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This review highlights new concepts in vascular patterning in the last 10 years, with emphasis on its beauty and complexity. Endothelial cell signaling pathways that respond to molecular or mechanical signals are described, and examples of vascular patterning that use these pathways in brain, skin, heart, and kidney are highlighted. The pathological consequences of patterning loss are discussed in the context of arteriovenous malformations (AVMs), and prospects for the next 10 years presented.

The zebrafish caudal fin has become a popular model to study cellular and molecular mechanisms of regeneration due to its high regenerative capacity, accessibility for experimental manipulations, and relatively simple anatomy. The formation of a regenerative epidermis and blastema are crucial initial events and tightly regulated. Both the regenerative epidermis and the blastema are highly organized structures containing distinct domains, and several signaling pathways regulate the formation and interaction of these domains. Bone is the major tissue regenerated from the progenitor cells of the blastema. Several cellular mechanisms can provide source cells for blastemal (pre-)osteoblasts, including dedifferentiation of differentiated osteoblasts and de novo formation from other cell types, providing intriguing examples of cellular plasticity. In recent years, omics analyses and single-cell approaches have elucidated genetic and epigenetic regulation, increasing our knowledge of the surprisingly complex coordination of various mechanisms to achieve successful restoration of a seemingly simple structure.

The capacity for regeneration is broad in the animal kingdom. Planarians are flatworms that can regenerate any missing body part and their regenerative powers have combined with ease of experimentation to make them a classic regeneration model for more than a century. Pluripotent stem cells called neoblasts generate missing planarian tissues. Fate specification happens in the neoblasts, and this can occur in response to regeneration instructions in the form of positional information. Fate specification can lead to differentiating cells in single steps rather than requiring a long lineage hierarchy. Planarians display constitutive expression of positional information from muscle cells, which is required for patterned maintenance of tissues in tissue turnover. Amputation leads to the rapid resetting of positional information in a process triggered by wound signaling and the resetting of positional information is required for regeneration. These findings suggest a model for planarian regeneration in which adult positional information resets after injury to regulate stem cells to bring about the replacement of missing parts.

Auxin signaling regulates growth and developmental processes in plants. The core of nuclear auxin signaling relies on just three components: TIR1/AFBs, Aux/IAAs, and ARFs. Each component is itself made up of several domains, all of which contribute to the regulation of auxin signaling. Studies of the structural aspects of these three core signaling components have deepened our understanding of auxin signaling dynamics and regulation. In addition to the structured domains of these components, intrinsically disordered regions within the proteins also impact auxin signaling outcomes. New research is beginning to uncover the role intrinsic disorder plays in auxin-regulated degradation and subcellular localization. Structured and intrinsically disordered domains affect auxin perception, protein degradation dynamics, and DNA binding. Taken together, subtle differences within the domains and motifs of each class of auxin signaling component affect signaling outcomes and specificity.

Proteases are signaling molecules that specifically control cellular functions by cleaving protease-activated receptors (PARs). The four known PARs are members of the large family of G protein-coupled receptors. These transmembrane receptors control most physiological and pathological processes and are the target of a large proportion of therapeutic drugs. Signaling proteases include enzymes from the circulation; from immune, inflammatory epithelial, and cancer cells; as well as from commensal and pathogenic bacteria. Advances in our understanding of the structure and function of PARs provide insights into how diverse proteases activate these receptors to regulate physiological and pathological processes in most tissues and organ systems. The realization that proteases and PARs are key mediators of disease, coupled with advances in understanding the atomic level structure of PARs and their mechanisms of signaling in subcellular microdomains, has spurred the development of antagonists, some of which have advanced to the clinic. Herein we review the discovery, structure, and function of this receptor system, highlight the contribution of PARs to homeostatic control, and discuss the potential of PAR antagonists for the treatment of major diseases.

The evolutionarily conserved leucine rich repeat (LRR) protein domain is a unique structural motif found in many viral, bacterial, archaeal, and eukaryotic proteins. The LRR domain serves many roles, including being a signaling domain and a pathogen recognition receptor. In the human innate immune system, it serves an essential role by recognizing fragments of bacterial cell walls. Interestingly, the human fungal pathogen

G protein-coupled receptors (GPCRs) are the largest family of membrane receptors in humans. They mediate nearly all aspects of human physiology and thus are of high therapeutic interest. GPCR signaling is regulated in space and time by receptor phosphorylation. It is believed that different phosphorylation states are possible for a single receptor, and each encodes for unique signaling outcomes. Methods to determine the phosphorylation status of GPCRs are critical for understanding receptor physiology and signaling properties of GPCR ligands and therapeutics. However, common proteomic techniques have provided limited quantitative information regarding total receptor phosphorylation stoichiometry, relative abundances of isomeric modification states, and temporal dynamics of these parameters. Here, we report a novel middle-down proteomic strategy and parallel reaction monitoring (PRM) to quantify the phosphorylation states of the C-terminal tail of metabotropic glutamate receptor 2 (mGluR2). By this approach, we found that mGluR2 is subject to both basal and agonist-induced phosphorylation at up to four simultaneous sites with varying probability. Using a PRM tandem mass spectrometry methodology, we localized the positions and quantified the relative abundance of phosphorylations following treatment with an agonist. Our analysis showed that phosphorylation within specific regions of the C-terminal tail of mGluR2 is sensitive to receptor activation, and subsequent site-directed mutagenesis of these sites identified key regions which tune receptor sensitivity. This study demonstrates that middle-down purification followed by label-free quantification is a powerful, quantitative, and accessible tool for characterizing phosphorylation states of GPCRs and other challenging proteins.

Abstract: Carbon monoxide (CO) is endogenously produced upon degradation of heme by heme oxygenases (HOs) and is suggested to act as a gaseous signaling molecule. The expression of HO-1 is triggered by the Nrf2-Keap1 signaling pathway which responds to exogenous stress signals and dietary constituents such as flavonoids and glucosinolates or reactive metabolic intermediates like 4-hydroxynonenal. Endogenous CO affects energy metabolism, regulates the utilization of glucose and addresses CYP450 enzymes. Using the CO releasing molecule-401 (CORM-401), we studied the effect of endogenous CO on ATP synthesis, AMP-signaling and activation of the AMPK pathway in cell culture. Upon exposure of cells to CORM-401, the mitochondrial ATP production rate was significantly decreased (P=0.007) to about 50%, while glycolytic ATP synthesis was unchanged (P=0.489). Total ATP levels were less affected as determined by mass spectrometry. Instead, levels of ADP and AMP were elevated following CORM-401 exposure by about two- (P=0.022) and four-fold (P=0.012) compared to control, respectively. Increased concentrations of AMP activate AMPK which was demonstrated by a 10 to 15-fold increased phosphorylation of Thr172 of the α-subunit of AMPK (P=0.025). A downstream target of AMPK is the kinase ULK1 which triggers autophagic and mitophagic processes. Activation of ULK1 after CO exposure was proven by a 3 to 5-fold elevated phosphorylation of ULK1 at Ser555 (P=0.004). The present data suggest that production of endogenous CO leads to increasing amounts of AMP which mediates AMPK-dependent downstream effects and likely triggers autophagic processes. Since dietary constituents and their metabolites induce the expression of the CO producing enzyme HO-1, CO signaling may also be involved in the cellular response to nutritional factors.

Acknowledgements: The authors are grateful to the other members of the Boletta laboratory for useful discussions and to L. Tronci for helpful suggestions on data analysis. This work was supported by the Italian Ministry of Health (RF-2018-12368254; RF-2016-02361267 to A.B.; GR-2016-02364851 to C.P. and R.P.), the Italian association of patients with PKD (AIRP to A.B.), the PKD Foundation (218G18 to A.B.), the European Community (H-2020-MSCA-ITN-2019#SCiLS to A.B. and R.R.), by the Italian association for research on cancer (AIRC, IG2019-23513 to A.B.), by Q12 grant (MRC_MC_UU_12022/6 to C.F.), the European Research Council (ERC819920 to C.F.), and the CRUK Programme Foundation (Award C51061/A27453 to C.F.). The authors are grateful to S. Bramani for her continuous support. Part of this work was carried out in ALEMBIC, an advanced microscopy laboratory established by IRCCS Ospedale San Raffaele and Università Vita-Salute San Raffaele. Part of the present work was performed by M.E.S. and A.K.N. in fulfilment of the requirements for obtaining a PhD degree at Vita-Salute San Raffaele University, Milano, Italy., Funder: EC | EC Seventh Framework Programm | FP7 People: Marie-Curie Actions (FP7-PEOPLE - Specific Programme "People" Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013)); doi: https://doi.org/10.13039/100011264; Grant(s): H2020-MSCA-ITN-2019#SCiLS, H2020-MSCA-ITN-2019#SCiLS, Funder: EC | EC Seventh Framework Programm | FP7 Ideas: European Research Council (FP7-IDEAS-ERC - Specific Programme: "Ideas" Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013)); doi: https://doi.org/10.13039/100011199; Grant(s): ERC819920, Depriving cells of nutrients triggers an energetic crisis, which is resolved by metabolic rewiring and organelle reorganization. Primary cilia are microtubule-based organelles at the cell surface, capable of integrating multiple metabolic and signalling cues, but their precise sensory function is not fully understood. Here we show that primary cilia respond to nutrient availability and adjust their length via glutamine-mediated anaplerosis facilitated by asparagine synthetase (ASNS). Nutrient deprivation causes cilia elongation, mediated by reduced mitochondrial function, ATP availability and AMPK activation independently of mTORC1. Of note, glutamine removal and replenishment is necessary and sufficient to induce ciliary elongation or retraction, respectively, under nutrient stress conditions both in vivo and in vitro by restoring mitochondrial anaplerosis via ASNS-dependent glutamate generation. Ift88-mutant cells lacking cilia show reduced glutamine-dependent mitochondrial anaplerosis during metabolic stress, due to reduced expression and activity of ASNS at the base of cilia. Our data indicate a role for cilia in responding to, and possibly sensing, cellular glutamine levels via ASNS during metabolic stress.

Infection with pathogenic microbes is a severe threat that hosts manage by activating the innate immune response. In Drosophila melanogaster, the Toll and Imd signaling pathways are activated by pathogen-associated molecular patterns to initiate cellular and humoral immune processes that neutralize and kill invaders. The Toll and Imd signaling pathways operate in organs such as fat body and gut that control host nutrient metabolism, and infections or genetic activation of Toll and Imd signaling also induce wide-ranging changes in host lipid, carbohydrate and protein metabolism. Metabolic regulation by immune signaling can confer resistance to or tolerance of infection, but it can also lead to pathology and susceptibility to infection. These immunometabolic phenotypes are described in this review, as are changes in endocrine signaling and gene regulation that mediate survival during infection. Future work in the field is anticipated to determine key variables such as sex, dietary nutrients, life stage, and pathogen characteristics that modify immunometabolic phenotypes and, importantly, to uncover the mechanisms used by the immune system to regulate metabolism.

Mitochondria are vital organelles with a central role in all aspects of cellular metabolism. As a means to support the ever-changing demands of the cell, mitochondria produce energy, drive biosynthetic processes, maintain redox homeostasis, and function as a hub for cell signaling. While mitochondria have been widely studied for their role in disease and metabolic dysfunction, this organelle has a continually evolving role in the regulation of development, wound repair, and regeneration. Mitochondrial metabolism dynamically changes as tissues transition through distinct phases of development. These organelles support the energetic and biosynthetic demands of developing cells and function as key structures that coordinate the nutrient status of the organism with developmental progression. This review will examine the mechanisms that link mitochondria to developmental processes. We will also examine the process of mitochondrial respiratory quiescence (MRQ), a novel mechanism for regulating cellular metabolism through the biochemical and physiological remodeling of mitochondria. Lastly, we will examine MRQ as a system to discover the mechanisms that drive mitochondrial remodeling during development.

A surge in the prevalence of obesity and metabolic syndrome, which promote systemic inflammation, underlies an increase in cardiometabolic disease. Free fatty acid receptor 4 is a nutrient sensor for long-chain fatty acids, like ω3-polyunsaturated fatty acids (ω3-PUFAs), that attenuates metabolic disease and resolves inflammation. Clinical trials indicate ω3-PUFAs are cardioprotective, and this review discusses the mechanistic links between ω3-PUFAs, free fatty acid receptor 4, and attenuation of cardiometabolic disease.

Heart regeneration is a remarkable process whereby regrowth of damaged cardiac tissue rehabilitates organ anatomy and function. Unfortunately, the human heart is highly resistant to regeneration, which creates a shortage of cardiomyocytes in the wake of ischemic injury, and explains, in part, why coronary artery disease remains a leading cause of death worldwide. Luckily, a detailed blueprint for achieving therapeutic heart regeneration already exists in nature because several lower vertebrate species successfully regenerate amputated or damaged heart muscle through robust cardiomyocyte proliferation. A growing number of species are being interrogated for cardiac regenerative potential, and several commonalities have emerged between those animals showing high or low innate capabilities. In this review, we provide a historical perspective on the field, discuss how regenerative potential is influenced by cardiomyocyte properties, mitogenic signals, and chromatin accessibility, and highlight unanswered questions under active investigation. Ultimately, delineating why heart regeneration occurs preferentially in some organisms, but not in others, will uncover novel therapeutic inroads for achieving cardiac restoration in humans.

Modulation of proteasome function by pharmacological interventions and molecular biology tools is an active area of research in cancer biology and neurodegenerative diseases. Curcumin (diferuloylmethane) is a naturally occurring polyphenol that affects multiple signaling pathways and known to modulate PKC activities. The therapeutic significance of curcumin is often considered to reside in its anti-inflammatory, antioxidant, anti-angiogenic, or anti-apoptotic properties. However, recent research suggests that the therapeutic efficacy of curcumin may be due to its activity as a potent inhibitor of the proteasome. In this study, we show that both curcumin and its synthetic polyphenolic derivative (didemethylcurcumin), CUIII modulated proteasome activity in a biphasic manner. Curcumin and CUIII increased proteasome activity at nanomolar concentrations but inhibited proteasome activity at micromolar concentrations. Curcumin was more effective than CUIII in relative proteasome activity increase at nanomolar concentrations. Also, curcumin was more effective than CUIII in relative proteasome activity inhibition at micromolar concentration. The docking study was conducted on the 20S proteasome catalytic subunit. Estimated Kd values for curcumin and didemethylcurcumin are 0.0054μM and 1.3167μM, respectively. These values correlate well with the results of the effectiveness of modulation by curcumin compare to CUIII. The small size of CUIII makes its dock to the narrow cavity of the active site, but the binding interaction is not strong compare to curcumin. This study suggests that curcumin and its didemethyl derivative can be used to modulate proteasome activity. This communication implicates the reason why curcumin and its didemethyl derivative can be used to two different disease mechanisms, neurodegeneration, and cancer.

Background: Endogenous and synthetic cannabinoids have been shown to induce cancer cell death through the accumulation of the sphingolipid, ceramide (Cer). Recently, we have demonstrated that Cer accumulation enhances the induction of immunogenic cell death (ICD). Objectives: The primary objective of this study was to demonstrate that (±) 5-epi CP 55,940 (5-epi), a by-product of the chemical synthesis of the synthetic cannabinoid CP 55,940, induces ICD in colorectal cancer (CRC) cells, and that modulation of the sphingolipid metabolic pathway through inhibition of the sphingosine kinases (SphKs) enhances these effects. Methods: A cell culture model system of human CRC cell lines was employed to measure the cell surface and intracellular production of markers of ICD. The effects of 5-epi, alone and in combination with SphK inhibitors, on production of Cer through the de novo sphingolipid synthesis pathway were measured by Liquid Chromatography - Tandem Mass Spectrometry (LC/MS/MS)-based sphingolipidomic analysis. Cell surface exposure of calreticulin (ectoCRT), a hallmark of ICD, was measured by flow cytometry. Examination of the effects of 5-epi, alone and in combination with SphK inhibitors, on the intracellular signaling pathway associated with ICD was conducted by immunoblot analysis of human CRC cell lines. Results: Sphingolipidomic analysis indicated that 5-epi induces the de novo sphingolipid synthetic pathway. 5-epi dose dependently induces cell surface exposure of ectoCRT, and inhibition of Cer metabolism through inhibition of the SphKs significantly enhances 5-epi-induced ectoCRT exposure in multiple CRC cell lines. 5-epi induces and SphK inhibition enhances activation of the cell death signaling pathway associated with ICD. Conclusions: This study is the first demonstration that cannabinoids can induce the cell surface expression of ectoCRT, and potentially induce ICD. Moreover, this study reinforces our previous observation of a role for Cer accumulation in the induction of ICD and extends this observation to the cannabinoids, agents not typically associated with ICD. Inhibition of SphKs enhanced the 5-epi-induced signaling pathways leading to ICD and production of ectoCRT. Overexpression of SphK1 has previously been associated with chemotherapy resistance. Thus, targeting the SphKs has the potential to reverse chemotherapy resistance and simultaneously enhance the antitumor immune response through enhancement of ICD induction.

Alcoholic liver disease (ALD) is caused by alcohol abuse and can progress to hepatitis, cirrhosis, and even hepatocellular carcinoma. Previous studies suggested that

Growing evidence from animal and human studies show that opioids have a major impact on the composition and function of gut microbiota. This leads to disruption in gut permeability and altered microbial metabolites, driving both systemic and neuroinflammation, which in turn impacts central nervous system (CNS) homeostasis. Tolerance and dependence are the major comorbidities associated with prolonged opioid use. Inflammatory mediators and signaling pathways have been implicated in both opioid tolerance and dependence. We provide evidence that targeting the gut microbiome during opioid use through prebiotics, probiotics, antibiotics, and fecal microbial transplantation holds the greatest promise for novel treatments for opioid abuse. Basic research and clinical trials are required to examine what is more efficacious to yield new insights into the role of the gut-brain axis in opioid abuse.

Cancer immunoediting progresses through elimination, equilibrium, and escape. Each of these phases is characterized by breaching, remodeling, and rebuilding tissue planes and structural barriers that engage extracellular matrix (ECM) components, in particular matrix proteoglycans. Some of the signals emanating from matrix proteoglycan remodeling are readily co-opted by the growing tumor to sustain an environment of tumor-promoting and immune-suppressive inflammation. Yet other matrix-derived cues can be viewed as part of a homeostatic response by the host, aiming to eliminate the tumor and restore tissue integrity. These latter signals may be harnessed for therapeutic purposes to tip the polarity of the tumor immune milieu toward anticancer immunity. In this review, we attempt to showcase the importance and complexity of matrix proteoglycan signaling in both cancer-restraining and cancer-promoting inflammation. We propose that the era of matrix diagnostics and therapeutics for cancer is fast approaching the clinic.

The nuclear compartment is delimited by a specialized expanded sheet of the endoplasmic reticulum (ER) known as the nuclear envelope (NE). Compared to the outer nuclear membrane and the contiguous peripheral ER, the inner nuclear membrane (INM) houses a unique set of transmembrane proteins that serve a staggering range of functions. Many of these functions reflect the exceptional position of INM proteins at the membrane-chromatin interface. Recent research revealed that numerous INM proteins perform crucial roles in chromatin organization, regulation of gene expression, genome stability, and mediation of signaling pathways into the nucleus. Other INM proteins establish mechanical links between chromatin and the cytoskeleton, help NE remodeling, or contribute to the surveillance of NE integrity and homeostasis. As INM proteins continue to gain prominence, we review these advancements and give an overview on the functional versatility of the INM proteome. © 2021, Cold Spring Harbor Perspectives in Biology by Cold Spring Harbor Laboratory Press ISSN:1943-0264

Leucine and insulin-like growth factor-1 (IGF-1) are important regulators of protein synthesis in skeletal muscle. The mechanistic target of rapamycin complex 1 (mTORC1) is of particular importance in their mechanism of action. In the present study, pathways through which leucine and IGF-1 converge to mediate activation of mTORC1 were examined in L6 myoblasts that were deprived of leucine and serum followed by readdition of either leucine or IGF-1. Compared with leucine- and serum-deprived myoblasts, IGF-1, but not leucine, promoted phosphorylation of protein kinase B (AKT), tuberous sclerosis complex 2 (TSC2), and the autophosphorylation site on mTOR (S2481) and also stimulated mTOR kinase activity in mTOR immunoprecipitated samples. Both leucine and IGF-1 promoted phosphorylation of mTOR on S2448. The association of mTOR with the regulatory-associated protein of mTOR (Raptor) was altered by IGF-1 treatment and trended ( P = 0.065) to be altered by leucine treatment. Alterations in the association of mTOR with Raptor were proportional to changes in phosphorylation of the mTOR substrates, eIF4E-binding protein 1 (4E-BP1), and ribosomal protein S6 Kinase-β1 (p70S6K1). Surprisingly, leucine, but not IGF-1, stimulated protein synthesis suggesting a unique role for nutrients in regulating protein synthesis. Overall, the results are consistent with a model whereby IGF-1 stimulates phosphorylation of 4E-BP1 and p70S6K1 in L6 myoblasts through an AKT-TSC2-mTORC1 signaling pathway that also involves changes in the interaction between mTOR and Raptor. In contrast, leucine signaling to mTOR results in alterations in certain mTOR phosphorylation sites and the interaction between mTOR and Raptor and stimulates protein synthesis.

Despite the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway being frequently altered in T-ALL/LBL, no specific therapy has been approved for T-ALL/LBL patients with constitutive signalling by JAK/STAT, so there is an urgent need to identify pathway members that may be potential therapeutic targets. In the present study, we searched for JAK/STAT pathway members potentially modulated through aberrant methylation and identified SOCS3 hypermethylation as a recurrent event in T-ALL/LBL. Additionally, we explored the implications of SOCS3 deregulation in T-ALL/LBL and demonstrated that SOCS3 counteracts the constitutive activation of the JAK/STAT pathway through different molecular mechanisms. Therefore, SOCS3 emerges as a potential therapeutic target in T-ALL/LBL, Comunidad de Madrid, Grant/Award Number: B2017/BMD-3778; LINFOMAS-CM; Fundación Científica Asociación Española Contra el Cáncer, Grant/Award Number: PROYE18054PIRI; Fundación Ramón Areces, Grant/Award Number: CIVP19S7917; Instituto de Investigación Sanitaria Fundación Jiménez Díaz; Ministerio de Ciencia, Innovación y Universidades, Grant/ Award Number: RTI2018- 093330-B-I00 and MCIU/FEDER; Ministerio de Economía y Competitividad, Grant/Award Number: SAF2015-70561-R and MINECO/FEDER

Understanding the composition and activation of multicomponent receptor complexes is a challenge in biology. To address this, we developed a synthetic approach based on nanobodies to drive assembly and activation of cell surface receptors and apply the concept by manipulating receptors that govern plant symbiosis with nitrogen-fixing bacteria. We show that the Lotus japonicus Nod factor receptors NFR1 and NFR5 constitute the core receptor complex initiating the cortical root nodule organogenesis program as well as the epidermal program controlling infection. We find that organogenesis signaling is mediated by the intracellular kinase domains whereas infection requires functional ectodomains. Finally, we identify evolutionarily distant barley receptors that activate root nodule organogenesis, which could enable engineering of biological nitrogen-fixation into cereals.

Oxaliplatin (OXA) is a usual chemotherapeutic agent applied in the colorectal cancer (CRC) clinical treatment. Interferon-alpha inducible protein 6 (IFI6) has been proved to promote proliferation and suppress apoptosis in several tumor cells, while the impacts of IFI6 on OXA resistance in CRC still need exploration. HCT116 and SW620 cells were used as the parental to obtain OXA-resistant cells. The influence of IFI6 on OXA sensitivity, cell proliferation and apoptosis were evaluated by overexpression or knockdown IFI6 in cells. In this work, we found that the level of IFI6 was significantly enhanced in HCT116/OXA and SW620/OXA cells as compared to the parental cells. Overexpression of IFI6 decreased the sensitivity of HCT116 and SW620 cells to OXA. However, knockdown of IFI6 enhanced the sensitivity of HCT116/OXA and SW620/OXA cells to OXA. And upregulated IFI6 promoted the proliferation and repressed apoptosis in HCT116 cells, while suppressed IFI6 markedly reduced proliferation and increased apoptosis in HCT116/OXA cells. Additionally, IFI6 suppressed the phosphorylation level of p38, and silenced IFI6 enhanced it. The addition of the p38 kinase inhibitor, SB203580, alleviated the decreased cell proliferation and increased apoptosis in HCT116/OXA cells. Suppressed IFI6 enhanced the reactive oxygen species (ROS) level in HCT116/OXA cells, and blockade of ROS with N-acetyl-L-cysteine (NAC) decreased the enhancement level of ROS and the phosphorylation level of the p38, which was induced by IFI6 down-regulation. We, therefore, implied that suppressed IFI6 reverses OXA-resistance of CRC cells via promoting the ROS-induced p38 mitogen-activated protein kinase (MAPK) signaling pathway.

Melanoma is one of the most dangerous types of cutaneous neoplasms, which are pigment-producing cells of neuroectodermal origin found all over the body. A great deal of research is focused on the mechanisms of melanoma to promote better diagnostic and treatment options for melanoma in its advanced stages. The progression of melanoma involves alteration in different levels of gene expression. With the successful implementation of next-generation sequencing technology, an increasing number of long noncoding RNAs (lncRNAs) sequences have been discovered, and a significant number of them have phenotypic effects in both in vitro and in vivo studies, implying that they play an important role in the occurrence and progression of human cancers, particularly melanoma. A number of evidence indicated that lncRNAs are important regulators in tumor cell proliferation, invasion, apoptosis, immune escape, energy metabolism, drug resistance, epigenetic regulation. To better understand the role of lncRNAs in melanoma tumorigenesis, we categorize melanoma-associated lncRNAs according to their cellular functions and associations with gene expression and signaling pathways in this review. Based on the mechanisms of lncRNA, we discuss the possibility of lncRNA-target treatments, and the application of liquid biopsies to detect lncRNAs in melanoma diagnosis and prognosis.

Salicylic acid (SA) plays a pivotal role in plant response to biotic and abiotic stress. Several core SA signaling regulators and key proteins in SA biosynthesis have been well characterized. However, much remains unknown about the origin, evolution, and early diversification of core elements in plant SA signaling and biosynthesis. In this study, we identified 10 core protein families in SA signaling and biosynthesis across green plant lineages. We found that the key SA signaling receptors, the nonexpresser of pathogenesis-related (NPR) proteins, originated in the most recent common ancestor (MRCA) of land plants and formed divergent groups in the ancestor of seed plants. However, key transcription factors for SA signaling, TGACG motif-binding proteins (TGAs), originated in the MRCA of streptophytes, arguing for the stepwise evolution of core SA signaling in plants. Different from the assembly of the core SA signaling pathway in the ancestor of seed plants, SA exists extensively in green plants, including chlorophytes and streptophyte algae. However, the full isochorismate synthase (ICS)-based SA synthesis pathway was first assembled in the MRCA of land plants. We further revealed that the ancient abnormal inflorescence meristem 1 (AIM1)-based β-oxidation pathway is crucial for the biosynthesis of SA in chlorophyte algae, and this biosynthesis pathway may have facilitated the adaptation of early-diverging green algae to the high-light-intensity environment on land. Taken together, our findings provide significant insights into the early evolution and diversification of plant SA signaling and biosynthesis pathways, highlighting a crucial role of SA in stress tolerance during plant terrestrialization.

Suplatast is a T helper 2 (Th2) cytokine inhibitor. Here, we tested its therapeutic effects using a mouse model of renal interstitial fibrosis caused by unilateral ureteral obstruction (UUO). In this model, suplatast was found to prevent the induced fibrosis in the obstructed kidney when given in the drinking water at 100 mg/kg/d. Mechanistically, suplaplast inhibited the phosphorylation of extracellular signal-regulated kinase (ERK) that was otherwise increased by UUO. Similarly, suplaplast reduced the increased accumulation of KIM-1, transforming growth factor β (TGF-β), type I collagen, interleukin-4 (IL-4), janus kinase (JAK)1 and signal transducer and activator of transcription (STAT)3 mRNA seen in the kidneys of UUO-treated mice. Furthermore, STAT3 phosphorylation, which was stimulated by UUO, was also significantly decreased by suplatast. Collectively, these data show that suplatast reduces UUO-induced renal interstitial fibrosis via mechanisms including a reduction of phosphorylation of ERK and JAK/STAT pathway signaling.

The possible anticancer activity of combination (M + E + F) of metformin (M), efavirenz (E), and fluoxetine (F) was investigated in normal HDF cells and HCT116 human colon cancer cells. Metformin increased cellular FOXO3a, p-FOXO3a, AMPK, p-AMPK, and MnSOD levels in HDFs but not in HCT116 cells. Cellular ATP level was decreased only in HDFs by metformin. Metformin increased ROS level only in HCT116 cells. Transfection of si-FOXO3a into HCT116 reversed the metformin-induced cellular ROS induction, indicating that FOXO3a/MnSOD is the key regulator for cellular ROS level. Viability readout with M, E, and F alone decreased slightly, but the combination of three drugs dramatically decreased cell survival in HCT116, A549, and SK-Hep-1 cancer cells but not in HDF cells. ROS levels in HCT116 cells were massively increased by M + E + F combination, but not in HDF cells. Cell cycle analysis showed that of M + E + F combination caused cell death only in HCT116 cells. The combination of M + E + F reduced synergistically mitochondrial membrane potential and mitochondrial electron transport chain complex I and III activities in HCT116 cells when compared with individual treatments. Western blot analysis indicated that DNA damage, apoptosis, autophagy, and necroptosis-realated factors increased in M + E + F-treated HCT116 cells. Oral administration with M + E + F combination for 3 weeks caused dramatic reductions in tumor volume and weight in HCT116 xenograft model of nude mice when compared with untreated ones. Our results suggest that M + E + F have profound anticancer activity both

Damage to Sertoli cell junction proteins caused by inflammation can lead to male infertility. Nerve growth factor (NGF) plays an important role in reproductive and inflammatory disease; however, whether and how NGF regulates Sertoli cell function remains unclear. Here, we aimed to assess the effect of NGF on the growth of Sertoli cells isolated from the testes of dairy goats and evaluate if NGF has a protective effect on these cells. We confirmed that Sertoli cell viability, proliferation, and ATP content increased following NGF treatment. In addition, qPCR results suggested that Sertoli cell apoptosis was inhibited after NGF treatment. To investigate the protective effect of NGF on Sertoli cells under pathological inflammatory conditions, LPS was used to induce inflammatory response in Sertoli cells. Post-treatment, the entangled filamentous pseudopodia of the cells loosened and no longer spanned adjacent cells. The expression of several junction proteins (ZO-1, occludin, CX-43, β-catenin, and N-cadherin), which was down-regulated after inflammatory response induction, was restored following NGF treatment. LPS-induced changes in cytotoxicity and transepithelial electrical resistance were reversed and the intercellular connections became tighter after NGF treatment. We further demonstrated that NGF prevented the inflammatory response of Sertoli cells via the PI3K/AKT/NFκB signaling pathway, similar to the effect of the PI3K-inhibitor, LY294002, which is modified by the PI3K activator, 740Y-P. These results provide insights for devising strategies for protecting the male reproductive system and curing or preventing associated pathological conditions.

Adolescence is a key phase of development for perturbations in fear extinction, with inability to adequately manage fear a potent factor for developing psychiatric disorders in adulthood. However, while behavioral correlates of adolescent fear regulation are established to a degree, molecular mediators of extinction learning in adolescence remain largely unknown. In this study, we observed fear acquisition and fear extinction (across 4 and 7 days) of adolescent and adult mice of both sexes and investigated how hippocampal levels of different plasticity markers relate to extinction learning. While fear was acquired evenly in males and females of both ages, fear extinction was found to be impaired in adolescent males. We also observed lower levels of GluA1, GLUN2A and GLUN2B subunits in male adolescents following fear acquisition, with an increase in their expression, as well as the activity of Erk-mTOR pathway over subsequent extinction sessions, which was paralleled with improved extinction learning. On the other hand, we detected no changes in plasticity-related proteins after fear acquisition in females, with alterations in GluA1, GluA4 and GLUN2B levels across fear extinction sessions. Additionally, we did not discern any pattern regarding the Erk-mTOR activity in female mice associated with their extinction performance. Overall, our research identifies sex-specific synaptic properties in the hippocampus that underlie developmentally regulated differences in fear extinction learning. We also point out hippocampal NMDA-Erk-mTOR signaling as the driving force behind successful fear extinction in male adolescents, highlighting this pathway as a potential therapeutic target for fear-related disorders in the adolescent population. © 2022 The Authors

Neuroinflammation (NF) is defined as the activation of brain glial cells that are found in neurodegenerative diseases including Alzheimer's disease (AD). It has been known that an increase in NF could reduce the memory process in the brain but the key factors, associated with NF, behind the dysregulation of memory remained elusive. We previously reported that the NF and aging processes reduced the special phospholipids, plasmalogens (Pls), in the murine brain by a mechanism dependent on the activation of transcription factors, NF-kB and c-MYC. A similar mechanism has also been found in postmortem human brain tissues with AD pathologies and in the AD model mice. Recent evidence showed that these phospholipids enhanced memory and reduced neuro-inflammation in the murine brain. Pls can stimulate the cellular signaling molecules, ERK and Akt, by activating the membrane-bound G protein-coupled receptors (GPCRs). Therefore, recent findings suggest that plasmalogens could be one of the key phospholipids in the brain to enhance memory and inhibit NF.

Hepatic fibrosis is caused by excessive accumulation of extracellular matrix (ECM) due to repeated liver injury. Hepatic stellate cells (HSCs) play a key role in the pathogenesis and progression of hepatic fibrosis. A study showed that CYP4A14 gene defect can inhibit hepatic fibrosis, but the specific mechanism was not clear. In this experiment, patients with hepatic fibrosis, LX-2 cells (a human HSCs line), and mice with liver fibrosis induced by carbon tetrachloride (CCl

Much effort has been employed to improve the quality of embryos obtained by in vitro production (IVP) given the relevance of this technology to current livestock systems. In this context, dynamic IVP systems have proved beneficial to the embryo once they mimic fluid flows and mechanical forces resulting from the movement of ciliated cells and muscle contraction in the reproductive tract. In the present study, we sought to confirm these initial findings as well as assess potential molecular consequences to the embryo by applying micro-vibration (45 Hz for 5 s once per 60 min) during both oocyte maturation and embryo culture in cattle. As a result, micro-vibration led to lower incidence of apoptosis in blastocysts following vitrification-thawing. Further analyses revealed epigenetic and transcriptional changes in blastocysts derived from the micro-vibration treatment, with a total of 502 differentially expressed genes. Enrichment analyses linked differentially expressed genes to 'Oxidative phosphorylation', 'Cytokine-cytokine receptor interaction', and 'Signaling pathways regulating pluripotency of stem cells'. Yet, a meta-analysis indicated that the transcriptional changes induced by micro-vibration were not toward that of in vivo-derived embryos. In conclusion, micro-vibration increases the cryoresistance of bovine embryos, but caution should be taken given the unclear consequences of epigenetic and transcriptional abnormalities induced by the treatment.

The lncRNA PVT1 reportedly functions as a competing endogenous RNA (ceRNA) of miR-186 and miR-26b in different tissue types. In this study, we investigated the possible involvement of the miR-186/Srf/Ctgf and miR-26b/Ctgf signaling pathways in the pathogenesis of hypoxia-induced PAH.Expression of PVT1, miR-186, miR-26b, and Srf and Ctgf mRNAs were evaluated by real-time polymerase chain reaction. Protein expression of SRF, CTGF, LC3B-I, LC3B-II, and Beclin-I was evaluated using western blotting. The regulatory relationship between the lncRNA, miRNAs, and target mRNAs was explored using luciferase assays. Immunohistochemistry was used to evaluate the expression of SRF and CTGF in situ. MTT assay was performed to assess the proliferation of PASMCs.Exposure to hypoxia markedly altered the expression of PVT1, Srf, Ctgf, miR-186, and miR-26b in a rat model. MiR-186 binding sites in the sequences of Srf mRNA and PVT1 were confirmed by luciferase assays, indicating that miR-186 may interact with both PVT1 and Srf mRNA. Additionally, miR-26b binding sites were identified in the sequences of Ctgf mRNA and PVT1, suggesting that miR-26b may interact with both PVT1 and Ctgf mRNA. In line with this, we found that overexpression of PVT1 reduced expression of miR-26b and miR-186 but activated expression of Srf, Ctgf, LC3B-II, and Beclin-I.Upregulation of PVT1 by exposure to hypoxia promoted the expression of CTGF, leading to deregulation of autophagy and abnormal proliferation of PASMCs. Dysregulation of the miR-186/Srf/Ctgf and miR-26b/Ctgf signaling pathways may be involved in the pathogenesis of hypoxia-induced PASMCs.

Everolimus was designed as a mammalian target of rapamycin (mTOR) inhibitor. It has been proven as a targeted drug for gastric cancer (GC) therapy. However, long-term treatment with everolimus may cause severe side effects for recipients. Decreasing the dosage and attenuating the associated risks are feasible to promote clinical translation of everolimus. This study aimed to identify the underlying mechanisms of responses to everolimus and develop novel regimens for GC treatment. Our findings proved that there was a significant dose-dependent relationship of everolimus-induced GC cell apoptosis and glycolysis inhibition. Then, we found that a member of glucose transporter (GLUT12) family, GLUT12, was actively upregulated to counteract the anticancer effects of everolimus. GLUT12 might be overexpressed in GC. High expression of GLUT12 might be correlated with tumor progression and short survival time of GC patients. Bioinformatic analysis suggested that GLUT12 might be involved in regulating cancer development and metabolism. The experiments proved that GLUT12 significantly promoted GC growth, glycolysis and impaired the anticancer effects of everolimus. Androgen receptor (AR) is a classical oncogenic factor in many types of cancer. Everolimus elevated GLUT12 expression in an AR-dependent manner. Inhibition of AR activity abrogated the promotive effects on GLUT12 expression. Both

Glioblastoma (GBM) is the most common and most lethal type of cancer of the central nervous system in adults. Despite aggressive treatment, which is based on surgical resection, if possible, followed by radiation and chemotherapy, a high recurrence rate and therapy resistance is observed. Thus, additional innovative therapies are urgently needed to improve the poor median survival of only 15 months. Treatment of solid tumours with non-invasive physical plasma (NIPP) represents such a novel and innovative anticancer procedure.In this study, we investigated the effect of NIPP, an ionized argon gas, on the in vitro growth of human GBM cell lines, LN-18 and U-87 MG. Proliferation was measured by live cell count. Subsequently, proliferative factors were analysed at the level of nucleic acids (polymerase chain reaction) and proteins (western blotting).For both GBM lines, a treatment time-dependent decrease in growth was observed compared to controls. Additionally, NIPP treatment resulted in reduced rates of AKT serine/threonine kinase 1 (AKT1) and extracellular-regulated kinase 1/2 ERK1/2 expression, whereas expression of p21, proliferating cell nuclear antigen, and heat-shock proteins 90α and 90β was not affected. In both cell lines, a strong increase in expression of tumour-suppressive microRNA-1 (miR-1) was detected after exposure to NIPP.Our results demonstrated that NIPP is able to efficiently attenuate growth of GBM cells and suggest AKT1, ERK1/2 and miR-1 to be pivotal factors of NIPP-modulated cellular signalling. Translated into the clinical setting, NIPP may represent a promising option for the treatment of GBM.

Immune responses are orchestrated by controlling the initiation, magnitude, and duration of various signaling pathways. Adaptor proteins act as positive or negative regulators by targeting critical molecules of signaling cascades. Signal-transducing adaptor protein-2 (STAP-2) contains typical features of adaptor proteins, like a pleckstrin homology (PH) domain in the N-terminal region and a Src homology 2 (SH2) domain in the central region. STAP-2 binds to a variety of signaling or transcriptional molecules to control multiple steps of inflammatory/immune responses. STAP-2 enhances T-cell receptor (TCR)-mediated signaling via the association with TCR-proximal CD3ζ immunoreceptor tyrosine-based activation motifs (ITAMs) and lymphocyte-specific protein tyrosine kinase (Lck). STAP-2 decreases adherence of T-cells to fibronectin (FN) through an association with focal adhesion kinase (Fak) and Casitas B-lineage Lymphoma (c-Cbl), and increases chemotaxis of T-cells toward stromal cell-derived factor-1α (SDF-1α) through interactions with Vav1 and Ras-related C3 botulinum toxin substrate 1 (Rac1). STAP-2 positively regulates activation-induced cell deathrough the association with Fas and caspase-8. This review describes the current knowledge of the roles of STAP-2 in T-cell-dependent immune responses and the possible clinical utility of STAP-2-targeting therapies.

The kinase LCK and CD4/CD8 co-receptors are crucial components of the T cell antigen receptor (TCR) signaling machinery, leading to key T cell fate decisions. Despite decades of research, the roles of CD4–LCK and CD8–LCK interactions in TCR triggering in vivo remain unknown. In this study, we created animal models expressing endogenous levels of modified LCK to resolve whether and how co-receptor-bound LCK drives TCR signaling. We demonstrated that the role of LCK depends on the co-receptor to which it is bound. The CD8-bound LCK is largely dispensable for antiviral and antitumor activity of cytotoxic T cells in mice; however, it facilitates CD8+ T cell responses to suboptimal antigens in a kinase-dependent manner. By contrast, the CD4-bound LCK is required for efficient development and function of helper T cells via a kinase-independent stabilization of surface CD4. Overall, our findings reveal the role of co-receptor-bound LCK in T cell biology, show that CD4- and CD8-bound LCK drive T cell development and effector immune responses using qualitatively different mechanisms and identify the co-receptor–LCK interactions as promising targets for immunomodulation.

Brevilin A (BA), a sesquiterpene lactone isolated from Centipeda minima herb, has been identified to exhibit potent anticancer activity. However, the potential pharmacological effect and mechanism of BA in regulating endothelial cell (EC) angiogenesis, a key event in tumor growth, is poorly understood. In this study, BA was shown to significantly prevent vascular endothelial growth factor (VEGF) induced EC angiogenic capacities in vitro, ex vivo, and in vivo. Subsequent functional assays revealed that BA dose dependently inhibited VEGF-stimulated survival, proliferation, migration, and triggered apoptosis activity in human umbilical vein endothelial cells (HUVECs), as well as suppressed the expression of antiapoptotic protein Bcl-2, increased the expression of proapoptotic protein caspase-3 and Bax, and suppressed PI3K/AKT pathway. Meanwhile, BA was also able to depolarize mitochondrial membranal permeability (MMP), accelerate mitochondrial superoxide accumulation, induce intracellular reactive oxygen species (ROS) production, and decreased intracellular glutathione (GSH) in HUVECs. Furthermore, both mitochondria-specific superoxide scavenger Mito-TEMPOL and broad-spectrum antioxidant N-acetyl-cysteine (NAC) dramatically abolished BA-induced mitochondrial dysfunction and mitochondrial ROS production, causing the reversion of PI3K/AKT pathway and repression of apoptosis, eventually correcting the impaired endothelial behavior in survival, growth, migration, and angiogenesis. Collectively, our data for the first time identified a new mechanism for antiangiogenic effect of BA in vascular EC, one that is based on the regulation of mitochondrial-dependent ROS overproduction.

The phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway is one of the most important intracellular pathways involved in cell proliferation, growth, differentiation, and survival. Therefore, this route is a prospective biological target for treating various human diseases, such as tumors, neurodegenerative diseases, pulmonary fibrosis, and diabetes. An increasing number of clinical studies emphasize the necessity of developing novel molecules targeting the PI3K/AKT/mTOR pathway. This review focuses on recent advances in ATP-competitive inhibitors, allosteric inhibitors, covalent inhibitors, and proteolysis-targeting chimeras against the PI3K/AKT/mTOR pathway, and highlights possible solutions for overcoming the toxicities and acquired drug resistance of currently available drugs. We also provide recommendations for the future design and development of promising drugs targeting this pathway.

Identification and analysis of small molecule bioactivity in target-agnostic cellular assays and monitoring changes in phenotype followed by identification of the biological target are a powerful approach for the identification of novel bioactive chemical matter in particular when the monitored phenotype is disease-related and physiologically relevant. Profiling methods that enable the unbiased analysis of compound-perturbed states can suggest mechanisms of action or even targets for bioactive small molecules and may yield novel insights into biology. Here we report the enantioselective synthesis of natural-product-inspired 8-oxotetrahydroprotoberberines and the identification of Picoberin, a low picomolar inhibitor of Hedgehog (Hh)-induced osteoblast differentiation. Global transcriptome and proteome profiling revealed the aryl hydrocarbon receptor (AhR) as the molecular target of this compound and identified a cross talk between Hh and AhR signaling during osteoblast differentiation.

Spätzle is a crucial ligand for Toll-like receptor (TLR) that triggers the activation of TLR signal pathway in insects. In this study, open reading frames (ORFs) of two spätzles were cloned from Portunus trituberculatus (PtSpz1 and PtSpz2). Both of PtSpzs contained the typical cystine-knot domain of spätzle. Tissue distribution analysis showed that both of PtSpzs were predominantly expressed in the gills. Transcriptional levels of the two PtSpzs in hemocytes and gill rapidly increased at 3 h and 6 h post Vibrio alginolyticus challenge, respectively. The two PtSpzs could bind to several pathogen-associated molecules including lipopolysaccharide (LPS), peptidoglycan (PGN) and envelope proteins of white spot syndrome virus (WSSV). Moreover, the two PtSpzs could directly interact with the extracellular leucine-rich repeats (LRR) domain of TLR. This study revealed that spätzle could interact with pathogen-associated molecules and TLR of host, which may be two important steps for spätzle to deliver signals into host cells.

Osteosarcoma (OS) is the most common primary malignant bone tumor and is mainly diagnosed in children. Toll-like receptor 9 (TLR9) is expressed in various tumor cells and was correlated with cancer progression. However, the underlying mechanism of TLR9 on the OS progression remains unclear. Our previous study demonstrated that the expression of TLR9 was positively correlated with the development stage of OS. Herein, we further evaluated the actual roles and the molecular mechanism of TLR9 on regulating OS cell proliferation and metastasis. Our data showed that TLR9 was upregulated in OS cells compared to normal osteoblastic cells, and knockdown of TLR9 inhibited OS cell proliferation and induced cell cycle arrest by the decreased expression of cyclin D1, CDK2, and p-Rb, while TLR9 overexpression exerted the inverse effects. Furthermore, TLR9 overexpression could enhance the migration and invasion activities of the OS cells by the upregulation of matrix metalloproteinases 2 (MMP2) and MMP9, and the opposite result was observed in TLR9-silenced cells. Moreover, the nuclear factor kappa B (NF-κB) signaling pathway was activated by TLR9, and TLR9-induced malignant phenotype of OS cells was abrogated by the NF-κB antagonist BAY11-7082. Our study indicated that TLR9 might play a critical role in facilitating OS progression by activating the NF-κB signaling pathway, which may provide a valuable therapeutic target for OS.

Abstract: Gliomas are the most common brain tumors. These tumors commonly exhibit continuous growth without invading surrounding brain tissues. Dominant remedial approaches suffer limited therapy and survival rates. Although some progress has been made in conventional glioma treat-ments, these breakthroughs have not yet proven sufficient for treating this malignancy. The remedi-al options are limited given gliomas' aggressive metastasis and drug resistance. Quercetin, a flavo-noid, is an anti-oxidative, anti-allergic, antiviral, anti-inflammatory, and anticancer compound. Mul-tiple lines of evidence have shown that Quercetin has anti-tumor effects, documenting this natural compound exerts its pharmacological effects by targeting a variety of cellular and molecular pro-cesses, i.e., apoptosis, metastasis, and autophagy. Herein, we summarize various cellular and mo-lecular pathways that are affected by Quercetin in gliomas.

Nononcogenic cancer drivers often impinge on complex signals that create new addictions and vulnerabilities. Protein kinase Cλ/ι (PKCλ/ι) suppresses tumorigenesis by blocking metabolic pathways that regulate fuel oxidation and create building blocks for the epigenetic control of cell differentiation. Reduced levels of PKCλ/ι unleash these pathways to promote tumorigenesis, but the simultaneous activation of the STING-driven interferon cascade prevents tumor initiation by triggering immunosurveillance mechanisms. However, depending on the context of other signaling pathways, such as WNT/β-catenin or PKCζ, and timing, PKCλ/ι deletion can promote or inhibit tumorigenesis. In this review, we discuss in detail the molecular and cellular underpinnings of PKCλ/ι functions in cancer with the perspective of the crosstalk between metabolism and inflammation in the tumor microenvironment.

Tissue-engineered corneal epithelium transplantation is effective treatment for severe limbal stem cell deficiency (LSCD), while epithelial terminal differentiation, tans-differentiation, and insufficient stem cell during construction affect the quality of tissue-engineered corneal epithelium. In this study, we applied SB203580 in the culture medium to downregulate the p38 mitogen-activated protein kinase (MAPK) signaling pathway during construction of tissue-engineered corneal epithelium. With application of SB203580, tissue-engineered corneal epithelium showed enhanced strength and condensed structure. The expression of progenitor cell markers ATP-binding cassette sub-family G member 2, tumor protein p63, keratin 14, and Wnt family member 7A was increased, differentiation markers keratin 12, paired box 6, keratin 10, and keratin 13 and trans-differentiation markers actin alpha 2, smooth muscle and snail family transcriptional repressor 1 was decreased, while cell junction markers claudin 1 and cadherin 1 was increased in the tissue-engineered corneal epithelium. The Wnt/catenin beta 1 signaling pathway was upregulated in the epithelium after p38 MAPK inhibition. Transplantation of tissue-engineered corneal epithelium treated with SB203580 to rabbit LSCD model showed faster wound healing and improved epithelial quality. We conclude that downregulation of p38 MAPK signaling pathway helps maintain the stemness and prevent terminal differentiation and abnormal differentiation of corneal epithelial cells during epithelium construction process, and thus can improve the quality of tissue-engineered corneal epithelium. Impact statement Downregulation of p38 MAPK signaling pathway helps maintain the self-renewal of limbal stem cells and prevents terminal differentiation and abnormal differentiation of corneal epithelial cells. Small molecules modulating p38 MAPK signaling pathway ameliorate tissue-engineered corneal epithelium.

During hypertension-induced endothelial dysfunction, periodic mechanical stretching (MS) activates related inflammatory pathways and leads to endothelial damage, but the underlying mechanisms remain unknown. The present study aimed to determine the injury of HUVECs caused by overstretching and the role of HMGB1-RAGE pathway in HUVECs after injury.Human umbilical vein endothelial cells (HUVECs) were cultured and seeded in BioFlex™ plates (six wells). Cells were exposed to 5% (physiological state) and 20% (pathological state) mechanical stretch at 1 Hz for 12 or 24 h in a Flexcell-5000™, with unstretched cells serving as controls. It was found that excessive MS can inhibit cell viability, proliferation, and tube-forming ability resulting in disordered cell arrangement and orientation, slowing cell migration. All these changes cause endothelial damage compared to physiological MS. Endothelial cells (ECs) promote cell migration and self-repair after injury by increasing the High-mobility group box 1 (HMGB1) expression. Experiments and protein prediction networks have shown that HMGB1 can also promote the expression of downstream protein bFGF by binding to receptor for advanced glycation end products (RAGE). Interestingly, VEGF protein expression did not change significantly during this repair process, implying that bFGF replaces the role of VEGF in vascular endothelial repair.The present study demonstrates that in the context of endothelial injury caused by excessive MS, the HMGB1/RAGE/bFGF pathway is activated and promotes endothelial repair after injury. Therefore, understanding these mechanisms will help find new therapies for diseases such as hypertension, atherosclerosis, and aneurysm formation.

Bisphenol A (BPA) exposure can be associated with neurodevelopmental disorders due to impairment of cell proliferation and synaptic development. Our study evaluated the effects of melatonin (MEL) on ambulatory activity, lipid peroxidation, cytokines, ERK/NF-kB signaling pathway in hippocampus and frontal lobe, and histopathological changes in the hippocampus of the BPA-treated rats. The animals were divided in 4 groups: control, BPA, BPA + MEL I, BPA + MEL II. MEL I (20 mg/kg b.w), and MEL II (40 mg/kg b.w.) were orally administered for 28 days. In the 29th day, BPA (1 mg/kg b.w) was intraperitoneally administered and, after 24 h, Open Field Test (OFT) and Elevated Plus Maze (EPM), were conducted. The results showed that MEL II group made significantly more entries in the open arms of EPM, travelled significantly greater distance and spent more time in the central part of OFT. Malondialdehyde levels were diminished by MEL II in the hippocampus and by MEL I in the frontal lobe. In the hippocampus, MAPK level was significantly lowered by both doses of MEL (p

Background: Colon cancer is a gastrointestinal malignancy with high incidence and poor prognosis. Objective: Saikosaponin B4 (SSB4) is a monomeric component of the Traditional Chinese medicine (TCM), Bupleurum. The current study investigates the therapeutic effect and mechanisms of SSB4 in colon cancer. Methods: The proliferation of two colon cancer cell lines, SW480 and SW620, were assessed using CCK8 and expression of regulatory molecules, including Bax, Caspase3, Caspase9, Cleaved Caspase3, Cleaved Caspase9 and Bcl2 by flow cytometry and Western blotting. Results: Survival rates, assessed by CCK8, of SW480 and SW620 cells decreased significantly when the SSB4 concentration was in the range 12.5–50 μg/ml. Flow cytometry measurements indicated apoptosis rates of 55.07% ± 1.63% for SW480 cells and 33.07% ± 1.28% for SW620 cells treated with 25 μg/ml SSB4. Western blotting revealed upregulation of the proapoptotic proteins, Bax, Caspase3, Caspase9, Cleaved Caspase3 and Cleaved Caspase9, and downregulation of the anti-apoptotic protein, Bcl2, in the presence of SSB4. Network pharmacology and molecular docking predicted that the PI3K/Akt/mTOR pathway might be the main regulatory target for the antitumor effect of SSB4. Further Western blotting experiments showed that SSB4 downregulated (p < 0.01) expression of PI3K, Akt, mTOR and the phosphorylated proteins, P-PI3K, P-Akt and P-MTOR. Expression of PI3K, Akt and mTOR mRNA was found to be downregulated by SSB4 (P < 0.01) as the result of RT-PCR measurements. Conclusion: SSB4 is a potent anti-colon cancer agent. Its effects are likely to be mediated by suppression of the PI3K/AKT/mTOR pathway.