Tamoxifen Exerts Anticancer Effects on Pituitary Adenoma Progression via Inducing Cell Apoptosis and Inhibiting Cell Migration.

Although pituitary adenomas are histologically benign, they are often accompanied by multiple complications, such as cardiovascular disease and metabolic dysfunction. In the present study, we repositioned the Food and Drug Administration -approved immune regulator tamoxifen to target STAT6 based on the genomics analysis of PAs. Tamoxifen inhibited the proliferation of GH3 and AtT-20 cells with respective IC50 values of 9.15 and 7.52 µM and increased their apoptotic rates in a dose-dependent manner. At the molecular level, tamoxifen downregulated phosphorylated PI3K, phosphorylated AKT and the anti-apoptotic protein Bcl-2 and increased the expression of pro-apoptotic proteins p53 and Bax in GH3 and AtT-20 cells. Furthermore, tamoxifen also inhibited the migration of both cell lines by reprogramming tumor-associated macrophages to the M1 phenotype through STAT6 inactivation and inhibition of the macrophage-specific immune checkpoint SHP1/SHP. Finally, administration of tamoxifen (20, 50, 100 mg·kg-1·d-1, for 21 days) inhibited the growth of pituitary adenomas xenografts in nude mice in a dose-dependent manner. Taken together, tamoxifen is likely to be a promising combination therapy for pituitary adenomas and should be investigated further.

Targeting Myadm to Intervene Pulmonary Hypertension on Rats Before Pregnancy Alleviates the Effect on Their Offspring's Cardiac-Cerebral Systems.

Pregnancy with pulmonary hypertension (PH) seriously threatens the life and safety of mothers and infants. Here, the long-term effect of maternal PH on the postpartum growth of rat offspring was focused for the first time, as well as explored the role of Myadm in PH rats before pregnancy based upon the previous findings. Patients with PH are prone to hypoxemia, leading to insufficient placental structure and function, which affects the organ function of fetuses, followed by evidence that differently expressed genes (DEGs) existed in the heart of maternal PH newborn rats and enriched in pathways related to cardiac and nerve development on human infants with similar birth outcome: low birth weight (LBW). LBW was one of the possible birth outcomes of pregnancy with PH, especially severe PH, accompanied by evidence that offspring derived from mothers with PH presented lower birth weights and slower growth rates than those derived from normal control mothers in a rat model. Besides, maternal PH rat offspring showed cardiac remodeling and a significant elevation of the expression levels of hypoxia- and inflammation-related markers in the cerebral cortex at both 10 and 14 weeks of age, respectively. What is more, the previous studies found that the overexpression of Myadm could result in the remodeling of the pulmonary artery. And targeting Myadm to intervene PH before pregnancy could alleviate sustained low weight growth in maternal PH rat offspring, and the pathological changes of the cardiac-cerebral system caused by maternal PH, including enlarged right heart cavity, loss of cardiomyocytes, abnormal heart index, as well as cerebral cortex hypoxia and the inflammatory state as they grew up to a certain extent. The findings show the pathological significance of maternal PH on offspring growth and the cardiac-cerebral development in a rat model, as well as point out the potential treatment target, which may provide a further reference for pregnancy outcomes in women with PH and healthy development of offspring to some extent.

A Novel Co-Crystal of Bexarotene and Ligustrazine Improves Pharmacokinetics and Tissue Distribution of Bexarotene in SD Rats.

Bexarotene (BEX), a specific retinoic acid X receptor (RXR) agonist granted by Food and Drug Administration (FDA) approval for the clinical treatment of T cell lymphoma, has now been found to exert pharmacological effects in the nervous system, with low bioavailability and poor cerebral distribution limiting its application in treatment on neurological disorders. Pharmaceutical co-crystal was a helpful method to improve the bioavailability and tissue distribution of active pharmaceutical ingredients (APIs). Here, 2bexarotene-ligustrazine (2BEX-LIG), a novel co-crystal system of BEX and ligustrazine (LIG) of which with BEX is an API, was constructed with satisfactory stability and enhanced solubility. The pharmacokinetics characteristics of BEX were detected, and the results showed that the absolute bioavailability and the cerebral concentration of BEX in rats administrated with 2BEX-LIG were enhanced from 22.89% to 42.86% and increased by 3.4-fold, respectively, compared with those in rats administrated an equivalent of BEX. Hence, our present study indicated that the novel co-crystal of 2BEX-LIG contributed to improving BEX oral bioavailability and cerebral distribution, thereby providing significant advantages for clinical application of brain tumors and other neurological diseases.

Circular RNAs: Promising Molecular Biomarkers of Human Aging-Related Diseases via Functioning as an miRNA Sponge.

Circular RNAs (circRNAs) are a new class of noncoding single-stranded RNAs that differ from linear microRNAs (miRNAs), since they form covalently closed loop structures without free 3' poly(A) tails or 5' caps. circRNAs are the competitive endogenous RNAs (ceRNAs) by binding to miRNA through miRNA response elements (MREs) (i.e., "miRNA sponge"), thereby reducing the quantity of miRNA available to target mRNA, subsequently promoting mRNA stability or protein expression, which involves the initiation and progress of human diseases. Owing to these features of abundance, stability, conservative property, and tissue and stage specificity, widely distributing in the extracellular space and in various bodily fluids, circRNAs can be considered as potential biomarkers for various diseases. Here, we reviewed the promising circRNAs being disease biomarkers, focused on their regulatory function by acting as miRNA sponges, and described their roles in cancer, cardiovascular or neurodegenerative diseases, osteoarthritis, rheumatoid arthritis, diabetes, and other human aging-related diseases, which provide a new direction for pathogenesis, diagnosis, and treatment of human aging-related diseases.

Tandem gene duplications drive divergent evolution of caffeine and crocin biosynthetic pathways in plants.

BACKGROUND: Plants have evolved a panoply of specialized metabolites that increase their environmental fitness. Two examples are caffeine, a purine psychotropic alkaloid, and crocins, a group of glycosylated apocarotenoid pigments. Both classes of compounds are found in a handful of distantly related plant genera (Coffea, Camellia, Paullinia, and Ilex for caffeine; Crocus, Buddleja, and Gardenia for crocins) wherein they presumably evolved through convergent evolution. The closely related Coffea and Gardenia genera belong to the Rubiaceae family and synthesize, respectively, caffeine and crocins in their fruits. RESULTS: Here, we report a chromosomal-level genome assembly of Gardenia jasminoides, a crocin-producing species, obtained using Oxford Nanopore sequencing and Hi-C technology. Through genomic and functional assays, we completely deciphered for the first time in any plant the dedicated pathway of crocin biosynthesis. Through comparative analyses with Coffea canephora and other eudicot genomes, we show that Coffea caffeine synthases and the first dedicated gene in the Gardenia crocin pathway, GjCCD4a, evolved through recent tandem gene duplications in the two different genera, respectively. In contrast, genes encoding later steps of the Gardenia crocin pathway, ALDH and UGT, evolved through more ancient gene duplications and were presumably recruited into the crocin biosynthetic pathway only after the evolution of the GjCCD4a gene. CONCLUSIONS: This study shows duplication-based divergent evolution within the coffee family (Rubiaceae) of two characteristic secondary metabolic pathways, caffeine and crocin biosynthesis, from a common ancestor that possessed neither complete pathway. These findings provide significant insights on the role of tandem duplications in the evolution of plant specialized metabolism.

miR-182-3p/Myadm contribute to pulmonary artery hypertension vascular remodeling via a KLF4/p21-dependent mechanism.

Rationale: There is a continued need for investigating the roles of microRNAs and their targets on the pathogenesis of pulmonary arterial hypertension (PAH) vascular remodeling. We recently identified the association of myeloid miR-182-3p and its new target, Myeloid-Associated Differentiation Marker (Myadm), with vascular remodeling. Here, we aimed to determine the role of miR-182-3p/Myadm on PAH vascular remodeling and the underlying molecular mechanism. Methods: The miR-182-3p/Myadm expression profiles were detected in PAH patients and experimental rodent models. Loss-of-function and gain-of-function studies using gene knock-in or gene knock-out and the combinations of the proteomic technology and genome-wide ChIP-Seq were employed to determine the downstream targets of miR-182-3p/Myadm in response to monocrotaline (MCT)-induced PAH. Results: The miR-182-3p/Myadm expression was altered in PAH patients and experimental rodent models. Both miR-182-3p inhibitor and overexpression of Myadm augmented the pathological progression in rats in response to MCT-induced PAH. In contrast, miR-182-3p mimic and Myadm gene knockout attenuated the changes in the hemodynamics and structure of the cardio-pulmonary system in MCT-induced PAH in rats. Myadm mediated the proliferation of pulmonary artery smooth muscle cells (PASMCs) by altering the cell cycle kinase inhibitor (p21/Cip1) expression through the transcription factor Krüppel-like factor 4 (KLF4) translocation into the cytoplasm. Conclusion: Our findings indicate the prognostic and therapeutic significance of miR-182-3p in PAH and provide a new regulatory model of the myeloid-derived miR-182-3p/Myadm/KLF4/p21 axis in PAH vascular remodeling.

Genome-Wide Characterization and Analysis of bHLH Transcription Factors Related to Crocin Biosynthesis in Gardenia jasminoides Ellis (Rubiaceae).

Crocins, enriched in Gardenia jasminoides fruits, have a pharmacological activity against central nervous system diseases, cardiovascular diseases, and cancer cell growth. The biosynthesis of crocins has been widely explored, but its regulatory mechanism remains unknown. Here, the basic helix-loop-helix (bHLH) transcription factors related to crocin biosynthesis were systematically identified on the basis of the genome of G. jasminoides. A total of 95 GjbHLH transcription factor genes were identified, and their phylogenetic analysis indicated that they could be classified into 23 subfamilies. The combination of gene-specific bHLH expression patterns, the coexpression analysis of biosynthesis genes, and the analysis of promoter sequences in crocin biosynthesis pathways suggested that nine bHLHs in G. jasminoides might negatively regulate crocin biosynthesis. This study laid a foundation for understanding the regulatory mechanism of crocin biosynthesis and the improvement and breeding of G. jasminoides varieties.

Genome Analysis of the Ancient Tracheophyte Selaginella tamariscina Reveals Evolutionary Features Relevant to the Acquisition of Desiccation Tolerance.

Resurrection plants, which are the "gifts" of natural evolution, are ideal models for studying the genetic basis of plant desiccation tolerance. Here, we report a high-quality genome assembly of 301 Mb for the diploid spike moss Selaginella tamariscina, a primitive vascular resurrection plant. We predicated 27 761 protein-coding genes from the assembled S. tamariscina genome, 11.38% (2363) of which showed significant expression changes in response to desiccation. Approximately 60.58% of the S. tamariscina genome was annotated as repetitive DNA, which is an almost 2-fold increase of that in the genome of desiccation-sensitive Selaginella moellendorffii. Genomic and transcriptomic analyses highlight the unique evolution and complex regulations of the desiccation response in S. tamariscina, including species-specific expansion of the oleosin and pentatricopeptide repeat gene families, unique genes and pathways for reactive oxygen species generation and scavenging, and enhanced abscisic acid (ABA) biosynthesis and potentially distinct regulation of ABA signaling and response. Comparative analysis of chloroplast genomes of several Selaginella species revealed a unique structural rearrangement and the complete loss of chloroplast NAD(P)H dehydrogenase (NDH) genes in S. tamariscina, suggesting a link between the absence of the NDH complex and desiccation tolerance. Taken together, our comparative genomic and transcriptomic analyses reveal common and species-specific desiccation tolerance strategies in S. tamariscina, providing significant insights into the desiccation tolerance mechanism and the evolution of resurrection plants.

Effects of calibration approaches on the accuracy for LC-MS targeted quantification of therapeutic protein.

LC-MS provides a promising alternative to ligand-binding assays for quantification of therapeutic proteins and biomarkers. As LC-MS methodology is based on the analysis of proteolytic peptides, calibration approaches utilizing various calibrators and internal standards (I.S.) have been developed. A comprehensive assessment of the accuracy and reliability of these approaches is essential but has yet been reported. Here we performed a well-controlled and systematic comparative study using quantification of monoclonal-antibody in plasma as the model system. Method development utilized a high-throughput orthogonal-array-optimization, and two sensitive and stable signature-peptides (SP) from different domains were selected based on extensive evaluations in plasma matrix. With the purities of all protein/peptide standards corrected by quantitative amino acid analysis (AAA), five calibration approaches using stable-isotope-labeled (SIL) I.S. were thoroughly compared, including those at peptide, extended-peptide, and protein levels and two "hybrid" approaches (i.e., protein calibrator with SIL-peptide or SIL-extended-peptide I.S.). These approaches were further evaluated in parallel for a 15 time point, preclinical pharmacokinetic study. All methods showed good precision (CV% < 20%). When examined with protein-spiked plasma QC, peptide-level calibration exhibited severe negative biases (-23 to -62%), highly discordant results between the two SP (deviations of 38-56%), and misleading pharmacokinetics assessments. Extended-peptide calibration showed significant improvements but still with unacceptable accuracy. Conversely, protein-level and the two hybrid calibrations achieved good quantitative accuracy (error < 10%), concordant results by two SP (deviations < 15%), and correct pharmacokinetic parameters. Hybrid approaches were found to provide a cost-effective means for accurate quantification without the costly SIL-protein. Other key findings include (i) using two SP provides a versatile gauge for method reliability; (ii) evaluation of peptide stability in the matrix before SP selection is critical; and (iii) using AAA to verify purities of protein/peptide calibrators ensures accurate quantitation. These results address fundamental calibration issues that have not been adequately investigated in published studies and will provide valuable guidelines for the "fit for purpose" development of accurate LC-MS assays for therapeutic proteins and biomarkers in biological matrices.

Environmental factors associated with disease progression after the first demyelinating event: results from the multi-center SET study.

OBJECTIVES: To investigate the associations of environmental MS risk factors with clinical and MRI measures of progression in high-risk clinically isolated syndromes (CIS) after the first demyelinating event. METHODS: We analyzed 211 CIS patients (age: 28.9±7.8 years) enrolled in the SET study, a multi-center study of high-risk CIS patients. Pre-treatment samples were analyzed for IgG antibodies against cytomegalovirus (anti-CMV), Epstein Barr virus (EBV) early nuclear antigen-1 (EBNA-1), viral capsid antigen (VCA), early antigen-diffuse (EA-D), 25 hydroxy-vitamin D3 and cotinine levels and HLA DRB1*1501 status. The inclusion criteria required evaluation within 4 months of the initial demyelinating event, 2 or more brain MRI lesions and the presence of two or more oligoclonal bands in cerebrospinal fluid. All patients were treated with interferon-beta. Clinical and MRI assessments were obtained at baseline, 6, 12, and 24 months. RESULTS: The time to first relapse decreased and the number of relapses increased with anti-CMV IgG positivity. Smoking was associated with increased number and volume of contrast-enhancing lesions (CEL) during the 2-year period. The cumulative number of CEL and T2 lesions during the 2-year period was greater for individuals in the highest quartile of anti-EBV VCA IgG antibodies. The percent loss of brain volume was increased for those in the highest quartile of with anti-EBV VCA IgG antibodies. CONCLUSIONS: Relapses in CIS patients were associated with CMV positivity whereas anti-EBV VCA positivity was associated with progression on MRI measures, including accumulation of CEL and T2 lesions and development of brain atrophy.

A rapid, reproducible, on-the-fly orthogonal array optimization method for targeted protein quantification by LC/MS and its application for accurate and sensitive quantification of carbonyl reductases in human liver.

Liquid chromatography (LC)/mass spectrometry (MS) in selected-reactions-monitoring (SRM) mode provides a powerful tool for targeted protein quantification. However, efficient, high-throughput strategies for proper selection of signature peptides (SP) for protein quantification and accurate optimization of their SRM conditions remain elusive. Here we describe an on-the-fly, orthogonal array optimization (OAO) approach that enables rapid, comprehensive, and reproducible SRM optimization of a large number of candidate peptides in a single nanoflow-LC/MS run. With the optimized conditions, many peptide candidates can be evaluated in biological matrixes for selection of the final SP. The OAO strategy employs a systematic experimental design that strategically varies product ions, declustering energy, and collision energy in a cycle of 25 consecutive SRM trials, which accurately reveals the effects of these factors on the signal-to-noise ratio of a candidate peptide and optimizes each. As proof of concept, we developed a highly sensitive, accurate, and reproducible method for the quantification of carbonyl reductases CBR1 and CBR3 in human liver. Candidate peptides were identified by nano-LC/LTQ/Orbitrap, filtered using a stringent set of criteria, and subjected to OAO. After evaluating both sensitivity and stability of the candidates, two SP were selected for quantification of each protein. As a result of the accurate OAO of assay conditions, sensitivities of 80 and 110 amol were achieved for CBR1 and CBR3, respectively. The method was validated and used to quantify the CBRs in 33 human liver samples. The mean level of CBR1 was 93.4 +/- 49.7 (range: 26.2-241) ppm of total protein, and of CBR3 was 7.69 +/- 4.38 (range: 1.26-17.9) ppm. Key observations of this study: (i) evaluation of peptide stability in the target matrix is essential for final selection of the SP; (ii) utilization of two unique SP contributes to high reliability of target protein quantification; (iii) it is beneficial to construct calibration curves using standard proteins of verified concentrations to avoid severe biases that may result if synthesized peptides alone are used. Overall, the OAO method is versatile and adaptable to high-throughput quantification of validated biomarkers identified by proteomic discovery experiments.

A straightforward and highly efficient precipitation/on-pellet digestion procedure coupled with a long gradient nano-LC separation and Orbitrap mass spectrometry for label-free expression profiling of the swine heart mitochondrial proteome.

For label-free expression profiling of tissue proteomes, efficient protein extraction, thorough and quantitative sample cleanup and digestion procedures, as well as sufficient and reproducible chromatographic separation, are highly desirable but remain challenging. However, optimal methodology has remained elusive, especially for proteomes that are rich in membrane proteins, such as the mitochondria. Here, we describe a straightforward and reproducible sample preparation procedure, coupled with a highly selective and sensitive nano-LC/Orbitrap analysis, which enables reliable and comprehensive expression profiling of tissue mitochondria. The mitochondrial proteome of swine heart was selected as a test system. Efficient protein extraction was accomplished using a strong buffer containing both ionic and nonionic detergents. Overnight precipitation was used for cleanup of the extract, and the sample was subjected to an optimized 2-step, on-pellet digestion approach. In the first step, the protein pellet was dissolved via a 4 h tryptic digestion under vigorous agitation, which nano-LC/LTQ/ETD showed to produce large and incompletely cleaved tryptic peptides. The mixture was then reduced, alkylated, and digested into its full complement of tryptic peptides with additional trypsin. This solvent precipitation/on-pellet digestion procedure achieved significantly higher and more reproducible peptide recovery of the mitochondrial preparation than observed using a prevalent alternative procedure for label-free expression profiling, SDS-PAGE/in-gel digestion (87% vs 54%). Furthermore, uneven peptide losses were lower than observed with SDS-PAGE/in-gel digestion. The resulting peptides were sufficiently resolved by a 5 h gradient using a nano-LC configuration that features a low-void-volume, high chromatographic reproducibility, and an LTQ/Orbitrap analyzer for protein identification and quantification. The developed method was employed for label-free comparison of the mitochondrial proteomes of myocardium from healthy animals versus those with hibernating myocardium. Each experimental group consisted of a relatively large number of animals (n = 10), and samples were analyzed in random order to minimize quantitative false-positives. With this approach, 904 proteins were identified and quantified with high confidence, and those mitochondrial proteins that were altered significantly between groups were compared with the results of a parallel 2D-DIGE analysis. The sample preparation and analytical strategy developed here represents an advancement that can be adapted to analyze other tissue proteomes.

Accurate localization and relative quantification of arginine methylation using nanoflow liquid chromatography coupled to electron transfer dissociation and orbitrap mass spectrometry.

Protein arginine (Arg) methylation serves an important functional role in eucaryotic cells, and typically occurs in domains consisting of multiple Arg in close proximity. Localization of methylarginine (MA) within Arg-rich domains poses a challenge for mass spectrometry (MS)-based methods; the peptides are highly charged under electrospray ionization (ESI), which limits the number of sequence-informative products produced by collision induced dissociation (CID), and loss of the labile methylation moieties during CID precludes effective fragmentation of the peptide backbone. Here the fragmentation behavior of Arg-rich peptides was investigated comprehensively using electron-transfer dissociation (ETD) and CID for both methylated and unmodified glycine-/Arg-rich peptides (GAR), derived from residues 679-695 of human nucleolin, which contains methylation motifs that are widely-represented in biological systems. ETD produced abundant information for sequencing and MA localization, whereas CID failed to provide credible identification for any available charge state (z = 2-4). Nevertheless, CID produced characteristic neutral losses that can be employed to distinguish among different types of MA, as suggested by previous works and confirmed here with product ion scans of high accuracy/resolution by an LTQ/Orbitrap. To analyze MA-peptides in relatively complex mixtures, a method was developed that employs nano-LC coupled to alternating CID/ETD for peptide sequencing and MA localization/characterization, and an Orbitrap for accurate precursor measurement and relative quantification of MA-peptide stoichiometries. As proof of concept, GAR-peptides methylated in vitro by protein arginine N-methyltransferases PRMT1 and PRMT7 were analyzed. It was observed that PRMT1 generated a number of monomethylated (MMA) and asymmetric-dimethylated peptides, while PRMT7 produced predominantly MMA peptides and some symmetric-dimethylated peptides. This approach and the results may advance understanding of the actions of PRMTs and the functional significance of Arg methylation patterns.

Ultra-sensitive quantification of paclitaxel using selective solid-phase extraction in conjunction with reversed-phase capillary liquid chromatography/tandem mass spectrometry.

The ability to quantify ultra-low concentrations of biologically active compounds in biological matrices is essential for the study of pharmacological/toxicological effects occurring at low doses. Selective solid-phase extraction (SPE) was combined with highly sensitive capillary LC (microLC)-MS/MS analysis to achieve ultra-sensitive quantification of the anti-cancer drug paclitaxel in cancer cells. The optimized SPE selectively extracted paclitaxel and eliminated undesirable matrix compounds, thus enabling a high sample loading volume on the microLC column without compromising chromatographic performance and operational robustness. The validated lower limit of quantification (LOQ) was 5pg/mL, approx. 20-fold more sensitive than published LC-MS/MS methods. The calibration curve was linear over the range of 5-6250pg/mL. Accuracy was 98-109% and the variation (CV%) was 2.3-7.4%. This method was applied successfully to quantify temporal drug accumulation by A121a ovarian cancer cells treated with sub-ng/mL concentrations of paclitaxel.