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Drug metabolite identification is an essential characterization process spanning multiple phases of drug discovery and development. Various data processing techniques have been employed in metabolite identification using high-resolution mass spectrometry. However, metabolite identification is not consistent among approaches. Thus, a more comprehensive approach to drug metabolite identification is required. This paper proposes two-dose difference in conjunction with stable isotope tracing (SIT) to identify pioglitazone (PIO) metabolites. The results of this study revealed thatincubating both native and isotope-labeled PIOs in the same tube led to more stable metabolite identification compared with separated incubation. Our approach offers a high accuracy rate in metabolite identification, with approximately 70 % of metabolites validated as potential PIO metabolites. We compared our developed approach with other 3 approaches, namely the dose-response technique coupled with SIT, mass defect filter coupled with SIT, and orthogonal partial least squares-discriminant analysis. The results revealed that our developed approach was able to identify not only all the potential PIO metabolites identified by the other 3 approaches but also additional metabolites. These results suggest that two-dose difference coupled with SIT is an effective and comprehensive approach for drug metabolite identification., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Thyroid cancer (TC) is one of the most prevalent endocrine malignancy with a steadily increasing incidence globally. Although standard treatments like thyroidectomy and radioiodine therapy effectively manage most cases of differentiated thyroid cancers (DTC), certain recurrent cases or those involving poorly differentiated thyroid cancers (PDTC) demand more specialized interventions. Follicular thyroid cancer (FTC) is the second most common type of DTC, and frequently metastasizes through the bloodstream to distant sites such as bones and lungs which is a leading cause of metastatic and recurrent DTC and significantly affects survival. However, existing drugs primarily address symptom management without offering a curative solution. Therefore, it is urgent to develop a new therapeutic agent for these challenging cases. Evodiamine (EVO), extracted from Evodia rutaecarpa , has shown potential as an anti-cancer agent in multiple types of human cancers including anaplastic thyroid cancer (ATC) and papillary thyroid cancer (PTC) cells. However, the anti-cancer effects of EVO on FTC have remained unclear. Therefore, the present study aims to investigate the anti-cancer effects of EVO in FTC cells. Our data showed that EVO effectively inhibits FTC cell growth, induces cell cycle arrest, and triggers apoptosis. Additionally, our study explored the underlying mechanisms through which EVO affects signaling pathways. To verify the anti-cancer effects of combination chemotherapy, EVO and doxorubicin were used together in FTC cells. In conclusion, this study demonstrates that EVO shows significant anti-human FTC activity, making it a promising therapeutic candidate for the treatment of follicular thyroid cancers., Competing Interests: None., (AJCR Copyright © 2024.)

Programmed death-1 (PD-1)/PD ligand-1 (PD-L1)-mediated immune escape contributes to cancer development and has been targeted as an anti-cancer strategy. Here, we show that inhibition of the RNA helicase DDX3 increased CD8 + T cell infiltration in syngeneic oral squamous cell carcinoma tumors. DDX3 knockdown compromised interferon-γ-induced PD-L1 expression and, in particular, reduced the level of cell-surface PD-L1. DDX3 promoted surface PD-L1 expression by recruiting the adaptor protein 2 (AP2) complex to the 3' UTR of PD-L1 mRNA. DDX3 depletion or 3' UTR truncation increased the binding of the coatomer protein complexes to PD-L1, leading to its intracellular accumulation. Therefore, this 3' UTR-dependent mechanism may counteract cellular negative effects on surface trafficking of PD-L1. Finally, pharmaceutic disruption of DDX3's interaction with AP2 reduced surface PD-L1 expression, supporting that the DDX3-AP2 pathway routes PD-L1 to the cell surface. Targeting DDX3 to modulate surface trafficking of immune checkpoint proteins may provide a potential strategy for cancer immunotherapy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Transcutaneous electrical nerve stimulator (TENS) has been demonstrated to be beneficial in glycemic control in animal models, but its application in humans has not been well studied. We randomly assigned 160 patients with type 2 diabetes on oral antidiabetic drugs 1:1 to the TENS study device (n = 81) and placebo (n = 79). 147 (92%) randomized participants (mean [SD] age 59 [10] years, 92 men [58%], mean [SD] baseline HbA 1c level 8.1% [0.6%]) completed the trial. At week 20, HbA 1c decreased from 8.1% to 7.9% in the TENS group (- 0.2% [95% CI - 0.4% to - 0.1%]) and from 8.1% to 7.8% in the placebo group (- 0.3% [95% CI - 0.5% to - 0.2%]) (P = 0.821). Glycemic variability, measured as mean amplitude of glycemic excursion (MAGE) at week 20 were significantly different in the TENS group vs. the placebo group (66 mg/dL [95% CI 58, 73] vs. 79 mg/dL [95% CI 72, 87]) (P = 0.009). Our study provides the clinical evidence for the first time in humans that TENS does not demonstrate a statistically significant HbA 1c reduction. However, it is a safe complementary therapy to improve MAGE in patients with type 2 diabetes., (© 2023. The Author(s).)

Skeletal stem cells residing in the suture mesenchyme are responsible for calvarial development, homeostatic maintenance, and injury-induced repair. These naïve cells exhibit long-term self-renewal, clonal expansion, and multipotency. They possess osteogenic abilities to regenerate bones in a cell-autonomous manner and can directly replace the damaged skeleton. Therefore, the establishment of reliable isolation and culturing methods for skeletal stem cells capable of preserving their stemness promises to further explore their use in cell-based therapy. Our research team is the first to isolate and purify skeletal stem cells from the calvarial suture and demonstrate their potent ability to generate bone at a single-cell level. Here, we describe detailed protocols for suture stem cell (SuSC) isolation and stemness maintenance in culture. These methods are extremely valuable for advancing our knowledge base of skeletal stem cells in craniofacial development, congenital deformity, and tissue repair and regeneration., Competing Interests: Competing interestsThe authors declare no competing financial interests., (Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC.)

Using antibody arrays, we found that the RNA helicase DDX3 modulates the expression of secreted signaling factors in oral squamous cell carcinoma (OSCC) cells. Ribo-seq analysis confirmed amphiregulin ( AREG ) as a translational target of DDX3. AREG exerts important biological functions in cancer, including promoting cell migration and paracrine effects of OSCC cells and reprogramming the tumor microenvironment (TME) of OSCC in mice. DDX3-mediated translational control of AREG involves its 3'-untranslated region. Proteomics identified the signal recognition particle (SRP) as an unprecedented interacting partner of DDX3. DDX3 and SRP54 were located near the endoplasmic reticulum, regulated the expression of a common set of secreted factors, and were essential for targeting AREG mRNA to membrane-bound polyribosomes. Finally, OSCC-associated mutant DDX3 increased the expression of AREG, emphasizing the role of DDX3 in tumor progression via SRP-dependent, endoplasmic reticulum-associated translation. Therefore, pharmacological targeting of DDX3 may inhibit the tumor-promoting functions of the TME., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

Thyroid cancer (TC) is the most common endocrine malignancy, and its global incidence has steadily increased over the past 15 years. TC is broadly divided into well-differentiated, poorly differentiated, and undifferentiated types, depending on the histological and clinical parameters. Thus far, there are no effective treatments for undifferentiated thyroid cancers or advanced and recurrent cancer. Therefore, the development of an effective therapeutic is urgently needed for such patients. Piperlongumine (PL) is a naturally occurring small molecule derived from long pepper; it is selectively toxic to cancer cells by generating reactive oxygen species (ROS). In this study, we demonstrate the potential anticancer activity of PL in four TC cell lines. For this purpose, we cultured TC cell lines and analyzed the following parameters: Cell viability, colony formation, cell cycle, apoptosis, and cellular ROS induction. PL modulated the cell cycle, induced apoptosis, and suppressed tumorigenesis in TC cell lines in a dose- and time-dependent manner through ROS induction. Meanwhile, an intrinsic caspase-dependent apoptosis pathway was observed in the TC cells under PL treatment. The activation of Erk and the suppression of the Akt/mTOR pathways through ROS induction were seen in cells treated with PL. PL-mediated apoptosis in TC cells was through the ROS-Akt pathway. Finally, the anticancer effect and safety of PL were also demonstrated in vivo. Our findings indicate that PL exhibits antitumor activity and has the potential for use as a chemotherapeutic agent against TC. This is the first study to show the sensitivity of TC cell lines to PL.

Skeletal stem cells from the suture mesenchyme, which are referred to as suture stem cells (SuSCs), exhibit long-term self-renewal, clonal expansion, and multipotency. These SuSCs reside in the suture midline and serve as the skeletal stem cell population responsible for calvarial development, homeostasis, injury repair, and regeneration. The ability of SuSCs to engraft in injury site to replace the damaged skeleton supports their potential use for stem cell-based therapy. Here, we identified BMPR1A as essential for SuSC self-renewal and SuSC-mediated bone formation. SuSC-specific disruption of Bmpr1a in mice caused precocious differentiation, leading to craniosynostosis initiated at the suture midline, which is the stem cell niche. We found that BMPR1A is a cell surface marker of human SuSCs. Using an ex vivo system, we showed that SuSCs maintained stemness properties for an extended period without losing the osteogenic ability. This study advances our knowledge base of congenital deformity and regenerative medicine mediated by skeletal stem cells., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Small ubiquitin-related modifier (SUMO)-specific protease 2 (SENP2) is essential for the development of healthy placenta. The loss of SENP2 causes severe placental deficiencies and leads to embryonic death that is associated with heart and brain deformities. However, tissue-specific disruption of SENP2 demonstrates its dispensable role in embryogenesis and the embryonic defects are secondary to placental insufficiency. SENP2 regulates SUMO1 modification of Mdm2, which controls p53 activities critical for trophoblast cell proliferation and differentiation. Here we use genetic analyses to examine the involvement of SUMO2 and SUMO3 for SENP2-mediated placentation. The results indicate that hyper-SUMOylation caused by SENP2 deficiency can be compensated by reducing the level of SUMO modifiers. The placental deficiencies caused by the loss of SENP2 can be alleviated by the inactivation of gene encoding SUMO2 or SUMO3. Our findings demonstrate that SENP2 genetically interacts with SUMO2 and SUMO3 pivotal for the development of three major trophoblast layers. The alleviation of placental defects in the SENP2 knockouts further leads to the proper formation of the heart structures, including atrioventricular cushion and myocardium. SUMO2 and SUMO3 modifications regulate placentation and organogenesis mediated by SENP2., (© 2019 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.)

Liver X receptor (LXR) is a nuclear receptor that regulates various biological processes, including de novo lipogenesis, cholesterol metabolism, and inflammation. Selective inhibition of LXR may aid the treatment of nonalcoholic fatty liver disease (NAFLD). Sesamin is a naturally occurring lignan in many dietary plants and has a wide range of beneficial effects on metabolism. The mechanism underlying sesamin action especially on the regulation of LXR remains elusive. Reporter assays, mRNA and protein expression, and in silico modeling were used to identify sesamin as an antagonist of LXR α . Sesamin was applied to the hepatic HepaRG and intestinal LS174T cells and showed that it markedly ameliorated lipid accumulation in the HepaRG cells, by reducing LXR α transactivation, inhibiting the expression of downstream target genes. This effect was associated with the stimulation of AMP-activated protein kinase (AMPK) signaling pathway, followed by decreased T0901317-LXR α -induced expression of SREBP-1c and its downstream target genes. Mechanistically, sesamin reduced the recruitment of SRC-1 but enhanced that of SMILE to the SREBP-1c promoter region under T0901317 treatment. It regulated the transcriptional control exerted by LXR α by influencing its interaction with coregulators and thus decreased mRNA and protein levels of genes downstream of LXR α and reduced lipid accumulation in hepatic cells. Additionally, sesamin reduced valproate- and rifampin-induced LXR α and pregnane X receptor (PXR) transactivation. This was associated with reduced expression of target genes and decreased lipid accumulation. Thus, sesamin is an antagonist of LXR α and PXR and suggests that it may alleviate drug-induced lipogenesis via the suppression of LXR α and PXR signaling., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2019 Tsai-Sung Tai et al.)

Aloperine, an alkaloid isolated from Sophora alopecuroides , exhibits multiple pharmacological activities including anti-inflammatory, antioxidant, antiallergic, antinociceptive, antipathogenic, and antitumor effects. Furthermore, it exerts protective effects against renal and neuronal injuries. Several studies have reported antitumor effects of aloperine against various human cancers, including multiple myeloma; colon, breast, and prostate cancers; and osteosarcoma. Cell cycle arrest, apoptosis induction, and tumorigenesis suppression have been demonstrated following aloperine treatment. In a previous study, we demonstrated antitumor effects of aloperine on human thyroid cancer cells through anti-tumorigenesis and caspase-dependent apoptosis induction via the Akt signaling pathway. In the present study, we demonstrated the modulation of the autophagy mechanism following the incubation of multidrug-resistant papillary and anaplastic human thyroid cancer cells with aloperine; we also illustrate the underlying mechanisms, including AMPK, Erk, JNK, p38, and Akt signaling pathways. Further investigation revealed the involvement of the Akt signaling pathway in aloperine-modulated autophagy in human thyroid cancer cells. These results indicate a previously unappreciated function of aloperine in autophagy modulation in human thyroid cancer cells.

Evodiamine is a natural product extracted from herbal plants such as Tetradium which has shown to have anti-fat uptake and anti-proliferation properties. However, the effects of evodiamine on the behavior of thyroid cancers are largely unknown. To determine if evodiamine might be useful in the treatment of thyroid cancer and its cytotoxic mechanism, we analyzed the impact of evodiamine treatment on differential protein expression in human thyroid cancer cell line ARO using lysine-labeling two-dimensional difference gel electrophoresis (2D-DIGE) combined with mass spectrometry (MS). This study demonstrated 77 protein features that were significantly changed in protein expression and revealed evodiamine-induced cytotoxicity in thyroid cancer cells involves dysregulation of protein folding, cytoskeleton, cytoskeleton regulation and transcription control. Our work shows that this combined proteomic strategy provides a rapid method to study the molecular mechanisms of evodiamine-induced cytotoxicity in thyroid cancer cells. The identified targets may be useful for further evaluation as potential targets in thyroid cancer therapy., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Objectives: The magnitude of muscle damage induced by maximal eccentric contractions (MaxEC) of the elbow flexors (EF) is reduced when it is preceded by low-intensity (10% of maximal voluntary isometric contraction strength) eccentric contractions (10%EC) of the same muscle, or by MaxEC of the opposite EF. This study investigated whether 10%EC would reduce the magnitude of muscle damage after MaxEC performed by the opposite arm., Design: Comparison among 6 groups for changes in indirect markers of muscle damage., Method: Young (21.0±1.8years) untrained men were assigned to five experimental groups (n=13/group) that performed 30, 10%EC followed by 30 MaxEC of the other arm performed at either 1 (1d), 2 (2d), 7 (1wk), 14 (2wk) or 21days (3wk) later, and one control group that performed 30 MaxEC without 10%EC (n=13). Changes in several indirect markers of muscle damage after MaxEC were compared among the groups by mixed-design two-way ANOVAs., Results: No significant changes in maximal voluntary concentric contraction torque, plasma creatine kinase activity and muscle soreness were evident after 10%EC. Changes in these variables after MaxEC were smaller (p<0.05) for the 1d, 2d and 1wk groups than control group, without significant differences between the 1d, 2d and 1wk groups. No significance differences in the changes were evident among the 2wk, 3wk and control groups, except for muscle soreness showing smaller (p<0.05) increases for the 2wk and 3wk groups than control group., Conclusions: These results showed that 10%EC conferred muscle damage protection to the contralateral arm that performed MaxEC., (Copyright © 2017. Published by Elsevier Ltd.)

Aims: Honokiol is a natural product extracted from herbal plants such as the Magnolia species which have been shown to exhibit anti-tumor and anti-metastatic properties. However, the effects of honokiol on thyroid cancers are largely unknown., Materials and Methods: To determine whether honokiol might be useful for the treatment of thyroid cancer and to elucidate the mechanism of toxicity of honokiol, we analyzed the impact of honokiol treatment on differential protein expression in human thyroid cancer cell line ARO using lysine-labeling two-dimensional difference gel electrophoresis (2D-DIGE) combined with mass spectrometry (MS)., Key Findings: This study revealed 178 proteins that showed a significant change in expression levels and also revealed that honokiol-induced cytotoxicity in thyroid cancer cells involves dysregulation of cytoskeleton, protein folding, transcription control and glycolysis., Significance: Our work shows that combined proteomic strategy provides a rapid method to study the molecular mechanisms of honokiol-induced cytotoxicity in thyroid cancer cells. The identified targets may be useful for further evaluation as potential targets in thyroid cancer therapy., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Mutated or dysregulated DDX3 participates in the progression and metastasis of cancer via its multiple roles in regulating gene expression and cellular signaling. Here, we show that the high expression levels of DDX3 in head and neck squamous cell carcinoma (HNSCC) correlate with lymph node metastasis and poor prognosis and demonstrate that DDX3 is essential for the proliferation, invasion, and metastasis of oral squamous cell carcinoma (OSCC) cells. Microarray analyses revealed that DDX3 is required for the expression of a set of pro-metastatic genes, including ATF4-modulated genes in an aggressive OSCC cell line. DDX3 activated translation of ATF4 and a set of its downstream targets, all of which contain upstream open reading frames (uORF). DDX3 promoted translation of these targets, likely by skipping the inhibitory uORF. DDX3 specifically enhanced the association of the cap-binding complex (CBC) with uORF-containing mRNAs and facilitated recruitment of the eukaryotic initiation factor 3 (eIF3). CBC and certain eIF3 subunits contributed to the expression of metastatic-related gene expression. Taken together, our results indicate a role for the novel DDX3-CBC-eIF3 translational complex in promoting metastasis. Significance: The discovery of DDX3-mediated expression of oncogenic uORF-containing genes expands knowledge on translational control mechanisms and provides potential targets for cancer therapy. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/16/4512/F1.large.jpg Cancer Res; 78(16); 4512-23. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Muscle damage after 30 maximal eccentric contractions of the elbow flexors (30MVEC) is reduced when the same exercise is performed by the opposite arm, and when two maximal voluntary isometric contractions at a long muscle length (2MVIC) are performed prior to 30MVEC by the same arm. This study investigated the hypothesis that 2MVIC would attenuate muscle damage after 30MVEC performed by the opposite arm. Untrained young (20-25 years) men were placed into 1 of 4 experimental groups that performed 2MVIC at 1 (1d), 2 (2d), 4 (4d), or 7 days (7d) before 30MVEC by the opposite arm, or one control group that performed 30MVEC only (n = 13/group). Changes in indirect muscle damage markers after 30MVEC were compared among the groups by mixed-design two-way ANOVA. Maximal voluntary concentric contraction torque, range of motion, plasma creatine kinase activity, and muscle soreness did not change significantly after 2MVIC. Changes in these variables after 30MVEC were smaller (P < .05) for 1d (eg, peak soreness: 45 ± 21 mm) and 2d groups (46 ± 20 mm) than control group (66 ± 18 mm), without significant differences between 1d and 2d groups. No significant differences in the changes were found among 4d, 7d, and control groups, except for soreness showing smaller (P < .05) increases for 4d group (54 ± 19 mm) than 7d (62 ± 17 mm) and control groups. These results supported the hypothesis and showed that muscle damage induced by 30MVEC was reduced by 2MVIC performed 1-2 days prior to 30MVIC by the contralateral arm., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Purpose: Eccentric exercise of the elbow flexors (EF) confers protective effect against muscle damage of the same exercise performed by the opposite arm at 1, 7, or 28 d later. This is known as the contralateral repeated bout effect (CL-RBE), but it is not known whether CL-RBE is evident for the knee flexors (KF). The present study tested the hypothesis that KF CL-RBE would be observed at 1, 7, and 28 d after the initial bout., Methods: Young untrained men were assigned to a control or one of three experimental groups (n = 13 per group). The experimental groups performed 60 maximal KF eccentric contractions (60MaxEC) using one leg followed by the same exercise using the opposite leg at 1, 7, or 28 d later. The control group used the nondominant leg to repeat 60MaxEC separated by 14 d. Changes in several indirect muscle damage markers after 60MaxEC were compared between bouts and among the groups by using a mixed-design, two-way ANOVA., Results: Changes in maximal voluntary isokinetic concentric contraction torque, range of motion, muscle soreness, and plasma creatine kinase activity after the first 60MaxEC were similar among the groups. These changes were smaller after the second than the first 60MaxEC for the control, 1-d, and 7-d groups, and the changes after the second 60MaxEC were smaller for the control than for both the 1- and 7-d groups (P < 0.05). When the KF CL-RBE was compared with the EF CL-RBE of the previous study, the magnitude was not significantly different., Conclusions: These results showed that CL-RBE was evident for KF in a similar manner to that for EF, but did not last for 28 d, and the CL-RBE was smaller than the ipsilateral RBE.

The global incidence of thyroid cancer, one of the most common endocrine malignancies, is especially high among women. Although most patients with thyroid cancers exhibit a good prognosis with standard treatment, there are no effective therapies for patients with anaplastic thyroid cancers or cancers that have reached an advanced or recurrent level. Therefore, it is important to develop highly effective compounds for treating such patients. Aloperine, a natural compound isolated from Sophora alopecuroides , has been reported to possess antioxidant, anti-inflammatory, anti-neuronal injury, anti-renal injury, antitumor, anti-allergic, and antiviral properties. In this study, we show that aloperine can inhibit cell growth in human anaplastic thyroid cancers and multidrug-resistant papillary thyroid cancers. Moreover, it could suppress in vitro tumorigenesis and promote cellular apoptosis. Further analysis demonstrated the involvement of caspase-dependent apoptosis, including intrinsic and/or extrinsic pathways, in aloperine-induced cellular apoptosis. However, cell cycle regulation was not detected with aloperine treatment. This study suggests the potential therapeutic use of aloperine in human anaplastic thyroid cancers and multidrug-resistant papillary thyroid cancers., Competing Interests: The authors declare no conflict of interest.

Recent identification and isolation of suture stem cells capable of long-term self-renewal, clonal expanding, and differentiating demonstrate their essential role in calvarial bone development, homeostasis, and injury repair. These bona fide stem cells express a high level of Axin2 and are able to mediate bone regeneration and repair in a cell autonomous fashion. The importance of Axin2 is further demonstrated by its genetic inactivation in mice causing skeletal deformities resembling craniosynostosis in humans. The fate determination and subsequent differentiation of Axin2+ stem cells are highly orchestrated by a variety of evolutionary conserved signaling pathways including Wnt, FGF, and BMP. These signals are often antagonistic of each other and possess differential effects on osteogenic and chondrogenic cell types. However, the mechanisms underlying the interplay of these signaling transductions remain largely elusive. Here we identify Rap1b acting downstream of Axin2 as a signaling interrogator for FGF and BMP. Genetic analysis reveals that Rap1b is essential for development of craniofacial and body skeletons. Axin2 regulates Rap1b through modulation of canonical BMP signaling. The BMP-mediated activation of Rap1b promotes chondrogenic fate and chondrogenesis. Furthermore, by inhibiting MAPK signaling, Rap1b mediates the antagonizing effect of BMP on FGF to repress osteoblast differentiation. Disruption of Rap1b in mice not only enhances osteoblast differentiation but also impairs chondrocyte differentiation during intramembranous and endochondral ossifications, respectively, leading to severe defects in craniofacial and body skeletons. Our findings reveal a dual role of Rap1b in development of the skeletogenic cell types. Rap1b is critical for balancing the signaling effects of BMP and FGF during skeletal development and disease. © 2017 American Society for Bone and Mineral Research., (© 2017 American Society for Bone and Mineral Research.)

Thyroid cancer is the most common endocrine malignancy, the global incidence rate of which is rapidly rising. Surgery and radioiodine therapies are common and effective treatments only for nonmetastasized primary tumors. Therefore, effective treatment modalities are imperative for patients with radioiodine-resistant thyroid cancer. Honokiol, a biophenolic compound derived from Magnolia spp., has been shown have diverse biological and pharmacological activities, including anti-inflammatory, antioxidative, antiangiogenic, and anticancer properties. In the present study, three human thyroid cancer cell lines, namely anaplastic, follicular, and poorly differentiated thyroid cancer cells, were used to evaluate the chemotherapeutic activity of honokiol. Cell viability, cell cycle, apoptosis, and autophagy induction were determined through flow cytometry and western blot analysis. We found that honokiol treatment can suppress cell growth, induce cell cycle arrest, and enhance the induction of caspase-dependent apoptosis and autophagy in cancer cells. Moreover, honokiol treatment modulated signaling pathways including Akt/mTOR, ERK, JNK, and p38 in the studied cells. In addition, the antitumorigenic activity of honokiol was also confirmed in vitro and in vivo. Our data provide evidence that honokiol has a unique application in chemotherapy for human thyroid cancers., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Purpose: This study compared the magnitude of the repeated bout effect (RBE) for different time intervals between two bouts of eccentric exercise of the elbow flexors to better understand the contralateral RBE (CL-RBE)., Methods: Untrained young men (22.0 ± 1.8 yr) were allocated to either a control or one of seven CL-RBE groups (n = 13 per group). The CL-RBE groups performed exercise consisting of 30 maximal isokinetic (30°·s) eccentric contractions of the elbow flexors (ECC1) with either dominant or nondominant arm followed 0.5 h, 6 h, 12 h, 24 h (1 d), 7 d (1 wk), 28 d (4 wk), or 56 d (8 wk) by the same exercise (ECC2) using the opposite arm. The control group used the nondominant arm for ECC1 and ECC2 separated by 2 wk., Results: Maximal voluntary concentric contraction torque, peak torque angle, range of motion, upper arm circumference, muscle soreness, ultrasound echo intensity, and plasma creatine kinase activity and myoglobin concentration changed (P < 0.05) after ECC1, without significant difference among the groups. Changes in all variables after ECC2 were smaller (P < 0.05) than those after ECC1 for the control, 1 d, 1 wk, and 4 wk groups, indicating the RBE. However, the changes were not significantly different between ECC1 and ECC2 for the 0.5 h, 6 h, 12 h, and 8 wk groups. The difference in the changes in all variables between ECC1 and ECC2 was smaller for the 1 d (70%), 1 wk (55%), and 4 wk (36%) than the control group (91%), and the magnitude of the CL-RBE was reduced with increasing the time between bouts from 1 d to 4 wk (P < 0.05)., Conclusion: These results suggest that the CL-RBE lasts shorter than the ipsilateral RBE (>8 wk) and requires a day to be conferred.

Unlabelled: The RNA helicase DDX3 is a component of neuronal granules, and its gene mutations are linked to intellectual disability (ID). Here we demonstrate that DDX3 depletion in neurons impairs neurite development by downregulating Rac1 level and activation. Moreover, DDX3 activates the translation of functionally coherent mRNAs involved in Rac1 activation including Rac1 Among the DDX3 regulon, Prkaca encodes the catalytic subunit of PKA, a potential activator of Rac1 in neurons. DDX3-modulated PKAcα and Rac1 expression tunes the strength of PKA-Rac1 signaling and thereby contributes to neurite outgrowth and dendritic spine formation. Inhibition of DDX3 activity or expression in neonatal mice impaired dendritic outgrowth and spine formation of hippocampal neurons, echoing neuronal deficits underling DDX3 mutation-associated ID. Finally, we provide evidence that DDX3 activates local protein synthesis through a 5' UTR-dependent mechanism in neurons. The novel DDX3 regulon may conduct a spatial and temporal control of Rac1 signaling to regulate neurite development., Significance Statement: DDX3X mutations are linked to intellectual disability (ID). We provide first evidence that DDX3 is required for neurite outgrowth and dendritic spine formation in vitro and in vivo We identified a DDX3 regulon constituting functionally cohesive mRNAs involved in Rac1 signaling, which contributes to DDX3-modulated neurite development. Inhibition or ablation of DDX3 in vivo shortened neurite lengths and impaired dendritic spine formation in hippocampal neurons, reflecting the prevalence of ID-associated DDX3X mutations in the helicase domain. Mechanistically, DDX3 activates local protein synthesis of mRNAs sharing similar 5' UTR structures and therefore controls Rac1 signaling strength in neurites., (Copyright © 2016 the authors 0270-6474/16/369792-13$15.00/0.)

RBM4 participates in cell differentiation by regulating tissue-specific alternative pre-mRNA splicing. RBM4 also has been implicated in neurogenesis in the mouse embryonic brain. Using mouse embryonal carcinoma P19 cells as a neural differentiation model, we observed a temporal correlation between RBM4 expression and a change in splicing isoforms of Numb, a cell-fate determination gene. Knockdown of RBM4 affected the inclusion/exclusion of exons 3 and 9 of Numb in P19 cells. RBM4-deficient embryonic mouse brain also exhibited aberrant splicing of Numb pre-mRNA. Using a splicing reporter minigene assay, we demonstrated that RBM4 promoted exon 3 inclusion and exon 9 exclusion. Moreover, we found that RBM4 depletion reduced the expression of the proneural gene Mash1, and such reduction was reversed by an RBM4-induced Numb isoform containing exon 3 but lacking exon 9. Accordingly, induction of ectopic RBM4 expression in neuronal progenitor cells increased Mash1 expression and promoted cell differentiation. Finally, we found that RBM4 was also essential for neurite outgrowth from cortical neurons in vitro. Neurite outgrowth defects of RBM4-depleted neurons were rescued by RBM4-induced exon 9-lacking Numb isoforms. Therefore our findings indicate that RBM4 modulates exon selection of Numb to generate isoforms that promote neuronal cell differentiation and neurite outgrowth., (© 2016 Tarn et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

The DEAD-box RNA helicase DDX3 is a versatile protein involved in multiple steps of gene expression and various cellular signaling pathways. DDX3 mutations have been implicated in the wingless (Wnt) type of medulloblastoma. We show here that small interfering RNA-mediated DDX3 knockdown in various cell lines increased cell-cell adhesion but decreased cell-extracellular matrix adhesion. Moreover, DDX3 depletion suppressed cell motility and impaired directional migration in the wound-healing assay. Accordingly, DDX3-depleted cells exhibited reduced invasive capacities in vitro as well as reduced metastatic potential in mice. We also examined the mechanism underlying DDX3-regulated cell migration. DDX3 knockdown reduced the levels of both Rac1 and β-catenin proteins, and consequentially downregulated the expression of several β-catenin target genes. Moreover, we demonstrated that DDX3-regulated Rac1 mRNA translation, possibly through an interaction with its 5'-untranslated region, and affected β-catenin protein stability in an Rac1-dependent manner. Taken together, our results indicate the DDX3-Rac1-β-catenin regulatory axis in modulating the expression of Wnt/β-catenin target genes. Therefore, this report provides a mechanistic context for the role of DDX3 in Wnt-type tumors.

Pedicularis longiflora experienced extensive populations' expansion in Quaternary, but the dispersal corridors were still unclear. According to the distribution patterns of haplotypes based on chloroplast DNA variation, the dispersal routes were predicted using species distribution models (MXENT) and the least-cost path method. Two possible dispersal routes from the southeastern part of Tibetan Plateau (TP) to interior were identified. The populations of East Himalayas-Hengduan Mountains region expanded to the western part of TP along with the Yarlung Zangbo River valley and the lower altitudes of the north slope of Himalayas. The expansion trend was also proved by SDMs based on two historical periods containing the Last Interglacial and the Last Glacial Maximum. In conclusion, identification of dispersal routes is significant to the evolutionary history of alpine plants and the protection of special species in TP.

To comprehend the sensorimotor control ability in diabetic hands, this study investigated the sensation, motor function and precision pinch performances derived from a pinch-holding-up activity (PHUA) test of the hands of diabetic patients and healthy subjects. The precision, sensitivity and specificity of the PHUA test in the measurements of diabetic patients were also analyzed. We hypothesized that the diabetic hands would have impacts on the sensorimotor functions of the hand performances under functionally quantitative measurements. One hundred and fifty-nine patients with clinically defined diabetes mellitus (DM) and 95 age- and gender-matched healthy controls were included. Semmes-Weinstein monofilament (SWM), static and moving two-point discrimination (S2PD and M2PD), maximal pinch strength and precision pinch performance tests were conducted to evaluate the sensation, motor and sensorimotor status of the recruited hands. The results showed that there were significant differences (all p<0.05) in SWM, S2PD, M2PD and maximum pinch strength between the DM and control groups. A higher force ratio in the DM patients than in the controls (p<0.001) revealed a poor ability of pinch force adjustment in the DM patients. The percentage of maximal pinch strength was also significantly different (p<0.001) between the DM and control groups. The sensitivity, specificity and area under the receiver operating characteristic curve were 0.85, 0.51, and 0.724, respectively, for the PHUA test. Statistically significant degradations in sensory and motor functions and sensorimotor control ability were observed in the hands of the diabetic patients. The PHUA test could be feasibly used as a clinical tool to determine the sensorimotor function of the hands of diabetic patients from a functional perspective.

The incurable differentiated thyroid cancer (DTC), poorly differentiated thyroid cancer (PDTC) and anaplastic thyroid cancer (ATC) are the most aggressive in all of the thyroid cancers. Unfortunately, there are almost no effective therapies. A novel and effective treatment is urgently needed to develop. Recently, reversine, a small synthetic purine analogue, has been reported to be effective in human thyroid cancer suppression through cell cycle arrest and apoptosis induction. In this study, we performed an in vitro evaluation of reversine on autophagy activation, one of the programmed cell death, and the related mechanisms in human follicular thyroid cancer cell line WRO. Incubation of WRO cells with reversine induced autophagosome formation in a short time treatment. LC3-II overexpression in a dosage-dependent manner with reversine treatment was demonstrated in the autophagy activation. Moreover, reversine suppressed Akt/mTOR related signaling pathway activation, a major pathway for autophagy activation, was also revealed in WRO cells. Our data demonstrated that reversine is effective to induce autophagy. Moreover, the LC3-II overexpression and the p62 protein were degraded in a time-dependent manner, indicating that the autophagic flux has happened in the reversine treated WRO cells. In addition, the activation of Akt/mTOR/p70S6K related pathways were shown to be reduced, suggesting these pathways may involve in the reversine mediated autophagy induction. Reversine is therefore worthy of further investigation in clinical therapeutics., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Purpose: A novel and effective treatment is urgently needed to deal with the current treatment dilemma in incurable differentiated thyroid cancer (DTC), poorly differentiated thyroid cancer (PDTC) and anaplastic thyroid cancer (ATC). Reversine, a small synthetic purine analogue (2,6-disubstituted purine), has been shown to be effective in tumor suppression., Methods: We performed in vitro evaluation of anti-tumor effects of reversine on proliferation, cell cycle, and apoptosis in human PDTC, ATC, and follicular thyroid cancer cell lines, respectively., Results: Treatment of these three lines with reversine inhibited proliferation in a time- and dose-dependent manner. G2/M accumulation was demonstrated in cell cycle analysis. Reversine induced apoptosis in PDTC cells with caspase-3 and caspase-8 activation, but not caspase-9. Use of a pan-caspase inhibitor before treatment with reversine attenuated cell death. Reversine also showed in vivo growth inhibitory effects on ATC cells in a xenograft nude mice model., Conclusions: Data demonstrated that reversine is effective in inhibiting the growth of thyroid cancer cells by cell cycle arrest or apoptosis, especially with the more aggressive ATC and PDTC. Apoptosis was induced by the mitochondria-independent pathway. Reversine is therefore worthy of further investigation in clinical therapeutics.

The RNA-binding protein hnRNP Q has been implicated in neuronal mRNA metabolism. Here, we show that knockdown of hnRNP Q increased neurite complexity in cultured rat cortical neurons and induced filopodium formation in mouse neuroblastoma cells. Reexpression of hnRNP Q1 in hnRNP Q-depleted cells abrogated the morphological changes of neurites, indicating a specific role for hnRNP Q1 in neuronal morphogenesis. A search for mRNA targets of hnRNP Q1 identified functionally coherent sets of mRNAs encoding factors involved in cellular signaling or cytoskeletal regulation and determined its preferred binding sequences. We demonstrated that hnRNP Q1 bound to a set of identified mRNAs encoding the components of the actin nucleation-promoting Cdc42/N-WASP/Arp2/3 complex and was in part colocalized with Cdc42 mRNA in granules. Using subcellular fractionation and immunofluorescence, we showed that knockdown of hnRNP Q reduced the level of some of those mRNAs in neurites and redistributed their encoded proteins from neurite tips to soma to different extents. Overexpression of dominant negative mutants of Cdc42 or N-WASP compromised hnRNP Q depletion-induced neurite complexity. Together, our results suggest that hnRNP Q1 may participate in localization of mRNAs encoding Cdc42 signaling factors in neurites, and thereby may regulate actin dynamics and control neuronal morphogenesis.

Introduction: Hyperparathyroidism often presents with asymptomatic hypercalcemia or minimal, unspecific symptoms and the middle-age female is the predominant population., Case Report: We reported a young Vietnamese female with primary hyperparathyroidism presented with chronic low back pain of about 1 year duration, similar to sacroiliitis. An initial study of bone scan raised the suspicion of sacroiliitis. However, careful review of her symptoms and signs did not demonstrate the presence of typical inflammatory low back pain instead of severe osteoporosis and osteitis fibrosa cystica. A computed tomography of pelvis showed subchondral bony resorption or erosive change with bilateral SI joint widening which more favored hyperparathyroidism than sacroiliitis., Discussion: This case highlights the point that primary hyperparathyroidism could be presented with sacroiliitis-like pain and severe bone disease in young patient from a developing country.

Renal transplant is the only curative treatment for end-stage renal disease. As diabetes and obesity are the major causes of graft failure and post-transplant complication, it is important to manage obesity in patients with renal transplant. Herein, we report a case of a morbidly obese renal-transplant patient with poorly controlled diabetes who received bariatric surgery. A 34-year-old obese Taiwanese man with type 2 diabetes had end-stage renal disease that had progressed since 2008, when he had commenced hemodialysis (January 2008) and had a renal transplant (July 2008). Because of persistent obesity and poorly controlled diabetes, he received LRYGB at Chiayi Christian hospital on 18 August 2010. In the month that followed, he lost 10 kg. His serum creatinine decreased to 1.11 mg/dL (1.4 mg/dL, preoperative) and his hemoglobin A1c decreased to 8.5% (10.4%, preoperative). These results indicate that, in obese renal transplant patients, LRYGB may be employed to treat obesity, control diabetes and stabilize or improve the renal function., (© 2011 Japan Society for Endoscopic Surgery, Asia Endosurgery Task Force and Blackwell Publishing Asia Pty Ltd.)

Aims and Background: Thyroid cancer is the most common endocrine neoplasm worldwide. Although differentiated thyroid cancers are associated with a favorable survival, the prognosis worsens dramatically for patients with distant metastasis. Metastases from follicular thyroid carcinoma (FTC) occur earlier and are more aggressive than those from papillary thyroid carcinoma. For FTC that is resistant to radioactive iodine, new treatments are urgently needed. Human alpha-fetoprotein (HAFP) is a tumor-associated fetal protein that has been demonstrated to regulate tumorigenesis. Growth inhibitory peptide (GIP), a synthetic 34-mer peptide isolated from the third domain of HAFP, has been shown to have antitumor growth ability in various human cancers. However, the effects of GIP in FTC have not yet been studied. The aim of this study was to investigate the antitumor ability of GIP in FTC., Methods and Study Design: Using both PBS and GIP control peptide as a negative control, the antiproliferative activity of GIP in the WRO human FTC cell line was determined using a tetrazolium-based colorimetric assay. In addition, cell migration and invasion assays were used to measure tumor metastasis inhibition effects in vitro., Results: GIP did not inhibit WRO cell proliferation in a time- or dose-dependent manner. However, in WRO cells treated with GIP for 4 days, migration was significantly inhibited at concentrations of 50 and 100 microM (33.3% and 19.5%, respectively; both P <0.05). Cell invasion was also significantly inhibited at 50 and 100 microM (67.1% and 39.0%, respectively; both P<0.05)., Conclusions: Although GIP failed to suppress FTC cell growth, it effectively interrupted both FTC cell migration and invasion abilities in vitro. Further validation in an animal model and elucidation of the underlying mechanisms will be required. GIP may potentially serve as an anti-FTC metastasis agent aiding current chemotherapy regimens.

Aims: To compare the importance of different inflammatory markers and traditional risk factors in predicting peripheral vascular disease (PVD) in patients with Type 2 diabetes mellitus., Methods: A cross-sectional analysis of 30 Type 2 diabetic patients with PVD defined by ankle-brachial index (ABI) < 0.9, and 60 Type 2 diabetic patients without PVD (ABI > 1.0). Overnight blood was drawn and sent for study., Results: Longer diabetic duration (15 +/- 8 vs. 10 +/- 7 years, P = 0.007), higher serum creatinine level (0.11 +/- 0.04 vs. 0.09 +/- 0.03 mmol/l, P = 0.001), higher total cholesterol/high-density lipoprotein-cholesterol (TC/HDL-C) ratio (5.2 +/- 1.6 vs. 4.3 +/- 1.1, P = 0.004) and increased hypertension status (70% vs. 52%, P = 0.014) and cerebral infarction (CI) history (23% vs. 3%, P = 0.009) were noted in Type 2 diabetes with PVD. Those with PVD also showed significantly higher serum levels of C-reactive protein (CRP) (median 0.282 vs. 0.102 mg/dl, P < 0.001) and interleukin (IL)-6 (10.6 +/- 1.81 vs 1.6 +/- 4.6 pg/ml, P = 0.001). Multivariate regression analysis showed that higher serum levels of C-reactive protein (CRP), longer diabetic duration, and use of angiotensin converting enzyme inhibitor (ACEI) were independently associated with PVD in Type 2 diabetes mellitus., Conclusions: Type 2 diabetic patients with PVD had longer diabetic duration, higher serum creatinine levels, higher TC/HDL-C ratio, higher hypertension and CI history and higher CRP and IL-6 levels. Only serum CRP level, diabetic duration, and use of ACEI were independently associated with PVD in Type 2 diabetes mellitus.

We strived to characterize the endothelial function status in type 2 diabetic patients with peripheral artery disease which was detected by ankle-brachial index by utilizing high frequency ultrasounds. Predictors of endothelial dysfunction were investigated. We chose 23 type 2 diabetic patients had ankle-brachial index <0.97 (0.15-0.95; mean=0.74+/-0.20), 31 diabetic patients had ankle-brachial index >/=1.0 and 28 non-diabetic subjects for study. Older age, a longer duration of diabetes, higher systolic blood pressure, higher prevalence of history of hypertension were observed in patients with peripheral vascular disease. Type 2 diabetic patients showed impaired flow-mediated dilatation than non-diabetic and it showed more impaired in patients with peripheral vascular disease. Nitroglyerin-induced dilatation showed a trend of impairment in patients with peripheral vascular disease but did not reach statistical significance. Age (r=-0.259, P=0.019), baseline brachial artery diameter (r=-0.321, P=0.003), ankle-brachial index (r=0.259, P=0.002) and hypertension history (P=0.01) were significantly associated with flow-mediated dilatation. However, after adjusting for age, only baseline diameter and ankle-brachial index were independent predictors of flow-mediated dilatation. In conclusion, we demonstrated flow-mediated dilatation was impaired in type 2 diabetic patients and it was further impaired in patients with peripheral vascular disease. Nitroglycerin-induced dilatation showed a trend of impairment but did not reach statistical significance.

In the current study, the contractile responses to muscarinic stimulation of urinary bladder strips from young rats and aged rats were compared. 1. The EC50 values of the two groups in response to bethanechol were similar. 2. The magnitude and the velocity of tension generation was significantly lower in strips from the aged group. 3. The magnitude as well as the velocity of tension generation in response to high K+ solution of the two models were similar. 4. The time to peak [Ca2+]i in response to bethanechol was prolonged in strips from the aged group as compared with the young group. 5. 45Ca2+ influx in response to bethanechol was significantly reduced in the aged group as compared with the young group. It is concluded that the reduced contractile response to muscarinic stimulation of isolated urinary bladder strips from aged rats is mediated at least in part by a decreased rate of Ca2+ entry.