Application of virome capture sequencing in shellfish sold at retail level in Singapore.

During the period from late 2019 to early 2020, we performed a foodborne virus detection from shellfish collected in Singapore at retail level. Multiple human enteric viruses were included as our targets including human noroviruses (NoVs) GI and GII, hepatitis A virus, hepatitis E virus and rotavirus. Out of the 60 shellfish samples, 23 (38·3%) were detected to be positive by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) with human enteric viruses. Six samples were selected to proceed with virome capture sequencing with positive control samples spiked with serially diluted NoV GII clinical samples in oyster extract. As a result, the natural sample with comparable Ct values (34·0-35·0) of the spiked sample as detected by RT-qPCR generated much lower read counts (>7-log2 cumulative sum scaling difference) and genome coverage (406 nt. vs 3715 nt.), suggesting that the RT-qPCR positive signals detected from the shellfish samples collected at the retail market were likely from degraded RNA derived from inactive virus particles.

Prospective evaluation of the cardiac safety of HER2-targeted therapies in patients with HER2-positive breast cancer and compromised heart function: the SAFE-HEaRt study.

PURPOSE: HER2-targeted therapies have substantially improved the outcome of patients with breast cancer, however, they can be associated with cardiac toxicity. Guidelines recommend holding HER2-targeted therapies until resolution of cardiac dysfunction. SAFE-HEaRt is the first trial that prospectively tests whether these therapies can be safely administered without interruptions in patients with cardiac dysfunction. METHODS: Patients with stage I-IV HER2-positive breast cancer candidates for trastuzumab, pertuzumab or ado-trastuzumab emtansine (TDM-1), with left ventricular ejection fraction (LVEF) 40-49% and no symptoms of heart failure (HF) were enrolled. All patients underwent cardiology visits, serial echocardiograms and received beta blockers and ACE inhibitors unless contraindicated. The primary endpoint was completion of the planned HER2-targeted therapies without developing either a cardiac event (CE) defined as HF, myocardial infarction, arrhythmia or cardiac death or significant asymptomatic worsening of LVEF. The study was considered successful if planned oncology therapy completion rate was at least 30%. RESULTS: Of 31 enrolled patients, 30 were evaluable. Fifteen patients were treated with trastuzumab, 14 with trastuzumab and pertuzumab, and 2 with TDM-1. Mean LVEF was 45% at baseline and 46% at the end of treatment. Twenty-seven patients (90%) completed the planned HER2-targeted therapies. Two patients experienced a CE and 1 had an asymptomatic worsening of LVEF to ≤ 35%. CONCLUSION: This study provides safety data of HER2-targeted therapies in patients with breast cancer and reduced LVEF while receiving cardioprotective medications and close cardiac monitoring. Our results demonstrate the importance of collaboration between cardiology and oncology providers to allow for delivery of optimal oncologic care to this unique population.

Antibacterial activity of Hibiscus rosa-sinensis L. red flower against antibiotic-resistant strains of Helicobacter pylori and identification of the flower constituents.

Utilization of plant resources for treatment of Helicobacter pylori infections is one of the appealing approaches as rapid emergence of antibiotic-resistant strains is occurring throughout the world. Ethanol extract and its fractions from Hibiscus rosa-sinensis red flower were assessed for antibacterial and urease inhibitory activities towards forty-three clinical strains and two reference strains of H. pylori. The ethyl acetate fraction exhibited the most potent bacteriostatic activity with minimum inhibitory concentrations (MICs) of 0.2-0.25 mg/mL and minimum bactericidal concentrations (MBCs) of 1.25-1.5 mg/mL against all test strains, including forty-three strains resistant to one to four antibiotics, azithromycin (MICs, 8-256 µg/mL), erythromycin (MICs, 8-128 µg/mL), levofloxacin (MICs, 8-256 µg/mL), and/or metronidazole (MICs, 8-256 µg/mL). The fraction had similar antibacterial activities toward these test strains suggesting the preparation and the antibiotics do not have a common mechanism of anti-H. pylori activity. The fraction also had stronger effects on biofilm formation, morphological conversion, and urease activity of H. pylori than the other fractions and the ethanol extract. These flower preparations were non-toxic to three human cell lines, and nine compounds were also isolated and identified from the ethyl acetate fraction. In vivo research needs to be conducted to confirm the potential usefulness of H. rosa-sinensis flower and its constituents for effective prevention and treatment of H. pylori disease.

Border disease virus (BDV) infections of small ruminants in Turkey: a new BDV subgroup?

Blood samples from sheep and/or goats from eight small ruminant flocks in the Turkish provinces of Aydin and Burdur were tested for the presence of Pestiviruses using an antigen-capture ELISA. From clinically affected animals, pathological and immunohistochemical findings were recorded. Post mortem examination of a virus-positive lamb showing abnormal fleece and paralysis of the hind legs revealed nonsuppurative meningoencephalomyelitis with hypomyelinogenesis. By immunohistochemistry Pestivirus antigen was detected in all parts of the brain including cerebellum, cerebral hemispheres and midbrain. Two Pestivirus isolates from a sheep and a goat kid, respectively, were isolated from samples that were positive in the antigen-capture ELISA. Genetic typing using the 5'-NTR (288bp) and N(pro) (738bp) showed that both were Border disease virus (BDV) isolates. By phylogenetic analysis, they formed a cluster clearly separated from the known clusters BDV-1 to BDV-6 and might therefore represent a new subgroup (BDV-7?). This is the first report confirming the occurrence and partial characterisation of BDV infection in small ruminants in Turkey.

Use of tiazofurin to enhance the metabolism and cytotoxic activities of analogues of guanine, guanosine, and deoxyguanosine.

An effective modulator of cellular guanine nucleotide pools, 2-beta-D-ribofuranosylthiazole-4-carboxamide (tiazofurin) was tested for its ability to affect utilization of certain guanine, guanosine, and deoxyguanosine analogues by Chinese hamster ovary cells and hypoxanthine guanine phosphoribosyltransferase (HGPRTase)-deficient variants. The nucleoside analogues investigated were chosen for their potential to be metabolized to the nucleotide level by pathways other than those requiring the action of HGPRTase. Exposure of tiazofurin-treated (500 microM for 3 h) cells to 3-deazaguanosine (200 microM for 3 h) resulted in enhanced 3-deazaGTP formation and an increase (5-10-fold) in the ratio 3-deazaGTP/GTP. Tiazofurin treatment also stimulated [3H]deoxyguanosine utilization (8-fold) by HGPRTase-deficient cells, and accordingly, greatly increased the cytotoxicity of 2'-deoxy-3-deazaguanosine and arabinosylguanine. This study emphasizes the potential usefulness of tiazofurin in sequential combination with appropriate analogues of guanosine and deoxyguanosine in a clinical setting and as a tool in studying the metabolism of these agents.

Phosphorylation of 3-deazaguanosine by nicotinamide riboside kinase in Chinese hamster ovary cells.

The growth inhibitory activity of 3-deazaguanosine toward a mutant line (TGR-3) of Chinese hamster ovary cells deficient in hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) was substantially reversed by the simultaneous addition of nicotinamide riboside. The activities of most other ribonucleoside analogues tested were unaffected. The formation of cellular 3-deazaGMP and 3-deazaGTP from the ribonucleoside analogue, as measured by high-pressure liquid chromatography, was inhibited by the presence of nicotinamide riboside. The inhibition was dependent on concentration of 3-deazaguanosine and could also be demonstrated by following the metabolism of 3-deazaguanosine, labeled with 14C in the ribose moiety, to [14C]3-deazaGTP. In the presence of 100 microM nicotinamide riboside formation of the labeled triphosphate derivative of 3-deazaguanosine was undetectable. A 3-deazaguanosine phosphorylating activity was separated from other cellular kinases by DEAE-cellulose chromatography. Contaminating purine nucleoside phosphorylase (EC 2.4.2.1) was subsequently removed by sucrose density gradient centrifugation. The resulting enzyme preparation demonstrated the greatest activities with nicotinamide riboside and 3-deazaguanosine and, in addition, could also phosphorylate tiazofurin and guanosine to lesser, but significant, degrees. These and other observations suggest that 3-deazaguanosine, and perhaps other agents such as tiazofurin, may, at least in part, be phosphorylated by a nicotinamide ribonucleoside kinase in these cells. If so, it is possible that the activity of this agent in other types of cells in vivo could be dependent upon the presence of this enzyme and that it could be influenced by cellular concentrations of the natural pyridine nucleoside.

Tiazofurin is phosphorylated by three enzymes from Chinese hamster ovary cells.

The growth inhibitory activity of tiazofurin toward adenosine kinase deficient Chinese hamster ovary (CHO) cells was partially reversed by the presence of nicotinamide riboside. Similarly, the formation of tiazofurin 5'-monophosphate and the active metabolite, tiazofurin 5'-adenine dinucleotide could be partially inhibited by 100 microM nicotinamide riboside in CHO cells and substantially inhibited (80-90%) in adenosine kinase deficient cells. Tiazofurin phosphorylating activity from CHO cell extracts was resolved into two peaks by DEAE-cellulose chromatography. The first peak of activity was identified as adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20). The second peak of activity correlated with a previously described 3-deazaguanosine phosphorylating activity that was identified as a nicotinamide ribonucleoside kinase. Contaminating purine nucleoside phosphorylase was removed by sedimentation through a sucrose density gradient which also resolved the tiazofurin phosphorylating activity into two peaks, one requiring just ATP and the other requiring both ATP and IMP. Of the substrates tested with the lower density peak, nicotinamide riboside was most efficient and was the only natural substance that competed well with tiazofurin for phosphorylation, substantiating its suggested identity as a nicotinamide ribonucleoside kinase. The apparent Km value for nicotinamide riboside (2 microM) was significantly less than that for tiazofurin (13.6 microM). ATP was the best phosphate donor; CTP and UTP were utilized less efficiently and IMP did not support the reaction. The best substrate for the higher density peak of tiazofurin phosphorylation was inosine and both ATP and IMP were required for the reaction, suggesting its identity as a 5'-nucleotidase. In summary, it appears that adenosine kinase, nicotinamide ribonucleoside kinase, and 5'-nucleotidase may all contribute to the phosphorylation of tiazofurin in CHO cells.

[Submandibular gland resection via subclavian into the road under the endoscope].

Objective:To evaluate resection of submandibular gland through a minimal skin incision under the endoscope. Method:A retrospective analysis of the clinical data from 28 cases of submandibular gland resection by endoscope surgery via subclavian approach, 14 cases of preoperative diagnosis of pleomorphic adenoma, submandibular gland of chronic inflammation in 11 cases, 3 cases of the submandibular gland stone,one case of lymphatic cyst,all cases were evaluated by preoperative imaging or 3 d sonography. Result:All patients' submandibular gland and tumors were resected totally under the endoscope, no open surgery, no surgical complications, and postoperative aesthetic outcome was good, patients were satisfied, pleomorphic adenoma patients were postoperative followed up of 4 to 24 months, and no recurrence. Conclusion:Under the cavity mirror via subclavian path submandibular gland resection is safe and feasible, and has a good cosmetic effect.

Antibacterial activity of Hibiscus rosa-sinensis L. red flower against antibiotic-resistant strains of Helicobacter pylori and identification of the flower constituents

Utilization of plant resources for treatment of Helicobacter pylori infections is one of the appealing approaches as rapid emergence of antibiotic-resistant strains is occurring throughout the world. Ethanol extract and its fractions from Hibiscus rosa-sinensis red flower were assessed for antibacterial and urease inhibitory activities towards forty-three clinical strains and two reference strains of H. pylori. The ethyl acetate fraction exhibited the most potent bacteriostatic activity with minimum inhibitory concentrations (MICs) of 0.2-0.25 mg/mL and minimum bactericidal concentrations (MBCs) of 1.25-1.5 mg/mL against all test strains, including forty-three strains resistant to one to four antibiotics, azithromycin (MICs, 8-256 µg/mL), erythromycin (MICs, 8-128 µg/mL), levofloxacin (MICs, 8-256 µg/mL), and/or metronidazole (MICs, 8-256 µg/mL). The fraction had similar antibacterial activities toward these test strains suggesting the preparation and the antibiotics do not have a common mechanism of anti-H. pylori activity. The fraction also had stronger effects on biofilm formation, morphological conversion, and urease activity of H. pylori than the other fractions and the ethanol extract. These flower preparations were non-toxic to three human cell lines, and nine compounds were also isolated and identified from the ethyl acetate fraction. In vivo research needs to be conducted to confirm the potential usefulness of H. rosa-sinensis flower and its constituents for effective prevention and treatment of H. pylori disease.

Bioactive glass stimulates in vitro osteoblast differentiation and creates a favorable template for bone tissue formation.

In this study, we have investigated the behavior of fetal rat osteoblasts cultured on bioactive glasses with 55 wt% silica content (55S) and on a bioinert glass (60S) used either in the form of granules or in the form of disks. In the presence of Bioglass granules (55 wt% silica content), phase contrast microscopy permitted step-by-step visualization of the formation of bone nodules in contact with the particles. Ultrastructural observations of undecalcified sections revealed the presence of an electron-dense layer composed of needle-shaped crystals at the periphery of the material that seemed to act as a nucleating surface for biological crystals. Furthermore, energy dispersive X-ray (EDX) analysis and electron diffraction patterns showed that this interface contains calcium (Ca) and phosphorus (P) and was highly crystalline. When rat bone cells were cultured on 55S disks, scanning electron microscopic (SEM) observations revealed that cells attached, spread to all substrata, and formed multilayered nodular structures by day 10 in culture. Furthermore, cytoenzymatic localization of alkaline phosphatase (ALP) and immunolabeling with bone sialoprotein antibody revealed a positive staining for the bone nodules formed in cultures on 55S. In addition, the specific activity of ALP determined biochemically was significantly higher in 55S cultures than in the controls. SEM observations of the material surfaces after scraping off the cell layers showed that mineralized bone nodules remained attached on 55S surfaces but not on 60S. X-ray microanalysis indicated the presence of Ca and P in this bone tissue. The 55S/bone interfaces also were analyzed on transverse sections. The interfacial analysis showed a firm bone bonding to the 55S surface through an intervening apatite layer, confirmed by the X-ray mappings. All these results indicate the importance of the surface composition in supporting differentiation of osteogenic cells and the subsequent apposition of bone matrix allowing a strong bond of the bioactive materials to bone.

Metabolism and action of neplanocin A in Chinese hamster ovary cells.

Neplanocin A is a naturally occurring carbocyclic analog of adenosine which contains a cyclopentene moiety in place of ribose and has demonstrated antitumor and antimicrobial activity. This compound was highly toxic to Chinese hamster ovary (CHO) cells; the approximate minimum inhibitory concentration of neplanocin A for inhibition of clone formation was 0.1 microM. The toxicity of the agent was greatly reduced by prior treatment with adenosine deaminase. [3H]Uridine incorporation into perchloric acid insoluble material in growing cells was inhibited by neplanocin A more dramatically than that of [3H]thymidine or [3H]leucine. Treatment with the drug resulted in a marked depression of ATP pool levels. High pressure liquid chromatographic analysis of cellular nucleotide pools from cells treated with neplanocin A revealed the formation of an apparent drug metabolite (NpcTP) that eluted in the triphosphate region of the chromatographic profile. Treatment of NpcTP with alkaline phosphatase produced a nucleoside with properties similar to neplanocin A. An adenosine-kinase-deficient cell line formed little, if any, NpcTP but demonstrated only slight resistance to the agent. These observations suggest that neplanocin A was efficiently metabolized to the triphosphate level but that this metabolite was responsible for only a fraction of the observed toxicity.

A comparative study on the diagnosis of maedi-visna infection in serum and colostrum samples using agar gel immunodiffusion (AGID) technique.

Serum/colostrum pairs were collected from 245 ewes in 6 sheep herds which had been determined previously to be infected with MV virus and were tested against maedi-visna infection using AGID test. Positive rates were detected as 3.8-41.2% in tested flocks. Serum and colostrum samples obtained from 53 sheep were positive for MV virus specific antibodies by AGID test. 16 colostrum samples were negative although serum samples obtained from the same animals were found to be positive for MV antibodies. Of the 245 sera and colostrum pairs tested, there was total agreement of results (+ or -) in 229 and disagreement in the results with the other 16 serum/colostrum pairs. Of the latter, all serum samples were positive and all colostrum samples were negative for MV antibodies. This study compared colostrum and serum samples for the determination of MV antibodies using AGID test under field conditions on naturally infected animals and on healthy animals. The results show that colostrum antibodies can be detected using AGID test and that colostrum is a reliable material to determine anti-MV virus antibodies. The procedure can be used for herd diagnosis.

Detection and sequence analysis of equine gammaherpesviruses from horses with respiratory tract disease in Turkey.

The equid herpesvirus 2 (EHV-2) and 5 (EHV-5), identified agents of respiratory infections and keratoconjunctivitis cases in some equids, comprise a high degree of antigenic heterogeneity. Prevalence and genetic characterization of EHV-2 and EHV-5 strains from Turkey were investigated in this study. A total of 73 nasal swabs and 54 blood specimens were sampled from horses with respiratory tract diseases characterized by mucopurulent nasal discharge and occasional coughing. Overall, EHV-2- and EHV-5-specific DNA amplicons were obtained from 19.2% (14/73) and 21.9% (16/73) of horses tested by multiplex nested PCR. Sequences of EHV-2 and EHV-5 glycoprotein B (gB) gene were used in a phylogenetic analysis that included six EHV-2 and three EHV-5 isolates, which showed that the Turkish EHV-2 and EHV-5 strains have marked sequence divergence from European strains and from each other. Turkish EHV-2 isolates were divided into two distinct subdivisions, and a few isolates were located on a separate branch. This study provides the first epidemiological and phylogenetical report about EHV-2 and EHV-5 infections in Turkey.

Human papillomavirus detection in self-collected vaginal specimens and matched clinician-collected cervical specimens.

Human papillomavirus (HPV) detection is an integral part of cervical cancer screening, and a range of specimen collection procedures are being tested. Preliminary studies have found that the majority of women prefer self-collection of vaginal specimens instead of clinician-collected specimens of the cervix. The purposes of the current study were to explore the social and behavioral predictors of acceptance of self-collection of vaginal specimens among patients and to assess concordance in detection of HPV between clinician-collected cervical specimens and self-collected vaginal specimens. The study was conducted at a university family medicine clinic using a cross-sectional study design, and enrollment of women presenting for routine gynecological examination consecutively in a period of 1 year, self-administered questionnaires, collection of paired vaginal and cervical specimens for HPV DNA using Hybrid Capture 2, and cytologic analysis. Most women (79.8% [398/499]) agreed to collect vaginal specimens. In our study, 76.6% (216/282) African American women (AA), 88.1% (156/176) white non-Hispanic (WNH) women, and 63.4% (26/41) women of other races (P < 0.0001) agreed to self-collect vaginal specimens. HPV was detected in 16.0% (80/499) of clinician-collected cervical specimens and 26.1% (104/398) of self-collected vaginal specimens (P < 0.001). HPV detection was concordant in 13.4% (53/398) women in both cervical and vaginal specimens. Self-collection of vaginal specimens for HPV DNA detection is acceptable to most women presenting for routine gynecological examination. WNH women were more likely to obtain self-collected specimens than AA women. Vaginal specimens were more likely to be positive for HPV than were cervical specimens.

3-Deazaguanosine is metabolized to the triphosphate derivative in Chinese hamster cells deficient in hypoxanthine-guanine phosphoribosyltransferase.

3-Deazaguanosine containing a 14C label in the ribose moiety was prepared using [U-14C]inosine as the [14C] ribose donor and commercial purine-nucleoside phosphorylase (EC 2.4.2.1) both to degrade the inosine, in the presence of phosphate, and to synthesize [14C-ribosyl]3-deazaguanosine in reduced phosphate and an excess of 3-deazaguanine. Purification was by high-pressure liquid chromatography (HPLC). [14C-ribosyl]3-Deazaguanosine was metabolized by Chinese hamster ovary cells to two metabolites, one major and one minor, eluting in the triphosphate region after HPLC analysis, and appeared to be incorporated into perchloric acid-insoluble material. Cell line TGR-3, deficient in hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) and resistant to 3-deazaguanine, also formed both metabolites. Line TGR-1/DGRR-9, deficient in hypoxanthine-guanine phosphoribosyltransferase and resistant to both 3-deazaguanine and 3-deazaguanosine, formed greatly reduced levels of the major metabolite. 3-Deazaguanosine 5'-triphosphate, prepared enzymically from authentic 3-deazaguanosine 5'-monophosphate, co-eluted with the major metabolite peak during HPLC analysis. Treatment of a metabolite-containing extract with bacterial alkaline phosphatase (EC 3.1.3.1) resulted in the formation of 3-deazaguanosine. These observations indicate that 3-deazaguanosine can be metabolized, in Chinese hamster ovary cells, to the triphosphate derivative in lieu of the action of hypoxanthine-guanine phosphoribosyltransferase.

Estrogen and progesterone receptor titers in primary epithelial ovarian carcinomas.

Only 69% (42 of 61) of the documented ovarian tumor specimens received between September 1981 and August 1982 contained 75% or more viable tumor. Of the 42 measured for estrogen receptors (ER) and progesterone receptors (PR), 34 (81%) had estrogen-binding titers. greater than or equal to I fmol/mg of cytosol protein, while 18 (43%) had progesterone-binding titers greater than or equal to 5 fmol/mg of cytosol protein. ER and PR titers were found to be independent of histologic grade, clinical stage, patient age, age at first pregnancy, age at menarche, age at menopause and number of pregnancies. The presence of ER and PR in malignant ovarian tumors suggests that assays positive for these hormones could aid in the selection of patients suitable for hormonal therapy.