size of 164.5 nm, a PDI of 0.199, an encapsulation efficiency of 98.5%, and a drug loading of 16.4%. For the solidification of HL-NLC (S-HL-NLC), the adsorbent was determined based on the physical properties of the S-HL-NLC, such as bulk density, tap density, angle of repose, Hausner ratio, Carr's index, and drug content. Florite R was chosen because of its excellent adsorption capacity, excellent physical properties, and solubility of the powder after manufacturing. Using an S-HL-NLC, the S-HL-NLC tablet with HPMC 4 K was prepared, which is showed a released extent of more than 90% at 24 h. Thus, we have developed the sustained release tablet containing the TCG-loaded HL-NLC. Moreover, the formulations have exhibited no cytotoxicity against Caco-2 cells and improved the cellular uptake of TCG. In pharmacokinetic study, compared with raw TCG, the bioavailability of HL-NLC and S-HL-NLC was increased by 293% and 323%, respectively. In conclusion, we successfully developed the TCG high-loaded NLC tablet, that exhibited a sustained release profile and enhanced oral bioavailability.

Mannosylated poly(acrylic acid)-coated mesoporous silica nanoparticles for anticancer therapy.

Although mesoporous silica nanoparticles (MSNs) are widely used as anticancer drug carriers, unmodified MSNs induce off-target effects and at high doses, there are adverse effects of hemolysis because of the interaction with the silanol group on the surface and cells. In this study, we developed doxorubicin (DOX)-loaded MSNs coated with mannose grafted poly (acrylic acid) copolymer (DOX@MSNs-man-g-PAA) to enhance the hemocompatibility and target efficacy to cancer cells. This uniform nanosized DOX@MSNs-man-g-PAA showed sustained and pH-dependent drug release with improved hemocompatibility over the bare MSNs. The uptake of the DOX@MSN-man-g-PAA in breast cancer cells was significantly improved by mannose receptor-mediated endocytosis, which showed significant increasing intracellular ROS and changes in mitochondrial membrane potential. This formulation exhibited superior tumor-suppressing activity in the MDA-MB-231 cells inoculated mice. Overall, the present study suggested the possibility of the copolymer-coated MSNs as drug carriers for cancer therapy.

Preparation and evaluation of rapid disintegrating formulation from coated microneedle.

Microneedles (MNs), one of the transdermal drug delivery systems, have received extensive interest as an alternative to parenteral or parenteral administrations. For the successful drug delivery of coated MNs, the coated drug or chemical of MNs should be dissolved by skin's interstitial fluid and completely released from the MNs. Thus, the rapid disintegration of the drug from MNs plays a crucial role in ideal drug delivery of MNs. In this study, we developed the rapid disintegration coating formulation to reduce the application time of MN. The rapid disintegration MN was developed using polymers (PVA or HPMC), glycerol, croscarmellose sodium, tween 80, and Brij, as thickener, plasticizer, disintegrating agent, and surfactants, respectively. HPMC MN showed the burst release and rapid disintegration. Moreover, the drug from HPMC MN was successfully delivered into porcine skin within 1 min. In toxicological evaluation, the HPMC MN did not alter the liver and kidney function. Besides, HPMC MN did not induce the acute inflammation and change of skin structure after the application on rat skin. Thus, the coating formulation in this study could be one of the options for the development of safe and rapid disintegration MN.

Risk factors for fatigue and stress among Korean police officers.

[Purpose] This study investigated factors that affect the health of police officers by analyzing job stress, psychosocial stress, and fatigue faced by police officers in order to provide basic data for the efficient management of police officers and future comparative research. [Subjects and Methods] Police officers admitted to the National Police Hospital from March to May 2013 were surveyed to investigate their degree of stress. The questionnaire consisted of 4 areas related to patient characteristics: general and demographic characteristics factors, job stress, psychosocial stress, and fatigue. [Results] The analysis of the relationships among job stress, psychosocial health, and fatigue showed the 0%, 44.7%, and 82% of those with healthy, potential, and high risks of stress had high job stress, respectively. Meanwhile, 40.8% and 77.9% of subjects with normal and high risks of fatigue had high job stress. [Conclusion] The studies can be used as basic and comparative data for the prevention and early control of job-related diseases for police officers.

Helicobacter apodemus sp. nov., a new Helicobacter species identified from the gastrointestinal tract of striped field mice in Korea.

A novel Helicobacter species was identified from the gastrointestinal tract of the Korean striped field mouse (Apodemus agrarius). Biochemical testing, ultrastructure characterization, and 16S rRNA gene sequence analysis suggested that this bacterium represents a distinct taxon. The bacterium was positive for urease activity, susceptible to cephalothin and nalidixic acid, and weakly positive for oxidase and catalase activity. Electron microscopy revealed that the bacterium has spirally curved rod morphology with singular bipolar nonsheathed flagella. Genotypically, the isolated bacterial strains (YMRC 000215, YMRC 000216, and YMRC 000419) were most closely related to a reference strain of Helicobacter mesocricetorum (97.25%, 97.32%, and 97.03% 16S rRNA sequence similarities, respectively). The 16S rRNA sequences of these strains were deposited into GenBank under accession numbers AF284754, AY009129, and AY009130, respectively. We propose the name Helicobacter apodemus for this novel species.

Prevalence of Musculoskeletal Disorders Among Korean Police Personnel.

This study was conducted to investigate efficient, systematic management of the Korean police and to examine the status and prevention of musculoskeletal disorders in Korean police officers. For police officers in special working environments, the importance of basic data is emphasized for human resources (HR) management and the prevention of industrial hazards from an industrial health care perspective. This study was conducted on police officers who visited the national police hospital and who underwent x-ray, computed tomography (CT), and magnetic resonance imaging (MRI) for the diagnosis of musculoskeletal disorders. The results revealed that examinations for the lower extremities and spine were most frequently conducted using x-ray, CT, and MRI. In particular, knee and lumbar examinations were most frequently conducted among all lower extremity and spine examinations, respectively.

Factors affecting the musculoskeletal symptoms of korean police officers.

] This study was conducted to investigate efficient, systematic management of the Korean police and to examine the status and prevention of musculoskeletal disorders in Korean police officers. [Subjects and Methods] A survey of police officers (353 subjects) who visited the National Police Hospital from March 2013 to May 2013 was conducted using a structured questionnaire. [Results] The incidence of pain was 44.2% in the shoulder, 41.4% in the waist, 31.2% in the neck, 26.1% in the legs/foot, 16.7% in the hand/wrist/finger, and 14.7% in the arm/elbow. The comparative risk of the relevant part factors was analyzed by multiple regression analysis. The shoulder had a 4.87 times higher risk in police lieutenants compared with those under the rank of corporal and a 1.78 times higher risk in people with chronic diseases than those without chronic diseases. The arm/elbow had a 2.37 times higher risk in people who exercised than those who did not exercise and a 1.78 times higher risk in people with a chronic disease than those without chronic diseases. Generally, people with a chronic disease showed a higher risk than those without chronic diseases. [Conclusion] The results of this study could be useful as basic data for improvement of police welfare, specialized treatment for the health safety of the police, and efficient management of police resources.

Preparation and diagnostic utility of a hemagglutination inhibition test antigen derived from the baculovirus-expressed hemagglutinin-neuraminidase protein gene of Newcastle disease virus.

A recombinant hemagglutinin-neuraminidase (rHN) protein from Newcastle disease virus (NDV) with hemagglutination (HA) activity was expressed in Spodoptera frugiperda cells using a baculovirus expression system. The rHN protein extracted from infected cells was used as an antigen in a hemagglutination inhibition (HI) test for the detection and titration of NDV-specific antibodies present in chicken sera. The rHN antigen produced high HA titers of 2(13) per 25 µL, which were similar to those of the NDV antigen produced using chicken eggs, and it remained stable without significant loss of the HA activity for at least 12 weeks at 4°C. The rHN-based HI assay specifically detected NDV antibodies, but not the sera of other avian pathogens, with a specificity and sensitivity of 100% and 98.0%, respectively, in known positive and negative chicken sera (n = 430). Compared with an NDV-based HI assay, the rHN-based HI assay had a relative sensitivity and specificity of 96.1% and 95.5%, respectively, when applied to field chicken sera. The HI titers of the rHN-based HI assay were highly correlated with those in an NDV-based HI assay (r = 0.927). Overall, these results indicate that rHN protein provides a useful alternative to NDV antigen in HI assays.

Characterization of clade 2.3.2.1 H5N1 highly pathogenic avian influenza viruses isolated from wild birds (mandarin duck and Eurasian eagle owl) in 2010 in Korea.

Starting in late November 2010, the H5N1 highly pathogenic avian influenza (HPAI) virus was isolated from many types of wild ducks and raptors and was subsequently isolated from poultry in Korea. We assessed the genetic and pathogenic properties of the HPAI viruses isolated from a fecal sample from a mandarin duck and a dead Eurasian eagle owl, the most affected wild bird species during the 2010/2011 HPAI outbreak in Korea. These viruses have similar genetic backgrounds and exhibited the highest genetic similarity with recent Eurasian clade 2.3.2.1 HPAI viruses. In animal inoculation experiments, regardless of their originating hosts, the two Korean isolates produced highly pathogenic characteristics in chickens, ducks and mice without pre-adaptation. These results raise concerns about veterinary and public health. Surveillance of wild birds could provide a good early warning signal for possible HPAI infection in poultry as well as in humans.

Anti-hyperglycemic effect of fermented ginseng in type 2 diabetes mellitus mouse model.

Ginseng has shown an efficacy in preventing and managing various health conditions. This study aimed to evaluate the effect of the fermented ginseng extract (FGE) in type 2 diabetes mellitus murine model. FGE was provided to male C57BL/ksJ-db/db mice for 8 weeks at 0.1% (w/w) dose in contrast to water for the control group. Potential anti-diabetic mechanisms were investigated with blood glucose, serum insulin, serum adiponectin, hemoglobin A1c (HbA1c), glucose tolerance, insulin secretion assay, quantitative real-time polymerase chain reaction, and hematoxylin-eosin staining. Compared with the control group, the FGE group had lower levels of blood glucose after 6 and 9 h fasting, HbA1c, and the area under the curve in an oral glucose tolerance test and higher levels of adiponectin and serum insulin (p < 0.05). The FGE group had higher levels of peroxisome proliferator-activated receptor gamma 2 and glucose transporter protein 2 mRNAs, a lower level of tumor necrosis factor-α (TNF-α) (p < 0.05), and less lymphocytes in pancreas than the control group had. The FGE exerted anti-diabetic effects in type 2 diabetic mice.

Molecular epidemiologic investigation of lentogenic Newcastle disease virus from domestic birds at live bird markets in Korea.

A Newcastle disease surveillance program was conducted at live bird markets in Korea to expand our epidemiologic understanding of the disease in Korea. During the surveillance program, 10 lentogenic Newcastle disease viruses (NDVs) were isolated and identified from apparently healthy chickens and ducks at live bird markets. The lentogenic viruses had sequence motifs of either 112GKQGRL117 (n = 8) or 112GRQGRL117 (n = 2) at the F0 cleavage site. Sequencing and phylogenetic analyses of NDV isolates based on the hypervariable region of the F protein revealed two different genotypes: genotypes I (n = 8) and II (n = 2). Genotype I viruses were most closely related to the NDV V4 strain (n = 7) or the NDV Ulster 2C strain (n = 1). In contrast, genotype II viruses clustered with the NDV vaccine strains (LaSota and VG/GA) that are commonly used as live vaccines in Korea. The epidemiologic importance of NDV at live bird markets in Korea is discussed.

Antigenic and immunogenic investigation of the virulence motif of the Newcastle disease virus fusion protein.

Newcastle disease (ND) caused by virulent Newcastle disease virus (NDV) is a highly contagious viral disease of poultry. Virulent NDVs characteristically have a multibasic amino acid sequence (virulence motif) such as (112)RRQKRF(117) at the cleavage site of the precusor fusion (F0) protein. The antigenic and immunogenic characteristics of the virulence motif (112)RRQKRF(117) in the F0 protein of virulent NDVs were investigated. Epitope mapping analysis revealed that a RRQKRF-specific monoclonal antibody 4G2 recognized the KRF section of the motif. A synthetic peptide bearing the RRQKRF motif reacted strongly with sera from virulent NDV (with RRQKRF motif)-infected chickens. These sera also showed reactivity to peptides bearing other virulence motifs ((112)KRQKRF(117), (112)RRQRRF(117) and (112)RRRKRF(117)) but not an avirulence motif ((112)GRQGRL(117)) by ELISA. The synthetic bearing RRQKRF motif reacted with 60% to 91% of sera taken from surviving chickens on ND outbreak farms but not with sera from vaccinated birds, even though most of the sera had antibody to NDV due to vaccination. This indicates that the virulence motif has the potential to differentiate virulent NDV infected birds from vaccinated birds.

Diagnostic utility of egg yolk for the detection of avian metapneumovirus antibodies in laying hens.

Surveillance and diagnosis of avian metapneumovirus (AMPV) infection typically involve measurement of serum antibodies. In the current study, eggs instead of serum samples were used for the detection of AMPV antibodies in egg-laying chicken hens by enzyme-linked immunosorbent assay (ELISA). AMPV-free commercial layer hens were experimentally challenged with AMPV strain SC1509 through intravenous or oculonasal administration. Antibody levels were determined by ELISA. AMPV antibodies were detected in egg yolks from challenged hens by 7 days postinoculation (dpi), with the peak titer at 16 dpi. Antibody levels in eggs laid at 28 dpi correlated well (r = 0.93) with sera taken 28 dpi from the same hens. In a field trial of the yolk ELISA, six broiler breeder farms were surveyed, and all tested positive for AMPV antibodies in hen eggs, although positivity varied from farm to farm. Abnormal discolored eggs collected from outbreak farms had significantly higher titers of AMPV yolk antibodies than normal eggs from the same farm, unlike clinically healthy farms, where normal and abnormal eggs had similar antibody titers. These results indicate that diagnosis of AMPV infection by yolk ELISA to detect anti-AMPV antibodies may be a suitable alternative to serologic testing.

Evaluation of a competitive ELISA for antibody detection against avian influenza virus.

Active serologic surveillance is necessary to control the spread of the avian influenza virus (AIV). In this study, we evaluated a commercially-available cELISA in terms of its ability to detect AIV antibodies in the sera of 3,358 animals from twelve species. cELISA detected antibodies against reference H1- through H15-subtype AIV strains without cross reactivity. Furthermore, the cELISA was able to detect antibodies produced following a challenge of the AIV H9N2 subtype in chickens, or following vaccination of the AIV H9 or H5 subtypes in chickens, ducks and geese. Next, we tested the sensitivity and specificity of the cELISA with sera from twelve different animal species, and compared these results with those obtained by the hemagglutination-inhibition (HI) test, the "gold standard" in AIV sera surveillance, a second commercially-available cELISA (IZS ELISA), or the agar gel precipitation (AGP) test. Compared with the HI test, the sensitivities and specificities of cELISA were 95% and 96% in chicken, 86% and 88% in duck, 97% and 100% in turkey, 100% and 87% in goose, and 91% and 97% in swine, respectively. The sensitivities and specificities of the cELISA in this study were higher than those of IZS ELISA for the duck, turkey, goose, and grey partridge sera samples. The results of AGP test against duck and turkey sera also showed significant correlation with the results of cELISA (R-value >0.9). In terms of flock sensitivity, the cELISA correlated better with the HI test than with commercially-available indirect ELISAs, with 100% flock sensitivity.

Pathogenicity and antigenicity of a new variant of Korean nephropathogenic infectious bronchitis virus.

Despite the existence of an active vaccination program, recently emerged strains of nephropathogenic infectious bronchitis virus (IBV) in Korea have caused significant economic losses in the poultry industry. In this study, we assessed the pathogenic and antigenic characteristics of a K-IIb type field strain of IBV that emerged in Korea since 2003, such as Kr/Q43/06. Specific pathogen free 1-week-old chickens exhibited severe respiratory symptoms (dyspnea) and nephropathogenic lesions (swollen kidneys with nephritis and urate deposits) following challenge with the recent IBV field strain. The antigenic relatedness (R value), based on a calculated virus neutralization index, of the K-IIb type field strain and K-IIa type strain KM91 (isolated in 1991) was 30%, which indicated that the recent strain, Kr/Q43/06, is a new variant that is antigenically distinct from strain KM91. This report is the first to document the emergence of a new antigenic variant of nephropathogenic IBV in chicken from Korea.

Molecular epizootiology of infectious bursal disease (IBD) in Korea.

We conducted a molecular epizootiological study of infectious bursal disease (IBD) in Korea by analyzing 85 IBD viruses (IBDVs) obtained from vaccinated or unvaccinated flocks between 1980 and 2007. Phylogenetic analysis of the partial nucleotide sequence of the hypervariable region of the VP2 gene (nucleotides 661-1020) and pathogenicity tests revealed more genetic and phenotypic diversity of IBDV in Korea than has been reported previously. We showed that very virulent IBDVs (vvIBDVs) were already present in Korea in 1986. Moreover, vvIBDVs were repeatedly detected in Korean poultry that had been vaccinated, which casts doubt on the IBD vaccine programs. We also identified novel putative antigenic variant (AV)-like IBDV isolates on the basis of their antigenic indices and the presence of amino acid changes (P222S or P222T-A321D) that are known to affect the antigenicity of VP2. These observations suggest that future studies examining the efficacy of conventional vaccines against atrophy of the bursa of Fabricius and vvIBDV shedding may be useful. Moreover, it will be of interest to determine the prevalence of putative Korean antigenic variants and whether these strains exert immunosuppressive effects in vaccinated birds.

A multiplex real-time PCR for differential detection and quantification of Salmonella spp., Salmonella enterica serovar Typhimurium and Enteritidis in meats.

Salmonella (S.) Typhimurium and S. Enteritidis are the major causative agents of food-borne illnesses worldwide. Currently, a rapid detection system using multiplex real-time polymerase chain reaction (PCR) has been applied for other food-borne pathogens such as Escherichia coli, Staphylococcus aureus and Streptococcus spp. A multiplex real-time PCR was developed for the simultaneous detection of Salmonella spp., especially S. Typhimurium and S. Enteritidis, in beef and pork. For the specific and sensitive multiplex real-time PCR, three representative primers and probes were designed based on sequence data from Genbank. Among the three DNA extraction methods (boiling, alkaline lysis, and QIAamp DNA Mini Kit), the QIAamp DNA Mini Kit was the most sensitive in this study. The optimized multiplex real-time PCR was applied to artificially inoculated beef or pork. The detection sensitivity of the multiplex real-time PCR was increased. The specificity of the multiplex real-time PCR assay, using 128 pure-cultured bacteria including 110 Salmonella isolates and 18 non-Salmonella isolates, was 100%, 100% and 99.1% for Salmonella spp., S. Typhimurium and S. Enteritidis, respectively. The sensitivity was 100%, 100% and 91.7% for Salmonella spp., S. Typhimurium and S. Enteritidis, respectively. The multiplex real-time PCR assay developed in this study could detect up to 0.54 +/- 0.09 and 0.65 +/- 0.07 log(10) CFU/ml for S. Typhimurium and S. Enteritidis for beef, 1.45 +/- 0.21 and 1.65 +/- 0.07 log(10) CFU/ml for S. Typhimurium and S. Enteritidis for pork, respectively, with all conditions optimized. Our results indicated that the multiplex real-time PCR assay developed in this study could sensitively detect Salmonella spp. and specifically differentiate S. Typhimurium from S. Enteritidis in meats.

Molecular epidemiological investigation of Newcastle disease virus from domestic ducks in Korea.

To expand the epidemiological understanding of Newcastle disease virus (NDV) found in domestic ducks in Korea, 14 NDV isolates from apparently healthy domestic ducks were biologically and genetically characterized. Thirteen and 1 isolates of NDV were categorized into lentogenic and velogenic viruses, respectively, based on in vivo pathogenicity tests. Twelve lentogenic viruses showed HA activity to horse RBCs, while 1 lentogenic virus and the velogenic virus were negative. Lentogenic viruses (n=13) had sequence motifs of (112)ERQERL(117) (n=1) or (112)GRQGRL(117) (n=12) at the F0 cleavage site, while the velogenic virus (n=1) had a sequence motif of (112)RRQKRF(117) at the same site. Phylogenetic analysis revealed that at least three distinct genotypes may exist in domestic ducks in Korea; one class I genotype (genotype 2), and two class II (genotypes I and VII) genotypes. The class I virus was most closely related to strains of genotype 2 which were isolated in birds from the USA, Germany and Denmark. Twelve lentogenic class II viruses were grouped together in genotype I, and were then divided into at least three clusters, namely Aomori-like, Ulster2C-like, and V4-like. The velogenic class II virus was assigned to genotype VII which represents viruses responsible for recent epidemics in many Asian countries including Korea. The epidemiological importance of domestic duck isolates of NDV in Korea is discussed.

Histomorphometric evaluation of immediately loaded SSII implants of different surface treatments in a dog model.

This study compared splint (experimental) and nonsplint (control) methods for immediately loaded implants and examined the bone-implant contact rate for smooth, oxidized, and resorbable blast medium (RBM) surfaces. The first through fourth mandibular premolars were extracted from six young adult dogs. Twelve weeks after extraction, implantation was performed at the extraction sites. The SSII OSSTEM implant had one of three surface treatments: smooth, oxidized, or RBM. Sixteen weeks after implantation, the dogs were euthanized; the hemimandibles were obtained and processed histologically to obtain nondecalcified sections. Longitudinal sections were made for each implant and analyzed using light microscopy. Independent of the splinting method, a significantly higher bone-implant contact was observed for implants with oxidized and RBM surfaces when compared with implants with smooth surfaces. Irrespective of the splinting method, immediately loaded implants with oxidized and RBM surfaces may result in higher bone-implant integration when compared with implants with smooth surfaces.