Changes In Cell Signaling Research Articles

Intracellular pH differentially regulates transcription of metabolic and signaling pathways in normal epithelial cells

Intracellular pH (pHi) dynamics regulate normal cell function, and dysregulated pHi dynamics is an emerging hallmark of cancer (constitutively increased pHi) and neurodegeneration (constitutively decreased pHi). However, the molecular mechanisms by which pHi dynamics regulate cell biology are poorly understood. Here, we discovered that altering pHi in normal human breast epithelial cells triggers global transcriptional changes. We identified 176 genes differentially regulated by pHi, with pHi-dependent genes clustering in signaling and glycolytic pathways. Using various normal epithelial cell models, we showed pH-dependent Notch homolog 1 protein expression, with increased protein abundance at high pHi. This resulted in pH-dependent downstream signaling, with increased Notch homolog 1 signaling at high pHi. We also found that high pHi increased the expression of glycolytic enzymes and regulators of pyruvate fate, including lactate dehydrogenase and pyruvate dehydrogenase kinase. These transcriptional changes were sufficient to alter lactate production, with high pHi shifting these normal epithelial cells toward a glycolytic metabolism and increasing lactate production. Thus, pHi dynamics transcriptionally regulate signaling and metabolic pathways in normal epithelial cells. Our data reveal new molecular regulators of pHi-dependent biology and a role for increased pHi in driving the acquisition of cancer-associated signaling and metabolic changes in normal human epithelial cells.

Application of Mesenchymal Stem Cells and Exosome alone or Combination Therapy as a Treatment Strategy for Wound Healing.

The process of wound healing consists of multiple phases, and any disruptions in these phases can lead to the wound becoming chronic and impose heavy financial and psychological costs on the patient and a huge economic burden on the country's healthcare system. Various treatments such as drugs, matrix and scaffolds, blood products, cell therapy, and a combination of these treatments are used for wound healing. The use of mesenchymal stem cells (MSCs) is one of these methods that have produced appropriate responses in the healing of patients' wounds. MSCs by secreting growth factors, cytokines, chemokines, and RNAs elicit changes in cell proliferation, migration, growth, signaling, immunomodulation, and wound re-epithelialization process, and as a result, accelerate wound closure and wound healing. These cells can be isolated from different body sources with different cell characteristics and used directly on the wound site or by injection. In addition, MSCs-derived exosomes have attracted growing attention due to circumventing concerns relating to the direct use of MSCs. To increase the performance of MSCs, they can be used together with other compounds such as platelets, matrices, or scaffolds. This study examined the functions of MSCs in wound healing, as well as the vesicles they secrete, cellular and molecular mechanisms, and combined treatments with MSCs for wound healing.

A single, maximal dose of celecoxib, ibuprofen, or flurbiprofen does not reduce the muscle signalling response to plyometric exercise in young healthy adults.

Non-steroidal anti-inflammatory drugs (NSAIDs) possess analgesic and anti-inflammatory properties by inhibiting cyclooxygenase (COX) enzymes. Conflicting evidence exists on whether NSAIDs influence signaling related to muscle adaptations and exercise with some research finding a reduction in muscle protein synthesis signaling via the AKT-mTOR pathway, changes in satellite cell signaling, reductions in muscle protein degradation, and reductions in cell proliferation. In this study, we determined if a single maximal dose of flurbiprofen (FLU), celecoxib (CEL), ibuprofen (IBU), or a placebo (PLA) affects the short-term muscle signaling responses to plyometric exercise. This was a block randomized, double-masked, crossover design, where 12 participants performed four plyometric exercise bouts consisting of 10 sets of 10 plyometric jumps at 40% 1RM. Two hours before exercise, participants consumed a single dose of celecoxib (CEL 200mg), IBU (800mg), FLU (100mg) or PLA with food. Muscle biopsy samples were collected before and 3-h after exercise from the vastus lateralis. Data were analyzed using a repeated measures (RM) ANOVA, ANOVA, or a Friedman test. Significance was considered at p < 0.05. We found no treatment effects on the mRNA expression of PTSG1, PTSG2, MYC, TBP, RPLOP, MYOD1, Pax7, MYOG, Atrogin-1, or MURF1 (all, p > 0.05). We also found no treatment effects on AKT-mTOR signaling or MAPK signaling measured through the phosphorylation status of mTORS2441, mTORS2448, RPS6 235/236, RPS 240/244, 4EBP1, ERK1/2, p38 T180/182 normalized to their respective total abundance (all, p > 0.05). However, we did find a significant difference between MNK1 T197/202 in PLA compared to FLU (p < .05). A single, maximal dose of IBU, CEL, or FLU taken prior to exercise did not affect the signaling of muscle protein synthesis, protein degradation, or ribosome biogenesis three hours after a plyometric training bout.

DIGEP-Pred 2.0: A web application for predicting drug-induced cell signaling and gene expression changes.

The analysis of drug-induced gene expression profiles (DIGEP) is widely used to estimate the potential therapeutic and adverse drug effects as well as the molecular mechanisms of drug action. However, the corresponding experimental data is absent for many existing drugs and drug-like compounds. To solve this problem, we created the DIGEP-Pred 2.0 web application, which allows predicting DIGEP and potential drug targets by structural formula of drug-like compounds. It is based on the combined use of structure-activity relationships (SARs) and network analysis. SAR models were created using PASS (Prediction of Activity Spectra for Substances) technology for data from the Comparative Toxicogenomics Database (CTD), the Connectivity Map (CMap) for the prediction of DIGEP, and PubChem and ChEMBL for the prediction of molecular mechanisms of action (MoA). Using only the structural formula of a compound, the user can obtain information on potential gene expression changes in several cell lines and drug targets, which are potential master regulators responsible for the observed DIGEP. The mean accuracy of prediction calculated by leave-one-out cross validation was 86.5 % for 13377 genes and 94.8 % for 2932 proteins (CTD data), and it was 97.9 % for 2170 MoAs. SAR models (mean accuracy-87.5 %) were also created for CMap data given on MCF7, PC3, and HL60 cell lines with different threshold values for the logarithm of fold changes: 0.5, 0.7, 1, 1.5, and 2. Additionally, the data on pathways (KEGG, Reactome), biological processes of Gene Ontology, and diseases (DisGeNet) enriched by the predicted genes, together with the estimation of target-master regulators based on OmniPath data, is also provided. DIGEP-Pred 2.0 web application is freely available at https://www.way2drug.com/digep-pred.

Abstract PR02: Aging-associated molecular and cellular alterations determine the biology and clinical characteristics of DLBCL in elderly patients

Abstract About 30% of DLBCLs occur in patients over 75 years old. Due to comorbidities, this potentially curable disease is treated with a reduced dose chemoimmunotherapy regimen, associated with worse response rates and outcomes than the standard approach. Our data indicates that even those able to tolerate full-dose treatments have comparatively worse outcomes. We found that DLBCLs in older patients are more frequently of the ABC-DLBCL subgroup, and their microenvironment (TME) has infiltration of inflammatory and immunosuppressed cells, both characteristics independently associated with chemoimmunotherapy resistance. These features may imply we are facing different biologically diseases. To investigate this, we developed aged DLBCL murine models by implanting three murine lymphoma cells (harboring Setd2/BCL2, Ezh2/BCL2 and Tp53 mutations) in the spleen of young (3-4 months) and aged (21-23 months) mice and monitoring tumor growth by in vivo imaging. We also performed an integrative characterization of the TME from young and aged mice by single cell RNA-sequencing (coding and BCR/TCR), immunophenotyping (flow cytometry) and (MS)-based proteomics. For these two different murine DLBCL cell lines utilized, aged mice had higher tumor burden and showed significantly worse survival than younger mice. In addition, DLBCL cells isolated from aged mice displayed higher clonality and the activation of pro-inflammatory transcriptional programs than DLBCL cells isolated from younger mice. Regarding non-malignant TME B cells, CD11c+ T-bet+ Zeb2+ age-associated B cells (AABs) were significantly more abundant in aged v young mice. AABs displayed a secretory phenotype and transcriptional up-regulation of genes associated with pro-inflammatory programs such as IL-6, Stat3 and Nfatc1. TME T cells showed profound changes including increased infiltration of Foxp3+ Tregs and Pd1+ Cd8+ T cells, as well as the appearance of Cd8+ Gzk+ aged-associated T cells (ATT). Aged mice presented alterations in the myeloid compartment with expansion of Cd45+ Cd11b+ cells. Moreover, the TME of aged mice was enriched in tumor-associated macrophages polarized towards a pro-inflammatory phenotype as well as more abundance of Ly6C+ Ly6G+ myeloid-derived suppressor cells (MDSC). Aged TMEs were also characterized by increased stiffness and extracellular matrix changes affecting collagen and proteoglycans. These TME changes, were associated with intrinsic signaling and transcriptional changes in lymphoma cells like increased activation of NF-kB pathway. Overall, these alterations were compatible with the TME of elderly DLBCL patients showing a global increase in the proportion of inflammatory and immunosuppressive (IN-LME) category (41% in 75-85 y.o. vs. 26% in 50-65 y.o.) and specifically in Tregs, MDSC and CD8 PD1HIGH cells in 75-85 y.o. This data indicates that low-grade chronic inflammation that has been associated with age-related diseases determines molecular and cellular characteristics of the DLBCL in the elderly population. Citation Format: Nahuel Zamponi, Maria Victoria Revuelta, Rossella Marullo, Nicolas Di Siervi, Nikita Kotlov, Wendy Béguelin, Leandro Cerchietti. Aging-associated molecular and cellular alterations determine the biology and clinical characteristics of DLBCL in elderly patients [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PR02.

Water-saving and water-spending strategy: The physiological, proteomic and metabolomic investigation of wheat response to drought and the following recovery

Drought as water deficit in the soil represents the most commonly occurring and the most variable environmental stress factor worldwide. Plants have evolved various strategies to cope with drought stress in relation to their other needs such as the necessity to supply carbon in plants with a C3 type of photosynthetic assimilation. In the present study, two wheat cultivars, Baletka and Tobak, representing two contrasting water management strategies, were studied at physiological, proteomic and metabolomic levels to provide a complex view on their phenotypic responses to drought treatment and subsequent recovery. Physiological characteristics clearly distinguished Baletka and Tobak and, moreover, in water-saving Baletka, both proteomics and metabolomics analyses revealed significant remodelling of cell wall, changes in endocytosis and cell signalling, and, especially, changes in synthesis of other defence proteins and LTI65kDa protein (also known as RD29B protein) which has been detected in wheat for the first time. LEA1 protein was proposed as a component of drought stress memory in Baletka. An amazing finding was the enhanced accumulation of oxalate oxidase implying enhanced oxalate oxidase activity leading to faster degradation of oxalate with the simultaneous gain of two molecules of CO2 which may represent an alternative source of CO2 for C3 plants exposed to drought stress.

Pifithrin-µ Induces Stress Granule Formation, Regulates Cell Survival, and Rewires Cellular Signaling.

(1) Background: Stress granules (SGs) are cytoplasmic protein-RNA condensates that assemble in response to various insults. SG production is driven by signaling pathways that are relevant to human disease. Compounds that modulate SG characteristics are therefore of clinical interest. Pifithrin-µ is a candidate anti-tumor agent that inhibits members of the hsp70 chaperone family. While hsp70s are required for granulostasis, the impact of pifithrin-µ on SG formation is unknown. (2) Methods: Using HeLa cells as model system, cell-based assays evaluated the effects of pifithrin-µ on cell viability. Quantitative Western blotting assessed cell signaling events and SG proteins. Confocal microscopy combined with quantitative image analyses examined multiple SG parameters. (3) Results: Pifithrin-µ induced bona fide SGs in the absence of exogenous stress. These SGs were dynamic; their properties were determined by the duration of pifithrin-µ treatment. The phosphorylation of eIF2α was mandatory to generate SGs upon pifithrin-µ exposure. Moreover, the formation of pifithrin-µ SGs was accompanied by profound changes in cell signaling. Pifithrin-µ reduced the activation of 5'-AMP-activated protein kinase, whereas the pro-survival protein kinase Akt was activated. Long-term pifithrin-µ treatment caused a marked loss of cell viability. (4) Conclusions: Our study identified stress-related changes in cellular homeostasis that are elicited by pifithrin-µ. These insights are important knowledge for the appropriate therapeutic use of pifithrin-µ and related compounds.

The Role of Selected Matrix Metalloproteinases in the Pathogenesis of Gliomas

Abstract The progression of tumor formation is a multifactorial process that involves changes at different levels. Within this intricate molecular and cellular landscape of tumorigenesis, specific enzymes, namely matrix metalloproteinases (MMPs), emerge as pivotal contributors. They may influence this process at the level of tissue remodelling, angiogenesis, changes in cell signalling, invasion and metastasis, but may also serve as prognostic markers or therapeutic targets. Although the function of MMPs has been known for a long time, their specific role in the pathogenesis of brain tumors has only begun to be investigated in the last three decades. Deregulation of MMPs expression, frequently observed in brain tumor tissue, is associated with malignant phenotype, dependent on the grade of malignancy and associated with a worse prognosis. They participate in tissue remodelling under physiological and pathological conditions, which predetermines their action especially in the process of invasion and migration of tumor cells into the surrounding tissue. Related to their potential in tumor progression, they are also being investigated as possible targets in anticancer therapy. It would be very difficult to characterize in detail the role of all known MMPs in the context of brain tumor pathogenesis, so we have selected those that have an essential effect in this issue.

Abstract 1537: CD63 acts as a key modulator of tumor progression and innate immune evasion in non-small cell lung cancer

Abstract Introduction: Lung cancer is a major reason for cancer-related mortality worldwide, causing more than two million cancer deaths per year. Over the last decade, new therapies have allowed long-term survival for patients even in advanced stages. However, only a minority of patients benefits from these therapies and patients often grow resistant over time. An important mechanism that contributes to cancer growth and the development of therapy resistances is the cross-talk between cancer cells and their tissue microenvironment, in particular fibroblasts and macrophages. In this context, we explored whether the tetraspanin CD63 - which is commonly found on intra- and extracellular membranes - stimulates cancer growth and mediates pro-tumorigenic functions of the tissue microenvironment in NSCLC. Methods: We analyzed patient tissue via immunohistochemistry and in-situ-hybridization. We deleted CD63 in cancer cell lines (A549, H810, LC19) and primary fibroblasts via CRISPR-Cas9. To assess phagocytosis, we used in vitro EdU assays and adaptive transfer models in vivo, and identified changes in cell signaling via mass spectrometry. We used an in vitro phagocytosis assay to determine the effect of CD63 on cancer cell phagocytosis through macrophages and analyzed the expression of M1 and M2-associated surface markers through flow cytometry upon co-culturing PBMC with cancer cells over one week. Results: In patient sections, cancer tissue did not only express more CD63 on the gene expression and protein level but a stronger CD63 expression also correlated with a denser macrophage infiltrate. Looking for pro-tumorigenic functions, deleting CD63 in three cancer cell lines reduced cancer cell growth both in vitro and in an adaptive transfer model in vivo. Additionally, deleting CD63 impaired the pro-tumorigenic focal adhesion signaling in primary fibroblasts. Concerning the interaction between cancer cells and macrophages, deleting CD63 reduced the macrophage-mediated phagocytosis of cancer cells and co-culturing primary monocytes with CD63-deficient lung cancer cells induced the expression of M1-associated surface markers on the PBMC. Conclusion: In conclusion, these results demonstrate that the tetraspanin CD63 drives NSCLC growth by stimulating cancer cell proliferation - in part by interfering with focal adhesion signaling - and facilitating immune evasion by preventing cancer cell phagocytosis through macrophages and inducing an immunosuppressive phenotype in macrophages. Citation Format: Tristan Lerbs, Lu Cui, Cristabelle De Souza, Reinhard Buettner, Gerlinde Wernig. CD63 acts as a key modulator of tumor progression and innate immune evasion in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1537.

Age at Injury as a Modifier of Preclinical TBI Behavioral, Neuropathological, and Inflammatory Outcomes.

Traumatic brain injury (TBI) is a leading cause of injury-related death and disability. In high-income countries, TBI is most prevalent among the older population (≥65years), commonly caused by falls. Though age at injury is associated with increased risk of mortality and poor outcome, the underlying mechanisms are unclear. Studies in animal models may yield insights into the intersection of TBI with age. Here we review recent studies in animal models where TBI induced in aged animals is associated with exacerbated behavioral deficits (e.g., mortality, thigmotaxis, and cognitive deficits), neuropathology (microgliosis and astrogliosis), neuroinflammation (e.g., cytokines and iNOS), microglial alterations (e.g., more cellular vesicles and adopting a damage-associated microglia gene signature), and cell signaling and pathway changes (e.g., complement, phagocytosis, autophagy, trophic factor signaling). As relatively few preclinical studies focus on aged animals, more research is needed to fully understand the pathophysiology of TBI in the aged population. Particularly, we recommend that (1) more aged animals should be used, (2) closed-head TBI models should be considered, and (3) animal models of comorbidity or polytrauma should be considered.

Co-Delivery of siNRF2 and Sorafenib by a "Click" Dual Functioned Hyperbranched Nanocarrier for Synergistically Inducing Ferroptosis in Hepatocellular Carcinoma.

In the course of antitumor therapy, the complex tumor microenvironment and drug-mediated changes in cell signaling and biological processes lead to drug resistance. The effect of sorafenib is greatly limited by the specific tumor microenvironment induced by antiangiogenic therapy and ferroptosis resistance induced by the upregulation of nuclear factor erythroid-2 related factor 2 (NRF2). In this study, a pH responsive and amphiphilic hyperbranched polyglycerol, HDP, is synthesized based on a co-graft click chemistry pathway. This nano-scale carrier provides excellent drug-loading capacity, storing stability and pH responsibility, and effectively co-delivery of sorafenib and siRNA. Sorafenib and siNRF2 plays a greatly synergistic effect in inducing reactive oxygen species (ROS), iron overloading, depleting glutathione (GSH), and promoting lipid peroxidation. Importantly, verified in two different animal experiments, HDP-ss (HDP loaded with both siNRF2 and sorafenib) presents a superior anti-tumor effect, by achieving a tumor inhibition rate of ≈94%. Thus, HDP can serve as an excellent targeted delivery nanocarrier with good biocompatibility in antitumor therapy, and combined application of siNRF2 effectively improves the antitumor effect of sorafenib by overcoming NRF2-mediated ferroptosis resistance. Taken together, this study provides a novel therapeutic strategy to combat the drug resistance in antiangiogenic therapy and ferroptosis.

Gastro-Esophageal Junction Precancerosis: Histological Diagnostic Approach and Pathogenetic Insights.

Barrett's esophagus (BE) was initially defined in the 1950s as the visualization of gastric-like mucosa in the esophagus. Over time, the definition has evolved to include the identification of goblet cells, which confirm the presence of intestinal metaplasia within the esophagus. Chronic gastro-esophageal reflux disease (GERD) is a significant risk factor for adenocarcinoma of the esophagus, as intestinal metaplasia can develop due to GERD. The development of adenocarcinomas related to BE progresses in sequence from inflammation to metaplasia, dysplasia, and ultimately carcinoma. In the presence of GERD, the squamous epithelium changes to columnar epithelium, which initially lacks goblet cells, but later develops goblet cell metaplasia and eventually dysplasia. The accumulation of multiple genetic and epigenetic alterations leads to the development and progression of dysplasia. The diagnosis of BE requires the identification of intestinal metaplasia on histologic examination, which has thus become an essential tool both in the diagnosis and in the assessment of dysplasia's presence and degree. The histologic diagnosis of BE dysplasia can be challenging due to sampling error, pathologists' experience, interobserver variation, and difficulty in histologic interpretation: all these problems complicate patient management. The development and progression of Barrett's esophagus (BE) depend on various molecular events that involve changes in cell-cycle regulatory genes, apoptosis, cell signaling, and adhesion pathways. In advanced stages, there are widespread genomic abnormalities with losses and gains in chromosome function, and DNA instability. This review aims to provide an updated and comprehensible diagnostic approach to BE based on the most recent guidelines available in the literature, and an overview of the pathogenetic and molecular mechanisms of its development.

Neural cell engraftment therapy for sporadic Creutzfeldt-Jakob disease restores neuroelectrophysiological parameters in a cerebral organoid model

BackgroundSporadic Creutzfeldt-Jakob disease (sCJD), the most common human prion disease, is a fatal neurodegenerative disease with currently no treatment options. Stem cell therapy for neurodegenerative diseases is emerging as a possible treatment option. However, while there are a few clinical trials for other neurodegenerative disorders such as Parkinson’s disease, prion disease cell therapy research has so far been confined to animal models.MethodsHere, we use a novel approach to study cell therapies in sCJD using a human cerebral organoid model. Cerebral organoids can be infected with sCJD prions allowing us to assess how neural precursor cell (NPC) therapy impacts the progression of sCJD. After 90 days of sCJD or mock infection, organoids were either seeded with NPCs or left unseeded and monitored for cellular composition changes, prion infection parameters and neuroelectrophysiological function at 180 days post-infection.ResultsOur results showed NPCs integrated into organoids leading to an increase in neuronal markers and changes in cell signaling irrespective of sCJD infection. Although a small, but significant, decrease in protease-resistant PrP deposition was observed in the CJD-infected organoids that received the NPCs, other disease-associated parameters showed minimal changes. However, the NPCs had a beneficial impact on organoid function following infection. sCJD infection caused reduction in neuronal spike rate and mean burst spike rate, indicative of reduced action potentials. NPC seeding restored these electrophysiological parameters to the uninfected control level.ConclusionsTogether with the previous animal studies, our results support that cell therapy may have some functional benefit for the treatment of human prion diseases.

Phosphoproteomic Changes Induced by Cell-Derived Matrix and Their Effect on Tumor Cell Migration and Cytoskeleton Remodeling.

Increased fibrotic extracellular matrix (ECM) deposition promotes tumor invasion, which is the first step of the metastatic cascade. Yet, the underlying mechanisms are poorly understood as conventional studies of tumor cell migration are often performed in 2D cultures lacking the compositional and structural complexity of native ECM. Moreover, these studies frequently focus on select candidate pathways potentially overlooking other relevant changes in cell signaling. Here, we combine a cell-derived matrix (CDM) model with phosphotyrosine phosphoproteomic analysis to investigate tumor cell migration on fibrotic ECM relative to standard tissue culture plastic (TCP). Our results suggest that tumor cells cultured on CDMs migrate faster and in a more directional manner than their counterparts on TCP. These changes in migration correlate with decreased cell spreading and increased cell elongation. While the formation of phosphorylated focal adhesion kinase (pFAK)+ adhesion complexes did not vary between TCP and CDMs, time-dependent phosphoproteomic analysis identified that the SRC family kinase LYN may be differentially regulated. Pharmacological inhibition of LYN decreased tumor cell migration and cytoskeletal rearrangement on CDMs and also on TCP, suggesting that LYN regulates tumor cell migration on CDMs in combination with other mechanisms. These data highlight how the combination of physicochemically complex in vitro systems with phosphoproteomics can help identify signaling mechanisms by which the fibrotic ECM regulates tumor cell migration.

Cranial Neural Crest Specific Deletion of Alpl (TNAP) via P0-Cre Causes Abnormal Chondrocyte Maturation and Deficient Cranial Base Growth.

Bone growth plate abnormalities and skull shape defects are seen in hypophosphatasia, a heritable disorder in humans that occurs due to the deficiency of tissue nonspecific alkaline phosphatase (TNAP, Alpl) enzyme activity. The abnormal development of the cranial base growth plates (synchondroses) and abnormal skull shapes have also been demonstrated in global Alpl-/- mice. To distinguish local vs. systemic effects of TNAP on skull development, we utilized P0-Cre to knockout Alpl only in cranial neural crest-derived tissues using Alpl flox mice. Here, we show that Alpl deficiency using P0-Cre in cranial neural crest leads to skull shape defects and the deficient growth of the intersphenoid synchondrosis (ISS). ISS chondrocyte abnormalities included increased proliferation in resting and proliferative zones with decreased apoptosis in hypertrophic zones. ColX expression was increased, which is indicative of premature differentiation in the absence of Alpl. Sox9 expression was increased in both the resting and prehypertrophic zones of mutant mice. The expression of Parathyroid hormone related protein (PTHrP) and Indian hedgehog homolog (IHH) were also increased. Finally, cranial base organ culture revealed that inorganic phosphate (Pi) and pyrophosphate (PPi) have specific effects on cell signaling and phenotype changes in the ISS. Together, these results demonstrate that the TNAP expression downstream of Alpl in growth plate chondrocytes is essential for normal development, and that the mechanism likely involves Sox9, PTHrP, IHH and PPi.

HCMV UL8 interaction with β-catenin and DVL2 regulates viral reactivation in CD34+ hematopoietic progenitor cells.

CD34+ hematopoietic progenitor cells (HPCs) are an important cellular reservoir for latent human cytomegalovirus (HCMV). Several HCMV genes are expressed during latency that are involved with the maintenance of the viral genome in CD34+ HPC. However, little is known about the process of viral reactivation in these cells. Here, we describe a viral protein, pUL8, and its interaction and stabilization with members of the Wnt/β-catenin pathway as an important component of viral reactivation. We further define that pUL8 and β-catenin interact with DVL2 via a PDZ-binding domain, and loss of UL8 interaction with β-catenin-DVL2 restricts viral reactivation. Our findings will be instrumental in understanding the molecular processes involved in HCMV reactivation in order to design new antiviral therapeutics.

Telomeric DNA breaks in human induced pluripotent stem cells trigger ATR-mediated arrest and telomerase-independent telomere damage repair.

Although mechanisms of telomere protection are well-defined in differentiated cells, how stem cells sense and respond to telomere dysfunction, in particular telomeric double-strand breaks (DSBs), is poorly characterized. Here, we report the DNA damage signaling, cell cycle, and transcriptome changes in human induced pluripotent stem cells (iPSCs) in response to telomere-internal DSBs. We engineer human iPSCs with an inducible TRF1-FokI fusion protein to acutely induce DSBs at telomeres. Using this model, we demonstrate that TRF1-FokI DSBs activate an ATR-dependent DNA damage response, which leads to p53-independent cell cycle arrest in G2. Using CRISPR-Cas9 to cripple the catalytic domain of telomerase reverse transcriptase, we show that telomerase is largely dispensable for survival and lengthening of TRF1-FokI-cleaved telomeres, which instead are effectively repaired by robust homologous recombination (HR). In contrast to HR-based telomere maintenance in mouse embryonic stem cells, where HR causes ZSCAN4-dependent extension of telomeres beyond their initial lengths, HR-based repair of telomeric breaks is sufficient to maintain iPSC telomeres at a normal length, which is compatible with sustained survival of the cells over several days of TRF1-FokI induction. Our findings suggest a previously unappreciated role for HR in telomere maintenance in telomerase-positive iPSCs and reveal distinct iPSC-specific responses to targeted telomeric DNA damage.

ERK1/2 modulation in the central nervous system of male and female thiamine-deficient mice with amprolium.

Thiamine deficiency experimental models focus on using the pyrithiamine analog in male rodents, making the thiamine deficiency effects in females and the use of other thiamine antagonists, such as amprolium, unknown. We investigated the impact of thiamine deficiency with amprolium in the cerebral cortex and thalamus of male and female mice by evaluating the modulation of ERK1/2 phosphorylation. The animals were exposed for 20 days to thiamine-deficient chow with different doses of amprolium (20, 40, 60 and 80 mg/kg) and at different treatment periods (five, 10, 15 or 20 days) at a dose of 60 mg/kg. After treatments, ERK1/2 phosphorylation was analyzed by western blot. In male mice, we observed a progressive increase in ERK1/2 phosphorylation in both the cerebral cortex and thalamus in response to the dose of amprolium. In females, ERK1/2 phosphorylation did not progressively increase in response to the amprolium dosage. However, an increase in phosphorylation at the higher doses of 60 and 80 mg/kg was observed. We observed a more intense increase in ERK1/2 phosphorylation in males' cerebral cortex and thalamus from 10 days onwards. In females, the ERK1/2 modulation profiles were similar. The results show that thiamine deficiency induction with amprolium is efficient, compatible with other recognized models that use pyrithiamine, showing changes in cell signaling in the nervous system. The study showed differences in response to thiamine deficiency with amprolium between male and female mice in relation to ERK1/2 phosphorylation and demonstrated that females respond positively to the method and can also be used as model animals.

Targeted Metabolomics in High Performance Sports: Differences between the Resting Metabolic Profile of Endurance- and Strength-Trained Athletes in Comparison with Sedentary Subjects over the Course of a Training Year.

Little is known about the metabolic differences between endurance and strength athletes in comparison with sedentary subjects under controlled conditions and about variation of the metabolome throughout one year. We hypothesized that (1) the resting metabolic profile differs between sedentary subjects and athletes and between perennially endurance- and strength-trained athletes and (2) varies throughout one year of training. We performed quantitative, targeted metabolomics (Biocrates MxP® Quant 500, Biocrates Life Sciences AG, Innsbruck, Austria) in plasma samples at rest in three groups of male adults, 12 strength-trained (weightlifters, 20 ± 3 years), 10 endurance-trained athletes (runners, 24 ± 3 years), and 12 sedentary subjects (25 ± 4 years) at the end of three training phases (regeneration, preparation, and competition) within one training year. Performance and anthropometric data showed significant (p < 0.05) differences between the groups. Metabolomic analysis revealed different resting metabolic profiles between the groups with acetylcarnitines, di- and triacylglycerols, and glycerophospho- and sphingolipids, as well as several amino acids as the most robust metabolites. Furthermore, we observed changes in free carnitine and 3-methylhistidine in strength-trained athletes throughout the training year. Regular endurance or strength training induces changes in the concentration of several metabolites associated with adaptations of the mitochondrial energy and glycolytic metabolism with concomitant changes in amino acid metabolism and cell signaling.

Pathophysiology of Stroke: A Molecular Mechanism Perspective

Stroke is a major cause of death and disability worldwide. The Resulting burden on the society continues to grow, with increase in the incidence of Stroke. Brain attack is a term introduced to describe the acute presentation of stroke, which emphasizes the need for urgent action to remedy the situation. While these observations underpinned key trials of thrombolysis, they also indicate that only patients who are likely to benefit should be exposed to its risks. This review explores the etiology and pathogenesis of ischemic stroke, and provides general information of model. This literature review examines the risk factors associated with ischemic stroke, changes in cell morphology and signaling in the brain after stroke. The Pathophysiology of cerebral ischemic injury is explained, and experimental animal models of global and focal ischemic stroke, and in vitro cellular stroke models, are described in details along with mechanism. This article is dedicated to acute ischemic strokes and its molecular mechanism. Classification systems for stroke a etiology are also discussed briefly, as well as current ischemic stroke therapies and new therapeutic strategies and clinical studies that focus on the potential of stroke recovery.