Construction and validation of a personalized prediction model for postpartum anxiety in pregnant women with preeclampsia.

BACKGROUND: Preeclampsia is a pregnancy-specific multi-system disease with multi-factor and multi-mechanism characteristics. The cure for preeclampsia is to terminate the pregnancy and deliver the placenta. However, it will reduce the perinatal survival rate, prolong the pregnancy cycle, and increase the incidence of maternal complications. With relaxation of the birth policy, the number of elderly pregnant women has increased significantly, and the prevalence rate of preeclampsia has increased. Inappropriate treatment can seriously affect the normal postpartum life of pregnant women. Studies have shown that postpartum anxiety in women with preeclampsia can affect physical and mental health, as well as infant growth and development. AIM: To analyze the factors influencing preeclampsia in pregnant women complicated with postpartum anxiety, and to construct a personalized predictive model. METHODS: We retrospectively studied 528 pregnant women with preeclampsia who delivered in Wenzhou Hospital of Integrated Traditional Chinese and Western Medicine between January 2018 and December 2021. Their basic data were collected, and various physiological and biochemical indicators were obtained by laboratory examination. The self-rating anxiety scale was used to determine whether the women had postpartum anxiety 42 d after delivery. The independent factors influencing postpartum anxiety in early pregnant women with eclampsia were analyzed with multifactor logistic regression and a predictive model was constructed. The Hosmer-Lemeshow test and receiver operating characteristic (ROC) curve were used to evaluate the calibration and discrimination of the predictive model. Eighty pregnant women with preeclampsia admitted to our hospital from January 2022 to May 2022 were retrospectively selected to verify the prediction model. RESULTS: We excluded 46 of the 528 pregnant women with preeclampsia because of loss to follow-up and adverse outcomes. A total of 482 cases completed the assessment of postpartum anxiety 42 d after delivery, and 126 (26.14%) had postpartum anxiety. Bad marital relationship, gender discrimination in family members, hematocrit (Hct), estradiol (E2) hormone and interleukin (IL)-6 were independent risk factors for postpartum anxiety in pregnant women with preeclampsia (P < 0.05). Prediction model: Logit (P) = 0.880 × marital relationship + 0.870 × gender discrimination of family members + 0.130 × Hct - 0.044 × E2 + 0.286 × IL-6 - 21.420. The area under the ROC curve of the model was 0.943 (95% confidence interval: 0.919-0.966). The threshold of the model was -1.507 according to the maximum Youden index (0.757), the corresponding sensitivity was 84.90%, and the specificity was 90.70%. Hosmer-Lemeshow χ2 = 5.900, P = 0.658. The sensitivity, specificity and accuracy of the model were 81.82%, 84.48% and 83.75%, respectively. CONCLUSION: Poor marital relationship, family gender discrimination, Hct, IL-6 and E2 are the influencing factors of postpartum anxiety in preeclampsia women. The constructed prediction model has high sensitivity and specificity.

Construction of a Matrix Cancer-Associated Fibroblast Signature Gene-Based Risk Prognostic Signature for Directing Immunotherapy in Patients with Breast Cancer Using Single-Cell Analysis and Machine Learning.

Cancer-associated fibroblasts (CAFs) are heterogeneous constituents of the tumor microenvironment involved in the tumorigenesis, progression, and therapeutic responses of tumors. This study identified four distinct CAF subtypes of breast cancer (BRCA) using single-cell RNA sequencing (RNA-seq) data. Of these, matrix CAFs (mCAFs) were significantly associated with tumor matrix remodeling and strongly correlated with the transforming growth factor (TGF)-ß signaling pathway. Consensus clustering of The Cancer Genome Atlas (TCGA) BRCA dataset using mCAF single-cell characteristic gene signatures segregated samples into high-fibrotic and low-fibrotic groups. Patients in the high-fibrotic group exhibited a significantly poor prognosis. A weighted gene co-expression network analysis and univariate Cox analysis of bulk RNA-seq data revealed 17 differential genes with prognostic values. The mCAF risk prognosis signature (mRPS) was developed using 10 machine learning algorithms. The clinical outcome predictive accuracy of the mRPS was higher than that of the conventional TNM staging system. mRPS was correlated with the infiltration level of anti-tumor effector immune cells. Based on consensus prognostic genes, BRCA samples were classified into the following two subtypes using six machine learning algorithms (accuracy > 90%): interferon (IFN)-γ-dominant (immune C2) and TGF-ß-dominant (immune C6) subtypes. Patients with mRPS downregulation were associated with improved prognosis, suggesting that they can potentially benefit from immunotherapy. Thus, the mRPS model can stably predict BRCA prognosis, reflect the local immune status of the tumor, and aid clinical decisions on tumor immunotherapy.

Low-dose metformin targets the lysosomal AMPK pathway through PEN2.

Metformin, the most prescribed antidiabetic medicine, has shown other benefits such as anti-ageing and anticancer effects1-4. For clinical doses of metformin, AMP-activated protein kinase (AMPK) has a major role in its mechanism of action4,5; however, the direct molecular target of metformin remains unknown. Here we show that clinically relevant concentrations of metformin inhibit the lysosomal proton pump v-ATPase, which is a central node for AMPK activation following glucose starvation6. We synthesize a photoactive metformin probe and identify PEN2, a subunit of γ-secretase7, as a binding partner of metformin with a dissociation constant at micromolar levels. Metformin-bound PEN2 forms a complex with ATP6AP1, a subunit of the v-ATPase8, which leads to the inhibition of v-ATPase and the activation of AMPK without effects on cellular AMP levels. Knockout of PEN2 or re-introduction of a PEN2 mutant that does not bind ATP6AP1 blunts AMPK activation. In vivo, liver-specific knockout of Pen2 abolishes metformin-mediated reduction of hepatic fat content, whereas intestine-specific knockout of Pen2 impairs its glucose-lowering effects. Furthermore, knockdown of pen-2 in Caenorhabditis elegans abrogates metformin-induced extension of lifespan. Together, these findings reveal that metformin binds PEN2 and initiates a signalling route that intersects, through ATP6AP1, the lysosomal glucose-sensing pathway for AMPK activation. This ensures that metformin exerts its therapeutic benefits in patients without substantial adverse effects.

Transient Receptor Potential V Channels Are Essential for Glucose Sensing by Aldolase and AMPK.

Fructose-1,6-bisphosphate (FBP) aldolase links sensing of declining glucose availability to AMPK activation via the lysosomal pathway. However, how aldolase transmits lack of occupancy by FBP to AMPK activation remains unclear. Here, we show that FBP-unoccupied aldolase interacts with and inhibits endoplasmic reticulum (ER)-localized transient receptor potential channel subfamily V, inhibiting calcium release in low glucose. The decrease of calcium at contact sites between ER and lysosome renders the inhibited TRPV accessible to bind the lysosomal v-ATPase that then recruits AXIN:LKB1 to activate AMPK independently of AMP. Genetic depletion of TRPVs blocks glucose starvation-induced AMPK activation in cells and liver of mice, and in nematodes, indicative of physical requirement of TRPVs. Pharmacological inhibition of TRPVs activates AMPK and elevates NAD+ levels in aged muscles, rejuvenating the animals' running capacity. Our study elucidates that TRPVs relay the FBP-free status of aldolase to the reconfiguration of v-ATPase, leading to AMPK activation in low glucose.

Hierarchical activation of compartmentalized pools of AMPK depends on severity of nutrient or energy stress.

AMPK, a master regulator of metabolic homeostasis, is activated by both AMP-dependent and AMP-independent mechanisms. The conditions under which these different mechanisms operate, and their biological implications are unclear. Here, we show that, depending on the degree of elevation of cellular AMP, distinct compartmentalized pools of AMPK are activated, phosphorylating different sets of targets. Low glucose activates AMPK exclusively through the AMP-independent, AXIN-based pathway in lysosomes to phosphorylate targets such as ACC1 and SREBP1c, exerting early anti-anabolic and pro-catabolic roles. Moderate increases in AMP expand this to activate cytosolic AMPK also in an AXIN-dependent manner. In contrast, high concentrations of AMP, arising from severe nutrient stress, activate all pools of AMPK independently of AXIN. Surprisingly, mitochondrion-localized AMPK is activated to phosphorylate ACC2 and mitochondrial fission factor (MFF) only during severe nutrient stress. Our findings reveal a spatiotemporal basis for hierarchical activation of different pools of AMPK during differing degrees of stress severity.

Fructose-1,6-bisphosphate and aldolase mediate glucose sensing by AMPK.

The major energy source for most cells is glucose, from which ATP is generated via glycolysis and/or oxidative metabolism. Glucose deprivation activates AMP-activated protein kinase (AMPK), but it is unclear whether this activation occurs solely via changes in AMP or ADP, the classical activators of AMPK. Here, we describe an AMP/ADP-independent mechanism that triggers AMPK activation by sensing the absence of fructose-1,6-bisphosphate (FBP), with AMPK being progressively activated as extracellular glucose and intracellular FBP decrease. When unoccupied by FBP, aldolases promote the formation of a lysosomal complex containing at least v-ATPase, ragulator, axin, liver kinase B1 (LKB1) and AMPK, which has previously been shown to be required for AMPK activation. Knockdown of aldolases activates AMPK even in cells with abundant glucose, whereas the catalysis-defective D34S aldolase mutant, which still binds FBP, blocks AMPK activation. Cell-free reconstitution assays show that addition of FBP disrupts the association of axin and LKB1 with v-ATPase and ragulator. Importantly, in some cell types AMP/ATP and ADP/ATP ratios remain unchanged during acute glucose starvation, and intact AMP-binding sites on AMPK are not required for AMPK activation. These results establish that aldolase, as well as being a glycolytic enzyme, is a sensor of glucose availability that regulates AMPK.

ULK1/2 Constitute a Bifurcate Node Controlling Glucose Metabolic Fluxes in Addition to Autophagy.

Metabolic reprogramming is fundamental to biological homeostasis, enabling cells to adjust metabolic routes after sensing altered availability of fuels and growth factors. ULK1 and ULK2 represent key integrators that relay metabolic stress signals to the autophagy machinery. Here, we demonstrate that, during deprivation of amino acid and growth factors, ULK1/2 directly phosphorylate key glycolytic enzymes including hexokinase (HK), phosphofructokinase 1 (PFK1), enolase 1 (ENO1), and the gluconeogenic enzyme fructose-1,6-bisphosphatase (FBP1). Phosphorylation of these enzymes leads to enhanced HK activity to sustain glucose uptake but reduced activity of FBP1 to block the gluconeogenic route and reduced activity of PFK1 and ENO1 to moderate drop of glucose-6-phosphate and to repartition more carbon flux to pentose phosphate pathway (PPP), maintaining cellular energy and redox homeostasis at cellular and organismal levels. These results identify ULK1/2 as a bifurcate-signaling node that sustains glucose metabolic fluxes besides initiation of autophagy in response to nutritional deprivation.

RHOBTB3 promotes proteasomal degradation of HIFα through facilitating hydroxylation and suppresses the Warburg effect.

Hypoxia-inducible factors (HIFs) are master regulators of adaptive responses to low oxygen, and their α-subunits are rapidly degraded through the ubiquitination-dependent proteasomal pathway after hydroxylation. Aberrant accumulation or activation of HIFs is closely linked to many types of cancer. However, how hydroxylation of HIFα and its delivery to the ubiquitination machinery are regulated remains unclear. Here we show that Rho-related BTB domain-containing protein 3 (RHOBTB3) directly interacts with the hydroxylase PHD2 to promote HIFα hydroxylation. RHOBTB3 also directly interacts with the von Hippel-Lindau (VHL) protein, a component of the E3 ubiquitin ligase complex, facilitating ubiquitination of HIFα. Remarkably, RHOBTB3 dimerizes with LIMD1, and constructs a RHOBTB3/LIMD1-PHD2-VHL-HIFα complex to effect the maximal degradation of HIFα. Hypoxia reduces the RHOBTB3-centered complex formation, resulting in an accumulation of HIFα. Importantly, the expression level of RHOBTB3 is greatly reduced in human renal carcinomas, and RHOBTB3 deficiency significantly elevates the Warburg effect and accelerates xenograft growth. Our work thus reveals that RHOBTB3 serves as a scaffold to organize a multi-subunit complex that promotes the hydroxylation, ubiquitination and degradation of HIFα.

The lysosomal v-ATPase-Ragulator complex is a common activator for AMPK and mTORC1, acting as a switch between catabolism and anabolism.

AMPK and mTOR play principal roles in governing metabolic programs; however, mechanisms underlying the coordination of the two inversely regulated kinases remain unclear. In this study we found, most surprisingly, that the late endosomal/lysosomal protein complex v-ATPase-Ragulator, essential for activation of mTORC1, is also required for AMPK activation. We also uncovered that AMPK is a residential protein of late endosome/lysosome. Under glucose starvation, the v-ATPase-Ragulator complex is accessible to AXIN/LKB1 for AMPK activation. Concurrently, the guanine nucleotide exchange factor (GEF) activity of Ragulator toward RAG is inhibited by AXIN, causing dissociation from endosome and inactivation of mTORC1. We have thus revealed that the v-ATPase-Ragulator complex is also an initiating sensor for energy stress and meanwhile serves as an endosomal docking site for LKB1-mediated AMPK activation by forming the v-ATPase-Ragulator-AXIN/LKB1-AMPK complex, thereby providing a switch between catabolism and anabolism. Our current study also emphasizes a general role of late endosome/lysosome in controlling metabolic programs.

AMP as a low-energy charge signal autonomously initiates assembly of AXIN-AMPK-LKB1 complex for AMPK activation.

The AMP-activated protein kinase (AMPK) is a master regulator of metabolic homeostasis by sensing cellular energy status. AMPK is mainly activated via phosphorylation by LKB1 when cellular AMP/ADP levels are increased. However, how AMP/ADP brings about AMPK phosphorylation remains unclear. Here, we show that it is AMP, but not ADP, that drives AXIN to directly tether LKB1 to phosphorylate AMPK. The complex formation of AXIN-AMPK-LKB1 is greatly enhanced in glucose-starved or AICAR-treated cells and in cell-free systems supplemented with exogenous AMP. Depletion of AXIN abrogated starvation-induced AMPK-LKB1 colocalization. Importantly, adenovirus-based knockdown of AXIN in the mouse liver impaired AMPK activation and caused exacerbated fatty liver after starvation, underscoring an essential role of AXIN in AMPK activation. These findings demonstrate an initiating role of AMP and demonstrate that AXIN directly transmits AMP binding of AMPK to its activation by LKB1, uncovering the mechanistic route for AMP to elicit AMPK activation by LKB1.

MDM2 Inhibits Axin-Induced p53 Activation Independently of its E3 Ligase Activity.

MDM2 plays a crucial role in negatively regulating the functions of tumor suppressor p53. Here we show that MDM2 can inhibit Axin-stimulated p53-dependent apoptosis by suppressing p53 phosphorylation at Ser 46 and apoptosis-related p53 transactivational activity. Interestingly, the ubiquitin E3 ligase activity of MDM2 is not required for this inhibitory effect. Mechanically, either wildtype MDM2 or its E3-dead mutant, disrupts the Axin-based HIPK2/p53 complex formation by blocking the binding of p53 and HIPK2 to Axin. MDM2Δp53, a deletion mutant that lacks p53 binding domain fails to exert the inhibitory effect, demonstrating that the interaction of MDM2 and p53, but not its E3 ligase activity toward p53 plays key role in suppressing Axin-stimulated p53 activation. Our results thus have revealed a novel aspect of the mechanism by which MDM2 regulates p53 activities.

The Axin/TNKS complex interacts with KIF3A and is required for insulin-stimulated GLUT4 translocation.

Insulin-stimulated glucose uptake by the glucose transporter GLUT4 plays a central role in whole-body glucose homeostasis, dysregulation of which leads to type 2 diabetes. However, the molecular components and mechanisms regulating insulin-stimulated glucose uptake remain largely unclear. Here, we demonstrate that Axin interacts with the ADP-ribosylase tankyrase 2 (TNKS2) and the kinesin motor protein KIF3A, forming a ternary complex crucial for GLUT4 translocation in response to insulin. Specific knockdown of the individual components of the complex attenuated insulin-stimulated GLUT4 translocation to the plasma membrane. Importantly, TNKS2(-/-) mice exhibit reduced insulin sensitivity and higher blood glucose levels when re-fed after fasting. Mechanistically, we demonstrate that in the absence of insulin, Axin, TNKS and KIF3A are co-localized with GLUT4 on the trans-Golgi network. Insulin treatment suppresses the ADP-ribosylase activity of TNKS, leading to a reduction in ADP ribosylation and ubiquitination of both Axin and TNKS, and a concurrent stabilization of the complex. Inhibition of Akt, the major effector kinase of insulin signaling, abrogates the insulin-mediated complex stabilization. We have thus elucidated a new protein complex that is directly associated with the motor protein kinesin in insulin-stimulated GLUT4 translocation.

ChIP-seq and Functional Analysis of the SOX2 Gene in Colorectal Cancers.

Abstract SOX2 is a high mobility group (HMG) box containing transcription factor that has been implicated in various types of cancer, but its role in colorectal cancers (CRC) has not been studied. Here we show that SOX2 is overexpressed in CRC tissues compared with normal adjacent tissues using immunohistochemical staining and RT-PCR. We also observed an increased SOX2 expression in nucleus of colorectal cancer tissues (46%, 14/30 cases vs. 7%, 2/30 adjacent tissues). Furthermore, knockdown of SOX2 in SW620 colorectal cancer cells decreased their growth rates in vitro cell line, and in vivo in xenograft models. ChIP-seq analysis of SOX2 revealed a consensus sequence of wwTGywTT. An integrated expression profiling and ChIP-seq analysis show that SOX2 is involved in the BMP signaling pathway, steroid metabolic process, histone modifications, and many receptor-mediated signaling pathways such as IGF1R and ITPR2 (Inositol 1,4,5-triphosphate receptor, type 2).

ChIP-seq and functional analysis of the SOX2 gene in colorectal cancers.

SOX2 is an HMG box containing transcription factor that has been implicated in various types of cancer, but its role in colorectal cancers (CRC) has not been studied. Here we show that SOX2 is overexpressed in CRC tissues compared with normal adjacent tissues using immunohistochemical staining and RT-PCR. We also observed an increased SOX2 expression in nucleus of colorectal cancer tissues (46%, 14/30 cases vs. 7%, 2/30 adjacent tissues). Furthermore, knockdown of SOX2 in SW620 colorectal cancer cells decreased their growth rates in vitro cell line, and in vivo in xenograft models. ChIP-Seq analysis of SOX2 revealed a consensus sequence of wwTGywTT. An integrated expression profiling and ChIP-seq analysis show that SOX2 is involved in the BMP signaling pathway, steroid metabolic process, histone modifications, and many receptor-mediated signaling pathways such as IGF1R and ITPR2 (Inositol 1,4,5-triphosphate receptor, type 2).

Recent patents and advances in genomic biomarker discovery for colorectal cancers.

Colorectal cancer (CRC) is the third most common cancer in the world. Early diagnosis of colorectal cancer is the key to reducing the death rate of CRC patients. Predicting the response to current therapeutic modalities of CRC will also have a great impact on patient care. This review summarizes recent advances and patents in biomarker discovery in CRC under five major categories; including genomic changes, expression changes, mutations, epigenetic changes and microRNAs. The interesting patents include: 1) a patent for a method to differentiate normal exfoliated cells from cancer cells based on whether they were subjected to apoptosis and DNA degradation; 2) A model (PM-33 multiple molecular marker model) based on expression changes of up-regulation of the MDM2, DUSP6, and NFl genes down-regulation of the RNF4, MMD and EIF2S3 genes, which achieved an 88% sensitivity, and an 82% specificity for CRC diagnosis; 3) gene mutations in PTEN, KRAS, PIK3CA for predicting the response to anti-EGFR therapies, a common drug used for CRC treatment; 4) patents on epigenetic changes of ITGA4, SEPT9, ALX4, TFAP2E FOXL2, SARM1, ID4 etc. and many key miRNAs. Finally, future directions in the fields were commented on or suggested, including the combination of multiple categories of biomarkers and pathway central or network-based biomarker panels.

Axin determines cell fate by controlling the p53 activation threshold after DNA damage.

Cells can undergo either cell-cycle arrest or apoptosis after genotoxic stress, based on p53 activity(1-6). Here we show that cellular fate commitment depends on Axin forming distinct complexes with Pirh2, Tip60, HIPK2 and p53. In cells treated with sublethal doses of ultra-violet (UV) radiation or doxorubicin (Dox), Pirh2 abrogates Axin-induced p53 phosphorylation at Ser 46 catalysed by HIPK2, by competing with HIPK2 for binding to Axin. However, on lethal treatment, Tip60 interacts with Axin and abrogates Pirh2-Axin binding, forming an Axin-Tip60-HIPK2-p53 complex that allows maximal p53 activation to trigger apoptosis. We also provide evidence that the ATM/ATR pathway mediates the Axin-Tip60 complex assembly. An axin mutation promotes carcinogenesis in Axin(Fu)/+ (Axin-Fused) mice, consistent with a dominantnegative role for Axin(Fu) in p53 activation. Thus, Axin is a critical determinant in p53-dependent tumour suppression in which Pirh2 and Tip60 have different roles in triggering cell-cycle arrest or apoptosis depending on the severity of genotoxic stress.

Gelsolin, but not its cleavage, is required for TNF-induced ROS generation and apoptosis in MCF-7 cells.

The tumor necrosis factor (TNF) can induce apoptosis in many cells including MCF-7 cells. To identify the genes responsible for TNF-induced apoptosis, we generated a series of TNF-resistant MCF-7 cell lines by employing retrovirus insertion-mediated random mutagenesis. In one of the resistant lines, gelsolin was found to be disrupted by viral insertion. Exogenous expression of gelsolin in this mutant cell line (Gel(mut)) restored the sensitivity to TNF-induced cell death and knock-down of gelsolin by siRNA conferred MCF-7 cells with resistance to TNF, indicating that gelsolin is required for TNF-induced apoptosis. Interestingly, the resistance of Gel(mut) cells to apoptosis induction is selective to TNF, since Gel(mut) and wild-type cells showed similar sensitivity to other death stimuli that were tested. Furthermore, TNF-induced ROS production in Gel(mut) cells was significantly decreased, demonstrating that gelsolin-mediated ROS generation plays a crucial role in TNF-induced apoptosis in MCF-7 cells. Importantly, caspase-mediated gelsolin cleavage is dispensable for TNF-triggered ROS production and subsequent apoptosis of MCF-7 cells. Our study thus provides genetic evidence linking gelsolin-mediated ROS production to TNF-induced cell death.

Cytosporone B is an agonist for nuclear orphan receptor Nur77.

Nuclear orphan receptor Nur77 has important roles in many biological processes. However, a physiological ligand for Nur77 has not been identified. Here, we report that the octaketide cytosporone B (Csn-B) is a naturally occurring agonist for Nur77. Csn-B specifically binds to the ligand-binding domain of Nur77 and stimulates Nur77-dependent transactivational activity towards target genes including Nr4a1 (Nur77) itself, which contains multiple consensus response elements allowing positive autoregulation in a Csn-B-dependent manner. Csn-B also elevates blood glucose levels in fasting C57 mice, an effect that is accompanied by induction of multiple genes involved in gluconeogenesis. These biological effects were not observed in Nur77-null (Nr4a1-/-) mice, which indicates that Csn-B regulates gluconeogenesis through Nur77. Moreover, Csn-B induced apoptosis and retarded xenograft tumor growth by inducing Nur77 expression, translocating Nur77 to mitochondria to cause cytochrome c release. Thus, Csn-B may represent a promising therapeutic drug for cancers and hypoglycemia, and it may also be useful as a reagent to increase understanding of Nur77 biological function.

CDK5 activator p35 downregulates E-cadherin precursor independently of CDK5.

Dysfunction of E-cadherins often results in metastasis of cancerous cells. Here we show that p35, a critical regulator of cyclin-dependent kinase 5 (CDK5), specifically depletes the precursor form of E-cadherin, but not the mature form, by using a precursor-specific antibody. Most intriguingly, this downregulation of precursor E-cadherin by p35 is unequivocally independent of CDK5. Moreover, we found that p35 forms complexes with E-cadherin proteins. We also found that p35 co-expression can target E-cadherin to lysosomes and that p35-triggered disappearance of E-cadherin precursor can be blocked specifically by lysosomal protease inhibitors, indicating that p35 induces endocytosis and subsequent degradation of precursor E-cadherin.