Developing a novel quasi-3D movable water phantom for radiation therapy workable in the magnetic resonance environment.

Background: The use of magnetic resonance linear accelerators (MR-LINACs) for clinical treatment has opened up new possibilities and challenges in the field of radiation oncology. However, annual quality assurance (QA) is relatively understudied due to practical considerations. Thus, to overcome the difficulty of measuring the dose with a small water phantom for TRS-398 or TG-51 in all external beam radiation treatment unit environments, such as MR compatibility, we designed a remote phantom with a three-axis changeable capacity for QA. Methods: The designed water phantom was tested under an MR environment. The water phantom system comprised of three parts: a phantom box, a dose measurement tool, and a PMD401 drive system. The UNIDOSE universal dosimeter was used to collect beam data. The manufacturer's developer tools were utilized to position the measurement. To ensure magnetic field homogeneity, a distortion phantom was prepared using sixty fish oil capsules aligned radially to distinguish the oil and free air. The phantom was scanned in both the MR simulator and computed tomography (CT), and the acquired images were analyzed to determine the position shift. Results: The dimensions of the device are 30 cm in the X-axis, 20 cm in the Y-axis, and 17 cm in the Z-axis. Total cost of materials was no more than $10,000 US dollars. Our results indicate that the device can function normally in a regular 1.5 T MR environment without interference from the magnetic field. The water phantom's traveling speed was found to be approximately 5 mm/s with a position difference confined within 6 cm intervals during normal use. The distortion test results showed that the prepared MR environment has uniform magnetic field homogeneity. Conclusions: In this study, we constructed a prototype water phantom device that can function in an MR simulator without interference between the magnetic field and electronic components. Compared to other commercially available MR-LINAC water phantoms, our device offers a more cost-effective solution for routine monthly QA. It can shorten the duration of QA tests and relieve the burden on medical physicists.

Development of a detector tube for screening tadalafil and its analogues in adulterated sexual enhancement products.

Sexual enhancement products adulterated with phosphodiesterase 5 inhibitors (PDE-5i) pose a serious public health concern. Tadalafil and its analogues (Tds) are PDE-5i frequently detected as adulterants. In this study, a Td detector tube for the rapid detection of Tds was developed based on the color change reaction between sulfuric acid and Tds. The specificity of this test method was evaluated using 13 Tds, all of which elicited positive results. Additionally, 30 commonly found adulterants in dietary supplements, 11 active pharmaceutical ingredients of psychotropic drugs and 18 food ingredients were tested and obtained no false-positive results, except levomepromazine. The test tube accurately detected the presence or absence of Tds in 54 commercially available products. The visual detection limit was 2-50 and 5-20 µg/ml for Tds and tadalafil-spiked samples with matrix, respectively. The applicability of the developed detector tube to a semiquantitative test using digital image analyses were investigated using red, green, and blue color values. The results of the recovery test suggested that the tube test was affected by the dark-colored matrix. The results of semiquantitative analyses of tadalafil for five marketed products were consistent with the liquid chromatographic quantification results, except for the blue value. The detector tube developed in this study can facilitate with the rapid screening of Tds in adulterated sexual enhancement products.

Effects of conjugated linoleic acid and exercise on body composition and obesity: a systematic review and meta-analysis.

CONTEXT: Conjugated linoleic acid (CLA) has been reported to have anti-obesity and antidiabetic effects. However, the benefits of CLA combined with exercise remain unclear, and studies report conflicting results. OBJECTIVE: A systematic review and meta-analysis were performed to investigate the synergistic effect of CLA and exercise on body composition, exercise-related indices, insulin resistance, and lipid profiles; and of the safety of CLA supplements. DATA SOURCES: In October 2021, the PubMed, Embase, and Cochrane Library databases were searched for reports on clinical trials of the combined intervention of CLA and exercise. DATA EXTRACTION: A total of 18 randomized controlled trials and 2 crossover trials were included. The methodological quality assessment was performed using the revised Cochrane risk-of-bias tool. Pooled effect sizes were reported as standardized mean difference (SMD) for continuous data and risk ratio for dichotomous data with their corresponding 95% confidence intervals (CIs). Heterogeneity was tested using the I2 statistic. DATA ANALYSIS: The combination of CLA and exercise resulted in significantly decreased body fat (SMD, -0.42 [95%CI, -0.70, -0.14]; P = 0.003; I2 = 65) and insulin resistance (SMD, -0.25 [95%CI, -0.44, -0.06]; P = 0.01; I2 = 0) than did exercise alone. In subgroup analysis, the following factors were associated with significant outcomes: (1) body mass index ≥25 kg/m2; (2) female sex; (3) follow-up time >4 weeks; and (4) intervention duration >4 weeks. Nevertheless, supplementation with CLA during exercise programs was not effective for body-weight control, exercise performance enhancement, or lipid-profile improvement. CLA in combination with exercise did not result in a higher risk of adverse events (risk ratio, 1.32 [95%CI, 0.94-1.84]; P > 0.05; I2 = 0). CONCLUSION: CLA combined with exercise is generally safe and can lower body fat and insulin resistance but does not reduce body weight, enhance exercise performance, or improve lipid profiles.

Decision Tree-Based Body Constitution Diagnosis System for Traditional Chinese Medicine.

This study aimed to establish a method for fast and accurate determination of body constitution types from the body constitution questionnaire (BCQ) by employing a decision tree model. The model was trained for 4 classes, namely, Yin-Xu, Yang-Xu, Phlegm and Blood Stasis, and Normal, and it achieved 67% accuracy for the testing dataset. The model also reduced the required number of BCQ questions from 44 to 3-6, depending on the responses. Lastly, we developed the Traditional Chinese Medicine (TCM) body constitution online diagnosis system using our model to collect data digitally and use it more practically and efficiently. This system can assist doctors to improve the diagnosis and treatment in TCM practice.

Current development of integrated web servers for preclinical safety and pharmacokinetics assessments in drug development.

In drug development, preclinical safety and pharmacokinetics assessments of candidate drugs to ensure the safety profile are a must. While in vivo and in vitro tests are traditionally used, experimental determinations have disadvantages, as they are usually time-consuming and costly. In silico predictions of these preclinical endpoints have each been developed in the past decades. However, only a few web-based tools have integrated different models to provide a simple one-step platform to help researchers thoroughly evaluate potential drug candidates. To efficiently achieve this approach, a platform for preclinical evaluation must not only predict key ADMET (absorption, distribution, metabolism, excretion and toxicity) properties but also provide some guidance on structural modifications to improve the undesired properties. In this review, we organized and compared several existing integrated web servers that can be adopted in preclinical drug development projects to evaluate the subject of interest. We also introduced our new web server, Virtual Rat, as an alternative choice to profile the properties of drug candidates. In Virtual Rat, we provide not only predictions of important ADMET properties but also possible reasons as to why the model made those structural predictions. Multiple models were implemented into Virtual Rat, including models for predicting human ether-a-go-go-related gene (hERG) inhibition, cytochrome P450 (CYP) inhibition, mutagenicity (Ames test), blood-brain barrier penetration, cytotoxicity and Caco-2 permeability. Virtual Rat is free and has been made publicly available at https://virtualrat.cmdm.tw/.

Dioscorea nipponica Makino suppresses TPA-induced migration and invasion through inhibition of matrix metalloproteinase-9 in human cervical cancer cells.

Dioscorea nipponica Makino has been used for the treatment of chronic bronchitis, rheumatoid arthritis, cough, and asthma. Several studies have established the antitumor effect of D. nipponica Makino extract (DNE). However, no investigations have considered the antimetastatic potential of DNE in cervical cancer cells. The present study examined the effects of DNE on cervical cancer cells treated with 12-O-tetradecanoylphorbol-13-acetate and characterized the possible molecular mechanisms. MTT assay results indicated that DNE exhibited very low cytotoxicity, and DNE significantly reduced the invasion and migration abilities of cervical cancer cells. Gelatin zymography analysis revealed that DNE significantly inhibited matrix metalloproteinase-9 (MMP-9) activity. Reverse transcription-polymerase chain reaction assay results revealed that DNE treatment inhibited the MMP-9 mRNA levels of HeLa and SiHa cells. Western blot results revealed that DNE significantly diminished the ERK1/2 phosphorylation. In conclusion, we revealed that the antimetastatic effects of DNE on cervical cancer cells are due to its inhibition of MMP-9 expression through the ERK1/2 pathway.

The Potential of Chinese Herbal Medicines in the Treatment of Cervical Cancer.

Cervical cancer is a global health issue and places a considerable economic and medical burden on society. Thus, a concerted effort to improve the treatment of cervical cancer is warranted. Although several treatment options are currently available for treating patients with cervical cancer, such as chemoradiation and neoadjuvant or adjuvant chemotherapy, more aggressive systemic therapies and newer therapeutic agents are under investigation. Medicinal herbs have long been used to treat diseases. In this review, we summarize studies analyzing the antitumor effects and underlying mechanisms of Chinese herbal medicines, including the effects of crude extracts and compounds in vitro or in animal models for inducing apoptosis and inhibiting invasion or metastasis. Chinese herbal medicines with therapeutic targeting, such as those that interfere with tumor growth and progression in cervical cancer, have been widely investigated. To apply Chinese herbal medicine in the treatment of cervical cancer, adequate clinical studies are required to confirm its clinical safety and efficiency. Further investigations focused on the purification, pharmacokinetics, and identification of compounds from Chinese herbal medicines in cervical cancer treatment are necessary to achieve the aforementioned treatment goals.

Long-acting muscarinic antagonist versus long-acting ß2 agonist/corticosteroid for moderate to severe chronic obstructive pulmonary disease patients: Exacerbation risk assessment.

BACKGROUND: Whether the beneficial effects of long-acting muscarinic antagonists (LAMA) are better than those of long-acting ß2 agonist/corticosteroids (LABA/ICS) in preventing exacerbations in chronic obstructive pulmonary disease (COPD) remains unclear. This study aimed to assess the risk of exacerbations in moderate to severe COPD patients receiving LAMA versus LABA/ICS. METHODS: We retrospectively reviewed the medical records of patients diagnosed with COPD (2008-2010). The inclusion criteria were age ≥ 40 years, forced expiratory volume in 1 second (FEV1) 30% to 80% of predicted value and at least three prescriptions for COPD medication, including LAMA or LABA/ICS. RESULTS: Of the 557 COPD patients screened, 90 patients were enrolled in the analysis. The demographic characteristics of patients receiving LABA/ICS or LAMA were similar. The all exacerbation rates was significantly higher in patients with global initiative for chronic obstructive lung disease stage II COPD treated with LABA/ICS than in those treated with LAMA (p = 0.001), regardless of previous exacerbation history. Patients with previous exacerbation history showed an independent increase in the risk of moderate or severe exacerbation compared with those without exacerbation history (hazard ratio 3.86, 95% CI 1.75-8.53, p = 0.001). CONCLUSION: In comparison with LABA/ICS, LAMA is beneficial in reducing exacerbation risk for moderate COPD. Previous exacerbation history independently predicts the future risk of exacerbation regardless of treatment.

Phloretin suppresses metastasis by targeting protease and inhibits cancer stemness and angiogenesis in human cervical cancer cells.

BACKGROUND: Phloretin, a dihydrochalcone flavonoid, possesses anti-inflammatory activity and inhibits the growth of various cancers. However, the flavonoid's effect on cervical cancer metastasis and angiogenesis remains unknown. PURPOSE: In this study, we provide molecular evidence associated with the antimetastatic and antiangiogenic effects of phloretin. METHODS: In this study, the anti-invasive effect of phloretin (0-60 µM) in cervical cancer cells was evaluated using the Matrigel invasion assay, gelatin zymography, cell-matrix adhesion assay, wound healing assay, and Western blotting. Antiangiogenic potential of phloretin (0-100 µM) was assessed by the Matrigel tube formation assay. The in vivo antitumor effect of phloretin (10 or 20 mg/kg) was fed by oral gavage and determined using subcutaneous inoculation and tail vein injection in immunodeficient nude mice. RESULTS: Phloretin (60 µM) showed marked suppression of invasion and migration through downregulation of matrix metalloproteinase (MMP)-2, MMP-3, and cathepsin S in human SiHa cervical cancer cells. Phloretin (60 µM) reversed the epithelial-mesenchymal transition induced by transforming growth factor-ß1 and downregulated mesenchymal markers, such as fibronectin, vimentin, and RhoA. Phloretin (100 µM) treatment significantly inhibited the aldehyde dehydrogenase 1 activity of SiHa cells, reduced the self-renewal properties and stemness signatures of CD44 and Sox-2 in sphere-forming cervical cancer-derived tumor-initiating cells, and inhibited the invasion, MMP-2 activity, and tube formation capacity of human umbilical vein endothelial cells. The ability of phloretin (20 mg/kg) to suppress lung metastasis and tumor growth in SiHa cells was evidenced by tail vein injection and subcutaneous inoculation in a tumor xenograft model. CONCLUSION: In summary, the findings indicate that phloretin inhibits the metastatic and angiogenic abilities and cancer stemness of SiHa cells, thereby suggesting that this flavonoid is a promising therapeutic agent for the treatment of human cervical cancer cells.

Hypocretin in median raphe nucleus modulates footshock stimuli-induced REM sleep alteration.

Stress is one of major factors that cause sleep problems. Hypocretin represents a stress-related neuropeptide and is well known in maintaining physiological wakefulness. The hypocretinergic neurons originate in the lateral hypothalamic area (LHA) and transmit to several brain regions, including the median raphe nuclei (MRNs). The MRNs modulate both fear responses and sleep-wake activity; however, it remains unclear whether stress alters the levels of hypocretin to regulate MRNs and consequently disrupt sleep. In this paper, we employed the inescapable footshock stimuli (IFS) as a stressor and hypothesized that the IFS-induced sleep disruption is mediated by increased hypocretins in the MRNs. Our results demonstrate that the concentrations of hypocretin in the hypothalamus increased after IFS. Rapid eye movement (REM) sleep was reduced after footshock, and microinjection of non-selective hypocretin receptor antagonist TCS-1102 into the MRNs blocked the IFS-induced decrease of REM sleep. Furthermore, administration of hypocretins into the MRNs mimicked the IFS-induced REM sleep reduction. These results conclude that the increased levels of hypocretins in the MRNs mediate the IFS-induced REM sleep reduction.

Antimetastatic effects of Terminalia catappa leaf extracts on cervical cancer through the inhibition of matrix metalloprotein-9 and MAPK pathway.

The effects of Terminalia catappa leaf extracts (TCE) have been widely investigated, including its antioxidative, anti-inflammatory, and antidiabetic activity, as well as its antimetastatic effects on several types of human cancer. However, no study has examined the antimetastatic potential of TCE in cervical cancer cells. This study aimed to elucidate the potential antimetastatic properties of ethanol extracts of Terminalia catappa in 12-O-tetradecanoylphorbol-13-acetate treated human cervical cancer cells and investigate the signaling pathway of this process. We demonstrated that TCE elicited very low cytotoxicity and significantly inhibited cellular migration and invasion in human HeLa and SiHa cervical cancer cells. Moreover, the gelatin zymography, reverse transcription-polymerase chain reaction (RT-PCR), and real-time PCR analysis revealed that the activity and mRNA level of matrix metalloproteinase-9 (MMP-9) were inhibited by TCE in a concentration-dependent manner. The Western blot results demonstrated that the highest concentration of TCE (100 µg/ml) reduced the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2) by 46% in the HeLa cell lines. In conclusion, it was revealed that TCE exerted antimetastatic effects on cervical cancer cells by inhibiting the expression of MMP-9 through the ERK1/2 pathway.

Hypericin inhibits hepatitis C virus replication via deacetylation and down-regulation of heme oxygenase-1.

BACKGROUND: Hepatitis C virus (HCV) is a globally prevalent pathogen and a leading cause of death and morbidity. Traditional therapy with pegylated interferon- and ribavirin has had only limited success, with some adverse effects. Direct-acting antivirals (DAAs) are effective in suppressing HCV replication, but are expensive. PURPOSE: Hypericin has been reported to be a good antiviral agent for inhibiting HCV replication, however, little is known about its mechanisms of action. The aim of this study is to elucidate the mode of action of hypericin in Ava5 human hepatoma cell line (Huh7 derivative) harboring HCV subgenomic replicon RNA. METHODS: To determine the non-structure protein 5A (NS5A) mRNA and NS3 protein expression levels, real-time PCR and Western blot analysis were performed, respectively. To investigate how hypericin inhibits HCV replication, 5-aza-2'-deoxycytidine (5-Aza-dC) and chidamide were used for determining histone modification. Furthermore, shRNA was applied to confirm the role of heme oxygenase (HO-1) in HCV repression. RESULTS: Hypericin in experiment were tested and showed no cytotoxicity. Hypericin reduced HO-1 and NS5A in a time- and dose- dependent manner. Chidamide, but not 5-Aza-dc, restored hypericin-induced reduction in HCV NS3 expression and reversed HO-1 expression in Ava5 cells. LY294002 inhibited HCV replication via HO-1 down-regulation. Constitutive expressed p-AKT was not involved in hypericin-induced reduction in HCV replication. In addition, shHO-1 inhibited HCV replication. CONCLUSION: In conclusion, hypericin inhibits HCV replication via down-regulation of HO-1 expression and deacetylation.

Glycopyrronium bromide inhibits lung inflammation and small airway remodeling induced by subchronic cigarette smoke exposure in mice.

The effects of long-acting muscarinic receptor antagonists (LAMAs) have not been evaluated in a model with simultaneous lung inflammation and small airway remodeling induced by cigarette smoke (CS). We exposed the mice to CS for four weeks with daily treatment with a LAMA (glycopyrronium bromide, NVA237) or its vehicle. Human bronchial epithelial cells (PBECs) and lung fibroblasts were exposed to CS extract (CSE) or acetylcholine with or without NVA237 treatment. We found that NVA237, but not its vehicle, suppressed elevations in inflammatory score, epithelial thickness, and peribronchial collagen deposition in CS-exposed mice. NVA237 alleviated CS-induced increased levels of chemokines, inflammatory cells, and total protein in the bronchoalveolar lavage fluid. NVA237 suppressed acetylcholine- or CSE-induced elevations in IL-8 production in PBECs and elevations in proliferation and collagen production in lung fibroblasts. These phenomena were also prevented by a p44/42 MAPK inhibitor. In conclusion, NVA237 exerted a potent suppressive effect on lung inflammation and small airway remodeling induced by subchronic CS exposure.

A standardized herbal extract mitigates tumor inflammation and augments chemotherapy effect of docetaxel in prostate cancer.

Activation of the NFκB pathway is often associated with advanced cancer and has thus been regarded as a rational therapeutic target. Wedelia chinensis is rich in luteolin, apigenin, and wedelolactone that act synergistically to suppress androgen receptor activity in prostate cancer. Interestingly, our evaluation of a standardized Wedelia chinensis herbal extract (WCE) concluded its efficacy on hormone-refractory prostate cancer through systemic mechanisms. Oral administration of WCE significantly attenuated tumor growth and metastasis in orthotopic PC-3 and DU145 xenografts. Genome-wide transcriptome analysis of these tumors revealed that WCE suppressed the expression of IKKα/ß phosphorylation and downstream cytokines/chemokines, e.g., IL6, CXCL1, and CXCL8. Through restraining the cytokines expression, WCE reduced tumor-elicited infiltration of myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs) and endothelial cells into the tumors, therefore inhibiting angiogenesis, tumor growth, and metastasis. In MDSCs, WCE also reduced STAT3 activation, downregulated S100A8 expression and prevented their expansion. Use of WCE in combination with docetaxel significantly suppressed docetaxel-induced NFκB activation, boosted the therapeutic effect and reduced the systemic toxicity caused by docetaxel monotherapy. These data suggest that a standardized preparation of Wedelia chinensis extract improved prostate cancer therapy through immunomodulation and has potential application as an adjuvant agent for castration-resistant prostate cancer.

Salidroside ameliorates sepsis-induced acute lung injury and mortality via downregulating NF-κB and HMGB1 pathways through the upregulation of SIRT1.

Sepsis is a life-threatening medical condition. Salidroside, a substance isolated from Rhodiola rosea, possesses antioxidant and anti-inflammatory properties. The effect and mechanism of salidroside on sepsis-induced acute lung injury still remains to be well clarified. Here, we investigated the effect and mechanism of salidroside on septic mouse models and explored the role of salidroside-upregulated SIRT1. Salidroside inhibited the inflammatory responses and HMGB1 productions in bacterial lipopolysaccharide (LPS)-treated macrophages and mice. Salidroside could also reverse the decreased SIRT1 protein expression in LPS-treated macrophages and mice. Salidroside also alleviated the sepsis-induced lung edema, lipid peroxidation, and histopathological changes and the mortality, and improved the lung PaO2/FiO2 ratio in cecal ligation and puncture (CLP)-induced septic mice. Salidroside significantly decreased the serum TNF-α, IL-6, NO, and HMGB1 productions, pulmonary inducible NO synthase (iNOS) and phosphorylated NF-κB-p65 protein expressions, and pulmonary HMGB1 nuclear translocation in CLP septic mice. Moreover, sepsis decreased the SIRT1 protein expression in the lungs of CLP septic mice. Salidroside significantly upregulated the SIRT1 expression and inhibited the inflammatory responses in CLP septic mouse lungs. These results suggest that salidroside protects against sepsis-induced acute lung injury and mortality, which might be through the SIRT1-mediated repression of NF-κB activation and HMGB1 nucleocytoplasmic translocation.

Risk and association of HLA with oxcarbazepine-induced cutaneous adverse reactions in Asians.

OBJECTIVE: To investigate the risk and genetic association of oxcarbazepine-induced cutaneous adverse reactions (OXC-cADRs), including Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN), in Asian populations (Chinese and Thai). METHODS: We prospectively enrolled patients with OXC-cADRs in Taiwan and Thailand from 2006 to 2014, and analyzed the clinical course, latent period, drug dosage, organ involvement, complications, and mortality. We also investigated the carrier rate of HLA-B*15:02 and HLA-A*31:01 of patients with OXC-cADRs and compared to OXC-tolerant controls. The incidence of OXC-SJS/TEN was compared with carbamazepine (CBZ)-induced SJS/TEN according to the nationwide population dataset from the Taiwan National Health Insurance Research Database. RESULTS: We enrolled 50 patients with OXC-cADRs, including 20 OXC-SJS/TEN and 6 drug reaction with eosinophilia and systemic symptoms, of Chinese patients from Taiwan and Thai patients from Thailand. OXC-cADRs presented with less clinical severity including limited skin detachment (all ≦5%) and no mortality. There was a significant association between HLA-B*15:02 and OXC-SJS (p = 1.87 × 10-10; odds ratio 27.90; 95% confidence interval [CI] 7.84-99.23) in Chinese and this significant association was also observed in Thai patients. The positive and negative predictive values of HLA-B*15:02 for OXC-SJS/TEN were 0.73% and 99.97%, respectively. HLA-A*31:01 was not associated with OXC-cADRs. The incidence and mortality of OXC-SJS/TEN was lower than CBZ-STS/TEN in new users (p = 0.003; relative risk 0.212; 95% CI 0.077-0.584). CONCLUSIONS: Our findings suggest that HLA-B*15:02 is significantly associated with OXC-SJS in Asian populations (Chinese and Thai). However, the severity and incidence of OXC-SJS/TEN are less than that of CBZ-SJS/TEN. The need for preemptive HLA-B*15:02 screening should be evaluated further.

Discovery of Indazoles as Potent, Orally Active Dual Neurokinin 1 Receptor Antagonists and Serotonin Transporter Inhibitors for the Treatment of Depression.

Combination studies of neurokinin 1 (NK1) receptor antagonists and serotonin-selective reuptake inhibitors (SSRIs) have shown promise in preclinical models of depression. Such a combination may offer important advantages over the current standard of care. Herein we describe the discovery and optimization of an indazole-based chemotype to provide a series of potent dual NK1 receptor antagonists/serotonin transporter (SERT) inhibitors to overcome issues of ion channel blockade. This effort culminated in the identification of compound 9, an analogue that demonstrated favorable oral bioavailability, excellent brain uptake, and robust in vivo efficacy in a validated depression model. Over the course of this work, a novel heterocycle-directed asymmetric hydrogenation was developed to facilitate installation of the key stereogenic center.

A High-Throughput Automated Microfluidic Platform for Calcium Imaging of Taste Sensing.

The human enteroendocrine L cell line NCI-H716, expressing taste receptors and taste signaling elements, constitutes a unique model for the studies of cellular responses to glucose, appetite regulation, gastrointestinal motility, and insulin secretion. Targeting these gut taste receptors may provide novel treatments for diabetes and obesity. However, NCI-H716 cells are cultured in suspension and tend to form multicellular aggregates, preventing high-throughput calcium imaging due to interferences caused by laborious immobilization and stimulus delivery procedures. Here, we have developed an automated microfluidic platform that is capable of trapping more than 500 single cells into microwells with a loading efficiency of 77% within two minutes, delivering multiple chemical stimuli and performing calcium imaging with enhanced spatial and temporal resolutions when compared to bath perfusion systems. Results revealed the presence of heterogeneity in cellular responses to the type, concentration, and order of applied sweet and bitter stimuli. Sucralose and denatonium benzoate elicited robust increases in the intracellular Ca(2+) concentration. However, glucose evoked a rapid elevation of intracellular Ca(2+) followed by reduced responses to subsequent glucose stimulation. Using Gymnema sylvestre as a blocking agent for the sweet taste receptor confirmed that different taste receptors were utilized for sweet and bitter tastes. This automated microfluidic platform is cost-effective, easy to fabricate and operate, and may be generally applicable for high-throughput and high-content single-cell analysis and drug screening.

Noninvasive, quantitative, spatiotemporal characterization of mineralization in three-dimensional collagen hydrogels using high-resolution spectral ultrasound imaging.

As tissue engineering products move toward the clinic, nondestructive methods to monitor their development and ensure quality are needed. In this study, high-resolution spectral ultrasound imaging (SUSI) was used to noninvasively characterize mineral content in collagen hydrogels. SUSI was used to generate three-dimensional (3D) grayscale (GS) images of construct morphology with submillimeter resolution. Spectral analysis of the backscattered radio frequency (RF) ultrasound signals was used to determine the midband fit (MBF) and slope of the linearized RF spectrum. These parameters are operator and instrument independent, and were used to characterize the spatial distribution of mineral in constructs supplemented with hydroxyapatite particles. GS and MBF correlated closely with mineral content, while slope was not dependent on concentration. SUSI also was used to monitor mineralization of collagen constructs by immersion in simulated body fluid (SBF) over 21 days. The construct surface was mineralized before the interior, and there was a dose-dependent effect of SBF concentration on degree of mineralization and deposited particle size. MBF density was closely correlated with the amount of calcium deposited. These data demonstrate that SUSI has utility as a noninvasive imaging method for quantitative analysis of mineralization in 3D protein constructs. Such techniques may assist the development of engineered orthopedic tissues.

Effect of cannabidiol on sleep disruption induced by the repeated combination tests consisting of open field and elevated plus-maze in rats.

Patients with post-traumatic stress disorder (PTSD) frequently complain of having sleep disturbances, such as insomnia and rapid eye movement (REM) sleep abnormality. Cannabidiol (CBD), a psycho-inactive constituent of marijuana, reduces physiological non-REM (NREM) sleep and REM sleep in normal rats, in addition to generating its anxiolytic effect. However, the effects of CBD on anxiety-induced sleep disturbances remain unclear. Because anxiety progression is caused by persistent stress for a period of time, we employed the repeated combination tests (RCT) consisting of a 50-min open field (OF) and a subsequent 10-min elevated plus-maze (EPM) for four consecutive days to simulate the development of anxiety. Time spent in the centre arena of OF and during open arms of the EPM was substantially decreased in latter days of RCT, suggesting the habituation, which potentially lessens anxiety-mediated behavioural responses, was not observed in current tests. CBD microinjected into the central nucleus of amygdala (CeA) significantly enhanced time spent in centre arena of OF, increased time during the open arms and decreased frequency of entry to the enclosed arms of EPM, further confirming its anxiolytic effect. The decrease of NREM sleep during the first hour and the suppression of REM sleep during hours 4-10 after the RCT represent the similar clinical observations (e.g. insomnia and REM sleep interruption) in PTSD patients. CBD efficiently blocked anxiety-induced REM sleep suppression, but had little effect on the alteration of NREM sleep. Conclusively, CBD may block anxiety-induced REM sleep alteration via its anxiolytic effect, rather than via sleep regulation per se. This article is part of a Special Issue entitled 'Anxiety and Depression'.