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The myeloma bone marrow microenvironment promotes proliferation of malignant plasma cells and resistance to therapy. Activation of JAK/STAT signaling is thought to be a central component of these microenvironment-induced phenotypes. In a prior drug repurposing screen, we identified tofacitinib, a pan-JAK inhibitor Food and Drug Administration (FDA) approved for rheumatoid arthritis, as an agent that may reverse the tumor-stimulating effects of bone marrow mesenchymal stromal cells. Herein, we validated in vitro, in stromal-responsive human myeloma cell lines, and in vivo, in orthotopic disseminated xenograft models of myeloma, that tofacitinib showed efficacy in myeloma models. Furthermore, tofacitinib strongly synergized with venetoclax in coculture with bone marrow stromal cells but not in monoculture. Surprisingly, we found that ruxolitinib, an FDA approved agent targeting JAK1 and JAK2, did not lead to the same anti-myeloma effects. Combination with a novel irreversible JAK3-selective inhibitor also did not enhance ruxolitinib effects. Transcriptome analysis and unbiased phosphoproteomics revealed that bone marrow stromal cells stimulate a JAK/STAT-mediated proliferative program in myeloma cells, and tofacitinib reversed the large majority of these pro-growth signals. Taken together, our results suggest that tofacitinib reverses the growth-promoting effects of the tumor microenvironment. As tofacitinib is already FDA approved, these results can be rapidly translated into potential clinical benefits for myeloma patients.

Germline mutations involving small mothers against decapentaplegic-transforming growth factor-β (SMAD-TGF-β) signaling are an important but rare cause of pulmonary arterial hypertension (PAH), which is a disease characterized, in part, by vascular fibrosis and hyperaldosteronism (ALDO). We developed and analyzed a fibrosis protein-protein network (fibrosome) in silico, which predicted that the SMAD3 target neural precursor cell expressed developmentally down-regulated 9 (NEDD9) is a critical ALDO-regulated node underpinning pathogenic vascular fibrosis. Bioinformatics and microscale thermophoresis demonstrated that oxidation of Cys18 in the SMAD3 docking region of NEDD9 impairs SMAD3-NEDD9 protein-protein interactions in vitro. This effect was reproduced by ALDO-induced oxidant stress in cultured human pulmonary artery endothelial cells (HPAECs), resulting in impaired NEDD9 proteolytic degradation, increased NEDD9 complex formation with Nk2 homeobox 5 (NKX2-5), and increased NKX2-5 binding to COL3A1 Up-regulation of NEDD9-dependent collagen III expression corresponded to changes in cell stiffness measured by atomic force microscopy. HPAEC-derived exosomal signaling targeted NEDD9 to increase collagen I/III expression in human pulmonary artery smooth muscle cells, identifying a second endothelial mechanism regulating vascular fibrosis. ALDO-NEDD9 signaling was not affected by treatment with a TGF-β ligand trap and, thus, was not contingent on TGF-β signaling. Colocalization of NEDD9 with collagen III in HPAECs was observed in fibrotic pulmonary arterioles from PAH patients. Furthermore, NEDD9 ablation or inhibition prevented fibrotic vascular remodeling and pulmonary hypertension in animal models of PAH in vivo. These data identify a critical TGF-β-independent posttranslational modification that impairs SMAD3-NEDD9 binding in HPAECs to modulate vascular fibrosis and promote PAH.

Dysregulation of YAP localization and activity is associated with pathological conditions such as cancer. Although activation of the Hippo phosphorylation cascade is known to cause cytoplasmic retention and inactivation of YAP, emerging evidence suggests that YAP can be regulated in a Hippo-independent manner. Here, we report that YAP is subject to non-proteolytic, K63-linked polyubiquitination by the SCFSKP2 E3 ligase complex (SKP2), which is reversed by the deubiquitinase OTUD1. The non-proteolytic ubiquitination of YAP enhances its interaction with its nuclear binding partner TEAD, thereby inducing YAP's nuclear localization, transcriptional activity, and growth-promoting function. Independently of Hippo signaling, mutation of YAP's K63-linkage specific ubiquitination sites K321 and K497, depletion of SKP2, or overexpression of OTUD1 retains YAP in the cytoplasm and inhibits its activity. Conversely, overexpression of SKP2 or loss of OTUD1 leads to nuclear localization and activation of YAP. Altogether, our study sheds light on the ubiquitination-mediated, Hippo-independent regulation of YAP.

Although tumour PD-L1 (CD274) expression had been used as a predictive biomarker in checkpoint immunotherapy targeting the PD1/PD-L1 axis in various cancers, the regulation of PD-L1 (CD274) expression is unclear. Yes-associated protein (YAP), an important oncogenic protein in Hippo signalling pathway, reportedly promotes cancer development. We investigated whether inhibition of YAP down-regulates PD-L1 (CD274) in human malignant pleural mesothelioma (MPM). Western blotting showed that 2 human MPM cell lines (H2052 and 211H) had increased PD-L1 protein expression compared to H290, MS-1 and H28 cells. In H2052 and 211H cells, PD-L1 mRNA expression was significantly increased compared to other MPM cell lines; YAP knockdown by small interfering RNA decreased PD-L1 protein and mRNA expression. Forced overexpression of the YAP gene increased PD-L1 protein expression in H2452 cells. Chromatin immunoprecipitation (ChIP) assay showed the precipitation of PD-L1 enhancer region encompassing 2 putative YAP-TEAD-binding sites in H2052 cells. We found that, in human MPM tissue microarray samples, YAP and PD-L1 concurrently expressed in immunohistochemistry stain (n = 70, P

Liver cancer comprises a group of malignant tumors, among which hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) are the most common. ICC is especially pernicious and associated with poor clinical outcome. Studies have shown that a subset of human ICCs may originate from mature hepatocytes. However, the mechanisms driving the trans-differentiation of hepatocytes into malignant cholangiocytes remain poorly defined. We adopted lineage tracing techniques and an established murine hepatocyte-derived ICC model by hydrodynamic injection of activated forms of AKT (myr-AKT) and Yap (YapS127A) proto-oncogenes. Wild-type, Notch1 flox/flox , and Notch2 flox/flox mice were used to investigate the role of canonical Notch signaling and Notch receptors in AKT/Yap-driven ICC formation. Human ICC and HCC cell lines were transfected with siRNA against Notch2 to determine whether Notch2 regulates biliary marker expression in liver tumor cells. We found that AKT/Yap-induced ICC formation is hepatocyte derived and this process is strictly dependent on the canonical Notch signaling pathway in vivo. Deletion of Notch2 in AKT/Yap-induced tumors switched the phenotype from ICC to hepatocellular adenoma-like lesions, while inactivation of Notch1 in hepatocytes did not result in significant histomorphological changes. Finally, in vitro studies revealed that Notch2 silencing in ICC and HCC cell lines down-regulates the expression of Sox9 and EpCAM biliary markers. Notch2 is the major determinant of hepatocyte-derived ICC formation in mice.

Recent reports indicate that hypoxia influences the circadian clock through the transcriptional activities of hypoxia-inducible factors (HIFs) at clock genes. Unexpectedly, we uncover a profound disruption of the circadian clock and diurnal transcriptome when hypoxic cells are permitted to acidify to recapitulate the tumor microenvironment. Buffering against acidification or inhibiting lactic acid production fully rescues circadian oscillation. Acidification of several human and murine cell lines, as well as primary murine T cells, suppresses mechanistic target of rapamycin complex 1 (mTORC1) signaling, a key regulator of translation in response to metabolic status. We find that acid drives peripheral redistribution of normally perinuclear lysosomes away from perinuclear RHEB, thereby inhibiting the activity of lysosome-bound mTOR. Restoring mTORC1 signaling and the translation it governs rescues clock oscillation. Our findings thus reveal a model in which acid produced during the cellular metabolic response to hypoxia suppresses the circadian clock through diminished translation of clock constituents.

Mutations in PKD1 and PKD2 are the leading cause of autosomal dominant polycystic kidney disease (ADPKD). In this issue of Genes & Development, a report by Cai and colleagues (pp. 781-793) reveals new insight into the molecular basis by which PKD1 deficiency leads to cystic kidney pathogenesis. By using extensive mouse genetic analyses coupled with in vitro cystic assays, the investigators delineate a RhoA-YAP-c-Myc signaling axis as a key downstream from PKD1 deficiency in ADPKD pathogenesis. Their findings provide evidence that the Hippo pathway could be a potential target for treating ADPKD.

Yes-associated protein (YAP) is a main mediator of the Hippo pathway and promotes cancer development and progression in human lung cancer. We sought to determine whether inhibition of YAP suppresses metastasis of human lung adenocarcinoma in a murine model. We found that metastatic NSCLC cell lines H2030-BrM3(K-rasG12C mutation) and PC9-BrM3 (EGFRΔexon19 mutation) had a significantly decreased p-YAP(S127)/YAP ratio compared to parental H2030 (K-rasG12C mutation) and PC9 (EGFRΔexon19 mutation) cells (P

Background and aimsRecent meta-analysis of genome-wide association studies have identified over 241 inflammatory bowel disease susceptibility loci. However, the known variants only account for a fraction of inflammatory bowel disease heritability. To identify additional susceptibility loci, we performed a trans-ethnic meta-analysis as well as an Asian-specific meta-analysis, using all published Immunochip association results of inflammatory bowel disease.MethodsAn inverse-variance fixed-effects meta-analysis was carried out across Korean and East Asian Immunochip datasets of 4156 cases and 4904 controls [Asian ancestry]. A trans-ethnic meta-analysis of inflammatory bowel disease was performed together with the European datasets of 38 155 cases and 48 485 controls genotyped on the immunochip using a Bayesian approach, Meta-Analysis of Trans-ethnic Association studies [MANTRA].ResultsWe identified seven novel associations, including three novel susceptibility loci at MYO10-BASP1, PPP2R3C/KIAA0391/PSMA6/NFKB1A and LRRK1 as well as four novel secondary associations within previously known loci at NCF4, TSPAN32, CIITA and VANGL2. The new loci further implicate alterations in B cell biology in Crohn's disease pathogenesis. The effects of five loci were universal across European and Asian ancestries, whereas the NCF4 and CIITA loci showed significant heterogeneity between European and East Asian populations. In addition, 103 previously known IBD loci showed supporting evidence of association with nominal significance [p < 0.05] in Asians.ConclusionsOur findings of new loci not previously associated with IBD support the importance of studying inflammatory bowel disease genetics in diverse populations.

How is protein synthesis initiated locally in neurons? We found that mTOR (mechanistic target of rapamycin) was activated and then up-regulated in injured axons, owing to local translation of mTOR messenger RNA (mRNA). This mRNA was transported into axons by the cell size-regulating RNA-binding protein nucleolin. Furthermore, mTOR controlled local translation in injured axons. This included regulation of its own translation and that of retrograde injury signaling molecules such as importin β1 and STAT3 (signal transducer and activator of transcription 3). Deletion of the mTOR 3' untranslated region (3'UTR) in mice reduced mTOR in axons and decreased local translation after nerve injury. Both pharmacological inhibition of mTOR in axons and deletion of the mTOR 3'UTR decreased proprioceptive neuronal survival after nerve injury. Thus, mRNA localization enables spatiotemporal control of mTOR pathways regulating local translation and long-range intracellular signaling.

Hippo/YAP signaling plays pleiotropic roles in the regulation of cell proliferation and differentiation during organogenesis and tissue repair. Herein we demonstrate increased YAP activity in respiratory epithelial cells in lungs of patients with idiopathic pulmonary fibrosis (IPF), a common, lethal form of interstitial lung disease (ILD). Immunofluorescence staining in IPF epithelial cells demonstrated increased nuclear YAP and loss of MST1/2. Bioinformatic analyses of epithelial cell RNA profiles predicted increased activity of YAP and increased canonical mTOR/PI3K/AKT signaling in IPF. Phospho-S6 (p-S6) and p-PTEN were increased in IPF epithelial cells, consistent with activation of mTOR signaling. Expression of YAP (S127A), a constitutively active form of YAP, in human bronchial epithelial cells (HBEC3s) increased p-S6 and p-PI3K, cell proliferation and migration, processes that were inhibited by the YAP-TEAD inhibitor verteporfin. Activation of p-S6 was required for enhancing and stabilizing YAP, and the p-S6 inhibitor temsirolimus blocked nuclear YAP localization and suppressed expression of YAP target genes CTGF, AXL, and AJUBA (JUB). YAP and mTOR/p-S6 signaling pathways interact to induce cell proliferation and migration, and inhibit epithelial cell differentiation that may contribute to the pathogenesis of IPF.

The Hippo pathway is a central regulator of tissue development and homeostasis, and has been reported to have a role during vascular development. Here we develop a bioluminescence-based biosensor that monitors the activity of the Hippo core component LATS kinase. Using this biosensor and a library of small molecule kinase inhibitors, we perform a screen for kinases modulating LATS activity and identify VEGFR as an upstream regulator of the Hippo pathway. We find that VEGFR activation by VEGF triggers PI3K/MAPK signaling, which subsequently inhibits LATS and activates the Hippo effectors YAP and TAZ. We further show that the Hippo pathway is a critical mediator of VEGF-induced angiogenesis and tumor vasculogenic mimicry. Thus, our work offers a biosensor tool for the study of the Hippo pathway and suggests a role for Hippo signaling in regulating blood vessel formation in physiological and pathological settings.

The activation of brown/beige adipose tissue (BAT) metabolism and the induction of uncoupling protein 1 (UCP1) expression are essential for BAT-based strategies to improve metabolic homeostasis. Here, we demonstrate that BAT utilizes actomyosin machinery to generate tensional responses following adrenergic stimulation, similar to muscle tissues. The activation of actomyosin mechanics is critical for the acute induction of oxidative metabolism and uncoupled respiration in UCP1+ adipocytes. Moreover, we show that actomyosin-mediated elasticity regulates the thermogenic capacity of adipocytes via the mechanosensitive transcriptional co-activators YAP and TAZ, which are indispensable for normal BAT function. These biomechanical signaling mechanisms may inform future strategies to promote the expansion and activation of brown/beige adipocytes.

Claudins, the integral tight junction (TJ) proteins that regulate paracellular permeability and cell polarity, are frequently dysregulated in cancer; however, their role in neoplastic progression is unclear. Here, we demonstrated that knockout of Cldn18, a claudin family member highly expressed in lung alveolar epithelium, leads to lung enlargement, parenchymal expansion, increased abundance and proliferation of known distal lung progenitors, the alveolar epithelial type II (AT2) cells, activation of Yes-associated protein (YAP), increased organ size, and tumorigenesis in mice. Inhibition of YAP decreased proliferation and colony-forming efficiency (CFE) of Cldn18-/- AT2 cells and prevented increased lung size, while CLDN18 overexpression decreased YAP nuclear localization, cell proliferation, CFE, and YAP transcriptional activity. CLDN18 and YAP interacted and colocalized at cell-cell contacts, while loss of CLDN18 decreased YAP interaction with Hippo kinases p-LATS1/2. Additionally, Cldn18-/- mice had increased propensity to develop lung adenocarcinomas (LuAd) with age, and human LuAd showed stage-dependent reduction of CLDN18.1. These results establish CLDN18 as a regulator of YAP activity that serves to restrict organ size, progenitor cell proliferation, and tumorigenesis, and suggest a mechanism whereby TJ disruption may promote progenitor proliferation to enhance repair following injury.

Mutations in several general pre-mRNA splicing factors have been linked to myelodysplastic syndromes (MDSs) and solid tumors. These mutations have generally been assumed to cause disease by the resultant splicing defects, but different mutations appear to induce distinct splicing defects, raising the possibility that an alternative common mechanism is involved. Here we report a chain of events triggered by multiple splicing factor mutations, especially high-risk alleles in SRSF2 and U2AF1, including elevated R-loops, replication stress, and activation of the ataxia telangiectasia and Rad3-related protein (ATR)-Chk1 pathway. We further demonstrate that enhanced R-loops, opposite to the expectation from gained RNA binding with mutant SRSF2, result from impaired transcription pause release because the mutant protein loses its ability to extract the RNA polymerase II (Pol II) C-terminal domain (CTD) kinase-the positive transcription elongation factor complex (P-TEFb)-from the 7SK complex. Enhanced R-loops are linked to compromised proliferation of bone-marrow-derived blood progenitors, which can be partially rescued by RNase H overexpression, suggesting a direct contribution of augmented R-loops to the MDS phenotype.

Disruption of hepatocyte growth hormone (GH) signaling through disruption of Jak2 (JAK2L) leads to fatty liver. Previously, we demonstrated that development of fatty liver depends on adipocyte GH signaling. We sought to determine the individual roles of hepatocyte and adipocyte Jak2 on whole-body and tissue insulin sensitivity and liver metabolism. On chow, JAK2L mice had hepatic steatosis and severe whole-body and hepatic insulin resistance. However, concomitant deletion of Jak2 in hepatocytes and adipocytes (JAK2LA) completely normalized insulin sensitivity while reducing liver lipid content. On high-fat diet, JAK2L mice had hepatic steatosis and insulin resistance despite protection from diet-induced obesity. JAK2LA mice had higher liver lipid content and no protection from obesity but retained exquisite hepatic insulin sensitivity. AKT activity was selectively attenuated in JAK2L adipose tissue, whereas hepatic insulin signaling remained intact despite profound hepatic insulin resistance. Therefore, JAK2 in adipose tissue is epistatic to liver with regard to insulin sensitivity and responsiveness, despite fatty liver and obesity. However, hepatocyte autonomous JAK2 signaling regulates liver lipid deposition under conditions of excess dietary fat. This work demonstrates how various tissues integrate JAK2 signals to regulate insulin/glucose and lipid metabolism.

Stem cells are critical for normal tissue homeostasis and injury‐induced regeneration, but how wounding affects tissue regeneration at the cellular and molecular levels is not yet fully understood. A new study by Yui et al (2018) demonstrates that the extracellular matrix (ECM) remodeling modulates intestinal stem cells in tissue repair and regeneration via activation of the Hippo pathway effectors YAP/TAZ.

Pex1 and Pex6 form a heterohexameric motor essential for peroxisome biogenesis and function, and mutations in these AAA-ATPases cause most peroxisome-biogenesis disorders in humans. The tail-anchored protein Pex15 recruits Pex1/Pex6 to the peroxisomal membrane, where it performs an unknown function required for matrix-protein import. Here we determine that Pex1/Pex6 from S. cerevisiae is a protein translocase that unfolds Pex15 in a pore-loop-dependent and ATP-hydrolysis-dependent manner. Our structural studies of Pex15 in isolation and in complex with Pex1/Pex6 illustrate that Pex15 binds the N-terminal domains of Pex6, before its C-terminal disordered region engages with the pore loops of the motor, which then processively threads Pex15 through the central pore. Furthermore, Pex15 directly binds the cargo receptor Pex5, linking Pex1/Pex6 to other components of the peroxisomal import machinery. Our results thus support a role of Pex1/Pex6 in mechanical unfolding of peroxins or their extraction from the peroxisomal membrane during matrix-protein import.

Apolipoprotein A-I binding protein (AIBP) has been shown to augment cholesterol efflux from endothelial cells and macrophages. In zebrafish and mice, AIBP-mediated regulation of cholesterol levels in the plasma membrane of endothelial cells controls angiogenesis. The goal of this work was to evaluate metabolic changes and atherosclerosis in AIBP loss-of-function and gain-of-function animal studies. Here, we show that Apoa1bp-/-Ldlr-/- mice fed a high-cholesterol, high-fat diet had exacerbated weight gain, liver steatosis, glucose intolerance, hypercholesterolemia, hypertriglyceridemia, and larger atherosclerotic lesions compared with Ldlr-/- mice. Feeding Apoa1bp-/-Ldlr-/- mice a high-cholesterol, normal-fat diet did not result in significant differences in lipid levels or size of atherosclerotic lesions from Ldlr-/- mice. Conversely, adeno-associated virus-mediated overexpression of AIBP reduced hyperlipidemia and atherosclerosis in high-cholesterol, high-fat diet-fed Ldlr-/- mice. Injections of recombinant AIBP reduced aortic inflammation in Ldlr-/- mice fed a short high-cholesterol, high-fat diet. Conditional overexpression of AIBP in zebrafish also reduced diet-induced vascular lipid accumulation. In experiments with isolated macrophages, AIBP facilitated cholesterol efflux to HDL, reduced lipid rafts content, and inhibited inflammatory responses to lipopolysaccharide.jlr Our data demonstrate that AIBP confers protection against diet-induced metabolic abnormalities and atherosclerosis.

Cadherin-mediated cell-cell adhesion plays an important role in organ development and changes in cadherin expression are often linked to morphogenetic and pathogenic events. Cadherins interact with other intracellular components to form adherens junctions (AJs) and provide mechanical attachments between adjacent cells. E-cadherin (Cdh1) represents an integral component of these intercellular junctions. To elucidate the function of E-cadherin in the developing pancreas, we generated and studied pancreas-specific Cdh1-knockout (Cdh1ΔPan/ΔPan) mice. Cdh1ΔPan/ΔPan mice exhibit normal body size at birth, but fail to gain weight and become hypoglycemic soon afterward. We found that E-cadherin is not required for the establishment of apical-basal polarity or pancreatic exocrine cell identity at birth. However, four days after birth, the pancreata of Cdh1ΔPan/ΔPan mutants display progressive deterioration of exocrine architecture and dysregulation of Wnt and YAP signaling. At this time point, the acinar cells of Cdh1ΔPan/ΔPan mutants begin to exhibit ductal phenotypes, suggesting acinar-to-ductal metaplasia (ADM) in the E-cadherin-deficient pancreas. Our findings demonstrate that E-cadherin plays an integral role in the maintenance of exocrine architecture and regulation of homeostatic signaling. The present study provides insights into the involvement of cadherin-mediated cell-cell adhesion in pathogenic conditions such as pancreatitis or pancreatic cancer.

Glioblastoma is the most common and aggressive adult brain cancer. Tumors show frequent dysregulation of the PI3K-mTOR pathway. Although a number of small molecules target the PI3K-AKT-mTOR axis, their preclinical and clinical efficacy has been limited. Reasons for treatment failure include poor penetration of agents into the brain and observations that blockade of PI3K or AKT minimally affects downstream mTOR activity in glioma. Clinical trials using allosteric mTOR inhibitors (rapamycin and rapalogs) to treat patients with glioblastoma have also been unsuccessful or uncertain, in part, because rapamycin inefficiently blocks the mTORC1 target 4EBP1 and feeds back to activate PI3K-AKT signaling. Inhibitors of the mTOR kinase (TORKi) such as TAK-228/MLN0128 interact orthosterically with the ATP- and substrate-binding pocket of mTOR kinase, efficiently block 4EBP1 in vitro, and are currently being investigated in the clinical trials. Preclinical studies suggest that TORKi have poor residence times of mTOR kinase, and our data suggest that this poor pharmacology translates into disappointing efficacy in glioblastoma xenografts. RapaLink-1, a TORKi linked to rapamycin, represents a drug with improved pharmacology against 4EBP1. In this review, we clarify the importance of 4EBP1 as a biomarker for the efficacy of PI3K-AKT-mTOR inhibitors in glioblastoma. We also review mechanistic data by which RapaLink-1 blocks p-4EBP1 and discuss future clinical strategies for 4EBP1 inhibition in glioblastoma. Clin Cancer Res; 24(1); 14-21. ©2017 AACR.

Purpose: To investigate the influence of dishevelled-2 (Dvl2) protein and the phosphorylation and ubiquitination of Dvl2 on dental implant osseointegration in hyperlipidemic rats. Materials and Methods: Thirty-six Wistar rats were divided equally into two groups: an experimental group fed with a high-fat diet for 8 weeks and a control group fed with a normal diet for 8 weeks. Serum lipids were detected, and titanium implants were placed into bilateral proximal metaphysis of rats’ femurs. At 1, 3, and 5 days after implantation, rats were sacrificed and 1 mm of bone around each implant was obtained. Real-time polymerase chain reaction of runt-related transcription factor 2 (Runx2), Cathepsin K, and Dvl2 was performed. Western blot of Dvl2, phosphorylation, and ubiquitination of Dvl2 was also performed. Results: In the experimental group, the expressions of Runx2, Dvl2, and phosphorylation of Dvl2 were lower (P < .05), while that of Cathepsin K and ubiquitination of Dvl2 were higher than those in the control group (P < .05). Conclusion: Hyperlipidemia suppressed the dental implant osseointegration. This inhibition may be modulated by downregulated Dvl2, phosphorylation of Dvl2 and upregulated ubiquitination of Dvl2. [ABSTRACT FROM AUTHOR]

Traditional drug screening methods lack features of the tumor microenvironment that contribute to resistance. Most studies examine cell response in a single biomaterial platform in depth, leaving a gap in understanding how extracellular signals such as stiffness, dimensionality, and cell-cell contacts act independently or are integrated within a cell to affect either drug sensitivity or resistance. This is critically important, as adaptive resistance is mediated, at least in part, by the extracellular matrix (ECM) of the tumor microenvironment. We developed an approach to screen drug responses in cells cultured on 2D and in 3D biomaterial environments to explore how key features of ECM mediate drug response. This approach uncovered that cells on 2D hydrogels and spheroids encapsulated in 3D hydrogels were less responsive to receptor tyrosine kinase (RTK)-targeting drugs sorafenib and lapatinib, but not cytotoxic drugs, compared to single cells in hydrogels and cells on plastic. We found that transcriptomic differences between these in vitro models and tumor xenografts did not reveal mechanisms of ECM-mediated resistance to sorafenib. However, a systems biology analysis of phospho-kinome data uncovered that variation in MEK phosphorylation was associated with RTK-targeted drug resistance. Using sorafenib as a model drug, we found that co-administration with a MEK inhibitor decreased ECM-mediated resistance in vitro and reduced in vivo tumor burden compared to sorafenib alone. In sum, we provide a novel strategy for identifying and overcoming ECM-mediated resistance mechanisms by performing drug screening, phospho-kinome analysis, and systems biology across multiple biomaterial environments.

One of the major biological functions of the retinal pigmented epithelium (RPE) is the clearance of shed photoreceptor outer segments (POS) through a multistep process resembling phagocytosis. RPE phagocytosis helps maintain the viability of photoreceptors that otherwise could succumb to the high metabolic flux and photo-oxidative stress associated with visual processing. The regulatory mechanisms underlying phagocytosis in the RPE are not fully understood, although dysfunction of this process contributes to the pathogenesis of multiple human retinal degenerative disorders, including age-related macular degeneration. Here, we present an integrated transcriptomic, proteomic, and phosphoproteomic analysis of phagocytosing RPE cells, utilizing three different experimental models: the human-derived RPE-like cell line ARPE-19, cultured murine primary RPE cells, and RPE samples from live mice. Our combined results indicated that early stages of phagocytosis in the RPE are mainly characterized by pronounced changes in the protein phosphorylation level. Global phosphoprotein enrichment analysis revealed involvement of PI3K/Akt, mechanistic target of rapamycin (mTOR), and MEK/ERK pathways in the regulation of RPE phagocytosis, confirmed by immunoblot analyses and in vitro phagocytosis assays. Most strikingly, phagocytosis of POS by cultured RPE cells was almost completely blocked by pharmacological inhibition of phosphorylation of Akt. Our findings, along with those of previous studies, indicate that these phosphorylation events allow the RPE to integrate multiple signals instigated by shed POS at different stages of the phagocytic process.

The vascular barrier that separates blood from tissues is actively regulated by the endothelium and is essential for transport, inflammation, and haemostasis. Haemodynamic shear stress plays a critical role in maintaining endothelial barrier function, but how this occurs remains unknown. Here we use an engineered organotypic model of perfused microvessels to show that activation of the transmembrane receptor NOTCH1 directly regulates vascular barrier function through a non-canonical, transcription-independent signalling mechanism that drives assembly of adherens junctions, and confirm these findings in mouse models. Shear stress triggers DLL4-dependent proteolytic activation of NOTCH1 to expose the transmembrane domain of NOTCH1. This domain mediates establishment of the endothelial barrier; expression of the transmembrane domain of NOTCH1 is sufficient to rescue defects in barrier function induced by knockout of NOTCH1. The transmembrane domain restores barrier function by catalysing the formation of a receptor complex in the plasma membrane consisting of vascular endothelial cadherin, the transmembrane protein tyrosine phosphatase LAR, and the RAC1 guanidine-exchange factor TRIO. This complex activates RAC1 to drive assembly of adherens junctions and establish barrier function. Canonical transcriptional signalling via Notch is highly conserved in metazoans and is required for many processes in vascular development, including arterial-venous differentiation, angiogenesis and remodelling. We establish the existence of a non-canonical cortical NOTCH1 signalling pathway that regulates vascular barrier function, and thus provide a mechanism by which a single receptor might link transcriptional programs with adhesive and cytoskeletal remodelling.

Background & aimsIntrahepatic cholangiocarcinoma (ICC) is a lethal malignancy without effective treatment options. MLN0128, a second generation pan-mTOR inhibitor, shows efficacy for multiple tumor types. We evaluated the therapeutic potential of MLN0128 vs. gemcitabine/oxaliplatin in a novel ICC mouse model.MethodsWe established a novel ICC mouse model via hydrodynamic transfection of activated forms of AKT (myr-AKT) and Yap (YapS127A) protooncogenes (that will be referred to as AKT/YapS127A). Genetic approaches were applied to study the requirement of mTORC1 and mTORC2 in mediating AKT/YapS127A driven tumorigenesis. Gemcitabine/oxaliplatin and MLN0128 were administered in AKT/YapS127A tumor-bearing mice to study their anti-tumor efficacy in vivo. Multiple human ICC cell lines were used for in vitro experiments. Hematoxylin and eosin staining, immunohistochemistry and immunoblotting were applied for the characterization and mechanistic study.ResultsCo-expression of myr-AKT and YapS127A promoted ICC development in mice. Both mTORC1 and mTORC2 complexes were required for AKT/YapS127A ICC development. Gemcitabine/oxaliplatin had limited efficacy in treating late stage AKT/YapS127A ICC. In contrast, partial tumor regression was achieved when MLN0128 was applied in the late stage of AKT/YapS127A cholangiocarcinogenesis. Furthermore, when MLN0128 was administered in the early stage of AKT/YapS127A carcinogenesis, it led to disease stabilization. Mechanistically, MLN0128 efficiently inhibited AKT/mTOR signaling both in vivo and in vitro, inducing strong ICC cell apoptosis and only marginally affecting proliferation.ConclusionsThis study suggests that mTOR kinase inhibitors may be beneficial for the treatment of ICC, even in tumors that are resistant to standard of care chemotherapeutics, such as gemcitabine/oxaliplatin-based regimens, especially in the subset of tumors exhibiting activated AKT/mTOR cascade. Lay summary: We established a novel mouse model of intrahepatic cholangiocarcinoma (ICC). Using this new preclinical model, we evaluated the therapeutic potential of mTOR inhibitor MLN0128 vs. gemcitabine/oxaliplatin (the standard chemotherapy for ICC treatment). Our study shows the anti-neoplastic potential of MLN0128, suggesting that it may be superior to gemcitabine/oxaliplatin-based chemotherapy for the treatment of ICC, especially in the tumors exhibiting activated AKT/mTOR cascade.

Simple Summary: Rhabdomyosarcoma accounts for more than 50% of all soft tissue sarcomas in childhood and adolescence. Despite intensified multimodality treatment, prognosis is extremely poor, with an overall survival rate of approximately 20% in the advanced stage. Therefore, there is an urgent need for targeted treatment options to improve overall survival rates, and to limit long-term side effects. Tumor-targeting peptides offer the possibility to deliver drugs selectively to the tumor site. Here, we select F3 as the best rhabdomyosarcoma-targeting peptide among a panel of 20 different tumor-targeting peptides. F3 peptide showed strong and specific binding to rhabdomyosarcoma, but not to normal cells, and efficient internalization with effective cytoplasmic delivery of the toxin Saporin. We show that nucleolin, the target of F3 peptide, is expressed on the surface of rhabdomyosarcoma cells. F3 peptide is a promising candidate for targeted drug delivery to rhabdomyosarcoma, e.g., by targeting drug-loaded nanoparticles. Active drug delivery by tumor-targeting peptides is a promising approach to improve existing therapies for rhabdomyosarcoma (RMS), by increasing the therapeutic effect and decreasing the systemic toxicity, e.g., by drug-loaded peptide-targeted nanoparticles. Here, we tested 20 different tumor-targeting peptides for their ability to bind to two RMS cell lines, Rh30 and RD, using quantum dots Streptavidin and biotin-peptides conjugates as a model for nanoparticles. Four peptides revealed a very strong binding to RMS cells: NCAM-1-targeting NTP peptide, nucleolin-targeting F3 peptide, and two Furin-targeting peptides, TmR and shTmR. F3 peptide showed the strongest binding to all RMS cell lines tested, low binding to normal control myoblasts and fibroblasts, and efficient internalization into RMS cells demonstrated by the cytoplasmic delivery of the Saporin toxin. The expression of the nucleophosphoprotein nucleolin, the target of F3, on the surface of RMS cell lines was validated by competition with the natural ligand lactoferrin, by colocalization with the nucleolin-binding aptamer AS1411, and by the marked sensitivity of RMS cell lines to the growth inhibitory nucleolin-binding N6L pseudopeptide. Taken together, our results indicate that nucleolin-targeting by F3 peptide represents a potential therapeutic approach for RMS. [ABSTRACT FROM AUTHOR]

Cancer is a fatal disease worldwide. Each year ten million people are diagnosed around the world, and more than half of patients eventually die from it in many countries. A majority of cancer remains asymptomatic in the earlier stages, with specific symptoms appearing in the advanced stages when the chances of adequate treatment are low. Cancer screening is generally executed by different imaging techniques like ultrasonography (USG), mammography, CT-scan, and magnetic resonance imaging (MRI). Imaging techniques, however, fail to distinguish between cancerous and non-cancerous cells for early diagnosis. To confirm the imaging result, solid and liquid biopsies are done which have certain limitations such as invasive (in case of solid biopsy) or missed early diagnosis due to extremely low concentrations of circulating tumor DNA (in case of liquid biopsy). Therefore, it is essential to detect certain biomarkers by a noninvasive approach. One approach is a proteomic or glycoproteomic study which mostly identifies proteins and glycoproteins present in tissues and serum. Some of these studies are approved by the Food and Drug Administration (FDA). Another non-expensive and comparatively easier method to detect glycoprotein biomarkers is by ELISA, which uses lectins of diverse specificities. Several of the FDA approved proteins used as cancer biomarkers do not show optimal sensitivities for precise diagnosis of the diseases. In this regard, expression of phosphoproteins is associated with a more specific stage of a particular disease with high sensitivity and specificity. In this review, we discuss the expression of different serum phosphoproteins in various cancers. These phosphoproteins are detected either by phosphoprotein enrichment by immunoprecipitation using phosphospecific antibody and metal oxide affinity chromatography followed by LC-MS/MS or by 2D gel electrophoresis followed by MALDI-ToF/MS analysis. The updated knowledge on phosphorylated proteins in clinical samples from various cancer patients would help to develop these serum phophoproteins as potential diagnostic/prognostic biomarkers of cancer. [ABSTRACT FROM AUTHOR]

Simple Summary: After initially responding to empiric radio-chemotherapy, most advanced thymomas and thymic carcinomas become refractory and require second-line therapies. The multi-target tyrosine kinase inhibitor, sunitinib, is one of few options, especially in patients with thymic carcinomas, and has resulted in partial remissions and prolonged overall survival. However, sunitinib shows limited activity, and not all patients benefit equally. A better understanding of its mode of action and the definition of predictive biomarkers would help select patients who profit most. Using a real-time multiplex tyrosine phosphorylation assay containing 144 kinase substrates in a defined set of sunitinib-sensitive and -resistant cell lines, we generated a sunitinib response index (SRI). Protein lysates from thymomas and thymic carcinomas with spike-in of sunitinib were then classified as potential responders vs. non-responders using the same SRI classifier. Bioinformatic prediction and further experimental analysis of activated upstream kinases identified TYRO3 as a potent mediator of sunitinib resistance, specifically in metastatic thymomas. TYRO3 could serve both as a biomarker of sunitinib resistance and a potential therapeutic target that could help to tailor treatment decisions and to overcome therapy resistance in advanced thymomas and thymic carcinomas. Background: After initially responding to empiric radio-chemotherapy, most advanced thymomas (TH) and thymic carcinomas (TC) become refractory and require second-line therapy. The multi-target receptor tyrosine kinase (RTK) inhibitor, sunitinib, is one of the few options, especially in patients with thymic carcinomas, and has resulted in partial remissions and prolonged overall survival. However, sunitinib shows variable activity in thymomas, and not all patients benefit equally. A better understanding of its mode of action and the definition of predictive biomarkers would help select patients who profit most. Methods: Six cell lines were treated with sunitinib in vitro. Cell viability was measured by MTS assay and used to define in vitro responders and non-responders. A quantitative real-time assay simultaneously measuring the phosphorylation of 144 tyrosine kinase substrates was used to correlate cell viability with alterations of the phospho-kinome, calculate a sunitinib response index (SRI), and impute upstream tyrosine kinases. Sunitinib was added to protein lysates of 29 malignant TH and TC. Lysates were analyzed with the same phosphorylation assay. The SRI tentatively classified cases into potential clinical responders and non-responders. In addition, the activation patterns of 44 RTKs were studied by phospho-RTK arrays in 37 TH and TC. Results: SRI application separated thymic epithelial tumors (TET) in potential sunitinib responders and resistant cases. Upstream kinase prediction identified multiple RTKs potentially involved in sunitinib response, many of which were subsequently shown to be differentially overexpressed in TH and TC. Among these, TYRO3/Dtk stood out since it was exclusively present in metastatic TH. The function of TYRO3 as a mediator of sunitinib resistance was experimentally validated in vitro. Conclusions: Using indirect and direct phosphoproteomic analyses to predict sunitinib response in malignant TET, we have shown that TH and TC express multiple important sunitinib target RTKs. Among these, TYRO3 was identified as a potent mediator of sunitinib resistance activity, specifically in metastatic TH. TYRO3 may thus be both a novel biomarker of sunitinib resistance and a potential therapeutic target in advanced thymomas and thymic carcinomas. [ABSTRACT FROM AUTHOR]

Introduction: Abnormal placental trophoblast function is the main cause of missed abortion (MA). Src kinase-associated phosphoprotein 2 (SKAP2) indirectly affects actin reunion, which is significantly associated with cell migration.Methods: Twenty women with MA and 20 healthy women who underwent voluntarily induced abortion were included in this study. Immunohistochemistry, qRT-PCR, and western blotting were used to determine SKAP2, WAVE2, and ARP2 expression in the villous tissues. We investigated the effects of SKAP2 and the W336K mutant (blocked SKAP2 Src homology 3 function) on growth and migration in HTR8/SVneo cells using the CCK8 assay, flow cytometry, and transwell assay. The effects of SKAP2 on the WAVE2-ARP2/3 signaling pathway in HTR8/SVneo cells were evaluated by western blotting and immunofluorescence.Results: Compared to the women in the voluntary abortion group, SKAP2 and WAVE2 expression levels were downregulated in the villous of patients with MA. In HTR8/SVneo cells, SKAP2 siRNA silencing regulated the growth and migration, while SKAP2 overexpression promoted growth and migration, and inhibited apoptosis. Additionally, SKAP2 regulated the expression of WAVE2 and ARP2, as well as the colocalization of actin with WAVE2. The SKAP2 W336K mutant could not alter WAVE2 and ARP2 expression, nor HTR8/SVneo cell growth and migration, with or without SKAP2 siRNA transfection.Discussion: SKAP2 could activate the WAVE2-ARP2/3 pathway resulting in an increase of growth and migration in trophoblasts. SKAP2 probably played an important role in MA by affecting the growth and migration of trophoblasts. [ABSTRACT FROM AUTHOR]

Objective: Nucleolar proteins have important functions in the regulation of cell homeostasis and play a crucial role in sensing various types of stress, such as genotoxic stress. Propolis has epithelial, analgesic, antibacterial, antifungal and antiviral effects. This study aimed to evaluate the gene expression levels of nucleolar proteins: nucleolin (NCL); nucleophosmin (NPM1); and upstream binding transcription factor (UBTF), as well as the benefits of propolis in wound healing. Method: This experimental study was conducted by creating clean and clean–contaminated wounds according to the Surgical Site Infection Guidelines, 2016. A total of seven animal groups were included in the study: control; laparotomy; anastomosis; fucidic acid with/without anastomosis; propolis with/without anastomosis Results: Statistically significant differences of levels of gene expression among the groups were detected for NCL (p=0.004), NPM1 (p=0.011) and UBTF (p=0.000). When the expression levels of the related genes and blood parameters are considered, the relationship between NCL, NPM1 and UBTF expression levels and blood parameters (NE, EO, NE(%), LY, LY(%), EO, EO(%), MO, MO(%), RBC, HB, HCT, MCV, MCHC, RDW, RDW(%), PLT, PDW and PCT) were statistically significant. Conclusion: The nucleolar proteins such as NCL, NPM1 and UBTF have important functions in cell viability and its maintenance under various condition such as stress and injury. Additionally, propolis has positive benefits in wound healing and in the prevention of wound infection. Our findings provide the first insights into the putative role of those proteins in wound healing. [ABSTRACT FROM AUTHOR]

Protein phosphorylation is a reversible post-translational modification (PTM) with major regulatory roles in many cellular processes. However, the analysis of phosphoproteins remains the most challenging barrier in the prevailing proteome research. Recent technological advances in two-dimensional electrophoresis (2-DE) coupled to mass spectrometry (MS) have enabled the identification, characterization, and quantification of protein phosphorylation on a global scale. Most research on phosphoproteins with 2-DE has been conducted using phosphostains. Nevertheless, low-abundant and low-phosphorylated phosphoproteins are not necessarily detected using phosphostains and/or MS. In this study, we report a comparative analysis of 2-DE phosphoproteome profiles using Pro-Q Diamond phosphoprotein stain (Pro-Q DPS) and chemical dephosphorylation of proteins with HF-P from longissimus thoracis (LT) muscle samples of the Rubia Gallega cattle breed. We found statistically significant differences in the number of identified phosphoproteins between methods. More specifically, we found a three-fold increase in phosphoprotein detection with the HF-P method. Unlike Pro-Q DPS, phosphoprotein spots with low volume and phosphorylation rate were identified by HF-P technique. This is the first approach to assess meat phosphoproteome maps using HF-P at a global scale. The results open a new window for 2-DE gel-based phosphoproteome analysis. [ABSTRACT FROM AUTHOR]

Simple Summary: Nucleolin (NCL) regulates tumour growth and angiogenesis, and its inhibition normalizes tumour vessels and impairs pancreatic ductal adenocarcinoma (PDAC) growth. Since tumour vessel normalization promotes immunostimulatory reprogramming, we investigated the effects of a selective inhibitor of NCL, the pseudopeptide N6L, on the immune microenvironment of PDAC. This work highlights a new therapeutic strategy that restrains immunosuppressive cells to promote T- cell recruitment and activation and to re-program the tumour stroma of PDAC. Background: The pancreatic ductal adenocarcinoma (PDAC) microenvironment is highly fibrotic and hypoxic, with poor immune cell infiltration. Recently, we showed that nucleolin (NCL) inhibition normalizes tumour vessels and impairs PDAC growth. Methods: Immunocompetent mouse models of PDAC were treated by the pseudopeptide N6L, which selectively inhibits NCL. Tumour-infiltrating immune cells and changes in the tumour microenvironment were analysed. Results: N6L reduced the proportion of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) and increased tumour-infiltrated T lymphocytes (TILs) with an activated phenotype. Low-dose anti-VEGFR2 treatment normalized PDAC vessels but did not modulate the immune suppressive microenvironment. RNAseq analysis of N6L-treated PDAC tumours revealed a reduction of cancer-associated fibroblast (CAF) expansion in vivo and in vitro. Notably, N6L treatment decreased IL-6 levels both in tumour tissues and in serum. Treating mPDAC by an antibody blocking IL-6 reduced the proportion of Tregs and MDSCs and increased the amount of TILs, thus mimicking the effects of N6L. Conclusions: These results demonstrate that NCL inhibition blocks the amplification of lymphoid and myeloid immunosuppressive cells and promotes T cell activation in PDAC through a new mechanism of action dependent on the direct inhibition of the tumoral stroma. [ABSTRACT FROM AUTHOR]

Simple Summary: Breast cancer remains the leading cause of cancer-related deaths in women worldwide. One particular subtype, triple negative breast cancer (TNBC), is known for being highly aggressive and metastatic, with a high prevalence of resistance to chemotherapy. Patients with chemotherapy-resistant TNBC tumors have virtually no treatment options. Here, we studied tyrosine phosphorylation-mediated signaling networks in chemotherapy-resistant TNBC xenografts, to identify potential therapeutic targets. We identified Src family kinases (SFK) as potential drug targets. Inhibition of SFKs radically reduced tumor growth in xenografts that had a signature of high SFK-activity. We assessed a small number of human tumor tissue specimens and found a low prevalence of SFK-driven tumors. These data may explain the previous unsuccessful clinical trials targeting SFKs in TNBC and warrant further studies with a higher number of patient samples. These results emphasize the importance of the characterization of phosphotyrosine signaling profiles for better patient stratification, in addition to gaining novel therapeutic insights. Neoadjuvant chemotherapy (NAC) remains the cornerstone of the treatment for triple negative breast cancer (TNBC), with the goal of complete eradication of disease. However, for patients with residual disease after NAC, recurrence and mortality rates are high and the identification of novel therapeutic targets is urgently needed. We quantified tyrosine phosphorylation (pTyr)-mediated signaling networks in chemotherapy sensitive (CS) and resistant (CR) TNBC patient-derived xenografts (PDX), to gain novel therapeutic insights. The antitumor activity of SFK inhibition was examined in vivo. Treated tumors were further subjected to phosphoproteomic and RNAseq analysis, to identify the mechanism of actions of the drug. We identified Src Family Kinases (SFKs) as potential therapeutic targets in CR TNBC PDXs. Treatment with dasatinib, an FDA approved SFK inhibitor, led to inhibition of tumor growth in vivo. Further analysis of post-treatment PDXs revealed multiple mechanisms of actions of the drug, confirming the multi-target inhibition of dasatinib. Analysis of pTyr in tumor specimens suggested a low prevalence of SFK-driven tumors, which may provide insight into prior clinical trial results demonstrating a lack of dasatinib antitumor activity in unselected breast cancer patients. Taken together, these results underscore the importance of pTyr characterization of tumors, in identifying new targets, as well as stratifying patients based on their activated signaling networks for therapeutic options. Our data provide a strong rationale for studying SFK inhibitors in biomarker-selected SFK-driven TNBC. [ABSTRACT FROM AUTHOR]

Simple Summary: Ribosome maturation in eukaryotes is a highly energy-consuming, evolutionarily conserved process, directed by a large number of auxiliary proteins (>200) called trans-acting factors (TAFs). TAFs direct the ordered and directional assembly of immature pre-40S and pre-60S complexes in multiple cellular compartments, but they are not themselves components of translationally competent ribosomes. Although the enzymatic properties of many TAFs are understood, others assist ribosome maturation without predicted enzymatic activity. An important late-acting 60S TAF is Mrt4, which initially binds to pre-60S complexes within the nucleus that must be released from pre-60S complexes following translocation into in the cytoplasm prior to the addition of a complex of acidic phosphoproteins denoting the ribosomal stalk, an essential component of the peptidyl transferase center (PTC) of ribosomes. Prior genetic studies implicated the dual-specificity phosphatase Yvh1 as the protein responsible for cytoplasmic Mrt4 removal, but how Yvh1 facilitates this is unknown. yvh1Δ strains are viable but grow slowly due to translational defects associated with improper ribosome assembly. Yvh1-dependent Mrt4 removal is independent of YVH1's protein phosphatase domain but instead maps to an evolutionarily conserved cysteine-rich domain (CRD) of unknown function. In order to better understand how YVH1's CRD facilitates Mrt4 removal and thus 60S maturation, we identified loss-of-function (LOF) variants of Yvh1 incapable of displacing Mrt4 precluding the addition of the ribosomal stalk. This approach additionally identified a variant of Yvh1 (Yvh1F283L) that functions as an expression-dependent, dominant-negative variant capable of perturbing ribosome assembly in cells containing wild-type YVH1. These findings are consistent with, and expand on, recent structural models for Yvh1-dependent Mrt4 removal from pre-60S complexes and generate novel first-generation probes that can be used to better understand eukaryotic ribosomal maturation. Ribosome assembly is a complex biological process facilitated by >200 trans-acting factors (TAFs) that function as scaffolds, place-holders or complex remodelers to promote efficient and directional ribosomal subunit assembly but are not themselves part of functional ribosomes. One such yeast TAF is encoded by Mrt4 which assembles onto pre-60S complexes in the nuclear compartment and remains bound to pre-60S complexes as they are exported into the cytoplasm. There, Mrt4 is displaced from pre-60S complexes facilitating the subsequent addition of the ribosomal stalk complex (P0/P1/P2). Ribosomal stalk proteins interact with translational GTPases (trGTPase) which facilitate and control protein synthesis on the ribosome. The rRNA-binding domain of Mrt4 is structurally similar to P0, with both proteins binding to the same interface of pre-60S subunits in a mutually exclusive manner; the addition of the ribosomal stalk therefore requires the displacement of Mrt4 from pre-60S subunits. Mrt4 removal requires the C-terminal cysteine-rich domain (CRD) of the dual-specificity phosphatase Yvh1. Unlike many other TAFs, yeast lacking Yvh1 are viable but retain Mrt4 on cytoplasmic pre-60S complexes precluding ribosomal stalk addition. Although Yvh1's role in Mrt4 removal is well established, how Yvh1 accomplishes this is largely unknown. Here, we report an unbiased genetic screen to isolate Yvh1 variants that fail to displace Mrt4 from pre-60S ribosomes. Bioorthogonal non-canonical amino acid tagging (BONCAT) approaches demonstrate that these YVH1 loss-of-function variants also display defects in nascent protein production. The further characterization of one LOF variant, Yvh1F283L, establishes it as an expression-dependent, dominant-negative variant capable of interfering with endogenous Yvh1 function, and we describe how this Yvh1 variant can be used as a novel probe to better understand ribosome maturation and potentially ribosome heterogeneity in eukaryotes. [ABSTRACT FROM AUTHOR]

Phosphorylation is one of the most important protein post‐translational modifications, which possesses dramatic regulatory effects on the function of proteins. In consideration of the low abundance and low stoichiometry of phosphorylation and non‐specific signal suppression, efficient capture of the phosphoproteins from complex biological samples is critical to meet the need for protein profiling. In this work, a facile preparation of titanium (IV)‐immobilized O‐phospho‐L‐tyrosine modified magnetic nanoparticles was developed for the enrichment of intact phosphoproteins. The prepared magnetic nanoparticles were characterized by various instruments and had a spherical shape with an average diameter of 300 nm. The adsorption isotherms were investigated and the maximum capacity for β‐casein was calculated to be 961.5 mg/g. Standard protein mixtures and biological samples (non‐fat milk and human serum) were selected to test the enrichment performance. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis analysis demonstrated the excellent enrichment performance with high selectivity. With the superparamagnetic property, titanium (IV)‐immobilized O‐phospho‐L‐tyrosine modified magnetic nanoparticles were convenient for the practical application and clinical promotion, thus having a promising prospect in the field of phosphoprotein research. [ABSTRACT FROM AUTHOR]

RationaleAutologous stem cell therapy using human c-Kit+ cardiac progenitor cells (hCPCs) is a promising therapeutic approach for treatment of heart failure (HF). However, hCPCs derived from aged patients with HF with genetic predispositions and comorbidities of chronic diseases exhibit poor proliferative and migratory capabilities, which impair overall reparative potential for injured myocardium. Therefore, empowering functionally compromised hCPCs with proregenerative molecules ex vivo is crucial for improving the therapeutic outcome in patients with HF.ObjectiveTo improve hCPC proliferation and migration responses that are critical for regeneration by targeting proregenerative P2Y2 nucleotide receptor (P2Y2R) activated by extracellular ATP and UTP molecules released following injury/stress.Methods and resultsc-Kit+ hCPCs were isolated from cardiac tissue of patients with HF undergoing left ventricular assist device implantation surgery. Correlations between P2 nucleotide receptor expression and hCPC growth kinetics revealed downregulation of select P2 receptors, including P2Y2R, in slow-growing hCPCs compared with fast growers. hCPC proliferation and migration significantly improved by overexpressing or stimulating P2Y2R. Mechanistically, P2Y2R-induced proliferation and migration were dependent on activation of YAP (yes-associated protein)-the downstream effector of Hippo signaling pathway.ConclusionsProliferation and migration of functionally impaired hCPCs are enhanced by P2Y2R-mediated YAP activation, revealing a novel link between extracellular nucleotides released during injury/stress and Hippo signaling-a central regulator of cardiac regeneration. Functional correlations exist between hCPC phenotypic properties and P2 purinergic receptor expression. Lack of P2Y2R and other crucial purinergic stress detectors could compromise hCPC responsiveness to presence of extracellular stress signals. These findings set the stage for subsequent studies to assess purinergic signaling modulation as a potential strategy to improve therapeutic outcome for use of hCPCs in patients with HF.

Malignant mesothelioma is an aggressive cancer that is resistant to current therapy. The poor prognosis of mesothelioma has been associated with elevated Yes-associated protein (YAP) activity. In this study, we evaluated the effect of targeting YAP in mesothelioma. First, we comprehensively studied YAP activity in five mesothelioma cell lines (211H, H2052, H290, MS-1 and H2452) and one normal mesothelial cell line (LP9). We found decreased phospho-YAP to YAP protein ratio and consistently increased GTIIC reporter activity in 211H, H2052 and H290 compared to LP9. The same three cell lines (IC50 s < 1 μM) were more sensitive than LP9 (IC50 = 3.5 μM) to the YAP/TEAD inhibitor verteporfin. We also found that verteporfin significantly reduced YAP protein level, mRNA levels of YAP downstream genes and GTIIC reporter activity in the same three cell lines, indicating inhibition of YAP signaling by verteporfin. Verteporfin also impaired invasion and tumoursphere formation ability of H2052 and H290. To validate the effect of specific targeting YAP in mesothelioma cells, we down-regulated YAP by siRNA. We found siYAP significantly decreased YAP transcriptional activity and impaired invasion and tumoursphere formation ability of H2052 and H290. Furthermore, forced overexpression of YAP rescued GTIIC reporter activity and cell viability after siYAP targeting 3'UTR of YAP. Finally, we found concurrent immunohistochemistry staining of ROCK2 and YAP (P < 0.05). Inhibition of ROCK2 decreased GTIIC reporter activity in H2052 and 211H suggesting that Rho/ROCK signaling also contributed to YAP activation in mesothelioma cells. Our results indicate that YAP may be a potential therapeutic target in mesothelioma.

Obtaining stable electrochemiluminescence (ECL) emissions from a hydrophobic luminophore in aqueous solutions and designing a method without the use of an exogenous coreactant are promising for ECL biosensing. Here, a highly sensitive signal-on ECL immunoassay for the SARS-CoV-2 N protein was developed using micelles as an ECL tag. The micelles were prepared by coencapsulating the luminophore hydrophobic CdSe/ZnS quantum dots and coreactant dibenzoyl peroxide within the hydrophobic core of micelles. The ECL probe was obtained by covalently bonding a SARS-CoV-2 N protein-binding aptamer onto the micelle surface. The construction of the immunosensor was initiated by the immobilization of the anti-SARS-CoV-2 N protein antibody onto the screen-printed carbon electrode (SPCE) with a -COOH-functionalized surface. The surface functionalization of SPCEs was achieved through paste-exfoliated graphene, which was modified with a -COOH group through supramolecular-covalent scaffolds on SPCE. Upon achieving sandwich complexes on the immunosensor, an efficient ECL signal response at -1.4 V versus Ag/AgCl was obtained in phosphate buffer solution. The ECL assay was used for the sensitive determination of SARS-CoV-2 N protein with the linear range from 0.01 to 50 ng mL -1 , and the detection limit was 3.0 pg mL -1 . The immunosensor showed good reproducibility and stability, and the ECL immunoassay was used to determine the SARS-CoV-2 N protein in serum samples. The proposed approach to obtain micelles is versatile for the preparation of stable ECL luminophores by using hydrophobic materials, and the strategy provides an alternative for ECL bioassays based on the coreactant route.

The production of eukaryotic mRNAs requires transcription by RNA polymerase (pol) II and co-transcriptional processing, including capping, splicing, and cleavage and polyadenylation. Pol II can positively affect co-transcriptional processing through interaction of factors with its carboxyl terminal domain (CTD), comprising 52 repeats of the heptapeptide Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7, and pol II elongation rate can regulate splicing. Splicing, in turn, can also affect transcriptional activity and transcription elongation defects are caused by some splicing inhibitors. Multiple small molecule inhibitors of splicing are now available, some of which specifically target SF3B1, a U2 snRNP component. SF3B1 inhibition results in a general downregulation of transcription elongation, including premature termination of transcription caused by increased use of intronic poly(A) sites. Here, we have investigated the effect of Madrasin and Isoginkgetin, two non-SF3B1 splicing inhibitors, on splicing and transcription. Surprisingly, we found that both Madrasin and Isoginkgetin affect transcription before any effect on splicing, indicating that their effect on pre-mRNA splicing is likely to be indirect. Both small molecules promote a general downregulation of transcription. Based on these and other published results, we conclude that these two small molecules should not be considered as primarily pre-mRNA splicing inhibitors., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Tellier et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cardiogenic shock (CS) is a devastating and fatal complication of acute myocardial infarction (AMI). CS can affect the pharmacokinetics and pharmacodynamics of medications. The unique properties of cangrelor make it the optimal P2Y12 inhibitor for CS-AMI, in terms of both efficacy and safety. The DAPT-SHOCK-AMI trial (ClinicalTrials.gov: NCT03551964; EudraCT: 2018-002161-19) will assess the benefits of cangrelor in patients with an initial CS-AMI undergoing primary angioplasty. This randomised, multicentre, placebo-controlled trial of approximately 550 patients (with an allowed 10% increase) in 5 countries using a double-blind design will compare initial P2Y12 inhibitor treatment strategies in patients with CS-AMI of (A) intravenous cangrelor and (B) ticagrelor administered as crushed tablets at a loading dose of 180 mg. The primary clinical endpoint is a composite of all-cause death, myocardial infarction (MI), or stroke within 30 days. The main secondary endpoints are (1) the net clinical endpoint, defined as death, MI, urgent revascularisation of the infarct-related artery, stroke, or major bleeding as defined by the Bleeding Academic Research Consortium criteria; (2) cardiovascular-related death, MI, urgent revascularisation, or heart failure; (3) heart failure; and (4) cardiovascular-related death, all (1-4) within 1 year after study enrolment. A platelet reactivity study that tests the laboratory antiplatelet benefits of cangrelor, when given in addition to standard antiplatelet therapy, will be conducted using vasodilator-stimulated phosphoprotein phosphorylation. The primary laboratory endpoints are the periprocedural rate of onset and the proportion of patients who achieve effective P2Y12 inhibition. The DAPT-SHOCK-AMI study is the first randomised trial to evaluate the benefits of cangrelor in patients with CS-AMI.

Objective: To assess the diagnostic value of anti-salivary gland protein-1 (SP1) antibody combined with anti-parotid secretory protein (PSP) antibody for Sjögren's syndrome (SS)., Methods: A total of 60 patients with primary SS (pSS) who were treated in the outpatient and inpatient department of Department of Rheumatology and Immunology of the Second Hospital of Hebei Medical University from January 2020 to December 2022 were collected. Thirty patients with other autoimmune diseases accompanied by dry mouth and/or dry eyes were collected as disease control group. Thirty healthy subjects from the physical examination center were collected for healthy control group, serum samples were obtained from all of them. Their general features and clinical information including clinical manifestations, laboratory examinations and other examinations were recorded. The 2016 American College of Rheumatology (ACR)/European League against Rheumatism (EULAR) classification criteria were adopted as the diagnostic standard of pSS. Immunoglobulin G (IgG) subtype of anti-SP1 antibody and anti-PSP antibody were detected by chemiluminescence immunoassay. The receiver operating characteristic (ROC) curve was used to evaluate the accuracy of anti-SP1 antibody and anti-PSP antibody in diagnosing pSS.The cli-nical characteristics of anti-SP1 antibody and anti-PSP antibody positive patients and negative patients in pSS group were further compared. Independent samples t test, Mann-Whitney U test, variance analysis, Kruskal-Wallis test, Chi-square test or Fisher's exact test and Spearman correlation analysis were used for statistical analysis., Results: There was no significant difference in age ( F =1.406, P =0.495) and gender ( χ 2 =2.105, P =0.349) among pSS group, disease control group and healthy control group. The expression levels of anti-SP1 antibody ( H =16.73, P < 0.001) and anti-PSP antibody ( H =26.09, P < 0.001) were statistically different among the three groups. An intergroup comparison of anti-SP1 antibody expression levels showed that there was a statistically significant difference between pSS and healthy control group ( P < 0.001), but no statistically significant difference between the other groups. Comparison of anti-PSP antibody expression levels between the groups showed that there were statistically significant differences between pSS and healthy control group ( P < 0.001), and between disease control group and healthy control group ( P =0.009), while no statistically significant differences between the other groups. The positive rate of anti-SP1 antibody in pSS group was significantly higher than that in disease control group and healthy control group (58.33% vs. 40.00% vs. 13.33%, P < 0.001). The positive rate of anti-PSP antibody in pSS group was significantly higher than that in disease control group and healthy control group (75.00% vs . 56.17% vs . 16.67%, P < 0.001). The area under the curve for anti-SP1 antibody was 0.688 ( P < 0.001). The sensitivity and specificity of anti-SP1 antibody were 58.33% (35/60) and 70.00% (42/60) respectively, the positive predictive value was 66.04% (35/53) and the negative predictive value was 54.55% (42/77) of anti-SP1 antibody.The area under the curve of anti-PSP antibody was 0.720 ( P < 0.001), with a sensitivity was 75.00% (45/60), and specificity was 63.33% (38/60).The positive predictive value and negative predictive value of anti-PSP antibody were 67.16% (45/67) and 71.70% (38/53) respectively. All the 13 pSS patients were negative for anti-Sjögren's syndrome A (SSA, including SSA52 and SSA60) antibody and anti- Sjögren's syndrome B (SSB) antibody. Among them, 11 patients were positive for both anti-SP1 antibody and anti-PSP antibody, 1 patient was positive for anti-SP1 antibody and 1 patient was positive for anti-PSP antibody. The clinical features of anti-SP1 antibody and anti-PSP antibody positive and negative groups were compared in pSS patients. The duration of disease in anti-SP1 antibody positive group was shorter ( Z =-2.277, P =0.023) when compared with the negative patients. The patients with positive anti-PSP antibody were younger than those in the negative group ( t =2.598, P < 0.05), the positive rate of rheumatoid factor ( P =0.002) and the serum level of IgG ( t =3.806, P =0.003) in anti-PSP antibody positive group were higher than in the negative group. Analysis of the correlation between anti-SP1 antibody and anti-PSP antibody in the pSS patients showed that there was significant correlation between them ( r =0.801, P < 0.001)., Conclusion: Both anti-SP1 antibody and anti-PSP antibody are valuable in the diagnosis of SS, and anti-SP1 antibody is helpful for the early diagnosis of pSS. The combined detection of anti-SP1 antibody and anti-PSP antibody is helpful for the early diagnosis of pSS patients with negative anti-SSA antibody and anti-SSB antibody.

Serological assays for antibody detection have contributed significantly to the diagnosis and control of infectious diseases. African swine fever is the most devastating infectious disease of domestic pigs and wild boars, severely threatening the global pig industry in recent years. Here, we developed a rapid, simple, and sensitive immunoassay based on the split-luciferase system to detect IgG antibodies against African swine fever virus (ASFV). In this assay, the p30 protein of ASFV was genetically coupled to the LgBiT and SmBiT subunits of nanoluciferase, which were used as fusion probes for specific antibodies. Target engagement of the probes results in the reconstitution of a functional nanoluciferase, which further catalyzes bioluminescent reactions. Different orientations of the LgBiT and SmBiT-p30 fusion sensors were designed and investigated, and N-LgBiT/p30 and N-SmBiT/p30 were identified as a promising sensor pair for reforming active nanoluciferase in the presence of specific antibodies. After optimization, this split-luciferase complementation assay showed high sensitivity and specificity for the detection of ASFV antibodies. The analytical sensitivity of the assay was 16 times greater than that of the blocking enzyme-linked immunosorbent assay (ELISA) by the detection of serial dilutions of serum, and no cross-reaction was observed with other swine pathogens. As demonstrated in clinical samples, its performance is highly consistent with that of a commercial ELISA kit, with a concordance rate of 98.19%. This assay is simple and easy to perform, providing a more flexible and efficient approach for the measurement of ASFV antibodies in clinical applications., Importance: The study is about a homogeneous split-luciferase assay for antibody detection. Split nanoluciferase biosensors for the detection of ASFV antibodies were designed. This sensor platform enables the sensitive and specific detection of antibodies. The split-luciferase assay is simple, rapid, and easy to use., Competing Interests: The authors declare no conflict of interest.

Background: Reliable biomarkers of coronavirus disease 2019 (COVID-19) outcomes are critically needed. We evaluated associations of spike antibody (Ab) and plasma nucleocapsid antigen (N Ag) with clinical outcomes in nonhospitalized persons with mild-to-moderate COVID-19., Methods: Participants were nonhospitalized adults with mild-to-moderate COVID-19 enrolled in ACTIV-2 between January and July 2021 and randomized to placebo. We used quantitative assays for severe acute respiratory syndrome coronavirus 2 spike Ab and N Ag in blood and determined numbers of hospitalization/death events within 28 days and time to symptom improvement., Results: Of 209 participants, 77 (37%) had quantifiable spike Ab and 139 (67%) quantifiable N Ag. Median age was 50 years; 111 (53%) were female, 182 (87%) White, and 105 (50%) Hispanic/Latino. Higher risk of hospitalization/death was seen with unquantifiable (22/132 [16.7%]) versus quantifiable (1/77 [1.3%]) spike Ab (risk ratio [RR], 12.83 [95% confidence interval {CI}, 1.76-93.34]) and quantifiable (22/139 [15.8%]) vs unquantifiable (1/70 [1.4%]) N Ag (RR, 11.08 [95% CI, 1.52-80.51]). Increasing risk of hospitalizations/deaths was seen with increasing N Ag levels. Time to symptom improvement was longer with unquantifiable versus quantifiable spike Ab (median, 14 [interquartile range {IQR}, 8 to >27] vs 8 [IQR, 4-22] days; adjusted hazard ratio [aHR], 0.66 [95% CI, .45-.96]) and with quantifiable versus unquantifiable N Ag (median, 12 [7 to >27] vs 10 [5-22] days; aHR, 0.79 [95% CI, .52-1.21])., Conclusions: Absence of spike Ab and presence of plasma N Ag predicted hospitalization/death and delayed symptom improvement in COVID-19 outpatients., Clinical Trials Registration: NCT04518410., Competing Interests: Potential conflicts of interest. N. J. received salary support to the institution from Sagent Pharmaceuticals. D. A. W. has received funding to his institution to support research and honoraria for advisory boards and consulting from Gilead Sciences. T. H. E. serves as a consultant for Tonix Pharmaceuticals. E. S. D. receives consulting fees from Gilead Sciences, Merck, and GSK/ViiV and research support through his institution from Gilead Sciences and GSK/ViiV. J. Z. L. has received research funding from Merck. J. J. E. is an ad hoc consultant to GSK/VIR, data monitoring committee chair for Adagio Phase 3 studies. J. S. C. has consulted for Merck and Company. D. M. S. has consulted for Fluxergy, Kiadis, Linear Therapies, Matrix BioMed, Arena Pharmaceuticals, VxBiosciences, Model Medicines, Bayer Pharmaceuticals, Signant Health, and Brio Clinical. K. W. C. received research funding to the institution from Merck Sharp & Dohme and is a consultant for Pardes Biosciences. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Odontoblasts are primarily responsible for synthesizing and secreting extracellular matrix proteins, which are crucial for dentinogenesis. Our previous single-cell profile and RNAscope for odontoblast lineage revealed that cyclic adenosine monophosphate responsive element-binding protein 3 like 1 (Creb3l1) was specifically enriched in the terminal differentiated odontoblasts. In this study, deletion of Creb3l1 in the Wnt1+ lineage led to insufficient root elongation and dentin deposition. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and RNA sequencing were performed to revealed that in CREB3L1-deficient mouse dental papilla cells (mDPCs), the genes near the closed chromatin regions were mainly associated with mesenchymal development and the downregulated genes were primarily related to biological processes including cell differentiation, protein biosynthesis and transport, all of which were evidenced by a diminished ability of odontoblastic differentiation, a significant reduction in intracellular proteins, and an even greater decline in extracellular supernatant proteins. Dentin matrix protein 1 (Dmp1), dentin sialophosphoprotein (Dspp), and transmembrane protein 30B (Tmem30b) were identified as direct transcriptional regulatory targets. TMEM30B was intensively expressed in the differentiated odontoblasts, and exhibited a significant decline in both CREB3L1-deficient odontoblasts in vivo and in vitro. Deletion of Tmem30b impaired the ability of odontoblastic differentiation, protein synthesis, and protein secretion in mDPCs. Moreover, overexpressing TMEM30B in CREB3L1-deficient mDPCs partially rescued the extracellular proteins secretion. Collectively, our findings suggest that CREB3L1 participates in dentinogenesis and facilitates odontoblastic differentiation by directly enhancing the transcription of Dmp1, Dspp, and other differentiation-related genes and indirectly promoting protein secretion partially via TMEM30B., (© 2024. The Author(s).)

With SARS-CoV-2 N protein as a model target, a signal-enhanced LFIA based on Au@Pt nanoparticles (NPs) as labels is proposed. This Au@Pt NPs combined the distinguished localized surface plasma resonance (LSPR) effect of Au NPs and the ultrahigh peroxidase-like catalytic activity of Pt NPs. Au@Pt NPs could trigger substrate chromogenic reaction, generating a color signal orders of magnitude darker than their intrinsic color. In the detection, after the coloration of the strips, 3,3',5,5'-tetramethylbenzidine (TMB) and H 2 O 2 were added, and a dark blue chelate (OxTMB) was produced soon, enhancing the band color significantly. After the signal amplification, the naked-eye detection limit for N protein reached 40 pg/mL. The detection sensitivity enhanced more than 1000 times than that without signal amplification. Compared with mainstream LFIA requiring complex readout instruments, the Au@Pt-based LFIA achieved a comparable sensitivity using naked eyes detection. This point is crucial, especially for unprofessional users or low-resource areas. Hence, this signal-enhanced LFIA may serve as a sensitive, cost-effective, and user-friendly detection method. It can shorten the testing window period and help identify early infections., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Na + -K + ATPase is an integral component of cardiac sarcolemma and consists of three major subunits, namely the α-subunit with three isoforms (α 1 , α 2 , and α 3 ), β-subunit with two isoforms (β 1 and β 2 ) and γ-subunit (phospholemman). This enzyme has been demonstrated to transport three Na and two K ions to generate a trans-membrane gradient, maintain cation homeostasis in cardiomyocytes and participate in regulating contractile force development. Na + -K + ATPase serves as a receptor for both exogenous and endogenous cardiotonic glycosides and steroids, and a signal transducer for modifying myocardial metabolism as well as cellular survival and death. In addition, Na + -K + ATPase is regulated by different hormones through the phosphorylation/dephosphorylation of phospholemman, which is tightly bound to this enzyme. The activity of Na + -K + ATPase has been reported to be increased, unaltered and depressed in failing hearts depending upon the type and stage of heart failure as well as the association/disassociation of phospholemman and binding with endogenous cardiotonic steroids, namely endogenous ouabain and marinobufagenin. Increased Na + -K + ATPase activity in association with a depressed level of intracellular Na + in failing hearts is considered to decrease intracellular Ca 2+ and serve as an adaptive mechanism for maintaining cardiac function. The slight to moderate depression of Na + -K + ATPase by cardiac glycosides in association with an increased level of Na + in cardiomyocytes is known to produce beneficial effects in failing hearts. On the other hand, markedly reduced Na + -K + ATPase activity associated with an increased level of intracellular Na + in failing hearts has been demonstrated to result in an intracellular Ca 2+ overload, the occurrence of cardiac arrhythmias and depression in cardiac function during the development of heart failure. Furthermore, the status of Na + -K + ATPase activity in heart failure is determined by changes in isoform subunits of the enzyme, the development of oxidative stress, intracellular Ca 2+ -overload, protease activation, the activity of inflammatory cytokines and sarcolemmal lipid composition. Evidence has been presented to show that marked alterations in myocardial cations cannot be explained exclusively on the basis of sarcolemma alterations, as other Ca 2+ channels, cation transporters and exchangers may be involved in this event. A marked reduction in Na + -K + ATPase activity due to a shift in its isoform subunits in association with intracellular Ca 2+ -overload, cardiac energy depletion, increased membrane permeability, Ca 2+ -handling abnormalities and damage to myocardial ultrastructure appear to be involved in the progression of heart failure.

Amelogenin is an intrinsically disordered protein essential to tooth enamel formation in mammals. Using advanced small angle X-ray scattering (SAXS) capabilities at synchrotrons and computational models, we revisited measuring the quaternary structure of murine amelogenin as a function of pH and phosphorylation at serine-16. The SAXS data shows that at the pH extremes, amelogenin exists as an extended monomer at pH 3.0 (R g  = 38.4 Å) and nanospheres at pH 8.0 (R g  = 84.0 Å), consistent with multiple previous observations. At pH 5.0 and above there was no evidence for a significant population of monomeric species. Instead, at pH 5.0, ∼80 % of the population is a heterogenous dimeric species that increases to ∼100 % at pH 5.5. The dimer population was observed at all pH > 5 conditions in dynamic equilibrium with a species in the pentamer range at pH < 6.5 and nanospheres at pH 8.0. At pH 8.0, ∼40 % of the amelogenin remained in the dimeric state. In general, serine-16 phosphorylation of amelogenin appears to modestly stabilize the population of the dimeric species., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Despite novel therapeutic strategies, advanced-stage prostate cancer (PCa) remains highly lethal, pointing out the urgent need for effective therapeutic strategies. While dysregulation of the splicing process is considered a cancer hallmark, the role of certain splicing factors remains unknown in PCa. This study focuses on characterizing the levels and role of SRSF6 in this disease. Comprehensive analyses of SRSF6 alterations (copy number/mRNA/protein) were conducted across eight well-characterized PCa cohorts and the Hi-MYC transgenic model. SRSF6 was up-regulated in PCa samples, correlating with adverse clinical parameters. Functional assays, both in vitro (cell proliferation, migration, colony, and tumorsphere formation) and in vivo (xenograft tumors), demonstrated the impact of SRSF6 modulation on critical cancer hallmarks. Mechanistically, SRSF6 regulates the splicing pattern of the histone-chaperone HIRA , consequently affecting the activity of H3.3 in PCa and breast cancer cell models and disrupting pivotal oncogenic pathways (AR and E2F) in PCa cells. These findings underscore SRSF6 as a promising therapeutic target for PCa/advanced-stage PCa.