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BackgroundOesophageal squamous cell carcinoma (SCC) causes the highest number of cancer deaths in some regions of Northern China. Previously, we narrowed down a critical region at 9q33-34, identified to be associated with tumour-suppressive function of deleted in oesophageal cancer 1 (DEC1) in oesophageal SCC.MethodsWe generated DEC1 antibody and constructed tissue microarrays (TMAs) utilising tissue specimens from Henan, a high-risk region for oesophageal SCC, to investigate the importance of DEC1 expression in this cancer.ResultsTissue microarray immunohistochemical staining reveals significant loss of DEC1 from hyperplasia, to tumour, and to lymph node metastasis. In addition, the loss of DEC1 in tumour is age-dependent. Interestingly, there is significant abrogation of DEC1 expression in patients with a family history of oesophageal SCC. Deleted in oesophageal cancer 1 localises to both the cytoplasm and nucleus. The vesicular pattern of DEC1 in the cytoplasm appears to localise at the Golgi and Golgi-endoplasmic reticulum intermediate compartment.ConclusionThis is the first TMA study to suggest a clinical association of DEC1 in lymph node metastatic oesophageal SCC, early onset oesophageal SCC and familial oesophageal SCC development. Subcellular localisation of DEC1 and its expression in oesophageal SCC tissues provide important insight for further deciphering the molecular mechanism of DEC1 in oesophageal SCC development.

Myeloid-derived suppressor cells (MDSCs), major components maintaining the immune suppressive microenvironment in lung cancer, are relevant to the invasion, metastasis, and poor prognosis of lung cancer, through the regulation of epithelial-mesenchymal transition, remodeling of the immune microenvironment, and regulation of angiogenesis. MDSCs regulate T-cell immune functions by maintaining a strong immunosuppressive microenvironment and promoting tumor invasion. This raises the question of whether reversing the immunosuppressive effect of MDSCs on T cells can improve lung cancer treatment. To understand this further, this review explores the interactions and specific mechanisms of different MDSCs subsets, including regulatory T cells, T helper cells, CD8 + T cells, natural killer T cells, and exhausted T cells, as part of the lung cancer immune microenvironment. Second, it focuses on the guiding significance confirmed via clinical liquid biopsy and tissue biopsy that different MDSC subsets improve the prognosis of lung cancer. Finally, we conclude that targeting MDSCs through action targets or signaling pathways can help regulate T-cell immune functions and suppress T-cell exhaustion. In addition, immune checkpoint inhibitors targeting MDSCs may serve as a new approach for enhancing the efficiency of immunotherapy and targeted therapy for lung cancer in the future, providing better comprehensive options for lung cancer treatment., (© 2023. Springer Science+Business Media, LLC.)

Background: The liver hemodynamic changes caused by portal hypertension (PH) are closely related to various complications such as gastroesophageal varices and portosys-temic shunts, which may lead to adverse clinical outcomes in these patients, so it is of great clinical significance to find treatment strategies with favorable clinical efficacy and low risk of complications., Aim: To study the clinical efficacy of total laparoscopic splenectomy (TLS) for PH and its influence on hepatic hemodynamics and liver function., Methods: Among the 199 PH patients selected from October 2016 to October 2020, 100 patients [observation group (OG)] were treated with TLS, while the remaining 99 [reference group (RG)] were treated with open splenectomy (OS). We observed and compared the clinical efficacy, operation indexes [operative time (OT) and intraoperative bleeding volume], safety (intraperitoneal hemorrhage, ascitic fluid infection, eating disorders, liver insufficiency, and perioperative death), hepatic hemodynamics (diameter, velocity, and flow volume of the portal vein system), and liver function [serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), and serum total bilirubin (TBil)] of the two groups., Results: The OT was significantly longer and intraoperative bleeding volume was significantly lesser in the OG than in the RG. Additionally, the overall response rate, postoperative complications rate, and liver function indexes (ALT, AST, and TBil) did not differ significantly between the OG and RG. The hepatic hemodynamics statistics showed that the pre- and postoperative blood vessel diameters in the two cohorts did not differ statistically. Although the postoperative blood velocity and flow volume reduced significantly when compared with the preoperative values, there were no significant inter-group differences., Conclusion: TLS contributes to comparable clinical efficacy, safety, hepatic hemodynamics, and liver function as those of OS in treating PH, with a longer OT but lesser intraoperative blood loss., Competing Interests: Conflict-of-interest statement: There is no conflict of interest., (©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved.)

Objective: To investigate the efficacy of integrated Chinese and Western medicine extending the progression-free survival (PFS) and overall survival (OS) of limited-stage small cell lung cancer (LS-SCLC) patients after the first-line chemoradiotherapy., Methods: The data of 67 LS-SCLC patients who received combined treatment of CM and Western medicine (WM) between January 2013 and May 2020 at the outpatient clinic of Guang'anmen Hospital were retrospectively analyzed. Thirty-six LS-SCLC patients who received only WM treatment was used as the WM control group. The medical data of the two groups were statistically analyzed. Survival analysis was performed using the product-limit method (Kaplan-Meier analysis). The median OS and PFS were calculated, and survival curves were compared by the Log rank test. The cumulative survival rates at 1, 2, and 5 years were estimated by the life table analysis. Stratified survival analysis was performed between patients with different CM administration time., Results: The median PFS in the CM and WM combination treatment group and the WM group were 19 months (95% CI: 12.357-25.643) vs. 9 months (95% CI: 5.957-12.043), HR=0.43 (95% CI: 0.27-0.69, P<0.001), respectively. The median OS in the CM and WM combination group and the WM group were 34 months (95% CI could not be calculated) vs. 18.63 months (95% CI: 16.425-20.835), HR=0.40 (95% CI: 0.24-0.66, P<0.001), respectively. Similar results were obtained in the further stratified analysis of whether the duration of CM administration exceeded 18 and 24 months (P<0.001)., Conclusion: The combination treatment of CM and WM with continuing oral administration of CM treatment after the first-line chemoradiotherapy for LS-SCLC patients produced better prognosis, lower risks of progression, and longer survival than the WM treatment alone. (Registration No. ChiCTR2200056616)., (© 2022. The Chinese Journal of Integrated Traditional and Western Medicine Press and Springer-Verlag GmbH Germany, part of Springer Nature.)

The γ-tubulin ring complex (γTuRC) is the principal nucleator of cellular microtubules, and the microtubule-nucleating activity of the complex is stimulated by binding to the γTuRC-mediated nucleation activator (γTuNA) motif. The γTuNA is part of the centrosomin motif 1 (CM1), which is widely found in γTuRC stimulators, including CDK5RAP2. Here, we show that a conserved segment within CM1 binds to the γTuNA and blocks its association with γTuRCs; therefore, we refer to this segment as the γTuNA inhibitor (γTuNA-In). Mutational disruption of the interaction between the γTuNA and the γTuNA-In results in a loss of autoinhibition, which consequently augments microtubule nucleation on centrosomes and the Golgi complex, the two major microtubule-organizing centers. This also causes centrosome repositioning, leads to defects in Golgi assembly and organization, and affects cell polarization. Remarkably, phosphorylation of the γTuNA-In, probably by Nek2, counteracts the autoinhibition by disrupting the γTuNA‒γTuNA-In interaction. Together, our data reveal an on-site mechanism for controlling γTuNA function., (© 2023 Yang et al.)

We had previously shown that THY1 (CD90) is a tumor suppressor in nasopharyngeal carcinoma (NPC) and that its down-regulation and loss of expression are associated with tumor metastasis, yet the mechanism leading to such effects remains unknown. In this study we show that tumor invasion could be suppressed by THY1 via adherens junction formation in a few NPC cell lines, and knockdown of THY1 would disrupt this cell-cell adhesion phenotype. Mechanistically, the activity of the SRC family kinase (SFK) member, SRC, and canonical Wnt signaling were dramatically reduced when THY1 was constitutively expressed. Previous studies by others have found that high levels of SRC activity in NPCs are associated with EMT and a poor prognosis. We hypothesized that THY1 can suppress tumor invasion in NPC via inhibition of SRC. By gene silencing of SRC, we found that the in vitro NPC cell invasion was significantly reduced and adherens junctions were restored. Through proteomic analysis, we identified that platelet-derived growth factor receptor β (PDGF-Rβ) and protein tyrosine phosphatase nonreceptor type 22 (PTPN22) are novel and potential binding partners of THY1, which were subsequently verified by co-immunoprecipitation (co-IP) analysis. The ligand of PDGF-Rβ (PDGF-BB) could highly induce SRC activation and NPC cell invasion, which could be almost completely suppressed by THY1 expression. On the other hand, the PTPN22 siRNA could enhance both the SRC activities and the cell invasion and could also disrupt the adherens junctions in the THY1-expressing NPC cells; the original THY1-induced phenotypes were reverted when the PTPN22 expression was reduced. Together, our results identified that PTPN22 is essential for THY1 to suppress cell invasion and SRC activity, maintain tight adherens junctions, and prevent NPC metastasis. These results suggested that PDGF-Rβ and SRC can be used as drug targets for suppressing NPC metastasis. Indeed, our in vivo assay using the SRC inhibitor KX2-391, clearly showed that inhibition of SRC signaling can prevent the metastasis of NPC, indicating that targeting SRC can be a promising approach to control the NPC progression.

Cochlear and vestibular hair cells are highly specialized sensory receptors for hearing and balance. Here, we report a serendipitous identification of a hair-cell-specific organelle in neonatal mouse inner ear, which we name "apicosome." The apicosome is ∼500 nm in diameter and shows itinerant nature and transient appearance during development in cochlear hair cells. In contrast to cochlear hair cells, the apicosome persists in vestibular hair cells even in adult. The timing of apicosome translocation and disappearance in cochlear hair cells during development is correlated with kinocilium development and maintenance. The apicosome is not seen in supporting cells despite the fact that nascent supporting cells have microvilli and a primary cilium. Interestingly, transdifferentiated hair cells from supporting cells also contain apicosome, suggesting that it is unique to hair cells. Thus, our study identifies a previously undescribed organelle in hair cells and lays the foundation for further characterization of this specialized structure., Competing Interests: The authors declare no competing interests., (© 2023 The Authors.)

In oral squamous cell carcinoma (OSCC), a highly aggressive and frequently lethal malignancy, the role and action mechanism of the microtubule regulatory protein CDK5RAP2 have not been fully understood. Here, we show that CDK5RAP2 is highly expressed in OSCC and its expression correlates with clinical stage and lymph node metastasis of the disease. The expression of CDK5RAP2 is regulated by the Wnt signaling pathway. Depletion of CDK5RAP2 inhibits the tumorigenesis and migration of OSCC cells and alters the OSCC cancer stem (-like) cell (CSC) signature. Notably, suppression of CDK5RAP2 expression disrupts spindle orientation during mitosis. Collectively, these results identify CDK5RAP2 as a potential CSC marker and reveal a mechanism that controls the CSC population in OSCC., (© 2023. The Author(s).)

Golgi-derived microtubules constitute an asymmetrical microtubule network that drives polarized transport of vesicles to support cell polarization and directional migration. Golgi-based microtubule nucleation requires the γ-tubulin ring complex (γTuRC), the principal microtubule nucleator in animal cells. In this chapter, we present methods for detecting γTuRC components and associated proteins on the Golgi, examining Golgi-based microtubule nucleation, and measuring the microtubule-nucleating activity of isolated γTuRCs. These approaches have been demonstrated to be effective for assessing the microtubule-organizing function of the Golgi complex., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

We present here a protocol to assay the centrosome separation events at late-G2 phase of the cell cycle by immunofluorescence microscopy. We describe the steps required for imaging and measurement of inter-centrosome distance. Here, we use GAS2L1 as an example, but the protocol can be used to test any protein for a role in centrosome separation and cohesion. The steps below are specific for hTERT RPE-1 cell lines, but other adherent cell lines (e.g., U2OS, MRC-5) are also amenable for this protocol. For complete details on the use and execution of this protocol, please refer to Au et al. (2017) and Au et al. (2020)., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Objective: To investigate the practicability and safety of transjugular liver biopsy (TJLB). Methods: Data of 53 cases with transjugular liver biopsy from June 2015 to June 2020 were collected. LABS-100 was used in all patients who underwent transjugular liver biopsy. Among them, 45 cases and eight were biopsied via hepatic vein and intrahepatic segment of the inferior vena cava. The surgical indications, related complications, and postoperative pathological diagnosis were analyzed and summarized. Results: TJLB was successful in all patients, with an average of 2.8 punctures per case. Satisfactory liver tissue and histopathological diagnosis was obtained in all patients. Two cases developed a cervical hematoma that was improved spontaneously, and one patient developed an intrahepatic hematoma that was improved after conservative treatment. Conclusion: TJLB is a practical and safe method for patients with contraindications to percutaneous liver biopsy.

Endogenous metabolites are a class of molecules playing diverse and significant roles in many metabolic pathways for disease. Honokiol (HNK), an active poly-phenolic compound, has shown potent anticancer activities. However, the detailed crucial mechanism regulated by HNK in colorectal cancer remains unclear. In the present study, we investigated the therapeutic effects and the underlying molecular mechanisms of HNK on colorectal cancer in a mouse model (ApcMin/+) by analyzing the urine metabolic profile based on metabolomics, which is a powerful tool for characterizing metabolic disturbances. We found that potential urine biomarkers were involved in the metabolism of compounds such as purines, tyrosines, tryptophans, etc. Moreover, we showed that a total of 27 metabolites were the most contribution biomarkers for intestinal tumors, and we found that the citrate cycle (TCA cycle) was regulated by HNK. In addition, it was suggested that the efficacy of HNK was achieved by affecting the multi-pathway system via influencing relevant metabolic pathways and regulating metabolic function. Our work also showed that high-throughput metabolomics can characterize the regulation of metabolic disorders as a therapeutic strategy to prevent colorectal cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chen, Shi, Qi, Chang and Zheng.)

Microtubule nucleation is mainly mediated by the γ-tubulin ring complex (γTuRC), whose core components are γ-tubulin and γ-tubulin complex proteins GCP2-6. A substantial fraction of γ-tubulin also exists with GCP2 and GCP3 in a tetramer called the γ-tubulin small complex (γTuSC). To date, the mechanisms underlying the turnover of γ-tubulin and GCPs have remained unclear. Here, we show that γ-tubulin, GCP2, and GCP3 are proteolyzed by the ubiquitin-proteasome system, and we identify cullin 1, cullin 4A, and cullin 4B as the E3 ligases that mediate the ubiquitination and, consequently, the degradation of γ-tubulin. Notably, we found that γTuSC disassembly promotes the degradation of γ-tubulin, GCP2, and GCP3, which indicates a role for γTuSCs in the stabilization of its components., (© 2021 Federation of European Biochemical Societies.)

Background: Transjugular intrahepatic portosystemic shunt (TIPS), splenectomy plus esophagogastric devascularization (SED) and endoscopic therapy + non-selective β-blockers (ET + NSBB) are widely applied in secondary prevention of recurrent gastroesophageal variceal bleeding in patients with liver cirrhosis. These different treatments, however, have not been compared in patients with idiopathic non-cirrhotic portal hypertension (INCPH)., Aim: To compare the outcomes of TIPS, SED and ET + NSBB in the control of variceal rebleeding in patients with INCPH., Methods: This retrospective study recruited patients from six centers across China. Demographic characteristics, baseline profiles and follow-up clinical outcomes were collected. Post-procedural clinical outcomes, including incidence of rebleeding, hepatic encephalopathy (HE), portal vein thrombosis (PVT) and mortality rates, were compared in the different groups., Results: In total, 81 patients were recruited, with 28 receiving TIPS, 26 SED, and 27 ET + NSBB. No significant differences in demographic and baseline characteristics were found among these three groups before the procedures. After treatment, blood ammonia was significantly higher in the TIPS group; hemoglobin level and platelet count were significantly higher in the SED group ( P < 0.01). Rebleeding rate was significantly higher in the ET + NSBB group ( P < 0.01). Mortality was 3.6%, 3.8% and 14.8% in the TIPS, SED and ET + NSBB groups, respectively, with no significant differences ( P = 0.082). Logistic regression analysis showed that mortality was significantly correlated with rebleeding, HE, portal thrombosis and superior mesenteric vein thrombosis ( P < 0.05)., Conclusion: In patients with INCPH, TIPS and SED were more effective in controlling rebleeding than ET + NSBB, but survival rates were not significantly different among the three groups. Mortality was significantly correlated with rebleeding, HE and PVT., Competing Interests: Conflict-of-interest statement: We have no conflict-of-interest to disclose., (©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.)

Centrosome disjunction occurs in late G2 to facilitate bipolar spindle formation and is mediated by the NIMA-related kinase Nek2. Here, we show that GAS2L1, a microtubule- and F-actin-binding protein required for centrosome disjunction, undergoes Nek2-mediated phosphorylation at Ser352 in G2/M. The phosphorylation is essential for centrosome disjunction in late G2 and for proper spindle assembly and faithful chromosome segregation in mitosis. GAS2L1 contains a calponin-homology (CH) domain and a GAS2-related (GAR) domain, which bind to F-actin and microtubules, respectively. Notably, the CH and GAR domains bind to each other to inhibit the functions of both domains, and Ser352 phosphorylation disrupts the interaction between the two domains and relieves the autoinhibition. We dissected the roles of the GAS2L1 phosphorylation and of centrosome-linker disassembly, which is another Nek2-mediated event, and found that these events together trigger centrosome disjunction. Therefore, our findings demonstrate the concerted Nek2 actions that split the centrosomes in late G2., (© 2020 Au et al.)

Crosstalk between the actin and microtubule cytoskeletons underlies cellular morphogenesis. Interactions between actin filaments and microtubules are particularly important for establishing the complex polarized morphology of neurons. Here, we characterized the neuronal function of growth arrest-specific 2-like 1 (Gas2L1), a protein that can directly bind to actin, microtubules and microtubule plus-end-tracking end binding proteins. We found that Gas2L1 promotes axon branching, but restricts axon elongation in cultured rat hippocampal neurons. Using pull-down experiments and in vitro reconstitution assays, in which purified Gas2L1 was combined with actin and dynamic microtubules, we demonstrated that Gas2L1 is autoinhibited. This autoinhibition is relieved by simultaneous binding to actin filaments and microtubules. In neurons, Gas2L1 primarily localizes to the actin cytoskeleton and functions as an actin stabilizer. The microtubule-binding tail region of Gas2L1 directs its actin-stabilizing activity towards the axon. We propose that Gas2L1 acts as an actin regulator, the function of which is spatially modulated by microtubules., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

The DNA polymerase δ catalytic subunit (PolD1) is a highly conserved protein with established functions in both the nucleus and the cytoplasm: whereas PolD1 participates in the replication and repair of nuclear DNA, it plays a role in the control of cytoplasmic microtubule growth by directly acting on microtubule-nucleator γ-tubulin ring complexes. Here, we show that PolD1 shuttles between the nucleus and the cytoplasm. PolD1 harbors two nuclear localization signals that mediate the active transport of PolD1 to the nucleus; conversely, PolD1 is exported from the nucleus by the exportin CRM1-dependent mechanism, a major nuclear-export pathway that mediates the export of various cargos. These findings suggest that the nucleocytoplasmic distribution of PolD1 is influenced by both the nuclear import and export activities of the protein., (Copyright © 2019. Published by Elsevier Inc.)

Endoplasmic reticulum (ER) membrane contact sites (MCSs) mark positions where endosomes undergo fission for cargo sorting. To define the role of ER at this unique MCS, we targeted a promiscuous biotin ligase to cargo-sorting domains on endosome buds. This strategy identified the ER membrane protein TMCC1, a member of a conserved protein family. TMCC1 concentrates at the ER-endosome MCSs that are spatially and temporally linked to endosome fission. When TMCC1 is depleted, endosome morphology is normal, buds still form, but ER-associated bud fission and subsequent cargo sorting to the Golgi are impaired. We find that the endosome-localized actin regulator Coronin 1C is required for ER-associated fission of actin-dependent cargo-sorting domains. Coronin 1C is recruited to endosome buds independently of TMCC1, while TMCC1/ER recruitment requires Coronin 1C. This link between TMCC1 and Coronin 1C suggests that the timing of TMCC1-dependent ER recruitment is tightly regulated to occur after cargo has been properly sequestered into the bud., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objective: To analyze the recent postoperative and long-term postoperative complications of open-splenectomy and disconnection in patients with portal hypertension. Methods: There were 1 118 cases with portal hypertension who underwent open splenectomy and azygoportal disconnection from April 2010 to September 2015 at Department of Surgery, People's Liberation Army 302 Hospital. Retrospective case investigation and telephone follow-up were conducted in October 2016. All patients had history of upper gastrointestinal bleeding before operation. Short-term complications after surgery were recorded including secondary laparotomy of postoperative abdominal hemostasis, severe infection, intake disorders, liver insufficiency, postoperative portal vein thrombosis and perioperative mortality. Long-term data including postoperative upper gastrointestinal rebleeding, postoperative survival rate and incidence of postoperative malignancy were recorded, too. GraphPad Prism 5 software for data survival analysis and charting. Results: Postoperative short-term complications in 1 118 patients included secondary laparotomy of postoperative abdominal hemostasis(1.8%, 21/1 118), severe infection(2.9%, 32/1 118), intake disorders(1.0%, 11/1 118), liver dysfunction (1.6%, 18/1 118), postoperative portal vein thrombosis(47.1%, 526/1 118)and perioperative mortality(0.5%, 5/1 118). After phone call following-up, 942 patients' long-term data were completed including 1, 3, 5 years postoperative upper gastrointestinal rebleeding rate(4.4%, 12.1%, 17.2%), 1, 3, 5-year postoperative survival rate(97.0%, 93.5%, 90.3%); the incidence of postoperative malignant tumors in 1, 3 and 5 years were 1.7%, 4.4% and 6.2%. Conclusions: Reasonable choosing of surgical indications and timing, proper performing the surgery process, effective conducting perioperative management of portal hypertension are directly related to the patient's short-term prognosis after portal hypertension. Surgical intervention can reduce the rates of patients with upper gastrointestinal rebleeding, improve survival, and do not increase the incidence of malignant tumors.

Recently, there have been many developments and improvements in portal hypertension surgery, but there are still many controversies regarding the surgical indications, the timing of surgery, and the choice of surgical procedures. Minimally invasive laparoscopy and robotics are the leading direction for the development of surgical techniques for portal hypertension. Surgical selection procedures should be based on evidence-based, but guidelines should not be blindly followed. Surgical development needs to strengthen multidisciplinary cooperation, and surgical reform is the driving force for surgical development.

End-binding proteins (EBs) are the core components of microtubule plus end tracking protein complexes, but it is currently unknown whether they are essential for mammalian microtubule organization. Here, by using CRISPR/Cas9-mediated knockout technology, we generated stable cell lines lacking EB2 and EB3 and the C-terminal partner-binding half of EB1. These cell lines show only mild defects in cell division and microtubule polymerization. However, the length of CAMSAP2-decorated stretches at noncentrosomal microtubule minus ends in these cells is reduced, microtubules are detached from Golgi membranes, and the Golgi complex is more compact. Coorganization of microtubules and Golgi membranes depends on the EB1/EB3-myomegalin complex, which acts as membrane-microtubule tether and counteracts tight clustering of individual Golgi stacks. Disruption of EB1 and EB3 also perturbs cell migration, polarity, and the distribution of focal adhesions. EB1 and EB3 thus affect multiple interphase processes and have a major impact on microtubule minus end organization., (© 2017 Yang et al.)

γ-Tubulin ring complexes (γTuRCs) initiate microtubule growth and mediate microtubule attachment at microtubule-organizing centers, such as centrosomes and the Golgi complex. However, the mechanisms that control γTuRC-mediated microtubule nucleation have remained mostly unknown. Here, we show that the DNA polymerase δ catalytic subunit (PolD1) binds directly to γTuRCs and potently inhibits γTuRC-mediated microtubule nucleation. Whereas PolD1 depletion through RNA interference does not influence centrosome-based microtubule growth, the depletion augments microtubule nucleation at the Golgi complex. Conversely, PolD1 overexpression inhibits Golgi-based microtubule nucleation. Golgi-derived microtubules are required for the assembly and maintenance of the proper Golgi structure, and we found that alteration of PolD1 levels affects Golgi structural organization. Moreover, suppression of PolD1 expression impairs Golgi reassembly after nocodazole-induced disassembly and causes defects in Golgi reorientation and directional cell migration. Collectively, these results reveal a mechanism that controls noncentrosomal γTuRC activity and regulates the organization of Golgi-derived microtubules.Microtubule organization requires γ-tubulin ring complexes (γTuRCs), but the mechanisms that control γTuRC-mediated microtubule nucleation are unclear. Here the authors show that the DNA polymerase δ catalytic subunit controls noncentrosomal γTuRC activity and regulates the organization of Golgi-derived microtubules.

Relaxin (RLX) has demonstrated diverse pharmacological effects in various scientific and clinical studies. The emergence of porcine relaxin (pRLX) has raised concerns on the doping potential of pRLX. There have also been speculations in the horseracing industry on its covert use. To control the abuse of pRLX in equine sports, a method to detect pRLX effectively and to provide its unequivocal identification in equine biological samples is required. This paper reports on the detection and confirmation of pRLX in equine plasma by liquid chromatography-high resolution mass spectrometry. pRLX was isolated from equine plasma by immunoaffinity purification using anti-pRLX antibody-coated magnetic beads. Anti-pRLX antibody was generated in-house by purifying antisera from rabbits immunized with pRLX. The isolated pRLX was subjected to reduction of their disulfide bonds to obtain their respective A- and B-chains. The extracts were then further purified and concentrated prior to reversed-phase LC separation and high resolution accurate mass measurement. As detection of the A-chains was far more sensitive than that of the B-chains, the A-chain of pRLX was set as the targets for detection and confirmation. The limit of detection for pRLX was around 0.005 ng/mL (~ 0.86 fM) and the limit of confirmation was around 0.02 ng/mL (~ 3.4 fM). It was observed that method sensitivity was improved at least 5-fold by using an EASY-spray column and emitter in place of the conventional ESI source. The applicability of this method was demonstrated by the identification of pRLX and its metabolites in equine plasma obtained after subcutaneous administration. To our knowledge, this is the first report of a validated mass spectrometry method for the unequivocal confirmation of pRLX in any biological fluid. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Like many complex human diseases, esophageal squamous cell carcinoma (ESCC) is known to cluster in families. Familial ESCC cases often show early onset and worse prognosis than the sporadic cases. However, the molecular genetic basis underlying the development of familial ESCC is mostly unknown. We reported that SLC22A3 is significantly down-regulated in nontumor esophageal tissues from patients with familial ESCC compared with tissues from patients with sporadic ESCCs. A-to-I RNA editing of the SLC22A3 gene results in its reduced expression in the nontumor esophageal tissues of familial ESCCs and is significantly correlated with lymph node metastasis. The RNA-editing enzyme ADAR2 , a familial ESCC susceptibility gene identified by our post hoc genome-wide association study, is positively correlated with the editing level of SLC22A3 Moreover, functional studies showed that SLC22A3 is a metastasis suppressor in ESCC, and deregulation of SLC22A3 facilitates cell invasion and filopodia formation by reducing its direct association with α-actinin-4 (ACTN4), leading to the increased actin-binding activity of ACTN4 in normal esophageal cells. Collectively, we now show that A-to-I RNA editing of SLC22A3 contributes to the early development and progression of familial esophageal cancer in high-risk individuals., Competing Interests: The authors declare no conflict of interest.

Microtubules are polar cytoskeleton filaments that extend via growth at their plus ends. Microtubule plus-end-tracking proteins (+TIPs) accumulate at these growing plus ends to control microtubule dynamics and attachment. The +TIP end-binding protein 1 (EB1) and its homologs possess an autonomous plus-end-tracking mechanism and interact with other known +TIPs, which then recruit those +TIPs to the growing plus ends. A major +TIP class contains the S X IP (Ser- X -Ile-Pro, with X denoting any amino acid residue) motif, known to interact with EB1 and its homologs for plus-end tracking, but the role of S X IP in regulating EB1 activities is unclear. We show here that an interaction of EB1 with the S X IP-containing +TIP CDK5 regulatory subunit-associated protein 2 (CDK5RAP2) regulates several EB1 activities, including microtubule plus-end tracking, dynamics at microtubule plus ends, microtubule and α/β-tubulin binding, and microtubule polymerization. The S X IP motif fused with a dimerization domain from CDK5RAP2 significantly enhanced EB1 plus-end-tracking and microtubule-polymerizing and bundling activities, but the S X IP motif alone failed to do so. An S X IP-binding-deficient EB1 mutant displayed significantly lower microtubule plus-end tracking than the wild-type protein in transfected cells. These results suggest that EB1 cooperates with CDK5RAP2 and perhaps other S X IP-containing +TIPs in tracking growing microtubule tips. We also generated plus-end-tracking chimeras of CDK5RAP2 and the adenomatous polyposis coli protein (APC) and found that overexpression of the dimerization domains interfered with microtubule plus-end tracking of their respective S X IP-containing chimeras. Our results suggest that disruption of S X IP dimerization enables detailed investigations of microtubule plus-end-associated functions of individual S X IP-containing +TIPs., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Mitotic spindle formation and chromosome segregation require timely separation of the two duplicated centrosomes, and this process is initiated in late G2 by centrosome disjunction. Here we report that GAS2L1, a microtubule- and actin-binding protein, associates with the proximal end of mature centrioles and participates in centriole dynamics and centrosome disjunction. GAS2L1 attaches microtubules and actin to centrosomes, and the loss of GAS2L1 inhibits centrosome disjunction in G2 and centrosome splitting induced by depletion of the centrosome linker rootletin. Conversely, GAS2L1 overexpression induces premature centrosome separation, and this activity requires GAS2L1 association with actin, microtubules, and the microtubule end-binding proteins. The centrosome-splitting effect of GAS2L1 is counterbalanced by rootletin, reflecting the opposing actions of GAS2L1 and the centrosome linker. Our work reveals a GAS2L1-mediated centriole-tethering mechanism of microtubules and actin, which provide the forces required for centrosome dynamics and separation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The Golgi apparatus controls the formation of non-centrosomal microtubule arrays important for Golgi organization, polarized transport, cell motility, and cell differentiation. Here, we show that CAMSAP2 stabilizes and attaches microtubule minus ends to the Golgi through a complex of AKAP450 and myomegalin. CLASPs stabilize CAMSAP2-decorated microtubules but are not required for their Golgi tethering. AKAP450 is also essential for Golgi microtubule nucleation, and myomegalin and CDK5RAP2 but not CAMSAP2 contribute to this function. In the absence of centrosomes, AKAP450- and CAMSAP2-dependent pathways of microtubule minus-end organization become dominant, and the presence of at least one of them is needed to maintain microtubule density. Strikingly, a compact Golgi can be assembled in the absence of both centrosomal and Golgi microtubules. However, CAMSAP2- and AKAP450-dependent Golgi microtubules facilitate Golgi reorientation and cell invasion in a 3D matrix. We propose that Golgi-anchored microtubules are important for polarized cell movement but not for coalescence of Golgi membranes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Purpose: To review clinical characteristics of pigmented villonodular synovitis (PVNS) in China., Methods: Electronic medical records (EMR) of four Chinese institutes were queried for patients with histologically proven PVNS between January 2005 and February 2014. Their data were collected including gender, age at diagnosis, clinical presentation, affected site, symptom duration, comorbidities, treatment strategy, recurrence and routine laboratories., Results: A total of 237 patients with biopsy-proven PVNS were investigated. The gender ratio was 1.35 for a female predominance (101 males and 136 females). The average age was 36 years (range, 2 to 83 years). The median delay from initial clinical symptom to diagnosis was 18 months. Main affected areas were the knee (73.84%) and the hip (18.14%). Forty patients had a clear history of joint trauma. Six patients were concurrently diagnosed with PVNS and avascular necrosis (AVN). Five patients suffered from PVNS following implantation of orthopaedic devices including artificial prosthesis, plate and wire. One hundred and twenty-nine patients underwent arthroscopic synovectomy and 108 open synovectomy. Altogether 48 patients (26 males and 22 females) had recurrence of disease. The relapse rate was 24% (knee) and 6.98% (hip), 20.93% (open surgery) and 19.44% (arthroscopy), respectively. Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) rate were elevated in 45.83% and 38.41% of the patients respectively., Conclusions: To our knowledge, this study is the largest sample size of PVNS patients reported as well as the largest sample of PVNS with concurrent AVN reported to date. Our outcomes suggest that PVNS shows a female predominance, occurs mostly between 20-40 years and favors the knee and hip. Recurrence is frequent, particularly in the knee. Serum ESR and CRP may be elevated in some patients. Additionally, the present study supports the theory of an association between PVNS and orthopedic surgery, which is not limited to joint replacement.

Ethnopharmacological Relevance: Eucommia ulmoides Oliv. bark (EU) is a common traditional Chinese herbal medicine for treatment of osteoarthritis (OA), but its therapeutic effect on OA and the underlying mechanisms have not been fully clarified. Our previous study showed that Eucommia ulmoides Oliv. bark aqueous extract (EUE) had a protective effect on cartilage, and this study was aimed to investigate the anti-osteoarthritis effect and mechanisms of EUE in a rat model of osteoarthritis., Materials and Methods: Thirty-two 5-week-old specific pathogen-free Sprague-Dawley rats which were randomized into four even groups (n=8). Group A received sham operation while the OA model was established using the modified Hulth technique in groups B, C and D. For eight weeks after operation, in addition to routine feeding, group A received gavage with deionized water, group B with deionized water, group C with 1.35 g/kg/day EUE, and group D with 2.7 g/kg/day EUE. Eight weeks postoperatively, all of the animals were euthanized for radiological, gross and histopathological observations to evaluate the effect of EUE on OA and to determine its potential mechanisms., Results: Radiological and histopathological observations showed that the articular degenerative changes were significantly more alleviated in groups C and D than in group B, while there were no obviously degenerative manifestations in group A. Mankin׳s scores in groups C and D were significantly lower than in group B (P<0.01). The severity of OA was significantly less in group D than in group C (P<0.01). The IL-1β and IL-6 contents in serum and MMP-3 secretion in articular cartilage were significantly lower in groups C and D than those in group B (P<0.01), and significantly lower in group D than those in group C (P<0.01). Compared with group B, phosphorylated Akt was significantly down-regulated in groups C and D., Conclusions: EUE may inhibit the progression of osteoarthritis by inhibiting the PI3K/Akt pathway to delay cartilage degeneration, reduce inflammatory cytokines and prevent MMP-3 secretion. Therefore, EU is a potential therapeutic agent for OA, but its efficacy is limited., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

The Golgi of mammalian cells is known to be a major microtubule-organizing site that requires microtubules for its organization and protein trafficking. However, the mechanisms underlying the microtubule organization of the Golgi remain obscure. We used immunoprecipitation coupled with mass spectrometry to identify a widely expressed isoform of the poorly characterized muscle protein myomegalin. This newly identified isoform, myomegalin variant 8 (MMG8), localized predominantly to cis-Golgi networks by interacting with AKAP450 (also known as AKAP9), and this interaction with AKAP450 was required for the stability of both proteins. Disrupting MMG8 expression affected endoplasmic reticulum (ER)-to-Golgi trafficking and caused Golgi fragmentation. Furthermore, MMG8 associated with γ-tubulin complexes and with the microtubule plus-end tracking protein EB1 (also known as MAPRE1), and was required for the Golgi localization of these two molecules. On the Golgi, γ-tubulin complexes mediated microtubule nucleation, whereas EB1 functioned in ER-to-Golgi trafficking. These results indicate that MMG8 participates in Golgi microtubule organization and thereby plays a crucial role in the organization and function of the Golgi., (© 2014. Published by The Company of Biologists Ltd.)

As the primary microtubule nucleator in animal cells, the γ-tubulin ring complex (γTuRC) plays a crucial role in microtubule organization, but little is known about how the activity of the γTuRC is regulated. Recently, isolated γTuRC was found to contain NME7, a poorly characterized member of the NME family. Here we report that NME7 is a γTuRC component that regulates the microtubule-nucleating activity of the γTuRC. NME7 contains two putative kinase domains, A and B, and shows autophosphorylating activity. Whereas domain A is involved in the autophosphorylation, domain B is inactive. NME7 interacts with the γTuRC through both A and B domains, with Arg-322 in domain B being crucial to the binding. In association with the γTuRC, NME7 localizes to centrosomes throughout the cell cycle and to mitotic spindles during mitosis. Suppression of NME7 expression does not affect γTuRC assembly or localization to centrosomes, but it does impair centrosome-based microtubule nucleation. Of importance, wild-type NME7 promotes γTuRC-dependent nucleation of microtubules, but kinase-deficient NME7 does so only poorly. These results suggest that NME7 functions in the γTuRC in a kinase-dependent manner to facilitate microtubule nucleation., (© 2014 Liu, Choi, 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).)

Effective treatment for metastatic prostate cancer is critically needed. The present study was aimed at identifying metastasis-driving genes as potential targets for therapy (oncotargets). A differential gene expression profile of metastatic LTL-313H and non-metastatic LTL-313B prostate cancer tissue xenografts, derived from one patient's specimen, was subjected to integrative analysis using the Ingenuity Upstream Regulator Analysis tool. Six candidate master regulatory genes were identified, including GATA2, a gene encoding a pioneer factor, a special transcription factor facilitating the recruitment of additional transcription factors. Elevated GATA2 expression in metastatic prostate cancer tissues correlated with poor patient prognosis. Furthermore, GATA2 gene silencing in human prostate cancer LNCaP cells led to a marked reduction in cell migration, tissue invasion, focal adhesion disassembly and to a dramatic change in cell transcriptomes, indicating that GATA2 plays a critical role in prostate cancer metastasis. As such, GATA2 could represent a prostate cancer metastasis-driving gene and a potential target for therapy of metastatic prostate cancer.

The endoplasmic reticulum (ER) is a continuous membrane network in eukaryotic cells comprising the nuclear envelope, the rough ER, and the smooth ER. The ER has multiple critical functions and a characteristic structure. In this study, we identified a new protein of the ER, TMCC1 (transmembrane and coiled-coil domain family 1). The TMCC family consists of at least 3 putative proteins (TMCC1-3) that are conserved from nematode to human. We show that TMCC1 is an ER protein that is expressed in diverse human cell lines. TMCC1 contains 2 adjacent transmembrane domains near the C-terminus, in addition to coiled-coil domains. TMCC1 was targeted to the rough ER through the transmembrane domains, whereas the N-terminal region and C-terminal tail of TMCC1 were found to reside in the cytoplasm. Moreover, the cytosolic region of TMCC1 formed homo- or hetero-dimers or oligomers with other TMCC proteins and interacted with ribosomal proteins. Notably, overexpression of TMCC1 or its transmembrane domains caused defects in ER morphology. Our results suggest roles of TMCC1 in ER organization.

Microtubule organization by microtubule-organizing centers such as the centrosome requires γ-tubulin, which exists in the γ-tubulin ring complex (γTuRC) that nucleates microtubules. The γTuRC is a ring-shaped, macromolecular complex whose core components are γ-tubulin and the γ-tubulin complex proteins. Despite the recent identification of additional γTuRC components, the molecular composition and regulatory properties of the complex remain poorly understood. The ability to purify the γTuRC at a large scale for characterization may hold a key to understanding the mechanism by which the γTuRC nucleates microtubules. In this chapter, we describe methods to isolate the γTuRC from human cell cultures and to perform assays on the purified γTuRC., (© 2014 Elsevier Inc. All rights reserved.)

The nucleus is a unique organelle that contains essential genetic materials in chromosome territories. The interchromatin space is composed of nuclear subcompartments, which are defined by several distinctive nuclear bodies believed to be factories of DNA or RNA processing and sites of transcriptional and/or posttranscriptional regulation. In this paper, we performed a genome-wide microscopy-based screening for proteins that form nuclear foci and characterized their localizations using markers of known nuclear bodies. In total, we identified 325 proteins localized to distinct nuclear bodies, including nucleoli (148), promyelocytic leukemia nuclear bodies (38), nuclear speckles (27), paraspeckles (24), Cajal bodies (17), Sam68 nuclear bodies (5), Polycomb bodies (2), and uncharacterized nuclear bodies (64). Functional validation revealed several proteins potentially involved in the assembly of Cajal bodies and paraspeckles. Together, these data establish the first atlas of human proteins in different nuclear bodies and provide key information for research on nuclear bodies.

CDK5RAP2 is a centrosomal protein known to be involved in the regulation of the γ-tubulin ring complex and thus the organization of microtubule arrays. However, the mechanism by which CDK5RAP2 is itself recruited to centrosomes is poorly understood. We report here that CDK5RAP2 displays highly dynamic attachment to centrosomes in a microtubule-dependent manner. CDK5RAP2 associates with the retrograde transporter dynein-dynactin and contains a sequence motif that binds to dynein light chain 8. Significantly, disruption of cellular dynein-dynactin function reduces the centrosomal level of CDK5RAP2. These results reveal a key role of the dynein-dynactin complex in the dynamic recruitment of CDK5RAP2 to centrosomes.

This study was aimed to investigate the effect of SNS-032 (C17 H24 N4O2S2) on cell cycle, apoptosis, differentiation and self-renewal of hematopoietic stem cells (HSC) in mice. The self-renewal capability of bone marrow cells was measured by cobblestone forming cell test. The expressions of self-renewal regulation genes, cell cycle-related genes, apoptosis-related genes were measured by real-time PCR. The cell cycle status and apoptosis of HSC and HPC were detected by flow cytometry. The results showed that there was no significant difference of the frequency of HSC between SNS-032 and control group. The expressions of CDK1, CDK2, CDK7 and p27 decreased in HSC (P < 0.05) while the expressions of CDK4, CDK6, p21, p18, p19, Bcl-2, Bax, Puma, p53, Bim1, Sall4 and Notch1 showed no difference between SNS-032 group and control group (P > 0.05). The fraction of viable HSC in each phase of cell cycle remained unchanged after the treatment of SNS-032 (P > 0.05). Furthermore, there was no statistical difference in the apoptotic fractions between control and drug-treated groups (P > 0.05). It is concluded that SNS-032 induce apoptosis of cancer cells. Interestingly, SNS-032 has no significant inhibitory effect on self-renewal and differentiation of normal HSC, as well as no obvious effect inducing apoptosis of normal HSC and HPC.