Quantification of AICAR and study of metabolic markers after administration.

Objectives: AICAR (5-amino-4-imidazolecarboxyamide ribonucleoside) was reported as the first pharmacological AMPK (adenosine 5'-monophosphate (AMP)-activated protein kinase) activator, and it has been confirmed to exhibit a significant endurance enhancement effect and prohibited for doping by the World Anti-Doping Agency. Due to the fact that the human body can produce such substances, in order to ensure fairness in sports competition, methods for rapid detection and multi-type identification of AICAR drugs taken orally should be established. Methods: to assess AICAR levels, a new rapid, sensitive, efficient, and selective method was reported for the quantitative detection of AICAR in urine using LC-MS/MS. The method was validated for quantitative purposes based on the elemental selectivity, intra- (1.0-15.6%) and inter-day precision (1.3-16.3%), accuracy (99.9-112.8%), matrix effects (88.9-103.6%), recovery (87.4-106.5%), and stability at four different concentrations. The calibration curve was linear over a wide concentration range of 10-10,000 ng mL-1 with a high coefficient of determination (R 2 > 0.998). The limit of detection (LOD) and limit of quantification (LOQ) for the experiment were determined to be 1 and 10 ng mL-1, respectively. Simultaneously, metabolomics analysis was used to obtain the metabolic fingerprint of different populations and biomarkers to distinguish administration cases through partial least squares discriminant analysis (PLS-DA) and a receiver operating characteristic (ROC) curve. Results: the method enables easy quantitation for LC-MS/MS analysis with the best recovery yield maintained, and the method was applied to 122 Asian biological samples with an average concentration of 1310.5 ± 1031.4 ng mL-1. Through drug metabolism research, 734 and 294 variables were extracted for data analysis respectively in the positive and negative ion modes, and more than 100 metabolites with significant up- and down-regulation were found after the test. Conclusions: this research developed a fast, precise, effective, and specific approach for the qualitative and quantitative identification of AICAR in urine. Meanwhile, administration metabolism studies found that there were significant changes in AICAR levels and other compounds, such as PC types PC(18:1/16:0), PC(16:0/18:0), and PC(16:0/16:0), PE types PE(18:0/20:4), and LPE-type 18:1, which could better distinguish samples before and after AICAR administration. The analysis provides a multi-perspective reference for WADA to determine a positive criterion.

Association Between Helicobacter pylori and Laryngopharyngeal Reflux Disease: A Systematic Review and Meta-Analysis.

BACKGROUND: It is controversial that Helicobacter pylori (H pylori) is involved in the pathogenesis or development of laryngopharyngeal reflux disease (LPRD). OBJECTIVE: To investigate the potential association between LPRD and H pylori infection. MATERIAL AND METHODS: A systematic review was performed of studies assessing the diagnosis or treatment of LPRD among patients with H pylori infection. Data sources are PubMed/MEDLINE, EMBASE[Ovid], Cochrane Library, and Web of Science, and ClinicalTrials.gov. RESULTS: Fifteen studies were analyzed in the review, with all eligible for the meta-analysis. A significant association between H pylori infection and LPRD was detected for higher rates of H pylori infection in patients with LPRD than in non-LPRD patients (relative risk (RR), 1.35; 95% CI, 1.12-1.63; P = 0.002), and H pylori-positive patients had a higher prevalence of LPRD than H pylori-negative patients (RR, 1.19; 95% CI, 1.07-1.31; P = 0.001). The prevalence of H pylori among patients with LPRD was 49% (95% CI, 36-61), the prevalence of H pylori among patients with non-LPRD was 35% (95% CI, 23-49). CONCLUSION AND SIGNIFICANCE: The limited evidence indicated the association between LPRD risk and increased H pylori infection. Different population races, diagnostic approach to LPRD, variant H pylori testing methods, age and sex may contribute to the heterogeneity. Further well-designed studies regarding the efficacy of H pylori eradication in the treatment of LPRD are strongly recommended in the future.

Selenomethionine Inhibits NF-κB-mediated Inflammatory Responses of Bovine Mammary Epithelial Cells Caused by Klebsiella pneumoniae by Increasing Autophagic Flux.

Klebsiella pneumoniae (K. pneumoniae) is one of the major pathogens causing bovine clinical mastitis. Autophagy maintains cellular homeostasis and resists excessive inflammation in eukaryotic organisms. Selenomethionine (Se-Met) is commonly used as a source of selenium supplementation for dairy cows. This study aimed to investigate the effects of Se-Met on inflammatory responses mediated by nuclear factor-kappa B (NF-κB) through autophagy. We infected bovine mammary epithelial cell line (MAC-T) with K. pneumoniae and examined the expression of autophagy-related proteins and changes in autophagic vesicles, LC3 puncta, and autophagic flux at various intervals. The results showed that K. pneumoniae activated the early-stage autophagy of MAC-T cells. The levels of LC3-II, Beclin1, and ATG5, as well as the number of LC3 puncta and autophagic vesicles, increased after 2 h post-treatment. However, the late-stage autophagic flux was blocked. Furthermore, the effect of autophagy on NF-κB-mediated inflammation was investigated with different autophagy levels. The findings showed that enhanced autophagy inhibited the K. pneumoniae-induced inflammatory responses of MAC-T cells. The opposite results were found with the inhibition of autophagy. Finally, we examined the effect of Se-Met on NF-κB-mediated inflammation based on autophagy. The results indicated that Se-Met alleviated K. pneumoniae-induced autophagic flux blockage, inhibited NF-κB-mediated inflammation, and decreased the adhesion of K. pneumoniae to MAC-T cells. The inhibitory effect of Se-Met on NF-κB-mediated inflammation could be partially blocked by the autophagy inhibitor chloroquine (CQ). Overall, Se-Met attenuated K. pneumoniae-induced NF-κB-mediated inflammatory responses by enhancing autophagic flux.

Sabatolimab (MBG453) model-informed drug development for dose selection in patients with myelodysplastic syndrome/acute myeloid leukemia and solid tumors.

Sabatolimab is a novel immunotherapy with immuno-myeloid activity that targets T-cell immunoglobulin domain and mucin domain-3 (TIM-3) on immune cells and leukemic blasts. It is being evaluated for the treatment of myeloid malignancies in the STIMULUS clinical trial program. The objective of this analysis was to support the sabatolimab dose-regimen selection in hematologic malignancies. A population pharmacokinetic (PopPK) model was fit to patients with solid tumors and hematologic malignancies, which included acute myeloid leukemia, myelodysplastic syndrome (including intermediate-, high-, and very high-risk per Revised International Prognostic Scoring System), and chronic myelomonocytic leukemia. The PopPK model, together with a predictive model of sabatolimab distribution to the bone marrow and binding to TIM-3 was used to predict membrane-bound TIM-3 bone marrow occupancy. In addition, the total soluble TIM-3 (sTIM-3) kinetics and the pharmacokinetic (PK) exposure-response relationship in patients with hematologic malignancies were examined. At intravenous doses above 240 mg Q2w and 800 mg Q4w, we observed linear PK, a plateau in the accumulation of total sTIM-3, and a flat exposure-response relationship for both safety and efficacy. In addition, the model predicted membrane-bound TIM-3 occupancy in the bone marrow was above 95% in over 95% of patients. Therefore, these results support the selection of the 400 mg Q2w and 800 mg Q4w dosing regimens for the STIMULUS clinical trial program.

Development of mRNA vaccines against respiratory syncytial virus (RSV).

Respiratory syncytial virus (RSV) is a single-stranded negative-sense RNA virus that is the primary etiologic pathogen of bronchitis and pneumonia in infants and the elderly. Currently, no preventative vaccine has been approved for RSV infection. However, advances in the characterization, and structural resolution, of the RSV surface fusion glycoprotein have revolutionized RSV vaccine development by providing a new target for preventive interventions. In general, six different approaches have been adopted in the development of preventative RSV therapeutics, namely, particle-based vaccines, vector-based vaccines, live-attenuated or chimeric vaccines, subunit vaccines, mRNA vaccines, and monoclonal antibodies. Among these preventive interventions, MVA-BN-RSV, RSVpreF3, RSVpreF, Ad26. RSV.preF, nirsevimab, clesrovimab and mRNA-1345 is being tested in phase 3 clinical trials, and displays the most promising in infant or elderly populations. Accompanied by the huge success of mRNA vaccines in COVID-19, mRNA vaccines have been rapidly developed, with many having entered clinical studies, in which they have demonstrated encouraging results and acceptable safety profiles. In fact, Moderna has received FDA approval, granting fast-track designation for an investigational single-dose mRNA-1345 vaccine against RSV in adults over 60 years of age. Hence, mRNA vaccines may represent a new, more successful, chapter in the continued battle to develop effective preventative measures against RSV. This review discusses the structure, life cycle, and brief history of RSV, while also presenting the current advancements in RSV preventatives, with a focus on the latest progress in RSV mRNA vaccine development. Finally, future prospects for this field are presented.

Selenomethionine protected BMECs from inflammatory injury and oxidative damage induced by Klebsiella pneumoniae by inhibiting the NF-κB and activating the Nrf2 signaling pathway.

Klebsiella pneumoniae (K. pneumoniae) is one of the main environmental pathogens causing bovine mastitis. The incidence of bovine mastitis caused by K. pneumoniae is increasing worldwide. Selenium is an essential trace element that has multiple physiological functions, such as antioxidant and anti-inflammatory activities. Therefore, this study aimed to verify whether selenomethionine (SeMet) could contribute to alleviating the inflammatory injury and oxidative damage induced by K. pneumoniae. Bovine mammary epithelial cells were cultured in vitro and pretreated with 4 µM SeMet before being infected with K. pneumoniae. Western blot analysis was used to detect the expression of the related proteins in the NF-κB and Nrf2 signaling pathways. The gene expression levels of IL-1ß, IL-6, IL-8, TNF-α, Nrf2, Keap1, NQO-1 and HO-1 were detected using RT-qPCR. The levels of MDA, GSH-PX, SOD, CAT and T-AOC were detected by commercial assay kits. Flow cytometry was used to determine the level of intracellular ROS, and immunofluorescence was used to detect the nuclear localization of Nrf2 protein. Briefly, SeMet downregulated the phosphorylation levels of IκBα and p65 proteins and the gene expression levels of IL-1ß, IL-6, IL-8 and TNF-α were also decreased. Moreover, the protein and gene expression levels of Nrf2, NQO-1 and HO-1 were upregulated, and the nuclear expression of Nrf2 protein was also promoted, which enhanced the activity of antioxidant enzymes. In conclusion, SeMet protected BMECs from inflammatory injury and oxidative stress induced by K. pneumoniae by inhibiting the NF-κB and activating the Nrf2 signaling pathway.

CRISPR/Cas9 Technique for Temperature, Drought, and Salinity Stress Responses.

Global warming and climate change have severely affected plant growth and food production. Therefore, minimizing these effects is required for sustainable crop yields. Understanding the molecular mechanisms in response to abiotic stresses and improving agricultural traits to make crops tolerant to abiotic stresses have been going on unceasingly. To generate desirable varieties of crops, traditional and molecular breeding techniques have been tried, but both approaches are time-consuming. Clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) and transcription activator-like effector nucleases (TALENs) are genome-editing technologies that have recently attracted the attention of plant breeders for genetic modification. These technologies are powerful tools in the basic and applied sciences for understanding gene function, as well as in the field of crop breeding. In this review, we focus on the application of genome-editing systems in plants to understand gene function in response to abiotic stresses and to improve tolerance to abiotic stresses, such as temperature, drought, and salinity stresses.

Multivariate analysis for organic milk authentication.

To differentiate organic milk (OM) from conventional milk (CM), an orthogonal projection to latent structure-discriminant analysis (OPLS-DA) model was constructed using δ13C, δ15N, δ18O, 51 elements and 35 fatty acids (FAs) as the variables. So far, most reported studies barely use three or more types of variables, but more variables could avoid one-sidedness and get stabler models. Our multivariate model combines geographical and nutritional parameters and displays better explanatory and predictive abilities (R2X = 0.647, R2Y = 0.962 and Q2 = 0.821) than models based on fewer variables for differentiating OM and CM. In particular, δ15N, Se, δ13C, Eu, K and α-Linolenic acid (ALA) are found to be critical parameters for the discrimination of OM. These results show that the multivariate model based on stable isotopes, elements and FAs can be used to identify OM, and can potentially expand the global databases for quality and authenticity of milk.

Correction: Determining the geographical origin of milk by multivariate analysis based on stable isotope ratios, elements and fatty acids.

Correction for 'Determining the geographical origin of milk by multivariate analysis based on stable isotope ratios, elements and fatty acids' by Siyan Xu et al., Anal. Methods, 2021, DOI: .

Determining the geographical origin of milk by multivariate analysis based on stable isotope ratios, elements and fatty acids.

To construct a reliable discrimination model for determining milk geographical origin, stable isotope ratios including δ13C, δ15N and δ18O, 51 elements and 35 fatty acids (FAs) in milk samples from Australia, New Zealand and Austria were detected and analyzed. It is found that all of the stable isotope ratios in the milk samples of Australia are the highest, followed by those of the samples from New Zealand and Austria. In addition, 14 elements and 8 FAs show different contents in the samples of different countries at the significance level of P < 0.05. Based on these results, a multivariate model with good robustness and predictive ability for authenticating milk origin (R2X = 0.693, Q2 = 0.854) was successfully constructed. Element contents and stable isotope ratios are more reliable variables for milk origin discrimination and Rb, δ18O, Tl, Ba, Mo, Sr, δ15N, Cs, As, Eu, C20:4n6, Sc, C13:0, K, Ca and C16:1n7 are the critical markers in the multivariate model for verifying milk origin.

Inhibition of IL1ß by Canakinumab May Be Effective against Diverse Molecular Subtypes of Lung Cancer: An Exploratory Analysis of the CANTOS Trial.

In the Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS), inhibition of the IL1ß inflammatory pathway by canakinumab has been shown to significantly reduce lung cancer incidence and mortality. Here we performed molecular characterization of CANTOS patients who developed lung cancer during the study, including circulating tumor DNA (ctDNA) and soluble inflammatory biomarker analysis. Catalogue of Somatic Mutations in Cancer (COSMIC) database ctDNA mutations were detected in 65% (46/71) of the CANTOS patients with lung cancer, with 51% (36/71) having detectable ctDNA at the time point closest to lung cancer diagnosis and 43% (29/67) having detectable ctDNA at trial randomization. Mutations commonly found in lung cancer were observed with no evidence of enrichment in any mutation following canakinumab treatment. Median time to lung cancer diagnosis in patients with (n = 29) versus without (n = 38) detectable COSMIC ctDNA mutations at baseline was 407 days versus 837 days (P = 0.011). For serum inflammatory biomarker analysis, circulating levels of C-reactive protein (CRP), IL6, IL18, IL1 receptor antagonist, TNFα, leptin, adiponectin, fibrinogen, and plasminogen activator inhibitor-1 were determined. Patients with the highest level of baseline CRP or IL6, both downstream of IL1ß signaling, trended toward a shorter time to lung cancer diagnosis. Other inflammation markers outside of the IL1ß pathway at baseline did not trend with time to lung cancer diagnosis. These results provide further evidence for the importance of IL1ß-mediated protumor inflammation in lung cancer and suggest canakinumab's effect may be mediated in part by delaying disease progression of diverse molecular subtypes of lung cancer. SIGNIFICANCE: These findings suggest that targeting the IL1ß inflammatory pathway might be critical in reducing tumor-promoting inflammation and lung cancer incidence.

Outcomes after primary open or endoscopic abductor tendon repair in the hip: a systematic review of the literature.

PURPOSE: The purpose of this study was to systematically appraise the evidence on primary open and endoscopic abductor tendon repair. METHODS: A systematic review of the literature was performed to (1) identify the demographic undergoing abductor tendon repair, (2) summarize the overall outcomes after primary surgical abductor tendon repair, (3) identify the type of tear most commonly encountered intraoperatively, (4) summarize the repair methods used, and (5) identify the published complication and tendon retear rates. RESULTS: A total of 8 articles were identified as eligible for inclusion. All studies were Level IV Evidence. Of the patients undergoing surgical repair, 90% were women. As assessed by a variety of outcome measures, most patients reported good to excellent functional outcomes and pain reduction after open or endoscopic repair. Intraoperatively, tears of the gluteus medius and partial-thickness tears were encountered most often. Tears involving both the gluteus medius and minimus occurred 29% of the time. Complication rates were low for both the open and endoscopic approaches. No tendon retears were documented after endoscopic repair, whereas the retear rate after open repair was 9%. CONCLUSIONS: Patients undergoing surgical repair for partial- and full-thickness tears are mostly women. Intraoperatively, tears almost always include the gluteus medius, with concomitant tearing of the gluteus minimus in approximately one-third of cases. Both open and endoscopic techniques are viable surgical approaches to repairing abductor tendon tears in the hip that produce good to excellent functional results and reduce pain; however, endoscopic repair appears to result in fewer postoperative complications including tendon retear. LEVEL OF EVIDENCE: Level IV, systematic review of Level IV studies.

Multiplicity adjustments in testing for bioequivalence.

Bioequivalence of two drugs is usually demonstrated by rejecting two one-sided null hypotheses using the two one-sided tests for pharmacokinetic parameters: area under the concentration-time curve (AUC) and maximum concentration (Cmax). By virtue of the intersection-union test, there is no need for multiplicity adjustment in testing the two one-sided null hypotheses within each parameter. However, the decision rule for bioequivalence often requires equivalence to be achieved simultaneously on both parameters that contain four one-sided null hypotheses together; without adjusting for multiplicity, the family wise error rate (FWER) could fail to be controlled at the nominal type-I error rate α. The multiplicity issue for bioequivalence in this regard is scarcely discussed in the literature. To address this issue, we propose two approaches including a closed test procedure that controls FWER for the simultaneous AUC and Cmax bioequivalence and requires no adjustment of the type-I error, and an alpha-adaptive sequential testing (AAST) that controls FWER by pre-specifying the significance level on AUC (α1) and obtaining it for Cmax (α2) adaptively after testing of AUC. While both methods control FWER, the closed test requires testing of eight intersection null hypotheses each at α, and AAST is at times accomplished through a slight deduction in α1 and no deduction in α2 relative to α. The latter considers equivalence reached in AUC a higher importance than that in Cmax. Illustrated with published data, the two approaches, although operate differently, can lead to the same substantive conclusion and are better than a traditional method like Bonferroni adjustment.

Inference of equivalence for the ratio of two normal means with unspecified variances.

Equivalence trials aim to demonstrate that new and standard treatments are equivalent within predefined clinically relevant limits. We focus on when inference of equivalence is made in terms of the ratio of two normal means. In the presence of unspecified variances, methods such as the likelihood-ratio test use sample estimates for those variances; Bayesian models integrate them out in the posterior distribution. These methods limit the knowledge on the extent to which equivalence is affected by variability of the parameter of interest. In this article, we propose a likelihood approach that retains the unspecified variances in the model and partitions the likelihood function into two components: F-statistic function for variances, and t-statistic function for the ratio of two means. By incorporating unspecified variances, the proposed method can help identify a numeric range of variances where equivalence is more likely to be achieved, which cannot be accomplished by current analysis methods. By partitioning the likelihood function into two components, the proposed method provides more inference information than a method that relies solely on one component. Using a published set of real example data, we show that the proposed method produces the same results as the likelihood-ratio test and is comparable to Bayesian analysis in the general case. In a special case where the ratio of two variances is directly proportional to the ratio of two means, the proposed method yields better results in inference about equivalence than either the likelihood-ratio test or the Bayesian method. Using a published set of real example data, the proposed likelihood method is shown to be a better alternative than current analysis methods for equivalence inference.

Covariate effect on constancy assumption in noninferiority clinical trials.

Noninferiority (NI) clinical trials are getting a lot of attention of late due to their direct application in biosimilar studies. Because of the missing placebo arm, NI is an indirect approach to demonstrate efficacy of a test treatment. One of the key assumptions in the NI test is the constancy assumption, that is, that the effect of the reference treatment is the same in current NI trials as in historical superiority trials. However, if a covariate interacts with the treatment arms, then changes in distribution of this covariate will likely result in violation of constancy assumption. In this article, we propose four new NI methods and compare them with two existing methods to evaluate the change of background constancy assumption on the performance of these six methods. To achieve this goal, we study the impact of three elements-(1) strength of covariate, (2) degree of interaction between covariate and treatment, and (3) differences in distribution of the covariate between historical and current trials-on both the type I error rate and power using three different measures of association: difference, log relative risk, and log odds ratio. Based on this research, we recommend using a modified covariate-adjustment fixed margin method.

Inference of bioequivalence for log-normal distributed data with unspecified variances.

Two drugs are bioequivalent if the ratio of a pharmacokinetic (PK) parameter of two products falls within equivalence margins. The distribution of PK parameters is often assumed to be log-normal, therefore bioequivalence (BE) is usually assessed on the difference of logarithmically transformed PK parameters (δ). In the presence of unspecified variances, test procedures such as two one-sided tests (TOST) use sample estimates for those variances; Bayesian models integrate them out in the posterior distribution. These methods limit our knowledge on the extent that inference about BE is affected by the variability of PK parameters. In this paper, we propose a likelihood approach that retains the unspecified variances in the model and partitions the entire likelihood function into two components: F-statistic function for variances and t-statistic function for δ. Demonstrated with published real-life data, the proposed method not only produces results that are same as TOST and comparable with Bayesian method but also helps identify ranges of variances, which could make the determination of BE more achievable. Our findings manifest the advantages of the proposed method in making inference about the extent that BE is affected by the unspecified variances, which cannot be accomplished either by TOST or Bayesian method.

Effect of radiofrequency use on hip arthroscopy irrigation fluid temperature.

PURPOSE: The purpose of this study was to determine operating parameters for joint fluid lavage using radiofrequency (RF) in maintaining intra-articular temperatures ≤50°C in the hip joint and to then quantify the influence of flow rate on maintaining safe intra-articular temperatures. METHODS: Fiberoptic intra-articular thermometers at radial distance intervals of 1, 2, 5, and 10 mm, spanning cross-sectional areas of 3.14, 12.56, 78.5, and 314.1 mm(2), respectively, from the RF probe were used in 3 human hip cadaveric specimens at room temperature, with 9 trials per variable, using a 3-portal technique with a capsule release. Using a new Dyonics RF System continuously at the superior capsulolabral junction for 90 seconds, continuous temperatures were recorded at 50 mm Hg inflow and variable outflow intervals: no flow and 5, 15, and 30 seconds. Lavages were 1 second in duration on suction. Statistical comparison was through multivariate regression analysis and a logistic model. RESULTS: Temperatures reached ≥50°C at 5-mm radial distance from the probe in all but the 5-second pulse lavage group. Elevated temperatures were reached within 1 to 2 seconds locally (1- to 2-mm radial distance) and at a radial distance of 5 mm in the 15-, 30-, and 0-second lavage groups. Logistic regression revealed a reduction in the odds that temperatures ≥50°C will occur as flow frequency increases every 30 (odds ratio = 0.68, P = .086); 15 (odds ratio = -1.22, P = .0067); and 5 (odds ratio = -4.26, P < .0001) seconds. CONCLUSIONS: Increasing-interval pulsed irrigation is effective in maintaining intra-articular temperature profiles below 50°C during use of continuous RF ablation. Five-second-interval pulsed lavage is the longest flow interval identified during which fluid 5 mm radially from the RF device never reached temperatures >50°C. CLINICAL RELEVANCE: Clinical guidelines for using the RF ablation include: meticulous technique, intermittent use, good inflow and outflow, and pulsed lavage at frequent intervals.