Tamoxifen resistance is often observed in the majority of estrogen receptor-positive breast cancers and it remains as a serious clinical problem in breast cancer management. Increased aerobic glycolysis has been proposed as one of the mechanisms for acquired resistance to chemotherapeutic agents in breast cancer cells such as adriamycin. Herein, we report that the glycolysis rates in LCC2 and LCC9--tamoxifen-resistant human breast cancer cell lines derived from MCF7--are higher than those in MCF7S, which is the parent MCF7 subline. Inhibition of key glycolytic enzyme such as hexokinase-2 resulted in cell growth retardation at higher degree in LCC2 and LCC9 than that in MCF7S. This implies that increased aerobic glycolysis even under O2-rich conditions, a phenomenon known as the Warburg effect, is closely associated with tamoxifen resistance. We found that HIF-1α is activated via an Akt/mTOR signaling pathway in LCC2 and LCC9 cells without hypoxic condition. Importantly, specific inhibition of hexokinase-2 suppressed the activity of Akt/mTOR/HIF-1α axis in LCC2 and LCC9 cells. In addition, the phosphorylated AMPK which is a negative regulator of mTOR was decreased in LCC2 and LCC9 cells compared to MCF7S. Interestingly, either the inhibition of mTOR activity or increase in AMPK activity induced a reduction in lactate accumulation and cell survival in the LCC2 and LCC9 cells. Taken together, our data provide evidence that development of tamoxifen resistance may be driven by HIF-1α hyperactivation via modulation of Akt/mTOR and/or AMPK signaling pathways. Therefore, we suggest that the HIF-1α hyperactivation is a critical marker of increased aerobic glycolysis in accordance with tamoxifen resistance and thus restoration of aerobic glycolysis may be novel therapeutic target for treatment of tamoxifen-resistant breast cancer.

Hematopoietic stem cells (HSCs) underlie the production of blood and immune cells for the lifetime of an organism. In vertebrate embryos, HSCs arise from the unique transdifferentiation of hemogenic endothelium comprising the floor of the dorsal aorta during a brief developmental window. To date, this process has not been replicated in vitro from pluripotent precursors, partly because the full complement of required signaling inputs remains to be determined. Here, we show that TNFR2 via TNF? activates the Notch and NF-?B signaling pathways to establish HSC fate, indicating a requirement for inflammatory signaling in HSC generation. We determine that primitive neutrophils are the major source of TNF?, assigning a role for transient innate immune cells in establishing the HSC program. These results demonstrate that proinflammatory signaling, in the absence of infection, is utilized by the developing embryo to generate the lineal precursors of the adult hematopoietic system.

Haematopoietic stem cells (HSCs) derive from haemogenic endothelial cells of the primitive dorsal aorta (DA) during vertebrate embryogenesis. The molecular mechanisms governing this unique endothelial to haematopoietic transition remain unclear. Here, we demonstrate a novel requirement for fibroblast growth factor (FGF) signalling in HSC emergence. This requirement is non-cell-autonomous, and acts within the somite to bridge the Wnt and Notch signalling pathways. We previously demonstrated that Wnt16 regulates the somitic expression of two Notch ligands, deltaC (dlc) and deltaD (dld), whose combined function is required for HSC fate. How Wnt16 connects to Notch function has remained an open question. Our current studies demonstrate that FGF signalling, via FGF receptor 4 (Fgfr4), mediates a signal-transduction pathway between Wnt16 and Dlc, but not Dld, to regulate HSC specification. Our findings demonstrate that FGF signalling acts as a key molecular relay within the developmental HSC niche to instruct HSC fate.

As FK506 binding proteins (FK506BPs) are known to play an important role in the regulation of a variety of biological processes related to cell survival, this study was designed to examined the protective effects of FK506 binding protein 12 (FK506BP) on low humidity air flow induced dry eye in a rat model using transduced PEP-1-FK506BP. After the topical application of PEP-1-FK506BP, tear volumes were markedly increased and significant prevention of cornea damage was observed compared with dry eye rats. Further, immunohistochemical analysis demonstrated that PEP-1-FK506BP markedly prevented damage to the cornea, the bulbar conjunctiva, and the palpebral conjunctiva epithelial lining compared with dry eye rats. In addition, caspase-3 and PARP expression levels were found to be decreased. These results demonstrated that topical application of PEP-1-FK506BP significantly ameliorates dry eye injury in an animal model. Thus, we suggest that PEP-1-FK506BP can be developed as a new ophthalmic drop to treat dry eye diseases.

Hematopoietic stem cells (HSCs) require multiple molecular inputs for proper specification, including activity of the Notch signaling pathway. A requirement for the Notch1 and dispensability of the Notch2 receptor has been demonstrated in mice, but the role of the remaining Notch receptors has not been investigated. Here, we demonstrate that three of the four Notch receptors are independently required for the specification of HSCs in the zebrafish. The orthologues of the murine Notch1 receptor, Notch1a and Notch1b, are each required intrinsically to fate HSCs, just prior to their emergence from aortic hemogenic endothelium. By contrast, the Notch3 receptor is required earlier within the developing somite to regulate HSC emergence in a non-cell-autonomous manner. Epistatic analyses demonstrate that Notch3 function lies downstream of Wnt16, which is required for HSC specification through its regulation of two Notch ligands, dlc and dld. Collectively, these findings demonstrate for the first time that multiple Notch signaling inputs are required to specify HSCs and that Notch3 performs a novel role within the somite to regulate the neighboring precursors of hemogenic endothelium.

Epithelial-mesenchymal transition (EMT) is associated with tumor hypoxia. EMT is regulated, in part, by the action of TWIST, which inhibits of E-cadherin expression and may interfere with the p53 tumor-suppressor pathway.

Paraoxonase 1 (PON1) is an antioxidant enzyme which plays a central role in various diseases. However, the mechanism and function of PON1 protein in inflammation are poorly understood. Since PON1 protein alone cannot be delivered into cells, we generated a cell permeable PEP-1-PON1 protein using protein transduction domains, and examined whether it can protect against cell death in lipopolysaccharide (LPS) or hydrogen peroxide (H2O2)-treated Raw 264.7 cells as well as mice with 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced skin inflammation. We demonstrated that PEP-1-PON1 protein transduced into Raw 264.7 cells and markedly protected against LPS or H2O2-induced cell death by inhibiting cellular reactive oxygen species (ROS) levels, the inflammatory mediator's expression, activation of mitogen-activated protein kinases (MAPKs) and cellular apoptosis. Furthermore, topically applied PEP-1-PON1 protein ameliorates TPA-treated mice skin inflammation via a reduction of inflammatory response. Our results indicate that PEP-1-PON1 protein plays a key role in inflammation and oxidative stress in vitro and in vivo. Therefore, we suggest that PEP-1-PON1 protein may provide a potential protein therapy against oxidative stress and inflammation.

One of the properties of human breast cancer cells is cancer stemness, which is characterized by self-renewal capability and drug resistance. Protein kinase D1 (PRKD1) functions as a key regulator of many cellular processes and is downregulated in invasive breast cancer cells. In this study, we found that PRKD1 was upregulated in MCF-7-ADR human breast cancer cells characterized by drug resistance. Additionally, we discovered that PRKD1 expression was negatively regulated by miR-34a binding to the PRKD1 3'-UTR. PRKD1 expression increased following performance of a tumorsphere formation assay in MCF-7-ADR cells. We also found that reduction of PRKD1 by ectopic miR-34a expression or PRKD1 siRNA treatment resulted in suppressed self-renewal ability in breast cancer stem cells. Furthermore, we confirmed that the PRKD1 inhibitor CRT0066101 reduced phosphorylated PKD/PKCμ, leading to suppression of breast cancer stemness through GSK3/β-catenin signaling. PRKD1 inhibition also influenced apoptosis initiation in MCF-7-ADR cells. Tumors from nude mice treated with miR-34a or CRT0066101 showed suppressed tumor growth, proliferation, and induced apoptosis. These results provide evidence that regulation of PRKD1, a novel miR-34a target, contributes to overcoming cancer stemness and drug resistance in human breast cancer.

The overall understanding of the molecular etiologies of intellectual disability (ID) and developmental delay (DD) is increasing as next-generation sequencing technologies identify genetic variants in individuals with such disorders. However, detailed analyses conclusively confirming these variants, as well as the underlying molecular mechanisms explaining the diseases, are often lacking. Here, we report on an ID syndrome caused by de novo heterozygous loss-of-function (LoF) mutations in SON. The syndrome is characterized by ID and/or DD, malformations of the cerebral cortex, epilepsy, vision problems, musculoskeletal abnormalities, and congenital malformations. Knockdown of son in zebrafish resulted in severe malformation of the spine, brain, and eyes. Importantly, analyses of RNA from affected individuals revealed that genes critical for neuronal migration and cortex organization (TUBG1, FLNA, PNKP, WDR62, PSMD3, and HDAC6) and metabolism (PCK2, PFKL, IDH2, ACY1, and ADA) are significantly downregulated because of the accumulation of mis-spliced transcripts resulting from erroneous SON-mediated RNA splicing. Our data highlight SON as a master regulator governing neurodevelopment and demonstrate the importance of SON-mediated RNA splicing in human development.

High intraoperative inspired oxygen concentration is applied to prevent desaturation during induction and recovery of anesthesia. However, high oxygen concentration may lead to postoperative pulmonary complications. The purpose of this study is to compare the postoperative pulmonary parameters according to intraoperative inspired oxygen fraction in patients undergoing general anesthesia. We identified all randomized controlled trials investigating postoperative differences in arterial gas exchange according to intraoperative fraction of inspired oxygen (FiO2). A total of 10 randomized controlled trials were included, and 787 patients were analyzed. Postoperative PaO2 was lower in the high FiO2 group compared with the low FiO2 group (mean difference (MD) -4.97 mmHg, 95% CI -8.21 to -1.72, p = 0.003). Postoperative alveolar-arterial oxygen gradient (AaDO2) was higher (MD 3.42 mmHg, 95% CI 0.95 to 5.89, p = 0.007) and the extent of atelectasis was more severe (MD 2.04%, 95% CI 0.14 to 3.94, p = 0.04) in high intraoperative FiO2 group compared with low FiO2 group. However, postoperative SpO2 was comparable between the two groups. The results of this meta-analysis suggest that high inspired oxygen fraction during anesthesia may impair postoperative pulmonary parameters. Cautious approach in intraoperative inspired oxygen fraction is required for patients susceptible to postoperative pulmonary complications.

During lumbar spine surgery, patients are placed in the prone position for surgical access. The prone position has various effects on cardiac and pulmonary function, including a decreased cardiac index (CI), decreased dynamic lung compliance (Cdyn), and increased peak inspiratory pressure (Ppeak). In this study, we compared the volume-controlled ventilation mode (VCV) and pressure-controlled ventilation with volume guaranteed mode (PCV-VG) based on hemodynamic and pulmonary variables in the prone position during lumbar spine surgery.

Although genetic testing is increasingly used in clinical nephrology, a large number of patients with congenital abnormalities of the kidney and urinary tract (CAKUT) remain undiagnosed with current gene panels. Therefore, careful curation of novel genetic findings is key to improving diagnostic yields. We recently described a novel intellectual disability syndrome caused by de novo heterozygous loss-of-function mutations in the gene encoding the splicing factor SON. Here, we show that many of these patients, including two previously unreported, exhibit a wide array of kidney abnormalities. Detailed phenotyping of 14 patients with SON haploinsufficiency identified kidney anomalies in 8 patients, including horseshoe kidney, unilateral renal hypoplasia, and renal cysts. Recurrent urinary tract infections, electrolyte disturbances, and hypertension were also observed in some patients. SON knockdown in kidney cell lines leads to abnormal pre-mRNA splicing, resulting in decreased expression of several established CAKUT genes. Furthermore, these molecular events were observed in patient-derived cells with SON haploinsufficiency. Taken together, our data suggest that the wide spectrum of phenotypes in patients with a pathogenic SON mutation is a consequence of impaired pre-mRNA splicing of several CAKUT genes. We propose that genetic testing panels designed to diagnose children with a kidney phenotype should include the SON gene.

Neutrophils are a type of white blood cell essential for the function of the innate immune system. To elucidate mechanisms of neutrophil biology, many studies are performed in vertebrate animal model systems. In Danio rerio (zebrafish), in vivo imaging of neutrophils is possible due to transgenic strains that possess fluorescently labeled leukocytes. However, due to the relative abundance of neutrophils, the counting process is laborious and subjective. In this article, we propose the use of a custom trained "you only look once" (YOLO) machine learning algorithm to automate the identification and counting of fluorescently labeled neutrophils in zebrafish. Using this model, we found the correlation coefficient between human counting and the model equals r = 0.8207 with an 8.65% percent error, while variation among human counters was 5% to 12%. Importantly, the model was able to correctly validate results of a previously published article that quantitated neutrophils manually. While the accuracy can be further improved, this model notably achieves these results in mere minutes compared with hours via standard manual counting protocols and can be performed by anyone with basic programming knowledge. It further supports the use of deep learning models for high throughput analysis of fluorescently labeled blood cells in the zebrafish model system.

No effective screening tools for ovarian cancer (OC) exist, making it one of the deadliest cancers among women. Considering little is known about the detailed progression and metastasis mechanism of OC at a molecular level, it is crucial to gain more insights on how metabolic and signaling alterations accompany its development. Herein, we present a comprehensive study using ultra-high-resolution Fourier transform ion cyclotron resonance matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) to investigate the spatial distribution and alterations of lipids in ovarian tissues collected from double knockout (n = 4) and a triple mutant mouse models (n = 4) of high-grade serous ovarian cancer (HGSC). Lipids belonging to a total of 15 different classes were annotated and their abundance changes compared to those in healthy mouse reproductive tissue (n = 4), mapping onto major lipid pathways involved in OC progression. From intermediate-stage OC to advanced HGSC, we provide a direct visualization of lipid distributions and their biological links to inflammatory response, cellular stress, cell proliferation, and other processes. We also show the ability to distinguish tumors at different stages from healthy tissues via a number of highly specific lipid biomarkers, providing targets for future panels that could be useful in diagnosis.

Owing to the remarkable advancements of artificial intelligence (AI) applications, AI-based detection of dental caries is continuously improving. We evaluated the efficacy of the detection of dental caries with quantitative light-induced fluorescence (QLF) images using a convolutional neural network (CNN) model.

No effective screening tools for ovarian cancer (OC) exist, making it one of the deadliest cancers among women. Considering that little is known about the detailed progression and metastasis mechanism of OC at a molecular level, it is crucial to gain more insights into how metabolic and signaling alterations accompany its development. Herein, we present a comprehensive study using ultra-high-resolution Fourier transform ion cyclotron resonance matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) to investigate the spatial distribution and alterations of lipids in ovarian tissues collected from double knockout (n = 4) and triple mutant mouse models (n = 4) of high-grade serous ovarian cancer (HGSOC). Lipids belonging to a total of 15 different classes were annotated and their abundance changes were compared to those in healthy mouse reproductive tissue (n = 4), mapping onto major lipid pathways involved in OC progression. From intermediate-stage OC to advanced HGSC, we provide direct visualization of lipid distributions and their biological links to inflammatory response, cellular stress, cell proliferation, and other processes. We also show the ability to distinguish tumors at different stages from healthy tissues via a number of highly specific lipid biomarkers, providing targets for future panels that could be useful in diagnosis.

The increasing prevalence of advanced maternal age (AMA) births necessitates the exploration of associated pregnancy outcomes within the healthcare-limited context of northern Tanzania to elucidate potential region-specific risks and implications. This study explored the influence of AMA on pregnancy outcomes in northern Tanzania, where healthcare resources and infrastructure are constrained in comparison to developed countries. This cross-sectional hospital-based study utilized maternally linked data from the Kilimanjaro Christian Medical Center (KCMC) Medical Registry and included 32,798 women who delivered single infants between 2004 and 2013. Multiple logistic regression models were used to determine adjusted odds ratios (aORs) and 95 % confidence intervals (CIs) for AMA-associated adverse pregnancy outcomes. A total of 16 % of mothers belonged to AMA with increased odds of undergoing a cesarean section (aOR: 1.32; 95%CI [1.24-1.41]; P < 0.001), gestational diabetes (aOR: 13.16; 95%CI [3.28-52.86]; P < 0.001) or pregestational diabetes (aOR: 3.15; 95%CI [1.87-5.31]; P < 0.000), and developing pre-eclampsia (aOR: 1.63; 95%CI [1.41-1.89]; P < 0.000). More women with AMA reported alcohol use during pregnancy and had preexisting conditions before conception than did younger women. Maternal education level, employment status, urban residency, and Christianity were statistically significant. This study establishes a connection between AMA and higher odds of cesarean section, gestational diabetes, pregestational diabetes, and pre-eclampsia. Women with AMA were more inclined to consume alcohol during pregnancy and exhibited preexisting conditions before conception. Moreover, AMA was linked to increased odds of low birth weight, stillbirths, and NICU transfers.

To investigate the role of α3 and α7 nicotinic acetylcholine receptor subunits (nAChRs) in the bladder, using a rat model with detrusor overactivity induced by partial bladder outlet obstruction (BOO).

Colorectal cancer is a major cause of cancer-related death in western countries, and thus there is an urgent need to elucidate the mechanism of colorectal tumorigenesis. A diet that is rich in fat increases the risk of colorectal tumorigenesis. Bile acids, which are secreted in response to the ingestion of fat, have been shown to increase the risk of colorectal tumors. The expression of cyclooxygenase (COX)-2, an inducible isozyme of cyclooxygenase, is induced by bile acids and correlates with the incidence and progression of cancers. In this study, we investigated the signal transduction pathways involved in the bile-acid-mediated induction of COX-2 expression. We found that staurosporine (sts), a potent protein kinase C (PKC) inhibitor, synergistically potentiated the deoxycholate-mediated induction of COX-2 expression. Sts did not increase the stabilization of COX-2 mRNA. The sts- and deoxycholate-mediated synergistic induction of COX-2 expression was suppressed by a membrane-permeable Ca(2+) chelator, a phosphoinositide 3-kinase inhibitor, a nuclear factor-κB pathway inhibitor, and inhibitors of canonical and stress-inducible mitogen-activated protein kinase pathways. Inhibition was also observed using PKC inhibitors, suggesting the involvement of certain PKC isozymes (η, θ, ι, ζ, or μ). Our results indicate that sts exerts its potentiating effects via the phosphorylation of p38. However, the effects of anisomycin did not mimic those of sts, indicating that although p38 activation is required, it does not enhance deoxycholate-induced COX-2 expression. We conclude that staurosporine synergistically enhances deoxycholate-induced COX-2 expression in RCM-1 colon cancer cells.

Cognitive impairment (CI) is reported in 29-57% of patients with neuromyelitis optica spectrum disorder (NMOSD). However, the pathophysiology underlying CI in NMOSD is poorly understood. The present study aims to investigate the predictive values of various conventional and quantitative MRI parameters for cognitive performance in patients with NMOSD. Neurological assessment and conventional, diffusion tensor, and volumetric MRI sequences were collected form 73 patients with NMOSD and 44 healthy controls (HCs). Patients with ≥3 failed tests were considered to have CI. Brain lesion load, gray matter (GM) and white matter (WM) atrophy, deep GM (DGM) atrophy, cortical thickness, and diffuse microstructural WM damage were assessed. Twenty-three (32%) patients with NMOSD had CI. Compared to cognitively preserved (CP) individuals, patients with CI had atrophy in the WM, thalamus, and caudate, decreased fractional anisotropy (FA) and increased mean diffusivity in their WM. A multivariate model indicated that mean FA values in the WM and volume in the nucleus accumbens (NAc) were associated with overall cognition (p = 0.002 and p = 0.008, respectively). Diffuse microstructural damage in the WM and DGM atrophy in the NAc are the strongest predictors of cognitive impairment in patients with NMOSD.