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Objective Dentin sialophosphoprotein ( Dspp ) is expressed in odontoblasts and transiently expressed in early ameloblasts. However, the origin of Dspp in ameloblasts remains unclear. Our previous studies demonstrated that copine-7 (CPNE7), a molecule that is secreted by the dental epithelium, is expressed in early ameloblasts and is then translocated to differentiating odontoblasts; its expression levels correlate with odontoblast differentiation under the control of Dspp expression. The objective of this study is to figure out the relationship between CPNE7 and Dspp during amelogenesis. Design The gene expression patterns of CPNE7 and dentin sialoprotein (DSP) were examined by immunohistochemistry, western blot analysis, and real-time polymerase chain reaction. The effects of CPNE7 on Dspp regulation were investigated using luciferase and chromatin immunoprecipitation assays in ameloblastic HAT-7 cells. Results The gene expression pattern of Cpne7 was similar to that of Dspp during ameloblast differentiation. Moreover, Gene expression omnibus profiles indicated that there is a close correlation between Cpne7 and Dspp expression in various normal human tissues. We also confirmed the effects of CPNE7 on the induction of Dspp in ameloblastic HAT-7 cells. Cpne7 overexpression promoted Dspp expression, whereas Dspp expression was down-regulated by Cpne7 inactivation. Conclusions These results suggest that the expression of Dspp in early amelogenesis is linked to CPNE7, a preameloblast-derived factor. [ABSTRACT FROM AUTHOR]

Objective MicroRNAs (miRNAs), small noncoding RNA molecules, are involved in the pathogenesis of various diseases such as cancer and arthritis. The aim of this study was to determine whether miR-127-5p regulates interleukin-1β (IL-1β)-induced expression of matrix metalloproteinase 13 (MMP-13) and other catabolic factors in human chondrocytes. Methods Expression of miR-127-5p and MMP-13 by normal and osteoarthritic (OA) human cartilage was determined using real-time polymerase chain reaction. The effect of miR-127-5p on MMP-13 expression was evaluated using transient transfection of human chondrocytes or chondrogenic SW-1353 cells with miR-127-5p or its antisense inhibitor (anti-miR-127-5p). MMP-13 protein production was quantified by enzyme-linked immunosorbent assay, and the involvement of miR-127-5p in IL-1β-mediated catabolic effects was examined by immunoblotting. MicroRNA-127-5p binding with the putative site in the 3′-untranslated region (3′-UTR) of MMP-13 messenger RNA (mRNA) was validated by luciferase reporter assay. Results There was a significant reduction in miR-127-5p expression in OA cartilage compared with normal cartilage. Up-regulation of MMP-13 expression by IL-1β was correlated with down-regulation of miR-127-5p expression in human chondrocytes. MicroRNA-127-5p suppressed IL-1β-induced MMP-13 production as well as the activity of a reporter construct containing the 3′-UTR of human MMP-13 mRNA. In addition, mutation of the miR-127-5p binding site in the 3′-UTR of MMP-13 mRNA abolished miR-127-5p-mediated repression of reporter activity. Conversely, treatment with anti-miR-127-5p remarkably increased reporter activity and MMP-13 production. Interestingly, the IL-1β-induced activation of JNK, p38, and NF-κB and expression of MMP-1 and cyclooxygenase 2 were significantly inhibited by miR-127-5p. Conclusion MicroRNA-127-5p is an important regulator of MMP-13 in human chondrocytes and may contribute to the development of OA. [ABSTRACT FROM AUTHOR]

Summary: Background:: Hair loss is a distressing condition for an increasing number of men and women. It is of great importance; therefore, to develop new therapies for the treatment of hair loss. Objective:: We examined the effects of 45 plant extracts that have been traditionally used for treating hair loss in oriental medicine in order to identify potential stimulants of hair growth. Methods:: Six-week-old female C57BL/6 and C3H mice were used for evaluating the hair growth-promoting effects of the plant extracts. Topical application onto the backs of the C57BL/6 and C3H mice was performed daily for 30 days and 45 days, respectively. Protein synthesis was measured by the cysteine uptake assay, using cultured murine vibrissae follicles. Proliferation of the immortalized human keratinocyte cell line (HaCaT) and human dermal papilla (DP) cells was evaluated by the MTT and thymidine incorporation assays. The mRNA levels of several growth factors that have been implicated in hair growth control were measured by reverse transcription-polymerase chain reaction (RT-PCR). Results:: Among the tested plant extracts, the extract of Asiasari radix showed the most potent hair growth stimulation in C57BL/6 and C3H mice experiments. In addition, this extract markedly increased the protein synthesis in vibrissae follicle cultures and the proliferation of both HaCaT and human DP cells in vitro. Moreover, the A. radix extract induced the expression of VEGF in human DP cells that were cultured in vitro. Conclusion:: These results suggest that the A. radix extract has hair growth-promoting potential, and that this effect may be due to its regulatory effects on both cell growth and growth factor gene expression. [Copyright &y& Elsevier]

For clinical applications of cells and tissue engineering products it is of importance to characterize the quality of the used cells in detail. Progenitor cells from the periosteum are already routinely applied in the clinics for the regeneration of the maxillary bone. Periosteal cells have, in addition to their potential to differentiate into bone, the ability to develop into cartilage and fat. However, the question arises whether all cells isolated from periosteal biopsies are able to differentiate into all three tissue types, or whether there are subpopulations. For an efficient and approved application in bone or cartilage regeneration the clarification of this question is of interest. Therefore, 83 different clonal cultures of freshly isolated human periosteal cells derived from mastoid periosteum biopsies of 4 donors were generated and growth rates calculated. Differentiation capacities of 51 clonal cultures towards the osteogenic, the chondrogenic, and the adipogenic lineage were investigated. Histological and immunochemical stainings showed that 100% of the clonal cultures differentiated towards the osteogenic lineage, while 94.1% demonstrated chondrogenesis, and 52.9% could be stimulated to adipogenesis. For osteogenesis real-time polymerase chain reaction (PCR) of BGLAP and RUNX2 and for adipogenesis of FABP4 and PPARG confirmed the results. Overall, 49% of the cells exhibited a tripotent potential, 45.1% showed a bipotent potential (without adipogenic differentiation), 3.9% bipotent (without chondrogenic differentiation), and 2% possessed a unipotent osteogenic potential. In FACS analyses, no differences in the marker profile of undifferentiated clonal cultures with bi- and tripotent differentiation capacity were found. Genome-wide microarray analysis revealed 52 differentially expressed genes for clonal subpopulations with or without chondrogenic differentiation capacity, among them DCN, NEDD9, TGFBR3, and TSLP. For clinical applications of periosteal cells in bone regeneration all cells were inducible. For a chondrogenic application a fraction of 6% of the mixed population could not be induced. [ABSTRACT FROM AUTHOR]

Abstract: The unclassified bovine enteric calicivirus (BEC) is a new bovine enteric calicivirus that is different from bovine norovirus, and causes diarrhea and pathologies in the small intestine of calves. This virus includes Nebraska (NB)- and Newbury agent 1 (NA1)-like strains. The prevalence of this BEC and its genetic characterization has only been reported in the UK and the USA. This study examined the prevalence and genetic diversity of these BECs in diarrheic calves in South Korea. Among a total of 645 diarrheic fecal specimens obtained from 629 cattle herds, these unclassified BECs were detected in 59 (9.1%) diarrheic fecal samples from 57 herds (9.3%) by either RT-PCR or nested PCR. Sequence and phylogenetic analyses of the partial RdRp gene showed that all the Korean BECs clustered together and were closely related to the NB-like viruses (80.9–88.1% nucleotide and 84.5–98.4% amino acid) but not to the NA1-like viruses (75.8–78.4% nucleotide and 79.7–82.8% amino acid). Although these viruses could not be classified into NA1- and NB-like viruses from the sequence and phylogenetic data of the entire capsid gene, all the Korean BECs clustered together on a branch separate from the other known BECs. These results show that these BEC infections are endemic in diarrheic calves in South Korea. The infecting strains are genetically closer to the NB-like viruses but have a distinct evolutionary pathway. [Copyright &y& Elsevier]

The aim was to determine whether various clinical specimens obtained from COVID-19 patients contain the infectious virus. To demonstrate whether various clinical specimens contain the viable virus, we collected naso/oropharyngeal swabs and saliva, urine and stool samples from five COVID-19 patients and performed a quantitative polymerase chain reaction (qPCR) to assess viral load. Specimens positive with qPCR were subjected to virus isolation in Vero cells. We also used urine and stool samples to intranasally inoculate ferrets and evaluated the virus titres in nasal washes on 2, 4, 6 and 8 days post infection. SARS-CoV-2 RNA was detected in all naso/oropharyngeal swabs and saliva, urine and stool samples collected between days 8 and 30 of the clinical course. Notably, viral loads in urine, saliva and stool samples were almost equal to or higher than those in naso/oropharyngeal swabs (urine 1.08 ± 0.16–2.09 ± 0.85 log 10 copies/mL, saliva 1.07 ± 0.34–1.65 ± 0.46 log 10 copies/mL, stool 1.17 ± 0.32 log 10 copies/mL, naso/oropharyngeal swabs 1.18 ± 0.12–1.34 ± 0.30 log 10 copies/mL). Further, viable SARS-CoV-2 was isolated from naso/oropharyngeal swabs and saliva of COVID-19 patients, as well as nasal washes of ferrets inoculated with patient urine or stool. Viable SARS-CoV-2 was demonstrated in saliva, urine and stool samples from COVID-19 patients up to days 11–15 of the clinical course. This result suggests that viable SARS-CoV-2 can be secreted in various clinical samples and respiratory specimens. [ABSTRACT FROM AUTHOR]

Background: Neurofibromatosis type 1 (NF1) is a frequent genetic disease characterized by multiple benign tumours with increased risk for malignancy. There is currently no biomarker for tumour load in NF1 patients. Methods: In situ hybridization and quantitative real-time polymerase reaction were applied to investigate expression of cartilage-specific genes in mice bearing conditional inactivation of NF1 in the developing limbs. These mice do not develop tumours but recapitulate aspects of NF1 bone dysplasia, including deregulation of cartilage differentiation. It has been recently shown that NF1 tumours require for their growth the master regulator of cartilage differentiation SOX9. We thus hypothesized that some of the cartilage-specific genes deregulated in an Nf1Prx1 mouse model might prove to be relevant biomarkers of NF1 tumours. We tested this hypothesis by analyzing expression of the SOX9 target gene product melanoma-inhibitory activity/cd-rap (MIA) in tumour and serum samples of NF1 patients. Results: Increased expression of Mia was found in Nf1-deficient cartilage in mice. In humans, MIA was expressed in all NF1-related tumours and its serum levels were significantly higher in NF1 patients than in healthy controls. Among NF1 patients, MIA serum levels were significantly higher in those with plexiform neurofibromas and in those with large number of cutaneous (> 1,000) or subcutaneous (> 100) neurofibromas than in patients without such tumours. Most notably, MIA serum levels correlated significantly with internal tumour burden. Conclusions: MIA is a potential serum biomarker of tumour load in NF1 patients which could be useful in following the disease course and monitoring the efficacy of therapies. [ABSTRACT FROM AUTHOR]

Abstract: Porcine group A rotavirus (GARV) is considered to be an important animal pathogen due to their economic impact in the swine industry and its potential to cause heterologous infections in humans. This study examined 475 fecal samples from 143 farms located in 6 provinces across South Korea. RT-PCR and nested PCR utilizing primer pairs specific for the GARV VP6 gene detected GARV-positive reactions in 182 (38.3%) diarrheic fecal samples. A total of 98 porcine GARV strains isolated from the GARV-positive feces were analyzed for G and P genotyping. Based on the sequence and phylogenetic analyses, the most predominant combination of G and P genotypes was G5P[7], found in 63 GARV strains (64.3%). The other combinations of G and P genotypes were G8P[7] (16 strains [16.3%]), G9P[7] (7 strains [7.1%]), G9P[23] (2 strains [2.0%]), and G8P[1] (1 strain [1.0%]). The counterparts of G or P genotypes were not determined in three G5, five P[7], and one P[1] strains. Interestingly, phylogenetic analysis indicated that all Korean G9 strains were more closely related to lineage VI porcine and human viruses than to other lineages (I–V) of GARVs and to Korean human G9 strains (lineage III). These results show that porcine GARV infections are common in diarrheic piglets in South Korea. The infecting strains are genetically diverse, and include homologous (G5P[7]), heterologous (G8P[1]), and reassortant (G8P[7]), as well as emerging G9 GARV strains. [ABSTRACT FROM AUTHOR]

Background Carbapenem-resistant Enterobacteriaceae (CRE) infection is an emerging clinical issue. One of the mechanisms of carbapenem-resistance is carbapenemase production. This study aimed to identify whether clinical outcomes differ by CRE resistance mechanism and to evaluate risk factors for mortality in patients with CRE bacteremia. Methods We conducted a retrospective cohort study comparing 14-day mortality between patients with carbapenemase-producing (CP)-CRE and non-CP-CRE bacteremia during January 2011 to October 2018. Only monomicrobial Escherichia coli or Klebsiella pneumoniae bacteremia were included in the study. A modified carbapenem inactivation method was used for phenotypic detection of carbapenemase production. The presence of a variety of carbapenemase genes was evaluated by PCR with specific primers. Results Of 134 patients with monomicrobial CRE bacteremia, 48 (35.8%) were infected with CP-CRE, and 86 (64.1%) were infected with non-CP-CRE. The most common carbapenemase in CP-CRE isolates was KPC (66.7%), followed by NDM-1 (18.8%), OXA-48-like (10.4%), and VIM (4.1%). Baseline characteristics were similar between the two groups (Table 1). However, the CP-CRE group was significantly more likely to undergo removal of eradicable foci and to have meropenem MIC >8 µg/mL. A total of 33 (24.6%) patients died within 14 days, including 9 (18.8%) in the CP-CRE group and 24 (27.9%) in the non-CP-CRE group. Deceased patients were more likely to have a higher Pitt bacteremia score, nosocomial acquisition, ineradicable or not-eradicated foci, immunosuppressant use, inappropriate definitive treatment (Table 2). Combination therapy for definitive treatment was associated with decreased mortality. In a multivariate analysis including carbapenemase production, a higher Pitt bacteremia score (aOR, 5.15), ineradicable or not-eradicated foci (aOR, 4.05) and combination therapy for definitive treatment (aOR, 0.35) were independent risk factors for mortality. Conclusion Our study suggests that carbapenemase production is not a mortality risk factor in CRE bacteremia and provides additional evidence for early source control and combination therapy. Disclosures All authors: No reported disclosures. [ABSTRACT FROM AUTHOR]