Stress-activated protein kinases are involved in the replication of porcine deltacoronavirus.

This study was conducted to examine the role of stress-activated protein kinases (SAPKs), including c-Jun NH2-terminal kinases (JNK1/2) and p38 mitogen-activated protein kinase (MAPK), in porcine deltacoronavirus (PDCoV) infection. Results demonstrated the activation of JNK1/2 and p38 MAPK in PDCoV-infected cells, which occurred concomitant with viral biosynthesis and irrespective of cell type. Pharmacological inhibition or knockdown of either SAPK significantly attenuated PDCoV replication, whereas addition of a signaling activator augmented virus infectivity. Moreover, pharmacological inhibition of JNK1/2 or p38 MAPK activation was innocuous to viral entry but significantly detrimental to post uncoating stages of the replication cycle. Remarkably, cytokine gene expression in PDCoV-infected IPEC-J2 cells was modified by inhibiting the activation of either SAPK. Collectively, these data indicate that JNK1/2 and p38 MAPK signaling pathways contribute to viral biosynthesis and regulate immune responses, thereby favoring the replication of PDCoV.

Preventive Intervention Program on the Outcomes of Very Preterm Infants and Caregivers: A Multicenter Randomized Controlled Trial.

Increased survival in the very preterm population results in a higher risk of developing neurodevelopmental and behavioral disabilities among survivors. We examined the outcomes of very preterm infants and parents after a preventive intervention program of four home visits by a specialized nurse, 5 days, 2 weeks, and 1 month after discharge, respectively, and at CA 2 months, followed by up to 12 times of group sessions between CA 3 and 6 months. Our multicenter randomized controlled trial assessed 138 preterm infants (gestational age ≤30 weeks or birth weight ≤1500 g) enrolled from the three participating hospitals. We randomly allocated the preterm babies to either the intervention or the control group. The primary outcome was the neurodevelopmental outcomes of Bayley-III scores at CA 10 and 24 months. At CA 10 months and 24 months, there were no significant differences between the intervention and control groups in the cognitive, motor, and language domains of Bayley-III scores. In addition, there were no significant differences in the mother's depression scale, mother-child attachment, and the modified Infant and Toddler Social and Emotional Assessment.

The Association of Ovarian Reserve with Exposure to Bisphenol A and Phthalate in Reproductive-aged Women.

BACKGROUND: Exposure to endocrine disrupting chemicals (EDCs) that influence the hormonal and homeostatic systems is known to be associated with gynecologic health risks in many countries. In this study, we evaluated exposure to EDCs associated with diminished ovarian reserve (DOR) and gynecologic health risks. METHODS: This cross-sectional study was performed from September 2014 to November 2014 and included 307 Korean reproductive-aged women. Anthropometric measurements, laboratory tests with urine and blood sampling and pelvic ultrasound examinations were performed. RESULTS: Urinary bisphenol A (BLA) level was significantly higher in the DOR group with anti-Müllerian hormone lower than 25 percentile (1.89 ± 2.17 ug/g and 1.58 ± 1.08 ug/g, P < 0.05). Urinary mono-(2-ethyl-5-hydroxyhexyl) phthalate, mono-(2-ethyl-5-oxohexyl) phthalate and mono-N-butyl phthalate, and substrates of phthalate were evaluated and no significant difference was observed between the DOR group and non-DOR group. Logistic regression analysis suggested an increase in infertility in high BPA exposure group and the odds ratio (OR, 4.248) was statistically significant after adjustment for age, birth control pills, and the age of menarche, parity, and waist circumference. High phthalate exposure was associated with endometrial polyp after adjustment (OR, 2.742). CONCLUSION: BPA exposure might be associated with DOR and infertility. Meanwhile, endometrial polyp is increased in women with high phthalate exposure. Therefore, the risk of exposures to EDCs for reproduction should be a matter of concern in reproductive-aged women.

Dietary supplement use in Korean children and adolescents, KNHANES 2015-2017.

OBJECTIVE: To date, there have been few studies on dietary supplement (DS) use in Korean children and adolescents, using nationally representative data. This study aimed to investigate the current status of DS use and its related factors, among Korean children and adolescents from the Korean National Health and Nutrition Examination Survey (KNHANES) data. DESIGN: A cross-sectional study. SETTING: Data from the KNHANES 2015-2017. Participants completed 24-h dietary recall interviews, including DS products that the subjects consumed. PARTICIPANTS: The study population was 4380 children and adolescents aged 1-18 years. RESULTS: Approximately 20.3 % of children and adolescents were using DS; the highest use was among children aged 1­3 years old, and the lowest use was among adolescents aged 16­18 years. The most frequently used DS was prebiotics/probiotics, followed by multivitamin/mineral supplements. Factors that were associated with DS use were lower birth weight in children aged <4 years; younger age, higher household income, regular breakfast intake and lower BMI in children aged 4-9 years; and regular breakfast intake and use of nutrition facts label in adolescents aged 10-18 years. Feeding patterns in infancy and having chronic diseases were not associated with DS use. CONCLUSIONS: We report that over 20 % of children and adolescents use DS. Nutritional education for parents and children about proper DS consumption is needed.

Novel lineage 1 recombinants of porcine reproductive and respiratory syndrome virus isolated from vaccinated herds: genome sequences and cytokine production profiles.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a widely disseminated, macrophage-tropic arterivirus that exhibits profound genetic and pathogenic heterogeneity. The present study was conducted to determine the complete genome sequences of two novel Korean lineage 1 PRRSV-2 strains, KNU-1901 and KNU-1902, which were isolated from vaccinated pig farms experiencing unusually high morbidity and mortality. Both isolates contained notable discontinuous 423-nucleotide deletions (DELs) within the genes encoding nonstructural protein 2 (nsp2) and GP3 when compared with the prototype strain VR-2332. In particular, the nsp2 DEL viruses had unique quadripartite discontinuous DEL signatures (111-1-19-9) in nsp2; this is an expanded version of the tripartite 111-1-19 DEL previously identified in virulent lineage 1 PRRSV-2 strains. Phylogenetic analysis revealed that both novel nsp2 DEL viruses belong to the Korean clade (KOR C) of lineage 1 isolates based on ORF5 but cluster with lineage KOR A strains based on the nsp2 or complete genome sequence. Recombination detection analysis suggested that both novel isolates are recombinants and may have evolved via natural inter-lineage recombination between circulating KOR A and KOR C strains. Interestingly, compared with the prototype VR-2332 virus, the novel nsp2 DEL variants were less efficient at promoting the expression of immune response genes in porcine alveolar macrophage culture. Taken together, we conclude that KNU-1901 and KNU-1902 are recently evolved recombinant variants of the virulent lineage 1 family that caused the regional severe PRRS outbreaks.

Porcine deltacoronavirus activates the Raf/MEK/ERK pathway to promote its replication.

Porcine deltacoronavirus (PDCoV) is a newly emerged swine coronavirus that causes acute enteritis in neonatal piglets. To date, little is known about the host factors or cellular signaling mechanisms associated with PDCoV replication. Since the Raf/MEK/ERK pathway is involved in modulation of various important cellular functions, numerous DNA and RNA viruses coopt this pathway for efficient propagation. In the present study, we found that PDCoV induces the activation of ERK1/2 and its downstream substrate Elk-1 early in infection irrespective of viral biosynthesis. Chemical inhibition or knockdown of ERK1/2 significantly suppressed viral replication, whereas treatment with an ERK activator increased viral yields. Direct pharmacological inhibition of ERK activation had no effect on the viral entry process but sequentially affected the post-entry steps of the virus life cycle. In addition, pharmacological sequestration of cellular or viral cholesterol downregulated PDCoV-induced ERK signaling, highlighting the significance of the cholesterol contents in ERK activation. However, ERK inhibition had no effect on PDCoV-triggered apoptosis through activation of the cytochrome c-mediated intrinsic mitochondrial pathway, suggesting the irrelevance of ERK activation to the apoptosis pathway during PDCoV infection. Altogether, our findings indicate that the ERK signaling pathway plays a pivotal role in viral biosynthesis to facilitate the optimal replication of PDCoV.

Trpm2 Ablation Accelerates Protein Aggregation by Impaired ADPR and Autophagic Clearance in the Brain.

TRPM2 a cation channel is also known to work as an enzyme that hydrolyzes highly reactive, neurotoxic ADP-ribose (ADPR). Although ADPR is hydrolyzed by NUT9 pyrophosphatase in major organs, the enzyme is defective in the brain. The present study questions the role of TRPM2 in the catabolism of ADPR in the brain. Genetic ablation of Trpm2 results in the disruption of ADPR catabolism that leads to the accumulation of ADPR and reduction in AMP. Trpm2-/- mice elicit the reduction in autophagosome formation in the hippocampus. Trpm2-/- mice also show aggregations of proteins in the hippocampus, aberrant structural changes and neuronal connections in synapses, and neuronal degeneration. Trpm2-/- mice exhibit learning and memory impairment, enhanced neuronal intrinsic excitability, and imbalanced synaptic transmission. These results respond to long-unanswered questions regarding the potential role of the enzymatic function of TRPM2 in the brain, whose dysfunction evokes protein aggregation. In addition, the present finding answers to the conflicting reports such as neuroprotective or neurodegenerative phenotypes observed in Trpm2-/- mice.

Cholesterol is important for the entry process of porcine deltacoronavirus.

The present study was conducted to examine whether cellular and/or viral cholesterol levels play a role in porcine deltacoronavirus (PDCoV) replication. Our results showed that depletion of cholesterol from cells or virions by treating them with methyl-ß-cyclodextrin (MßCD) diminished PDCoV infection in a dose-dependent manner. The addition of exogenous cholesterol to MßCD-treated cells or virions moderately restored PDCoV infectivity. Furthermore, the pharmacological sequestration of cellular or viral cholesterol efficiently blocked both virus attachment and internalization. Taken together, the current data indicate that the cholesterol present in the cell membrane and viral envelope contributes to PDCoV replication by acting as a key component in viral entry.

Clinical experience of long-term use of dienogest after surgery for ovarian endometrioma.

OBJECTIVE: Endometriosis is a common and recurring gynecologic disease which have afflicting females of reproductive age. We investigated the efficacy of long-term, post-operative use of dienogest for ovarian endometrioma. METHODS: We studied 203 patients who had undergone laparoscopic or robotic surgery for ovarian endometrioma, and were administrated dienogest 2 mg/day beginning in July of 2013, and continuing. We evaluated side effects of dienogest and ultrasonography was performed every 6 months to detect potential recurrence of endometrioma (2 cm) in these post-surgical patients. RESULTS: The follow-up observation periods were 30.2±20.9 months from surgery. The mean age was 34.1±7.2 years old. The mean diameter of pre-operative endometrioma was 5.6±3.0 cm2. One hundred eighty-two (89.7%) women received dienogest continuously for 12.0±7.1 months. Of the subjects, 21 (10.3%) patients discontinued dienogest at 2.4±1.0 months. The most common side effect when dienogest was discontinued was abnormal uterine bleeding. The occurrence rate of vaginal bleeding was 15.8%, a number which did not differ significantly in patients with/without post-operative gonadotropin releasing hormone agonist administration. The other side effects were gastrointestinal trouble including constipation, acne, headache, depression, hot flush, weight gain, and edema. However, no serious adverse events or side effects were documented and recurrent endometriomas were diagnosed in 3 patients (1.5%). CONCLUSION: The data indicates that dienogest was both tolerable and safe for long-term use as prophylaxis in an effort to obviate the recurrence of ovarian endometrioma post-operatively, as well as potential need for surgical re-intervention.

Cellular cholesterol is required for porcine nidovirus infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine epidemic diarrhea virus (PEDV) are porcine nidoviruses that are considered emerging and re-emerging viral pathogens of pigs that pose a significant economic threat to the global pork industry. Although cholesterol is known to affect the replication of a broad range of viruses in vitro, its significance and role in porcine nidovirus infection remains to be elucidated. Therefore, the present study was conducted to determine whether cellular or/and viral cholesterol levels play a role in porcine nidovirus infection. Our results showed that depletion of cellular cholesterol by treating cells with methyl-ß-cyclodextrin (MßCD) dose-dependently suppressed the replication of both nidoviruses. Conversely, cholesterol depletion from the viral envelope had no inhibitory effect on porcine nidovirus production. The addition of exogenous cholesterol to MßCD-treated cells moderately restored the infectivity of porcine nidoviruses, indicating that the presence of cholesterol in the target cell membrane is critical for viral replication. The antiviral activity of MßCD on porcine nidovirus infection was found to be predominantly exerted when used as a treatment pre-infection or prior to the viral entry process. Furthermore, pharmacological sequestration of cellular cholesterol efficiently blocked both virus attachment and internalization and, accordingly, markedly affected subsequent post-entry steps of the replication cycle, including viral RNA and protein biosynthesis and progeny virus production. Taken together, our data indicate that cell membrane cholesterol is required for porcine nidovirus entry into cells, and pharmacological drugs that hamper cholesterol-dependent virus entry may have antiviral potential against porcine nidoviruses.

A New Instrument for Measuring Tibial Torsion in Pediatric Patients.

OBJECTIVE: To develop and test the validity and reliability of a new instrument for measuring the thigh-foot angle (TFA) for the patients with in-toeing and out-toeing gait. METHODS: The new instrument (Thigh-Foot Supporter [TFS]) was developed by measuring the TFA during regular examination of the tibial torsional status. The study included 40 children who presented with in-toeing and out-toeing gaits. We took a picture of each case to measure photographic-TFA (P-TFA) in the proper position and to establish a criterion. Study participants were examined by three independent physicians (A, B, and C) who had one, three and ten years of experience in the field, respectively. Each examiner conducted a separate classical physical examination (CPE) of every participant using a gait goniometer followed by a TFA assessment of each pediatric patient with or without the TFS. Thirty minutes later, repeated in the same way was measured. RESULTS: Less experienced examiner A showed significant differences between the TFA values depending on whether TFS used (left p=0.003 and right p=0.008). However, experienced examiners B and C did not show significant differences. Using TFS, less experienced examiner A showed a high validity and all examiner's inter-test and the inter-personal reliabilities increased. CONCLUSION: TFS may increase validity and reliability in measuring tibial torsion in patients who has a rotational problem in lower extremities. It would be more useful in less experienced examiners.

Atypical Neonatal Marfan Syndrome with p.Glu1073Lys Mutation of FBN1: the First Case in Korea.

Neonatal Marfan syndrome (nMFS) is considered to be on the most severe end of the spectrum of type I fibrillinopathies. The common features of nMFS include ascending aortic dilatation, severe mitral and/or tricuspid valve insufficiency, ectopia lentis, arachnodactyly, joint contractures, crumpled ear, loose skin, and pulmonary emphysema.We describe a newborn male diagnosed with nMFS. He presented several atypical features, such as diaphragmatic eventration, severe hydronephrosis with hydroureter, and dilated cisterna magna. Molecular analysis revealed a missense mutation at nucleotide 3217 (c.3217G>A) in exon 26 of the fibrillin-1 (FBN1) gene, resulting in the substitution of a glutamate for a lysine at codon 1073 (E1073K) in the 12th calcium binding epidermal growth factor-like domain of the FBN1 protein. Here we report a rare case of Nmfs with several combined atypical features, such as diaphragmatic eventration, severe hydronephrosis with hydroureter, and dilated cisterna magna. Our report is the first atypical nMFS case with p.Glu1073Lys mutation of FBN1 in Korea and may help clinicians with the diagnosis and follow-up of atypical nMFS.

Relationship Between HbA1c and Complex Regional Pain Syndrome in Stroke Patients With Type 2 Diabetes Mellitus.

OBJECTIVE: To investigate the relationship between glycosylated hemoglobin A (HbA1c) and complex regional pain syndrome (CRPS) in stroke patients with type 2 diabetes mellitus (T2DM). METHODS: A retrospective chart review was performed of stroke patients from January 2012 to December 2013. We reviewed 331 patients and included 200 in the analysis. We divided them into CRPS and non-CRPS groups and compared them by age, gender, stroke lesion, cause of stroke, duration of T2DM, HbA1c (%), National Institutes of Health Stroke Scale score, affected shoulder flexor muscle strength, Fugl-Meyer Assessment score, motricity index, Functional Independence Measure, Korean version of Modified Barthel Index, blood glucose level on admission day, duration from stroke onset to HbA1c check, and duration from stroke onset to three-phase bone scan for CRPS diagnosis. Thereafter, we classified the patients into five groups by HbA1c level (group 1, 5.0%-5.9%; group 2, 6.0%-6.9%; group 3, 7.0%-7.9%; group 4, 8.0%-8.9%; and group 5, 9.0%-9.9%) and we investigated the difference in CRPS prevalence between the two groups. RESULTS: Of the 200 patients, 108 were in the CRPS group and 92 were in the non-CRPS group. There were significant differences in HbA1c (p<0.05) between the two groups but no significant differences in any other factors. Across the five HbA1c groups, there were significant differences in CRPS prevalence (p<0.01); specifically, it increased as HbA1c increased. CONCLUSION: This study suggests that higher HbA1c relates to higher CRPS prevalence and thus that uncontrolled blood glucose can affect CRPS occurrence in stroke patients with diabetes.

The Risk Factors and Pregnancy Outcomes of 48 Cases of Heterotopic Pregnancy from a Single Center.

The purpose of this study was to investigate risk factors that are associated with heterotopic pregnancy (HP) following in vitro fertilization (IVF)-embryo transfer (ET) and to demonstrate the outcomes of HP after the surgical treatment of ectopic pregnancies. Forty-eight patients from a single center, who were diagnosed with HP between 1998 and 2012 were included. All of the patients had received infertility treatments, such as Clomid with timed coitus (n = 1, 2.1%), superovulation with intrauterine insemination (n = 7, 14.6%), fresh non-donor IVF-ET (n = 33, 68.8%), and frozen-thawed cycles (n = 7, 14.6%). Eighty-four additional patients were randomly selected as controls from the IVF registry database. HP was diagnosed at 7.5 ± 1.2 weeks (range 5.4-10.3) gestational age. In six cases (12.5%), the diagnosis was made three weeks after the patients underwent treatment for abortion. There were significant differences in the history of ectopic pregnancy (22.5% vs. 3.6%, P = 0.002). There were no significant differences in either group between the rates of first trimester intrauterine fetal loss (15.0% vs. 13.1%) or live birth (80.0% vs. 84.1%) after the surgical treatment for ectopic pregnancy. The risk factors for HP include a history of ectopic pregnancy (OR 7.191 [1.591-32.513], P = 0.010), abortion (OR 3.948 [1.574-9.902], P = 0.003), and ovarian hyperstimulation syndrome (OHSS) (OR 10.773 [2.415-48.060], P = 0.002). In patients undergoing IVF-ET, history of ectopic pregnancy, abortion, and OHSS may be risk factors for HP as compared to the control group of other IVF patients. The surgical treatment of HP does not appear to affect the rates of first trimester fetal loss or live birth.

JNK and p38 mitogen-activated protein kinase pathways contribute to porcine epidemic diarrhea virus infection.

The mitogen-activated protein kinase (MAPK) pathways, which are central building blocks in the intracellular signaling network, are often manipulated by viruses of diverse families to favor their replication. Among the MAPK family, the extracellular signal-regulated kinase (ERK) pathway is known to be modulated during the infection with porcine epidemic diarrhea virus (PEDV); however, involvement of stress-activated protein kinases (SAPKs) comprising p38 MAPK and c-Jun NH2-terminal kinase (JNK) remains to be determined. Therefore, in the present study, we investigated whether activation of p38 MAPK and JNK cascades is required for PEDV replication. Our results showed that PEDV activates p38 MAPK and JNK1/2 up to 24h post-infection, whereas, thereafter their phosphorylation levels recede to baseline levels or even fall below them. Notably, UV-irradiated inactivated PEDV, which can enter cells but cannot replicate inside them, failed to induce phosphorylation of p38 MAPK and JNK1/2 suggesting that viral biosynthesis is essential for activation of these kinases. Treatment of cells with selective p38 or JNK inhibitors markedly impaired PEDV replication in a dose-dependent manner and these antiviral effects were found to be maximal during the early times of the infection. Furthermore, direct pharmacological inhibition of p38 MAPK or JNK1/2 activation resulted in a significant reduction of viral RNA synthesis, viral protein expression, and progeny release. However, independent treatments with either SAPK inhibitor did not inhibit PEDV-induced apoptotic cell death mediated by activation of mitochondrial apoptosis-inducing factor (AIF) suggesting that SAPKs are irrelevant to the apoptosis pathway during PEDV infection. In summary, our data demonstrated critical roles of the p38 and JNK1/2 signaling pathways in facilitating successful viral infection during the post-entry steps of the PEDV life cycle.

Measuring levels of biogenic amines and their metabolites in rat brain tissue using high-performance liquid chromatography with photodiode array detection.

We developed a method to detect biogenic amines and their metabolites in rat brain tissue using simultaneous high-performance liquid chromatography and a photodiode array detection. Measurements were made using a Hypersil Gold C-18 column (250 × 2.1 mm, 5 µm). The mobile phase was 5 mM perchloric acid containing 5 % acetonitrile. The correlation coefficient was 0.9995-0.9999. LODs (S/N = 3) and LOQs (S/N = 10) were as follows: dopamine 0.4 and 1.3 pg, 3, 4-dihydroxyphenylacetic acid 8.4 and 28.0 pg, serotonin 0.4 and 1.3 pg, 5-hydroxyindolacetic acid 3.4 and 11.3 pg, and homovanillic acid 8.4 and 28.0 pg. This method does not require derivatization steps, and is more sensitive than the widely used HPLC-UV method.