Impact of Supplementing a Backgrounding Diet with Nonprotein Nitrogen on In Vitro Methane Production, Nutrient Digestibility, and Steer Performance.

Two experiments were conducted to evaluate the effect of nonprotein nitrogen (NPN) supplementation on in vitro fermentation and animal performance using a backgrounding diet. In experiment 1, incubations were conducted on three separate days (replicates). Treatments were control (CTL, without NPN), urea (U), urea-biuret (UB), and urea-biuret-nitrate (UBN) mixtures. Except for control, treatments were isonitrogenous using 1% U inclusion as a reference. Ruminal fluid was collected from two Angus-crossbred steers fed a backgrounding diet plus 100 g of a UBN mixture for at least 35 d. The concentration of volatile fatty acids (VFA) and ammonia nitrogen (NH3-N), in vitro organic matter digestibility (IVOMD), and total gas and methane (CH4) production were determined at 24 h of incubation. In experiment 2, 72 Angus-crossbred yearling steers (303 ±â€…29 kg of body weight [BW]) were stratified by BW and randomly allocated in nine pens (eight animals/pen and three pens/treatment). Steers consumed a backgrounding diet formulated to match the diet used in the in vitro fermentation experiment. Treatments were U, UB, and UBN and were isonitrogenous using 1% U inclusion as a reference. Steers were adapted to the NPN supplementation for 17 d. Then, digestibility evaluation was performed after 13 d of full NPN supplementation for 4 d using 36 steers (12 steers/treatment). After that, steer performance was evaluated for 56 d (24 steers/treatment). In experiment 1, NPN supplementation increased the concentration of NH3-N and VFA (P < 0.01) without affecting the IVOMD (P = 0.48), total gas (P = 0.51), and CH4 production (P = 0.57). Additionally, in vitro fermentation parameters did not differ (P > 0.05) among NPN sources. In experiment 2, NPN supplementation did not change dry matter and nutrient intake (P > 0.05). However, UB and UBN showed lower (P < 0.05) nutrient digestibility than U, except for starch (P = 0.20). Dry matter intake (P = 0.28), average daily gain (P = 0.88), and gain:feed (P = 0.63) did not differ among steers receiving NPN mixtures. In conclusion, tested NPN mixtures have the potential to be included in the backgrounding diets without any apparent negative effects on animal performance and warrant further studies to evaluate other variables to fully assess the response of feeding these novel NPN mixtures.

Nonprotein nitrogen (NPN) supplements can be used as a nitrogen source for ruminants fed low-protein diets. The most common NPN source is urea, included typically at a range between 0.5% and 1% of the diet dry matter in growing beef cattle. Although other NPN sources and mixtures are available, there is scarce information regarding their use in ruminant production. Two experiments were conducted to evaluate the effect of NPN sources on in vitro fermentation and animal performance using a backgrounding diet. In experiment 1, three different incubations were performed for 24 h. Treatments were control (without NPN), urea (U), urea­biuret (UB), and urea­biuret­nitrate (UBN) mixtures. In experiment 2, 72 crossbred yearling steers were randomly assigned to one of the following treatments: U, UB, and UBN mixtures. Diets were formulated to contain the same nitrogen concentration in both experiments. In experiment 1, supplementation of NPN increased the in vitro fermentation, but there were no differences among NPN sources. In experiment 2, steers performed similarly among NPN sources. These findings suggest that NPN mixtures have the potential to be included in the backgrounding diets without detrimental effects. Further studies should evaluate other variables (e.g., fermentation dynamic and microbial protein supply) when using these novel mixtures.

Orexin-A attenuated motion sickness through modulating neural activity in hypothalamus nuclei.

BACKGROUND AND PURPOSE: We evaluated the hypothesis that central orexin application could counteract motion sickness responses through regulating neural activity in target brain areas. EXPERIMENTAL APPROACH: Thec effects of intracerebroventricular (i.c.v.) injection of orexin-A and SB-334867 (OX1 antagonist) on motion sickness-induced anorexia, nausea-like behaviour (conditioned gaping), hypoactivity and hypothermia were investigated in rats subjected to Ferris wheel-like rotation. Orexin-A responsive brain areas were identified using Fos immunolabelling and were verified via motion sickness responses after intranucleus injection of orexin-A, SB-334867 and TCS-OX2-29 (OX2 antagonist). The efficacy of intranasal application of orexin-A versus scopolamine on motion sickness symptoms in cats was also investigated. KEY RESULTS: Orexin-A (i.c.v.) dose-dependently attenuated motion sickness-related behavioural responses and hypothermia. Fos expression was inhibited in the ventral part of the dorsomedial hypothalamus (DMV) and the paraventricular nucleus (PVN), but was enhanced in the ventral part of the premammillary nucleus ventral part (PMV) by orexin-A (20 µg) in rotated animals. Motion sickness responses were differentially inhibited by orexin-A injection into the DMV (anorexia and hypoactivity), the PVN (conditioned gaping) and the PMV (hypothermia). SB-334867 and TCS-OX2-29 (i.c.v. and intranucleus injection) inhibited behavioural and thermal effects of orexin-A. Orexin-A (60 µg·kg-1) and scopolamine inhibited rotation-induced emesis and non-retching/vomiting symptoms, while orexin-A also attenuated anorexia with mild salivation in motion sickness cats. CONCLUSION AND IMPLICATIONS: Orexin-A might relieve motion sickness through acting on OX1 and OX2 receptors in various hypothalamus nuclei. Intranasal orexin-A could be a potential strategy against motion sickness.

Omecamtiv mecarbil does not prolong QTc intervals at therapeutic concentrations.

AIMS: Omecamtiv mecarbil (OM) is a novel selective cardiac myosin activator under investigation for the treatment of heart failure. This study aimed to evaluate the effect of therapeutic concentrations of OM on electrocardiogram (ECG) parameters and exclude a clinically concerning effect on the rate-corrected QT (QTc) interval. METHODS: In part A, 70 healthy subjects received a 25 mg oral dose of OM, and pharmacokinetics were assessed. Only subjects with maximum observed plasma concentration ≤ 350 ng/mL (n = 60) were randomized into part B, where they received a single oral dose of placebo, 50 mg OM and 400 mg moxifloxacin in a 3-period, 3-treatment, 6-sequence crossover study with continuous ECG collection. RESULTS: After a 50-mg dose of OM, mean placebo-corrected change from baseline QTcF (∆∆QTcF; Fridericia correction) ranged from -6.7 ms at 1 hour postdose to -0.8 ms at 4 hours postdose. The highest upper bound of the 1-sided 95% confidence interval (CI) was 0.7 ms (4 h postdose). Moxifloxacin resulted in a clear increase in mean ∆∆QTcF, with a peak value of 13.1 ms (90% CI: 11.71-14.57) at 3 hours; lower bound of the 1-sided 95% CI was > 5 ms at all of the 3 prespecified time points. Based on a concentration-QTc analysis, an effect on ∆∆QTcF exceeding 10 ms can be excluded up to OM plasma concentrations of ~800 ng/mL. There were no serious or treatment-emergent adverse events leading to discontinuation from the study. CONCLUSION: OM does not have a clinically relevant effect on the studied ECG parameters.

Comprehensive in vitro pro-arrhythmic assays demonstrate that omecamtiv mecarbil has low pro-arrhythmic risk.

Omecamtiv mecarbil (OM) is a myosin activator (myotrope), developed as a potential therapeutic agent for heart failure with reduced ejection fraction. To characterize the potential pro-arrhythmic risk of this novel sarcomere activator, we evaluated OM in a series of International Conference on Harmonization S7B core and follow-up assays, including an in silico action potential (AP) model. OM was tested in: (i) hERG, Nav1.5 peak, and Cav1.2 channel assays; (ii) in silico computation in a human ventricular AP (hVAP) population model; (iii) AP recordings in canine cardiac Purkinje fibers (PF); and (iv) electrocardiography analysis in isolated rabbit hearts (IRHs). OM had low potency in the hERG (half-maximal inhibitory concentration [IC50 ] = 125.5 µM) and Nav1.5 and Cav1.2 assays (IC50  > 300 µM). These potency values were used as inputs to investigate the occurrence of repolarization abnormalities (biomarkers of pro-arrhythmia) in an hVAP model over a wide range of OM concentrations. The outcome of hVAP analysis indicated low pro-arrhythmia risk at OM concentration up to 30 µM (100-fold the effective free therapeutic plasma concentration). In the isolated canine PF assay, OM shortened AP duration (APD)60 and APD90 significantly from 3 to 30 µM. In perfused IRH, ventricular repolarization (corrected QT and corrected JT intervals) was decreased significantly at greater than or equal to 1 µM OM. In summary, the comprehensive proarrhythmic assessment in human and non-rodent cardiac models provided data indicative that OM did not delay ventricular repolarization at therapeutic relevant concentrations, consistent with clinical findings.

Pharmacophore-based virtual screening, synthesis, biological evaluation, and molecular docking study of novel pyrrolizines bearing urea/thiourea moieties with potential cytotoxicity and CDK inhibitory activities.

In the current study, virtual screening of a small library of 1302 pyrrolizines bearing urea/thiourea moieties was performed. The top-scoring hits were synthesised and evaluated for their cytotoxicity against three cancer (MCF-7, A2780, and HT29) and one normal (MRC-5) cell lines. The results of the MTT assay revealed potent cytotoxic activities for most of the new compounds (IC50 = 0.16-34.13 µM). The drug-likeness study revealed that all the new compounds conform to Lipinski's rule. Mechanistic studies of compounds 18 b, 19a, and 20a revealed the induction of apoptosis and cell cycle arrest at the G1 phase in MCF-7 cells. The three compounds also displayed potent inhibitory activity against CDK-2 (IC50 = 25.53-115.30 nM). Moreover, the docking study revealed a nice fitting of compound 19a into the active sites of CDK-2/6/9. These preliminary results suggested that compound 19a could serve as a promising scaffold in the discovery of new potent anticancer agents.

Fluorescence detection of malachite green in fish tissue using red emissive Se,N,Cl-doped carbon dots.

Facile detection of malachite green (MG), a toxic dye, in aquaculture is urgently demanded for environment and food safety. Herein, we design a novel fluorescent probe, namely red emissive Se,N,Cl-doped carbon dots (CDs), to accurately determinate MG. CDs are prepared by hydrothermal treatment of selenourea and o-phenylenediamine in HCl solution. This material exhibits excitation-independent dual emissions at 625 and 679 nm, with a high quantum yield of 23.6%. A selective and sensitive fluorescent sensor toward MG is established based on inner filter effect, because both the excitation and emission light of CDs can be strongly absorbed by MG. The fluorescence quenching of CDs is linear to the MG concentration over the range of 0.07-2.50 µM with a low detection limit of 21 nM. Trace-level analysis of MG in fish tissue is successfully explored, demonstrating the great potential of the proposed sensor for MG monitoring in aquatic products.

CKD-5, a novel pan-histone deacetylase inhibitor, synergistically enhances the efficacy of sorafenib for hepatocellular carcinoma.

BACKGROUND: Histone deacetylase inhibitors (HDACIs) have distinctive epigenetic targets involved in hepatocarcinogenesis and chemoresistance. A recent phase I/II study reported the possibility of HDACI as a chemosensitizer in sorafenib-resistant patients. In this study, we evaluated whether CKD-5, a novel pan-HDACI, can potentiate the efficacy of sorafenib. METHODS: The anticancer effect of CKD-5 with and without sorafenib was evaluated in vitro using an MTS assay with human HCC cells (SNU-3058 and SNU-761) under both normoxic and hypoxic conditions. Microarray analysis was performed to investigate the mechanism of cell death, which was also evaluated by small interfering RNA (siRNA) transfection and subsequent immunoblot assays. In vivo experiments were conducted using two different murine HCC models. C3H mice implanted with MH134 cells and C57BL/6 mice implanted with RIL-175 cells were treated with weekly CKD-5 with and without sorafenib for 2 weeks. RESULTS: CKD-5 treatment significantly suppressed human HCC cell growth in both normoxic and hypoxic conditions. Microarray analysis and real-time PCR showed that CKD-5 treatment significantly increased peripherin expression in HCC cells and that downregulation of peripherin by siRNA decreased CKD-5-induced apoptosis. The combination of CKD-5 and sorafenib decreased cell viability more effectively than sorafenib or CKD-5 monotherapy in human and murine HCC cells. The effectiveness of the combination therapy was consistently demonstrated in the animal models. Histological and biochemical analyses demonstrated good tolerance of CKD-5 plus sorafenib in vivo. CONCLUSION: CKD-5 may enhance sorafenib efficacy through epigenetic regulation. The combination of CKD-5 and sorafenib might be a novel therapeutic option for the treatment of HCC.

High NRF2 Levels Correlate with Poor Prognosis in Colorectal Cancer Patients and with Sensitivity to the Kinase Inhibitor AT9283 In Vitro.

Aberrant hyperactivation of nuclear factor erythroid 2 (NF-E2) p45-related factor 2 (NRF2) is a common event in many tumour types and associates with resistance to therapy and poor patient prognosis; however, its relevance in colorectal tumours is not well-established. Measuring the expression of surrogate genes for NRF2 activity in silico, in combination with validation in patients' samples, we show that the NRF2 pathway is upregulated in colorectal tumours and that high levels of nuclear NRF2 correlate with a poor patient prognosis. These results highlight the need to overcome the protection provided by NRF2 and present an opportunity to selectively kill cancer cells with hyperactive NRF2. Exploiting the CRISPR/Cas9 technology, we generated colorectal cancer cell lines with hyperactive NRF2 and used them to perform a drug screen. We identified AT9283, an Aurora kinase inhibitor, for its selectivity towards killing cancer cells with hyperactive NRF2 as a consequence to either genetic or pharmacological activation. Our results show that hyperactivation of NRF2 in colorectal cancer cells might present a vulnerability that could potentially be therapeutically exploited by using the Aurora kinase inhibitor AT9283.

Single-Cell Mechanical Analysis of Human Pluripotent Stem Cell-Derived Cardiomyocytes for Drug Testing and Pathophysiological Studies.

Current platforms for studying the mechanical properties of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) as single cells do not measure forces directly, require numerous assumptions, and cannot study cell mechanics at different loading conditions. We present a method for directly measuring the active and passive forces generated by single-cell hPSC-CMs at different stretch levels. Utilizing this technique, single hPSC-CMs exhibited positive length-tension relationship and appropriate inotropic, klinotropic, and lusitropic changes in response to pharmacological treatments (isoproterenol and verapamil). The unique potential of the approach for drug testing and disease modeling was exemplified by doxorubicin and omecamtiv mecarbil drug studies revealing their known actions to suppress (doxorubicin) or augment (omecamtiv mecarbil at low dose) cardiomyocyte contractility, respectively. Finally, mechanistic insights were gained regarding the cellular effects of these drugs as doxorubicin treatment led to cellular mechanical alternans and high doses of omecamtiv mecarbil suppressed contractility and worsened the cellular diastolic properties.

Metal-organic frameworks derived magnetic porous carbon for magnetic solid phase extraction of benzoylurea insecticides from tea sample by Box-Behnken statistical design.

Ferric oxide/carbon (Fe2O3@C) was fabricated via direct carbonization of metal-organic framework of iron (MOF-235) under argon atmosphere. The magnetic Fe2O3 nanoparticles are evenly embedded in porous carbon matrix, while original morphology of MOF-235 was well-maintained. The synthesized Fe2O3@C was used as magnetic sorbent for extracting five benzoylurea insecticides (BUs). The materials exhibited excellent extraction performance, which benefited not only from the strong π-π interaction and hydrophobic interaction (π-conjugated system), but also to the abundant adsorption sites and flexible transport channel (the interconnected 3D porous structure). A three-factor-three-level Box-Behnken design (BBD) was selected to optimize three greatly influential parameters: amount of adsorbent (A), desorption time (B) and volume of desorption solvent (C) by response surface methodology. The established method coupled to HPLC-UV detection showed wide linearity with the range of 0.2-450 µg•L-1, relatively low limits of detection (0.05-0.10 µg•L-1) with the relative standard deviation (RSD) (n = 7) lower t than 5.47%. Moreover, the proposed method was successfully applied to analyze BUs in tea samples and investigate the removal effect of different washing on BUs residues from tea leaf. These results indicated that the synthesized Fe2O3@C is a promising adsorbent material for magnetic solid phase extraction of BUs at trace concentrations from tea samples.

Evaluating Biochemically Recurrent Prostate Cancer: Histologic Validation of 18F-DCFPyL PET/CT with Comparison to Multiparametric MRI.

Background Prostate cancer recurrence is found in up to 40% of men with prior definitive (total prostatectomy or whole-prostate radiation) treatment. Prostate-specific membrane antigen PET agents such as 2-(3-{1-carboxy-5-[(6-[18F]fluoro-pyridine 3-carbonyl)-amino]-pentyl}-ureido)-pentanedioic acid (18F-DCFPyL) may improve detection of recurrence compared with multiparametric MRI; however, histopathologic validation is lacking. Purpose To determine the sensitivity, specificity, and positive predictive value (PPV) of 18F-DCFPyL PET/CT based on histologic analysis and to compare with pelvic multiparametric MRI in men with biochemically recurrent prostate cancer. Materials and Methods Men were prospectively recruited after prostatectomy and/or radiation therapy with rising prostate-specific antigen level (median, 2.27 ng/mL; range, 0.2-27.45 ng/mL) and a negative result at conventional imaging (bone scan and/or CT). Participants underwent 18F-DCFPyL PET/CT imaging and 3.0-T pelvic multiparametric MRI. Statistical analysis included Wald and modified χ2 tests. Results A total of 323 lesions were visualized in 77 men by using 18F-DCFPyL or multiparametric MRI, with imaging detection concordance of 25% (82 of 323) when including all lesions in the MRI field of view and 53% (52 of 99) when only assessing prostate bed lesions. 18F-DCFPyL depicted more pelvic lymph nodes than did MRI (128 vs 23 nodes). Histologic validation was obtained in 80 locations with sensitivity, specificity, and PPV of 69% (25 of 36; 95% confidence interval [CI]: 51%, 88%), 91% (40 of 44; 95% CI: 74%, 98%), and 86% (25 of 29; 95% CI: 73%, 97%) for 18F-DCFPyL and 69% (24 of 35; 95% CI: 50%, 86%), 74% (31 of 42; 95% CI: 42%, 89%), and 69% (24 of 35; 95% CI: 50%, 88%) for multiparametric MRI (P = .95, P = .14, and P = .07, respectively). In the prostate bed, sensitivity, specificity, and PPV were 57% (13 of 23; 95% CI: 32%, 81%), 86% (18 of 21; 95% CI: 73%, 100%), and 81% (13 of 16; 95% CI: 59%, 100%) for 18F-DCFPyL and 83% (19 of 23; 95% CI: 59%, 100%), 52% (11 of 21; 95% CI: 29%, 74%), and 66% (19 of 29; 95% CI: 44%, 86%) for multiparametric MRI (P = .19, P = .02, and P = .17, respectively). The addition of 18F-DCFPyL to multiparametric MRI improved PPV by 38% overall (P = .02) and by 30% (P = .09) in the prostate bed. Conclusion Findings with 2-(3-{1-carboxy-5-[(6-[18F]fluoro-pyridine 3-carbonyl)-amino]-pentyl}-ureido)-pentanedioic acid (18F-DCFPyL) were histologically validated and demonstrated high specificity and positive predictive value. In the pelvis, 18F-DCFPyL depicted more lymph nodes and improved positive predictive value and specificity when added to multiparametric MRI. © RSNA, 2020 Online supplemental material is available for this article. See also the editorial by Zukotynski and Rowe in this issue.

Investigating the role of the amygdala orexin receptor 1 in memory acquisition and extinction in a rat model of PTSD.

Understanding the mechanisms underlying memory is essential for the treatment of post-traumatic stress disorder (PTSD). Orexin, as a lateral hypothalamic (LH) neuropeptide, interferes with the stages of memory, primarily through the orexin receptor1 (Orx1R). The aim of this study was to evaluate the effects of amygdala Orx1R in the acquisition and extinction processes of PTSD modeled in animals. In three experiments, rats were divided into three groups: control (Naïve), shock (receiving a foot shock), and PTSD (experiencing Single prolonged stress (SPS) method). The first experiment aimed to evaluate LH activity in PTSD modeled rats. The second and third experiments aimed to evaluate the effects of Orx1R in the acquisition and extinction of fear memory in PTSD modeled animals. SB334867 (SB) or its solvent was microinjected into the amygdala and the rats were subjected to conditioning thereafter. In the second group, we used a single injection after conditioning. In the third group, we used three consecutive injections (one after each memory test). Some behaviors and Orx1R expression were evaluated. The freezing response was significantly longer in the PTSD group than on the control. Similarly, anxiety and sensitized fear were also intensified. CFos expression levels in LH was higher in the PTSD group. Inhibition of Orx1R in the amygdala significantly decreased memory acquisition, diminished anxiety, and decreased the sensitized fear in the SB group. Applying SB to the amygdala after each fear memory test significantly decreased freezing. Expression of Orx1R was significantly higher following fear conditioning. These results indicate a likely involvement of the orexin and amygdalar Orx1R in memory acquisition and in extinction of PTSD.

Central neuropeptide-S administration alleviates stress-induced impairment of gastric motor functions through orexin-A.

BACKGROUND/AIMS: The novel brain peptide neuropeptide-S (NPS) is produced exclusively by a small group of cells adjacent to the noradrenergic locus coeruleus. The NPSR mRNA has been detected in several brain areas involved in stress response and autonomic outflow, such as amygdala and hypothalamus, suggesting that central NPS may play a regulatory role in stress-induced changes in gastrointestinal (GI) motor functions. In rodents, exogenous central NPS was shown to inhibit stress-stimulated fecal output. Moreover, exogenous NPS was demonstrated to activate hypothalamic neurons that produce orexin-A (OXA), which has been shown to stimulate postprandial gastric motor functions via central vagal pathways. Therefore, we tested whether OXA mediates the NPS-induced alterations in gastric motor functions under stressed conditions. MATERIALS AND METHODS: We investigated the effect of central exogenous NPS on solid gastric emptying (GE) and gastric postprandial motility in acute restraint stress (ARS)-loaded conscious rats. The OXA receptor antagonist SB-334867 was administered centrally prior to the central NPS injection. The expression of NPSR in the hypothalamus and dorsal vagal complex was analyzed by immunofluorescence. RESULTS: Central administration of NPS restored the ARS-induced delayed GE and uncoordinated postprandial antro-pyloric contractions. The alleviative effect of NPS on GE was abolished by pretreatment of the OX1R antagonist SB-334867. In addition to hypothalamus, NPSR was detected in the dorsal motor nucleus of vagus, which suggest a direct stimulatory action of exogenous NPS on gastric motility. CONCLUSION: NPS may be a novel candidate for the treatment of stress-related gastric disorders.

Force and Calcium Transients Analysis in Human Engineered Heart Tissues Reveals Positive Force-Frequency Relation at Physiological Frequency.

Force measurements in ex vivo and engineered heart tissues are well established. Analysis of calcium transients (CaT) is complementary to force, and the combined analysis is meaningful to the study of cardiomyocyte biology and disease. This article describes a model of human induced pluripotent stem cell cardiomyocyte-derived engineered heart tissues (hiPSC-CM EHTs) transduced with the calcium sensor GCaMP6f followed by sequential analysis of force and CaT. Average peak analysis demonstrated the temporal sequence of the CaT preceding the contraction twitch. The pharmacological relevance of the test system was demonstrated with inotropic indicator compounds. Force-frequency relationship was analyzed in the presence of ivabradine (300 nM), which reduced spontaneous frequency and unmasked a positive correlation of force and CaT at physiological human heart beating frequency with stimulation frequency between 0.75 and 2.5 Hz (force +96%; CaT +102%). This work demonstrates the usefulness of combined force/CaT analysis and demonstrates a positive force-frequency relationship in hiPSC-CM EHTs.

The arousal effect of hyperbaric oxygen through orexin/hypocretin an upregulation on ketamine/ethanol-induced unconsciousness in male rats.

Approaches that facilitate the recovery from coma would have enormous impacts on patient outcomes and medical economics. Orexin-producing neurons release orexins (also known as hypocretins) energy-dependently to maintain arousal. Hyperbaric oxygen (HBO) could increase ATP levels by preserving mitochondrial function. We investigated, for the first time, the arousal effects of HBO and orexins mechanisms in a rat model of unconsciousness induced by ketamine or ethanol. A total of 120 Sprague-Dawley male rats were used in this study. Unconsciousness was induced either by intraperitoneal injection of ketamine or ethanol. The HBO treatment (100% O2 at 3 ATA) was administered immediately after unconsciousness induction for 1 hr. SB334867, orexin-1 receptor (OX1R) inhibitor, or JNJ10397049, orexin-2 receptor (OX2R) inhibitor was administered 30 min intraperitoneally before unconsciousness induction. Loss of righting reflex test (LORR) and Garcia test were used to evaluate the unconsciousness duration and neurological deficits after recovering from unconsciousness, respectively. Enzyme-linked immunosorbent assay was used to measure brain tissue ATP and orexin A levels. Ketamine or ethanol injection resulted in LORR immediately and neurological deficits 6 hr after unconsciousness induction. HBO treatment significantly reduced the LORR duration, improved Garcia scores and unregulated ATP and orexin A levels in the brain tissue. Administration of OX1R inhibitor or OX2 R inhibitor abolished arousal and neurological benefits of HBO. In conclusion, HBO exerted arousal-promoting effects on unconscious rats induced by ketamine or ethanol. The underlying mechanism was via, at least in part, ATP/orexin A upregulation. HBO may be a practical clinical approach to accelerate unconsciousness recovery in patients.

RKI-1447 suppresses colorectal carcinoma cell growth via disrupting cellular bioenergetics and mitochondrial dynamics.

Accumulated evidence suggested the importance of the Rho/Rho-kinase (ROCK) signaling pathway in cancer proliferation and invasion. However, its role in colorectal carcinoma (CRC) is not well understood. This study evaluated the effect of ROCK signaling pathway on CRC behavior on the basis of a novel Rho/ROCK inhibitor RKI-1447. Here, we found RKI-1447 could drastically suppress HCT-8 and HCT-116 cell growth and promoted apoptosis. Our in vitro data indicated suppressed cytoskeletal dynamics induced by RKI-1447 inhibition on mitochondrial respiration, which was evidenced by basal and maximal respiration rates, and ATP production. Simultaneously, cellular basal and maximal glycolytic rates, and glycolytic capacity were also reduced in response to RKI-1447. Moreover, RKI-1447 caused excessive reactive oxygen species generation and membrane depolarization as well as activated ER-stress. We also demonstrated CHOP is essential for RKI-1447 induced cell apoptosis. Finally, we proved inhibition of ROCK by RKI-1447 could effectively inhibit CRC growth in vivo. Taken together, this study demonstrated that inhibition of ROCK signaling pathway by RKI-1447 could suppress CRC via cytoskeleton associated mitochondrial dysfunction and cellular bioenergetics disruption. Our data suggest RKI-1447 may be an attractive antitumor drug candidate for the treatment of CRC.

Development and application of a HPLC-ICP-MS method to determine selenium speciation in muscle of pigs treated with different selenium supplements.

Dietary selenium deficiency is recognized as a global problem. Pork is the most widely consumed meat throughout the world and an important source of selenium for humans. In this study, a reliable approach was developed for analyzing selenium and its speciation in the muscles of pigs after different selenium treatments. The selenium source deposition efficiency was ranked as: selenomethionine > methylselenocysteine > selenite, and the muscle selenium content had a dose effect with selenomethionine supplementation. In total, four species of selenium were detected in the muscles of pigs and the distributions of these selenium species were greatly affected by the dietary selenium supplementation forms and levels. Selenomethionine (>70% of total selenium) and selenocystine (>11%) were the major selenium species, followed by methylselenocysteine and selenourea. Therefore, selenium-enriched pork produced from selenomethionine is a good source for improving human dietary selenium intake.

Changing the treatment paradigm for Parkinson's disease psychosis with pimavanserin.

Introduction: Parkinson's disease psychosis (PDP) may affect up to 60% of patients with Parkinson's disease over the course of their disease, and is associated with poor prognosis, including increased risks of mortality and nursing home placement. PDP treatments have been limited to off-label use of atypical antipsychotics, most of which pose risks of worsened motor symptoms and other potential adverse events (AEs) due to their dopamine receptor blockade and additional off-target receptor affinities. Pimavanserin is a highly selective 5-HT2A inverse agonist and poses no known risks for worsening of parkinsonism or other off-target receptor AEs. Pimavanserin is the first and only medication approved for PDP treatment. Areas covered: This review covers estimated prevalence, clinical characteristics, diagnostic criteria, and risk factors for PDP; the hypothetical progression of PDP; management of PDP including use of antipsychotics; pharmacology and clinical trial data on pimavanserin; and expert opinion on PDP treatment. The NLM/PubMed database was searched for papers using the search terms of "PDP" AND "treatment" AND "pimavanserin" for the last 10 years. Expert opinion: The recent insights into PDP pathophysiology and approval of the only medication specifically to treat PDP are key advances that should improve the recognition, diagnosis, and management of PDP.

Discovery of Conformational Control Inhibitors Switching off the Activated c-KIT and Targeting a Broad Range of Clinically Relevant c-KIT Mutants.

Drug resistance due to acquired mutations that constitutively activate c-KIT is a significant challenge in the treatment of patients with gastrointestinal stromal tumors (GISTs). Herein, we identified 1-(5-ethyl-isoxazol-3-yl)-3-(4-{2-[6-(4-ethylpiperazin-1-yl)pyrimidin-4-ylamino]-thiazol-5-yl}phenyl)urea (10a) as a potent inhibitor against unactivated and activated c-KIT. The binding of 10a induced rearrangements of the DFG motif, αC-helix, juxtamembrane domain, and the activation loop to switch the activated c-KIT back to its structurally inactive state. To the best of our knowledge, it is the first structural evidence demonstrating how a compound can inhibit the activated c-KIT by switching back to its inactive state through a sequence of conformational changes. Moreover, 10a can effectively inhibit various c-KIT mutants and the proliferation of several GIST cell lines. The distinct binding features and superior inhibitory potency of 10a, together with its excellent efficacy in the xenograft model, establish 10a as worthy of further clinical evaluation in the advanced GISTs.