Pursuing Impactful Quantitative Proteomics Using QC-Channels in Every Spectrum and Trend-Design in Experiment.

False changes discovered by quantitative proteomics reduce the trust of biologists in proteomics and limit the applications of proteomics to unlock biological mechanisms, which suppresses the application of proteomics techniques in the pharmaceutical industry more than it does in academic research. To remove false changes that arise during LC-MS/MS data acquisition, we evaluated the contributions of peptide abundance and number of unique peptides on reproducibility. Lower abundance and only one unique peptide have a higher risk of generating a higher coefficient of variation (CV), resulting in less accurate quantification. However, the abundance of peptides in samples is not adjustable and discarding proteins quantified by only one unique peptide is not a choice either. Indeed, a large percentage of proteins are accurately quantified by only one unique peptide. Therefore, to improve the calculations of the CV, we leverage a new function in PEAKS called QC-channels which enables technical replicates of each spectrum to be evaluated prior to calculation of the CV. While the QC-channels function in PEAKS significantly reduced the false quantification, random false changes still exist due to known or unknown reasons. To address this challenge, we present the idea of Trend-design to track trend changes rather than changes from two points to remove false quantifications and reveal consequential changes responding to a treatment or condition. The idea was confirmed by molecules with different affinity and dose in the current study. The combination of QC-channels and Trend-design enables a more impactful quantitative proteomics to allow unlocking biological mechanisms using proteomics.

Establishment and comparison of piecewise linear regression models to measure thyroid volume by 2D and 3D ultrasound.

OBJECTIVE: Compared thyroid volumes measured by 2-D and 3-D US with those of resected specimens and proposed new models to improve measurement accuracy. METHODS: This study included 80 patients who underwent total thyroidectomy. One 2D_model and one 3D_model were developed using piecewise linear regression analysis. The accuracy of these models was compared using an ellipsoid model (2-D_US value × 0.5), 3-D_US value, and Ying's model [1.76 + (2-D_US value × 0.38)]. RESULTS: The new 2D_model was: V=2.66 + (0.71 * X1) - (1.51 * X2). In this model, if 2-D_US value <= 228.39, X1 = 2-D_US value and X2 = 0; otherwise, X1 = 2-D_US value and X2 = 2-D_US value - 228.39. The 3D_model was: V= 2.90 + (1.08 * X1) + (2.43 * X2). In this model, if 3-D_US value <= 102.06, X1 = 3-D_US value and X2 = 0; otherwise, X1 = 3-D_US value and X2 = 3-D_US value - 102.06. The accuracy of the new models was higher than that of the 3-D_US value, the ellipsoid model, and Ying's model (P<0.05). CONCLUSION: The models established are more accurate than the traditional ones and can accurately measure thyroid volume.

Processing negative emotion in two languages of bilinguals: Accommodation and assimilation of the neural pathways based on a meta-analysis.

Numerous functional magnetic resonance imaging (fMRI) studies have examined the neural mechanisms of negative emotional words, but scarce evidence is available for the interactions among related brain regions from the functional brain connectivity perspective. Moreover, few studies have addressed the neural networks for negative word processing in bilinguals. To fill this gap, the current study examined the brain networks for processing negative words in the first language (L1) and the second language (L2) with Chinese-English bilinguals. To identify objective indicators associated with negative word processing, we first conducted a coordinate-based meta-analysis on contrasts between negative and neutral words (including 32 contrasts from 1589 participants) using the activation likelihood estimation method. Results showed that the left medial prefrontal cortex (mPFC), the left inferior frontal gyrus (IFG), the left posterior cingulate cortex (PCC), the left amygdala, the left inferior temporal gyrus (ITG), and the left thalamus were involved in processing negative words. Next, these six clusters were used as regions of interest in effective connectivity analyses using extended unified structural equation modeling to pinpoint the brain networks for bilingual negative word processing. Brain network results revealed two pathways for negative word processing in L1: a dorsal pathway consisting of the left IFG, the left mPFC, and the left PCC, and a ventral pathway involving the left amygdala, the left ITG, and the left thalamus. We further investigated the similarity and difference between brain networks for negative word processing in L1 and L2. The findings revealed similarities in the dorsal pathway, as well as differences primarily in the ventral pathway, indicating both neural assimilation and accommodation across processing negative emotion in two languages of bilinguals.

Construction and Validation of a Predictive Nomogram Based on Ultrasound for Lymph Node Metastasis of Papillary Thyroid Carcinoma in the Cervical Central Region.

ABSTRACT: To establish and validate a nomogram for predicting lymph node metastasis (LNM) of papillary thyroid carcinoma (PTC) in the cervical central region. This retrospective study included 287 PTC patients with 309 nodules treated from December 2018 to May 2020 at our hospital. The cohort was divided randomly into a training set and a testing set according to a 7:3 ratio. The training set contained 216 nodules, and the testing set contained 93 nodules. The nomogram was developed using the training set, and the data of the testing set were used to validate the performance of nomogram. The predictive accuracy and discriminative ability of the nomogram were determined by concordance index (C-index) and calibration curve. The study showed multifocality, thyroid lesion size, and American College of Radiology Thyroid Imaging, Reporting and Data System (TI-RADS) score were significantly independently associated with LNM in the cervical central region. In the testing set, the calibration curve showed that the nomogram had good discrimination with a C-index of 0.775 (95% confidence interval, 0.680-0.869) and adequate calibration ( P = 0.808). By decision curve analysis and clinical impact curve analysis, the nomogram was shown to have a satisfactory net benefit between thresholds of 0.40 and 0.75. The nomogram can be used for predicting LNM of PTC in the cervical central region and may provide valuable guidance for planning the surgical treatment of PTC patients.

Transcriptomics and quantitative proteomics reveal changes after second stimulation of bone marrow-derived macrophages from lupus-prone MRL/lpr mice.

Innate immune memory can cause the occurrence and exacerbation of autoimmune diseases, and it is as well as being strongly associated with the pathogenesis of systemic lupus erythematosus (SLE), however, the specific mechanism remains to be further studied. We learned that IFN-γ stimulation generated innate immune memory in bone marrow-derived macrophages (BMDMs) and activated memory interferon-stimulated genes (ISGs). This research used IFN-γ and lipopolysaccharide (LPS) to treat BMDMs with lupus-prone MRL/lpr mice and showed that particular memory ISGs were substantially elevated in prestimulated macrophages. In order to identify the differentially expressed genes (DEGs), researchers turned to RNA-seq. GO and KEGG analysis showed that up-regulated DEGs were enriched in defense and innate immune responses, and were related to the expression of pattern recognition receptors (PRRs)-related pathways in macrophages. TMT-based proteome analysis revealed differentially expressed proteins (DEPs) up-regulated in BMDMs were abundant in metabolic pathways such as glucose metabolism. Our study found that after the secondary stimulation of MRL/lpr mice, the expression of PRRs in innate immune cells was changed, and IFN-related pathways were activated to release a large number of ISGs to promote the secondary response. At the same time, related metabolic modes such as glycolysis were enhanced, and epigenetic changes may occur. Therefore, SLE is brought on, maintained, and worsened by a variety of factors that work together to produce innate immune memory.

A predictive model for first-trimester pregnancy inception after IVF-ET based on multimodal ultrasound evaluation of endometrial receptivity.

BACKGROUND: In-vitro fertilization-embryo transfer (IVF-ET) is a commonly used assisted reproductive technology. Its success depends on many factors, including endometrial receptivity. Endometrial receptivity can be evaluated by ultrasound, endometrial biopsy, and magnetic resonance imaging. Compared with the latter two methods, ultrasound has the advantages of wide availability, non-invasiveness, and low cost. Three-dimensional (3D) ultrasound imaging examines endometrial thickness, morphology, and blood vessels, which are associated with the success of embryo implantation. However, there are no reports of endometrial receptivity assessment by 3D ultrasound. Therefore, we aimed to evaluate endometrial receptivity using 3D ultrasound and construct a predictive model for first-trimester pregnancy inception following IVF-ET. METHODS: We performed a prospective observational study on infertile women who underwent IVF-ET between December 2019 and February 2021. These women had 3D ultrasound evaluations, measuring endometrial thickness, volume, pattern, morphology, peristalsis, uterine artery blood flow index, sub-endometrial blood flow index, and distribution pattern. We recorded the occurrence of first-trimester pregnancies in these women. Using Akaike information criterion (AIC) and backward stepwise regression, a first-trimester pregnancy prediction model was constructed based on the minimum AIC value and validated internally and externally. RESULTS: 111 women were enrolled, with 103 included in the analysis. Univariate and multiple logistic regression analyses showed that endometrial thickness and vascularization flow index (VFI) were independent factors associated with the occurrence of a pregnancy. The final prediction model corresponding to the minimum AIC value (65.166) was Y = - 6.131-0.182endometrial thickness + 0.542endometrial volume + 4.374VFI + 0.132age. In the test set, modeling cohort, and external validation cohort, the model showed satisfactory differentiation, with C index of 0.841 (95%CI 0.699-0.817), 0.727 (95%CI 0.619-0.815), and 0.745 (95%CI 0.671-0.840), respectively. The Hosmer-Lemeshow goodness of fit tests reported P = 0.865, 0.139, and 0.070, respectively, indicating a high agreement with the actual IVF-ET outcome. This model reached the highest diagnostic efficiency (sensitivity 88.9%, specificity 75%, Youden index 0.639) at a diagnostic cut-off value of ≥ 0.360. CONCLUSIONS: The predictive model based on endometrial receptivity evaluations by 3D ultrasound had high diagnostic efficiency and could be a simple and effective tool to predict first-trimester pregnancy inception after IVF-ET.

[Establishment of a predictive model for central cervical lymph node metastasis of papillary thyroid carcinoma based on ACR TI-RADS score and evaluation of its diagnostic efficacy].

Objective:To establish a predictive model for central lymph node metastasis（CLNM） of papillary thyroid carcinoma（PTC） based on ACR TI-RADS grades（ATR model） and evaluate its diagnostic efficacy. Methods:A total of 319 patients with PTC diagnosed from January 2019 to May 2020 were included, including 366 nodules were used as the modeling cohort to construct the risk prediction model. A total of 105 PTC patients with 121 nodules from June to August 2020 were included as the external validation cohort. The C-index of the model was calculated and the Hosmer-Lemeshow goodness-of-fit test was performed to compare the diagnostic efficiency of ACR model and those conventional imaging models. Results:The ATR model, Y=-3.719+0.765×gender+1.094×multifocality+0.08×maximum diameter+0.266×ACR TI-RADS score. In the training set, validation set and external validation cohort, the model C-index was 0.758（95%CI: 0.699-0.817）, 0.717（95%CI: 0.619-0.815） and 0.756（95%CI: 0.671-0.840）, respectively. The Hosmer-Lemeshow goodness of fit test showed that the prediction rate of the model was consistent with the actual incidence rate（P=0.918; P=0.581; P=0.366）. With ≥0.434 as the diagnostic threshold, the model had the highest diagnostic efficacy （sensitivity: 86.0%, specificity: 56.3%, Youden index: 0.423）. In the external validation cohort, there was no significant difference between C-US and CT（P>0.05）. Compared with C-US and CT, the sensitivity（66.1% vs 16.1%, P<0.001; 66.1% vs 9.7%, P<0.001） and accuracy（68.6% vs 55.4%, P=0.041; 68.6% vs 52.9%, P=0.012） of ATR model were higher, and the negative predictive value was higher than that of CT（66.7% vs 50.9%, P=0.042）, but there was no difference between ATR model and C-US（66.7% vs 52.3%, P=0.066）; There was no significant difference among the three positive predictive values（70.7% vs 83.3%, P=0.211; 70.7% vs 85.7%, P=0.319）, but the specificity of the model was low （71.2% vs 96.6%, P=0.001; 71.2% vs 98.3%, P<0.001）. Conclusion:The predictive model based on ACR TI-RADS grades can predict CLNM of PTC more accurately and sensitively than traditional imaging examination.

Investigating the duality of Inpp4b function in the cellular transformation of mouse fibroblasts.

Inositol Polyphosphate 4-Phosphatase, Type II (INPP4B) is a tumour suppressor in breast, ovarian, prostate, thyroid and other cancers, attributed to its ability to reduce oncogenic Akt-signaling. However, emerging studies show that INPP4B also has tumour-promoting properties in cancers including acute myeloid leukemia, colon cancer, melanoma and breast cancer. Together these findings suggest that INPP4B may be a context dependent cancer gene. Whether INPP4B functions solely in a tumour suppressing or tumour promoting manner, or both in non-transformed cells is currently not clear. In this study, consequences of deficiency and overexpression of INPP4B on cellular transformation was investigated using a mouse embryonic fibroblast (MEF) model of cellular transformation. We observed that neither deficiency nor overexpression of INPP4B was sufficient to induce neoplastic transformation, alone or in combination with H-Ras V12 or E1A overexpression. However, Inpp4b-deficiency did cooperate with SV40 T-Large-mediated cellular transformation, a finding which was associated with increased phosphorylated-Akt levels. Transformation and phosphorylated-Akt levels were dampened upon overexpression of INPP4B in SV40 T-Large-MEF. Together, our findings support a model where INPP4B function suppresses transformation mediated by SV40 T-Large, but is inconsequential for Ras and E1A mediated transformation.

Therapeutic luminal coating of the intestine.

The gastrointestinal tract is the site of most drug delivery and therapeutic interventions for the management and treatment of numerous diseases. However, selective access to its mucosa, especially in the small bowel, is challenging. Here we develop an orally administered gut-coating formulation that provides a transient coating of the bowel. Through a materials screening campaign, we identified a sucrose octasulfate aluminium complex and further engineered the pH-dependent material into a complex coacervate formulation linked via pH-independent electrostatic interaction, which allowed an effective transient physical coating on the gastrointestinal mucosa, independent of gastric acid exposure. We tested the therapeutic values of this technology in two settings. Oral administration of this gut-coating formulation modulated the nutrient contact with bowel mucosa, which lowered the glucose responses in rodent models indicating a potential therapeutic utility in diabetes. Furthermore, the formulation protected biological agents from gastric acid exposure and degradation, which enabled oral delivery to the small bowel mucosa.

Discovery of LY3104607: A Potent and Selective G Protein-Coupled Receptor 40 (GPR40) Agonist with Optimized Pharmacokinetic Properties to Support Once Daily Oral Treatment in Patients with Type 2 Diabetes Mellitus.

As a part of our program to identify potent GPR40 agonists capable of being dosed orally once daily in humans, we incorporated fused heterocycles into our recently disclosed spiropiperidine and tetrahydroquinoline acid derivatives 1, 2, and 3 with the intention of lowering clearance and improving the maximum absorbable dose (Dabs). Hypothesis-driven structural modifications focused on moving away from the zwitterion-like structure. and mitigating the N-dealkylation and O-dealkylation issues led to triazolopyridine acid derivatives with unique pharmacology and superior pharmacokinetic properties. Compound 4 (LY3104607) demonstrated functional potency and glucose-dependent insulin secretion (GDIS) in primary islets from rats. Potent, efficacious, and durable dose-dependent reductions in glucose levels were seen during glucose tolerance test (GTT) studies. Low clearance, volume of distribution, and high oral bioavailability were observed in all species. The combination of enhanced pharmacology and pharmacokinetic properties supported further development of this compound as a potential glucose-lowering drug candidate.

The Discovery, Preclinical, and Early Clinical Development of Potent and Selective GPR40 Agonists for the Treatment of Type 2 Diabetes Mellitus (LY2881835, LY2922083, and LY2922470).

The G protein-coupled receptor 40 (GPR40) also known as free fatty acid receptor 1 (FFAR1) is highly expressed in pancreatic, islet ß-cells and responds to endogenous fatty acids, resulting in amplification of insulin secretion only in the presence of elevated glucose levels. Hypothesis driven structural modifications to endogenous FFAs, focused on breaking planarity and reducing lipophilicity, led to the identification of spiropiperidine and tetrahydroquinoline acid derivatives as GPR40 agonists with unique pharmacology, selectivity, and pharmacokinetic properties. Compounds 1 (LY2881835), 2 (LY2922083), and 3 (LY2922470) demonstrated potent, efficacious, and durable dose-dependent reductions in glucose levels along with significant increases in insulin and GLP-1 secretion during preclinical testing. A clinical study with 3 administered to subjects with T2DM provided proof of concept of 3 as a potential glucose-lowering therapy. This manuscript summarizes the scientific rationale, medicinal chemistry, preclinical, and early development data of this new class of GPR40 agonists.

Molecular targets of androgen signaling that characterize skeletal muscle recovery and regeneration.

The high regenerative capacity of adult skeletal muscle relies on a self-renewing depot of adult stem cells, termed muscle satellite cells (MSCs). Androgens, known mediators of overall body composition and specifically skeletal muscle mass, have been shown to regulate MSCs. The possible overlapping function of androgen regulation of muscle growth and MSC activation has not been carefully investigated with regards to muscle regeneration.Therefore, the aim of this study was to examine coinciding androgen-mediated genetic changes in an in vitro MSC model and clinically relevant in vivo models. A gene signature was established via microarray analysis for androgen-mediated MSC engagement and highlighted several markers including follistatin (FST), IGF-1, C-X-C chemokine receptor 4 (CXCR4), hepatocyte growth factor (HGF) and glucocorticoid receptor (GR). In an in vivo muscle atrophy model, androgen re-supplementation significantly increased muscle size and expression of IGF-1, FST, and HGF, while significantly decreasing expression of GR. Biphasic gene expression profiles over the 7-day re-supplementation period identified temporal androgen regulation of molecular targets involved in satellite cell engagement into myogenesis. In a muscle injury model, removal of androgens resulted in delayed muscle recovery and regeneration. Modifications in the androgen signaling gene signature, along with reduced Pax7 and MyoD expression, suggested that limited MSC activation and increased inflammation contributed to the delayed regeneration. However, enhanced MSC activation in the androgen-deplete mouse injury model was driven by an androgen receptor (AR) agonist. These results provide novel in vitro and in vivo evidence describing molecular targets of androgen signaling, while also increasing support for translational use of AR agonists in skeletal muscle recovery and regeneration.

Benzopyrans are selective estrogen receptor beta agonists with novel activity in models of benign prostatic hyperplasia.

Benzopyran selective estrogen receptor beta agonist-1 (SERBA-1) shows potent, selective binding and agonist function in estrogen receptor beta (ERbeta) in vitro assays. X-ray crystal structures of SERBA-1 in ERalpha and beta help explain observed beta-selectivity of this ligand. SERBA-1 in vivo demonstrates involution of the ventral prostate in CD-1 mice (ERbeta effect), while having no effect on gonadal hormone levels (ERalpha effect) at 10x the efficacious dose, consistent with in vitro properties of this molecule.

The selective estrogen receptor modulator trioxifene (LY133314) inhibits metastasis and extends survival in the PAIII rat prostatic carcinoma model.

Trioxifene (LY133314) is a selective estrogen receptor modulator (SERM) with competitive binding activity against estradiol for estrogen receptor alpha (ERalpha) and antagonistic activity against ERalpha-mediated gene expression. The PAIII rat prostatic adenocarcinoma (PCa) is an androgen receptor-negative, ERalpha- and ERbeta-positive, spontaneously metastatic rodent tumor cell line. After s.c. implantation of 10(6) PAIII cells in the tail, s.c. administration of trioxifene (2.0, 4.0, 20.0, or 40.0 mg/kg-day) for 30 days produced significant (P < 0.05) inhibition of PAIII metastasis from the primary tumor in the tail to the gluteal and iliac lymph nodes (maximum nodal weight decreases, 86% and 88% from control values, respectively). PAIII metastasis to the lungs was significantly inhibited by trioxifene treatment. Numbers of pulmonary foci in PAIII-bearing rats were significantly (P < 0.05) reduced by trioxifene administration in a dose-related manner (maximal reduction, 98% from control values). Continual administration of the compound significantly (P < 0.05) extended survival of PAIII-bearing rats. Trioxifene inhibited the proliferation of PAIII cells at micromolar levels in vitro but did not slow growth of the primary tumor growth in the tail. Trioxifene administration also produced regression of male accessory sex organs. In PAIII-tumor-bearing animals, trioxifene administration produced a maximal regression of 76% for ventral prostate and 64% for seminal vesicle (P < 0.05 for both). SERMs may be preferable to estrogens given their efficacy in experimental PCa models and relative lack of side effects observed in clinical trials. Our data support the contention that trioxifene represents a SERM with potential antimetastatic efficacy for the treatment of androgen-independent, metastatic PCa.