Stachyonic Acid: A Dengue Virus Inhibitor from Basilicum polystachyon.

Stachyonic acid A, arising from the first in-depth phytochemical investigation of the herb Basilicum polystachyon, was found to display potent inhibitory activity against dengue virus, with limited cytotoxicity. Andrographolide, a known dengue virus inhibitor and closely related labdane-type diterpene, is structurally more complex but displayed poor antiviral activity in the PRNT assay, and increased cytotoxicity in comparison. Furthermore, a Diels-Alder reaction with PTAD identified the active pharmacophore of stachyonic acid to be the conjugated diene.

Basimarols A, B, and C, Highly Oxygenated Pimarane Diterpenoids from Basilicum polystachyon.

The highly oxygenated pimarane diterpenoids basimarols A, B, and C (3-5) were isolated from the plant species Basilicum polystachyon, which was collected within the Australian arid zone. Structure elucidation was performed using a suite of spectroscopic techniques, including X-ray crystallography. Anticancer and anti-DENV activity of 3-5 was explored, but only limited activity was observed. More extensive antiviral evaluation of stachyonic acid A (1), which was also isolated from B. polystachyon, revealed broad spectrum antiviral activity against West Nile virus (Kunjin strain, WNVKun) and human influenza viruses H1N1 and H3N2.

Reappraisal of endometrial thickness for the detection of endometrial cancer in postmenopausal bleeding: a retrospective cohort study.

OBJECTIVE: To estimate the accuracy of transvaginal ultrasound (TVS) measurement of endometrial thickness (ET) in diagnosing endometrial cancer in postmenopausal women with vaginal bleeding (PMB). DESIGN: Retrospective cohort study. SETTING: One-stop PMB clinic in a Hong Kong teaching hospital. POPULATION: A cohort of 4383 women with PMB. METHODS: Transvaginal ultrasonic measurement of ET and endometrial biopsies were obtained in women presenting with PMB between 2002 and 2013. Endometrial histology was used as the reference standard to calculate accuracy estimates. MAIN OUTCOME MEASURES: Accuracy data for TVS ET presented as sensitivity, specificity, and area under the receiver operator characteristic (ROC) curve. RESULTS: Endometrial cancer was diagnosed in 3.8% of women. The median ET in those with endometrial cancer was significantly higher than those with benign conditions (15.7 versus 3.2 mm, P < 0.001). The area under the ROC curve was 0.92 (95% CI 0.89-0.94). The sensitivity for the detection of endometrial cancer at 3-, 4-, and 5-mm cut-offs were 97.0% (95% CI 94.5-99.6%), 94.1% (95% CI 90.5-97.6%), and 93.5% (95% CI 89.7-97.2%), respectively. The corresponding estimates of specificity at these thresholds were 45.3% (95% CI 43.8-46.8%), 66.8% (65.4-68.2%), and 74.0% (72.7-75.4%). CONCLUSIONS: Transvaginal ultrasound using a 3-mm cut-off has high sensitivity for detecting endometrial cancer and can identify women with PMB who are highly unlikely to have endometrial cancer, thereby avoiding more invasive endometrial biopsy.

Wasabi (Eutrema japonicum) Reduces Obesity and Blood Pressure in Diet-Induced Metabolic Syndrome in Rats.

6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC) has several biological functions. The present study aimed to evaluate the composition of hydroponically grown Tasmanian wasabi (Eutrema japonicum (Miq.) Koidz.) for 6-MSITC in all plant tissues and investigate the influence of wasabi (rhizome and stem blend) in high-carbohydrate, high-fat (H) diet-fed rats. Male Wistar rats were fed either a corn starch (C) or H diet. After the initial 8 weeks, half of the animals on the C and H diets were given 5% (w/w) wasabi powder in their respective diets for an 8-week duration (CW and HW). The control animals received diets without supplementation throughout the 16-week experiment. Our findings demonstrated that wasabi grown under hydroponic conditions contained 6-MSITC in all parts of the plant such as the stem, leaf and flower, as well as the commonly used rhizome, albeit at lower concentrations. Rats treated with wasabi showed reductions in body weight (H, 460.0 ± 9.5; HW, 416.0 ± 3.6 g), fat mass (H, 178 ± 14; HW, 120 ± 23 g), plasma triglycerides (H, 1.7 ± 0.3; HW, 0.9 ± 0.3 mmol/L) and total cholesterol (H, 1.5 ± 0.1; HW, 1.0 ± 0.04 mmol/L), and the plasma activities of aspartate transaminase. Systolic blood pressure and the area under the curve of blood glucose concentration were decreased by wasabi treatment. Thus, wasabi may be a novel alternative treatment to assist in the management of obesity and related metabolic disorders.

Newcastle disease virus-infected splenocytes express the proopiomelanocortin gene.

Infection of murine splenocytes with Newcastle disease virus results in expression of the proopiomelanocortin gene as determined by dot and northern blot analysis of total cellular and poly(A)+ cytoplasmic RNA. These data provide the first evidence that the precursor protein for adrenocorticotropin and beta-endorphin can be synthesized by lymphoid cells.

Exosomes from human saliva as a source of microRNA biomarkers.

OBJECTIVE: The aim of this study was to examine the presence of microRNAs (miRNAs) within exosomes isolated from human saliva and to optimize and test methods for successful downstream applications. DESIGN: Exosomes isolated from fresh and frozen glandular and whole human saliva were used as a source of miRNAs. The presence of miRNAs was validated with TaqMan quantitative PCR and miRNA microarrays. RESULTS: We successfully isolated exosomes from human saliva from healthy controls and a patient with Sjögren's syndrome. microRNAs extracted from the exosomal fraction were sufficient for quantitative PCR and microarray profiling. CONCLUSIONS: The isolation of miRNAs from easily and non-invasively obtained salivary exosomes with subsequent characterization of the miRNA expression patterns is promising for the development of future biomarkers of the diagnosis and prognosis of various salivary gland pathologies.

Evaluation of polymyxin B in combination with 13 other antibiotics against carbapenemase-producing Klebsiella pneumoniae in time-lapse microscopy and time-kill experiments.

OBJECTIVES: This study aimed to explore the interactions of polymyxin B in combination with 13 other antibiotics against carbapenemase-producing Klebsiella pneumoniae. METHODS: Five clinical isolates of multidrug-resistant K. pneumoniae producing KPC-2, KPC-3, NDM-1, OXA-48 and VIM-1 carbapenemases were used. Polymyxin B was tested alone and in combination with amikacin, aztreonam, cefepime, chloramphenicol, ciprofloxacin, fosfomycin, linezolid, meropenem, minocycline, rifampicin, temocillin, thiamphenicol and trimethoprim. Inhibition of growth during antibiotic exposure was evaluated in 24-hr automated time-lapse microscopy experiments. Combinations that showed positive interactions were subsequently evaluated in static time-kill experiments. RESULTS: All strains carried multiple (≥9) resistance genes as determined by whole-genome sequencing. In the initial screening the combination of polymyxin B and minocycline was most active with enhanced activity compared with the single antibiotics detected against all strains. Positive interactions were also observed with polymyxin B in combination with rifampicin and fosfomycin against four of five strains and less frequently with other antibiotics. Time-kill experiments demonstrated an additive or synergistic activity (1-2 log10 or ≥2 log10 CFU/mL reduction, respectively, compared with the most potent single antibiotic) with 21 of 23 tested combinations. However, because of regrowth, only 13 combinations were synergistic at 24 hr. Combinations with minocycline or rifampicin were most active, each showing synergy and bacteriostatic or bactericidal effects resulting in 1.93-3.97 and 2.55-5.91 log10 CFU/mL reductions, respectively, after 24 hr against four strains. DISCUSSION: Polymyxin B in combination with minocycline, rifampicin or fosfomycin could be of potential clinical interest. Time-lapse microscopy showed some discrepancy in results compared with the time-kill data but was useful for screening purposes.

Heyde's syndrome: diagnosis and management by the novel single-balloon enteroscopy.

We present a case of obscure gastro-intestinal bleeding due to small bowel angiodysplasia in a 68-year-old woman with severe aortic valve stenosis. The diagnosis was confirmed and subsequently managed using single-balloon enteroscopy.

Menstrual problems and health-seeking behaviour in Hong Kong Chinese girls.

OBJECTIVE: To ascertain the prevalence of menstrual problems in adolescent girls and their health-seeking behaviour. DESIGN: Questionnaire survey on menstruation, menstrual problems, medical consultation, and factors influencing girls seeking medical care. SETTING: Secondary schools in the catchment area of a tertiary teaching hospital in Hong Kong. PARTICIPANTS: A total of 5609 girls from 10 secondary schools. MAIN OUTCOME MEASURES: Prevalence of menstrual problems and health-seeking behaviour of adolescent girls. RESULTS: The mean age of the girls and their mean age at menarche were 15.1 (standard deviation, 2.0) years and 12.3 (1.1) years, respectively. The prevalence of menorrhagia, dysmenorrhoea, and menstrual symptoms were 17.9% (95% confidence interval, 16.9-19.1%), 68.7% (67.7-70.3%), and 37.7% (36.7-39.3%), respectively. The prevalence of menstrual symptoms (P<0.001) and dysmenorrhoea (P<0.001) increased with gynaecological age (calendar age minus age at menarche), whilst the proportion having short or long cycles decreased (P=0.002 and P=0.009). One in eight girls reported having been absent from school, whilst only 6.4% had sought medical care because of menses. Multivariate analysis indicated that seeking medical care for menorrhagia was dependent on the opinion of a family member (P=0.005), and for dysmenorrhoea on its severity (P=0.046) and anxiety about embarrassing questions (P=0.039). CONCLUSIONS: The prevalence of menstrual problems in Hong Kong Chinese girls is high and causes significant disruption to their school and daily activities. However, only a minority seek medical advice.

Setting the stage for acute-on-chronic kidney injury.

Acute-on-chronic kidney disease will be familiar to many nephrologists. Hsu et al. quantify the risk of acute-on-chronic disease across the stages of preexisting chronic kidney disease. Their study demonstrates the valuable insights that large epidemiological studies can bring to the field of acute kidney injury.

AP214, an analogue of alpha-melanocyte-stimulating hormone, ameliorates sepsis-induced acute kidney injury and mortality.

Sepsis remains a serious problem in critically ill patients with the mortality increasing to over half when there is attendant acute kidney injury. alpha-Melanocyte-stimulating hormone is a potent anti-inflammatory cytokine that inhibits many forms of inflammation including that with acute kidney injury. We tested whether a new alpha-melanocyte-stimulating hormone analogue (AP214), which has increased binding affinity to melanocortin receptors, improves sepsis-induced kidney injury and mortality using a cecal ligation and puncture mouse model. In the lethal cecal ligation-puncture model of sepsis, severe hypotension and bradycardia resulted and AP214 attenuated acute kidney injury of the lethal model with a bell-shaped dose-response curve. An optimum AP214 dose reduced acute kidney injury even when it was administered 6 h after surgery and it significantly improved blood pressure and heart rate. AP214 reduced serum TNF-alpha and IL-10 levels with a bell-shaped dose-response curve. Additionally; NF-kappaB activation in the kidney and spleen, and splenocyte apoptosis were decreased by the treatment. AP214 significantly improved survival in both lethal and sublethal models. We have shown that AP214 improves hemodynamic failure, acute kidney injury, mortality and splenocyte apoptosis attenuating pro- and anti-inflammatory actions due to sepsis.

Furofuran lignans from the Simpson Desert species Eremophila macdonnellii.

The Eremophila plant family, which occurs in the arid zones of Australia, have witnessed extensive investigation, mostly inspired by Aboriginal traditional medicine. A wide and varied biological and phytochemical profile has been reported for over 18 individual species of Australian Eremophila, although E. macdonnellii from the Simpson Desert has not yet been investigated. Isolation and elucidation of one new and six known furofuran lignans are reported. The new lignan, epimethoxypiperitol, displayed moderate anti-cancer activity against the breast cancer cell line (MCF-7).

Differential expression of mRNA for guanylyl cyclase-linked endothelium-derived relaxing factor receptor subunits in rat kidney.

Endothelium-derived relaxing factor (EDRF) has profound effects on the renal vasculature, the glomerular mesangium, and also affects renal salt excretion. EDRF stimulates guanylyl cyclases, which are thought to be heterodimers comprised of alpha and beta subunits. Two alpha and two beta isoforms have been identified thus far. However, the molecular composition of in vivo guanylyl cyclase-linked EDRF receptors is unknown. We used polymerase chain reaction to clone a portion of the rat alpha 2 subunit. Guanylyl cyclase-linked EDRF receptor mRNA was detected in microdissected renal structures using a reverse transcription/polymerase chain reaction assay. The interlobular artery/afferent arteriole contained mRNA for the alpha 1, alpha 2, and beta 1 subunits; a faint beta 2 band was found in 29% of experiments. In contrast, the cortical collecting duct contained mRNA only for alpha 1 and beta 2 subunits. We conclude that guanylyl cyclase-linked EDRF receptor subunit isoforms are independently and heterogeneously expressed in the renal vasculature and cortical collecting duct, suggesting that several different EDRF receptors exist in vivo. These data suggest that the tubule receptor is composed of alpha 1/beta 2. The vasculature may contain at least two different EDRF receptors (alpha 1/beta 1 and alpha 2/beta 1). Some beta 2 may also be expressed, allowing for even greater heterogeneity.

A randomised controlled trial of prophylactic levonorgestrel intrauterine system in tamoxifen-treated women.

OBJECTIVE: To study the prophylactic use of levonorgestrel intrauterine system (LNG-IUS) in the prevention of endometrial pathology in women having breast cancer treated with tamoxifen. DESIGN: Randomised controlled trial. SETTING: A tertiary teaching hospital. POPULATION: One hundred and thirteen women (66 premenopausal/47 postmenopausal) who required adjuvant tamoxifen for breast cancer after the completion of postoperative radiotherapy and chemotherapy. METHODS: Women were randomised to treatment group (prophylactic LNG-IUS insertion before the commencement of tamoxifen) or control group. Uterine cavity was examined by outpatient hysteroscopy and endometrial biopsy before and at 12 months after commencement of tamoxifen. MAIN OUTCOME MEASURES: De novo endometrial pathology at 1 year of tamoxifen. RESULTS: Women in the treatment group had a much lower incidence of endometrial polyp (1.8 versus 15.5%, P= 0.017) (relative risk: 0.12; 95% CI: 0.02-0.91) at 12 months. There was no significant difference in the incidence of submucosal fibroid between the two groups (1.8 versus 3.4%, P= 1.0). LNG-IUS was retained in 95% women in the treatment group at 1 year. CONCLUSION: LNG-IUS reduces the occurrence of de novo endometrial polyp in women treated with tamoxifen for breast cancer.

An audit on hysterectomy for benign diseases in public hospitals in Hong Kong.

OBJECTIVE: To assess the standard of hysterectomy in public hospitals in Hong Kong, so as to improve the quality of patient care and outcome. DESIGN: Clinical audit. SETTING: Twelve Hong Kong Hospital Authority public hospitals. PATIENTS: All patients undergoing hysterectomy for benign gynaecological conditions during the period from 1 July 2002 to 31 December 2002 inclusive. RESULTS: A total of 1330 patients were included for analysis: 934 (70.2%) having abdominal hysterectomies, 184 (13.8%) having laparoscopic hysterectomies, and 212 (15.9%) undergoing vaginal hysterectomies. Uterine fibroids constituted the commonest indication for abdominal (73.7%) and laparoscopic (61.4%) hysterectomies, while genital prolapse was the most common indication (96.2%) for vaginal hysterectomy. The majority of patients undergoing laparoscopic and vaginal hysterectomy (86.3% and 84.8% respectively) were given prophylactic antibiotics, in contrast to only 45.8% of those undergoing abdominal hysterectomy. In all, 85.8% of the abdominal and vaginal hysterectomies performed by trainees were supervised, while for trainees performing laparoscopic hysterectomy, all had specialists as their first assistant. The overall incidence of complications for vaginal hysterectomy was lower than that for both abdominal hysterectomy (P<0.001) and laparoscopic hysterectomy (P<0.05). Infectious morbidity was significantly higher in patients undergoing abdominal hysterectomy without prophylactic antibiotics. CONCLUSION: The overall incidence of complications was lower for vaginal hysterectomies, as compared to both abdominal and laparoscopic hysterectomies, whereas the risk of urinary tract injury was significantly higher for laparoscopic hysterectomy. According to our audit, the level of supervision for the trainees was high. However, routine antibiotic prophylaxis should be more consistently used in the territory.

Urine Exosomes: An Emerging Trove of Biomarkers.

Exosomes are released by most cells and can be isolated from all biofluids including urine. Exosomes are small vesicles formed as part of the endosomal pathway that contain cellular material surrounded by a lipid bilayer that can be traced to the plasma membrane. Exosomes are potentially a more targeted source of material for biomarker discovery than unfractionated urine, and provide diagnostic and pathophysiological information without an invasive tissue biopsy. Cytoplasmic contents including protein, mRNA, miRNA, and lipids have all been studied within the exosomal fraction. Many prospective urinary exosomal biomarkers have been successfully identified for a variety of kidney or genitourinary tract conditions; detection of systemic conditions may also be possible. Isolation and analysis of exosomes can be achieved by several approaches, although many require specialized equipment or involve lengthy protocols. The need for timely analysis in the clinical setting has driven considerable innovation with several promising options recently emerging. Consensus on exosome isolation, characterization, and normalization procedures would resolve critical clinical translational bottlenecks for existing candidate exosomal biomarkers and provide a template for additional discovery studies.

Laboratory parameter-based machine learning model for excluding non-alcoholic fatty liver disease (NAFLD) in the general population.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) affects 20%-40% of the general population in developed countries and is an increasingly important cause of hepatocellular carcinoma. Electronic medical records facilitate large-scale epidemiological studies, existing NAFLD scores often require clinical and anthropometric parameters that may not be captured in those databases. AIM: To develop and validate a laboratory parameter-based machine learning model to detect NAFLD for the general population. METHODS: We randomly divided 922 subjects from a population screening study into training and validation groups; NAFLD was diagnosed by proton-magnetic resonance spectroscopy. On the basis of machine learning from 23 routine clinical and laboratory parameters after elastic net regulation, we evaluated the logistic regression, ridge regression, AdaBoost and decision tree models. The areas under receiver-operating characteristic curve (AUROC) of models in validation group were compared. RESULTS: Six predictors including alanine aminotransferase, high-density lipoprotein cholesterol, triglyceride, haemoglobin A1c , white blood cell count and the presence of hypertension were selected. The NAFLD ridge score achieved AUROC of 0.87 (95% CI 0.83-0.90) and 0.88 (0.84-0.91) in the training and validation groups respectively. Using dual cut-offs of 0.24 and 0.44, NAFLD ridge score achieved 92% (86%-96%) sensitivity and 90% (86%-93%) specificity with corresponding negative and positive predictive values of 96% (91%-98%) and 69% (59%-78%), and 87% of overall accuracy among 70% of classifiable subjects in the validation group; 30% of subjects remained indeterminate. CONCLUSIONS: NAFLD ridge score is a simple and robust reference comparable to existing NAFLD scores to exclude NAFLD patients in epidemiological studies.

Multipotent neural precursors express neural and hematopoietic factors, and enhance ex vivo expansion of cord blood CD34+ cells, colony forming units and NOD/SCID-repopulating cells in contact and noncontact cultures.

In view of the possible crosstalks between hematopoiesis and neuropoiesis, we evaluated two microenvironments, murine neonatal neural cell line C17.2 and primary embryonic aorta-gonad-mesonephros (AGM) stromal cells, on the ex vivo expansion of CD34+ cells from human cord blood. In a contact culture system, C17.2 or AGM cells significantly enhanced the expansion of CD34+ cells to a panel of early and committed hematopoietic progenitor cells. In a noncontact transwell system, pre-established C17.2 cells significantly increased the expansion of total nucleated cells, CD34+ cells and multilineage colony forming cells (P<0.01). Expanded cells were infused into nonobese diabetic/severe-combined immunodeficient mice. The engraftment of human (hu)CD45+ cells in the bone marrow of these mice was consistently higher in all the 10 experiments conducted with the support of C17.2 cells when compared with those in respective control groups (11.9 vs 2.43%, P=0.03). Using RT-PCR and Southern blot analysis, we showed that AGM and C17.2 cells expressed a panel of hematopoietic, bone morphogenetic and neurotrophic factors. Our data provided the first evidence on the promoting effects of a neural progenitor cell line on hematopoiesis at a noncontact condition. The mechanism could be mediated by the expression of multilineage regulatory factors.

RNA: a method to specifically inhibit PCR amplification of known members of a multigene family by degenerate primers.

The polymerase chain reaction (PCR) is a versatile method to amplify specific DNA with oligonucleotide primers. By designing degenerate PCR primers based on amino acid sequences that are highly conserved among all known gene family members, new members of a multigene family can be identified. The inherent weakness of this approach is that the degenerate primers will amplify previously identified, in addition to new, family members. To specifically address this problem, we synthesized a specific RNA for each known family member so that it hybridized to one strand of the template, adjacent to the 3'-end of the primer, allowing the degenerate primer to bind yet preventing extension by DNA polymerase. To test our strategy, we used known members of the soluble, nitric oxide-sensitive guanylyl cyclase family as our templates and degenerate primers that discriminate this family from other guanylyl cyclases. We demonstrate that amplification of known members of this family is effectively and specifically inhibited by the corresponding RNAs, alone or in combination. This robust method can be adapted to any application where multiple PCR products are amplified, as long as the sequence of the desired and the undesired PCR product(s) is sufficiently distinct between the primers.