Water chemistry and stable isotope characteristics of subsidence lakes in coal mining areas, Eastern China.

Many subsidence lakes have formed in eastern China as a result of underground coal mining. These coal mining-related subsidence lakes vary in their formation time and connectivity with rivers. These factors may influence the water chemistry and hydrogen and oxygen stable isotope characteristics of the lake water. This study collected and tested subsidence lake water, atmospheric precipitation, river water, and shallow groundwater in the study area. The results showed that the water chemical types of the subsidence lake water and river water are Cl-Na and HCO3·Cl-Na and that the water chemical types of the shallow groundwater are mainly HCO3·Cl-Na and HCO3·Cl-Ca. There are no significant differences in the water chemical characteristics of subsidence lakes with different subsidence ages and types. The major ions in each water body mainly come from evaporite dissolution and silicate weathering, and ion exchange occurs. Reverse ion exchange occurs in some shallow groundwater samples. The stable isotopes of hydrogen and oxygen in the subsidence lake water, river water, and shallow groundwater are distributed along a straight line with a slope less than that of the LMWL, indicating that these water bodies have a common source, namely, precipitation. With increases in the formation time of the subsidence lakes, the heavy isotopes in the lake water gradually become depleted, and the d value gradually increases, mainly driven by precipitation dilution, weakening evaporation, river recharge, and groundwater recharge. The isotopic values of different types of lakes with the same subsidence time differ little. The research results may provide scientific guidance for the rational development and utilization of water resources in coal mining subsidence areas, enrich the study of the hydrological cycle in the area, and are of great significance for the protection of the local water balance and water environment.

MAP3K2-regulated intestinal stromal cells define a distinct stem cell niche.

Intestinal stromal cells are known to modulate the propagation and differentiation of intestinal stem cells1,2. However, the precise cellular and molecular mechanisms by which this diverse stromal cell population maintains tissue homeostasis and repair are poorly understood. Here we describe a subset of intestinal stromal cells, named MAP3K2-regulated intestinal stromal cells (MRISCs), and show that they are the primary cellular source of the WNT agonist R-spondin 1 following intestinal injury in mice. MRISCs, which are epigenetically and transcriptomically distinct from subsets of intestinal stromal cells that have previously been reported3-6, are strategically localized at the bases of colon crypts, and function to maintain LGR5+ intestinal stem cells and protect against acute intestinal damage through enhanced R-spondin 1 production. Mechanistically, this MAP3K2 specific function is mediated by a previously unknown reactive oxygen species (ROS)-MAP3K2-ERK5-KLF2 axis to enhance production of R-spondin 1. Our results identify MRISCs as a key component of an intestinal stem cell niche that specifically depends on MAP3K2 to augment WNT signalling for the regeneration of damaged intestine.

Trends of Bead Counting-Based Technologies Toward the Detection of Disease-Related Biomarkers.

Nowadays, the biomolecular assay platforms built-up based on bead counting technologies have emerged to be powerful tools for the sensitive and high-throughput detection of disease biomarkers. In this mini-review, we classified the bead counting technologies into statistical counting platforms and digital counting platforms. The design principles, the readout strategies, as well as the pros and cons of these platforms are introduced in detail. Finally, we point out that the digital bead counting technologies will lead the future trend for the absolute quantification of critical biomarkers, and the integration of new signal amplification approaches and routine optical/clinical instruments may provide new opportunities in building-up easily accessible digital assay platforms.

Prognostic value of metabolic tumor volume and total lesion glycolysis from ¹8F-FDG PET/CT in lymph node metastases and risk stratification of endometrial carcinoma.

OBJECTIVE: To investigate the prognostic value of metabolic tumor volume (MTV) and total lesion glycolysis (TLG), measured by preoperative ¹8F-fluorodeoxyglucose positron emission tomography/computed tomography (¹8F-FDG PET/CT), in risk stratification of patients with endometrial carcinoma (EC). METHODS: The patients with pathological diagnosis of EC who underwent preoperative ¹8F-FDG PET/CT imaging were retrospectively selected for analysis of the prognostic values of PET parameters in risk classification and lymph node metastases (LNMs). Receiver-operating-characteristic analysis was used to analyze the correlation of PET parameters cutoff values with deep myometrial invasion (MI), lymphovascular space involvement and LNM for prognostic values in risk stratification. RESULTS: The sensitivity, specificity, positive predictive value, negative predictive value and accuracy for detection of LNM are 83.3%, 99.7%, 90.9%, 99.5% and 99.2%, respectively. The MTV and TLG of primary lesion of EC in the patients with LNM are notably higher than those in patients without LNM, p<0.010. The MTV and TLG of the EC primary lesions in high-risk patients are significantly higher than those in low-risk patients (p<0.010), but the maximum standardized uptake value (SUVmax) is not. The MTV and TLG of primary lesions were superior to SUVmax for predicting of deep MI, LNM and high-risk of EC (p<0.005). CONCLUSION: MTV and TLG of primary lesions are more valuable in predicting risk stratification of EC patients. Preoperative ¹8F-FDG PET/CT imaging is useful in predicting the LNM of EC and may help guide pelvic lymphadenectomy to avoid unnecessary pelvic lymphadenectomy in EC patients with low-risk stratification.

Author Correction: Sin1 phosphorylation impairs mTORC2 complex integrity and inhibits downstream Akt signalling to suppress tumorigenesis.

In the version of this Article originally published, the labels for Rictor and mTOR in the whole cell lysate (WCL) blots were swapped in Fig. 3b and the mTOR blot was placed upside down. Unprocessed blots of mTOR were also missing from Supplementary Fig. 9. The corrected Figs are shown below. In addition, control blots for the mTOR antibody (Cell Signalling Technology #2972) were also missing. These are now provided below, as Fig. 9, and show that the lower band is likely non-specific.

Sin1/mTORC2 regulate B cell growth and metabolism by activating mTORC1 and Myc.

Proper control of B cell growth and metabolism is crucial for B-cell-mediated immunity, but the underlying molecular mechanisms remain incompletely understood. In this study, Sin1, a key component of mTOR complex 2 (mTORC2), specifically regulates B cell growth and metabolism. Genetic ablation of Sin1 in B cells reduces the cell size at either the transitional stage or upon antigen stimulation and severely impairs metabolism. Sin1 deficiency also severely impairs B-cell proliferation, antibody responses, and anti-viral immunity. At the molecular level, Sin1 controls the expression and stability of the c-Myc protein and maintains the activity of mTORC1 through the Akt-dependent inactivation of GSK3 and TSC1/2, respectively. Therefore, our study reveals a novel and specific role for Sin1 in coordinating the activation of mTORC2 and mTORC1 to control B cell growth and metabolism.

Metabolic regulation of T cell development by Sin1-mTORC2 is mediated by pyruvate kinase M2.

Glucose metabolism plays a key role in thymocyte development. The mammalian target of rapamycin complex 2 (mTORC2) is a critical regulator of cell growth and metabolism, but its role in early thymocyte development and metabolism has not been fully studied. We show here that genetic ablation of Sin1, an essential component of mTORC2, in T lineage cells results in severely impaired thymocyte development at the CD4-CD8- double negative (DN) stages but not at the CD4+CD8+ double positive (DP) or later stages. Notably, Sin1-deficient DN thymocytes show markedly reduced proliferation and glycolysis. Importantly, we discover that the M2 isoform of pyruvate kinase (PKM2) is a novel and crucial Sin1 effector in promoting DN thymocyte development and metabolism. At the molecular level, we show that Sin1-mTORC2 controls PKM2 expression through an AKT-dependent PPAR-γ nuclear translocation. Together, our study unravels a novel mTORC2-PPAR-γ-PKM2 pathway in immune-metabolic regulation of early thymocyte development.

Oxidative NHC catalysis: direct activation of ß sp3 carbons of saturated acid chlorides.

The first activation of saturated acid chlorides by oxidative N-heterocyclic carbene catalysis has been successfully utilized to synthesize enantio-enriched spirooxindole lactones and δ-lactones. The reaction involves the transformation of the ß sp3 carbon of saturated acid chlorides into an electrophilic carbon as a key step. The product was obtained in excellent yield and stereoselectivity.

Active mTORC2 Signaling in Naive T Cells Suppresses Bone Marrow Homing by Inhibiting CXCR4 Expression.

Recirculation of naive T cells between secondary lymphoid organs to receive survival cues and scan for signs of infection or other pathologic conditions is important for immune homeostasis and effective immune responses. Although the mechanisms that specifically guide the entry of naive T cells into secondary lymphoid organs are well studied, the mechanisms that keep them from fluxing into inappropriate or undesirable compartments, such as healthy tissues or bone marrow, are less well understood. In this study, we report an unexpected finding that under steady state, bone marrow homing of naive T cells is actively suppressed by mTORC2 signaling. We found that in mice, T cell-specific deletion of an essential mTORC2 component Sin1 results in increased accumulation of naive T cells in the bone marrow. Mechanistically, we show that loss of mTORC2 signaling in naive T cells results in enhanced FOXO1 activity, which leads to increased CXCR4 expression and chemotactic response to CXCL12, a key chemokine that promotes bone marrow homing and retention of T cells. Together, the results of our study reveal a novel role of mTORC2 in T cell homeostasis via active suppression of naive T cell bone marrow homing by the mTORC2-FOXO1-CXCR4 axis.

Disrupted in schizophrenia 1 (DISC1) inhibits glioblastoma development by regulating mitochondria dynamics.

Glioblastoma(GBM) is one of the most common and aggressive malignant primary tumors of the central nervous system and mitochondria have been proposed to participate in GBM tumorigenesis. Previous studies have identified a potential role of Disrupted in Schizophrenia 1 (DISC1), a multi-compartmentalized protein, in mitochondria. But whether DISC1 could regulate GBM tumorigenesis via mitochondria is still unknown. We determined the expression level of DISC1 by both bioinformatics analysis and tissue analysis, and found that DISC1 was highly expressed in GBM. Knocking down of DISC1 by shRNA in GBM cells significantly inhibited cell proliferation both in vitro and in vivo. In addition, down-regulation of DISC1 decreased cell migration and invasion of GBM and self renewal capacity of glioblastoma stem-like cells. Furthermore, multiple independent rings or spheres could be observed in mitochondria in GBM depleted of DISC1, while normal filamentous morphology was observed in control cells, demonstrating that DISC1 affected the mitochondrial dynamic. Dynamin-related protein 1 (Drp1) was reported to contribute to mitochondrial dynamic regulation and influence glioma cells proliferation and invasion by RHOA/ ROCK1 pathway. Our data showed a significant decrease of Drp1 both in mRNA and protein level in GBM lack of DISC1, indicating that DISC1 maybe affect the mitochondrial dynamic by regulating Drp1. Taken together, our findings reveal that DISC1 affects glioblastoma cell development via mitochondria dynamics partly by down regulation of Drp1.

MEKK2 mediates an alternative ß-catenin pathway that promotes bone formation.

Proper tuning of ß-catenin activity in osteoblasts is required for bone homeostasis, because both increased and decreased ß-catenin activity have pathologic consequences. In the classical pathway for ß-catenin activation, stimulation with WNT ligands suppresses constitutive phosphorylation of ß-catenin by glycogen synthase kinase 3ß, preventing ß-catenin ubiquitination and proteasomal degradation. Here, we have found that mitogen-activated protein kinase kinase kinase 2 (MAP3K2 or MEKK2) mediates an alternative pathway for ß-catenin activation in osteoblasts that is distinct from the canonical WNT pathway. FGF2 activates MEKK2 to phosphorylate ß-catenin at serine 675, promoting recruitment of the deubiquitinating enzyme, ubiquitin-specific peptidase 15 (USP15). USP15 in turn prevents the basal turnover of ß-catenin by inhibiting its ubiquitin-dependent proteasomal degradation, thereby enhancing WNT signaling. Analysis of MEKK2-deficient mice and genetic interaction studies between Mekk2- and ß-catenin-null alleles confirm that this pathway is an important physiologic regulator of bone mass in vivo. Thus, an FGF2/MEKK2 pathway mediates an alternative nonclassical pathway for ß-catenin activation, and this pathway is a key regulator of bone formation by osteoblasts.

Structure and vascular function of MEKK3-cerebral cavernous malformations 2 complex.

Cerebral cavernous malformations 2 (CCM2) loss is associated with the familial form of CCM disease. The protein kinase MEKK3 (MAP3K3) is essential for embryonic angiogenesis in mice and interacts physically with CCM2, but how this interaction is mediated and its relevance to cerebral vasculature are unknown. Here we report that Mekk3 plays an intrinsic role in embryonic vascular development. Inducible endothelial Mekk3 knockout in neonatal mice is lethal due to multiple intracranial haemorrhages and brain blood vessels leakage. We discover direct interaction between CCM2 harmonin homology domain (HHD) and the N terminus of MEKK3, and determine a 2.35 Å cocrystal structure. We find Mekk3 deficiency impairs neurovascular integrity, which is partially dependent on Rho-ROCK signalling, and that disruption of MEKK3:CCM2 interaction leads to similar neurovascular leakage. We conclude that CCM2:MEKK3-mediated regulation of Rho signalling is required for maintenance of neurovascular integrity, unravelling a mechanism by which CCM2 loss leads to disease.

Metabolic mechanism of phenyllactic acid naturally occurring in Chinese pickles.

Phenyllactic acid, a phenolic acid phytochemical with the antimicrobial activity, was rarely reported in food besides honey and sourdough. This study evidenced a new food source of phenyllactic acid and elucidated its metabolic mechanism. Phenyllactic acid naturally occurred in Chinese pickles with concentrations ranged from 0.02 to 0.30 mM in 23 pickle samples including homemade and commercial ones. Then, lactic acid bacteria capable of metabolizing phenyllactic acid were screened from each homemade pickle and a promising strain was characterized as Lactobacillus plantarum. Moreover, the investigation of the metabolic mechanism of phenyllactic acid in pickles suggested that the yield of phenyllactic acid was positively related to the content of phenylalanine in food, and the addition of phenylalanine as precursor substance could significantly promote the production of phenyllactic acid. This investigation could provide some insights into the accumulation of phenyllactic acid in pickle for long storage life.

Preparation of polysaccharides from cyanobacteria Nostoc commune and their antioxidant activities.

In this study, water soluble polysaccharides were prepared from cyanobacteria Nostoc commune by water extraction. Factors affecting the polysaccharide yields were investigated, and the optimum extraction conditions were determined as follows: time, 4h; temperature, 90 °C; the ratio of liquid to solid, 60:1 (v/w); and extraction times, 4. The extract was filtered, concentrated to ∼10% (w/v), precipitated with 3 volumes of ethanol, freeze-dried, and ground to yield a water soluble power. The polysaccharide content of the product was 96.7%, and the yield was 9.18% (w/w). Fourier transform infrared spectra demonstrated that the product samples were mainly composed of polysaccharides. The polysaccharides showed high hydroxyl radical scavenging activity (92.71%) and reducing capacity (0.445) at the concentration of 10 mg/mL.

Sin1 phosphorylation impairs mTORC2 complex integrity and inhibits downstream Akt signalling to suppress tumorigenesis.

The mechanistic target of rapamycin (mTOR) functions as a critical regulator of cellular growth and metabolism by forming multi-component, yet functionally distinct complexes mTORC1 and mTORC2. Although mTORC2 has been implicated in mTORC1 activation, little is known about how mTORC2 is regulated. Here we report that phosphorylation of Sin1 at Thr 86 and Thr 398 suppresses mTORC2 kinase activity by dissociating Sin1 from mTORC2. Importantly, Sin1 phosphorylation, triggered by S6K or Akt, in a cellular context-dependent manner, inhibits not only insulin- or IGF-1-mediated, but also PDGF- or EGF-induced Akt phosphorylation by mTORC2, demonstrating a negative regulation of mTORC2 independent of IRS-1 and Grb10. Finally, a cancer-patient-derived Sin1-R81T mutation impairs Sin1 phosphorylation, leading to hyper-activation of mTORC2 by bypassing this negative regulation. Together, our results reveal a Sin1-phosphorylation-dependent mTORC2 regulation, providing a potential molecular mechanism by which mutations in the mTORC1-S6K-Sin1 signalling axis might cause aberrant hyper-activation of the mTORC2-Akt pathway, which facilitates tumorigenesis.

Preparation of xanthan-derived oligosaccharides and their hydroxyl radical scavenging activity.

In this study, the xanthan-derived oligosacchrides were prepared by hydrolysis of xanthan using hydrogen peroxide (H(2)O(2)) in alkaline solution. The hydrolysis process was monitored by the dextrose equivalent values of the hydrolysates. The optimal hydrolysis conditions were found to be reaction time 24 h, temperature 65 °C, H(2)O(2) concentration 1.6% (v/v), and NaOH concentration, 3 M. Under these optimized conditions, the maximum dextrose equivalent value (7.53%) was observed. The structure of the hydrolysates were characterized by Fourier-transform infrared spectroscopy. The xanthan-derived oligosacchrides showed high hydroxyl radical scavenging activity. The xanthan-derived oligosacchrides content of the product and the yield were 96.8% and 95.7% (w/w), respectively.

mTOR complex 2 regulates proper turnover of insulin receptor substrate-1 via the ubiquitin ligase subunit Fbw8.

The mammalian target of rapamycin (mTOR) integrates signals from nutrients and insulin via two distinct complexes, mTORC1 and mTORC2. Disruption of mTORC2 impairs the insulin-induced activation of Akt, an mTORC2 substrate. Here, we found that mTORC2 can also regulate insulin signaling at the level of insulin receptor substrate-1 (IRS-1). Despite phosphorylation at the mTORC1-mediated serine sites, which supposedly triggers IRS-1 downregulation, inactive IRS-1 accumulated in mTORC2-disrupted cells. Defective IRS-1 degradation was due to attenuated expression and phosphorylation of the ubiquitin ligase substrate-targeting subunit, Fbw8. mTORC2 stabilizes Fbw8 by phosphorylation at Ser86, allowing the insulin-induced translocation of Fbw8 to the cytosol where it mediates IRS-1 degradation. Thus, mTORC2 negatively feeds back to IRS-1 via control of Fbw8 stability and localization. Our findings reveal that in addition to persistent mTORC1 signaling, heightened mTORC2 signals can promote insulin resistance due to mTORC2-mediated degradation of IRS-1.

Sin1 regulates Treg-cell development but is not required for T-cell growth and proliferation.

Mammalian Sin1 plays key roles in the regulation of mitogen-activated protein kinase (MAPK) and mammalian target of rapamycin (mTOR) signaling. Sin1 is an essential component of mTOR complex 2 (mTORC2). The functions of Sin1 and mTORC2 remain largely unknown in T cells. Here, we investigate Sin1 function in T cells using mice that lack Sin1 in the hematopoietic system. Sin1 deficiency blocks the mTORC2-dependent Akt phosphorylation in T cells during development and activation. Sin1-deficient T cells exhibit normal thymic cellularity and percentages of double-negative, double-positive, and single-positive CD4(+) and CD8(+) thymocytes. Sin1 deficiency does not impair T-cell receptor (TCR) induced growth and proliferation. Sin1 appears dispensable for in vitro CD4(+) helper cell differentiation. However, Sin1 deficiency results in an increased proportion of Foxp3(+) natural T-regulatory (nTreg) cells in the thymus. The TGF-ß-dependent differentiation of CD4(+) T cells in vitro is enhanced by the inhibition of mTOR but not by loss of Sin1 function. Our results reveal that Sin1 and mTORC2 are dispensable for the development and activation of T cells but play a role in nTreg-cell differentiation.

Inhibition of the mTORC2 and chaperone pathways to treat leukemia.

Constitutive activation of the kinases Akt or protein kinase C (PKC) in blood cancers promotes tumor-cell proliferation and survival and is associated with poor patient survival. The mammalian target of rapamycin (mTOR) complex 2 (mTORC2) regulates the stability of Akt and conventional PKC (cPKC; PKCα and PKCß) proteins by phosphorylating the highly conserved turn motif of these proteins. In cells that lack mTORC2 function, the turn motif phosphorylation of Akt and cPKC is abolished and therefore Akt and cPKC protein stability is impaired. However, the chaperone protein HSP90 can stabilize Akt and cPKC, partially rescuing the expression of these proteins. In the present study, we investigated the antitumor effects of inhibiting mTORC2 plus HSP90 in mouse and human leukemia cell models and show that the HSP90 inhibitor 17-allylaminogeldanamycin (17-AAG) preferentially inhibits Akt and cPKC expression and promotes cell death in mTORC2 deficient pre-B leukemia cells. Furthermore, we show that 17-AAG selectively inhibits mTORC2 deficient leukemia cell growth in vivo. Finally, we show that the mTOR inhibitors rapamycin and pp242 work together with 17-AAG to inhibit leukemia cell growth to a greater extent than either drug alone. These studies provide a mechanistic and clinical rationale to combine mTOR inhibitors with chaperone protein inhibitors to treat human blood cancers.

Protein implicated in nonsyndromic mental retardation regulates protein kinase A (PKA) activity.

Mutation of the coiled-coil and C2 domain-containing 1A (CC2D1A) gene, which encodes a C2 domain and DM14 domain-containing protein, has been linked to severe autosomal recessive nonsyndromic mental retardation. Using a mouse model that produces a truncated form of CC2D1A that lacks the C2 domain and three of the four DM14 domains, we show that CC2D1A is important for neuronal differentiation and brain development. CC2D1A mutant neurons are hypersensitive to stress and have a reduced capacity to form dendrites and synapses in culture. At the biochemical level, CC2D1A transduces signals to the cyclic adenosine 3',5'-monophosphate (cAMP)-protein kinase A (PKA) pathway during neuronal cell differentiation. PKA activity is compromised, and the translocation of its catalytic subunit to the nucleus is also defective in CC2D1A mutant cells. Consistently, phosphorylation of the PKA target cAMP-responsive element-binding protein, at serine 133, is nearly abolished in CC2D1A mutant cells. The defects in cAMP/PKA signaling were observed in fibroblast, macrophage, and neuronal primary cells derived from the CC2D1A KO mice. CC2D1A associates with the cAMP-PKA complex following forskolin treatment and accumulates in vesicles or on the plasma membrane in wild-type cells, suggesting that CC2D1A may recruit the PKA complex to the membrane to facilitate signal transduction. Together, our data show that CC2D1A is an important regulator of the cAMP/PKA signaling pathway, which may be the underlying cause for impaired mental function in nonsyndromic mental retardation patients with CC2D1A mutation.