AE-GCN transfers the AE-specific representations to the corresponding GCN-specific layers and unifies these two types of deep neural networks for spatial clustering via the clustering-aware contrastive mechanism. In this way, AE-GCN accommodates the strengths of both AE and GCN for learning an effective representation. We validate the effectiveness of AE-GCN on spatial domain identification and data denoising using multiple SRT datasets generated from ST, 10x Visium, and Slide-seqV2 platforms. Particularly, in cancer datasets, AE-GCN identifies disease-related spatial domains, which reveal more heterogeneity than histological annotations, and facilitates the discovery of novel differentially expressed genes of high prognostic relevance. These results demonstrate the capacity of AE-GCN to unveil complex spatial patterns from SRT data.Spatially resolved transcriptomics (SRT) provides an unprecedented opportunity to investigate the complex and heterogeneous tissue organization. However, it is challenging for a single model to learn an effective representation within and across spatial contexts. To solve the issue, we develop a novel ensemble model, AE-GCN ( Spatially resolved transcriptomics (SRT) technologies, such as spatial transcriptomics (ST) , 10x VisCurrently, the spatial domain detection methods could be mainly divided into two categories: non-spatial and spatial clustering methods. Some non-spatial methods originally developed for single-cell RNA-sequencing (scRNA-seq) studies, e.g., Seurat  and ScanThough these methods have provided useful information on the usage of expression profiles and spatial information, they usually depend on single models, which center on either expression data itself or spatially neighboring structure, thus probably resulting in the preferred usage of the focused data type. For example, the non-spatial clustering methods only models the gene expression itself, while the spatial clustering methods often take spatial neighbors prior as a hard constraint to ensure spatial clustering continuity, which may lead to over-smoothing of expression  and missautoencoder-assisted graph convolutional neural network), which combines the autoencoder (AE) and graph convolutional neural network (GCN), for accurate and fine-grained spatial domain identification. Specifically, AE-GCN relies on AE for learning expression data-based representations and GCN for spatial graph-constrained learning. AE-GCN orderly transfers the AE-specific representations to GCN-specific layers and unifies these two types of neural networks for spatial clustering via a clustering-aware contrastive mechanism. In this way, AE-GCN combines the advantages of the two models and takes full integration of the expression data and spatial information during the representation learning process.Here, we develop a novel combined model, AE-GCN  based on gene expression and select the top Assume that there are original expression matrix Then, the weighted adjacency matrix Limited by the transcript capture rate of SRT technologies, expression data are often sparse and noisy. AE-GCN generates the enhanced expression data We employ AE to learn the useful representations from the expression data itself and assume that there are The output  for 13 10x Visium datasets, one ST dataset, and one Slide-seqV2 dataset are selected using scanpy.pp.highly_variable_genes from the Scanpy Python package. The log-transformation of the expression profiles is performed using scanpy.pp.log1p on the original gene expression data.AE-GCN uses the combined latent representation y Leiden  algorithFor the enhanced expression matrix We use adjusted Rand index (ARI)  and clusdatasets .We use bulk expression data with patient survival information to evaluate the prognostic significance of genes via the Kaplan–Meier plotter  in the IWe evaluated the ability of AE-GCN to detect spatial domains using 12 human dorsolateral prefrontal cortex (DLPFC) slices generated using 10x Visium. The DLPFC dataset obtained from spatialLIBD  is manuaGenerally, most of the spatial clustering methods performed better than non-spatial algorithms . Slide-seqV2 can measure gene expression at near-cellular resolution  image , ventricle (AE-GCN domain 12), and habenula (AE-GCN domain 11) sections . These sPcp4 ) and indH) image . For venentricle  and Gabbhabenula ) or staihabenula . Thus, fn = 428 spots). The histopathological image and annotations were taken as references  ST dataset (ferences . We asseferences .S100P and TNS4) appeared more spatially smoothed on spots in situ . In this manner, we used the AE-GCN-reconstructed data to denoise the low-quality measurements and evaluated the effectiveness in recovering gene spatial expression patterns. Based on the denoised data, we selected the top 50 DEGs of each domain from the reconstructed data enoising . Overall in situ . These t in situ . For exarognosis  . The histopathological annotations from the original paper  Visium dataset  demonstrates that AE-GCN reveals more functional regions and novel cancer prognostic genes for interpreting cancer heterogeneity, suggesting that AE-GCN has great capability of unveiling tissue heterogeneity from SRT data.The effectively combined modeling is key to the superiority of AE-GCN in the SRT study. Generally, AE models learn the representations from expression data itself, while GCN models learn the structured representations from the sample graph structure by providing an approximate second-order graph regularization, which may suffer from over-smoothing issues. AE-GCN combines the characteristics of these two deep learning methods and integrates them to learn effective representations so that AE is used to weaken the problem of overfitting while simultaneously learning the structured representations in GCN. Additionally, the proposed clustering-aware contrastive module in AE-GCN further promotes the combined model from processes independent of clustering targets to the model that achieves effective spatial clustering. Thus, AE-GCN can not only effectively use the information of the expression data itself but also reasonably regularize the learned information from expression data by spatial structure between spots, which has better advantages than the spatial domain detection methods based on a single-model design in SRT studies.Currently, AE-GCN only models gene expression and spatial information from SRT data and cannot utilize histological images which are also provided by several SRT technologies, e.g., 10x Visium. Although some methods have used histological images in spatial domain detection, histological images are mainly used to enhance the quality of expression data and lack of modeling image data separately, e.g., stLearn . Compare


Tumor cells from the same specimen are functionally heterogeneous. Cancer stem cells (CSCs) are populations of tumor cells with self-renewal and differentiation properties. CSCs are found in nearly all solid and hematological tumors and are characterized by various surface or intracellular markers. These markers can be used to develop tumor-specific antibodies, cytotoxic immune cells, vaccines, and direct immune responses to the tumor cells, including CSC populations. This review discusses the emerging CSC-directed immunotherapies, the current state of their clinical development, the approaches to improve their safety and efficacy, and future strategies to strengthen anti-CSC immunotherapy.The generally accepted view is that CSCs hijack the signaling pathways attributed to normal stem cells that regulate the self-renewal and differentiation processes. Therefore, the development of selective targeting strategies for CSC, although clinically meaningful, is associated with significant challenges because CSC and normal stem cells share many important signaling mechanisms for their maintenance and survival. Furthermore, the efficacy of this therapy is opposed by tumor heterogeneity and CSC plasticity. While there have been considerable efforts to target CSC populations by the chemical inhibition of the developmental pathways such as Notch, Hedgehog (Hh), and Wnt/β-catenin, noticeably fewer attempts were focused on the stimulation of the immune response by CSC-specific antigens, including cell-surface targets. Cancer immunotherapies are based on triggering the anti-tumor immune response by specific activation and targeted redirecting of immune cells toward tumor cells. This review is focused on CSC-directed immunotherapeutic approaches such as bispecific antibodies and antibody-drug candidates, CSC-targeted cellular immunotherapies, and immune-based vaccines. We discuss the strategies to improve the safety and efficacy of the different immunotherapeutic approaches and describe the current state of their clinical development. Cancer remains a leading cause of death worldwide, despite advancements in its treatment . ConventAs the tumor develops, the tumor microenvironment (TME) becomes progressively more crucial to maintaining the growth and functions of CSCs through the interplay with cellular components and modification of the extracellular matrix (ECM). The cellular components of TME, such as endothelial cells (ECs), mesenchymal cells (MSCs), immune cells, and cancer-associated fibroblasts (CAFs), play a role in therapeutic resistance by activating CSC-related signaling pathways such as Wnt, Notch, and nuclear factor kappa B (NF-κB) pathways ,16,17,18Some CSCs can withstand conventional treatments such as chemo- and radiotherapy, which effectively destroys a large portion of the tumor bulk, causing tumor shrinkage. However, standard treatment often fails to prevent disease recurrence if the CSCs are not completely eradicated ,22,23. S+ CSCs compared to CD44− non-CSCs in head and neck squamous cell carcinoma (HNSCC) [Targeted therapies against CSCs are promising strategies to prevent cancer development and reduce the risk of recurrence . Therefo (HNSCC)  and regu (HNSCC) . CSCs, d (HNSCC) , are shi (HNSCC) ,76. A hi (HNSCC) . However (HNSCC) ,79. TherThe generally accepted view is that CSCs hijack the signaling pathways, attributed to normal stem cells, that regulate the self-renewal and differentiation processes. Therefore, the development of selective targeting strategies for CSCs, although clinically meaningful, is associated with significant challenges because CSCs and normal stem cells share many important signaling mechanisms for their maintenance and survival. Many chemical inhibitors used to target the CSC regulating signaling pathways described earlier, such as Notch, Hh, and Wnt/β-catenin signalings, also have a toxic effect on normal stem cells ,81,82. W+ leukemic stem-like cells and accumulation of CD68+ macrophages [In 1993, Seiter and colleagues published a seminal study describing the anti-tumor effect of anti-CD44v antibody using preclinical syngeneic rat xenograft models . The firrophages .vβ3, and activating integrin-dependent signaling molecules such as focal adhesion kinase (FAK) [Integrin associated protein (IAP), or CD47, is another promising target overexpressed on cancer cells, including CSCs, in different types of hematological and solid tumors. CD47 plays a critical role in regulating the homeostasis of immune cells ), including their activation, differentiation, migration, and death. It is also crucial for tumor growth, as CD47 expression on solid tumors results in evasion of the innate immune response ,91. CD47se (FAK) . CD47 alse (FAK) . Of impose (FAK) . Breast se (FAK) . Furtherse (FAK) . Of impose (FAK) . Recent se (FAK) . IBI188,se (FAK) . IBI188 Another attractive target is the interleukin-3 receptor (IL-3R) alpha chain, or CD123, which is highly expressed in the undifferentiated precursor (blast) cells, but present at a low level on normal hematopoietic stem cells, and is associated with the development of AML, acute lymphoblastic leukemia (ALL), B-lymphoid leukemia (BLL), hairy cell leukemia (HCL), Hodgkin lymphoma, blastic plasmacytoid dendritic neoplasms (BPDCN), and MDS ,101. The+ blasts in samples from AML patients [These clinical investigations fueled the further development of the anti-CD123 treatment using immunological approaches. In particular, a humanized anti-CD123 mAb Talacotuzumab  was engineered to have increased affinity for CD16 on NK cells and target CD123 positive cells through NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) . The prepatients  and in Apatients ,106. Howpatients . Furtherpatients .To improve the clinical effectiveness of the CD123-targeted antibody therapy, an antibody-drug conjugate was developed. To this end, a humanized high-affinity anti-CD123 antibody was linked to the DNA-alkylating cytotoxic compound from the class of indolinobenzodiazepine pseudodimer (IGN) . This anIn contrast to the conventional monospecific antibody, bsAB are artificially engineered antibodies with dual specificity for different epitopes attributed to the same or different antigens, therefore offering a variety of therapeutic opportunities, including retargeting of immune cells and modulations of the ligand and receptor action with an efficacy that is hard to achieve for single antibodies . The appSeveral CD47-targeted bsABs are currently being tested in early-phase clinical trials, including HX009, PD-1 × CD47 bsAB, tested in patients with relapsed or refractory lymphoma (NCT05189093). HX009 binds to PD-1 expressed on T cells and CD47 on tumor cells. By this, HX009 blocks the binding of CD47 on tumor cells with SIRPα on macrophages and DCs and, therefore, activates macrophage-mediated phagocytosis of the CD47-expressing tumor cells. On the other hand, the binding of HX009 to PD-1 prevents the interaction between PD-1 and its ligands, PD-L1 and PD-L2, and inhibits the downstream signaling pathways. This signal inhibition recovers effector T cell functions and activates cytotoxic T cell-mediated antitumor immunity . Prelimi+ positive lung CSC populations [Another example is Amivantamab, a bsAB targeting EGFR and c-Met driving tumor growth in patients with non-small cell lung cancer (NSCLC). NSCLC progression is frequently associated with activating mutations in the kinase domain of EGFR. Some of these mutations, such as in-frame base pair insertions in exon 20 (ex20-ins), result in tumor resistance to conventional EGFR tyrosine kinase inhibitors . EGFR anulations .+/EpCAM+ CSCs from malignant ascites in patients with the ovarian, pancreatic, and gastric cancer [EpCAM is another marker highly expressed in tumor cells, including CSCs . Catumaxc cancer .Antibody-based cancer immunotherapy, particularly monospecific and bsAB therapy, is a promising strategy for cancer treatment , and mosIn contrast to the antibody-based immunotherapy available off-the-shelf, adoptive cell therapy (ACT) has been developed as more individualized anti-cancer immunotherapy when cytotoxic lymphocytes, such as T cells or NK cells, are customized for each patient. The cytotoxic activities of T cells and NK cells are mediated by releasing the pore-forming protein perforin, granzyme serine protease, and pro-inflammatory cytokines, making them attractive candidates for the ACT ,156,157.The idea for CAR design was coined by Gross and colleagues in 1989 . The CARThe manufacturing of CAR-T cells consists of multiple steps, including the collection of peripheral blood mononuclear cells (PBMCs) from the patient, T cell isolation, activation, genetic manipulation for CAR expression, expansion, and quality checks for further applications . CAR-T cAlthough several CAR-based therapies for blood cancers have been clinically successful, CAR-modified immune cells still face several hurdles in clinical application for solid tumors. Different biological factors might decrease CAR-T cell efficacy, including a loss of tumor antigen (antigen escape) associated with the development of therapy resistance, lack of antigen specificity resulting in the “on-target off-tumor” toxicity against normal tissues, CAR-T exhaustion , immunosThe growing awareness of these limitations has driven the development of new generations of CAR T cells, and various strategies are being pursued to overcome the existing difficulties. Bispecific CARs, multi-CARs, and logic-gated CAR T cells are being developed to enhance the specificity as well as efficiency, and thereby reduce off-tumor effects of CAR T cell products ,185,186.In contrast to CAR-T cells, CAR-NK cells are less likely to induce off-tumor toxicities and adverse side effects, such as CRS and neurotoxicity, that could be partially attributed to their short lifespan in the bloodstream and different types of secreted cytokines . The preAn additional promising approach to boost immune responses against CSCs is to use CSCs as a source of antigens to pulse antigen-presenting DCs and develop anti-CSC DC vaccines. DCs were first discovered in 1973 by Ralph Steinmann and Zanvil A. Cohn as a phagocytic cell population in the murine spleen . DCs areDendritic CSC vaccination (CSC-DC) is a promising form of DC-mediated immunotherapy. The administration of DCs loaded with MUC1-derived peptide, alone or in combination with other tumor-specific antigens, has been tested in several clinical trials for patients with refractory NSCLC , pancreaAnother approach employing the peptide-loaded DCs is the ALDH peptide-based DC vaccine ,241. ALDhigh) can be identified and isolated quickly by fluorescence-activated cell sorting (FACS), providing a source of antigens for developing CSC-targeted therapeutic approaches. Ning and coauthors first confirmed high tumorigenicity of ALDHhigh cell populations compared to ALDHnegative cells using murine melanoma D5 and squamous cell carcinoma SCC7 syngeneic xenograft tumor models in the immunocompetent C3H and C57BL/6 mice. Next, they evaluated the antitumor immunity induced by vaccination with murine bone-marrow-derived DCs pulsed with the lysate of ALDHhigh cells  compared with the lysate of whole unsorted heterogeneous tumor cells (H-TPDC). They demonstrated that the vaccination of DCs pulsed with the lysate of ALDHhigh cells induced significantly higher protective immunity against tumors than H-TPDCs, as well as DCs pulsed with the lysate of ALDHnegative cells [+/CD24− CSC population. This finding revealed that CSC mRNA induces a more potent cytotoxic T cell response as compared to mRNA isolated from the entire tumor cell population [Similar to other immunotherapy directed against a single antigen, the efficacy of the peptide-based DC vaccines could be compromised by heterogeneity and plasticity of antigen expression in tumor cells, including CSCs. A pulsing of DCs with the entire tumor cell lysate, potentially including the whole repertoire of the tumor antigens, is another promising strategy for developing CSC-specific DC vaccines. The tumor cells with high ALDH activity . In particular, the phase I clinical trial assessed the safety and tolerability of the autologous DC vaccine pulsed with lysate derived from allogeneic GBM stem-like cells. The glioblastoma cells used for the vaccine preparation were isolated from a single patient and propagated as neurospheres in serum-free conditions for CSC enrichment. For the autologous vaccine preparation, PBMCs were collected by leukapheresis and used to isolate monocytes, which were differentiated to DCs and pulsed with GBM CSC lysates. DC vaccines were administered intradermally. The therapy was safe and well tolerated. A subset of the patients (9 of the assessed 25 patients) developed a cytotoxic T cell immune response. The study was not powered to evaluate treatment efficacy, although a comparison of the progression-free survival (PFS) and OS with historical control suggested that CSC-targeted immunotherapy can be a treatment of patients with GBM .high CSCs in tumor tissue when combined with anti-PD-L1 therapy, and it exerted strong inhibitory effects on tumor growth in the syngeneic D5 melanoma and 4T1 breast cancer models [high tumor cells, and inhibited D5 tumor growth in vivo. The dual ALDH1A1 and ALDH1A3 peptide DC vaccine possessed significantly higher antitumor activity as compared to the single ALDH1A1 or ALDH1A3 peptide vaccines [high-DC vaccines for the treatment of the syngeneic melanoma B16-F10 tumors growing in the immunocompetent C57BL/6 mice. Importantly, this study revealed that animals treated with the dual blockade of PD-L1 and CTLA-4 and CSC-DC vaccine conferred significantly more tumor regression than the CSC-DC vaccine alone. They also showed that the combination of the CSC-DC vaccine and immune checkpoint blockade significantly induced CD8+ T cell proliferation and CSC-specific cytotoxic T cell activity compared with the CSC-DC vaccine alone. This study provided the scientific basis for a clinical trial involving the combination of the CSC-DC vaccine and simultaneous PD-L1 and CTLA-4 blockades for improved tumor control in patients with cancer [Results of the clinical studies for DC-based vaccines demonstrated that their combination with therapies that stimulate immune responses and inhibit immune suppression might be more effective for cancer treatment than vaccine administration alone . ICI repr models . Liao etvaccines . Of impovaccines . Zheng eh cancer .high-CSC lysate-pulsed DCs in combination with local tumor radiation therapy (RT) given in 6 doses of 8.5 Gy. They employed syngeneic D5 melanoma and squamous sarcoma SCC7 tumor model in immunocompetent C57BL/6 and C3H mice. This study confirmed the therapeutic efficacy of the CSC-DC vaccine and demonstrated that it has a higher anti-cancer efficiency in the adjuvant setting when administered after RT. The studies conducted by Hu et al. also assessed the therapeutic potential of CSC-DC vaccination in adjuvant setting [high-DC treatment after surgical tumor resection significantly reduced local recurrence, prevented lung metastases, and reduced tumor ALDHhigh CSC populations in the immunocompetent C3H mice bearing syngeneic squamous carcinoma SCC7 tumors. The study of El-Ashmawy et al. suggested that the efficacy of the cisplatin-based chemotherapy for the treatment of murine Ehrlich carcinoma can be improved by its combination with the DC vaccine developed against the CD44+/CD24− CSC-like cell population [A combination of CSC-targeted DC vaccines with conventional therapy is a promising approach to target both CSC and non-CSC cell subsets and to prevent the interconversion between the cell populations. Lu et al. evaluated the therapeutic efficiency of ALDH setting . They repulation .Taken together, the immune targeting of CSCs represents a promising approach to cancer treatment. Furthermore, its combination with ICIs and conventional therapy such as surgery and chemo- and radiotherapy could be a strategy to optimize its therapeutic effectiveness.ClinicalTrials.gov website yet (NCT03085992). Of importance, small-molecule inhibitors of fat mass and obesity-associated protein (FTO) demonstrated to suppress AML stem cell self-renewal as well as inhibit immune checkpoint expression, and immune evasion, hinting at the broad potential of anti-CSC therapy [Conventional treatment strategies for patients with malignant diseases include surgery, chemotherapy, and radiotherapy, referred to as the traditional “three pillars” of cancer treatment. Combining immunotherapies with conventional treatments has recently become a cornerstone of cancer therapy . It has  therapy . The CSCThe therapeutic success of anti-cancer immunotherapy depends on the ability of the immune system to detect and destroy tumors as foreign tissue. However, the efficiency of cancer immunotherapy can be limited by immune evasion. CSCs possess multiple mechanisms to escape immune surveillance and create an immunosuppressive microenvironment. Results of the preclinical studies demonstrated that a combination of CSC-targeted immunotherapies with an immune checkpoint blockade stimulates anti-tumor immune responses and might be a more promising cancer therapy for further clinical studies. Tumor cell plasticity and heterogeneity also reduce the efficacy of anti-CSC therapy. Developing immunotherapy against more than one CSC antigen might lower the risk of tumor escape and non-specific toxicity. A combination of CSC-specific immunotherapy with conventional treatments, such as radio- and chemotherapy targeting the bulk tumor cells and ICI enhancing the anti-tumor immune response, could be a strategy to prevent CSC replenishment by non-CSC de-differentiation. Furthermore, conventional therapy such as radiotherapy and chemotherapy can activate multiple immune-stimulating mechanisms, highlighting the consideration for their combination with anti-CSC immunotherapy ,269,270.


The organ culture of thymus from immunized IBMI-huNSG mice with a neutralizing antibody to HLA-DR showed the HLA-DR-dependent expansion of CD4 single positive thymocytes. Mature peripheral T-cells exhibited alloreactive proliferation when co-cultured with human peripheral blood mononuclear cells. Live imaging of the thymus from immunized IBMI-huNSG mice revealed dynamic adhesive contacts of human-derived thymocytes and DCs accompanied by Rap1 activation. These findings demonstrate that an increase in HLA-DR+ cells by immunization promotes HLA-restricted thymocyte expansion in humanized mice, offering a unique opportunity to generate humanized mice with ease.Immune responses in humanized mice are generally inefficient without co-transplantation of human thymus or HLA transgenes. Previously, we generated humanized mice via the intra-bone marrow injection of CD133+ cord blood cells into irradiated adult immunodeficient mice (IBMI-huNSG mice), which could mount functional immune responses against HTLV-1, although the underlying mechanisms were still unknown. Here, we investigated thymocyte development in IBMI-huNSG mice, focusing on the roles of human and mouse MHC restriction. IBMI-huNSG mice had normal developmental profiles but aberrant thymic structures. Surprisingly, the thymic medulla-like regions expanded after immunization due to enhanced thymocyte expansion in association with the increase in HLA-DR+ cells, including CD205 Engraftment of CD34+ HSCs from human cord blood into such immunodeficient mice successfully reconstitutes and maintains the human hemato-lymphoid system over the long term, leading to the establishment of humanized mice; this approach has been used frequently. However, it has become apparent that T-cell functions and humoral immune responses in those mice are severely compromised -thymidine was added to each well, and the sample was incubated for an additional 8 h before harvest and measurement using a scintillation counter.T-cells were isolated from the spleen of humanized mice using a negative selection kit . Syngeneic stimulator cells were obtained from bone marrow cells derived from the same humanized mice. Allogeneic stimulator cells from human PBMCs were obtained from blood of a healthy control. Splenocytes from C57BL/6 mice were used as xenogeneic antigen-presenting cells. Stimulator cells were exposed to 30 Gy γ-irradiation. After 72 h, a mixture of each type of stimulator cell and 1 × 10Statistical analysis was performed using GraphPad Prism 8  and the Excel software 2019 .


The advent of mRNA vaccines represents a significant advance in the field of vaccinology. While several vaccine approaches  had been investigated at the start of the COVID-19 pandemic, mRNA vaccines quickly gained popularity due to superior immunogenicity at a low dose, strong safety/tolerability profiles, and the possibility of rapid vaccine mass manufacturing and deployment to rural regions. In addition to inducing protective neutralizing antibody responses, mRNA vaccines can also elicit high-magnitude cytotoxic T-cell responses comparable to natural viral infections; thereby, drawing significant interest from cancer immunotherapy experts. This mini-review will highlight key developmental milestones and lessons we have learned from mRNA vaccines during the COVID-19 pandemic, with a specific emphasis on clinical trial data gathered so far for mRNA vaccines against melanoma and other forms of cancer. Reversal of the suppressive tumor microenvironment by checkpoint blockade against PD-1, PD-L1, and CTLA4 could potentiate immunosurveillance which may significantly impact clinical outcomes for patients , 3. mRNA2In vitro transcribed (IVT) mRNAs were first used as a vector for gene transfer, whereby Wolff et al. first reported in vivo expression of transgenes in mouse muscles inoculated with the mRNA vector -methyltransferase  could be used alternatively to force RNA polymerase to incorporate ARCA in the forward orientation and produce fully translatable mRNA transcripts with ARCA at the 5’-end  domain can be directly incorporated into the template DNA plasmid to precisely control the number of nucleotide bases in the Poly-A tail  occur at different frequencies in different target tissues. Therefore, design of the mRNA sequence should carefully consider the mode of vaccine delivery  to fully utilize the endogenous tRNA pool (codon usage). In addition, mRNA sequence is frequently optimized for both mouse and humans to enable preclinical evaluation of the vaccine candidates in animal models . In addiThe 5’ and 3’ untranslated regions (UTR) not only affect thermal stability of mRNAs but also could regulate translation of the transcripts . Incorpo2.3in vitro generated transcripts through High Performance Liquid Chromatography (HPLC) can thereby reduce local reactivity of mRNA vaccines (in vitro generated mRNA transcripts (Double stranded RNA (dsRNA) can be either recognized by TLR3 in the endosome or RIG-I in the cytosol to trigger local type I interferon responses . Carefulvaccines . More renscripts .in vivo delivery. Currently, there are several protamine-formulated mRNA vaccines under clinical investigations. CV-9201, for example, is a vaccine encoding five non-small cell lung cancer (NSCLC) tumor-associated antigens (TAAs) used to treat Stage IIIb or IV NSCLC in a Phase 1/2 study. The study demonstrates that CV-9201 was well-tolerated and could induce antigen-specific T cell responses but failed to improve overall survival in vaccine recipients as compared to historical controls  derivatives to form micelles that can stabilize negatively charged mRNA transcripts, be preferentially taken up by antigen-presenting cells (APCs) such as dendritic cells and macrophages, and offload cargos in acidic endosomes (fh) cell maturation , transgene uptake by the target tissues, and the vaccine’s adverse effect profiles. Non-formulated (naked) mRNAs have previously been studied in several clinical trials. However, they demonstrated limited uptake and immunogenicity due to low thermal stability and poor transit across cellular membrane secondary to the negative charges on the RNA backbone . Self-ascontrols . Finallyndosomes . In addituration . Furtherne model .3An unprecedented opportunity was created for the development of mRNA vaccines during the COVID-19 pandemic. As the original Wuhan strain was extremely contagious and associated with high mortality, academic institutions and pharmaceutical companies rapidly designed, produced and tested vaccine candidates at record speed. For the Pfizer/BioNTech vaccine, animal studies were commenced in Jan 2020 following the release of the SARS-CoV-2 genome. Phase 1/2 study initiated in April whereas Phase 2/3 study commenced in July of 2020, with the vaccine approved by the US FDA under Emergency Use Authorization (EUA) in December 2020. It was rapidly deployed to healthcare workers fighting at the frontlines against COVID-19 . Pre-app4Currently several mRNA cancer vaccine candidates are under clinical investigation. These vaccines may target tumor associated antigens , tumor-specific antigens (TSA or antigens that spontaneously arise in tumors and are therefore unique to cancer cells), or seek to prime the endogenous immune system (immunostimulants).a priori knowledge of tumor transcriptomic signatures. However, as TAAs are also expressed in healthy tissues, vaccines have the theoretical risk of inducing autoimmunity , ovarian cancer (NCT04163094), and NSCLC  with or without CPI  and with or without adjuvant/neoadjuvant chemotherapies . NSCLC-specific mRNA vaccines CV9201 (NCT00923312) and CV9202 (NCT01915524) were found to be safe, and induced antigen-specific T-cell responses in 63% and 84% subjects respectively. However, CV9201 was not found to improve progression-free survival or overall survival in trial participants , 38. As immunity . Centralimmunity .in silico binding algorithms. For example, the mRNA-4650 (NCT03480152) was a personalized neo-antigen vaccine encoding up-to 20 neo-epitopes against metastatic gastrointestinal tumors. In a Phase 1 study, the vaccine induced neoantigen-specific CD8+ and CD4+ T-cell responses in three of four subjects but did not induce significant clinical responses. Further analysis showed predominant elicitation of CD4+, as opposed to CD8+, T cell responses by vaccines despite selection of HLA-I restricted epitopes during vaccine design, highlighting challenges with the in silico prediction algorithm , which are only expressed in infected and transformed cells. BNT113 is an HPV E6/7 mRNA vaccine used for treatment of HPV-positive head and neck squamous cell carcinoma currently being studied in Phase 1 NCT03418480) and Phase 2 NCT04534205) trials along with PD-1 inhibitor pembrolizumab  intra-tumoral injection of mRNA-2752 encoding three cytokines OX40L/IL23/IL36γ along with anti-PD-L1 antibody durvalumab in patients with solid tumors or lymphoma. Preliminary analyses showed dose-limiting toxicity (due to cytokine release syndrome) in one of thirty patients receiving 8mg mRNA-2752, increased cytokine IFNγ and TNFα expression in both tumor and plasma, and partial responses in two of the forty-five patients, highlighting some potential (as well as limitations) of this approach  and host  factors   . For mRNal (RFS) . mRNA-41al (RFS) .6Since their invention three decades ago, mRNA vaccines have come a long way with multiple advances. These include modification of nucleotides, capping, sequence engineering, purification and LNP formulation, that collectively help to overcome key barriers  and empower the platform to be a promising tool in our fight against cancer. The COVID-19 pandemic significantly expedited RNA vaccine development, producing a deep appreciation for its immune/adverse effect profile and comfort in designing novel vaccines for quick first-in-human studies. mRNA vaccines are unique in their ability to activate multiple arms of the immune system , and preliminary (not yet published) data with the Moderna melanoma neoantigen vaccine appears promising in the Phase 2 study.in- silico prediction algorithms or novel in vitro HLA-binding assays will likely improve antigen design and best utilize the RNA cassettes. Finally, even the best-designed vaccine might not adequately overcome the suppressive tumor microenvironment at distant metastatic sites. A multimodal approach involving vaccines, CPI and immunostimulants might work synergistically (However, key challenges in the field still remain. While CD8+ T cell responses are induced by the mRNA vaccines, the magnitudes of responses in humans appear to be significantly lower than those in animal studies, corresponding to more limited anti-tumor efficacy in the context of advanced disease studies to date. Further attempts to amplify cellular responses through self-amplifying RNAs  or antigstically , and shoZX was involved in conceptualization, writing, and revision of the manuscript. DF was involved in conceptualization, writing, and revision of the manuscript. All authors contributed to the article and approved the submitted version.


Although clinical applications of SARS-CoV-2 antibody tests during the COVID-19 pandemic were limited to identifying recent/prior infection, how these tests were used for clinical management of COVID-19 patients is unknown. We consider US infectious disease (ID) physicians’ perceptions about SARS-CoV-2 antibody tests to inform preparedness for future events.In March 2022, we surveyed members of the Emerging Infections Network (EIN), a national network of ID physicians on use of SARS-CoV-2 antibody assays, interpretation of results, and clinical scenarios for which such tests were considered. We analyzed comments provided in a free text field for key themes.Overall, 96 respondents provided non-mutually exclusive free-text comments. Fourteen used serology to assess patients with suspected MIS-C. Another 8 used serology in individuals with immunocompromising conditions, including organ transplant recipients and those with human immunodeficiency virus (HIV) infection, for example, when making decisions to utilize anti-SARS-CoV-2 mAbs (available at the time of the survey but no longer authorized for PReP or treatment.) Twenty-one respondents shared that the most important need for SARS-CoV-2 antibody assays was to discern a correlate of immune protection. Twenty-six considered that serology was not useful for clinical decision-making, and 23 recognized the limitations of the antibody tests, called for more studies, and indicated that additional guidance would be beneficial, noting for example, “Many clinicians order antibody tests but have no idea how to interpret or misinterpret, so guidelines on usage and when not to use would be extremely helpful.”This analysis provides historical insights into challenges practicing ID physicians faced in the midst of a pandemic. During March 2022, some respondents reported use of SARS-CoV-2 antibody assays including when making treatment decisions. As diagnostic and treatment modalities continue to evolve, federal agencies, medical societies, and academic partners can consider providing guidance on appropriate use of novel tests in clinical practice for near term and future responses.Philip M. Polgreen, MD, Eli Lilly: Case adjudication for a clinical trial


To determine the role and rational application of epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) adjuvant therapy in patients with completely resected stage IB-IIIA EGFR-mutant non-small-cell lung cancer (NSCLC).Randomized controlled trials (RCTs) that compared the survival outcomes between adjuvant EGFR-TKIs and adjuvant chemotherapy or a placebo, or between different EGFR-TKI treatment durations for resected NSCLC, were eligible for inclusion. Disease-free survival (DFS) and overall survival (OS) with hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated as effective measures using random-effect or fixed-effect models. Subgroup analysis was also performed.Eleven RCTs involving 2102 EGFR-mutant NSCLC patients with or without EGFR-TKI adjuvant therapy were included. For all stage IB-IIIA NSCLC patients, EGFR-TKIs adjuvant therapy could not only significantly improve DFS  and 2- and 3-year DFS rates, but also improve OS , compared with chemotherapy or the placebo. Further subgroup analyses indicated prolonged OS from first-generation EGFR-TKI adjuvant therapy in stage III patients, compared with chemotherapy or the placebo . Of note, osimertinib adjuvant therapy led to the OS benefit expanding from stage III to stage II-III patients, with significantly improved DFS and a lower risk of brain recurrence, compared with the placebo. A 2-year treatment duration with EGFR-TKI adjuvant therapy showed a significantly lower recurrence risk than a ≤ 1-year duration.The DFS advantage from first-generation EGFR-TKI adjuvant therapy can translate into an OS benefit in stage III NSCLC patients. Osimertinib might be more suitable for adjuvant therapy than first-generation EGFR-TKIs, because of the lower recurrence rate and the potential OS benefit even in early-stage patients. The optimal treatment duration for EGFR-TKIs at different stages of disease needs to be validated.The online version contains supplementary material available at 10.1186/s12885-023-11194-6. Surgery is the most effective treatment for patients with resectable non-small-cell lung cancer (NSCLC), including those with a positive oncogenic driver such as epidermal growth factor receptor (EGFR) mutations. Compared with surgery alone, adjuvant platinum-based chemotherapy can further reduce the risk of disease recurrence and prolong survival in patients with stage II-IIIA or partial high recurrence risk stage IB NSCLC after complete resection . HoweverThe success achieved with EGFR tyrosine kinase inhibitors (EGFR-TKIs) in advanced NSCLC patients has promoted the investigation of EGFR-TKIs as adjuvant treatment for resected EGFR-mutant NSCLC patients , 5. To dHowever, the long-term survival results of these trials are not consistent. Two phase III RCTs (ADJUVANT and IMPACT) that reported their final OS analysis at the American Society of Clinical Oncology (ASCO) Annual Meeting 2021 showed that adjuvant gefitinib appeared to prevent early relapse in patients with completely resected stage II-III EGFR-mutant NSCLC but did not significantly prolong OS in these patients , 14. TheThe present study was conducted according to the 2020 Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) guidelines . The cheThe PubMed, Embase, Cochrane Library, and Chinese National Knowledge Infrastructure databases were searched systematically for relevant publications. The detailed search strategy of each database was shown in Supplementary Table 2 Table . The folStudies that met the following inclusion criteria were included in the meta-analysis: (1) phase II or III RCTs that compared the survival outcomes (DFS and/or OS) in stage I-IIIA NSCLC patients treated with adjuvant EGFR-TKIs versus adjuvant chemotherapy or a placebo or compared the efficacy for different durations of adjuvant EGFR-TKI therapy; (2) included patients who underwent primary radical resection harboring EGFR mutations (exon 19 deletion or exon 21 Leu858Arg); (3) hazard ratios (HRs) for survival analysis (DFS or OS) or the number of events for relevant clinical endpoint events from the overall patient population or subgroup analysis were available; and (4) contained original data sufficient to calculate HRs or P values. All of the included studies were published in English regardless of the publication status .The exclusion criteria were as follows: (1) letters, case reports, reviews, editorials, commentaries; duplicates, single-arm trials, observational studies, or nonclinical studies; (2) studies with irretrievable or insufficient data for statistical analysis; (3) studies written in languages other than English; and (4) low sample size (n < 10).For each included trial, we extracted the corresponding information, including the trial name, year of publication or conference presentation, first author, trial phase, number of participants, pathological stage, treatment regimen, generation of adjuvant EGFR-TKIs, and outcomes . Two of our authors (SLZ and LTH) extracted data and assessed the quality of the included studies using the Cochrane Risk of Bias (RoB) 2 tool  independhttps://github.com/markummitchell/engauge-digitizer) to extract data from the statistical graphs independently. Data relating to DFS and OS were pooled and analyzed by HRs and 95% confidence intervals (CIs) ; data relating to survival rates and adverse events (AEs) were pooled and analyzed by relative risk (RR) and 95% CIs; and P values of less than 5% (P < 0.05) were considered statistically significant. I2 tests were used to test for heterogeneity, and no heterogeneity was assumed if P > 0.1 or I2 < 50% using the fixed-effects model (Mantel–Haenszel method). Otherwise, a random-effect model (DerSimonian–Laird method) was applied. In addition, sensitivity analysis for the primary outcomes was conducted based on the leave-one-out approach, and publication bias was evaluated by a funnel plot and Begg’s and Egger’s tests.Data were analyzed using STATA 14.0  statistical software. For each eligible study, if the associated information was present merely in figures, two reviewers (SLZ and LTH) used Engauge Digitizer 10.8 software ; thus, a random-effects model was applied. Our results showed that adjuvant therapy with EGFR-TKIs compared with non-EGFR-TKIs was significantly associated with a 57% reduction in the risk of disease progression or death . Also, a significant reduction in the risk of disease progression or death  were included in the analysis of DFS for the comparison between the EGFR-TKI group (TKI only/TKI plus chemotherapy) and the non-EGFR-TKI (control) group (placebo/chemotherapy) in an adjuvant setting for patients with resected EGFR-mutant NSCLC. As shown in Fig. . 1 Fig. ) was fou. 1 Fig. , no signA series of subgroup analyses were performed to explore the effects of variables on the efficacy of EGFR inhibitors in resected EGFR-mutant NSCLC patients. The subgroup included interventions (adjuvant EGFR-TKI versus placebo or chemotherapy), different generations of EGFR-TKIs, clinical stage, brain recurrence and EGFR mutation status (exon 19 deletion and L858R mutation).2 = 79.5%; Fig. To further explore the effects of different interventions on DFS, subgroup analyses of DFS in patients treated with adjuvant EGFR-TKIs vs. a placebo and in those treated with adjuvant EGFR-TKIs vs. chemotherapy were performed. Five RCTs compared adjuvant first-generation EGFR-TKIs with a placebo. The ADAURA study compared the adjuvant third-generation EGFR-TKI osimertinib with a placebo. Four other studies compared adjuvant chemotherapy followed by first-generation EGFR-TKIs (gefitinib/erlotinib/icotinib) with adjuvant chemotherapy alone or a placebo –9, 18. TData on DFS based on stage IB, II, or III disease were available in five RCTs Fig. C and the2 = 60.2%; Fig. 2 = 55.4%; Fig. Based on the available data on the brain recurrence rate reported from five trials , an analysis of the effects of EGFR-TKIs on brain recurrence was conducted. EGFR-TKI therapy reduced the risk of brain recurrence  or L858For all patients with stage IB-III NSCLC, the pooled OS analysis from eight RCTs with available or updated data showed a significant improvement in long-term survival in the adjuvant EGFR-TKI group compared with the control group in resected EGFR-mutant NSCLC  who received subsequent EGFR-TKI therapy than in the EGFR-TKI adjuvant treatment group , with an RR value of 0.79  with those who received first- or second-generation EGFR-TKI therapy for 2 years , 20. AltThe most common severe AEs (grade ≥ 3) documented in 10 of the 11 selected studies, including 1064 EGFR-mutated patients treated with EGFR-TKIs, were elevated alanine transaminase , elevated aspartate transaminase , rash , stomatitis , diarrhea , pneumonia , anorexia , dry skin , and asthenia . The pooled data of EGFR-TKI-related grade ≥ 3 AEs are shown in Supplementary Table 3 Table . In addiSensitivity analysis was used to assess the impact of each individual study on the pooled HRs of the median DFS and OS by sequentially removing each eligible study. For DFS analysis, the HR value in each trial was similar to the pooled HR , except for the ADAURA study, which resulted in a higher HR value when this study was omitted  the use of first-generation EGFR-TKIs may induce some biological behavior changes in NSCLC patients, resulting in the induction of drug resistance mechanisms ; (2) theIt is worth noting whether the DFS advantage can be translated to a significant improvement in OS because this can determine the ultimate value of adjuvant therapy with EGFR-TKIs. The present meta-analysis suggested that first-generation EGFR-TKI adjuvant therapy can improve OS in patients with stage III EGFR-mutant NSCLC, but had no impact on OS for stage IB-II NSCLC patients. The lack of OS benefit may be due to several reasons.First, the minor DFS gap between prior adjuvant EGFR-TKIs and chemotherapy/placebo may not correlate with a statistically significant OS difference. In theory, longer EGFR-TKI treatment or a more potent EGFR-TKI may increase this difference. As the results reported in ICOMPARE study  suggesteSecond, different follow-up treatments in different groups may narrow the OS gap. Our pooled data from four RCTs , 13, 14 Third, early-stage EGFR-mutant lung cancers may benefit less from adjuvant EGFR-TKIs, reducing the overall population survival benefit. Based on the sensitivity analysis Fig. B and theSeveral questions regarding EGFR-TKI adjuvant therapy remain to be solved. First, circulating tumor DNA (ctDNA) has been considered a potential predictor of minimal residual disease or disease recurrence and is used to identify molecular residual disease in localized lung cancer . FurtherThe potential bias of this meta-analysis was that the inhomogeneous study design included patients with different treatment regimens, at different stages of disease, and with different treatment durations. To reduce the treatment regimen bias, we performed the present analysis grouped by two comparisons: adjuvant EGFR-TKIs versus placebo and adjuvant EGFR-TKIs versus chemotherapy. The different generations of EGFR-TKIs were also categorized and analyzed. Although we performed specific subgroup analyses of stage I, II, and III patients in terms of DFS and OS, the results were compromised by a lack of information on the stage of disease for each patient and the limited survival data. In addition, the median duration of EGFR-TKI treatment differed in each study, which could also cause some selection bias.To the best of our knowledge, this meta-analysis includes the largest sample size analyzed to date and determined the role of EGFR-TKIs in an adjuvant setting in patients with completely resected stage IB-IIIA EGFR-mutant NSCLC. Patients with resected EGFR-mutant NSCLC showed significantly improved DFS when treated with adjuvant EGFR-TKIs compared with non-EGFR-TKI treatment, regardless of EGFR mutation type or EGFR-TKI generation and stage, with tolerated AEs. The DFS advantage from first-generation EGFR-TKI adjuvant therapy can translate to an OS benefit in stage III NSCLC patients. Osimertinib may be a better candidate for adjuvant therapy than first-generation EGFR-TKIs, due to the lower recurrence rate and the potent OS benefit, even in early-stage patients. In addition, a 2-year treatment time with EGFR-TKI adjuvant therapy was found to be superior to ≤ 1-year adjuvant therapy; however, the optimal treatment duration of EGFR-TKIs at different stages of disease needs to be validated. Further studies are warranted to identify the optimal adjuvant treatment regimen, duration, and agent for patients with different clinical characteristics.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3Supplementary Material 4Supplementary Material 5Supplementary Material 6Supplementary Material 7Supplementary Material 8


Positive psychology theory sustains that the construct of the Self and its components, such as self-evaluation, social self-esteem, and self-coherence, determine our behavior. Personal daily habits and lifestyle modalities lay on these personality components. Problematic and addictive behavior is also strongly influenced by our Self and its main elements.This study aims to determine those personality components related to the central Self-construct that actuates problematic smartphone use.Our further objective is to identify targeted, self-enhancing activities that prevent problematic smartphone use.Participants were teenagers and adults (N=147) from the 17-73 age group (mean age 37.5 years), 31 male and 116 female.Respondents provided self-reported data on their demographic characteristics, perceived self-esteem, social self-esteem, sense of coherence, and problematic smartphone use through an online survey attainable on a web-based platform.Instruments were the Core Self-Evaluation Scale , the MOS-SSS Social Support Assessing Scale , the Sense of Coherence Scale , and the Smartphone Application-Based Addiction Scale .Respondents who reported being more familiar with smartphone applications and spending more time online scored higher on the problematic smartphone use scale. Our study found significant associations between age and problematic smartphone use, with those from younger groups scoring higher.Participants characterized by lower self-esteem proved a more pronounced problematic smartphone use. In our sample, social self-image and social support did not show relevant correlations with the total score of problematic smartphone use.However, a high sense of coherence showed a significant negative association with problematic smartphone use.A more mature Self-construct characterized by a positive self-evaluation and increased sense of coherence act as protective factors against problematic smartphone use.Providing adequate self-evaluation and social support among young through targeted activities will have a higher role in younger age groups, preventing problematic smartphone use.None Declared


While an understanding of the human interactome is within attainable reach , an impeIn this work, we explored the validity of extracting context-specific PPI subnetworks by analyzing the differential coexpression of interacting protein pairs. We first compared highly differentially coexpressed genes (“high-DC genes”) with highly differentially expressed genes (“high-DE genes”) in terms of their fit within a PPI-network analysis. Then, in a human PPI network overlaid with gene-gene differential correlation (DC) values calculated from the microarray gene expression dataset GSE6764, we sought high-DC subnetworks for each disease stage transition of hepatocellular carcinoma (HCC).The validity of integrating DCA with a PPI network was demonstrated in two lines of evidence. First, higher expression correlations were associated with PPI pairs than non-PPI pairs, and exceptionally high DC values were observed within part of the PPI pairs. Second, compared with the high-DE genes, high-DC genes were more enriched with HCC-related genes and were more condensed in the reference network. Then, we extracted DC-PPI subnetworks for the four transitions over five HCC stages. All subnetworks turned out to be significantly enriched with HCC-related genes, HCV-targeted genes, and cancer genes. A comparison of the multiple transition-wise subnetworks gene by gene enabled us to identify the recurrent hub proteins, while comparing them edge by edge allowed us to identify protein pairs with constantly-changing relationships.We demonstrated a differential coexpression workflow within the context of a human PPI reference network. As applied to a multi-stage HCC expression dataset, our approach has generated a set of differentially coexpressed genes and network modules with promising candidates for follow-up HCC investigation.


Therefore, we suggested a hypothesis that UT inhibitors might be novel diuretics to excrete water without disturbing electrolyte metabolism.Diuretics are used widely to raise renal salt and water clearance in a variety of conditions, such as oedema, as well in non-edematous states such as hypertension, which can reduce morbidity and mortality of cardiovascular and cerebrovascular diseases, especially the frequency of stroke and congestive heart failure. However, long-term use of conventional diuretics has several adverse effects including electrolyte disorders, hyperuricemia, hyperlipidemia, and glucose tolerance decrease. Electrolyte abnormalities can induce cardiac arrythmias and sudden death. Therefore, discovering a new diuretic that does not cause electrolyte disturbance becomes a hot issue. Phenotype analysis of knockout mice lacking urea transporter UT-B or various UT-A isoforms has provided evidence for the involvement of UTs in the urinary concentrating mechanism. Functional deletion of UT-B or UT-A isoforms markedly caused polyuria and urea selective low urine concentrating ability. However, deletion of UT-B or UT-As did not affect GFR and clearance rate of the principal solutes (Nainh-14 has inhibition activity on human, rabbit, rat and mouse UT-B. We used rats as an in vivo test model for determining the diuretic activity of UT-A4. Interestingly, UT-A4 caused dose-dependent polyuria, low urinary osmolality and urea concentration in rats. 18-h water deprivation raised urine concentrating ability in rats with or without UT-A4 treatment. However, urine osmolality and urea concentration remained significantly less in UT-A4 treated rats than that in control rats, except of unchanged non-urea solutes. Osmolality and urea concentration was significantly decreased in inner medullary tissue of UTinh-14 treated rats, but not in HCTZ treated rats. All these results suggest that UTinh-14 caused urea selective diuresis.Present study discovered a potent small-molecular urea transporter inhibitor, UT-A4, using an erythrocyte osmotic lysis assay. Stopped flow light scattering experiment, a classical assay for measuring water and urea permeability, confirmed that UT-A4 reversibly inhibited UT-B activity. The experiments also showed that UT-A4 targeted the intracellular part of UT-B protein and had the same inhibition activity on influx and efflux of urea across membrane. UTinh-14 treated rats. However, HCTZ treated rats had significant higher excretion of osmole and non-urea solutes than control and UTinh-14 treated rats, which made lower blood Na+, K+ and Cl-. These results indicate that UT-A4 is a selective UT inhibitor and has urea selective diuretic activity without disturbing excretion of electrolytes. It might have potential value on drug discovery as a new diuretic without electrolyte imbalance and metabolic disorder. It might also be used as a tool drug to study the physiological roles of UTs in big animal models.The excretion of osmoles, urea and non-urea solutes had no significant difference between control and UT


MMR) genes, we recently found that cancer-affected MMR gene mutation carriers had shorter telomeres and more pronounced shortening of telomere length with age than controls and unaffected MMR gene mutation carriers. Here we evaluate blood telomere length in MMR-proficient hereditary non-polyposis CRC, i.e. familial CRC type X (fCRC-X). A total of 57 cancer-affected and 57 cancer-free individuals from 34 Amsterdam-positive fCRC-X families were analyzed and compared to the data previously published on 144 cancer-affected and 100 cancer-free MMR gene mutation carriers, and 234 controls. Relative telomere length was measured using a monochrome multiplex quantitative PCR method, following strict measures to avoid sources of bias and adjusting by age. Despite the retrospective nature of our study, the results show that longer telomeres associate with cancer risk in fCRC-X, thus identifying different patterns of telomere length according to the status of the MMR system.Aberrant telomere length measured in blood has been associated with increased risk of several cancer types. In the field of hereditary non-polyposis colorectal cancer (CRC), and more particularly in Lynch syndrome, caused by germline mutations in the mismatch repair ( MMR gene, i.e. MLH1, MSH2, MSH6 or PMS2 . The remaining 40% do not show MMR defects and the genetic cause of the familial CRC aggregation is still unknown, having been grouped as familial CRC type X (fCRC-X) Family history is one of the strongest risk factors for the development of colorectal cancer (CRC) and is involved in approximately 20% of all CRC cases. However, only 2–6% of all CRCs are explained by germline mutations in known high-penetrance CRC genes. The Amsterdam criteria were defined to identify hereditary non-polyposis CRC cases, considering young age (<50 years) at cancer diagnosis and high familial aggregation of CRC (Amsterdam I) or other related tumors (Amsterdam II). Approximately 60% of the families that fulfill the Amsterdam criteria show DNA mismatch repair (MMR) deficiency as a consequence of a germline mutation or epimutation in a Chromosome telomeres consist of multiple short repeats (TTTAGG) that protect against large-scale genomic rearrangements. Telomeres shorten with cell division, eventually leading to cellular senescence. On rare occasions, cells that aberrantly bypass replicative senescence with critically short telomeres may develop genomic instability and potentially become tumorigenic. In cancer cells, however, as in stem cells, telomerase, the enzyme that adds telomeric repeats to the chromosome ends, is expressed, compensating for telomere erosion and preventing senescence/apoptosis Germline mutations in the components of the telomerase complex cause dyskeratosis congenita. Patients with this disorder have short telomeres, which lead to bone marrow failure and increased cancer risk MMR gene mutation carriers had shorter telomeres and showed faster telomere attrition with age, measured in blood, than controls and cancer-free MMR gene mutation carriers Regarding hereditary CRC, our group recently reported that cancer-affected Here we report the first study of the behavior of blood telomere length in MMR-proficient hereditary non-polyposis CRC, i.e. fCRC-X, and compare it to the behavior observed in controls and in hereditary non-polyposis CRC with a MMR defect, i.e. LS .A total of 114 individuals, 57 cancer-affected and 57 cancer-free, from 34 fCRC-X families were included in the study. These families fulfilled the Amsterdam criteria but did not show MMR defects . In all, 76.5% (26/34) of the families fulfilled Amsterdam I criteria and 23.5% (8/34) Amsterdam II. For comparative purposes, previously published telomere length data from individuals belonging to LS families and from controls were included in the analyses Relative telomere length (RTL) was assessed using a monochrome multiplex quantitative PCR method RTL was adjusted by age based on the telomere length shortening occurring with age in the general population (controls), as previously reported MMR gene mutation carriers, 100 unaffected MMR gene mutation carriers and 234 controls Age-adjusted RTL was evaluated in 57 cancer-affected fCRC-X patients and 57 unaffected individuals from 34 fCRC-X families. The results were compared to the results we published in a previous study of 144 cancer-affected MMR gene mutation carriers were shorter than those in unaffected mutation carriers (p = 0.032) MMR gene mutation carriers (p = 0.015), but of similar length to controls  and cancer-free controls (p = 0.013) . These rcontrols .We found that longer telomeres are associated with cancer in fCRC-X. Moreover, based on data previously published by our group Several epidemiological studies have investigated the association of telomere length with CRC risk in the general population, producing conflicting results MMR gene mutation carriers). Therefore, longer telomeres are associated with cancer risk in MMR-proficient hereditary non-polyposis CRC, as observed in large prospective population-based CRC series Despite the retrospective nature of our study, we found that cancer-affected fCRC-X individuals had longer telomeres than cancer-free members of the same type X families, controls, and cancer-affected members of LS families (Previous evidence supports the hypothesis that long telomeres might increase cancer risk: first, long telomeres may delay cellular senescence and apoptosis, increasing the chance that genetic abnormalities will accumulate Our findings, together with those observed in large population-based CRC series In conclusion, in contrast to in the observations for LS but in line with the results of large CRC prospective studies, elongated telomeres are associated with increased cancer risk in Amsterdam-positive MMR-proficient hereditary non-polyposis CRC.


MAPK (mitogen-activated protein kinase) phospho-serine/threonine/tyrosine-binding protein], resulted in potent chemoresistance. Our follow-up studies demonstrated that knockdown of MK-STYX prevents cells from undergoing apoptosis through a block in cytochrome c release, but that MK-STYX does not localize proximal to the molecular machinery currently known to control this process. In an effort to define its molecular mechanism, we utilized an unbiased proteomics approach to identify proteins that interact with MK-STYX. We identified the mitochondrial phosphatase, PTPMT1 , as the most significant and unique interaction partner of MK-STYX. We previously reported that knockdown of PTPMT1, an important component of the cardiolipin biosynthetic pathway, is sufficient to induce apoptosis and increase chemosensitivity. Accordingly, we hypothesized that MK-STYX and PTPMT1 interact and serve opposing functions in mitochondrial-dependent cell death. We confirmed that MK-STYX and PTPMT1 interact in cells and, importantly, found that MK-STYX suppresses PTPMT1 catalytic activity. Furthermore, we found that knockdown of PTPMT1 resensitizes MK-STYX knockdown cells to chemotherapeutics and restores the ability to release cytochrome c. Taken together, our data support a model in which MK-STYX controls apoptosis by negatively regulating PTPMT1. Given the important role of PTPMT1 in the production of cardiolipin and other phospholipids, this raises the possibility that dysregulated mitochondrial lipid metabolism may facilitate chemoresistance.We previously performed an RNA interference (RNAi) screen and found that the knockdown of the catalytically inactive phosphatase, MK-STYX [ Chemoresistance in primary and recurrent tumors is a significant challenge commonly encountered in the clinic, often resulting in patient mortality. The majority of currently utilized chemotherapeutic agents function through the induction of an intrinsic cell death program termed apoptosis. Evasion of apoptosis has been recognized as a hallmark of cancer, and as such, is critical to disease manifestation and progression In an attempt to identify novel regulators of the apoptotic pathway, we previously performed an RNAi screen targeting all known and putative kinases and phosphatases in the human genome B-Cell CLL/Lymphoma 2) family of proteins BCL2-associated X Protein) or BAK (BCL2-Antagonist/Killer 1) homooligomerize, destabilizing the outer mitochondrial membrane (OMM) and allowing the efflux of pro-apoptotic proteins normally localized within the inner mitochondrial membrane space (IMS) In a follow-up study, we demonstrated that small interfering RNA (siRNA)-mediated knockdown of MK-STYX induces robust chemoresistance to multiple cytotoxic death-inducing agents, such as paclitaxel, cisplatin, and etoposide To determine whether MK-STYX directly affects BAX/BAK oligomerization, we determined its subcellular localization, which we found to be mitochondrial in vivo. Interestingly, other catalytically inactive phosphatases (pseudophosphatases) in the human genome often regulate the catalysis, localization, and/or function of active phosphatases Due to the non-canonical nature of this regulation, we sought to identify interaction partners of MK-STYX to help define its molecular function. To do this, we utilized an unbiased proteomics approach to identify proteins interacting with MK-STYX. This study identified the mitochondrial phosphatase, PTPMT1, as the most significant and unique interaction partner of MK-STYX. PTPMT1 is a catalytically active DUSP located within the IMM, with its phosphatase domain facing the matrix in vitro. To determine the genetic nature of this interaction, we performed single and combinatorial siRNA transfections of PTPMT1 and MK-STYX. Knockdown of PTPMT1 resensitized MK-STYX-depleted cells to chemotherapeutic treatment and cytochrome c release. Overall, these data suggest a model whereby MK-STYX inhibits PTPMT1 phosphatase activity to modulate cellular viability. Because PTPMT1 is a key regulator of phospholipid biosynthesis, in particular cardiolipin, these data suggest that dysregulated mitochondrial lipid metabolism could be a novel mechanism by which cancer cells can resist therapeutic treatments.Here, we demonstrate that MK-STYX and PTPMT1 interact on a genetic, physical, and functional level. We verify the initial proteomics-based identification of the MK-STYX – PTPMT1 interaction using co-immunoprecipitation. Furthermore, we show that MK-STYX directly suppresses the catalytic activity of PTPMT1 using purified proteins To more completely understand its cellular and molecular function, we sought to identify novel protein interaction partners of MK-STYX. We utilized an unbiased proteomics approach in which tandem affinity purification (TAP) was followed by mass spectrometry (MS). The TAP-MS approach is appropriate to identify candidate interaction partners, as it has been shown to identify multiprotein complexes under near physiological conditions Table S1). MS data was also used to compile an ‘interactor score’, which evaluated the number of experiments in which the peptide was found ; the number of peptides correlating with a protein (to increase confidence in the interactor); and MASCOT scores (Matrix Science) of those peptides (demonstrating the reliability of the protein identification). Thus, a larger interactor score correlates with a more robust proteomic interaction. The protein with the highest interactor score in our experiment was MK-STYX, which was expected as TAP experiments commonly show the most peptide coverage from the baited protein. The most significant (highest interactor score) and unique (p<0.0006) interaction partner of MK-STYX was the mitochondrial dual specificity phosphatase, PTPMT1  in triplicate for a total of six independent experimental conditions. Upon expression of these constructs in HeLa cells, MK-STYX was isolated by a two-step affinity purification method, interacting proteins were resolved by SDS-PAGE, and gel fragments were digested and identified by liquid chromatography coupled to mass spectrometry (LC-MS/MS). Unique interactors were identified as those peptides corresponding to proteins identified in at least one of three replicates across the experimental conditions. To determine specific interactors (relative to non-specific binding proteins), all co-interacting peptides identified were bioinformatically filtered against a database of approximately 350 control (non-MK-STYX) TAP-tagging experiments. Statistical analysis eliminated these non-specific interacting proteins and prioritized the most unique interacting proteins with MK-STYX P) Next, we sought to determine if MK-STYX alters the catalytic activity of PTPMT1. To this end, we generated pure recombinant GST-tagged PTPMT1 and FLAG-tagged MK-STYX proteins (max (maximum enzyme velocity) and Km (substrate concentration at 1/2 Vmax) were determined for each condition , while leaving Vmax largely unchanged The significant increase in Km caused by MK-STYX, evidenced by the steep trajectory in the Lineweaver-Burk plot (red line) when compared to the FLAG control (blue), is consistent with competitive inhibition. Through this mode of inhibition, MK-STYX is predicted to disrupt the interaction of OMFP with PTPMT1 We found that in the presence of a FLAG protein control, PTPMT1 had a Kin vitro, we hypothesized that MK-STYX functions upstream of PTPMT1, downregulating its activity to promote apoptosis.We have previously characterized MK-STYX as a ‘death phosphatase’, demonstrating that its overexpression promotes apoptosis while its depletion causes striking resistance to cell death From this hypothesis, we first predicted that dual knockdown of MK-STYX and PTPMT1 would result in decreased basal cell viability, as MK-STYX depletion would not protect against cell death caused by the downregulation of its cellular target, PTPMT1 Cells transfected with MK-STYX siRNA are significantly chemoresistant 50 of 24 nM , particularly when compared to the EC50 value of MK-STYX knockdown cells  Binding Protein 1), was abrogated when MK-STYX was mutated back to an active enzyme We have confirmed a physical interaction between MK-STYX and PTPMT1 in cancer cells. MK-STYX lacks catalytic activity due to a naturally occurring cysteine-to-serine substitution, rendering the enzyme incapable of dephosphorylation in vitro, supporting the conclusion that this inhibitory relationship is direct. Specifically, the data best supports a competitive mode of PTPMT1 inhibition conferred by MK-STYX m caused by co-incubation with MK-STYX suggests MK-STYX can interfere with substrate binding to free PTPMT1. While competitive inhibitors  often bind an enzyme in the same location as the substrate  and thereby directly compete with it, this is not always the case in vivo remains to be determined. Further studies, including structural modeling and assays that more closely reflect the endogenous activity of PTPMT1, are required to further clarify the nature of this relationship.In addition to physically interacting in cells, we discovered that MK-STYX significantly reduces the phosphatase activity of PTPMT1. This data was obtained using purified recombinant proteins Consistent with the direct repression of PTPMT1 activity by MK-STYX, we have demonstrated that MK-STYX and PTPMT1 dually function to regulate apoptosis and chemosensitivity, with PTPMT1 functioning downstream of MK-STYX  and 293FT cells (Invitrogen) were cultured in DMEM with a final concentration of 10% FBS. Paclitaxel (Sigma-Aldrich), as well as cisplatin and the pan-caspase inhibitor zVAD  were resuspended in DMSO according to manufacturer directions. 3-O-Methylfluorescein phosphate (OMFP) was purchased from Sigma-Aldrich, diluted initially in DMSO to 10 mM, then to final concentration in phosphatase assay buffer. Recombinant proteins were obtained from BPS Bioscience (see below). FLAG peptide was obtained from Sigma-Aldrich.Control (AllStars Negative Control), MK-STYX , or PTPMT1  (Qiagen) were transfected into HeLa cells at a concentration of 50 nM per siRNA with Oligofectamine (Invitrogen) for the time indicated in each figure legend. Knockdown efficiency was assayed using RT-PCR and the delta-delta Ct method using HRPT1 as a reference gene, as previously described GST-PTPMT1 and FLAG-MK-STYX (Styxl1) were obtained from BPS Bioscience . Briefly, PTPMT1 ) fused to an amino-terminal GST tag was produced using a baculovirus infected Sf9 insect cell system. High yield (0.6 mg per ml) and purity (≥85%) was obtained. Similarly, MK-STYX ) fused to an amino-terminal FLAG tag was produced using a baculovirus infected Sf9 insect cell system. High yield (0.15 mg per ml) and purity (≥90%) was obtained.A 2× stock of phosphatase assay buffer, pH 7.0 was prepared in water  and 2× (10 mM) dithiothreitol (DTT) added fresh before use (modified from TAP-epitope-tagged MK-STYX was expressed in HeLa cells by retrovirus-mediated gene transfer. N- (NTAP) and C- (CTAP) terminal fusions were constructed, and viral stocks were generated in HEK293 Gag-Pol packaging cell lines. Expression levels of MK-STYX were gauged close to endogenous levels by adjusting the multiplicity of infection. MK-STYX was immunopurified in complex with its endogenous associated proteins using a modified TAP protocol 50 values were calculated with GraphPad PRISM using a log inhibitor vs. response (3-parameter) algorithm and a bottom constraint of 0% viability.CellTiter-Glo (Promega) was used to detect cellular viability according to the manufacturer's directions. Real-time cell proliferation and viability was determined using the xCELLigence system, as previously described Immunofluorescence for cytochrome c release was performed as previously described p-values were generated by unpaired, two-tailed t-tests in which equal variance was not assumed (using Microsoft Excel or GraphPad PRISM). Brackets on figures indicate which two values were compared in each test and astericks indicate the following p-values: *  = p≤0.05; **  = p≤0.01; ***  = p≤0.001.The open reading frame (ORF) of human MK-STYX  was cloned into pRK7 with an N-terminal V5 tag (introduced in the forward primer). The ORF of human PTPMT1 (NM_175732.2) was cloned into pRK7 with a C-terminal FLAG tag (introduced in the forward primer). Cysteine 132 of PTPMT1 was mutated Serine (C132S) using a QuikChange Site-Directed Mutagenesis kit (Agilent) and sequence-verified.V5-MK-STYX and FLAG-PTPMT1 constructs were transfected into 293FT cells using FuGENE 6 according to the manufacturer's directions. 48 hours post-transfection, cells were lysed and cleared as previously described Table S1MK-STYX interactors identified by LC-MS/MS. Endogenous interaction partners were identified by LC-MS/MS and bioinformatic filters applied against a large control dataset (∼350 non-MK-STYX TAP tag experiments) to identify unique and significant interactions with MK-STYX (STYXL1 gene symbol). The gene name of each protein identified is listed in order of significance (the most significant hits listed at the top and decreasing in significance down the list). Control hits designates how many peptides of that protein were found in any other non-MK-STYX TAP experiment, which is converted to a fraction of total TAP experiments in the next column. STYXL1 hits shows the amount of peptides from the identified protein were found in the TAP-MK-STYX experiment, which is also converted to a fraction relative to number of replicates in the experiment. Number of IDs shows in how many experiments (of the triplicate performed) the peptides corresponding to the interacting protein was found. These data were compiled to create a p-value, demonstrating the significance of each interaction partner. Additionally, an interactor score was calculated based on this p-value, the number of replicates in which the interactor was identified, the uniqueness of the MK-STYX interaction relative to the control interactions, as well as the MASCOT scores and total coverage of the peptides identified from each interaction protein. A lower p-value and/or a higher interactor score correlates with a stronger molecular interaction with MK-STYX.(DOCX)Click here for additional data file.


The purpose of this study is to compare exam time and quality of images obtained using standard segmented k-space acquisitions versus single-shot and real time techniques that require no breath-holds.The typical CMR exam utilizes segmented k-space acquisitions that require repeated breath-holds, regular cardiac rhythm, and total exam times on the order of forty-five minutes. Widespread utilization of CMR has been hampered by these prolonged exam times and limited reliability in patients with irregular rhythm and/or inability to breath-hold.Cardiac MRI studies performed at our lab over a twenty-five day period were retrospectively reviewed and 16 consecutive studies identified that did not involve stress imaging or MR angiography; the majority included cine, late gadolinium enhancement (LGE), and velocity mapping. Indications for CMR were: cardiomyopathy 11, cardiac mass 2, valvular 1, viability 1, pericardial 1. Studies were separated into two groups: eight were performed using standard segmented k-space acquisitions and eight in which cardiac arrhythmia or inability to breath-hold led to the use of real-time and single-shot methods exclusively. All images were qualitatively assessed for image quality, ability to estimate function.Total exam time for patients scanned using the standard breath-hold protocols was (mean+/- SD) 37.1+/- 13.5 minutes compared with 26.6 +/- 7.6 minutes for patients scanned using real-time cine and velocity-mapping and single-shot LGE methods. In patients where segmented acquisitions were attempted but abandoned due to arrhythmia or inability to breath-hold, real-time imaging significantly reduced artifacts and cardiac endocardial borders were better identified compared to segmented scans under these circumstances. LV wall motion assessment was feasible in seven out of the eight patients who had to undergo the real time cine imaging (87.5%) vs. all eight patients (100%) who underwent standard segmented cine imaging.Real-time and single-shot imaging not only reduces scan time, but also improves reliability in patients with arrhythmias or inability to breath-hold. The use of real time cine in these patients did not compromise the ability to analyze function and morphology and provided proper diagnostic answers. A prospective cohort study is underway to evaluate the reliability and cost-effectiveness of such an approach.None.


The behavioral consequences of the mutation have not been fully explored due to the complexity in studies of mutant receptors in vivo. To facilitate the determination of the contribution of phosphorylation sites in MOR to opioid-induced analgesic behaviors, we expressed mutant and wild-type human MORs (hMORs) in sensory dorsal root ganglion (DRG) neurons, a major site for nociceptive (pain) signaling and determined morphine- and the full MOR agonist, DAMGO,-induced effects on heat-induced hyperalgesic behaviors and potassium current (IK) desensitization in these rats.Phosphorylation sites in the C-terminus of mu-opioid receptors (MORs) are known to play critical roles in the receptor functions. Our understanding of their participation in opioid analgesia is mostly based on studies of opioid effects on mutant receptors expressed in K desensitization in both rat groups. In contrast, DAMGO increased analgesic potency and elicited IK desensitization to a significantly less extent in hMOR-T than in hMOR rats. The development and extent of acute and chronic tolerance induced by repeated morphine or DAMGO applications were not altered by the T394A mutation.A mutant hMOR DNA with the putative phosphorylation threonine site at position 394 replaced by an alanine (T394A), i.e., hMOR-T, or a plasmid containing wild type hMOR (as a positive control) was intrathecally delivered. The plasmid containing GFP or saline was used as the negative control. To limit the expression of exogenous DNA to neurons of DRGs, a neuron-specific promoter was included in the plasmid. Following a plasmid injection, hMOR-T or hMOR receptors were expressed in small and medium DRG neurons. Compared with saline or GFP rats, the analgesic potency of morphine was increased to a similar extent in hMOR-T and hMOR rats. Morphine induced minimum IK desensitization, but has limited effect on morphine-induced responses. On the other hand, the mutation contributes minimally to both DAMGO- and morphine-induced behavioral tolerance. Furthermore, the study shows that plasmid gene delivery of mutant receptors to DRG neurons is a useful strategy to explore nociceptive behavioral consequences of the mutation.These results indicate that phosphorylation of T394 plays a critical role in determining the potency of DAMGO-induced analgesia and I Nearly all the opioids used in clinics, including morphine and fentanyl, exert their analgesic effects through the activation of mu-opioid receptors (MORs). Once activated by opioids, the MOR undergoes G-protein receptor kinase (GRK)-dependent phosphorylation. Within minutes of agonist exposure, MOR starts to lose coupling with its effectors, including adenylyl cyclase, G protein-coupled inwardly rectifying potassium (GIRK) channels and voltage dependent Ca2+ channels, and reduces its agonist sensitivity, a phenomenon referred to as “desensitization” [2-MePhe4-Gly-ol] enkephalin (DAMGO), is much reduced, and DAMGO-induced inhibition of adenylyl cyclase activity is also decreased  where min and max are minimal and maximal responses, the potency, ED50, is the effective morphine dose producing 50% of the maximal analgesic effect, and n is the Hill coefficient. A P < 0.05 was considered significant.All data were recorded as mean ± SEM. Analgesic effects were shown as MPE by using the relation MPE = (postdrug PWL - baseline PWL) / (cutoff PWL - baseline PWL) × 100%. To assess the significance of changes between two means, the Student’s 2-MePhe4-Gly-ol] enkephalin; DRG: Dorsal root ganglion; hMOR: Wild-type human mu-opioid receptor; hMOR-T: T394A mutant human mu-opioid receptor; MOR: Mu-opioid receptor; MPE: Maximum possible effect; PWL: Paw withdrawal latency.CHO: Chinese hamster ovary cells; DAMGO: [D-AlaThe authors declare that they have no competing interests.GL designed and performed behavioral and electrophysiological experiments, analyzed the data and edited the manuscript, FM designed and performed immunocytochemical experiments, analyzed the data and prepared the manuscript. YG analyzed the data and edited the manuscript. LMH conceived and coordinated the study, designed experiments, prepared plasmids, analyzed the data and prepared the manuscript. All authors read and approved the final manuscript.


C. elegans have revealed that prohibitin differentially influences aging by moderating fat metabolism and energy production, in response to both intrinsic signalling events and extrinsic cues. These findings indicate that prohibitin is a context-dependent modulator of longevity. The tight evolutionary conservation and ubiquitous expression of prohibitin proteins suggest a similar role for the mitochondrial prohibitin complex during aging in other organisms.While specific signalling cascades involved in aging, such as the insulin/IGF-1 pathway, are well-described, the actual metabolic changes they elicit to prolong lifespan remain obscure. Nevertheless, the tuning of cellular metabolism towards maximal survival is the molecular basis of longevity. The eukaryotic mitochondrial prohibitin complex is a macromolecular structure at the inner mitochondrial membrane, implicated in several important cellular processes such as mitochondrial biogenesis and function, molecular signalling, replicative senescence, and cell death. Recent studies in  Caenorhabditis elegans and mammals [Prohibitins are ubiquitous, evolutionarily strongly conserved proteins that localize to mitochondria. The eukaryotic mitochondrial prohibitin (PHB) complex comprises two highly homologous subunits, PHB-1 and PHB-2 (around 50% amino acid sequence identity and 60% similarity). PHB-1 and PHB-2, with molecular weights of 32 and 34 kDa respectively, associate to form a ring-like macromolecular structure of approximately 1 MD [ mammals . The PHBSeveral roles have been proposed for mitochondrial prohibitins. First, the PHB complex was suggested to regulate membrane protein degradation by the mitochondrial m-AAA protease . Later, C. elegans, and 53/79% identity/similarity between C. elegans and Saccharomyces cerevisiae) suggests an essential cellular function. Although disruption of the PHB complex in S. cerevisiae does not result in any observable growth phenotype under laboratory conditions [C. elegans and in mice, prohibitins are required for embryonic development [C. elegans results in pronounced germline defects such as diminished oocyte production with smaller brood size [The high degree of evolutionary conservation of the PHB proteins  cells. However, phb-null mutants in stationary phase tend to lose respiratory capacity due to deletions of the mitochondrial genome (mtDNA) [phb-null mother cells [phb-null mother cells at the end of their replicative lifespan [phb-null yeast cells undergo premature aging, probably due to a cumulative decline in cellular metabolic capacity.Disruption of the PHB complex in st cells ,23,29, w (mtDNA) . Loss ofer cells ,31. Similifespan . This suC. elegans, depletion of prohibitins by RNAi reduces the lifespan of wild type animals [C. elegans mutants [C. elegans cells, given that neurons are particularly refractory to RNAi[In  animals , recapit animals . Surpris mutants . These iy to RNAi. Since nC. elegans transmembrane insulin receptor kinase DAF-2 doubles the lifespan on wild type animals [daf-2 mutants by 150% [daf-2 mutants is dependent on the transcription factor DAF-16/FOXO [daf-2 mutants [C. elegans [daf-7 mutant and the transmembrane TGF-β receptor serine/threonine kinase daf-4 mutant. The highly conserved IGF-1 and TGF-β signaling pathways also regulate diapause entry (dauer larva formation) in the nematode [phb-1 or phb-2 extends the lifespan of animals carrying a lesion in the daf-11 gene. Thus, depending on the genetic background, prohibitin function has opposing effects on C. elegans aging. Although depletion of prohibitin compromises survival of wild-type animals, it substantially extends the lifespan of mutants defective in diapause signaling. Thus, the PHB complex plays an important role in modulating the rate of aging by coordinating mitochondrial energy metabolism in response to diapause signaling and energy demands.The Insulin/Insulin Growth Factor 1 (IGF-1) signaling pathway (IIS) regulates aging in worms, flies and mammals . Mutatio animals ,35, and  by 150% . The lif-16/FOXO . Similar mutants . In addi elegans . Prohibinematode . The tranematode . Knock-dgas-1 mutants which carry a lesion in the 49-kDa iron-sulphur subunit of complex I of the ETC. Similarly, prohibitin depletion extends the lifespan of short-lived mev-1 mutants, which are defective in succinate dehydrogenase cytochrome b, a component of complex II. Mutations in the isp-1 gene, encoding the Rieske iron-sulfur subunit of complex III, and in the clk-1 gene, involved in ubiquinone biosynthesis, extend the lifespan of C. elegans [isp-1 and clk-1 mutant animals [eat-2 mutants. Whether PHB effects on longevity are mediated by the effects of DR on mitochondrial metabolism, on cellular signaling or on both is currently unknown.Mitochondria are involved in key aging associated processes, such as cellular metabolism, ATP synthesis and the production and detoxification of reactive oxygen species (ROS). Unpaired mitochondrial function has been shown to increase lifespan across phyla . However elegans ,40. Proh animals . Therefo animals . ProhibiC. elegans nuclear hormone receptor NHR-49 regulates fat mobilization by regulating the expression of a wide variety of genes, including mitochondrial fatty acid β-oxidation enzymes and fatty acid desaturases, such as FAT-7. Prohibitin deficiency extends the lifespan of both nhr-49 and fat-7 mutants [Mitochondrial function is strongly linked to fat metabolism and altered fat metabolism accompanies the aging process. The  mutants . Taken tAll the above data indicates that the metabolic state will determine whether prohibitin will promote or compromise longevity. In agreement with this idea, a temperature shift is sufficient to revert the aging phenotype of PHB depletion. At higher temperature (25°C versus the standard 20°C growing conditions), where energy demands are higher and metabolic activity is elevated, prohibitin deficiency is beneficial for survival. Prohibitin deficiency is also beneficial under acute thermal stress (35°C), since elimination of PHB proteins render wild type animals strongly thermo-tolerant.C. elegans. Many loss-of-function mutations in mitochondrial genes increase longevity [The link between mitochondrial energy metabolism and longevity has been most extensively studied in the nematode ongevity ,40,42-45ongevity -50. ProhProhibitin depletion results in reduced fat content, in all genetic backgrounds where PHB deficiency extends lifespan . In wildnhr-49 gene result in fat accumulation due to the reduced expression of mitochondrial fatty acid β-oxidation enzymes and nhr-49 mutants are short-lived. PHB knockdown in nhr-49 mutants dramatically reduces fat content and extends longevity. NHR-49 regulates the expression of the delta-9 stearoyl-CoA desaturase FAT-7, which is required for the synthesis of monounsaturated fatty acids [fat-7 mutants. These observations suggest that prohibitin deficiency engages fat metabolism to promote longevity in sensitized genetic backgrounds.Mutations in the ty acids . PHB depHow does PHB depletion affect mitochondrial activity to modulate aging? In wild type animals, prohibitin knockdown results in adult-onset mitochondrial over-proliferation in the intestine. This is plausibly due to the activation of a cellular retrograde response to mitochondrial deficiency, which has been shown in yeast to induce mitochondrial biogenesis . Howeverdaf-2, daf-7, isp-1 and gas-1 mutants. However, no ATP increase is observed in clk-1, eat-2 and nhr-49 mutants. In particular, eat-2 and nhr-49 mutants depleted of prohibitins show less ATP content. Nevertheless, ATP content does not provide information about the capacity of ATP generation, since differences could be due to reduced or increased ATP consumption. ATP flux measurements should be much more informative.Reduced fat and mitochondrial content upon PHB depletion correlate with extended longevity, however ATP content does not always correlate with extended longevity. Prohibitin knockdown significantly increases total ATP content in mev-1 mutants) or thermal stress (wild-type nematodes grown at 25°C). Therefore, mitohormesis is unlikely to mediate the effects of prohibitin elimination on aging.To date, there is no clear mechanistic explanation for the observed increased longevity of mitochondrial mutants. Mitochondria are major producers of ROS as a result of electron misplacement along the ETC. The free radical theory of aging postulates that ROS cause aging by inflicting cellular damage. In view of this theory, one possibility is that mitochondrial mutations might result in reduced rate of living and decreased ROS production . In othedaf-2(e1370) mutants. Although old daf-2(e1370) adults are more resistant to paraquat upon PHB depletion, paraquat treatment results in the same level of ROS production in daf-2(e1370) adult animals regardless of the presence or the absence of the PHB complex. Moreover, surpriseingly, L4 daf-2(e1370) larvae are clearly more sensitive to paraquat in the absence of the PHB complex, despite the final output being extraordinarily increased lifespan.Although a handful of data support the oxidative damage theory of aging, recent data from diverse organisms put the correlation between oxidative stress and aging into question . ProhibiCertainly, mitochondrial dysfunction upon prohibitin depletion results in more intricate physiological responses than increasing or reducing ROS levels and oxidative damage and unveiling the mechanisms implicated in mitochondrial-mediated life extension is crucial to understand how lifespan is regulated.daf-2 mutants [C. elegans mutants. Although depletion of PHB does not affect the lifespan of the glyoxylate mutant gei-7 (our unpublished observation) it would be interesting to see if gei-7 is required in the mutant genetic backgrounds where PHB elimination extends lifespan.A feasible mechanism involved in the lifespan extension conferred by prohibitin depletion is cellular signaling and the activation of alternative metabolic routes that will counter the mitochondrial defect. Long lived yeast mitochondrial mutants activate a retrograde signaling pathway that results in the activation of specific transcription factors that will shift metabolism away from the Krebs cycle towards the glyoxylate cycle. This metabolic shift has also been observed in dauer larvae and long-lived  mutants ,56. It idaf-2 mutations and extend lifespan independently of DAF-16 [It is generally believed that mitochondrial dysfunctions exert their effect on lifespan independently of the IIS pathway, because they show a synergistic effect with f DAF-16 ,40,47,50f DAF-16 ,50. MitoC. elegans aak-2/AMPK is partially required for the life extension of daf-2 and mitochondrial mutants [aak-2/AMPK kinase is required for the reduced fat content phenotype elicited by PHB depletion, and knockdown of prohibitins shortens the lifespan of aak-2 mutants. On the other hand, aak-2 mutants show increased mitochondrial biogenesis, which is further enhanced by PHB elimination [cep-1) mutant animals, however, it would be necessary to examine the requirement of p53 in the cases where PHB deficiency extends lifespan.Reduced AMP/ATP ratios activate the AMP-activated protein kinase (AMPK). The  mutants ,58. The mination . AMPK tamination  and it hmination . Prohibijnk-1 mutants and remains unchanged after prohibitin removal. On the other hand, overexpression of JNK-1 in wild-type animals reduces mitochondrial content and suppresses mitochondrial proliferation upon prohibitin depletion. phb gene knock-down shortens the lifespan of animals lacking JNK-1 and extends the lifespan of animals overexpressing JNK-1. Overexpression of JNK-1 promotes DAF-16/FOXO nuclear localization under conditions of stress [phb deficient animals [The mitogen activated protein kinase (MAPK) JNK-1, is also required for the reduction of fat content in animals lacking prohibitins. On the one hand mitochondrial content is higher in f stress . Similarf stress , result  animals .There is an urgent need to gain more insight into the transcriptional regulation and the signaling cascades involved in the regulation of lipid metabolism and mitochondrial biogenesis in response to PHB depletion in wild type animals and in the mutants where PHB depletion extends lifespan. This will allow delineating the molecular genetic pathways of PHB function as well as the molecular pathways involved in the crosstalk between mitochondria and cellular signaling networks in the regulation of aging.Mitochondrial mutations result in pleiotrophic effects and possibly, different mutations will affect the aging rate differently and in a tissue specific manner. Aging is a hormonally regulated process; therefore, it is essential to understand when and in which specific tissues PHB depletion exerts its beneficial effects on longevity. The striking longevity phenotype observed upon prohibitin elimination in diapause mutants is, to our knowledge, the first example of a genetic manipulation with context-dependent, opposing effects on aging Figure . Thus, p