Mesenchymal stem cell detachment with trace trypsin is superior to EDTA for in vitro chemotaxis and adhesion assays.

Trypsin is frequently used to dissociate mesenchymal stem cells (MSCs) for in vitro adhesion and chemotaxis assays. However, its potential impact on surface receptor degradation is poorly understood. The purpose of this study was to evaluate the effect of trypsin-EDTA exposure versus PBS-EDTA on MSC surface receptor integrity and function. Primary human MSCs were detached with PBS-EDTA alone, or Cell Dissociation Buffer followed by 30 s exposure to 0.05% w/v trypsin-EDTA (trace trypsin method, TT), or 0.25% w/v trypsin exposure for 2 or 5 min. Cells were characterized for surface integrity of ß1 integrin (CD29) and PDGF Receptor (PDGF-R), and assessed in vitro for adhesion to atelocollagen-coated surfaces and migration to PDGF-BB. PBS-EDTA detachment fully preserved receptor integrity but routinely detached only half of the adherent cells and led to cell aggregates that failed to adhere evenly across the Transwell migration insert. Both CD29 and PDGF-R were significantly degraded by 0.25% trypsin detachment for 2 or 5 min compared to the TT method or PBS-EDTA (p < 0.05). Cells migrated optimally to PDGF-BB when detached with the TT method (3.1-fold vs α-MEM, p = 0.01). Cells attached optimally to atelocollagen when detached using the TT method or PBS-EDTA (6- to 10-fold vs 0.25% trypsin, p < 0.01). CDB followed by trace trypsin-EDTA exposure is recommended over PBS-EDTA to produce a single-cell MSC suspension that preserves receptor integrity and more reproducible receptor-mediated responses.

Efficacy of high dose Vitamin D supplementation in improving serum 25(OH)D among migrant and non migrant population: a retrospective study.

BACKGROUND: Higher dose of vitamin D supplementation 50000 IU is required for those whose serum 25(OH)D levels are 50 nmol/L and below. The increment in serum 25(OH)D though not significantly affected by race, sex or age it is negatively correlated to the baseline 25(OH)D concentration. This study investigated whether the mean increase in serum 25(OH)D will be higher among participants with lower baseline 25(OH)D levels and whether the duration of supplementation has an influence on the serum 25(OH)D achieved. METHODS: A clinical audit of patients' medical records from a community health centre in Melbourne for the period 01.01.2010 to 31-12.2012 was undertaken. Paired sample t test was used to determine difference in pre and post dose serum 25(OH)D. Simple and multiple linear regressions were used to examine the association between the difference in pre and post dose serum 25(OH)D and duration of supplementation and baseline serum 25(OH)D, adjusting for socio-demographic factors. RESULTS: A total of 205 patients were included in the study. Mean difference in serum 25(OH)D was highest 52.8 nmol/L (95 % CI: 46.63-58.92) among those whose serum 25(OH)D was below 25 nmol/L at baseline. Baseline 25(OH)D alone accounted for 13.7 % of variance in the effect size (F(2, 202) = 16.0. p < 0.001), with the effect size significantly higher among participants with a baseline 25(OH)D level of 25-49 nmol/L (ß = 11.93, 95 % CI: 0.48, 23.40, p < 0.05). Mean serum 25(OH)D difference was highest, 47.53 nmol/L (95 % CI: 40.95-54.11) when measured within 3 months of supplementation. Duration of supplementation explained 2.9 % of the variance in the effect size (F (1, 203) = 6.11, p < 0.05) and there was an inverse relationship between the length of supplementation and mean pre and post supplementation serum 25(OH)D difference (ß = -1.45, 95 % CI: -2.62, -0.29, p = 0.014). CONCLUSION: Following 50000 IU vitamin D3 for 12 months mean serum 25(OH)D increase was highest among those whose baseline serum 25(OH)D was lower. Migrants especially dark-skinned are at a high risk for vitamin D deficiency in Australia. High dose vitamin D3 50000 IU (cholecalciferol) is effective in achieving sufficient serum 25(OH)D among these populations who tend to have lower baseline serum 25(OH)D.

UHPLC UHD Q-TOF MS/MS analysis of the impact of sulfur fumigation on the chemical profile of Codonopsis Radix (Dangshen).

Over recent decades sulfur fumigation has been becoming abused in processing some freshly harvested Chinese medicinal herbs, although it is questioned whether sulfur fumigation can result in changes in efficacy and safety of the herbs. One of the herbs commonly processed by sulfur fumigation is Codonopsis Radix (Dangshen). A report showed that lobetyolin content in sulfur-fumigated Dangshen was lower than in air-dried Dangshen. Whereas there is no investigation designed to compare the chemical profiles of the sulfur-fumigated Dangshen and the air-dried Dangshen. In the present study, a rapid and versatile ultra-high-performance liquid chromatography coupled with ultra-high resolution quadrupole time-of-flight mass spectrometry (UHPLC UHD Q-TOF MS/MS) method was developed for comprehensive analysis of the chemical profiles of sulfur-fumigated and air-dried Dangshen samples. Principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) demonstrated that there were significant chemical differences between sulfur-fumigated and air-dried Dangshen samples. Among the changed components, 57 compounds were identified, in which 15 sulfur-containing compounds were detected only in sulfur-fumigated samples. The established methods were successfully applied to discriminate sulfur-fumigated Dangshen among commercial samples. Whether the chemical changes caused by sulfur fumigation affect the clinical efficacy and safety of Dangshen needs to be further investigated.

Assessment of vitamin D and its association with cardiovascular disease risk factors in an adult migrant population: an audit of patient records at a Community Health Centre in Kensington, Melbourne, Australia.

BACKGROUND: Vitamin D deficiency is a global public health problem associated with increased risk of cardio-metabolic diseases and osteoarthritis. Migrants with dark skin settled in temperate climates are at greater risk of both vitamin D deficiency and cardiovascular diseases. This study aims to identify the risk of vitamin D deficiency and associations with cardiovascular disease in a migrant population in Australia. METHODS: An audit was carried out at a Community Health Service in Kensington, Melbourne which, services a large migrant population. Data from the clinical records of all adults who visited the medical centre at least once during the period from 1st January 2010 to 31st December 2012 was extracted. The future (10 year) coronary heart disease risk was estimated using Framingham Risk Score. RESULTS: The centre has given higher priority to vitamin D testing in migrants, those middle-aged, females and those with diabetes and osteoarthritis. Migrants from countries located in lower latitude regions (Latitude N230 to S230) were 1.48 (95% C.I. 1.32-1.65) times more likely to develop vitamin D deficiency post migration and 0.44 (95% C.I. 0.31-0.62) times less likely to have a >15% 10-year risk of coronary heart disease when compared to their Australian-born counterparts. CONCLUSIONS: Adherence to a high risk strategy for vitamin D testing was observed in the centre. Pre-migration latitude is an important factor for vitamin D deficiency (lower the latitude higher the risk) and in predicting future risk of cardiovascular disease in migrants. These findings suggest that a targeted approach for vitamin D testing, including zone of origin might better identify individuals at higher risk of both vitamin D deficiency and cardiovascular disease.

Leukapheresis and Tisagenlecleucel Manufacturing Outcomes in Patients Age <3 Years with Relapsed/Refractory Acute Lymphoblastic Leukemia.

Tisagenlecleucel is approved for the treatment of relapsed/refractory (r/r) B cell acute lymphoblastic leukemia (B-ALL) in patients up to age 25 years based on the results of a pivotal trial (ELIANA) in pediatric and young adult patients. However, that trial did not include patients age <3 years because of the challenges posed by leukapheresis of very young and low-weight patients. Data on leukapheresis material and manufacturing outcomes among patients age <3 years have been collected since the time of global regulatory approval. Here we report leukapheresis characteristics and manufacturing outcomes for tisagenlecleucel produced for patients age <3 years in US and non-US commercial settings. Qualified patients with r/r B-ALL were age <3 years at the time of request for commercial tisagenlecleucel, with manufacturing data starting after August 30, 2017 (date of first US Food and Drug Administration approval). Leukapheresis and manufacturing outcomes data were stratified by age and weight. CD3+ cell count and CD3+/total nucleated cell (TNC) percentages were obtained from the leukapheresis material; leukocyte subpopulations were obtained via quality control vials. Of the 146 tisagenlecleucel quality control batches analyzed for CD3+ cell count and CD3+/TNC%, 86 batches (84 patients) were from US sites and 60 batches were from non-US sites. The median patient age and weight were 1.2 years and 10.4 kg at US sites and 1.5 years and 10.5 kg at non-US sites. Globally, 137 of 146 batches (94%) were manufactured within specifications across 16 countries. Among tisagenlecleucel batches manufactured in the United States between 2017 and 2021, there was a trend toward increasing CD3+ counts, CD3+/TNC%, and manufactured dose of chimeric antigen receptor (CAR) T cells; there was no difference in median days of collection by patient age or weight. Globally, a trend toward 1 or more potential additional collection days was observed for patients weighing ≤10 kg. Leukapheresis and tisagenlecleucel manufacturing in pediatric patients with r/r B-ALL age <3 years, including infants (<1 year), and low weight are feasible. As global experience with leukapheresis and patient identification for CAR-T cell therapy increased over time, a corresponding improvement in tisagenlecleucel manufacturing success has been observed. Clinical outcome data for these patients are currently being explored.

UDP-glucose dehydrogenase (UGDH) activity is suppressed by peroxide and promoted by PDGF in fibroblast-like synoviocytes: Evidence of a redox control mechanism.

UDP-glucose dehydrogenase (UGDH) generates essential precursors of hyaluronic acid (HA) synthesis, however mechanisms regulating its activity are unclear. We used enzyme histostaining and quantitative image analysis to test whether cytokines that stimulate HA synthesis upregulate UGDH activity. Fibroblast-like synoviocytes (FLS, from N = 6 human donors with knee pain) were cultured, freeze-thawed, and incubated for 1 hour with UDP-glucose, NAD+ and nitroblue tetrazolium (NBT) which allows UGDH to generate NADH, and NADH to reduce NBT to a blue stain. Compared to serum-free medium, FLS treated with PDGF showed 3-fold higher UGDH activity and 6-fold higher HA release, but IL-1beta/TGF-beta1 induced 27-fold higher HA release without enhancing UGDH activity. In selected proliferating cells, UGDH activity was lost in the cytosol, but preserved in the nucleus. Cell-free assays led us to discover that diaphorase, a cytosolic enzyme, or glutathione reductase, a nuclear enzyme, was necessary and sufficient for NADH to reduce NBT to a blue formazan dye in a 1-hour timeframe. Primary synovial fibroblasts and transformed A549 fibroblasts showed constitutive diaphorase/GR staining activity that varied according to supplied NADH levels, with relatively stronger UGDH and diaphorase activity in A549 cells. Unilateral knee injury in New Zealand White rabbits (N = 3) stimulated a coordinated increase in synovial membrane UGDH and diaphorase activity, but higher synovial fluid HA in only 2 out of 3 injured joints. UGDH activity (but not diaphorase) was abolished by N-ethyl maleimide, and inhibited by peroxide or UDP-xylose. Our results do not support the hypothesis that UGDH is a rate-liming enzyme for HA synthesis under catabolic inflammatory conditions that can oxidize and inactivate the UGDH active site cysteine. Our novel data suggest a model where UGDH activity is controlled by a redox switch, where intracellular peroxide inactivates, and high glutathione and diaphorase promote UGDH activity by maintaining the active site cysteine in a reduced state, and by recycling NAD+ from NADH.

Development of a QPatch-Automated Electrophysiology Assay for Identifying TMEM16A Small-Molecule Inhibitors.

The calcium-activated chloride channel, TMEM16A, is involved in airway hydration and bronchoconstriction and is a promising target for respiratory disease. Drug development efforts around channels require an electrophysiology-based assay for identifying inhibitors or activators. TMEM16A has proven to be a difficult channel to record on automated electrophysiology platforms due to its propensity for rundown. We developed an automated, whole-cell, electrophysiology assay on the QPatch-48 to evaluate small-molecule inhibitors of TMEM16A. In this assay, currents remained stable for a duration of roughly 11 min, allowing for the cumulative addition of five concentrations of compounds and resulted in reproducible IC50s. The absence of rundown was likely due to a low internal free-calcium level of 250 nM, which was high enough to produce large currents, but also maintained the voltage dependence of the channel. Current amplitude averaged 6 nA using the single-hole QPlate and the channel maintained outward rectification throughout the recording. Known TMEM16A inhibitors were tested and their IC50s aligned with those reported in the literature using manual patch-clamp. Once established, this assay was used to validate novel TMEM16A inhibitors that were identified in our high-throughput fluorescent-based assay, as well as to assist in structure-activity relationship efforts by the chemists. Overall, we demonstrate an easy to operate, reproducible, automated electrophysiology assay using the QPatch-48 for TMEM16A drug development efforts.

Liposomes modulate docetaxel-induced lipid oxidization and membrane damage in human hepatoma cells.

The aim of this study was to investigate the effect of liposomes on docetaxel-induced lipid oxidization and membrane damage in human hepatoma cells. Cytotoxicity of free docetaxel and docetaxel-containing liposomes was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay in human hepatoma cell lines HepG2 and SMMC-7721. To the cell lines, blank liposomes prepared with soybean phosphatidylcholine (SPC), dimyristoylphosphocholine (DMPC), and dioleoylphosphocholine (DOPC) did not show any significant toxicity below a 0.02-mg/mL phospholipid concentration. On the other hand, free docetaxel showed IC(50) values of 9.13 x 10(-6) +/- 1.54 x 10(-5) and 1.58 x 10(-2) +/- 2.71 x 10(-2) mg/mL in HepG2 cells and SMMC-7721 cells, respectively, after of 24 hours of incubation. IC(50) values of docetaxel-encapsulating liposomes, measured in terms of total docetaxel concentration, were at least 1.5-fold higher than those of free docetaxel. SPC liposomes reduced cellular damage caused by free docetaxel, as evidenced by the attenuation of docetaxel-induced lactate dehydrogenase (LDH) leakage by over 11% after liposome encapsulation at each dosage. Docetaxel-induced oxidative membrane damage was monitored by the formation of the lipid peroxidation product, malondialdehyde (MDA), and the antioxidative property of SPC liposome was monitored by the suppression of superoxide dismutase (SOD). These data demonstrated that free docetaxel facilitated MDA formation and suppressed SOD, and that these membrane-damaging effects were reduced by SPC liposomes.

Anti-inflammatory and analgesic effects of the ethanol extract of Rosa multiflora Thunb. hips.

This study aimed to assess the anti-inflammatory and analgesic effects of Fructus Rosae Multiflorae (FRM, hips of Rosa multiflora Thunb.). FRM was extracted with 75% ethanol and the dried extract (FRME) was administered intragastrically (i.g.) at 100, 200 and 400mg/kg. The anti-inflammatory effect was evaluated in four experimental animal models and analgesic effect in two animal models. Pretreatment with a single dose of FRME produced significant dose-dependent anti-inflammatory effects on carrageenin-induced rat hind paw edema, xylene-induced mouse ear edema and acetic acid-induced mouse vascular permeation. In a 7-day study, daily administration of FRME suppressed cotton pellet-induced rat granuloma formation. Pretreatment with a single dose of FRME also produced dose-dependent anti-nociceptive effects in thermally- and chemically induced mouse pain models. In addition, a single dose of FRME at 2.4g/kg body weight (equivalent to 87.6g of dried hips per kg body weight) produced no observable acute toxicity in mice within seven days. These results demonstrate that FRME possesses anti-inflammatory and analgesic effects and has no obvious acute toxicity, which advanced our understanding of the folk use of FRM in treating various inflammatory disorders.

Lysosomal rupture induced by structurally distinct chitosans either promotes a type 1 IFN response or activates the inflammasome in macrophages.

Chitosan is a family of glucosamine and N-acetyl glucosamine polysaccharides with poorly understood immune modulating properties. Here, functional U937 macrophage responses were analyzed in response to a novel library of twenty chitosans with controlled degree of deacetylation (DDA, 60-98%), molecular weight (1 to >100 kDa), and acetylation pattern (block vs. random). Specific chitosan preparations (10 or 190 kDa 80% block DDA and 3, 5, or 10 kDa 98% DDA) either induced macrophages to release CXCL10 and IL-1ra at 5-50 µg/mL, or activated the inflammasome to release IL-1ß and PGE2 at 50-150 µg/mL. Chitosan induction of these factors required lysosomal acidification. CXCL10 production was preceded by lysosomal rupture as shown by time-dependent co-localization of galectin-3 and chitosan and slowed autophagy flux, and specifically depended on IFN-ß paracrine activity and STAT-2 activation that could be suppressed by PGE2. Chitosan induced a type I IFN paracrine response or inflammasome response depending on the extent of lysosomal rupture and cytosolic foreign body invasion. This study identifies the structural motifs that lead to chitosan-driven cytokine responses in macrophages and indicates that lysosomal rupture is a key mechanism that determines the endogenous release of either IL-1ra or IL-1ß.

Antiangiogenic activity of a concentrated effective microorganism fermentation extract.

We have previously demonstrated the possible growth inhibitory activity of both first generation of the effective microorganism fermentation extract (EM-X) as well as the second generation (EM-X2) on cancer cell lines in vitro. The possible anti-angiogenic potential of EM-X has not been reported. Herein we show that using the concentrated EM-X, the growth of human umbilical cord endothelial cells (HUCE) was significantly inhibited in vitro. Enzyme linked immunosorbent assay suggested that the concentrated EM-X is able to reduce the level of vascular endothelial growth factor (VEGF) from Hep3B hepatocellular carcinoma (HCC) cells. The conditioned culture medium obtained from the concentrated EM-X incubated Hep3B HCC cells possessed significant antiproliferative effect on the HUCE cells. Moreover, in vivo chick chorioallantoic membrane assay further demonstrated that the concentrated EM-X is able to greatly inhibit the basic fibroblast growth factor induced angiogenesis from chick embryo experiment. We speculate that the anti-cancer potential of this concentrated EM-X involved growth inhibition on cancer cell and antiangiogenic effect on HUCE cells.

Apoptotic potential of the concentrated effective microorganism fermentation extract on human cancer cells.

The effective microorganism fermentation extract (EM-X, the first generation) was claimed to possess strong anti-oxidation property. On the other hand, we have shown that the second generation of the effective microorganism fermentation extract (EM-X2) possessed growth inhibition on human cancer cells involving MDA-MB231 breast cancer and K-562 chronic myelogenous leukaemia cells. Elevation of super oxide dismutase activity from EM-X2 treated cancer cell extract was observed. However, the possible anti-cancer activity of the first generation of the EM-X was not reported. Here we demonstrate that the concentrated form of the EM-X from its original fluid also possess antiproliferation ability together with induction of apoptosis on the human cancer cell lines including Hep3B hepatocellular carcinoma (HCC) and KG1a acute myelogenous leukaemia (AML). Similar effect could also be demonstrated on primary cultured bone marrow samples isolated from patients with AML. Morphological inspection revealed that common apoptotic feature was found on these concentrated EM-X treated cancer cells. Both the anchorage-dependent clonogenicity assay on Hep3B HCC and methyl-cellulose colony formation assay on KG1a cells and bone marrow cells from AML patients further revealed the ability of the concentrated EM-X on reducing their colony formation ability. Incubating KG1a with concentrated EM-X readily induced apoptosis as demonstrated by flow cytometric analysis. Interestingly, few growth inhibition effect of the concentrated EM-X was observed on both the SV40 transformed THLE-2 liver epithelial cells and primary cultured non-malignant haematological disordered bone marrow. Collectively, this concentrated EM-X is effective in inducing cell death and reducing the regeneration potential of both Hep3B HCC and KG1a AML cells in vitro.

Vitamin D testing patterns among general practitioners in a major Victorian primary health care service.

OBJECTIVE: To examine 25(OH)D testing patterns and frequency among general practitioners in a major community health service. METHOD: A clinical audit of patient records at a community health centre in Melbourne was undertaken. Patients aged 18 years and above were included. Univariate and multivariate logistic regression was used to examine the association between vitamin D testing and socio-demographic characteristics while Poisson regression was used for the frequency of testing. RESULTS: There were 1,217 patients tested for serum 25(OH)D. The community health centre was served by 12 general practitioners and an infectious disease specialist. The odds of vitamin D testing showed a positive, albeit weak, association with age (OR 1.01, 95%CI 1.00-1.02, p<0.05), were higher among females than males (OR 1.42, 95%CI 1.18-1.70, p<0.05) and higher among migrants compared to non-migrants (OR 2.57, 95%CI 2.14-3.09, p<0.05). The frequency of testing was also higher among females than males (IRR 1.17, 95%CI 1.07-1.28, p<0.05) and higher among migrants than non-migrants (IRR 1.19, 95%CI 1.08-1.31, p<0.05). CONCLUSION: Advancing age, being female and being a migrant were associated with an increased likelihood of vitamin D testing. IMPLICATIONS: Development of evidence-based policies and guidelines are needed to manage over-testing of vitamin D in Australia. Studies that include health services from different areas are required to understand vitamin D testing patterns among the general practitioners.

Biodegradable chitosan microparticles induce delayed STAT-1 activation and lead to distinct cytokine responses in differentially polarized human macrophages in vitro.

Current data suggest that chitosan activates wound macrophages to release endogenous factors that guide mesenchymal stem cell (MSC) to bone fractures. We tested the hypothesis that chitosan, a polymer containing glucosamine and N-acetyl glucosamine, stimulates macrophages in different polarization states to release functional MSC chemokines and mainly anabolic factors. Low-serum conditioned medium was collected from M0, M1 and M2a U937 macrophages previously differentiated with phorbol myristate acetate (PMA) and exposed or not for 24h to chitosan microparticles (80% degree of deacetylation, DDA, 130kDa). Chitosan particles were highly phagocytosed. Chitosan enhanced anabolic factor release from M0 and M2a macrophages (MCP-1, IP-10, MIP-1beta, IL-1ra, IL-10, PDGF), and IL-1beta release, with 25- to 400-fold excess IL-1ra over IL-1beta. In M1 macrophages, chitosan enhanced IL-1beta without enhancing or suppressing inflammatory factor release (IL-6, IP-10, IL-8). M0 and M2a macrophages, with or without chitosan stimulation, produced conditioned medium that promoted 2-fold more MSC chemotaxis than low-serum control medium, while M1-conditioned medium failed to induce MSC chemotaxis. Acetylated chitosan induced U937 macrophages to release IL-1ra without STAT-6 activation, and also induced a delayed STAT-1 activation/IP-10 release response that was not observed using non-biodegradable chitosan (98% DDA, 130kDa). In primary human macrophages, acetylated chitosan enhanced IL-1ra release without inducing IL-1beta, and required PMA priming to elicit STAT-1 activation and IP-10 release. We conclude that biodegradable chitosan particles enhance M0 and M2a macrophage anabolic responses independent of the IL4/STAT-6 axis, by inducing excess IL-1ra over IL-1beta and more chemokine release, without altering their inherent capacity to attract MSCs.

Placental histopathology of congenital syphilis.

OBJECTIVE: To evaluate the contribution of placental histopathology to the diagnosis of congenital syphilis. METHODS: From January 1, 1986, through December 31, 1998, all pregnant women presenting to a large, urban Dallas County labor and delivery unit with untreated syphilis at delivery and who had placental evaluation performed were identified. Women were clinically staged, and the infants were evaluated for congenital syphilis using a standard protocol. Each placenta was evaluated by two independent pathologists. Histologic characteristics of the placenta related to congenital syphilis in live-born and stillborn infants were then analyzed. RESULTS: Sixty-seven women met the study criteria: 33 (49%) stillborn and 18 (27%) live-born infants with congenital syphilis, 15 (22%) uninfected live-born infants, and one uninfected stillborn fetus diagnosed by current criteria. There were no differences between the groups with regard to demographic characteristics, prenatal care, or stage of syphilis. Stillborn infants were more likely to deliver preterm (P <.001). Controlling for gestational age, histopathology revealed necrotizing funisitis, villous enlargement, and acute villitis associated with congenital syphilis. Erythroblastosis was more common in stillborn infants with congenital syphilis than all live-born infants (odds ratio 16, 95% confidence interval 1, 370). The addition of histologic evaluation to conventional diagnostic evaluations improved the detection rate for congenital syphilis from 67% to 89% in live-born infants, and 91% to 97% in stillborn infants. CONCLUSION: Our results show that histopathologic examination of the placenta is a valuable adjunct to the contemporary diagnostic criteria used to diagnose congenital syphilis.

Usage of complementary medicine among children.

OBJECTIVE: To determine the prevalence of and parental attitudes toward the usage of complementary medicine among the paediatric population of a large regional public hospital in Victoria. Relationships between complementary medicine usage and sociological or medical data of the surveyed families are explored. DESIGN: One hundred and twenty surveys were handed out and returned from parents of nonsurgical inpatients of the children's ward of the Bendigo Base Hospital. Survey data was supplemented by information available from the hospital medical record. RESULTS: Thirty-three percent of respondents indicated they used complementary medicine for their inpatient child, and 41% for at least one of their children. Vitamins were more popular and acupuncture less popular than complementary medicine modalities used by their parents. Complementary medicine use was not correlated with: the patient's age; presenting complaint; duration of inpatient stay; or number of previous admissions. Families with children using complementary medicine were more likely to have skilled or professional parents who also used complementary medicine. There was a correlation between children using complementary medicine and inadequate vaccination. CONCLUSION: A significant proportion of children are exposed to complementary medicine. Parent, rather than child, characteristics were most strongly correlated with complementary usage.

Expression of ORAII, a plasma membrane resident subunit of the CRAC channel, in rodent and non-rodent species.

We determined the expression of ORAI1 protein in rodent and non-rodent tissues using a monoclonal antibody directed against an extracellular loop of the protein. Previous reports using antibodies directed at the C-terminus of ORAI1 have not detected central nervous system (CNS) expression. Our results demonstrate broad tissue expression that includes the CNS using a unique monoclonal antibody specific to an extracellular loop of ORAI1. In addition, we present in situ hybridization (ISH) results using a probe within the middle of the mouse coding region showing CNS expression of Orai1 RNA. We contrast the patterns of rodent and human tissue expression and conclude that rodents have similar expression of ORAI1 in most tissue types when compared to primates, with an important exception being the male reproductive system, where human-specific expression is observed.

Bone morphogenetic protein-9 activates Smad and ERK pathways and supports human osteoclast function and survival in vitro.

BMP-9 is a potent osteogenic factor; however, its effects on osteoclasts, the bone-resorbing cells, remain unknown. To determine the effects of BMP-9 on osteoclast formation, activity and survival, we used human cord blood monocytes as osteoclast precursors that form multinucleated osteoclasts in the presence of RANKL and M-CSF in long-term cultures. BMP-9 did not affect osteoclast formation, but adding BMP-9 at the end of the culture period significantly increased bone resorption compared to untreated cultures, and reduced both the rate of apoptosis and caspase-9 activity. BMP-9 also significantly downregulated the expression of pro-apoptotic Bid, but only after RANKL and M-CSF, which are both osteoclast survival factors, had been eliminated from the culture medium. To investigate the mechanisms involved in the effects of BMP-9, we first showed that osteoclasts expressed some BMP receptors, including BMPR-IA, BMPR-IB, ALK1, and BMPR-II. We also found that BMP-9 was able to induce the phosphorylation of Smad-1/5/8 and ERK 1/2 proteins, but did not induce p38 phosphorylation. Finally, knocking down the BMPR-II receptor abrogated the BMP-9-induced ERK-signaling, as well as the increase in bone resorption. In conclusion, these results show for the first time that BMP-9 directly affects human osteoclasts, enhancing bone resorption and protecting osteoclasts against apoptosis. BMP-9 signaling in human osteoclasts involves the canonical Smad-1/5/8 pathway, and the ERK pathway.

Chemical differentiation of two taste variants of Gynostemma pentaphyllum by using UPLC-Q-TOF-MS and HPLC-ELSD.

To differentiate the sweet and bitter taste variants of a Chinese medicinal tea Gynostemma pentaphyllum (GP), a method for the quantitative analysis of ginsenosides Rb(1), Rb(3), Rd, and F(2) in GP by using UPLC-Q-TOF-MS was developed. According to the different contents of the four ginsenosides, chemical differentiation of the two taste variants of GP was achieved by principal component analysis (PCA). A supplementary quantitative analysis method of using HPLC-ELSD for determination of 20(S)-panaxadiol in the hydrolysates of GP was also developed. Similarly, chemical differentiation based on different amounts of 20(S)-panaxadiol was established and the result was well consistent with that based on the analysis of the four ginsenosides. It was found that the amounts of the four ginsenosides and 20(S)-panaxadiol in the sweet taste variant were significantly higher than those in the bitter one. The significant difference between the sweet and bitter taste variants of GP was easily visualized in 3D-PCA score plots. The PCA loading plot also indicated the contributions among the four ginsenosides (Rd > Rb(3) > F(2) > Rb(1)) for distinguishing the two taste variants. This is the first report to describe the use of these two quantitative methods (UPLC-Q-TOF-MS and HPLC-ELSD) for the accurate authentication and quality control of GP.