Serum starvation enhances nonsense mutation readthrough Abstract Of all genetic mutations causing human disease, premature termination codons (PTCs) that result from splicing defaults, insertions, deletions, and point mutations comprise around 30%. From these mutations, around 11% are a substitution of a single nucleotide that change a codon into a premature termination codon. These types of mutations affect several million patients suffering from a large variety of genetic diseases, ranging from relatively common inheritable cancer syndromes to muscular dystrophy or very rare neuro-metabolic disorders. Over the past three decades, genetic and biochemical studies have revealed that certain antibiotics and other synthetic molecules can act as nonsense mutation readthrough-inducing drugs. These compounds bind a specific site on the rRNA and, as a result, the stop codon is misread and an amino acid (that may or may not differ from the wild-type amino acid) is inserted and translation occurs through the premature termination codon. This strategy has great therapeutic potential. Unfortunately, many readthrough agents are toxic and cannot be administered over the extended period usually required for the chronic treatment of genetic diseases. Furthermore, readthrough compounds only restore protein production in very few disease models and the readthrough levels are usually low, typically achieving no more than 5% of normal protein expression. Efforts have been made over the years to overcome these obstacles so that readthrough treatment can become clinically relevant. Here, we present the creation of a stable cell line system that constitutively expresses our dual-reporter vector harboring two cancer initiating nonsense mutations in the adenomatous polyposis coli (APC) gene. This system will be used as an improved screening method for isolation of new nonsense mutation readthrough inducers. Using these cell lines as well as colorectal cancer cell lines, we demonstrate that serum starvation enhances drug-induced readthrough activity, an observation which may prove beneficial in a therapeutic scenario that requires higher levels of the restored protein. Key messages Nonsense mutations affects millions of people worldwide. We have developed a nonsense mutation read-through screening tool. We find that serum starvation enhances antibiotic-induced nonsense mutation read-through. Our results suggest new strategies for enhancing nonsense mutation read-through that may have positive effects on a large number of patients.

Keratinocyte-specific ablation of Mcpip1 impairs skin integrity and promotes local and systemic inflammation Abstract MCPIP1 (Regnase-1, encoded by the ZC3H12A gene) regulates the mRNA stability of several inflammatory cytokines. Due to the critical role of this RNA endonuclease in the suppression of inflammation, Mcpip1 deficiency in mice leads to the development of postnatal multiorgan inflammation and premature death. Here, we generated mice with conditional deletion of Mcpip1 in the epidermis (Mcpip1EKO). Mcpip1 loss in keratinocytes resulted in the upregulated expression of transcripts encoding factors related to inflammation and keratinocyte differentiation, such as IL-36α/γ cytokines, S100a8/a9 antibacterial peptides, and Sprr2d/2h proteins. Upon aging, the Mcpip1EKO mice showed impaired skin integrity that led to the progressive development of spontaneous skin pathology and systemic inflammation. Furthermore, we found that the lack of epidermal Mcpip1 expression impaired the balance of keratinocyte proliferation and differentiation. Overall, we provide evidence that keratinocyte-specific Mcpip1 activity is crucial for the maintenance of skin integrity as well as for the prevention of excessive local and systemic inflammation. Key messages Loss of murine epidermal Mcpip1 upregulates transcripts related to inflammation and keratinocyte differentiation. Keratinocyte Mcpip1 function is essential to maintain the integrity of skin in adult mice. Ablation of Mcpip1 in mouse epidermis leads to the development of local and systemic inflammation.

Heterologous prime-boost vaccination against tuberculosis with recombinant Sendai virus and DNA vaccines Abstract In an earlier study, a novel Sendai virus–vectored anti-tuberculosis vaccine encoding Ag85A and Ag85B (SeV85AB) was constructed and shown to elicit antigen-specific T cell responses and protection against Mycobacterium tuberculosis (Mtb) infection in a murine model. In this study, we evaluate whether the immune responses induced by this novel vaccine might be elevated by a recombinant DNA vaccine expressing the same antigen in a heterologous prime-boost vaccination strategy. The results showed that both SeV85AB prime-DNA boost (SeV85AB-DNA) and DNA prime-SeV85AB boost (DNA-SeV85AB) vaccination strategies significantly enhanced the antigen-specific T cell responses induced by the separate vaccines. The SeV85AB-DNA immunization regimen induced higher levels of recall T cell responses after Mtb infection and conferred better immune protection compared with DNA-SeV85AB or a single immunization. Collectively, our study lends strong evidence that a DNA vaccine boost might be included in a novel SeV85AB immunization strategy designed to enhance the immune protection against Mtb. Key messages A heterologous prime-boost regimen with a novel recombinant SeV85AB and a DNA vaccine increase the T cell responses above those from a single vaccine. The heterologous prime-boost regimen provided protection against Mtb infection. The DNA vaccine might be included in a novel SeV85AB immunization strategy designed to enhance the immune protection against Mtb.

Dehydroepiandrosterone on metabolism and the cardiovascular system in the postmenopausal period Abstract Dehydroepiandrosterone (DHEA), mostly present as its sulfated ester (DHEA-S), is an anabolic hormone that naturally declines with age. Furthermore, it is the most abundant androgen and estrogen precursor in humans. Low plasma levels of DHEA have been strongly associated with obesity, insulin resistance, dyslipidemia, and high blood pressure, increasing the risk of cardiovascular disease. In this respect, DHEA could be regarded as a promising agent against metabolic syndrome (MetS) in postmenopausal women, since several age-related metabolic diseases are reported during aging. There are plenty of experimental evidences showing beneficial effects after DHEA therapy on carbohydrate and lipid metabolism, as well as cardiovascular health. However, its potential as a therapeutic agent appears to attract controversy, due to the lack of effects on some symptoms related to MetS. In this review, we examine the available literature regarding the impact of DHEA therapy on adiposity, glucose metabolism, and the cardiovascular system in the postmenopausal period. Both clinical studies and in vitro and in vivo experimental models were selected, and where possible, the main cellular mechanisms involved in DHEA therapy were discussed. Schematic representation showing some of the general effects observed after administration DHEA therapy on target tissues of energy metabolism and the cardiovascular system. ↑ represents an increase, ↓ represents a decrease, – represents a worsening and ↔ represents no change after DHEA therapy

FGA isoform as an indicator of targeted therapy for EGFR mutated lung adenocarcinoma Abstract Epidermal growth factor receptor (EGFR) gene is frequently mutated in non-small cell lung cancer (NSCLC), which can be targeted by EGFR tyrosine kinase inhibitors (TKIs). It is hard, however, to monitor the performance of EGFR-TKI therapy dynamically. Therefore, therapeutic indicators are urgently needed. Novel antibody microarray, containing 41,472 antibodies, was used for comprehensive analyzing of serum samples from 9 normal subjects and 9 EGFR mutated lung adenocarcinoma patients at three EGFR-TKI treatment time points, including before treatment (Baseline), partial response (PR) during treatment, and disease progression (PD) after resistance. Through microarray data analysis, five candidate antibodies were screened out for confirmation in serum samples and the verified one was utilized for candidate protein identification through immunoprecipitation-mass spectrometry strategy. A novel protein, isoform 2 of fibrinogen alpha chain (FGA2), was revealed and verified in the discovery sample set. Its performance as therapy indicator was further evaluated in another pre-validation sample set (n = 60). Our data confirmed that serum FGA2 level was correlated with EGFR-TKI response (p < 0.05). The expression and secretion of FGA2 in hepatocytes were inhibited by EGFR-TKI, partially explaining the downregulation of FGA2 in serum. Our results demonstrate that FGA2 is an indicator of targeted therapy for EGFR mutated lung adenocarcinoma. Key messages Antibody microarray was coupled with mass spectrometry for proteomics research. FGA2 was discovered as an indicator of EGFR-TKI targeted therapy. FGA2’s expression/secretion in hepatocytes was dramatically inhibited by EGFR-TKI.

Insights in the immunobiology of glioblastoma Abstract Glioblastoma, a grade IV astrocytoma, is considered as the most malignant intracranial tumor, characterized by poor prognosis and therapy resistance. Tumor heterogeneity that often leads to distinct functional phenotypes contributes to glioblastoma (GB) indispensable growth and aggressiveness. The complex interaction of neoplastic cells with tumor microenvironment (TME) along with the presence of cancer stem-like cells (CSCs) largely confers to extrinsic and intrinsic GB heterogeneity. Recent data indicate that glioma cells secrete a variety of soluble immunoregulatory factors to attract different cell types to TME including astrocytes, endothelial cells, circulating stem cells, and a range of immune cells. These further induce a local production of cytokines, chemokines, and growth factors which upon crosstalk with extracellular matrix (ECM) components reprogram immune cells to inflammatory or anti-inflammatory phenotypes and manipulate host’s immune response in favor of cancer growth and metastasis. Herein, we provide an overview of the immunobiologic factors that orchestrate the complex network of glioma cells and TME interactions in an effort to identify potential therapeutic targets for GB malignancy. Current therapeutic schemes and advances in targeting GB-TME crosstalk are further discussed. Key messages • Intrinsic and extrinsic tumor heterogeneity affects GB growth and aggressiveness. • GB cells secrete growth factors and chemoattractants to recruit immune cells to TME. • GAMs are a critical cell type in promoting GB growth. • GAMs change from pro-inflammatory, anti-tumor M1 phenotype to pro-tumorigenic M2. • Novel therapeutic agents target the crosstalk of neoplastic cells with TME.

Characterisation of transcription factor profiles in polycystic kidney disease (PKD): identification and validation of STAT3 and RUNX1 in the injury/repair response and PKD progression Abstract Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic renal disease, caused in the majority of the cases by a mutation in either the PKD1 or the PKD2 gene. ADPKD is characterised by a progressive increase in the number and size of cysts, together with fibrosis and distortion of the renal architecture, over the years. This is accompanied by alterations in a complex network of signalling pathways. However, the underlying molecular mechanisms are not well characterised. Previously, we defined the PKD Signature, a set of genes typically dysregulated in PKD across different disease models from a meta-analysis of expression profiles. Given the importance of transcription factors (TFs) in modulating disease, we focused in this paper on characterising TFs from the PKD Signature. Our results revealed that out of the 1515 genes in the PKD Signature, 92 were TFs with altered expression in PKD, and 32 of those were also implicated in tissue injury/repair mechanisms. Validating the dysregulation of these TFs by qPCR in independent PKD and injury models largely confirmed these findings. STAT3 and RUNX1 displayed the strongest activation in cystic kidneys, as demonstrated by chromatin immunoprecipitation (ChIP) followed by qPCR. Using immunohistochemistry, we showed a dramatic increase of expression after renal injury in mice and cystic renal tissue of mice and humans. Our results suggest a role for STAT3 and RUNX1 and their downstream targets in the aetiology of ADPKD and indicate that the meta-analysis approach is a viable strategy for new target discovery in PKD. Key messages We identified a list of transcription factors (TFs) commonly dysregulated in ADPKD. Out of the 92 TFs identified in the PKD Signature, 35% are also involved in injury/repair processes. STAT3 and RUNX1 are the most significantly dysregulated TFs after injury and during PKD progression. STAT3 and RUNX1 activity is increased in cystic compared to non-cystic mouse kidneys. Increased expression of STAT3 and RUNX1 is observed in the nuclei of renal epithelial cells, also in human ADPKD samples.

Traumatic brain injury-induced downregulation of Nrf2 activates inflammatory response and apoptotic cell death Abstract Recent studies from our group and others have demonstrated that oxidative stress, Ca2+ signaling, and neuroinflammation are major mechanisms contributing to post-traumatic neurodegeneration. The present study investigated the mechanisms of regulation of nuclear factor E2-related factor 2 (Nrf2) and its role in regulating antioxidant genes and oxidative stress-induced neuroinflammation and neurodegeneration following TBI. Nrf2 transcriptional system is the major regulator of endogenous defense mechanisms operating within the cells. Wild-type (Nrf2+/+) and Nrf2-deficient mice (Nrf2−/−) were subjected to 15 psi fluid percussion injury and demonstrated the regulatory role of Nrf2 in the expression antioxidant genes and oxidative stress, neuroinflammation, and cell death. Immunohistochemistry, q-RT-PCR, and western blotting techniques detected downregulation of Nrf2 and antioxidant proteins such as HO-1, GPx1, GSTm1, and NQO1 in mouse brain samples. Further, our study demonstrated that the downregulation of Nrf2 and antioxidant genes in TBI correlated with the induction of free radical-generating enzyme NADPH oxidase 1 and inducible nitric oxide synthase and their corresponding oxidative/nitrosative stress markers 4-hydroxynonenal and 3-nitrotyrosine. The decrease in Nrf2 with subsequent increase in oxidative stress markers led to the activation of MMP3/9, TGF-β1, and NF-kB that further led to neuroinflammation and apoptosis. The absence of Nrf2 function in mice resulted in exacerbated brain injury as shown by the increased oxidative stress markers, pro-inflammatory cytokines, and apoptosis markers at 24 h after TBI. In conclusion, this study could establish the significance of Nrf2 in transforming into a novel preventive approach against the pathophysiology of TBI. Key messages • Traumatic brain injury impairs Nrf2 signaling in mouse. • Nrf2-mediated activation of antioxidant genes are altered after TBI. • Impairment of Nrf2 signaling leads to oxidative stress. • TBI-induced downregulation of Nrf2 activates MMPs, TGF-β1, and NF-kB. • Nrf2 regulates neuroinflammation and apoptotic cell death in TB.

Correction to: Fis1 depletion in osteoarthritis impairs chondrocyte survival and peroxisomal and lysosomal function In Fig. 1C, the wrong si-control image was used by mistake.

Anti-oxidative effects of superoxide dismutase 3 on inflammatory diseases Abstract Free radicals and other oxidants are critical determinants of the cellular signaling pathways involved in the pathogenesis of several human diseases including inflammatory diseases. Numerous studies have demonstrated the protective effects of antioxidant enzymes during inflammation by elimination of free radicals. The superoxide dismutase (SOD), an antioxidant enzyme, plays an essential pathogenic role in the inflammatory diseases by not only catalyzing the conversion of the superoxide to hydrogen peroxide and oxygen but also affecting immune responses. There are three distinct isoforms of SOD, which distribute in different cellular compartments such as cytosolic SOD1, mitochondrial SOD2, and extracellular SOD3. Many studies have investigated the anti-oxidative effects of SOD3 in the inflammatory diseases. Herein, in this review, we focus on the current understanding of SOD3 as a therapeutic protein in inflammatory diseases such as skin, autoimmune, lung, and cardiovascular inflammatory diseases. Moreover, the mechanism(s) by which SOD3 modulates immune responses and signal initiation in the pathogenesis of the diseases will be further discussed.