Prospectively Isolated Tetraspanin+ Neoblasts Are Adult Pluripotent Stem Cells Underlying Planaria Regeneration.

Proliferating cells known as neoblasts include pluripotent stem cells (PSCs) that sustain tissue homeostasis and regeneration of lost body parts in planarians. However, the lack of markers to prospectively identify and isolate these adult PSCs has significantly hampered their characterization. We used single-cell RNA sequencing (scRNA-seq) and single-cell transplantation to address this long-standing issue. Large-scale scRNA-seq of sorted neoblasts unveiled a novel subtype of neoblast (Nb2) characterized by high levels of PIWI-1 mRNA and protein and marked by a conserved cell-surface protein-coding gene, tetraspanin 1 (tspan-1). tspan-1-positive cells survived sub-lethal irradiation, underwent clonal expansion to repopulate whole animals, and when purified with an anti-TSPAN-1 antibody, rescued the viability of lethally irradiated animals after single-cell transplantation. The first prospective isolation of an adult PSC bridges a conceptual dichotomy between functionally and molecularly defined neoblasts, shedding light on mechanisms governing in vivo pluripotency and a source of regeneration in animals. VIDEO ABSTRACT.

Shared and Distinctive Neighborhoods of Emerin and Lamin B Receptor Revealed by Proximity Labeling and Quantitative Proteomics.

Emerin and lamin B receptor (LBR) are abundant transmembrane proteins of the nuclear envelope that are concentrated at the inner nuclear membrane (INM). Although both proteins interact with chromatin and nuclear lamins, they have distinctive biochemical and functional properties. Here, we have deployed proximity labeling using the engineered biotin ligase TurboID (TbID) and quantitative proteomics to compare the neighborhoods of emerin and LBR in cultured mouse embryonic fibroblasts. Our analysis revealed 232 high confidence proximity partners that interact selectively with emerin and/or LBR, 49 of which are shared by both. These included previously characterized NE-concentrated proteins, as well as a host of additional proteins not previously linked to emerin or LBR functions. Many of these are TM proteins of the ER, including two E3 ubiquitin ligases. Supporting these results, we found that 11/12 representative proximity relationships identified by TbID also were detected at the NE with the proximity ligation assay. Overall, this work presents methodology that may be used for large-scale mapping of the landscape of the INM and reveals a group of new proteins with potential functional connections to emerin and LBR.

Mitochondrial oxidative stress in the tumor microenvironment and cancer immunoescape: foe or friend?

The major concept of "oxidative stress" is an excess elevated level of reactive oxygen species (ROS) which are generated from vigorous metabolism and consumption of oxygen. The precise harmonization of oxidative stresses between mitochondria and other organelles in the cell is absolutely vital to cell survival. Under oxidative stress, ROS produced from mitochondria and are the major mediator for tumorigenesis in different aspects, such as proliferation, migration/invasion, angiogenesis, inflammation, and immunoescape to allow cancer cells to adapt to the rigorous environment. Accordingly, the dynamic balance of oxidative stresses not only orchestrate complex cell signaling events in cancer cells but also affect other components in the tumor microenvironment (TME). Immune cells, such as M2 macrophages, dendritic cells, and T cells are the major components of the immunosuppressive TME from the ROS-induced inflammation. Based on this notion, numerous strategies to mitigate oxidative stresses in tumors have been tested for cancer prevention or therapies; however, these manipulations are devised from different sources and mechanisms without established effectiveness. Herein, we integrate current progress regarding the impact of mitochondrial ROS in the TME, not only in cancer cells but also in immune cells, and discuss the combination of emerging ROS-modulating strategies with immunotherapies to achieve antitumor effects.

Prediction of vaginal birth after cesarean delivery in Southeast China: a retrospective cohort study.

BACKGROUND: We aimed to develop and validate a nomogram for effective prediction of vaginal birth after cesarean (VBAC) and guide future clinical application. METHODS: We retrospectively analyzed data from hospitalized pregnant women who underwent trial of labor after cesarean (TOLAC), at the Fujian Provincial Maternity and Children's Hospital, between October 2015 and October 2017. Briefly, we included singleton pregnant women, at a gestational age above 37 weeks who underwent a primary cesarean section, in the study. We then extracted their sociodemographic data and clinical characteristics, and randomly divided the samples into training and validation sets. We employed the least absolute shrinkage and selection operator (LASSO) regression to select variables and construct VBAC success rate in the training set. Thereafter, we validated the nomogram using the concordance index (C-index), decision curve analysis (DCA), and calibration curves. Finally, we adopted the Grobman's model to perform comparisons with published VBAC prediction models. RESULTS: Among the 708 pregnant women included according to inclusion criteria, 586 (82.77%) patients were successfully for VBAC. Multivariate logistic regression models revealed that maternal height (OR, 1.11; 95% CI, 1.04 to 1.19), maternal BMI at delivery (OR, 0.89; 95% CI, 0.79 to 1.00), fundal height (OR, 0.71; 95% CI, 0.58 to 0.88), cervix Bishop score (OR, 3.27; 95% CI, 2.49 to 4.45), maternal age at delivery (OR, 0.90; 95% CI, 0.82 to 0.98), gestational age (OR, 0.33; 95% CI, 0.17 to 0.62) and history of vaginal delivery (OR, 2.92; 95% CI, 1.42 to 6.48) were independently associated with successful VBAC. The constructed predictive model showed better discrimination than that from the Grobman's model in the validation series (c-index 0.906 VS 0.694, respectively). On the other hand, decision curve analysis revealed that the new model had better clinical net benefits than the Grobman's model. CONCLUSIONS: VBAC will aid in reducing the rate of cesarean sections in China. In clinical practice, the TOLAC prediction model will help improve VBAC's success rate, owing to its contribution to reducing secondary cesarean section.

Cellular, ultrastructural and molecular analyses of epidermal cell development in the planarian Schmidtea mediterranea.

The epidermis is essential for animal survival, providing both a protective barrier and cellular sensor to external environments. The generally conserved embryonic origin of the epidermis, but the broad morphological and functional diversity of this organ across animals is puzzling. We define the transcriptional regulators underlying epidermal lineage differentiation in the planarian Schmidtea mediterranea, an invertebrate organism that, unlike fruitflies and nematodes, continuously replaces its epidermal cells. We find that Smed-p53, Sox and Pax transcription factors are essential regulators of epidermal homeostasis, and act cooperatively to regulate genes associated with early epidermal precursor cell differentiation, including a tandemly arrayed novel gene family (prog) of secreted proteins. Additionally, we report on the discovery of distinct and previously undescribed secreted organelles whose production is dependent on the transcriptional activity of soxP-3, and which we term Hyman vesicles.

AXL phosphorylates and up-regulates TNS2 and its implications in IRS-1-associated metabolism in cancer cells.

BACKGROUND: TNS2 is a focal adhesions protein and a binding partner for many proteins, including the receptor tyrosine kinase Axl. Although TNS2 can bind with Axl, the details of their interactions have not been elucidated. TNS2 is involved in IRS-1 signaling pathway. In this study, we confirmed the relationship between TNS2 expression and the expression of Axl, IRS-1, PDK1 and Glut4 in pancreatic cancer patients. METHODS: The expression levels of TNS2, Axl, IRS-1, PDK1 and Glut4 in human cancer cells were measured by Western blot and/or IP-Western blot assays. Paired samples of pancreatic cancer and non-cancer tissues were obtained from 33 patients and were used to construct tissue microarrays. The expression levels of these markers in the tissue microarrays were measured by enzyme-linked Immunohistochemistry assay, and the relationships were analyzed by Pearson's chi-square test and two-tailed t-test analysis. RESULTS: We demonstrated for the first time that TNS2 is a phosphorylation substrate of Axl. Moreover, we found a positive relationship between TNS2 expression and the expression of Axl, IRS-1, PDK1 and Glut4 in pancreatic cancer patients. Based on these results, we suggest that Axl modulates glucose metabolism potentially through TNS2 and IRS-1. We hypothesize that there exists a novel mechanism whereby Axl binds to and phosphorylates TNS2, releasing TNS2 from interaction with IRS-1 and resulting in increased stability of IRS-1. The two key enzymes of aerobic glycolysis (Glut4 and PDK1) were found to be up-regulated by Axl/TNS2/IRS-1 cross-talk and may play a critical role in glucose metabolism of cancer cells. CONCLUSIONS: Our results revealed for the first time that Axl binds to and phosphorylates TNS2 and that Axl/TNS2/IRS-1 cross-talk may potentially play a critical role in glucose metabolism of cancer cells.

ARLTS1 polymorphism is associated with an increased risk of familial cancer: evidence from a meta-analysis.

Adenosine diphosphate (ADP)-ribosylation factor-like tumour suppressor gene 1(ARLTS1) might be associated with an increased risk of several types of familial cancers. However, previous studies have shown that cancer susceptibility is not completely consistent with ARLTS1 polymorphisms, and the precise mechanism remains unknown. Therefore, we conducted a meta-analysis of case-control studies by searching the PubMed, Embase, OVID, Science Direct and Chinese National Knowledge Infrastructure (CNKI) databases. In total, 12 studies met the inclusion criteria and were included in this meta-analysis. Statistical analyses were performed using STATA 11.0 software. Overall, the Cys148Arg T > C variant significantly increased cancer risk (CC vs. TT: OR = 1.27, 95% CI = 1.15-1.41, P < 0.05). The stratification indicated that the Cys148Arg variant is significantly associated with sporadic cancer (CC vs. TT: OR = 1.36, 95% CI = 1.18-1.55) and familial cancer (CC vs. TT: OR = 1.26, 95% CI = 1.12-1.43). Trp149Stop, Pro131Leu, Ser99Ser and Leu132Leu were not correlated with cancer susceptibility. Based on these results, we demonstrated that the ARLTS1 Cys148Arg polymorphism is associated with an increased risk of sporadic cancer and familial cancer, and there were no associations between the other four SNPs (i.e., Trp149Stop, Pro131Leu, Ser99Ser and Leu132Leu) and cancer risk.

Lower frequencies of circulating suppressive regulatory T cells and higher frequencies of CD4+ naïve T cells at baseline are associated with severe immune-related adverse events in immune checkpoint inhibitor-treated melanoma.

BACKGROUND: Immune-related adverse events (irAEs) are major barriers of clinical management and further development of immune checkpoint inhibitors (ICIs) for cancer therapy. Therefore, biomarkers associated with the onset of severe irAEs are needed. In this study, we aimed to identify immune features detectable in peripheral blood and associated with the development of severe irAEs that required clinical intervention. METHODS: We used a 43-marker mass cytometry panel to characterize peripheral blood mononuclear cells from 28 unique patients with melanoma across 29 lines of ICI therapy before treatment (baseline), before the onset of irAEs (pre-irAE) and at the peak of irAEs (irAE-max). In the 29 lines of ICI therapy, 18 resulted in severe irAEs and 11 did not. RESULTS: Unsupervised and gated population analysis showed that patients with severe irAEs had a higher frequency of CD4+ naïve T cells and lower frequency of CD16+ natural killer (NK) cells at all time points. Gated population analysis additionally showed that patients with severe irAEs had fewer T cell immunoreceptor with Ig and ITIM domain (TIGIT+) regulatory T cells at baseline and more activated CD38+ CD4+ central memory T cells (TCM) and CD39+ and Human Leukocyte Antigen-DR Isotype (HLA-DR)+ CD8+ TCM at peak of irAEs. The differentiating immune features at baseline were predominantly seen in patients with gastrointestinal and cutaneous irAEs and type 1 diabetes. Higher frequencies of CD4+ naïve T cells and lower frequencies of CD16+ NK cells were also associated with clinical benefit to ICI therapy. CONCLUSIONS: This study demonstrates that high-dimensional immune profiling can reveal novel blood-based immune signatures associated with risk and mechanism of severe irAEs. Development of severe irAEs in melanoma could be the result of reduced immune inhibitory capacity pre-ICI treatment, resulting in more activated TCM cells after treatment.

Disrupting CD38-driven T cell dysfunction restores sensitivity to cancer immunotherapy.

A central problem in cancer immunotherapy with immune checkpoint blockade (ICB) is the development of resistance, which affects 50% of patients with metastatic melanoma1,2. T cell exhaustion, resulting from chronic antigen exposure in the tumour microenvironment, is a major driver of ICB resistance3. Here, we show that CD38, an ecto-enzyme involved in nicotinamide adenine dinucleotide (NAD+) catabolism, is highly expressed in exhausted CD8+ T cells in melanoma and is associated with ICB resistance. Tumour-derived CD38hiCD8+ T cells are dysfunctional, characterised by impaired proliferative capacity, effector function, and dysregulated mitochondrial bioenergetics. Genetic and pharmacological blockade of CD38 in murine and patient-derived organotypic tumour models (MDOTS/PDOTS) enhanced tumour immunity and overcame ICB resistance. Mechanistically, disrupting CD38 activity in T cells restored cellular NAD+ pools, improved mitochondrial function, increased proliferation, augmented effector function, and restored ICB sensitivity. Taken together, these data demonstrate a role for the CD38-NAD+ axis in promoting T cell exhaustion and ICB resistance, and establish the efficacy of CD38 directed therapeutic strategies to overcome ICB resistance using clinically relevant, patient-derived 3D tumour models.

Evaluation of effect of reducing additives during vitrification via simulation and experiment.

This study investigates how reducing additives governed the vitrification of prepared specimens. In the experiments, pure CaO/CaCO3 and SiO2 served as the major components of glassy matrix (basicity = mass ratio of CaO/SiO2 = 2/3) with doping of hazardous metals (Cr Cu, and Ni). The substitution ratio of CaCO3 for CaO was used as an operating parameter. The specimens were vitrified at 1400 degrees C and a sequential extraction protocol was used to determine the phase distribution of Cr, Cu, and Ni. The volume fractions of crystalline and amorphous phases were measured using semiquantitative x-ray diffraction (XRD) analysis. A commercial software package (HSC Chemistry 6.0) was used to simulate the experiment to acquire additional information. The simulation results showed the addition of CaCO3 generated CO and CO2 at high temperature. This reducing atmosphere might enhance Cu and Ni to be easily separated from slags and elevated the levels of Cu and Ni in ingots. At higher CaCO3 mol(%), the polymerization of silicate (from sorosilicate to inosilicate) in slag rose and the CaSiO3 amount increased. In addition, the immobilization of metals and the acid resistance of slags were improved. The results indicate that CaCO3 addition is favorable for increasing the metal level in ingots and the metal encapsulation in slag in vitrification.

Aldolase B-driven lactagenesis and CEACAM6 activation promote cell renewal and chemoresistance in colorectal cancer through the Warburg effect.

Colorectal cancer (CRC) is a prevalent malignancy worldwide and is associated with a high mortality rate. Changes in bioenergy metabolism, such as the Warburg effect, are often observed in CRC. Aldolase B (ALDOB) has been identified as a potential regulator of these changes, but its exact role in CRC cell behavior and bioenergetic homeostasis is not fully understood. To investigate this, two cohorts of CRC patients were analyzed independently. The results showed that higher ALDOB expression was linked to unfavorable prognosis, increased circulating carcinoembryonic antigen (CEA) levels, and altered bioenergetics in CRC. Further analysis using cell-based assays demonstrated that ALDOB promoted cell proliferation, chemoresistance, and increased expression of CEA in CRC cells. The activation of pyruvate dehydrogenase kinase-1 (PDK1) by ALDOB-induced lactagenesis and secretion, which in turn mediated the effects on CEA expression. Secreted lactate was found to enhance lactate dehydrogenase B (LDHB) expression in adjacent cells and to be a crucial modulator of ALDOB-mediated phenotypes. Additionally, the effect of ALDOB on CEA expression was downstream of the bioenergetic changes mediated by secreted lactate. The study also identified CEA cell adhesion molecule-6 (CEACAM6) as a downstream effector of ALDOB that controlled CRC cell proliferation and chemoresistance. Notably, CEACAM6 activation was shown to enhance protein stability through lysine lactylation, downstream of ALDOB-mediated lactagenesis. The ALDOB/PDK1/lactate/CEACAM6 axis plays an essential role in CRC cell behavior and bioenergetic homeostasis, providing new insights into the involvement of CEACAM6 in CRC and the Warburg effect. These findings may lead to the development of new treatment strategies for CRC patients.

Comparative membrane proteomics reveals diverse cell regulators concentrated at the nuclear envelope.

The nuclear envelope (NE) is a subdomain of the ER with prominent roles in nuclear organization, largely mediated by its distinctive protein composition. We developed methods to reveal novel, low abundance transmembrane (TM) proteins concentrated at the NE relative to the peripheral ER. Using label-free proteomics that compared isolated NEs to cytoplasmic membranes, we first identified proteins with apparent NE enrichment. In subsequent authentication, ectopically expressed candidates were analyzed by immunofluorescence microscopy to quantify their targeting to the NE in cultured cells. Ten proteins from a validation set were found to associate preferentially with the NE, including oxidoreductases, enzymes for lipid biosynthesis and regulators of cell growth and survival. We determined that one of the validated candidates, the palmitoyltransferase Zdhhc6, modifies the NE oxidoreductase Tmx4 and thereby modulates its NE levels. This provides a functional rationale for the NE concentration of Zdhhc6. Overall, our methodology has revealed a group of previously unrecognized proteins concentrated at the NE and additional candidates. Future analysis of these can potentially unveil new mechanistic pathways associated with the NE.

Comparative membrane proteomics reveals diverse cell regulators concentrated at the nuclear envelope.

The nuclear envelope (NE) is a subdomain of the ER with prominent roles in nuclear organization, which are largely mediated by its distinctive protein composition. We developed methods to reveal low-abundance transmembrane (TM) proteins concentrated at the NE relative to the peripheral ER. Using label-free proteomics that compared isolated NEs with cytoplasmic membranes, we first identified proteins with apparent NE enrichment. In subsequent authentication, ectopically expressed candidates were analyzed by immunofluorescence microscopy to quantify their targeting to the NE in cultured cells. Ten proteins from a validation set were found to associate preferentially with the NE, including oxidoreductases, enzymes for lipid biosynthesis, and regulators of cell growth and survival. We determined that one of the validated candidates, the palmitoyltransferase Zdhhc6, modifies the NE oxidoreductase Tmx4 and thereby modulates its NE levels. This provides a functional rationale for the NE concentration of Zdhhc6. Overall, our methodology has revealed a group of previously unrecognized proteins concentrated at the NE and additional candidates. Future analysis of these can potentially unveil new mechanistic pathways associated with the NE.

Simultaneous prospective purification of adult subventricular zone neural stem cells and their progeny.

The ability to prospectively isolate adult neural stem cells and their progeny is crucial to study their biology and therapeutic potential. Stem cells in adult mammalian neurogenic niches are a subset of astrocytes. A major limitation in the field has been the inability to distinguish stem cell astrocytes from niche astrocytes. Here, we show that epidermal growth factor receptor (EGFR)-positive subventricular-zone (SVZ) astrocytes are activated stem cells that are eliminated by antimitotic treatment. We developed a simple strategy to simultaneously purify cells at different stages of the adult SVZ stem cell lineage by using FACS. This method combines the use of fluorescent EGF ligand, CD24, and GFP expression in GFAP::GFP transgenic mice and allows the simultaneous purification of activated stem cell astrocytes (GFP(+)EGFR(+)CD24(-)), niche astrocytes (GFP(+)EGFR(-)CD24(-)), transit amplifying cells (GFP(-)EGFR(+)CD24(-)), and neuroblasts (GFP(-)EGFR(-)CD24(low)). One in three EGFR(+) astrocytes gives rise to neurospheres in vitro, a 20-fold enrichment over unsorted cells. Importantly, these cells constitute the neurosphere-forming population among SVZ astrocytes. This approach will be of great utility for future functional and molecular studies of the SVZ stem cell lineage.

Tanshinone IIA Microemulsion Protects against Cerebral Ischemia Reperfusion Injury via Regulating H3K18ac and H4K8ac In Vivo and In Vitro.

Tanshinone IIA (TanIIA) has neuroprotective effects against cerebral ischemia reperfusion injury (CIRI), but its clinical application is limited due to poor water solubility and robust first pass elimination property. In this study, we developed microemulsion loaded with TanIIA (TanIIA ME) to break through these limitations, and explored the neuroprotective effect of TanIIA ME against CIRI and the epigenetic regulation mechanism of this neuroprotection. In vivo, middle cerebral artery occlusion (MCAO) models were treated with TanIIA ME and TanIIA solution or sodium valproate as a control. The effect of TanIIA ME on HDAC activity was determined by ELISA assay. In addition, we used primary hippocampal neurons to establish oxygen-glucose deprivation and reoxygenation (OGD/R) models. Lactate dehydrogenase (LDH) assay and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were performed to investigate the neuroprotective efficacy of TanIIA ME. Subsequently, the expression of H3K18ac, H4K8ac, NMDAR1, caspase-3, and MAP-2 were investigated in MCAO or OGD/R models treated with TanIIA ME, TanIIA solution or sodium valproate. In vivo experimental results indicated that TanIIA ME significantly reduced neurological scores, infarction volume, and HDAC activity compared with TanIIA solution and MCAO group, accompanied by upregulation of H3K18ac, H4K8ac, and MAP-2 expression and downregulation of NMDAR1 and caspase-3 expression. Additionally, in OGD/R models, the results demonstrated that TanIIA ME treatment had a better neuroprotective effect along with increased H3K18ac, H4K8ac, and MAP-2 expression and decreased NMDAR1 and caspase-3 expression, compared with the other treatments except sodium valproate. Overall, TanIIA ME treatment exhibited superior efficacy in protecting against CIRI through mechanisms that might involve the inhibition of NMDAR1 and caspase-3 expression and the enhancement of MAP-2 expression by regulating histone H3K18 and H4K8 acetylation. Thus, TanIIA ME could be potentially used to develop a promising drug for the treatment of ischemic stroke.

Biosorption of lead, Mercury, and cadmium ions by Aspergillus terreus immobilized in a natural matrix.

Our aim was to investigate the biosorption of Pb2+, Hg2+, Cd2 from aqueous solution by Aspergillus terreus (both free and immobilized on loofa sponge discs). Our results show that the adsorption capacity of fungal biomass on loofa sponge (FBLS) is superior to free fungal biomass (FFB). The adsorption selectivity by FBLS was in the order Pb2+>Hg2+>Cd2+. The maximum metal ions adsorbed was 247.2, 37.7, 23.8 mg/g FBLS for Pb2+, Hg2+ and Cd2+, respectively. Metal uptake by FBLS was affected by the pH of the metal solution, but independent of temperature (10-50 degrees C). The Langmuir model was more suitable than the Freundlich model to describe the biosorption process of FBLS. The regenerated FBLS was found to be effective for repeated use for five cycles without significant loss in adsorption capacity. This research demonstrates that FBLS possesses excellent capacity for Pb2+ biosorption from aqueous solution and industrial wastewaters.

Mitochondrial Lon-induced mtDNA leakage contributes to PD-L1-mediated immunoescape via STING-IFN signaling and extracellular vesicles.

BACKGROUND: Mitochondrial Lon is a chaperone and DNA-binding protein that functions in protein quality control and stress response pathways. The level of Lon regulates mitochondrial DNA (mtDNA) metabolism and the production of mitochondrial reactive oxygen species (ROS). However, there is little information in detail on how mitochondrial Lon regulates ROS-dependent cancer immunoescape through mtDNA metabolism in the tumor microenvironment (TME). METHODS: We explored the understanding of the intricate interplay between mitochondria and the innate immune response in the inflammatory TME. RESULTS: We found that oxidized mtDNA is released into the cytosol when Lon is overexpressed and then it induces interferon (IFN) signaling via cGAS-STING-TBK1, which upregulates PD-L1 and IDO-1 expression to inhibit T-cell activation. Unexpectedly, upregulation of Lon also induces the secretion of extracellular vehicles (EVs), which carry mtDNA and PD-L1. Lon-induced EVs further induce the production of IFN and IL-6 from macrophages, which attenuates T-cell immunity in the TME. CONCLUSIONS: The levels of mtDNA and PD-L1 in EVs in patients with oral cancer function as a potential diagnostic biomarker for anti-PD-L1 immunotherapy. Our studies provide an insight into the immunosuppression on mitochondrial stress and suggest a therapeutic synergy between anti-inflammation therapy and immunotherapy in cancer.

Stem cells: from epigenetics to microRNAs.

The complexity and cellular diversity of the adult brain arises from the proliferation and differentiation of a small number of stem cells. The intrinsic state of stem cells depends on their spatial and temporal history and affects their responsiveness to extrinsic signals from the microenvironment. Stem cell self-renewal and differentiation along neuronal and glial lineages are defined by the dynamic interplay between transcription, epigenetic control, and posttranscriptional regulators, including microRNAs, whose key role in stem cell biology is just emerging.

Identification of new transmembrane proteins concentrated at the nuclear envelope using organellar proteomics of mesenchymal cells.

The double membrane nuclear envelope (NE), which is contiguous with the ER, contains nuclear pore complexes (NPCs) - the channels for nucleocytoplasmic transport, and the nuclear lamina (NL) - a scaffold for NE and chromatin organization. Since numerous human diseases linked to NE proteins occur in mesenchyme-derived cells, we used proteomics to characterize NE and other subcellular fractions isolated from mesenchymal stem cells and from adipocytes and myocytes. Based on spectral abundance, we calculated enrichment scores for proteins in the NE fractions. We demonstrated by quantitative immunofluorescence microscopy that five little-characterized proteins with high enrichment scores are substantially concentrated at the NE, with Itprip exposed at the outer nuclear membrane, Smpd4 enriched at the NPC, and Mfsd10, Tmx4, and Arl6ip6 likely residing in the inner nuclear membrane. These proteins provide new focal points for studying the functions of the NE. Moreover, our datasets provide a resource for evaluating additional potential NE proteins.

PTBP1-mediated regulation of AXL mRNA stability plays a role in lung tumorigenesis.

AXL is expressed in many types of cancer and promotes cancer cell survival, metastasis and drug resistance. Here, we focus on identifying modulators that regulate AXL at the mRNA level. We have previously observed that the AXL promoter activity is inversely correlated with the AXL expression levels, suggesting that post-transcriptional mechanisms exist that down-regulate the expression of AXL mRNA. Here we show that the RNA binding protein PTBP1 (polypyrimidine tract-binding protein) directly targets the 5'-UTR of AXL mRNA in vitro and in vivo. Moreover, we also demonstrate that PTBP1, but not PTBP2, inhibits the expression of AXL mRNA and the RNA recognition motif 1 (RRM1) of PTBP1 is crucial for this interaction. To clarify how PTBP1 regulates AXL expression at the mRNA level, we found that, while the transcription rate of AXL was not significantly different, PTBP1 decreased the stability of AXL mRNA. In addition, over-expression of AXL may counteract the PTBP1-mediated apoptosis. Knock-down of PTBP1 expression could enhance tumor growth in animal models. Finally, PTBP1 was found to be negatively correlated with AXL expression in lung tumor tissues in Oncomine datasets and in tissue micro-array (TMA) analysis. In conclusion, we have identified a molecular mechanism of AXL expression regulation by PTBP1 through controlling the AXL mRNA stability. These findings may represent new thoughts alternative to current approaches that directly inhibit AXL signaling and may eventually help to develop novel therapeutics to avoid cancer metastasis and drug resistance.