The development of a multiplex polymerase chain reaction (PCR) method for rapid and accurate detection and typing of herpes simplex virus type 1 (HSV-1), and type-2 (HSV-2), cytomegalovirus (CMV) and Epstein-Barr virus (EBV) is very important for clinical diagnosis to allow the deliver of therapy as early as possible. Large scale amplifications by multiplex PCR of viral DNA can lower the cost and time for viral diagnosis. In this study, therefore sensitive quadruplex PCR was achieved by optimizing parameters such as primers, and 1.5 mM magnesium and 200 uM dNTPs concentrations. The concentrations of HSV-1, HSV-2, CMV and EBV primers were 0.5, 0.3, 0.25 and 0.25 pmoles, respectively. Optimal annealing temperature was 54 degrees C. Employing these conditions, we could detect 10 copies of reconstructed template plasmid DNA, which were cloned to vectors containing target sequences of viral DNA. PCR products of 271 bp for HSV-1, 231 bp for HSV-2, 368 bp for CMV, and 326 bp for EBV were separated on 5.0% polyacrylamide gel electrophoresis and confirmed by direct sequencing. The present study showed that the quadruplex PCR assay described herein has potential application in clinical diagnosis, when rapid, accurate detection and typing of viruses HSV-1, HSV-2, CMV or EBV are necessary.
Cytomegalovirus/classification/*isolation & purification ; Herpesvirus 1, Human/classification/*isolation & purification ; Herpesvirus 2, Human/classification/*isolation & purification ; Herpesvirus 4, Human/classification/*isolation & purification ; Human ; *Polymerase Chain Reaction/methods ; Sensitivity and Specificity ; Support, Non-U.S. Gov't

BACKGROUND: The development of new drugs or alternative therapies effective against methicillin-resistant Staphylococcus aureus (MRSA) is of great importance, and various natural anti-MRSA products are good candidates for combination therapies. We evaluated the antibacterial activities of a Phellinus baumii ethyl acetate extract (PBEAE) and its synergistic effects with beta-lactams against MRSA. METHODS: The broth microdilution method was used to determine the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of the PBEAE. The PBEAE synergistic effects were determined by evaluating the MICs of anti-staphylococcal antibiotic mixtures, with or without PBEAE. Anti-MRSA synergistic bactericidal effects of the PBEAE and beta-lactams were assessed by time-killing assay. An ELISA was used to determine the effect of the PBEAE on penicillin binding protein (PBP)2a production. RESULTS: The MICs and MBCs of PBEAE against MRSA were 256-512 and 1,024-2,048 microg/mL, respectively. The PBEAE significantly reduced MICs of all beta-lactams tested, including oxacillin, cefazolin, cefepime, and penicillin. However, the PBEAE had little or no effect on the activity of non-beta-lactams. Time-killing assays showed that the synergistic effects of two beta-lactams (oxacillin and cefazolin) with the PBEAE were bactericidal in nature (Deltalog10 colony forming unit/mL at 24 hr: 2.34-2.87 and 2.10-3.04, respectively). The PBEAE induced a dose-dependent decrease in PBP2a production by MRSA, suggesting that the inhibition of PBP2a production was a major synergistic mechanism between the beta-lactams and the PBEAE. CONCLUSIONS: PBEAE can enhance the efficacy of beta-lactams for combined therapy in patients infected with MRSA.
Acetates/chemistry ; Agaricales/*chemistry/metabolism ; Anti-Infective Agents/chemistry/*pharmacology ; Drug Synergism ; Enzyme-Linked Immunosorbent Assay ; Methicillin-Resistant Staphylococcus aureus/*drug effects/metabolism ; Microbial Sensitivity Tests ; Penicillin-Binding Proteins/analysis/metabolism ; Plant Extracts/chemistry/*pharmacology ; beta-Lactams/*pharmacology

As of September 2020, SARS-CoV-2 has infected over 30 million people worldwide, and the death toll has now risen to 950,000. Given that Povidone-iodine (PVP-I) had consistently been showing the virucidal efficacy against various types of viruses, such as SARS-CoV, MERS-CoV, and Ebola, we conducted this study to figure out the virucidal effect against SARS-CoV-2 by using a viral plaque assay. We performed Kill-Time assays to assess the viral inactivation of SARS-CoV-2 contaminants after the application of the PVP-I product (Betadine® Throat Spray, PVP-I 0.45%). This test consisted of clean and dirty conditions and was designed to check the viral titers at a contact time of 60 seconds, which were evaluated by plaque-reduction rates in Vero cells. This PVP-I product fully achieved ≥4 log 10 reductions in viral titers under both clean and dirty conditions. This level of reduction, ≥4 log 10 (99.99%), in viral titers presented to be effective in terms of virucidal efficacy, according to the European standards, EN14476. This study revealed the virucidal efficacy of Betadine® Throat Spray against SARS-CoV-2 virus. Given that the convenience and availability of this product, we think that it may contribute to inhibit viral infection and transmissibility as an active type of personal protective equipment (PPE) by managing the hygiene of patients and medical professionals.

C-Terminal carboxyl methylation of a human placental 23 kDa protein catalyzed by membrane-associated methyltransferase has been investigated. The 23 kDa protein substrate methylated was partially purified by DEAE-Sephacel, hydroxyapatite and Sephadex G-100 gel filtration chromatographies. The substrate protein was eluted on Sephadex G-100 gel filtration chromatography as a protein of about 29 kDa. In the absence of Mg2+, the methylation was stimulated by guanine nucleotides (GTP, GDP and GTPgammaS), but in the presence of Mg2+, only GTPgammaS stimulated the methylation which was similar to the effect on the G25K/rhoGDI complex. AFC, an inhibitor of C-terminal carboxyl methylation, inhibited the methylation of human placental 23 kDa protein. These results suggests that the substrate is a small G protein different from the G25K and is methylated on C-terminal isoprenylated cysteine residue. This was also confirmed by vapor phase analysis. The methylated substrate protein was redistributed to membrane after in vitro methylation, suggesting that the methylation of this protein is important for the redistribution of the 23 kDa small G protein for its putative role in intracellular signaling.
Cysteine/metabolism* ; Female ; GTP-Binding Proteins/metabolism* ; Guanine Nucleotides/pharmacology ; Human ; Methylation ; Placenta/metabolism* ; Placenta/enzymology ; Pregnancy ; Pregnancy Proteins/metabolism* ; Protein Methyltransferases/metabolism*

Synovial fluid (SF) aspiration cytology is a useful diagnostic tool. For patients with gouty arthritis, the diagnosis is confirmed by the presence of monosodium uric acid (MSU) crystals in the SF, and these crystals are long, pointed ended and needle-shaped and they show strongly negative birefringence. Sometimes, it is difficult to diagnosis between gouty arthritis and other type of inflammatory arthritis. We experienced two unusual cases of gouty arthritis that we performed SF analysis for. The first patient was a 35 year old male who presented with relatively typical clinical symptoms with hyperuricemia, but the SF showed acute inflammatory cells without crystals on light microscopy. Only a few suspected crystals of MSU were identified on polarizing microscopy. The second patient was a 45 year old male who presented with atypical symptoms and pain and swelling of the left ankle and knee joint for 3 weeks. The uric acid level in the serum and urine was increased, but not over the normal limit. However, on light and polarizing microscopy, there were numerous MSU crystals in the SF. Conclusively, in some cases of gouty arthritis, the crystals are not identified on light microscopy or the uric acid level is not dramatically increased. So, the polarizing microscopy, the clinical information and the laboratory findings are all included in the work-up when evaluating the SF cytology of arthritis patients.
Animals ; Ankle ; Arthritis ; Arthritis, Gouty ; Birefringence ; Gout ; Humans ; Hyperuricemia ; Knee Joint ; Light ; Male ; Microscopy ; Synovial Fluid ; Uric Acid

Recently it was shown that single nucleotide polymorphisms (SNPs) can explain individual variation because of the small changes of the gene expression level and that the 50% decreased expression of an allele might even lead to predisposition to cancer. In this study, we found that a decreased expression of an allele might cause predisposition to genetic disease. Dopa responsive dystonia (DRD) is a dominant disease caused by mutations in GCH1 gene. The sequence analysis of the GCH1 in a patient with typical DRD symptoms revealed two novel missense mutations instead of a single dominant mutation. Family members with either of the mutations did not have any symptoms of DRD. The expression level of a R198W mutant allele decreased to about 50%, suggesting that modestly decreased expression caused by an SNP should lead to predisposition of a genetic disease in susceptible individuals.
Child ; Clubfoot/genetics ; Dopamine/deficiency ; Dystonic Disorders/drug therapy/enzymology/*genetics/physiopathology ; GTP Cyclohydrolase/*genetics/metabolism ; Genes, Recessive ; *Genetic Predisposition to Disease ; Humans ; Levodopa/administration & dosage ; Male ; Mutation, Missense ; Pedigree ; Polymorphism, Genetic

The methylation of a 23-kDa nuclear protein increased after partial hepatectomy and methylation returned to basal levels after the initial stage of regeneration. The methylating enzyme was partially purified from rat liver by ammonium sulfate precipitation, DEAE-anion exchange chromatography and Butyl-Sepharose chromatography. The 23-kDa protein was purified from a nuclear fraction of liver tissue with SP-Sepharose. When the 23-kDa protein was methylated with the partially purified methyltransferase and analyzed on C18 high performance liquid chromatography (HPLC), the methylated acceptor amino acid was monomethyl lysine (MML). Previously, only arginine N-methylation of specific substrate proteins has been reported during liver regeneration. However, in this report, we found that lysine N-methylation increased during early hepatic regeneration, suggesting that lysine N-methylation of the 23-kDa nuclear protein may play a functional role in hepatic regeneration. The methyltransferase did not methylate other proteins such as histones, hnRNPA1, or cytochrome C, suggesting the enzyme is a 23-kDa nuclear protein- specific lysine N-methyltransferase.
Animals ; Cytochromes c/metabolism ; DNA Helicases/metabolism ; Hepatectomy ; Histone-Lysine N-Methyltransferase/*metabolism ; Histones/metabolism ; Liver ; Liver Regeneration/*physiology ; Lysine/*metabolism ; Methylation ; Proteins/*metabolism ; Rats ; Rats, Sprague-Dawley ; Research Support, Non-U.S. Gov't

BACKGROUND: We examined cervicovaginal smears that contained definite low-grade squamous intraepithelial lesion (LSIL) cells and rare atypical cells suggestive of high-grade SIL (HSIL) (ASC-H) or contained borderline dysplastic cells between LSIL and HSIL. Such lesions were classified as LSIL-H. This study aimed to investigate the cytologic and histologic characteristics of LSIL-H category and we evaluated the associated clinical risk. METHODS: The histologic outcomes of LSIL-H were compared with those of LSIL and ASC-H. Both the cytologic and histologic findings of LSIL-H that were confirmed as cervical intraepithelial neoplasia 2 (CIN2) or greater (CIN2+) were investigated. RESULTS: LSIL-H accounted for 0.09% of the Pap tests. On the follow-up histology, the most frequent outcome was CIN2, and the risk of CIN2+ was higher than that for ASC-H. In the cases of LSIL-H that was histologically confirmed as CIN2+, most of the atypical cells suggestive of HSIL were cytologically similar to those of CIN2, and the corresponding cervical tissues were characterized by small CIN2+ lesions in a large background of flat condyloma/CIN1. The LSIL-H cases not confirmed on initial colposcopically-directed biopsy required further follow-up. CONCLUSIONS: LSIL-H may be a valid diagnostic category with distinctive features that are different from LSIL or ASC-H. LSIL-H needs further follow-up for the proper management.
Biopsy ; Cervical Intraepithelial Neoplasia ; Cervix Uteri ; Female ; Follow-Up Studies ; Vaginal Smears

BACKGROUND: We examined cervicovaginal smears that contained definite low-grade squamous intraepithelial lesion (LSIL) cells and rare atypical cells suggestive of high-grade SIL (HSIL) (ASC-H) or contained borderline dysplastic cells between LSIL and HSIL. Such lesions were classified as LSIL-H. This study aimed to investigate the cytologic and histologic characteristics of LSIL-H category and we evaluated the associated clinical risk. METHODS: The histologic outcomes of LSIL-H were compared with those of LSIL and ASC-H. Both the cytologic and histologic findings of LSIL-H that were confirmed as cervical intraepithelial neoplasia 2 (CIN2) or greater (CIN2+) were investigated. RESULTS: LSIL-H accounted for 0.09% of the Pap tests. On the follow-up histology, the most frequent outcome was CIN2, and the risk of CIN2+ was higher than that for ASC-H. In the cases of LSIL-H that was histologically confirmed as CIN2+, most of the atypical cells suggestive of HSIL were cytologically similar to those of CIN2, and the corresponding cervical tissues were characterized by small CIN2+ lesions in a large background of flat condyloma/CIN1. The LSIL-H cases not confirmed on initial colposcopically-directed biopsy required further follow-up. CONCLUSIONS: LSIL-H may be a valid diagnostic category with distinctive features that are different from LSIL or ASC-H. LSIL-H needs further follow-up for the proper management.
Biopsy ; Cervical Intraepithelial Neoplasia ; Cervix Uteri ; Female ; Follow-Up Studies ; Vaginal Smears

Brugada syndrome, an autosomal dominantly inherited form of ventricular fibrillation, is characterized by ST-segment elevation in leads V1-3 and right bundle-branch block on surface electrocardiogram. It is caused by mutations in the cardiac sodium channel gene, SCN5A, and to the best of our knowledge, there has been no report of this mutation in Korea. Three members of a family were heterozygous for a G to T substitution at the nucleotide position 5851 in exon 28 of the SCN5A gene. This nucleotide alteration makes a missense mutation, leading to a valine to leucine substitution (V1951L), in the carboxy terminal region of the sodium channel a subunit. We report here a missense mutation in a Korean family with Brugada-type electrocardiogram.
Brugada Syndrome ; Bundle-Branch Block ; Electrocardiography ; Exons ; Humans ; Korea ; Leucine ; Mutation, Missense* ; Sodium Channels ; Valine ; Ventricular Fibrillation