Saliva-based Proteinase K method: A rapid and reliable diagnostic tool for the detection of SARS-COV-2 in children.

Early and accurate detection of viruses in children might help prevent transmission and severe diseases. In this study, the severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) detection in children was evaluated using saliva specimens with a Proteinase K (PTK)-based RNA preparation, as saliva collection is a simple and noninvasive procedure, even in young children, with fewer concerns about sample contamination. The saliva-based PTK and the conventional paired nasopharyngeal aspiration (NPA)-based detection methods were compared between COVID-19-positive and -negative children. In addition, the detection rate for SARS-COV-2 and the difference between admission and discharge by the saliva-based PTK method was tested in COVID-19 patients. The diagnostic accuracy of the saliva-based PTK method was 98.8% compared to NP swab-based reverse transcriptase polymerase chain reaction. Saliva samples showed high sensitivity (94.1%) and specificity (100%) when using the PTK method. Furthermore, the saliva-based PTK method significantly reduced the test processing time by 2 h. Notably, Ct values at discharge increased in saliva samples compared with those at admission, which might indicate patients' clinical conditions or virus activity. In conclusion, the saliva-based PTK implemented in this study streamlines RNA extraction, making the process faster, safer, and more cost-effective, demonstrating that this method is a rapid and reliable diagnostic tool for SARS-CoV-2 detection in children.

Cross-Protection against MERS-CoV by Prime-Boost Vaccination Using Viral Spike DNA and Protein.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory illness and has a high mortality of ∼34%. However, since its discovery in 2012, an effective vaccine has not been developed for it. To develop a vaccine against multiple strains of MERS-CoV, we targeted spike glycoprotein (S) using prime-boost vaccination with DNA and insect cell-expressed recombinant proteins for the receptor-binding domain (RBD), S1, S2, SΔTM, or SΔER. Our S subunits were generated using an S sequence derived from the MERS-CoV EMC/2012 strain. We examined humoral and cellular immune responses of various combinations with DNA plasmids and recombinant proteins in mice. Mouse sera immunized with SΔER DNA priming/SΔTM protein boosting showed cross-neutralization against 15 variants of S-pseudovirions and the wild-type KOR/KNIH/002 strain. In addition, these immunizations provided full protection against the KOR/KNIH/002 strain challenge in human DPP4 knock-in mice. These findings suggest that vaccination with the S subunits derived from one viral strain can provide cross-protection against variant MERS-CoV strains with mutations in S. DNA priming/protein boosting increased gamma interferon production, while protein-alone immunization did not. The RBD subunit alone was insufficient to induce neutralizing antibodies, suggesting the importance of structural conformation. In conclusion, heterologous DNA priming with protein boosting is an effective way to induce both neutralizing antibodies and cell-mediated immune responses for MERS-CoV vaccine development. This study suggests a strategy for selecting a suitable platform for developing vaccines against MERS-CoV or other emerging coronaviruses.IMPORTANCE Coronavirus is an RNA virus with a higher mutation rate than DNA viruses. Therefore, a mutation in S-protein, which mediates viral infection by binding to a human cellular receptor, is expected to cause difficulties in vaccine development. Given that DNA-protein vaccines promote stronger cell-mediated immune responses than protein-only vaccination, we immunized mice with various combinations of DNA priming and protein boosting using the S-subunit sequences of the MERS-CoV EMC/2012 strain. We demonstrated a cross-protective effect against wild-type KOR/KNIH/002, a strain with two mutations in the S amino acids, including one in its RBD. The vaccine also provided cross-neutralization against 15 different S-pseudotyped viruses. These suggested that a vaccine targeting one variant of S can provide cross-protection against multiple viral strains with mutations in S. The regimen of DNA priming/Protein boosting can be applied to the development of other coronavirus vaccines.

Pharmacologic Activation of Lytic Epstein-Barr Virus Gene Expression without Virion Production.

Several therapeutic strategies targeting Epstein-Barr virus (EBV)-associated tumors involve upregulation of viral lytic gene expression. Evidence has been presented that the unfolded protein response (UPR) leads to EBV lytic gene expression. Clofoctol, an antibacterial antibiotic, has been reported to upregulate the UPR in prostate cancer cell lines and to slow their growth. We investigated the effects of clofoctol on an EBV-positive Burkitt lymphoma cell line and confirmed the upregulation of all three branches of the UPR and activation of EBV lytic gene expression. While immediate early, early, and late EBV RNAs were all upregulated, immediate early and early viral proteins but not late viral proteins were expressed. Furthermore, infectious virions were not produced. The use of clofoctol in combination with a protein kinase R-like endoplasmic reticulum kinase inhibitor led to expression of late viral proteins. The effects of clofoctol on EBV lytic protein upregulation were not limited to lymphoid tumor cell lines but also occurred in naturally infected epithelial gastric cancer and nasopharyngeal cancer cell lines. An agent that upregulates lytic viral protein expression but that does not lead to the production of infectious virions may have particular value for lytic induction strategies in the clinical setting.IMPORTANCE Epstein-Barr virus is associated with many different cancers. In these cancers the viral genome is predominantly latent; i.e., most viral genes are not expressed, most viral proteins are not synthesized, and new virions are not produced. Some strategies for treating these cancers involve activation of lytic viral gene expression. We identify an antibacterial antibiotic, clofoctol, that is an activator of EBV lytic RNA and protein expression but that does not lead to virion production.

Microneedle Array Patch (MAP) Consisting of Crosslinked Hyaluronic Acid Nanoparticles for Processability and Sustained Release.

AIMS: Crosslinked hyaluronic acid (X-linked HA) is not suitable for making microneedles because of the low fluidity of X-linked HA hydrogel. Microneedles were fabricated using X-linked HA nanoparticles (X-linked HA-NPs) to utilize the sustained drug delivery capability of X-linked HA-NPs and to obtain the processability advantages of X-linked HA. METHOD: The puncture performance of a microneedle array patch (MAP) made of crosslinked hyaluronic acid nanoparticles (X-linked HA-NP-MAP) was evaluated by insertion in vitro into porcine skin. After a predetermined attachment time, the remaining height of the X-linked HA-NP-MAP was measured to determine the dissolution rate. X-linked HA-NP-MAP and free HA-MAP containing Rhodamine B isothiocyanate-dextran were administered into the back skin of mice, and the relative fluorescent intensity in the back skin was measured over time. RESULTS: The puncture performance of the X-linked HA-NP-MAP was over 90%. The diameter of redispersed X-linked HA-NPs was same as that of the premolded X-linked HA-NPs. The dissolution rate was not different from that of free HA-MAP. In an in vivo experiment, X-linked HA-NP-MAP was administered into the mouse's back skin successfully and the relative fluorescent intensity of X-linked HA-NP-MAP lasted longer than that of HA-MAP. CONCLUSION: X-linked HA-NPs provide the biocompatibility, the processability of micromolding, sustained drug release, successful penetration into the skin, and relatively short insertion time for full disintegration of NPs in the skin. X-linked HA-NP-MAP can be used for various applications that require several days of sustained drug release.

Chitinase 3-like 1 protein plays a critical role in respiratory syncytial virus-induced airway inflammation.

BACKGROUND: Chitinase 3-like 1 protein (CHI3L1) (YKL-40 in humans and breast regression protein [BRP]-39 in mice) is required for optimal allergen sensitization and Th2 inflammation in various chronic inflammatory diseases including asthma. However, the role of CHI3L1 in airway inflammation induced by respiratory viruses has not been investigated. The aim of this study was to investigate the relationship between CHI3L1 and airway inflammation caused by respiratory syncytial virus (RSV) infection. METHODS: We measured YKL-40 levels in human nasopharyngeal aspirate (NPA) from hospitalized children presenting with acute respiratory symptoms. Wild-type (WT) and BRP-39 knockout (KO) C57BL/6 mice were inoculated with live RSV (A2 strain). Bronchoalveolar lavage fluid and lung tissue samples were obtained on day 7 after inoculation to assess lung inflammation, airway reactivity, and expression of cytokines and BRP-39. RESULTS: In human subjects, YKL-40 and IL-13 levels in NPA were higher in children with RSV infection than in control subjects. Expression of BRP-39 and Th2 cytokines, IL-13 in particular, was increased following RSV infection in mice. Airway inflammation caused by RSV infection was reduced in BRP-39 KO mice as compared to WT mice. Th2 cytokine levels were not increased in the lungs of RSV-infected BRP-39 KO mice. BRP-39 regulated M2 macrophage activation in RSV-infected mice. Additionally, treatment with anti-CHI3L1 antibody attenuated airway inflammation and Th2 cytokine production in RSV-infected WT mice. CONCLUSION: These findings suggest that CHI3L1 could contribute to airway inflammation induced by RSV infection. CHI3L1 could be a potential therapeutic candidate for attenuating Th2-associated immunopathology during RSV infection.

Development of Botulinum Toxin A-Coated Microneedles for Treating Palmar Hyperhidrosis.

Hyperhidrosis is a disorder that is characterized by the production of excess amounts of sweat. The botulinum neurotoxin A (BoNT/A) has been used to treat hyperhidrosis through multiple intradermal injections at the site of the condition. However, because of BoNT/A toxicity, it is important to precisely deliver the proper dose of the toxin to the target site. In addition, the use of a conventional hypodermic needle for multiple injections in the palm makes the approach undesirable and painful. Here, we designed a BoNT/A-coated microneedle (BoNT-MN) array and tested its efficacy as a substitute pain-free method to treat hyperhidrosis. BoNT-MNs were prepared by coating polylactic acid microneedles with a BoNT/A formulation and were found to successfully penetrate into a thick skin in vitro. The coating formulations were then tested for their stability at 4, 25, and 37 °C for 24 h. BoNT-MNs were found to be much more stable than BoNT/A in a liquid state. Additionally, we carried out in vivo experiments by treating the right paws of mice with BoNT-MNs and found that the treatment induced a significant reduction in the sweating response in the mouse foot pad. Thus, BoNT/A treatment using microneedles is beneficial and may be used as a more efficient and less painful approach to treat hyperhidrosis.

Activated Leukocyte Cell Adhesion Molecule Stimulates the T-Cell Response in Allergic Asthma.

RATIONALE: The activated leukocyte cell adhesion molecule (ALCAM) is a cluster of differentiation 6 ligand that is important for stabilizing the immunological synapse and inducing T-cell activation and proliferation. OBJECTIVES: In this study, we investigated the role of ALCAM in the development of inflammation in allergic asthma. METHODS: An ovalbumin (OVA)-induced allergic asthma model was established in wild-type (WT) and ALCAM-deficient (ALCAM-/-) mice. T-cell proliferation was evaluated in cocultures with dendritic cells (DCs). Bone marrow-derived dendritic cells (BMDCs) from WT and ALCAM-/- mice were cultured and adoptively transferred to OT-II mice for either OVA sensitization or challenge. An anti-ALCAM antibody was administered to assess its therapeutic potential. ALCAM concentrations in the sputum and serum of children with asthma were quantified by ELISA. MEASUREMENTS AND MAIN RESULTS: Inflammatory responses were lower in ALCAM-/- mice than in WT mice, and T cells cocultured with DCs from ALCAM-/- mice showed reduced proliferation relative to those cocultured with DCs from WT mice. A decreased inflammatory response was observed upon adoptive transfer of BMDCs from ALCAM-/- mice as compared with that observed after transfer of BMDCs from WT mice. In addition, anti-ALCAM antibody-treated mice showed a reduced inflammatory response, and sputum and serum ALCAM concentrations were higher in children with asthma than in control subjects. CONCLUSIONS: ALCAM contributes to OVA-induced allergic asthma by stimulating T-cell activation and proliferation, suggesting it as a potential therapeutic target for allergic asthma.

Curcumin Treatment in Combination with Glucose Restriction Inhibits Intracellular Alkalinization and Tumor Growth in Hepatoma Cells.

Dysregulation of cellular energy metabolism is closely linked to cancer development and progression. Calorie or glucose restriction (CR or GR) inhibits energy-dependent pathways, including IGF-1/PI3K/Akt/mTOR, in cancer cells. However, alterations in proton dynamics and reversal of the pH gradient across the cell membrane, which results in intracellular alkalinization and extracellular acidification in cancer tissues, have emerged as important etiopathogenic factors. We measured glucose, lactate, and ATP production after GR, plant-derived CR-mimetic curcumin treatment, and curcumin plus GR in human hepatoma cells. Intracellular pH regulatory effects, in particular, protein-protein interactions within mTOR complex-1 and its structural change, were investigated. Curcumin treatment or GR mildly inhibited Na+/H+ exchanger-1 (NHE1). vATPase, monocarboxylate transporter (MCT)-1, and MCT4 level. Combination treatment with curcumin and GR further enhanced the inhibitory effects on these transporters and proton-extruding enzymes, with intracellular pH reduction. ATP and lactate production decreased according to pH change. Modeling of mTOR protein revealed structural changes upon treatments, and curcumin plus GR decreased binding of Raptor and GßL to mTOR, as well as of Rag A and Rag B to Raptor. Consequently, 4EBP1 phosphorylation was decreased and cell migration and proliferation were inhibited in a pH-dependent manner. Autophagy was increased by curcumin plus GR. In conclusion, curcumin treatment combined with GR may be a useful supportive approach for preventing intracellular alkalinization and cancer progression.

Severe Fever with Thrombocytopenia Syndrome Virus Infection, South Korea, 2010.

Severe fever with thrombocytopenia syndrome (SFTS) was reported in China in 2009 and in South Korea in 2012. We found retrospective evidence of SFTS virus infection in South Korea in 2010, suggesting that infections in South Korea occurred before previously reported and were more concurrent with those in China.

Host Cell Mimic Polymersomes for Rapid Detection of Highly Pathogenic Influenza Virus via a Viral Fusion and Cell Entry Mechanism.

Highly pathogenic avian influenza virus (HPAIV) infections have occurred continuously and crossed the species barrier to humans, leading to fatalities. A polymerase chain reaction based molecular test is currently the most sensitive diagnostic tool for HPAIV; however, the results must be analyzed in centralized diagnosis systems by a trained individual. This requirement leads to delays in quarantine and isolation. To control the spread of HPAIV, rapid and accurate diagnostics suitable for field testing are needed, and the tests must facilitate a differential diagnosis between HPAIV and low pathogenic avian influenza virus (LPAIV), which undergo cleavage specifically by trypsin- or furin-like proteases, respectively. In this study, a differential avian influenza virus rapid test kit is developed and evaluated in vitro and using clinical specimens from HPAIV H5N1-infected animals. It is demonstrated that this rapid test kit provides highly sensitive and specific detection of HPAIV and LPAIV and is thus a useful field diagnostic tool for H5N1 HPAIV outbreaks and for rapid quarantine control of the disease.

A high-throughput assay of NK cell activity in whole blood and its clinical application.

Natural killer (NK) cells are lymphocytes of the innate immune system and have the ability to kill tumor cells and virus-infected cells without prior sensitization. Malignant tumors and viruses have developed, however, strategies to suppress NK cells to escape from their responses. Thus, the evaluation of NK cell activity (NKA) could be invaluable to estimate the status and the outcome of cancers, viral infections, and immune-mediated diseases. Established methods that measure NKA, such as (51)Cr release assay and CD107a degranulation assay, may be used to determine NK cell function, but they are complicated and time-consuming because they require isolation of peripheral blood mononuclear cells (PBMC) or NK cells. In some cases these assays require hazardous material such as radioactive isotopes. To overcome these difficulties, we developed a simple assay that uses whole blood instead of PBMC or isolated NK cells. This novel assay is suitable for high-throughput screening and the monitoring of diseases, because it employs serum of ex vivo stimulated whole blood to detect interferon (IFN)-γ secreted from NK cells as an indicator of NKA. After the stimulation of NK cells, the determination of IFNγ concentration in serum samples by enzyme-linked immunosorbent assay (ELISA) provided a swift, uncomplicated, and high-throughput assay of NKA ex vivo. The NKA results microsatellite stable (MSS) colorectal cancer patients was showed significantly lower NKA, 263.6 ± 54.5 pg/mL compared with healthy subjects, 867.5 ± 50.2 pg/mL (p value <0.0001). Therefore, the NKA could be utilized as a supportive diagnostic marker for microsatellite stable (MSS) colorectal cancer.

A DNA vaccine against GII.4 human norovirus VP1 induces blocking antibody production and T cell responses.

Human noroviruses (HuNoVs) are highly contagious and a leading cause of epidemics of acute gastroenteritis worldwide. Among the various HuNoV genotypes, GII.4 is the most prevalent cause of outbreaks. However, no vaccines have been approved for HuNoVs to date. DNA vaccines are proposed to serve as an ideal platform against HuNoV since they can be easily produced and customized to express target proteins. In this study, we constructed a CMV/R vector expressing a major structural protein, VP1, of GII.4 HuNoV (CMV/R-GII.4 HuNoV VP1). Transfection of CMV/R-GII.4 HuNoV VP1 into human embryonic kidney 293T (HEK293T) cells resulted in successful expression of VP1 proteins in vitro. Intramuscular or intradermal immunization of mice with the CMV/R-GII.4 HuNoV VP1 construct elicited the production of blocking antibodies and activation of T cell responses against GII.4 HuNoV VP1. Our collective data support the utility of CMV/R-GII.4 HuNoV VP1 as a promising DNA vaccine candidate against GII.4 HuNoV.

Lipo-pam™ adjuvanted herpes zoster vaccine induces potent gE-specific cellular and humoral immune responses.

Herpes zoster (HZ), also known as shingles, is caused by the reactivation of latent varicella-zoster virus (VZV). Decreased VZV-specific T-cell immune responses significantly contribute to the development of HZ. Shingrix is a recombinant zoster vaccine that is currently used to prevent HZ. However, Shingrix has high reactogenicity and pain at the injection site due to QS21, one of the adjuvant components. In this study, we developed a new herpes zoster vaccine formulation called CVI-VZV-001, containing gE protein and a novel liposome-based adjuvant Lipo-pam™, which consists of two TLR agonists. We evaluated the immunogenicity of CVI-VZV-001 in mouse and rabbit models. CVI-VZV-001 elicited robust gE-specific T-cell immune responses and gE-specific antibody production. Specifically, CVI-VZV-001 induced polyfunctional CD4+ T cell populations that secrete multiple cytokines. Furthermore, CVI-VZV-001 sustained the gE-specific immune responses for up to six months after immunization. To ensure CVI-VZV-001's safety for further development, we conducted a good laboratory practice (GLP) toxicity test, which confirmed that CVI-VZV-001 is safe for use. At present, CVI-VZV-001 is undergoing phase I clinical trials. This study suggests that CVI-VZV-001 can be a potent candidate for the HZ vaccine with high immunogenicity and safety.

Identification and characterization of recent retrovirus in Rhinolophus ferrumequinum bats.

An investigation into retrovirus was conducted in six species of bats (Myotis aurascens, Myotis petax, Myotis macrodactylus, Miniopterus fuliginosus, Rhinolophus ferrumequinum, and Pipistrellus abramus) inhabiting South Korea. Exogenous retroviruses (XRVs) were detected in the tissue samples of R. ferrumequinum individuals by PCR assay. Proviruses were identified in all tissue samples through viral quantification using a digital PCR assay per organ (lung, intestine, heart, brain, wing, kidney, and liver), with viral loads varying greatly between each organ. In phylogenetic analysis based on the whole genome, the Korean bat retroviruses and the R. ferrumequinum retrovirus (RfRV) strain formed a new clade distinct from the Gammaretrovirus clade. The phylogenetic results determined these viruses to be RfRV-like viruses. In the Simplot comparison, Korean RfRV-like viruses exhibited relatively strong fluctuated patterns in the latter part of the envelope gene area compared to other gene areas. Several point mutations within this region (6,878-7,774 bp) of these viruses were observed compared to the RfRV sequence. One Korean RfRV-like virus (named Y4b strain) was successfully recovered in the Raw 264.7 cell line, and virus particles replicated in the cells were confirmed by transmission electron microscopy. RfRVs (or RfRV-like viruses) have been spreading since their first discovery in 2012, and the Korean RfRV-like viruses were assumed to be XRVs that evolved from RfRV.IMPORTANCER. ferrumequinum retrovirus (RfRV)-like viruses were identified in greater horseshoe bats in South Korea. These RfRV-like viruses were considered exogenous retroviruses (XRVs) that emerged from RfRV. Varying amounts of provirus detected in different organs suggest ongoing viral activity, replication, and de novo integration in certain organs. Additionally, the successful recovery of the virus in the Raw 264.7 cell line provides strong evidence supporting their status as XRVs. These viruses have now been identified in South Korea and, more recently, in Kenya since RfRV was discovered in China in 2012, indicating that RfRVs (or RfRV-like viruses) have spread worldwide.

Synergistic toxicity with copper contributes to NAT2-associated isoniazid toxicity.

Anti-tuberculosis (AT) medications, including isoniazid (INH), can cause drug-induced liver injury (DILI), but the underlying mechanism remains unclear. In this study, we aimed to identify genetic factors that may increase the susceptibility of individuals to AT-DILI and to examine genetic interactions that may lead to isoniazid (INH)-induced hepatotoxicity. We performed a targeted sequencing analysis of 380 pharmacogenes in a discovery cohort of 112 patients (35 AT-DILI patients and 77 controls) receiving AT treatment for active tuberculosis. Pharmacogenome-wide association analysis was also conducted using 1048 population controls (Korea1K). NAT2 and ATP7B genotypes were analyzed in a replication cohort of 165 patients (37 AT-DILI patients and 128 controls) to validate the effects of both risk genotypes. NAT2 ultraslow acetylators (UAs) were found to have a greater risk of AT-DILI than other genotypes (odds ratio [OR] 5.6 [95% confidence interval; 2.5-13.2], P = 7.2 × 10-6). The presence of ATP7B gene 832R/R homozygosity (rs1061472) was found to co-occur with NAT2 UA in AT-DILI patients (P = 0.017) and to amplify the risk in NAT2 UA (OR 32.5 [4.5-1423], P = 7.5 × 10-6). In vitro experiments using human liver-derived cell lines (HepG2 and SNU387 cells) revealed toxic synergism between INH and Cu, which were strongly augmented in cells with defective NAT2 and ATP7B activity, leading to increased mitochondrial reactive oxygen species generation, mitochondrial dysfunction, DNA damage, and apoptosis. These findings link the co-occurrence of ATP7B and NAT2 genotypes to the risk of INH-induced hepatotoxicity, providing novel mechanistic insight into individual AT-DILI susceptibility. Yoon et al. showed that individuals who carry NAT2 UAs and ATP7B 832R/R genotypes are at increased risk of developing isoniazid hepatotoxicity, primarily due to the increased synergistic toxicity between isoniazid and copper, which exacerbates mitochondrial dysfunction-related apoptosis.

Reactive oxygen species induce antiviral innate immune response through IFN-λ regulation in human nasal epithelial cells.

This study sought to explore the role of the IFN-related innate immune responses (IFN-ß and IFN-λ) and of reactive oxygen species (ROS) after influenza A virus (IAV) infection for antiviral innate immune activity in normal human nasal epithelial (NHNE) cells that are highly exposed to IAV. Passage-2 NHNE cells were inoculated with the IAV WSN/33 for 1, 2, and 3 days to assess the capacity of IFN and the relationship between ROS generation and IFN-λ secretion for controlling IAV infection. Viral titers and IAV mRNA levels increased after infection. In concert with viral titers, we found that the generation of IFNs, such as IFN-ß, IFN-λ1, and IFN-λ2/3, was induced after IAV infection until 3 days after infection. The induction of IFN-λ gene expression and protein secretion may be predominant after IAV infection. Similarly, we observed that intracellular ROS generation increased 60 minutes after IAV infection. Viral titers and mRNA levels of IAV were significantly higher in cases with scavenging ROS, in cases with an induced IFN-λ mRNA level, or where the secreted protein concentration of IFN-λ was attenuated after the suppression of ROS generation. Both mitochondrial and dual oxidase (Doux)2-generated ROS were correlated with IAV mRNA and viral titers. The inhibition of mitochondrial ROS generation and the knockdown of Duox2 gene expression highly increased IAV viral titers and decreased IFN-λ secretion. Our findings suggest that the production of ROS may be responsible for IFN-λ secretion to control IAV infection. Both mitochondria and Duox2 are possible sources of ROS generation, which is required to initiate an innate immune response in NHNE cells.

LOXL2 expression is associated with invasiveness and negatively influences survival in breast cancer patients.

Lysyl oxidase-like 2 (LOXL2) is associated with invasiveness and metastasis in breast cancer. We analyzed the prognostic impact of LOXL2 for breast cancer patients and investigated the role of LOXL2 in breast cancer cell lines. Immunohistochemical study of LOXL2 expression was done in samples from 309 patients. Survival analysis was performed using log-rank test and Cox regression hazard model. After identification of LOXL2 expression in breast cancer cell lines, we performed matrigel invasion and wound-healing assays with LOXL2-silenced cell lines. In the human study, LOXL2 was expressed in 16.2 % of patients. Comparing the LOXL2-positive versus negative groups, there was a significantly higher proportion of estrogen receptor-negative patients (54.0 vs. 37.0 %, respectively; p = 0.029) and triple-negative patients (34.0 vs. 18.0 %; p = 0.022) in the positive group. In multivariate analysis for overall survival and metastasis-free survival, positive LOXL2 was demonstrated as a poor prognostic factor (HR 2.27 and 2.10, respectively). In vitro study indicated that LOXL2 silencing induces a mesenchymal-epithelial transition-like process in basal cell lines (MDA-MB-231 and BT549) associated with decreased invasive and migratory properties. These clinical and preclinical data confirm that higher LOXL2 expression is associated with invasiveness of basal-like breast cancer cells and lower survival of breast cancer patients. Our results suggest the clinical value of LOXL2 as a therapeutic target in breast cancer.

Zinc inhibits osteoclast differentiation by suppression of Ca2+-Calcineurin-NFATc1 signaling pathway.

BACKGROUND: Zinc, an essential trace element, inhibits osteoclast differentiation in vitro and in vivo. The molecular mechanism for the inhibitory effect of zinc, however, is poorly understood. The purpose of this study was to investigate the effect of zinc and determine its molecular mechanism on receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis in mouse bone marrow-derived monocyte cells (BMMs) and RAW264.7 cells. RESULTS: In BMMs, zinc treatment during osteoclast differentiation decreased RANKL-induced osteoclast formation in a dose-dependent manner. We show that zinc suppressed the mRNA levels of nuclear factor of activated T-cells, cytoplasmic 1 (Nfatc1). Zinc also accumulated phospho-Nfatc1 (p-Nfatc1) in the cytosol in a dose-dependent manner and inhibited the translocation of Nfatc1 to the nucleus in RAW264.7 cells. Zinc suppressed the activities of Nfatc1 in the nucleus without changing the activities of NF-κB in RAW264.7 cells. In contrast, calcineurin activity decreased in response to zinc but its protein level was unchanged. RANKL-induced Ca2+ oscillations were inhibited by zinc treatment, but phospho-phospholipase Cγ1 (p-PLCγ1), the upstream signaling molecule of Ca2+ oscillations, was unaffected. Moreover, a constitutively active form of Nfatc1 obviously rescued suppression of osteoclastogenesis by zinc. CONCLUSIONS: Taken together, these results demonstrate for the first time that the inhibitory effect of zinc during osteoclastogesis is caused by suppressing the Ca2+-Calcineurin-NFATc1 signaling pathway. Thus, zinc may be a useful therapeutic candidate for the prevention of bone loss caused by NFATc1 activation in osteoclasts.

PCR-Based Detection and Genetic Characterization of Parainfluenza Virus 5 Detected in Pigs in Korea from 2016 to 2018.

This study applied a molecular-based method to detect parainfluenza virus 5 (PIV5) collected from 2016 to 2018 in nine provinces of Republic of Korea. We demonstrated that PIV5 was detectable in both serum and pooled organs at an average positive rate of 1.78% (99/5566). Among these, the complete genome sequence of 15,246 nucleotides was obtained for 12 field strains. Three out of the 12 strains had the lowest genetic identity (96.20-96.68%) among the 21 porcine PIV5 genomes collected in Germany, China, India, and Republic of Korea from 1998 to 2017. By analyzing a large collection of complete genome sequences of the structural protein-coding F and HN genes, this study proposed a classification of PIV5 into two lineages, 1 and 2, and identified that group 2.2.2 within sub-lineage 2.2 was substantially divergent. The evolution of two structural protein-coding genes was largely under purifying selection. A few codons (6/9 for the F gene, 7/8 for the HN gene) had elevated dN/dS values, which were loaded on internal branches and were predicted to be related to beneficial trait(s) of the virus.

Cell-to-cell contact with hepatitis C virus-infected cells reduces functional capacity of natural killer cells.

The distinct feature of hepatitis C virus (HCV) infection is a high incidence of chronicity. The reason for chronic HCV infection has been actively investigated, and impairment of innate and adaptive immune responses against HCV is proposed as a plausible cause. Whereas functional impairment of HCV-specific T cells is well characterized, the role and functional status of natural killer (NK) cells in each phase of HCV infection are still elusive. We therefore investigated whether direct interaction between NK cells and HCV-infected cells modulates NK cell function. HCV-permissive human hepatoma cell lines were infected with cell culture-generated HCV virions and cocultured with primary human NK cells. Cell-to-cell contact between NK cells and HCV-infected cells reduced NK cells' capacity to degranulate and lyse target cells, especially in the CD56(dim) NK cell subset, which is characterized by low-density surface expression of CD56. The decrease in degranulation capacity was correlated with downregulated expression of NK cell-activating receptors, such as NKG2D and NKp30, on NK cells. The ability of NK cells to produce and secrete gamma interferon (IFN-γ) also diminished after exposure to HCV-infected cells. The decline of IFN-γ production was consistent with the reduction of NK cell degranulation. In conclusion, cell-to-cell contact with HCV-infected cells negatively modulated functional capacity of NK cells, and the inhibition of NK cell function was associated with downregulation of NK-activating receptors on NK cell surfaces. These observations suggest that direct cell-to-cell interaction between NK cells and HCV-infected hepatocytes may impair NK cell function in vivo and thereby contribute to the establishment of chronic infection.