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.

TAK1 negatively regulates NF-κB and p38 MAP kinase activation in Gr-1+CD11b+ neutrophils.

Stringent control of NF-κB and mitogen-activated protein kinase (MAPK) signaling is critical during innate immune responses. TGF-ß activated kinase-1 (TAK1) is essential for NF-κB activation in T and B cells but has precisely the opposite activity in myeloid cells. Specific deletion of TAK1 (Map3k7(ΔM/ΔM)) led to development of splenomegaly and lymphomegaly associated with neutrophilia. Compared with wild-type cells, TAK1-deficient neutrophils enhanced the phosphorylation of the kinases IKK, p38, and JNK and the production of interleukin-1ß (IL-1ß), IL-6, tumor necrosis factor-α (TNF-α), and reactive oxygen species (ROS) after lipopolysaccharide (LPS) stimulation. Map3k7(ΔM/ΔM) mice were significantly more susceptible to LPS-induced septic shock and produced higher amounts of IL-1ß, IL-6, and TNF-α in plasma than do wild-type mice. Specific ablation of p38 rescued the phenotype and functional properties of Map3k7(ΔM/ΔM) mice. Our findings identify a previously unrecognized role of TAK1 as a negative regulator of p38 and IKK activation in a cell type-specific manner.

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.

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.

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.

Sin1-mTORC2 suppresses rag and il7r gene expression through Akt2 in B cells.

Mammalian target of rapamycin (mTOR) is an important mediator of phosphoinositol-3-kinase (PI3K) signaling. PI3K signaling regulates B cell development, homeostasis, and immune responses. However, the function and molecular mechanism of mTOR-mediated PI3K signaling in B cells has not been fully elucidated. Here we show that Sin1, an essential component of mTOR complex 2 (mTORC2), regulates B cell development. Sin1 deficiency results in increased IL-7 receptor (il7r) and RAG recombinase (rag1 and rag2) gene expression, leading to enhanced pro-B cell survival and augmented V(D)J recombinase activity. We further show that Akt2 specifically mediates the Sin1-mTORC2 dependent suppression of il7r and rag gene expression in B cells by regulating FoxO1 phosphorylation. Finally, we demonstrate that the mTOR inhibitor rapamycin induces rag expression and promotes V(D)J recombination in B cells. Our study reveals that the Sin1/mTORC2-Akt2 signaling axis is a key regulator of FoxO1 transcriptional activity in B cells.

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.

Feasibility of low-cost particulate matter sensors for long-term environmental monitoring: Field evaluation and calibration.

Low-cost sensor networks offer the potential to reduce monitoring costs while providing high-resolution spatiotemporal data on pollutant levels. However, these sensors come with limitations, and many aspects of their field performance remain underexplored. During October to December 2023, this study deployed two identical low-cost sensor systems near an urban standard monitoring station to record PM2.5 and PM10 concentrations, along with environmental temperature and humidity. Our evaluation of the monitoring performance of these sensors revealed a broad data distribution with a systematic overestimation; this overestimation was more pronounced in PM10 readings. The sensors showed good consistency (R2 > 0.9, NRMSE<5 %), and normalization residuals were tracked to assess stability, which, despite occasional environmental influences, remained generally stable. A lateral comparison of four calibration models (MLR, SVR, RF, XGBoost) demonstrated superior performance of RF and XGBoost over others, particularly with RF showing enhanced effectiveness on the test set. SHAP analysis identified sensor readings as the most critical variable, underscoring their pivotal role in predictive modeling. Relative humidity consistently proved more significant than dew point and temperature, with higher RH levels typically having a positive impact on model outputs. The study indicates that, with appropriate calibration, sensors can supplement the sparse networks of regulatory-grade instruments, enabling dense neighborhood-scale monitoring and a better understanding of temporal air quality trends.

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.

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.

MEKK3 regulates IFN-gamma production in T cells through the Rac1/2-dependent MAPK cascades.

MEKK3 is a conserved Ser/Thr protein kinase belonging to the MAPK kinase kinase (MAP3K) family. MEKK3 is constitutively expressed in T cells, but its function in T cell immunity has not been fully elucidated. Using Mekk3 T cell conditional knockout (T-cKO) mice, we show that MEKK3 is required for T cell immunity in vivo. Mekk3 T-cKO mice had reduced T cell response to bacterial infection and were defective in clearing bacterial infections. The Ag-induced cytokine production, especially IFN-γ production, was impaired in Mekk3-deficient CD4 T cells. The TCR-induced ERK1/2, JNK, and p38 MAPKs activation was also defective in Mekk3-deficient CD4 T cells. In vitro, MEKK3 is not required for Th1 and Th2 cell differentiation. Notably, under a nonpolarizing condition (Th0), Mekk3 deficiency led to a significant reduction of IFN-γ production in CD4 T cells. Furthermore, the IL-12/IL-18-driven IFN-γ production and MAPK activation in Mekk3-deficient T cells was not affected suggesting that MEKK3 may selectively mediate the TCR-induced MAPK signals for IFN-γ production. Finally, we found that MEKK3 activation by TCR stimulation requires Rac1/2. Taken together, our study reveals a specific role of MEKK3 in mediating the TCR signals for IFN-γ production.

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.

mTOR complex 2 targets Akt for proteasomal degradation via phosphorylation at the hydrophobic motif.

The protein kinase Akt (also known as protein kinase B) is a critical signaling hub downstream of various cellular stimuli such as growth factors that control cell survival, growth, and proliferation. The activity of Akt is tightly regulated, and the aberrant activation of Akt is associated with diverse human diseases including cancer. Although it is well documented that the mammalian target of rapamycin complex 2 (mTORC2)-dependent phosphorylation of the Akt hydrophobic motif (Ser-473 in Akt1) is essential for full Akt activation, it remains unclear whether this phosphorylation has additional roles in regulating Akt activity. In this study, we found that abolishing Akt Ser-473 phosphorylation stabilizes Akt following agonist stimulation. The Akt Ser-473 phosphorylation promotes a Lys-48-linked polyubiquitination of Akt, resulting in its rapid proteasomal degradation. Moreover, blockade of this proteasomal degradation pathway prolongs agonist-induced Akt activation. These data reveal that mTORC2 plays a central role in regulating the Akt protein life cycle by first stabilizing Akt protein folding through the turn motif phosphorylation and then by promoting Akt protein degradation through the hydrophobic motif phosphorylation. Taken together, this study reveals that the Akt Ser-473 phosphorylation-dependent ubiquitination and degradation is an important negative feedback regulation that specifically terminates Akt activation.

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.

The effect of root exudates from two transgenic insect-resistant cotton lines on the growth of Fusarium oxysporum.

The attenuation of disease resistance in transgenic insect-resistant cotton has become one of the important factors restricting cotton production in China. Two transgenic insect-resistant cotton lines and their parental conventional cotton lines were used as the testing materials. The effects of root exudates of these cotton lines on the spore germination and mycelial growth of Fusarium oxysporum were studied and the components, contents of amino acids and sugars were determined. The results showed that the resistance of the two insect-resistant cotton lines to F. oxysporum was inferior to the parental lines, and that their root exudates promoted fungal spore germination and mycelial growth. Considerable differences in the components and contents of both, amino acids and sugars were found between the root exudates of transgenic cotton lines and their parental lines, where the disease indices were highly correlated with the total amount of sugars in the root exudates.

A prospective study of dietary intakes and influential factors from pregnancy to postpartum on maternal weight retention in Taipei, Taiwan.

Excessive gestational weight gain and postpartum weight retention are risk factors for female obesity. The present study was to examine dietary intakes and weight history from a prospective follow-up study from early pregnancy to 1 year postpartum. A total of 151 pregnant women within 20 weeks of pregnancy in Taipei, Taiwan were interviewed periodically to collect dietary and lifestyle information. The participants had an average age of 30 years and the average gestational weight gain was 14 kg, with an average daily intake of 7830 kJ (1870 kcal) in the 1 year following parturition. By bivariate analyses, maternal age, pre-pregnancy BMI and breast-feeding were not related to postpartum weight retention, but gestational weight gain had significant positive correlations (r 0.54 at 6 months, r 0.44 at 1 year; P < 0.05). The generalised estimating equations showed that the average weight before pregnancy, at 6 months and 1 year postpartum was 53.35 kg, 55.75 kg (weight retention 2.36 kg; P < 0.01) and 54.75 kg (weight retention 1.48 kg; P < 0.01), respectively. After controlling for age, pre-pregnancy BMI, gestational weight gain and parity, we found at 6 months that the adjusted weight retention at postpartum was 0.79 kg (P < 0.01), but at 1 year it was - 0.08 kg (P>0.05). From multivariate analyses, dietary energy intake and energy intake per kg body weight as a long-term physical activity index could explain 24 % of the variation at 6 months and 27 % of the variation at 1 year in postpartum weight retention. These results suggest that pregnant women should be advised to control gestational weight gain, decrease energy intakes after child-bearing and maintain regular exercise in order to prevent postpartum obesity.

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.

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.

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.

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.