24-h Time Point Research Articles

Role of saturated and unsaturated fatty acids on dicarbonyl-albumin derived advanced glycation end products in vitro.

Glycation is a non-enzymatic reaction that occurs between the free amino group of proteins and reducing sugars and/or lipids, leading to the formation of advanced glycation end products (AGEs). The reaction also produces reactive oxygen species that have detrimental effects on cellular and extracellular proteins. Aminoguanidine is a known inhibitor of AGEs, and some fatty acids are known to have a beneficial role in vivo by reducing inflammation and oxidative stress. However, the role of fatty acids on AGE formation has not been thoroughly reported. We investigated the role of a range of fatty acids in the formation of AGEs and their reactive intermediates using an in vitro BSA-dicarbonyl model. The model assessed a time-dependent (0-72h) and dicarbonyl concentration (0-2mM) -dependent studies for the optimal formation of AGEs. A 72h time point was found to be optimal for the reaction of BSA with either methylglyoxal (MGO) or glyoxal (GO) to generate AGE-BSA complexes. When arachidonic, eicosapentaenoic or docosahexaenoic acids were included in the reaction, a significant decrease in protein-bound fluorescent AGEs was seen compared totherespective controls. In contrast, saturated and 18 carbon polyunsaturated fatty acids showed no significant activity. Liquid chromatography-mass spectrometry (LC-MS/MS) analysis showed saturated fatty acids significantly decreased the production of Nε-carboxymethyllysine (CML) and Nε-carboxyethyllysine (CEL) from GO and MGO models, respectively, whilst increasing methylglyoxal-derived hydroimidazolone (MG-H1). In contrast, arachidonic, eicosapentaenoic and docosahexaenoic acids did not significantly change either CEL or MG-H1 compared to no treatment controls whilst significantly reducing CML levels.

Quantitative magnetic resonance imaging assessment of brain injury after successful cardiopulmonary resuscitation in a rat model of asphyxia cardiac arrest.

The aim of this study was to use dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and diffusion-weighted magnetic resonance imaging (DWI) to measure changes in blood-brain barrier (BBB) permeability and cerebral edema over time in a rat model of asphyxial cardiac arrest (ACA). ACA was established by endotracheal tube clamping. Male rats were randomized into a sham group (n = 5) and three ACA groups (n = 18). After return of spontaneous circulation (ROSC), the rats were randomized to perform DWI and DCE-MRI exam in the 6h, 24h and 72h timepoint (ROSC + 6h, ROSC + 24h, and ROSC + 72h). Results shows that fifteen of 18 animals achieved successful resuscitation in the ACA groups. The average apparent diffusion coefficient(ADC) value of the whole brain in ROSC + 6h was markedly lower than those of the sham, ROSC + 24h, and ROSC + 72h. The aquaporin-4(AQP4) score in ROSC + 6h was significantly higher than those in the other groups, which were negatively correlated with the ADC values. The ratio of whole brain to masseter muscle of volume transfer constant (rKtrans), tissue interstitium-to-plasma rate constant(rKep), and fractional extra-cellular space volume(rVe) in ROSC + 6h were all significantly higher than those in the sham, ROSC + 24h, and ROSC + 72h. The transforming growth factor β1(TGF-β1) and vascular endothelial growth factor A(VEGF-a) scores in ROSC + 6h were significantly higher than those in the other groups, which were all positively correlated with rKtrans and rKep. In conclusions, brain injury is a frequent complication after CA and resuscitation. DWI and DCE-MRI can quantitatively evaluate brain injury in term of cerebral edema and BBB permeability after successful CPR.

Predictors of Early and Late Infarct Growth in DEFUSE 3.

Introduction: The goal of this study is to explore the impact of reperfusion and collateral status on infarct growth in the early and late time windows.Materials and Methods: Seventy patients from the DEFUSE 3 trial (Endovascular Therapy Following Imaging Evaluation for Ischemic Stroke) with baseline, 24-h, and late follow-up scans were evaluated. Scans were taken with DWI or CTP at time of enrollment (Baseline), with DWI or CT 24-h after enrollment (24-h), and with DWI or CT 5 days after enrollment (Late). Early infarct growth (between baseline and 24-h scans) and late infarct growth (between 24-h and late scans) was assessed for each patient. The impact of collateral and reperfusion status on infarct growth was assessed in univariate and multivariate regression.Results: The median early infarct growth was 30.3 ml (IQR 16.4–74.5) and the median late infarct growth was 6.7 ml (IQR −3.5–21.6) in the overall sample. Patients with poor collaterals showed greater early infarct growth (Median 58.5 ml; IQR 18.6–125.6) compared to patients with good collaterals (Median 28.4 ml; IQR 15.8–49.3, unadjusted p = 0.04, adjusted p = 0.06) but showed no difference in late infarct growth. In contrast, patients who reperfused showed no reduction in early infarct growth but showed reduced late infarct growth (Median 1.9 ml; IQR −6.1–8.5) compared to patients without reperfusion (Median 11.2 ml; IQR −1.1–27.2, unadjusted p < 0.01, adjusted p = 0.04).Discussion: In the DEFUSE 3 population, poor collaterals predict early infarct growth and absence of reperfusion predicts late infarct growth. These results highlight the need for timely reperfusion therapy, particularly in patients with poor collaterals and indicate that the 24-h timepoint is too early to assess the full impact of reperfusion therapy on infarct growth.Clinical Trial Registration: http://www.clinicaltrials.gov, Unique identifier [NCT02586415].

FCER1G and PTGS2 Serve as Potential Diagnostic Biomarkers of Acute Myocardial Infarction Based on Integrated Bioinformatics Analyses.

This study aimed to explore the potential diagnostic biomarkers and mechanisms underlying acute myocardial infarction (AMI). We downloaded four datasets (GSE19339, GSE48060, GSE66360, and GSE97320) from the Gene Expression Omnibus database and combined them as an integrated dataset. A total of 153 differentially expressed genes (DEGs) were analyzed by the linear models for microarray analysis (LIMMA) package. Weighted gene co-expression network analysis was used to screen for the significant gene modules. The intersection of DEGs and genes in the most significant module was termed "common genes" (CGs). CGs were mainly enriched in "inflammatory response," "neutrophil chemotaxis," and "IL-17 signaling pathway" through functional enrichment analyses. Subsequently, 15 genes were identified as the hub genes in the protein-protein interaction network. The Fc fragment of IgE receptor Ig (FCER1G) and prostaglandin-endoperoxide synthase 2 (PTGS2) showed significantly increased expression in AMI patients and mice at the 12-h time point in our experiments. The receiver operating characteristic (ROC) curve was used to evaluate the diagnostic value of FCER1G and PTGS2. The area under ROC curve of FCER1G and PTGS2 was 77.6% and 80.7%, respectively. Moreover, the micro (mi)RNA-messenger (m)RNA network was also visualized; the results showed that miRNA-143, miRNA-144, and miRNA-26 could target PTGS2 in AMI progression.

Analysis of permeability and biological properties of dentin treated with experimental bioactive glasses

ObjectivesTo evaluate obliterating capability and biological performance of desensitizing agents. Methods50 dentin blocks were distributed according to the desensitizing agent used (n = 10): Control (Artificial saliva); Ultra EZ (Ultradent); Desensibilize Nano P (FGM); T5-OH Bioactive Glass (Experimental solution); F18 Bioactive Glass (Experimental solution). Desensitizing treatments were performed for 15 days. In addition, specimens were subjected to acid challenge to simulate oral environment demineralizing conditions. Samples were subjected to permeability analysis before and after desensitizing procedures and acid challenge. Cytotoxicity analysis was performed by using Alamar Blue assay and complemented by total protein quantification by Pierce Bicinchoninic Acid assay at 15 min, 24-h and 48-h time points. Scanning electron microscopy and energy dispersion X-ray spectroscopy were performed for qualitative analysis. Data of dentin permeability was analyzed by two-way repeated measures ANOVA and Tukey's test. For cytotoxicity, Kruskal-Wallis and Newman-Keuls tests. Resultsfor dentin permeability there was no significant difference among desensitizing agents after treatment, but control group presented highest values (0.131 ± 0.076 Lp). After acid challenge, control group maintained highest values (0.044 ± 0.014 Lp) with significant difference to other groups, except for Desensibilize Nano P (0.037 ± 0.019 Lp). For cytotoxicity, there were no significant differences among groups. ConclusionBioglass-based desensitizers caused similar effects to commercially available products, regarding permeability and dentin biological properties. Clinical significanceThere is no gold standard protocol for dentin sensitivity. The study of novel desensitizing agents that can obliterate dentinal tubules in a faster-acting and long-lasting way may help meet this clinical need.

Anti-Cancer Properties of Nicotinic Cid-Alpha Linolenic Acid Derivative on A375 Melanoma Cell Line: Assessment of Apoptosis and WNT Signaling Pathways

Background: Malignant melanoma is aggressive skin cancer whose survival rate is extremely low. Commencing apoptosis is believed to be a significant issue in cancer treatment and targeting the apoptosis and WNT signaling pathways, which is probably a potentially successful strategy to overcome tumor plasticity in melanoma. Method: We conducted the present in vitro study to investigate anti-proliferative and apoptotic effects of Nic-ALA, as a new compound, on A375 melanoma cell line using MTT assay and flow cytometry, respectively. The gene expression profiles of the cancer cells were obtained for Bcl-2 and Bax as the main genes of the apoptosis signaling pathway and WIF1 and beta-catenin genes from the WNT signaling pathway with qRT-PCR. Results: Nic-ALA's cytotoxicity on A375 melanoma cell line from MTT assay was obtained with IC50 166.7, 144.2, and 146.1µM. This novel derivative induced 11.3, 46.1, and 85.7% of apoptosis in 24, 48, and 72h time points, respectively. In the treated cells, the expression of Bax, beta-catenin, and WIF1 genes increased while the expression of Bcl-2 decreased significantly at 200µM concentration and the treated times of 48 and 72h. Conclusion: The anti-proliferation of Nic-ALA at a lower value than what we found in nicotinic acid alone represented the higher bioavailability and transport efficiency of this novel derivative through A375 melanoma cell line. Its antipoetic effects were obtained by increasing the apoptosis rate and expression of the Bax gene and reducing Bcl-2 gene expression. Up-regulation of WIF1 and beta-catenin in the WNT signaling pathway emphasized Nic-ALA's anti-cancer effect on A375 melanoma cell line.

Gulf War Agent Exposure Causes Dose‐Dependent Impairment of Memory Formation and Exercise Intolerance in a Mouse Model of Gulf War Illness

Gulf War Illness (GWI) is a chronic multi-symptom condition that still affects a third of veterans who served in the 1991 Persian Gulf War (GW). Latently-emerging and persistent symptoms fall into 5 general domains: neurological/cognition/mood, muscle/joint pain, respiratory, gastrointestinal disturbances and especially chronic fatigue. In particular, veterans exhibit CNS deficits in the neuropsychological domains of visuospatial abilities, attention/executive functioning, and learning/memory. GWI remains untreated due its poorly understood etiology and pathophysiology. It is suggested that GWI is a result of continuous exposure of military personnel to GW agents (pyridostigmine bromide, (PB), permethrin (PER), diethyltoluamide (DEET) and deployment/combat stress. To further investigate the cognitive manifestations of GWI pathophysiology, we studied the effects of chronic GW agent exposure in adult C57Bl/6N males utilizing a repeated measures design. The dosing paradigm consisted of 5 d/week for 28d of exposure to two doses of PB (6.5 mg/kg/d, 2x/d and 8.7 mg/kg/d, 1x/d; po), PER (1.3 mg/kg/d; top) in 70% ETOH, DEET (1650 mg/kg/d in DMSO) and stress 5 min/d (n=5–7/group). Two controls were included: sham (saline po, 70% ETOH and DMSO top) and sham/stress. Behavioral endpoints were measured at different timepoints post treatment (PT) up to PT160d. The 8.7 mg/kg/d dose was more effective in modeling cognitive deficits on the passive avoidance test since this group failed to avoid the aversive dark chamber at the 24h, and 7d and/or 3d retention timepoints at PT70. A repeat test at PT120 indicated that the 8.7 and sham/stress (P<.01) but not the 6.5 mg/kg/d group displayed retention of emotional reactivity memory at all test timepoints. In the novel object recognition test, performed at PT17, GW groupsbut not sham/stress displayed deficient memory retention at 24h (P<.01). On the Barnes Maze, no GW exposure effects on reference memory were noted during acquisition training nor a 24h acquisition probe trial. Since the neuropsychological deficits seen in GW chemical exposures can be exacerbated by comorbid mood-related conditions, animals were tested for anxiety and depression but no GW-specific effects were found. To model chronic fatigue, using a 5-day exercise endurance test we found that 8.7 mg/kg/d showed decreased latency to exhaustion at PT23 and PT161 compared to sham/stress (P<.05, P<.01). This effect could not be attributed to problems with motor coordination as measured on Suok and rotarod tests. In summary, the GW agent exposure paradigm produces specific cognitive deficits in associative aversive learning and novel object recognition memory but not visuospatial (reference) memory. GW agents also cause mice to tire faster during exercise without deficits in motor coordination. This animal model may be useful in examining common mechanisms underlying deficient cognitive performance and chronic fatigue in GWI.

ASICs are required for immediate exercise‐induced muscle pain (IEIP), but not delayed‐onset muscle soreness (DOMS)

Introduction IEIP and DOMS are types of muscle pain induced by exercise. IEIP occurs during and immediately after, whereas DOMS occurs days after exercise, and are attributed to metabolites and inflammation in muscle, respectively. Acid sensing ion channels (ASICs), H+-gated channels expressed in muscle afferents, play a role in different pain conditions. ASICs are activated by H+ and other chemicals released during intense muscle contraction and microinjuries. Recently, we showed that ASICs are required for IEIP. PURPOSE Here, we tested if ASICs are also required for DOMS. METHODS C57BL/6 wild type (WT) and ASIC3-/- mice underwent an exhaustive exercise (exercise groups), or were placed on a non-moving treadmill for a same amount of time (control groups). Exercise-induced muscle pain was assessed by muscle withdrawal threshold (MWT) at baseline, immediately and 24h after exercise. Locomotor movement, grip strength and exercise performance were tested at baseline and 24hrs after exercise for WT and ASIC3-/- to evaluate DOMS. RESULTS ASIC3-/- had similar baseline muscle pain, locomotor activity, grip strength, and exercise performance as WT mice. WT showed diminished MWT immediately after exercise indicated they developed IEIP (P < 0.001), but ASIC3-/- mice did not. However, both, ASIC3-/- and WT had similarly lower MWT (P < 0.05) and grip strength (P < 0.01) 24hrs after baseline-exercise. On the other hand, ASIC3-/- displayed significantly lower locomotor activity and repeat exercise performance at 24h timepoint compared to WT mice­ (P < 0.05). CONCLUSION While WT and ASIC3-/- had a same exercise performance, they had different pain perception immediately after, but not a day after exercise. Unlike WT mice, ASIC3-/- did not develop IEIP. At 24h after exercise, ASIC3-/- developed similar DOMS features (hyperalgesia and lower strength) as WT mice. Interestingly, ASIC3 -/- mice had diminished locomotor movement and repeat exercise performance at 24h. These results show ASICs are required for IEIP, but not DOMS, and they might play a protective role from muscle injury during metabolic perturbations like high intensity exercise.

Development and validation of the TGx-HDACi transcriptomic biomarker to detect histone deacetylase inhibitors in human TK6 cells.

Transcriptomic biomarkers can be used to inform molecular initiating and key events involved in a toxicant's mode of action. To address the limited approaches available for identifying epigenotoxicants, we developed and assessed a transcriptomic biomarker of histone deacetylase inhibition (HDACi). First, we assembled a set of ten prototypical HDACi and ten non-HDACi reference compounds. Concentration-response experiments were performed for each chemical to collect TK6 human lymphoblastoid cell samples after 4h of exposure and to assess cell viability following a 20-h recovery period in fresh media. One concentration was selected for each chemical for whole transcriptome profiling and transcriptomic signature derivation, based on cell viability at the 24-h time point and on maximal induction of HDACi-response genes (RGL1, NEU1, GPR183) or cellular stress-response genes (ATF3, CDKN1A, GADD45A) analyzed by TaqMan qPCR assays after 4h of exposure. Whole transcriptomes were profiled after 4h exposures by Templated Oligo-Sequencing (TempO-Seq). By applying the nearest shrunken centroid (NSC) method to the whole transcriptome profiles of the reference compounds, we derived an 81-gene toxicogenomic (TGx) signature, referred to as TGx-HDACi, that classified all 20 reference compounds correctly using NSC classification and the Running Fisher test. An additional 4 HDACi and 7 non-HDACi were profiled and analyzed using TGx-HDACi to further assess classification performance; the biomarker accurately classified all 11 compounds, including 3 non-HDACi epigenotoxicants, suggesting a promising specificity toward HDACi. The availability of TGx-HDACi increases the diversity of tools that can facilitate mode of action analysis of toxicants using gene expression profiling.

Erector spinae block for postoperative pain management in lumbar disc hernia repair.

Lumbar disc herniation is the most common spinal disorder and various less invasive techniques such as microdiscectomy have been described. However, postoperative pain management in patients undergoing discectomy is still commonly inadequate. Erector spinae plane (ESP) block is a relatively easier technique with lower risks of complications, and can be performed to provide postoperative analgesia for various procedures. The current study aimed to determine the effect of ESP block on postoperative analgesia in patients who underwent elective lumbar disc herniation repair surgeries. Fifty-four ASA I-II patients aged 18-65years scheduled for elective discectomy surgery were included in the study. Patients were randomized either to the ESP or control group. Ultrasound-guided ESP block with 20mL of 0.25% bupivacaine was performed preoperatively in the ESP group patients and a sham block was performed with 20mL normal saline in the control group patients. All the patients were provided with intravenous patient-controlled analgesia devices containing morphine. Morphine consumption and numeric rating scale (NRS) scores for pain were recorded 1, 6, 12, and 24h after surgery. A significantly lower morphine consumption was observed at 6, 12, and 24h timepoints in the ESP group (p < 0.05 for each timepoint). Total morphine consumption at 24h after surgery decreased by 57% compared to that of the control group (11.3 ± 9.5mg in the ESP group and 27 ± 16.7mg in the control group). NRS scores were similar between the two groups. This study showed that ESP block provided effective analgesia in patients who underwent lumbar disc herniation surgery. NCT03744689.

Nebulized Magnesium Sulfate for Treatment of Persistent Pulmonary Hypertension of Newborn: A Pilot Randomized Controlled Trial.

ObjectivesTo investigate the effectiveness of nebulized magnesium sulfate in treating persistent pulmonary hypertension of newborn (PPHN).MethodsTwenty-eight mechanically ventilated term neonates with severe PPHN were randomized into two groups: NebMag group (n = 14), who receiving nebulized isotonic magnesium (1024 mg/h), and IVMag group (n = 14), who received intravenous magnesium (200 mg/kg over 30 min, followed by 50 mg/kg/h). The study time frame was 24 h. Outcome measures were the changes in oxygenation index (OI), mean arterial blood pressure (MABP), vasoactive inotropic score (VIS), and serum magnesium level.ResultsBaseline demographic, ventilatory, and hemodynamic characteristics were comparable between the two groups. At the end of the study, the OI decreased by 44.3% in the NebMag group compared with 35.3% in the IVMag group (mean difference −3.14; 95%CI −5.08, −1.19; p 0.003). The NebMag group had a higher MABP (mean difference 2.29 mmHg; 95% CI 1.80, 2.77; p 0.000) and lower VIS (mean difference −14.64; 95% CI −16.52, −12.77; p 0.000) at the 24-h study time point. The increase in serum magnesium level, measured at 12-h study time point, was lower in the NebMag group (mean difference −2.26 mmol/L; 95% CI −2.58, −1.96; p 0.000).ConclusionNebulized magnesium sulfate may be an effective therapeutic modality for neonates with severe PPHN on mechanical ventilation, but this should be confirmed by larger studies.Retrospectively registered at www.clinicaltrials.gov (identifier: NCT04328636).

Analysis of the bacterial biofilm formation in different models of the in vitro culture

Introduction. Microtiter plate assay (MPA) remains one of workhorses of in vitro biofilm research but it requires optimization of experimental conditions to fulfill the biofilm formation requirements of different bacterial pathogens. Aim. The aim was to determine the effect of TSB and RPMI1640 culture media and selected culture variables (O2 vs. 5% CO2, extended incubation time) on the biofilm production by bacteria commonly involved in biofilm-related infections: Enterococcus faecalis (EF), Escherichia coli (EC), Staphylococcus aureus (SA), Pseudomonas aeruginosa (PA), Klebsiella pneumoniae (KP). Material and methods. The investigation was performed using the MPA with crystal violet. Results. Statistically significant (p&lt;0.05) increase in biofilm production between 24h and 72h time points was observed for EF (TSB o2, RPMIo2 and RPMIco2), EC (TSBo2), SA (TSBo2, TSBco2), KP (TSBo2, TSBco2), PA (RPMIco2, TSBco2). The TSB caused a significantly greater stimulation of biofilm production compared to RPM1640. It outcompeted RPMI1640 irrespective of the atmospheric conditions for SA and KP and under aerobic conditions for EF. Conclusion. Although the TSB provided the most optimal conditions for biofilm production, the process was influenced by the strain type, atmospheric conditions and period of cultivation which limits the ability to design a single universal model of the in vitro biofilm investigation.

Role of Adamts-1 in Pleomorphic Xanthoastrocytoma Tumor Cells Progression.

To analyze the expression of ADAMTS-1, NF-?B, and STAT3 in human pleomorphic xanthoastrocytoma specimens, and their correlation with glioma advancement. Pleomorphic xanthoastrocytoma tumor cell lines were treated with low and high doses of cytokines at 24 and 48 hours (h) to replicate the inflammatory environment. The effects of IL-1 were assessed with the scratch wound-healing assay, and the expression levels of ADAMTS-1, NF-?B, and STAT3 of the groups were determined by western blot analysis. Cytokine treatment significantly increased the migration of PXA glioma cells after scratching at 24h and 48h time points. Similarly, 10 and 30 ng/mL IL-1 induced 1.86 and 1.94 fold increases, respectively, in ADAMTS-1 expression after 24h, and 3 and 3.27 fold increases, respectively, after 48h, compared with the non-treatment control group.10 and 30 ng/mL IL-1 doses caused 2.5 and 2.6 fold increase, in NF-?B protein levels after 24h, and 3.16 and 3.41 fold increases after 48h, compared with the non-treatment group. The protein levels of STAT3 after 24h were 2.62 and 2.43 fold higher, and 3.78 and 3.84 fold higher after 48 hours, with 10 and 30 ng/mL IL-1, compared with the non-treatment group. The proliferation and progression of glioma cells were proportional to the increased expression levels of ADAMTS-1, NF-?B, and STAT3. Our findings indicate that the proteolytic function of ADAMTS-1 may be associated with the malignant transformation of low-grade gliomas.

The spectrum of 124I uptake in the lacrimal gland and nasolacrimal sac/duct on PET/CT imaging.

124I PET/CT in 31 DTC patients was performed at 2, 24, 48, 72, and 96 h after oral administration of 31.5 or 62.9 MBq (0.85 or 1.7 mCi) of 124I after either recombinant human thyroid-stimulating hormone injections or thyroid hormone withdrawal. All but two patients had a history of prior 131I therapy. Patterns of 124I uptake in the lacrimal glands and nasolacrimal sac/ducts (NLD) were assessed. A total of 173 individual 124I PET/CT scans (forming 35 sets of scans) were reviewed for 31 patients. Lacrimal glands were visualized bilaterally in only 4 patients. The focal mild uptake (grade 2), best seen on the 2-h images, was crescent-shaped and located in the lateral upper quadrant of the orbit. In contrast, the NLDs were identified in all patients (bilateral in 29 of 31 patients) with high focal uptake (grade 4) peaking on the 2- and 24-h timepoints; however, the overall pattern of uptake was variable. Of the 29 patients with prior 131I therapy, three patients had a relatively fixed and unchanging pattern of uptake on at least one side of the NLDs. In patients with DTC, 124I activity in the NLDs is more frequently visualized, more intense, more prolonged, and more variable than in the lacrimal glands. The lack of clearance may suggest possible obstruction or stasis of an NLD.

Characterization of the nutritive value of perennial ryegrass (Lolium perenne L.) dominated pastures using updated chemical methods with application for the Cornell Net Carbohydrate and Protein System

Ruminant nutrition models, such as the Cornell Net Carbohydrate and Protein System (CNCPS), have evolved over time resulting in increased precision and accuracy with which they can predict animal performance. In tandem with this evolution, more precise and accurate methodologies have been developed to describe the feed chemistry inputs required to conduct model simulations. However, these updated methods have not been applied to pasture-based systems. The objective of this study was to characterize the nutritive value of perennial ryegrass (Lolium perenne L.; PRG) dominated pastures using comprehensive feed chemistry methods. Fifty-five samples from three independent experiments were used in this study. The data generated demonstrated that the amylase- and sodium sulphite-treated NDF corrected for ash residue (aNDFom) method was significantly lower than the amylase- and sodium sulphite-treated NDF (aNDF) method; hence, due to ash contamination, aNDF consistently overestimated the true measure of cell wall content in PRG. Results from multiple time point in vitro aNDFom digestibility analysis indicated that immature PRG comprises of a large potentially digestible pool that degrades at a rapid rate. A 12-h time point combined with 30, 120, and 240 h better describes the aNDFom degradation curve of PRG. In addition, undigested aNDFom at 240 h of fermentation was affected by PRG category; whereby, spring and summer were lowest (98 and 111 g/kg of aNDFom, respectively), autumn intermediate (155 g/kg of aNDFom), and drought stressed pastures the highest (200 g/kg of aNDFom). The extent of in vitro aNDFom digestion was not reached prior to 240 h; however, a strong Pearson correlation coefficient between 120-h and 240-h time points (0.98) indicated the potential to predict undigested aNDFom from the 120-h time point. Based on the N fractionation scheme employed by the CNCPS, a large proportion of PRG N is soluble and likely highly degradable in the rumen. An assay capable of quantitatively describing the rumen degradation of PRG N is required. Using a single 21-h HCl hydrolysis, to determine amino acid concentration, underestimated the concentration of Ile and Val indicating that multiple-hydrolysis time procedures are required to predict the true amino acid concentration of PRG. In conclusion, the updated feed chemistry procedures conducted in this study, allowed a more accurate characterization of the nutritive value of PRG. This increased understanding in combination with formulation models, such as the CNCPS, can provide more robust information on how PRG interacts with the ruminant animal, in order to increase the efficiency and productivity of pasture-based systems.

Cellular and Transcriptional Adaptation of Bovine Granulosa Cells Under Ethanol-Induced Stress In Vitro.

Granulosa cells (GCs) are the major cellular component in a follicular microenvironment and play an indispensable role in ovarian function. This study was conducted to investigate the effects of ethanol exposure on the cellular and transcriptional changes of ovarian GCs. For this purpose, bovine GCs were exposed to different concentrations of ethanol (0, 50, 100, 200, 500 and 1000) to mimic the effects of alcohol in in vitro. Subsequently, 100 and 1000mM concentrations were discarded from further experiments, as 100mM was not different from 50mM, and 1000mM was supertoxic to the cells. The results showed that there was a gradual loss of cell viability with the increase of the ethanol concentration, i.e. lowest viability was observed at the highest concentration (1000mM), which is further supported by cell proliferation assay. Mitochondrial activity decreased significantly at higher concentrations. The expression of NRF2 decreased significantly (P<0.05) in ethanol-exposed cells compared with the cells in the control group at the 6-h time point, whereas the expression was increased in 500mM concentration at the 24-h time point. The expression of antioxidant genes, downstream to Nrf2-pathway activation, showed that overall expression pattern similar to NRF2. The result of this study prompted us to postulate that ethanol exposure decreases the ability of GCs to handle stress by downregulating the expression of genes involved in Nrf2-pathway.

TAK-242 Attenuates Crush Injury Induced Acute Kidney Injury through Inhibiting TLR4/NF-κB Signaling Pathways in Rats.

To investigate if toll-like receptor (TLR) 4/nuclear factor-kappa B (NF-κB) signaling pathways mediated crush injury induced acute kidney injury (AKI) in rats, and if TAK-242 (a specific inhibitor of TLR4) attenuates the injury through inhibiting the signaling pathways. This study was divided into two parts: (1) Establish the crush injury model: 50 rats were randomly divided into control group and four crush injury groups (n = 10/group). Crush injury groups were given 3kg pressure for eight hours and were sacrificed at the time points of 0h, 6h, 12h, and 24h after relieving pressure. And (2) Select the most obvious injury group (12h group) for drug intervention group. Thirty rats were randomly divided into control group, 12h group, and 12h+TAK-242 group (n = 10/group). Two parts detection were as follows: pathological changes of kidney tissues were observed in Haematoxylin and Eosin (HE) staining. Serum creatinine, blood urea nitrogen (BUN), myoglobin (Mb), and blood potassium were examined by automatic biochemical analysis instrument. Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured by enzyme-linked immunosorbent assay (ELISA). The TLR4 messenger ribonucleic acid (mRNA), TLR4, and P65 were detected by real-time polymerase chain reaction (PCR), western blot, immunohistochemistry staining. Compared with the control group, kidney tissues were damaged in crush injury groups, and most obvious in the 12h group. The level of serum creatinine, BUN, Mb, blood potassium, IL-6, TNF-α, and TLR4mRNA were increased in the crush injury groups and significantly increased in the 12h group (P <.05). The TLR4 and P65 were significantly increased in the 12h group (P <.05). Compared with the 12h group, kidney tissue damage was significantly reduced in the TAK-242 group (P <.05). The level of serum creatinine, BUN, Mb, blood potassium, IL-6, TNF-α, TLR4mRNA, TLR4, and P65 in the TAK-242 group were significantly reduced (P <.05). The present findings conclude that TLR4/NF-κB signaling pathways mediated crush injury induced AKI in rats, and TAK-242 attenuates the injury through inhibiting the signaling pathways.

Radiation and Gold Nanoparticle Increase the Expression of Immunogenic Cell Death Markers in MDA MB 231 Breast Cancer Model

Tumor immunogenic cell death (ICD) plays a major role in stimulating host anti-cancer immune response that can determine the success of cancer radiotherapy (RT). Additionally, ICD can also be induced by radiation itself. The main feature of ICD is the release of immunogenic molecules by dying cells, termed damage associated molecular patterns (DAMPs) that act on innate and adaptive immune components to induce long-lasting antitumor immunity. Two breast cancer (BCa) DAMPs involved in phagocytosis and dendritic cell antigen presentation are Calreticulin (CLR) and HMGB1; loss of HMGB1 expression is linked to poor clinical prognosis. Therefore, these molecules are being studied for their therapeutic and prognostic potential. As RT, either alone or in combination, is often part of standard BCa therapies, the effect of RT and radiosensitizers, such as gold nanoparticles (AuNPs), on DAMPs expression must be considered when designing new protocols, especially if combining RT with an adjuvant mode such as immunotherapy. The goal of this study was to measure the effect of radiation on CLR and HMGB-1 expression in the presence and absence of a novel AuNP radiosensitizer in MDA MB 231 BCa in vitro and in vivo models. We hypothesized that AuNP modulates radiation induced ICD by altering CLR and HMGB1 expression. In vitro: MDA MB 231 cells were incubated for 3h with 14nm AuNPs and irradiated. After 48h and 72h the percentages of CLR and HMGB1 positive cells were determined by flow cytometry. In vivo: Female nude mice bearing MDA MB 231 tumors received intratumoral injections of 14 nm AuNPs. After 24h mice were irradiated with 15 Gy dose using 160 kV photons. Mice were euthanized, histological sections prepared, stained with anti CLR antibodies and analyzed by light microscopy. In vitro, radiation induced increase in the percentages of CLR and HMGB1 positive cells was potentiated by AuNP at 48h and 72h time points. In control cells, AuNP alone increased the percent of HMGB1 positive cells at both time points. The percent of CLR positive cells was increased at 72h only. In vivo, in animals receiving RT or AuNP only, CLR expression was increased relative to control animals and CLR was detected extracellularly after RT, and extra- and intracellularly after AuNP alone. However, after combined RT and AuNP, CLR expression was higher than after single treatments and was detected both, extra- and intracellularly. In BCa patients, induction of ICD may play a critical role in improving clinical outcomes. Here we show that AuNP enhanced the immunogenic effect of a single irradiation dose in BCa cells and mouse model. This effect was measured by an increase in the expression of CLR and HMGB1, DAMPs critical to the response of BCa. In addition, AuNP alone exhibited an immunogenic effect similar to RT alone. These findings support that BCa depends on ICD and provide a platform for designing multimodal BCa RT formulations with novel radiosensitizers or immunotherapy.

Pharmacokinetics of Sustained-release, Oral, and Subcutaneous Meloxicam over 72 Hours in Male Beagle Dogs.

In cynomolgus macaques, plasma levels of sustained-release formulations of meloxicam meet or exceed efficacious concentrations for 48 to 72 h, thereby allowing less animal handling and providing more consistent efficacy than standard formulations of meloxicam. The goal of this study was to compare the pharmacokinetics of a single subcutaneous dose of a sustained-release formulation of meloxicam (Melox-SR) with those of oral (Melox-PO) and standard subcutaneous (Melox-SC) formulations dosed every 24 h for 3 consecutive days. Dogs (5 or 6 adult male Beagles) each received the following 3 treat- ments: first, Melox-SR (10 mg/mL, 0.6 mg/kg SC once), next Melox-SC (0.2 mg/kg SC once, followed by 0.1 mg/kg SC every 24 h), and finally Melox-PO (same dosage as Melox-SC), with a washout period of at least 2 wk between formulations. Blood was collected at 0 (baseline), 1, 4, 8, 12, 24, 48, and 72 h after the initial administration of each formulation for comparison of meloxicam plasma concentrations. Blood was also collected before administration and at 48 h after Melox-SR injection for CBC and chemistry analysis. Plasma concentrations (mean ± 1 SD) of Melox-SR peaked at the 1-h time point (2180 ± 359 ng/ mL), whereas those of Melox-PO (295 ± 55 ng/mL) and Melox-SC (551 ± 112 ng/mL) peaked at the 4-h time point. Melox-SR yielded significantly higher plasma concentrations than Melox-PO and Melox-SC until the 48 and 72-h time points, respec- tively. Melox-SC plasma concentrations were significantly higher than those of Melox-PO at 4, 8, 12, 24, 48 and 72 h. No lesions were noted at the Melox-SR injection sites, and Melox-SR administration was not associated with changes in the CBC and serum chemistry panels. A single 0.6-mg/kg dose of Melox-SR can yield plasma concentrations that exceed 350 ng/mL for at least 72 h in adult male dogs.

Potential therapeutic effects of electrogram-guided cardioneuroablation in long QT syndrome: case series

A significant shortening of the corrected QT interval (QTc) in addition to parasympathetic denervation after cardioneuroablation (CNA) was recently demonstrated in patients with vagally mediated bradyarrhythmias and normal QTc range. This study assessed the effects of CNA on ventricular repolarization and heart rate by using QTc measurements in 2 patients with long QT syndrome (LQTS). The case series included 2 consecutive patients with significant sinus bradycardia and refractory paroxysmal atrial fibrillation (AF). All atrial ganglionated plexus (GP) sites in addition to pulmonary vein isolation were successively targeted by using electrogram-guided strategy. QTc was calculated on 12-lead ECG before the procedure (time point 1), at post-ablation 24h (time point 2), and at the last follow-up visit (time point 3), respectively. In the first case, QTc (Bazett) shortened from 612 to 551msec between time points 1 and 2 and was 419msec in time point 3. Similarly, QTc (Bazett) shortened from 480 to 401msec between time points 1 and 3 in the second case. In both cases, minimum and mean heart rates were significantly increased after ablation. The parameters of which are used to estimate both sympathetic and parasympathetic changes in heart rate variability were significantly decreased after ablation. There were no arrhythmia-related symptoms during follow-up. The present case series reports a new ablation strategy systematically targeting autonomic GPs in LQTS patients. CNA shortens QTc (through sympathetic modulation) and increases heart rate. Although promising, these preliminary results need to be confirmed in the larger prospective study.