Clinical outcomes and treatment necessity in patients with toxin-negative Clostridioides difficile stool samples.

PURPOSE: The clinical significance of negative toxin enzyme immunoassays (EIA) for Clostridioides difficile infections (CDIs) is unclear. Our study aimed to investigate the significance of toxin EIA-negative in the diagnosis and prognosis of CDI. METHODS: All stool specimens submitted for C. difficile toxin EIA testing were cultured to isolate C. difficile. In-house PCR for tcdA, tcdB, cdtA, and cdtB genes were performed using C. difficile isolates. Stool specimens were tested with C. difficile toxins A and B using EIA kit (RIDASCREEN Clostridium difficile toxin A/B, R-Biopharm AG, Darmstadt, Germany). Characteristics and subsequent CDI episodes of toxin EIA-negative and -positive patients were compared. RESULTS: Among 190 C. difficile PCR-positive patients, 83 (43.7%) were toxin EIA-negative. Multivariate analysis revealed independent associations toxin EIA-negative results and shorter hospital stays (OR = 0.98, 95% CI 0.96-0.99, p = 0.013) and less high-risk antibiotic exposure in the preceding month (OR = 0.38, 95% CI 0.16-0.94, p = 0.035). Toxin EIA-negative patients displayed a significantly lower white blood cell count rate (11.0 vs. 35.4%, p < 0.001). Among the 54 patients who were toxin EIA-negative and did not receive CDI treatment, three (5.6%) were diagnosed with CDI after 7-21 days without complication. CONCLUSION: Our study demonstrates that toxin EIA-negative patients had milder laboratory findings and no complications, despite not receiving treatment. Prolonged hospitalisation and exposure to high-risk antibiotics could potentially serve as markers for the development of toxin EIA-positive CDI.

Neurotrophic phenolic glycosides from the roots of Armoracia rusticana.

Armoracia rusticana P. G. Gaertner. belongs to the Brassicaceae family and has aroused scientific interest for its anti-inflammatory and anticancer activities. In a continuing investigation to discover bioactive constituents from A. rusticana, we isolated 19 phenolic glycosides including three undescribed flavonol glycosides and one undescribed neolignan glycoside from MeOH extract of this plant. Their structures were elucidated based on NMR spectroscopic analysis (1H, 13C, 1H-1H COSY, HSQC, and HMBC), HRESIMS, and chemical methods. The determination of their absolute configuration was accomplished by ECD and LC-MS analysis. All the compounds were assessed for their potential neurotrophic activity through induction of nerve growth factor in C6 glioma cell lines and for their anti-neuroinflammatory activity based on the measurement of inhibition levels of nitric oxide production and pro-inflammatory cytokines (i.e., IL-1ß, IL-6, and TNF-α) in lipopolysaccharide-activated microglia BV-2 cells.

Ravidomycin Analogs from Streptomyces sp. Exhibit Altered Antimicrobial and Cytotoxic Selectivity.

Six new ravidomycin analogs (1-4, 6, and 7) were isolated from Streptomyces sp. Am59 using UV- and LCMS-guided separation based on Global Natural Products Social (GNPS) molecular networking analysis. Furthermore, we isolated fucomycin V (9), which possesses the same chromophore as ravidomycin but features a d-fucopyranose instead of d-ravidosamine. This is the first report of 9 as a natural product. Four new analogs (10-13) of 9 were also isolated. The structures were elucidated by combined spectroscopic and computational methods. We also found an inconsistency with the published [α]D25 of deacetylravidomycin, which is reported to have a (-) sign. Instead, we observed a (+) specific rotation for the reported absolute configuration of deacetylravidomycin (containing d-ravidosamine). We confirmed the positive sign by reisolating deacetylravidomycin from S. ravidus and by deacetylating ravidomycin. Finally, antibacterial, antifungal, and cytotoxicity activities were determined for the compounds. Compared to deacetylravidomycin, the compounds 4-6, 9, 11, and 12 exhibited greater antibacterial selectivity.

Anti-Inflammatory, Neurotrophic, and Cytotoxic Oxylipins Isolated from Chaenomeles sinensis Twigs.

Oxylipins are important biological molecules with diverse roles in human and plants such as pro-/anti-inflammatory, antimicrobial, and regulatory activity. Although there is an increasing number of plant-derived oxylipins, most of their physiological roles in humans remain unclear. Here, we describe the isolation, identification, and biological activities of four new oxylipins, chaenomesters A-D (1-4), along with a known compound (5), obtained from Chaenomeles sinensis twigs. Their chemical structures were determined by spectroscopic (i.e., NMR) and spectrometric (i.e., HRMS) data analysis including 1H NMR-based empirical rules and homonuclear-decoupled 1H NMR experiments. Chaenomester D (4), an omega-3 oxylipin, showed a potent inhibitory effect on nitric oxide (NO) production in lipopolysaccharide (LPS)-activated BV-2 cells (NO production, 8.46 ± 0.68 µM), neurotrophic activity in C6 cells through the induction of the secretion of nerve growth factor (NGF, 157.7 ± 2.4%), and cytotoxicity in A549 human cancer cell lines (IC50 = 27.4 µM).

Clinical Interpretation Challenges of Germline-Shared Somatic Variants in Cancer.

OBJECTIVE: To investigate the interpretation differences of germline-shared somatic variants. METHODS: A total of 123,302 COSMIC variants associated with hematologic malignant neoplasms were used. The pathogenicity and actionability of shared variants were analyzed based on the standardized guidelines. RESULTS: The overall frequency of variants shared in ClinVar/HGMD and COSMIC was 10%. The pathogenicity of 54 shared variants was pathogenic/likely pathogenic (P/LP; n = 30), variants of unknown significance (n = 3), and benign/likely benign (n = 21). In total, 30 P/LP variants were reclassified to tier I/tier II (83%) and tier III (17%) variants. CONCLUSIONS: This is the first study about different clinical interpretations of shared variants based on the current standard guidelines. This study takes a meaningful step in bridging the interpretation gap between the somatic and germline variants.

Inhibitory Effect of LGS and ODE Isolated from the Twigs of Syringa oblata subsp. dilatata on RANKL-Induced Osteoclastogenesis in Macrophage Cells.

Osteoblasts and osteoclasts play a pivotal role in maintaining bone homeostasis, of which excessive bone resorption by osteoclasts can cause osteoporosis and various bone diseases. However, current osteoporosis treatments have many side effects, and research on new treatments that can replace these treatments is ongoing. Therefore, in this study, the roles of ligustroside (LGS) and oleoside dimethylester (ODE), a natural product-derived compound isolated from Syringa oblata subsp. dilatata as a novel, natural product-derived osteoporosis treatments were investigated. In the results of this study, LGS and ODE inhibited the differentiation of receptor activator of nuclear factor kappa-Β ligand (RANKL)-induced RAW264.7 cells into osteoclasts without cytotoxicity, and down-regulated the activity of TRAP, a specific biomarker of osteoclasts. In addition, it inhibited bone resorption and actin ring formation, which are important functions and features of osteoclasts. Also, the effects of LGS and ODE on the mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) and phosphoinositide 3-kinases (PI3K)/ protein kinase B (Akt)/mechanistic target of rapamycin (mTOR) signaling pathways that play important roles in osteoclast differentiation were evaluated. In the results, LGS and ODE downregulated the phosphorylation of RANKL-induced MAPK and PI3K/Akt/mTOR proteins in a concentration-dependent manner, translocation of NF-κB into the nucleus was inhibited. As a result, the compounds LGS and ODE isolated from S. oblate subsp. dilatata effectively regulated the differentiation of RANKL-induced osteoclasts and inhibited the phosphorylation of signaling pathways that play a pivotal role in osteoclast differentiation. Therefore, these results suggest the possibility of LGS and ODE as new natural product treatments for bone diseases caused by excessive osteoclasts.

Calcific tendinitis of the shoulder in the Korean population: demographics and its relation with coexisting rotator cuff tear.

BACKGROUND: To evaluate the demographics, clinical and radiographic features of calcific tendinitis of the shoulder in the Korean population, specifically focusing on the incidence of coexisting rotator cuff tear. METHODS: Between October 2014 and January 2015, we performed a prospective multicenter study with 506 patients from 11 training hospitals in Korea. We collected data of demographics and radiographic analysis based on simple radiographs, clinical assessments based on visual analog scale (VAS) and the American Shoulder Elbow Surgeons (ASES) score, and treatment modalities that are used currently. We also evaluated coexisting rotator cuff tear by ultrasonography (US) or magnetic resonance imaging (MRI) images. RESULTS: There were 402 female patients (79%) with mean age of 55 years (range, 31-87 years). Mean duration of symptoms was 16 months. Mean size of calcific materials was 11.4 mm (range, 0-35 mm). Mean value of VAS and ASES scores were 6.5 (range, 1-10) and 47 (range, 8-95), respectively. Of 383 patients (76%), 59 (15%) had rotator cuff tear including 15 full-thickness tears on US or MRI. Patients with rotator cuff tears were significantly associated with older age, recurrent symptoms, menstrual disorders in females, and having undergone calcification removal surgery and rotator cuff repair (all p<0.05). CONCLUSIONS: This study reported demographic, radiographic, and clinical features of calcific tendinitis of the shoulder in Korean population, which were not different from those of Western population. Coexisting rotator cuff tear was found with 15% incidence in this large series, suggesting that further radiographic study to evaluate rotator cuff tear might be needed in some calcific tendinitis patients of older age and presenting with recurrent symptoms.

Morolic Acid 3-O-Caffeate Inhibits Adipogenesis by Regulating Epigenetic Gene Expression.

Obesity causes a wide range of metabolic diseases including diabetes, cardiovascular disease, and kidney disease. Thus, plenty of studies have attempted to discover naturally derived compounds displaying anti-obesity effects. In this study, we evaluated the inhibitory effects of morolic acid 3-O-caffeate (MAOC), extracted from Betula schmidtii, on adipogenesis. Treatment of 3T3-L1 cells with MAOC during adipogenesis significantly reduced lipid accumulation and decreased the expression of adiponectin, a marker of mature adipocytes. Moreover, the treatment with MAOC only during the early phase (day 0-2) sufficiently inhibited adipogenesis, comparable with the inhibitory effects observed following MAOC treatment during the whole processes of adipogenesis. In the early phase of adipogenesis, the expression level of Wnt6, which inhibits adipogenesis, increased by MAOC treatment in 3T3-L1 cells. To identify the gene regulatory mechanism, we assessed alterations in histone modifications upon MAOC treatment. Both global and local levels on the Wnt6 promoter region of histone H3 lysine 4 trimethylation, an active transcriptional histone marker, increased markedly by MAOC treatment in 3T3-L1 cells. Our findings identified an epigenetic event associated with inhibition of adipocyte generation by MAOC, suggesting its potential as an efficient therapeutic compound to cure obesity and metabolic diseases.

Antineurodegenerative Labdane Diterpenoid Glycosides from the Twigs of Pinus koraiensis.

Eleven new labdane-type diterpenoid glycosides, koraiensides A-K (1-11), together with two known analogues were isolated from the twigs of Pinus koraiensis. Their structures were elucidated via NMR, HRMS, and ECD data, DP4+ statistical analysis, and hydrolysis. The metabolites were tested for induction of nerve growth factor in C6 glioma cells to evaluate their potential neuroprotective activity. The compounds were measured for production of nitric oxide levels in lipopolysaccharide (LPS)-activated murine microglia BV2 cells to assess their antineuroinflammatory activity. Compounds 10 and 13 showed NGF secretion inducing effects from C6 glioma cells (162.3 ± 13.9% and 162.7 ± 6.9%, respectively). Compound 6 showed an IC50 value of 24.1 µM, implying significant inhibition of NO production.

Mutation analysis in Korean patients with T-cell acute lymphoblastic leukemia.

Genomic studies have illuminated the alterations in pathways underlying T-cell acute lymphoblastic leukemia (T-ALL) pathogenesis, but detailed mutation data by next-generation sequencing have not been reported in Korean patients. We aimed to investigate mutation frequency, spectrum, and pattern in the Korean patients with T-ALL. We designed a multigene panel targeting 101 genes and validated it using 10 reference materials. The mutation analysis was done in a total of 10 patients with T-ALL. Clinical data and laboratory tests including immunophenotyping, cytogenetics, and molecular genetic tests were also investigated. All of the 10 patients harbored at least one mutation (range 1-6 per patient). A total of 34 clinically significant mutations including 15 novel mutations were identified in 23 genes. The median of variant allelic frequencies (VAFs) and blasts were counted upto 33% (range 5-91%) and 79% (range 38-90%), respectively. Recurrent mutations were involved in epigenetic regulators (60%), NOTCH1 signaling (40%), PI3K-AKT (40%), JAK-STAT (30%), and transcription factors (30%). We found that both NOTCH signaling and JAK-STAT signaling were positively associated with epigenetic regulators, while showed mutually exclusive patterns with PI3K-AKT pathway. This study showed that the frequency of mutations in epigenetic regulators in Korean patients was significantly higher than expected. Distribution of VAF as well as mutation spectrum is considerably heterogeneous in Korean patients with T-ALL. Although from a limited number of patients, this study provides the first detailed mutational portrait of T-ALL of Korean patients, and gives additional insight into molecular pathogenesis of the disease.

Securinega Alkaloids from the Twigs of Securinega suffruticosa and Their Biological Activities.

Seven new Securinega alkaloids, securingines A-G (1-7), together with seven known analogues (8-14), were isolated from the twigs of Securinega suffruticosa. Their chemical structures were elucidated by a combined approach of spectroscopic analysis, chemical methods, ECD calculations, and DP4+ probability analysis. The full NMR assignments and the absolute configuration of compound 8 are also reported. In addition, all the isolated phytochemicals (1-14) were assessed for their cytotoxic, anti-inflammatory, and potential neuroprotective activities. Compound 4 showed cytotoxic activity (IC50 values of 1.5-6.8 µM) against four human cell lines (A549, SK-OV-3, SK-MEL-2, and HCT15). Compounds 3, 10, 12, and 13 showed potent inhibitory effects on nitric oxide production (IC50 values of 12.6, 12.1, 1.1, and 7.7 µM, respectively) in lipopolysaccharide-stimulated murine microglia BV-2 cells. Compound 5 exhibited a nerve growth factor production effect (172.6 ± 1.2%) in C6 glioma cells at 20 µg/mL.

Identification of protoberberine alkaloids as novel histone methyltransferase G9a inhibitors by structure-based virtual screening.

The protein lysine methyltransferase G9a, which controls gene expression by epigenetic regulation of H3K9 methylation, is related to various human diseases, including cancer, drug addiction, and mental retardation. In recent years, genetic, biological, and physiological evidence has established G9a inhibitors as potential chemotherapeutic agents for cancer treatment. In this study, we identified protoberberine alkaloid pseudodehydrocorydaline (CT13) as a novel G9a inhibitor, by structure-based virtual screening of in-house library containing natural product compounds. The activity of CT13 was determined by biophysical analyses involving MALDI-TOF mass spectrometry and western blot analysis. CT13 showed selective inhibitory activity against G9a and suppressed the level of H3K9me2 in MCF7 human breast cancer cells. Molecular docking analysis suggested the binding mode of CT13 which occupies the binding site of histone H3 substrate. CT13 provides a novel scaffold for further development of analogous synthetic G9a inhibitors.

Bioactive triterpenoids from twigs of Betula schmidtii.

Investigation of the MeOH extract of Betula schmidtii twigs resulted in the isolation and identification of three new triterpenoids (1-3), along with ten known ones (4-13). The structures of new compounds (1-3) were elucidated by spectroscopic methods, including 1D, 2D NMR (1H and 13C NMR, COSY, HSQC, HMBC, and NOESY), HR-MS, and chemical methods. All the isolated compounds were evaluated for their cytotoxicity against A549, SK-OV-3, SK-MEL-2, and HCT-15 cell lines. Compound 11 exhibited potent cytotoxic activities against four cell lines, and compounds 5 and 13 significantly induced nerve growth factor secretion in a C6 rat glioma cell line. Their anti-inflammatory effects were also assessed by measuring nitric oxide production in lipopolysaccharide-activated BV-2 cells. Compounds 7 and 12 displayed potent inhibition of nitric oxide production, without significant cell toxicity.

Germline BRCA mutations in Asian patients with pancreatic adenocarcinoma: a prospective study evaluating risk category for genetic testing.

Introduction Germline BRCA mutations may have therapeutic implications as surrogate markers of DNA-damage repair status in pancreatic ductal adenocarcinoma (PDAC). We performed a prospective study to evaluate the efficiency of risk criteria based on personal or family history of breast and ovarian cancer for determining germline BRCA mutations in PDAC patients with Asian ethnicity. Methods Between November 2015 and May 2016, we screened consecutive PDAC patients with locally advanced unresectable or metastatic disease who were referred for systemic chemotherapy. Analyses for germline BRCA mutations were performed if patients had one or more first-degree or second-degree relatives with breast or ovarian cancers or had a personal medical history of these diseases. DNA was extracted from whole blood, and all coding exons and their flanking intron regions of BRCA1 and BRCA2 were sequenced. Results A total of 175 patients were screened for personal and family history and 10 (5.7%) met the inclusion criteria for genetic sequencing. Pathogenic germline BRCA2 mutation [c.7480C>T (p.Arg2494*)] was identified in one male patient, resulting in a frequency of 10% for the risk-stratified patients and 0.6% for the unselected PDAC population. Two patients had germline BRCA2 variants of uncertain significance [c.1744A>C (p.Thr582Pro) and c.68-7T>A]. Conclusion Personal or family history of breast or ovarian cancers is a feasible, cost-effective risk categorization for screening germline BRCA mutations in Asian PDAC patients as 10% of this population had the pathogenic mutation herein. Future validation from a large, prospective cohort is needed.

Rapid Progressive Osteonecrosis of the Humeral Head After Arthroscopic Rotator Cuff Surgery.

PURPOSE: To verify the clinical features and the risk factors of rapid progressive osteonecrosis of the humeral head after arthroscopic rotator cuff surgery (ARCS). METHODS: Data and medical records of 24 patients who had rapid progressive collapse of the humeral head after ARCS performed from October 2012 to March 2016 were retrospectively analyzed. Among them, 8 patients demonstrated no evidence of osteonecrosis of the humeral head preoperatively yet developed rapid progressive collapse of the humeral head within 12 months after ARCS. RESULTS: All patients were women with a mean age of 64.0 years (range, 52-74 years), and all of them had surgery on their dominant side. Sudden pain developed at a mean 4 months (range, 0-6 months) after index surgery. The rapid progressive collapse of the humeral head occurred within 12 months after index surgery. No clear risk factor or evidence supporting an association between ARCS and humeral head osteonecrosis was found. CONCLUSIONS: Although the cause of the rapid progressive humeral head osteonecrosis after ARCS still needs to be established, surgeons should be aware of the possible development of humeral head osteonecrosis after ARCS, especially in older women with dominant arm involvement. LEVEL OF EVIDENCE: Level IV, prognostic case series.

Large-scale clinical validation of biomarkers for pancreatic cancer using a mass spectrometry-based proteomics approach.

We performed an integrated analysis of proteomic and transcriptomic datasets to develop potential diagnostic markers for early pancreatic cancer. In the discovery phase, a multiple reaction monitoring assay of 90 proteins identified by either gene expression analysis or global serum proteome profiling was established and applied to 182 clinical specimens. Nine proteins (P < 0.05) were selected for the independent validation phase and quantified using stable isotope dilution-multiple reaction monitoring-mass spectrometry in 456 specimens. Of these proteins, four proteins (apolipoprotein A-IV, apolipoprotein CIII, insulin-like growth factor binding protein 2 and tissue inhibitor of metalloproteinase 1) were significantly altered in pancreatic cancer in both the discovery and validation phase (P < 0.01). Moreover, a panel including carbohydrate antigen 19-9, apolipoprotein A-IV and tissue inhibitor of metalloproteinase 1 showed better performance for distinguishing early pancreatic cancer from pancreatitis (Area under the curve = 0.934, 86% sensitivity at fixed 90% specificity) than carbohydrate antigen 19-9 alone (71% sensitivity).Overall, we present the panel of robust biomarkers for early pancreatic cancer diagnosis through bioinformatics analysis that combined transcriptomic and proteomic data as well as rigorous validation on a large number of independent clinical samples.

A biphenyl derivative from the twigs of Chaenomeles speciosa.

In our continuing search for bioactive constituents of Korean medicinal sources, we investigated an 80% MeOH extract of the twigs of Chaenomeles speciosa. Column chromatographic purification of the CHCl3 fraction resulted in the isolation of a new biphenyl derivative (1), along with four known biphenyl compounds (2-5) and six triterpenes (6-11). The chemical structure of the new compound was determined on the basis of spectroscopic analyses including 1D and 2D NMR data. Among isolates, compound 3 exhibited potent cytotoxic activities against SK-OV-3, SK-MEL-2, and XF498 cell lines (IC50=5.91, 4.22, and 6.28µM, respectively). Also, Compounds 9 and 10 showed strong anti-neuroinflammatory activities (IC50 2.38, and 6.70µM, respectively).

Secoiridoid Glucosides from the Twigs of Syringa oblata var. dilatata and Their Neuroprotective and Cytotoxic Activities.

Phytochemical investigation of the twigs of Syringa oblata var. diatata led to the isolation of two new secoiridoid glucosides, dilatioside A-B (1-2), along with thirteen known ones (3-15). The structures were determined by spectroscopic methods including one and two dimensional (1- and 2D-) NMR techniques, high resolution (HR)-FAB-MS, and chemical methods. The isolated compounds (1-15) were tested for the induction of nerve growth factor (NGF) secretion in a C6 rat glioma cell line and their cytotoxicity against four human cancer cell lines (A549, SK-OV-3, SK-MEL-2, HCT15) in vitro using a sulforhodamine B bioassay. Compounds 5, 7, 8, 10, and 14 were found to induce upregulation of NGF secretion without causing significant cell toxicity.

Outcomes of the Distal Metatarsal Dorsiflexion Osteotomy for Advanced Hallux Rigidus.

BACKGROUND: Hallux rigidus can be treated using several different methods and the best treatment option depends on the severity of degenerative changes of the metatarsophalangeal (MTP) joint. However, the ideal operative option for advanced hallux rigidus remains debatable. This prospective study was performed to evaluate the intermediate-term clinical outcomes of distal metatarsal osteotomy used as a joint-preserving method for the treatment of advanced hallux rigidus. METHODS: Forty-two cases (39 patients) were followed for more than 3 years after distal metatarsal dorsiflexion osteotomy for advanced hallux rigidus of grade III-IV. Clinical evaluations included the American Orthopaedic Foot & Ankle Society (AOFAS) scores, Foot and Ankle Ability Measure (FAAM) scores, and patient subjective satisfaction scores. Range of motion (ROM) of great toe, complications, reoperation rates, width of the MTP joint space, and times to union were evaluated. RESULTS: Mean AOFAS hallux and mean FAAM scores significantly improved from 56.4 and 61.2 points preoperatively to 87.6 and 88.7 points at final follow-up, respectively ( P < .001). Grade III and IV groups had significantly different AOFAS and FAAM scores at final follow-up. Mean dorsiflexion of great toe significantly improved from 14.8° preoperatively to 35.5° at final follow-up ( P < .001). Mean patient satisfaction score at final follow-up was 92.8 points. There were 4 cases (9.5%) of subsequent fusion and 2 cases (4.8%) of transfer metatarsalgia. CONCLUSIONS: Distal metatarsal dorsiflexion osteotomy using bio-compression screws appears to be an effective operative option for grade III advanced hallux rigidus with viable cartilage on >50% of the first metatarsal articular surface, as it restored joint motion, provided reliable pain relief, and did not require implant removal. However, based on the unsatisfactory clinical results and the high rate of reoperation observed, the authors cannot recommend this operative method for the treatment of end-stage (grade IV) hallux rigidus. LEVEL OF EVIDENCE: Level IV, prospective case series.

Allimacrosides A-E, new steroidal glycosides from Allium macrostemon Bunge.

A new pregnane-type steroidal glycoside (1), two new spirostane-type steroidal glycosides (2, 3), and two new furostane-type steroidal glycosides (4, 5), named allimacrosides A-E, together with four known compounds (6-9) were isolated from a 80% MeOH extract of Allium macrostemon Bunge. The identification and structural elucidation of these compounds were based on their 1D- and 2D-NMR spectra, and HR-FAB-MS data analysis. The isolated compounds were tested for cytotoxicity against four human tumor cell lines in vitro using the sulforhodamine B bioassay.