Evaluation of the Variability of the ORF34, ORF68, and MLST Genes in EHV-1 from South Korea.

Equine herpesvirus-1 (EHV-1) is an important pathogen in horses. It affects horses worldwide and causes substantial economic losses. In this study, for the first time, we characterized EHV-1 isolates from South Korea at the molecular level. We then aimed to determine the genetic divergences of these isolates by comparing them to sequences in databases. In total, 338 horse samples were collected, and 12 EHV-1 were isolated. We performed ORF30, ORF33, ORF68, and ORF34 genetic analysis and carried out multi-locus sequence typing (MLST) of 12 isolated EHV-1. All isolated viruses were confirmed as non-neuropathogenic type, showing N752 of ORF30 and highly conserved ORF33 (99.7-100%). Isolates were unclassified using ORF68 analysis because of a 118 bp deletion in nucleotide sequence 701-818. Seven EHV-1 isolates (16Q4, 19R166-1, 19R166-6, 19/10/15-2, 19/10/15-4, 19/10/18-2, 19/10/22-1) belonged to group 1, clade 10, based on ORF34 and MLST analysis. The remaining 5 EHV-1 isolates (15Q25-1, 15D59, 16Q5, 16Q40, 18D99) belonged to group 7, clade 6, based on ORF34 and MLST analysis.

Effects of Dangguixu-san in patients with acute lateral ankle sprain: a randomized controlled trial.

BACKGROUND: Dangguixu-san (DS), a herbal extract, is widely used in Korean medicine to treat pain and swelling caused by ankle sprain. However, there is insufficient evidence regarding the effects of DS on ankle sprains. Accordingly, we assessed the efficacy and safety of DS for the treatment of acute lateral ankle sprain (ALAS). METHODS: This study was a multicenter (two Korean hospitals), randomized, double-blind, placebo-controlled, parallel-arm clinical trial with a 1:1 allocation ratio that included a per-protocol analysis and sub-analysis based on symptom severity. Forty-eight participants (n = 28 at Semyung University Korean Medicine Hospital in Chungju; n = 20 at DongShin University Gwangju Korean Medicine Hospital) with grade I or II ALAS that occurred within 72 h before enrollment were randomized to a DS (n = 24) or placebo (n = 24) group. Both groups received acupuncture treatment once daily for 5 consecutive days and the trial medication (DS/placebo capsule) three times a day for 7 consecutive days. Primary (visual analog scale [VAS] scores for pain) and secondary (Foot and Ankle Outcome Scores [FAOS], edema, and European Quality of Life Five-Dimension-Five-Level Scale [EQ-5D-5L] scores) outcome measures were recorded at baseline (week 0), the end of the intervention (week 1), and 4 weeks after treatment completion (week 5). RESULTS: Forty-six participants completed the trial (n = 23 each). Changes in VAS scores, FAOS Symptom/Rigidity, and FAOS Ache from week 1 to week 5 showed significant differences between the two groups. Sub-analyses showed significant differences in changes of FAOS Ache (week 0 to week 5) and VAS scores, total FAOS, and EQ-5D-5L scores (week 1 to week 5) between the two subgroups (grade II). There were no adverse events and significant negative changes in clinical laboratory parameters in both groups. CONCLUSIONS: Overall, the results of this study are in favor of DS combined with acupuncture and suggest that DS combined with acupuncture is a safe treatment with positive long-term effects in terms of pain reduction and symptom alleviation in patients with grade I or II ALAS. TRIAL REGISTRATION: Clinical Research Information Service KCT0002374 . Registered on July 11, 2017; retrospectively registered.

Efficacy of nano-particulated, water-soluble erlotinib against intracranial metastases of EGFR-mutant lung cancer.

Central nervous system (CNS) metastasis is one of the serious complications of epidermal growth factor receptor (EGFR)-mutant lung cancer, which arises due to poor penetration of the brain-blood barrier by EGFR-tyrosine kinase inhibitors (EGFR-TKIs). Although osimertinib, a third-generation EGFR-TKI, has efficacy against CNS metastases, further treatment modalities are still needed as some of these lesions do not respond to osimertinib, or undergo progression after an initial response to this drug if radiotherapy has already been conducted. Here, we investigated the efficacy of water-soluble erlotinib (NUFS-sErt) against these metastases. This agent was synthesized using a nano-particulation platform technology utilizing fat and supercritical fluid (NUFS™) to resolve the low solubility problem that typically prevents the creation of injectable forms of EGFR-TKIs. The average NUFS-sErt particle size was 236.4 nm, and it showed time-dependent dissolution in culture media. The effects of NUFS-sErt were similar to those of conventional erlotinib in terms of inhibiting the proliferation of EGFR-mutant lung cancer cells and suppressing EGFR signaling. In an intraperitoneal xenograft model of HCC827 cells, intraperitoneal administration of NUFS-sErt produced a dose-dependent inhibition of tumor growth and enhanced survival rate. Notably, the injection of NUFS-sErt into the brain ventricle caused significant tumor growth inhibition in an intracranial xenograft model. Hence, our current findings indicate that NUFS-sErt is a novel, water-soluble form of erlotinib that can be administered using intraventricular or intrathecal injections. The target cases would be patients with a progressive CNS metastasis and no other therapeutic options. This drug could also be given intravenously to patients with swallowing difficulties or an inability to ingest due to a medical condition.

Effects of Dangguixu-san on acute lateral ankle sprain: study protocol for a randomized controlled trial.

BACKGROUND: Ankle sprain is a common musculoskeletal injury. In Korean medicine, blood stasis is thought to be the main cause of pain and swelling in patients with ankle sprain. Dangguixu-san (DS), a herbal extract, is widely used in Korean medicine for the treatment of traumatic ecchymosis and pain by promoting blood circulation and relieving blood stasis. However, the effects of DS on ankle sprain have not been evaluated in a randomized clinical trial. Here, we describe the protocol for a randomized controlled trial that will evaluate the efficacy and safety of DS for the treatment of ankle sprain. METHODS/DESIGN: In this randomized, double-blinded, placebo-controlled, parallel-arm clinical trial with a 1:1 allocation ratio, participants (n = 48) with acute lateral ankle sprain (ALAS) that occurred within 72 h before enrollment will be randomly assigned to a DS (n = 24) or a placebo (n = 24) group. Both groups will receive acupuncture treatment once a day for 5 days a week (excluding Saturday and Sunday) and the trial medication (DS/placebo capsule) three times a day for seven consecutive days. The primary outcome measure will be pain relief evaluated using a Visual Analog Scale (VAS). Secondary outcome measures will include Foot and Ankle Outcome Scores (FAOS), edema, European Quality of Life Five-Dimension-Five-Level Scale (EQ-5D-5 L) scores, and the number of recurrent ankle sprains. VAS, FAOS, edema, and EQ-5D-5 L scores will be recorded before, at the end of, and at 4 weeks after treatment completion. EQ-5D-5 L scores will be additionally recorded at 26 weeks after treatment completion. The number of recurrent ankle sprains will be recorded at 4, 8, 12, and 26 weeks after treatment completion. DISCUSSION: This study is expected to provide evidence regarding the efficacy, safety, and usefulness of DS for the treatment of ALAS. TRIAL REGISTRATION: cris.nih.go.kr, registration number: KCT 0002374 . Registered on 11 July, 2017 and approved by the Ministry of Food and Drug Safety (registration number, 31244).

The Electrical Properties for Phenolic Isocyanate-Modified Bisphenol-Based Epoxy Resins Comprising Benzoate Group.

Epoxy resin has been required to have a low dielectric constant (D(k)), low dissipation factor (Df), low coefficient of thermal expansion (CTE), low water absorption, high mechanical, and high adhesion properties for various applications. A series of novel phenolic isocyanate-modified bisphenol-based epoxy resins comprising benzoate group were prepared for practical electronic packaging applications. The developed epoxy resins showed highly reduced dielectric constants (D(k)-3.00 at 1 GHz) and low dissipation values (Df-0.014 at 1 GHz) as well as enhanced thermal properties.

A Retrospective Analysis of Patients' Conditions Using Acupuncture in a Traditional Korean Medicine Hospital.

Objective. The aim of this study was to identify the patient demographics, health issues, and type of acupuncture treatments who visited a traditional Korean medical hospital for acupuncture treatment. Methods. We retrospectively analysed the data using the electronic medical records (EMRs) of patients treated with at least one treatment of acupuncture from 1 January 2010 to December 2012 in the Chung-Ju Korean hospital at Semyung University. Results. The total number of identified patients was 1189 inpatients and 10138 outpatients. The 50-59 age group received acupuncture treatment in the hospital the most, followed by the 40-49 age group. Among the patients undergoing acupuncture treatment because of a diagnosis of pain, 82.74% were outpatients and 72.85% were inpatients. Additionally, all patients with a spine condition received acupuncture treatment. The most common musculoskeletal conditions of patients at the traditional Korean medicine (TKM) hospital were associated with spine conditions, such as low back pain and neck pain. Various treatments have been performed at the hospital in conjunction with acupuncture. The study results show a high prevalence of acupuncture treatment for diagnosed diseases. Conclusion. Our study suggests the need to investigate additional TKM hospitals to analyse characteristics of patients who received specific treatments. Analysis of the characteristics of patients treated with Korean acupuncture at the TKM hospital in this study will help future researchers who want to implement strong clinical evidence. However, we cannot completely discount all symptoms because of the retrospective nature of this study, and only one hospital was used, which limits the generalisation of our findings.

Seroprevalence of five arboviruses in sentinel cattle as part of nationwide surveillance in South Korea, 2009-2012.

To investigate the possible circulation of arboviruses in South Korea, nationwide surveillance of five arbovirues was conducted in sentinel calves during 2009-2012. We used serum neutralization tests to investigate the presence of antibodies for the Aino virus, Akabane virus, bovine ephemeral fever virus, Chuzan virus and Ibaraki virus. In 2009, 2011 and 2012, the seropositive rates for these five arboviruses were all less than 14.1%. In 2010, however, the seropositive rates for Aino virus and Akabane virus were 33.2% and 40.2%, respectively. High seropositive rates were also associated with a large-scale outbreak of Akabane viral encephalomyelitis in cattle in southern Korea in 2010. Continued seroprevalence surveillance will be useful for monitoring natural arboviral diseases.

Effects of Wonli Acupuncture Procedure in Patients with LSS: A Clinical, Retrospective Study.

Background. Lumbar spinal stenosis (LSS) is a disease with increasing prevalence due to prolongation of average life span. Despite various treatment methods, many limitations remain unsolved. Objective. We are reporting cases of patients who have been treated with Wonli Acupuncture, a method of treating LSS by directly approaching the intervertebral foramen and interlaminar space with acupuncture needles different from those used in original acupuncture. Methods. A total of 82 patients with LSS were treated with Wonli Acupuncture, and out of those, 47 patients without exclusion criteria were selected for the following research. We compared the pretreatment VAS and ODI scores based on 1-year follow-up measurements. Results. The ODI value dropped by 15.3 ± 24.8 on average (from 35.2 ± 19.9 at the baseline to 19.8 ± 20.6 at the reading) (P < 0.01) and the average VAS also dropped by 19.2 ± 37.2 (from 60.7 ± 23.1 at baseline to 41.5 ± 31.9 at the reading) (P < 0.01). Conclusions. Wonli Acupuncture was found to have clinical efficacy for lumbar spinal stenosis.

Effects of Salviae Miltiorrhizae Radix Hot Aqueous Extract on Nitric Oxide and Prostaglandin E2 Production and on 1,1-diphenyl-2-picryl hydrazyl Radical Scavenging in Macrophages.

OBJECTIVES: The objective of this study is to investigate the effects of Salviae Miltiorrhizae Radix hot aqueous extract on nitric oxide (NO) and prostaglandin E2 (PGE2 ) production and on 1,1-diphenyl-2-picryl hydrazyl (DPPH) free-radical scavenging in macrophages. METHODS: Salviae Miltiorrhizae Radix(300 g) was heated at 100℃ with distilled water (2 L) for 4 hours. The extract was filtered and concentrated to 100 mL by using a rotary evaporator, was frozen at -80℃, and was then freeze-dried by using a freezing-drying system. The RAW 264.7 macrophage was subcultured by using 10-㎍/mL lipopolysaccharide (LPS). In order to evaluate cytotoxicity, we performed (3-(4,5-dimrthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assays and measured the cell viability. The NO production was measured by using Griess assays, and the PGE2 production was measured by using enzyme immunoassays. The antioxidant activity, the DPPH free-radical scavenging capability, was measured by using the DPPH method. RESULTS: Cell viability with the 1-, 5-, 25-, 125- and 625-㎍/mL Salviae Miltiorrhizae Radix hot aqueous extract was not significantly decreased compared to the cell viability without the extract. When 125 and 625 ㎍/mL of Salviae Miltiorrhizae Radix hot aqueous extract were used, NO production in LPS-stimulated RAW 264.7 macrophages was significantly inhibited compared to that in the control group. When 25, 125, and 625 ㎍/mL of Salviae Miltiorrhizae Radix hot aqueous extract were used, PGE2 production in LPS-stimulated RAW 264.7 macrophages was significantly inhibited compared to that in the control group. The 125- and 625-㎍/mL Salviae Miltiorrhizae Radix hot aqueous extracts had high DPPH free-radical scavenging capabilities in RAW 264.7 macrophages. CONCLUSION: This study indicates that Salviae Miltiorrhizae Radix hot aqueous extract suppresses NO and PGE2 production and improves DPPH free-radical scavenging capability. Thus, it seems that Salviae Miltiorrhizae Radix hot aqueous extract may have an anti-inflammation effect and antioxidant activity.

Single Dose Toxicity of Chukyu (spine-healing) Pharmacopuncture Injection in the Muscle of Rats.

OBJECTIVES: This study was performed to analyze the single dose toxicity of Chukyu (spine-healing) pharmacopuncture. METHODS: All experiments were conducted at the Biotoxtech, an institution authorized to perform non-clinical studies under the regulations of Good Laboratory Practice (GLP) regulations. Sprague-Dawley rats were chosen for the pilot study. Doses of Chukyu (spine-healing) pharmacopuncture, 0.1, 0.5 and 1.0 mL, were administered to the experimental groups, and a dose of normal saline solution, 1.0 mL, was administered to the control group. This study was conducted under the approval of the Institutional Animal Ethic Committee. RESULTS: No deaths or abnormalities occurred in any of the four groups. No significant changes in weight, hematological parameters or clinical chemistry between the control group and the experimental groups were observed. To check for abnormalities in organs and tissues, we used microscopy to examine representative histological sections of each specified organ; the results showed no significant differences in any of the organs or tissues except in one case, where interstitial infiltrating macrophages were found in one female rat in the 0.5-mL/animal experimental group. CONCLUSION: The above findings suggest that treatment with Chukyu (spine-healing) pharmacopuncture is relatively safe. Further studies on this subject are needed to yield more concrete evidence.

Single-dose Intramuscular Toxicity of Neutral Natured Blood Stasis Pharmacopuncture in Sprague-Dawley Rats.

OBJECTIVES: This study was performed to analyze the single-dose toxicity of neutral natured blood stasis pharmacopuncture extracts. METHODS: All experiments were conducted at Biotoxtech, an institution authorized to perform non-clinical studies, under the regulations of Good Laboratory Practice (GLP). Sprague-Dawley rats were chosen for the pilot study. Doses of neutral natured blood stasis pharmacopuncture extracts, 0.1, 0.5 and 1.0 mL, were administered to the experimental group, and the same doses of normal saline solution were administered to the control group. This study was conducted under the approval of the Institutional Animal Ethics Committee. RESULTS: In all 4 groups, no deaths occurred, and the neutral natured blood stasis pharmacopuncture extracts administered by intramuscular (IM) injection was over 1.0 mL/animal. No significant changes in the body weights between the control group and the experimental group were observed. To check for abnormalities in organs and tissues, we used microscopy to examine representative histological sections of each specified organ; the results showed no significant differences in any organs or tissues. CONCLUSION: The above findings suggest that treatment with neutral natured blood stasis pharmacopuncture extracts is relatively safe. Further studies on this subject should be conducted to yield more concrete evidence.

A Pilot Study on Single-dose Toxicity Testing of Scolopendrid Pharmacopuncture in Sprague-Dawley Rats.

OBJECTIVES: This study was performed to analyze single dose toxicity and the lethal dose of Scolopendrid Pharmacopuncture in rats. METHODS: All experiments were conducted at the Korea Testing & Research Institute (KTR), an institution authorized to perform non-clinical studies, under the regulations of Good Laboratory Practice (GLP). Sprague-Dawley rats were chosen for the pilot study. Doses of Scolopendrid pharmacopuncture, 0.1, 0.5, and 1.0 mL, were administered to the experimental group, and 1.0 mL doses of normal saline solution were administered to the control group. This study was conducted under the approval of the Institutional Animal Ethic Committee. RESULTS: No deaths or abnormalities occurred in any of the groups. No significant changes in the weight, hematological parameters or clinical chemistry were noted between the control group and the experimental group. To check for abnormalities in organs and tissues, we used microscopy to examine representative histological sections of each specified organ; the results showed no significant differences in any of the organs or tissues. CONCLUSION: The above findings suggest Scolopendrid Pharmacopuncture is a relatively safe to use for treatment. Further studies on the subject should be conducted to yield more concrete evidence.

Comparing the effects of individualized, standard, sham and no acupuncture in the treatment of knee osteoarthritis: a multicenter randomized controlled trial.

BACKGROUND: Acupuncture is an effective yet complex therapy, integrating syndrome differentiation, selection of appropriate acupoints and skillful needling techniques. Clinicians carefully tailor acupuncture treatment to each patient. However, most clinical trials of acupuncture have been based on a standardized formula of points for every patient without properly accounting for individualdifferences and, as a result, have not been reflective of the true efficacy of clinical practice. To determine the efficacy of meridian-based syndrome differentiation and Sa-am acupuncture, we have designed a simple pragmatic trial providing individualized treatments while working within a general framework. METHODS/DESIGN: The study is designed to be a parallel, patient- and assessor-blind, randomized controlled trial (RCT). A total of250 patients with knee osteoarthritis (OA) will be recruited from two independent hospitals, Semyung University Oriental Medicine Hospital in Chung-ju and Dongguk University Oriental Hospital in Ilsan, South Korea. Patients will be randomly allocated into four treatment groups: 1. individualized, meridian-based syndrome differentiation and Sa-am acupuncture treatment;2. standard acupuncture treatment;3. sham acupuncture treatment; and 4. no acupuncture treatment. Patients in groups 1 to 3 will be treated by certified oriental medicine doctors twice a week for 6 weeks. The primary outcome measure will be the self-reported total Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score change. The trial will also include secondary outcome measures. DISCUSSION: This trial is designed to determine the efficacy of individualized acupuncture treatment in patients with knee OA by comparing the differences between individualized, standard, sham and no acupuncture treatments. The results of this trial may validate the efficacy of individualized acupuncture therapy, encouraging its widespread use. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01569230.

Bovine papular stomatitis virus (BPSV) infections in Korean native cattle.

An outbreak of a disease with parapox-like symptoms was reported in South Korea in April 2012. Three of 45 Korean native cattle, age 20-24 months, were affected. Parapoxviruses were detected and identified by electron microscopy and polymerase chain reaction (PCR). To determine the genetic characteristics of the Korean strains, the sequence of the major envelope protein (B2L) was determined and compared with published reference sequences. Phylogenetic analysis revealed that the parapoxvirus strains were closely related to not only isolates from Japan, but also isolates from Germany, Sudan and the United states. This is the first report on an outbreak and the molecular characterization of BPSV in Korea.

Study on a Single-Dose Toxicity Test of D-Amino Acid Oxidase (DAAO) Extracts Injected into the Tail Vein of Rats.

OBJECTIVE: This study was performed to analyze the single-dose toxicity of D-amino acid oxidase (DAAO) extracts. METHODS: All experiments were conducted at the Korea Testing & Research Institute (KTR), an institution authorized to perform non-clinical studies, under the regulations of Good Laboratory Practice (GLP). Sprague-Dawley rats were chosen for the pilot study. Doses of DAAO extracts, 0.1 to 0.3 cc, were administered to the experimental group, and the same doses of normal saline solution were administered to the control group. This study was conducted under the approval of the Institutional Animal Ethics Committee. RESULTS: In all 4 groups, no deaths occurred, and the LD50 of DAAO extracts administered by IV was over 0.3 ml/kg. No significant changes in the weight between the control group and the experimental group were observed. To check for abnormalities in organs and tissues, we used microscopy to examine representative histological sections of each specified organ, the results showed no significant differences in any organs or tissues. CONCLUSION: The above findings suggest that treatment with D-amino acid oxidase extracts is relatively safe. Further studies on this subject should be conducted to yield more concrete evidence.

Molecular characterization of a Korean bovine parainfluenza virus type 3 isolate.

Bovine parainfluenza virus type 3 (BPIV-3) was isolated from Korean native cattle that presented clinical signs of mild pneumonia. The complete genome of a representative isolate (12Q061) was sequenced. The newly identified strain, which was found to be distinct from the previously reported genotypes A (BPIV-3a) and B (BPIV-3b) and closely related to the Chinese strain SD0835, was tentatively classified as genotype C (BPIV-3c). Our results suggest a relationship between BPIV-3 genetic variation and the geographic location of its isolation. Identification of these new BPIV-3 genotypes may facilitate the development of improved diagnostic methods and vaccines. This is to our knowledge the first report of the identification and molecular characterization of BPIV-3 in Korea.

Comparison of the modified subvastus and medial parapatellar approaches in total knee arthroplasty.

In this paper, we evaluated the difference between the modified subvastus approach and the medial parapatellar approach in total knee arthroplasty(TKA). We assessed the time of active straight-leg raise (SLR) post-operatively and the range of flexion of the operated knee at the tenth post-operative day, 6 weeks and 6 months, 12 months and 3 years. We investigated the degree of the patellar tilt and subluxation 24 months post-operatively. The patients who underwent the modified subvastus approach performed active SLR earlier (mean 0.5 days) than the medial parapatellar approach patients (mean 2.2 days). Knee flexion was better at the tenth post-operative day in the modified subvastus approach group compared to the medial parapatellar approach group. There was no statistical difference between the two groups with regard to the patellar tilt and subluxation. We conclude that the modified subvastus approach is recommendable in primary TKA.

Tetra-µ-benzoato-bis-[(quinoxaline)copper(II)].

The paddlewheel-type centrosymmetric dinuclear title complex, [Cu(2)(C(7)H(5)O(2))(4)(C(8)H(6)N(2))(2)], contains four bridging benzoate groups and two terminal quinoxaline ligands. The octa-hedral coordination around each Cu atom, with four O atoms in the equatorial plane, is completed by an N atom of a quinoxaline mol-ecule [Cu-N = 2.2465 (18) Å] and by the second Cu atom [Cu⋯Cu = 2.668 (5) Å]. The Cu atom is 0.216 Šout of the plane of the four O atoms.

Biomimetic hydrocarbon oxidation catalyzed by nonheme iron(III) complexes with peracids: evidence for an Fe(V)=O species.

Mononuclear nonheme iron(III) complexes of tetradentate ligands containing two deprotonated amide moieties, [Fe(Me(2)bpb)Cl(H(2)O)] (3 a) and [Fe(bpc)Cl(H(2)O)] (4 a), were prepared by substitution reactions involving the previously synthesized iron(III) complexes [Et(3)NH][Fe(Me(2)bpb)Cl(2)] (3) and [Et(3)NH][Fe(bpc)Cl(2)] (4). Complexes 3 a and 4 a were characterized by IR and elemental analysis, and complex 3 a also by X-ray crystallography. Nonheme iron(III) complexes 3, 3 a, 4, and 4 a catalyze olefin epoxidation and alcohol oxidation on treatment with m-chloroperbenzoic acid. Pairwise comparisons of the reactivity of these complexes revealed that the nature of the axial ligand (Cl(-) versus H(2)O) influences the yield of oxidation products, whereas an electronic change in the supporting chelate ligand has little effect. Hydrocarbon oxidation by these catalysts was proposed to involve an iron(V) oxo species which is formed on heterolytic O-O bond cleavage of an iron acylperoxo intermediate (FeOOC(O)R). Evidence for this iron(V) oxo species was derived from KIE (k(H)/k(D)) values, H(2) (18)O exchange experiments, and the use of peroxyphenylacetic acid (PPAA) as the peracid. Our results suggest that an Fe(V)=O moiety can form in a system wherein the supporting chelate ligand comprises a mixture of neutral and anionic nitrogen donors. This work is relevant to the chemistry of mononuclear nonheme iron enzymes that are proposed to oxidize organic substrates via reaction pathways involving high-valent iron oxo species.