Cells, Volume 8, Issue 4 (April 2019) – 94 articles

Inducible cyclization recombinase (Cre) transgenic mouse strains are powerful tools for cell lineage tracing and tissue-specific knockout experiments. However, low efficiency or leaky expression can be important pitfalls. Here, we compared the efficiency and specificity of two commonly used cholangiocyte-specific Cre drivers, the Opn-iCreER^T2 and Ck19-CreER^T drivers, using a tdTomato reporter strain. We found that Opn-iCreER^T2 triggered recombination of the tdTomato reporter in 99.9% of all cholangiocytes while Ck19-CreER^T only had 32% recombination efficiency after tamoxifen injection. In the absence of tamoxifen, recombination was also induced in 2% of cholangiocytes for the Opn-iCreER^T2 driver and in 13% for the Ck19-CreER^T driver. For both drivers, Cre recombination was highly specific for cholangiocytes since recombination was rare in other liver cell types. Toxic liver injury ectopically activated Opn-iCreER^T2 but not Ck19-CreER^T expression in hepatocytes. However, ectopic recombination in hepatocytes could be avoided by applying a three-day long wash-out period between tamoxifen treatment and toxin injection. Therefore, the Opn-iCreER^T2 driver is best suited for the generation of mutant bile ducts, while the Ck19-CreER^T driver has near absolute specificity for bile duct cells and is therefore favorable for lineage tracing experiments. Full article

Mitochondrion harbors its own DNA (mtDNA), which encodes many critical proteins for the assembly and activity of mitochondrial respiratory complexes. mtDNA is packed by many proteins to form a nucleoid that uniformly distributes within the mitochondrial matrix, which is essential for mitochondrial functions. Defects or mutations of mtDNA result in a range of diseases. Damaged mtDNA could be eliminated by mitophagy, and all paternal mtDNA are degraded by endonuclease G or mitophagy during fertilization. In this review, we describe the role and mechanism of mtDNA distribution and elimination. In particular, we focus on the regulation of paternal mtDNA elimination in the process of fertilization. Full article

Fascinating about classical major histocompatibility complex (MHC) molecules is their polymorphism. The present study is a review and discussion of the fish MHC situation. The basic pattern of MHC variation in fish is similar to mammals, with MHC class I versus class II, and polymorphic classical versus nonpolymorphic nonclassical. However, in many or all teleost fishes, important differences with mammalian or human MHC were observed: (1) The allelic/haplotype diversification levels of classical MHC class I tend to be much higher than in mammals and involve structural positions within but also outside the peptide binding groove; (2) Teleost fish classical MHC class I and class II loci are not linked. The present article summarizes previous studies that performed quantitative trait loci (QTL) analysis for mapping differences in teleost fish disease resistance, and discusses them from MHC point of view. Overall, those QTL studies suggest the possible importance of genomic regions including classical MHC class II and nonclassical MHC class I genes, whereas similar observations were not made for the genomic regions with the highly diversified classical MHC class I alleles. It must be concluded that despite decades of knowing MHC polymorphism in jawed vertebrate species including fish, firm conclusions (as opposed to appealing hypotheses) on the reasons for MHC polymorphism cannot be made, and that the types of polymorphism observed in fish may not be explained by disease-resistance models alone. Full article

Investigating adaptive potential and understanding the relative roles of selection and genetic drift in populations of endangered species are essential in conservation. Major histocompatibility complex (MHC) genes characterized by spectacular polymorphism and fitness association have become valuable adaptive markers. Herein we investigate the variation of all MHC class I and II genes across seven populations of an endangered bird, the crested ibis, of which all current individuals are offspring of only two pairs. We inferred seven multilocus haplotypes from linked alleles in the Core Region and revealed structural variation of the class II region that probably evolved through unequal crossing over. Based on the low polymorphism, structural variation, strong linkage, and extensive shared alleles, we applied the MHC haplotypes in population analysis. The genetic variation and population structure at MHC haplotypes are generally concordant with those expected from microsatellites, underlining the predominant role of genetic drift in shaping MHC variation in the bottlenecked populations. Nonetheless, some populations showed elevated differentiation at MHC, probably due to limited gene flow. The seven populations were significantly differentiated into three groups and some groups exhibited genetic monomorphism, which can be attributed to founder effects. We therefore propose various strategies for future conservation and management. Full article

Hepatitis C (HCV) is a major cause of liver disease, in which a third of individuals with chronic HCV infections may develop liver cirrhosis. In a chronic HCV infection, host immune factors along with the actions of HCV proteins that promote viral persistence and dysregulation of the immune system have an impact on immunopathogenesis of HCV-induced hepatitis. The genome of HCV encodes a single polyprotein, which is translated and processed into structural and nonstructural proteins. These HCV proteins are the target of the innate and adaptive immune system of the host. Retinoic acid-inducible gene-I (RIG-I)-like receptors and Toll-like receptors are the main pattern recognition receptors that recognize HCV pathogen-associated molecular patterns. This interaction results in a downstream cascade that generates antiviral cytokines including interferons. The cytolysis of HCV-infected hepatocytes is mediated by perforin and granzyme B secreted by cytotoxic T lymphocyte (CTL) and natural killer (NK) cells, whereas noncytolytic HCV clearance is mediated by interferon gamma (IFN-γ) secreted by CTL and NK cells. A host–HCV interaction determines whether the acute phase of an HCV infection will undergo complete resolution or progress to the development of viral persistence with a consequential progression to chronic HCV infection. Furthermore, these host–HCV interactions could pose a challenge to developing an HCV vaccine. This review will focus on the role of the innate and adaptive immunity in HCV infection, the failure of the immune response to clear an HCV infection, and the factors that promote viral persistence. Full article

Breast cancer (BC) is the leading cause of cancer-related mortality in women, only followed by lung cancer. Given the importance of BC in public health, it is essential to identify biomarkers to predict prognosis, predetermine drug resistance and provide treatment guidelines that include personalized targeted therapies. The Hedgehog (Hh) signaling pathway plays an essential role in embryonic development, tissue regeneration, and stem cell renewal. Several lines of evidence endorse the important role of canonical and non-canonical Hh signaling in BC. In this comprehensive review we discuss the role of Hh signaling in breast development and homeostasis and its contribution to tumorigenesis and progression of different subtypes of BC. We also examine the efficacy of agents targeting different components of the Hh pathway both in preclinical models and in clinical trials. The contribution of the Hh pathway in BC tumorigenesis and progression, its prognostic role, and its value as a therapeutic target vary according to the molecular, clinical, and histopathological characteristics of the BC patients. The evidence presented here highlights the relevance of the Hh signaling in BC, and suggest that this pathway is key for BC progression and metastasis. Full article

Chronic hepatitis C virus (HCV) infection causes generalized CD8⁺ T cell impairment, not limited to HCV-specific CD8⁺ T-cells. Liver-infiltrating monocyte-derived macrophages (MDMs) contribute to the local micro-environment and can interact with and influence cells routinely trafficking through the liver, including CD8⁺ T-cells. MDMs can be polarized into M1 (classically activated) and M2a, M2b, and M2c (alternatively activated) phenotypes that perform pro- and anti-inflammatory functions, respectively. The impact of chronic HCV infection on MDM subset functions is not known. Our results show that M1 cells generated from chronic HCV patients acquire M2 characteristics, such as increased CD86 expression and IL-10 secretion, compared to uninfected controls. In contrast, M2 subsets from HCV-infected individuals acquired M1-like features by secreting more IL-12 and IFN-γ. The severity of liver disease was also associated with altered macrophage subset differentiation. In co-cultures with autologous CD8⁺ T-cells from controls, M1 macrophages alone significantly increased CD8⁺ T cell IFN-γ expression in a cytokine-independent and cell-contact-dependent manner. However, M1 macrophages from HCV-infected individuals significantly decreased IFN-γ expression in CD8⁺ T-cells. Therefore, altered M1 macrophage differentiation in chronic HCV infection may contribute to observed CD8⁺ T-cell dysfunction. Understanding the immunological perturbations in chronic HCV infection will lead to the identification of therapeutic targets to restore immune function in HCV⁺ individuals, and aid in the mitigation of associated negative clinical outcomes. Full article

We previously established a method for a directed differentiation of human pluripotent stem cells into classical brown adipocytes (BA) by forming aggregates via massive floating culture in the presence of a specific cytokine cocktail. However, use of recombinant cytokines requires significant cost. Moreover, an enforced differentiation by exogenously added cytokines may amend skewed differentiation propensity of patient’s pluripotent stem cells, providing unsatisfactory disease models. Therefore, an exogenous cytokine-free method, where cytokines required for differentiation are provided in an auto/paracrine manner mimicking natural developmental process, is beneficial. Here we show that, if human pluripotent stem cells are cultured as size-controlled spheroids (100–120 µm radius, 2000–2500 cells/spheroid) in a mutually segregated manner with half-change of the medium every other day, they differentiate into classical BA via an authentic MYF5-positive myoblast route in the absence of exogenous cytokines. Differentiated BA exerted thermogenic activity in transplanted mice in response to beta-adrenergic receptor agonist stimuli. The cytokine-free differentiation method has further advantages in exploring BATokines, BA-derived physiologically active substances. Indeed, we have found that BA produces an unknown small (<1000 Da), highly hydrophilic molecule that augments insulin secretion from pancreatic beta cells. Our upgraded technique will contribute to an advancement of stem cell study for diverse purposes. Full article

Leptin is an important secretory protein that regulates the body’s intake and energy consumption, and the functions of the Hh signaling pathway related to white adipocyte browning are controversial. It has been reported that leptin plays a critical role in adipogenesis by regulating the Hh signaling pathway, but whether there is a functional relationship between leptin, the Hh signaling pathway, and adipocyte browning is not clear. In this research, mouse white pre-adipocytes were isolated to explore the influence of the Hh signal pathway and leptin during the process described above. This showed that leptin decreased high fat diet-induced obese mice body weight and inhibited the Hh signaling pathway, which suggested that leptin and the Hh signaling pathway have an important role in obesity. After activation of the Hh signaling pathway, significantly decreased browning fat-relative gene expression levels were recorded, whereas inhibition of the Hh signaling pathway significantly up-regulated the expression of these genes. Similarly, leptin also up-regulated the expression of these genes, and increased mitochondrial DNA content, but decreased the expression of Gli, the key transcription factors of the Hh signaling pathway. In short, the results show that leptin promotes white adipocyte browning through inhibiting the Hh signaling pathway. Overall, these results demonstrate that leptin serves as a potential intervention to decrease obesity by inhibiting the Hh signaling pathway. Full article

Small RNAs (sRNAs), a class of regulatory non-coding RNAs around 20~30-nt long, including small interfering RNAs (siRNAs) and microRNAs (miRNAs), are critical regulators of gene expression. Recently, accumulating evidence indicates that sRNAs can be transferred not only within cells and tissues of individual organisms, but also across different eukaryotic species, serving as a bond connecting the animal, plant, and microbial worlds. In this review, we summarize the results from recent studies on cross-kingdom sRNA communication. We not only review the horizontal transfer of sRNAs among animals, plants and microbes, but also discuss the mechanism of RNA interference (RNAi) signal transmission via cross-kingdom sRNAs. We also compare the advantages of host-induced gene silencing (HIGS) and spray-induced gene silencing (SIGS) technology and look forward to their applicable prospects in controlling fungal diseases. Full article

Despite recent efforts, prostate cancer (PCa) remains one of the most common cancers in men. Currently, there is no effective treatment for castration-resistant prostate cancer (CRPC). There is, therefore, an urgent need to identify new therapeutic targets. The Hippo pathway and its downstream effectors—the transcriptional co-activators, Yes-associated protein (YAP) and its paralog, transcriptional co-activator with PDZ-binding motif (TAZ)—are foremost regulators of stem cells and cancer biology. Defective Hippo pathway signaling and YAP/TAZ hyperactivation are common across various cancers. Here, we draw on insights learned from other types of cancers and review the latest advances linking the Hippo pathway and YAP/TAZ to PCa onset and progression. We examine the regulatory interaction between Hippo-YAP/TAZ and the androgen receptor (AR), as main regulators of PCa development, and how uncontrolled expression of YAP/TAZ drives castration resistance by inducing cellular stemness. Finally, we survey the potential therapeutic targeting of the Hippo pathway and YAP/TAZ to overcome PCa. Full article

Mesenchymal stem cells (MSCs) are promising tools for cell-based therapies due to their homing to injury sites, where they secrete bioactive factors such as cytokines, lipids, and nucleic acids, either free or conveyed within extracellular vesicles (EVs). Depending on the local environment, MSCs’ therapeutic value may be modulated, determining their fate and cell behavior. Inflammatory signals may induce critical changes on both the phenotype and secretory portfolio. Intriguingly, in animal models resembling joint diseases as osteoarthritis (OA), inflammatory priming enhanced the healing capacity of MSC-derived EVs. In this work, we selected miRNA reference genes (RGs) from the literature (let-7a-5p, miR-16-5p, miR-23a-3p, miR-26a-5p, miR-101-3p, miR-103a-3p, miR-221-3p, miR-423-5p, miR-425-5p, U6 snRNA), using EVs isolated from adipose-derived MSCs (ASCs) primed with IFNγ (iASCs). geNorm, NormFinder, BestKeeper, and ΔCt methods identified miR-26a-5p/16-5p as the most stable, while miR-103a-rp/425-5p performed poorly. Our results were validated on miRNAs involved in OA cartilage trophism. Only a proper normalization strategy reliably identified the differences between donors, a critical factor to empower the therapeutic value of future off-the-shelf MSC-EV isolates. In conclusion, the proposed pipeline increases the accuracy of MSC-EVs embedded miRNAs assessment, and help predicting donor variability for precision medicine approaches. Full article

Type 2 diabetes mellitus (T2DM) is a prevalent chronic metabolic disorder accompanied by high blood glucose, insulin resistance, and relative insulin deficiency. Endoplasmic reticulum (ER) stress induced by high glucose and free fatty acids has been suggested as one of the main causes of β-cell dysfunction and death in T2DM. Stem cell-derived insulin-secreting cells were recently suggested as a novel therapy for diabetes. In the present study, we demonstrate the therapeutic potential of tonsil-derived mesenchymal stem cells (TMSCs) to treat high-fat diet (HFD)-induced T2DM. To explore whether TMSC administration can alleviate T2DM, TMSCs were intraperitoneally injected in HFD-induced T2DM mice once every 2 weeks. TMSC injection markedly improved glucose tolerance and glucose-stimulated insulin secretion and prevented HFD-induced pancreatic β-cell hypertrophy and cell death. In addition, TMSC injection relieved the ER-stress response and preserved gene expression related to glucose sensing and insulin secretion. Moreover, administration of TMSC-derived conditioned medium induced similar therapeutic outcomes, suggesting paracrine effects. Finally, proteomic analysis revealed high secretion of insulin-like growth factor-binding protein 5 by TMSCs, and its expression was critical for the protective effects of TMSCs against HFD-induced glucose intolerance and ER-stress response in pancreatic islets. TMSC administration can alleviate HFD-induced-T2DM via preserving pancreatic islets and their function. These results provide novel evidence of TMSCs as an ER-stress modulator that may be a novel, alternative cell therapy for T2DM. Full article

Autistic Spectrum Disorder (ASD) is characterized by the impairment of socio-communicative skills and the presence of restricted and stereotyped behavior patterns. Recent researches have revealed the influence of mitochondrial physiology on the development of ASD. Several research groups have identified defects in respiratory complexes, coenzyme-Q10 deficiency, increased oxidative damage, decreased of superoxide dismutase (SOD2). A study on the influence of mitochondrial physiology on the development of ASD can provide new alternatives and challenges. That is why we set ourselves the general objective to initiate studies of mitochondrial physiology in Chilean children with ASD. A sample of oral mucosa was collected in a group of 12 children diagnosed with ASD and 12 children without ASD. In children with ASD, we found a significant increase in mitochondrial DNA levels. Likewise, in these children, an increase in the protein oxidation was observed. Finally, a downward trend in the expression of the HIGD2A and SOD2 genes was observed, while DRP1, FIS1, MFN1, MFN2, and OPA1 gene expression show an upward trend. The increment of mitochondrial DNA, high oxidative stress, and high expression of the MFN2 gene could help as a scanner of the mitochondrial function in children with ASD. Full article

Background: Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease with high mortality. The identification of specific HNSCC biomarkers will increase treatment efficacy and limit the toxicity of current therapeutic strategies. Long non-coding RNAs (lncRNAs) are promising biomarkers. Accordingly, here we investigate the biological role of ZFAS1 and its potential as a biomarker in HNSCC. Methods: The expression level of ZFAS1 in HNSCC cell lines was analyzed using qRT-PCR. Based on the HNSCC TCGA data, the ZFAS1 expression profile, clinicopathological features, and expression of correlated genes were analyzed in patient tissue samples. The selected genes were classified according to their biological function using the PANTHER tool. The interaction between lncRNA:miRNA and miRNA:mRNA was tested using available online tools. All statistical analyses were accomplished using GraphPad Prism 5. Results: The expression of ZFAS1 was up-regulated in the metastatic FaDu cell line relative to the less aggressive SCC-25 and SCC-040 and dysplastic DOK cell lines. The TCGA data indicated an up-regulation of ZFAS1 in HNSCCs compared to normal tissue samples. The ZFAS1 levels typically differed depending on the cancer stage and T-stage. Patients with a lower expression of ZFAS1 presented a slightly longer disease-free survival and overall survival. The analysis of genes associated with ZFAS1, as well its targets, indicate that they are linked with crucial cellular processes. In the group of patients with low expression of ZFAS1, we detected the up-regulation of suppressors and down-regulation of genes associated with epithelial-to-mesenchymal transition (EMT) process, metastases, and cancer-initiating cells. Moreover, the negative correlation between ZFAS1 and its host gene, ZNFX1, was observed. The analysis of interactions indicated that ZFAS1 has a binding sequence for miR-150-5p. The expression of ZFAS1 and miR-150-5p is negatively correlated in HNSCC patients. miR-150-5p can regulate the 3′UTR of EIF4E mRNA. In the group of patients with high expression of ZFAS1 and low expression of miR-150-5p, we detected an up-regulation of EIF4E. Conclusions: In HNSCC, ZFAS1 displays oncogenic properties, regulates important processes associated with EMT, cancer-initiating cells, and metastases, and might affect patients’ clinical outcomes. ZFAS1 likely regulates the cell phenotype through miR-150-5p and its downstream targets. Following further validation, ZFAS1 might prove a new and valuable biomarker. Full article

Reduced risk of HIV-1 infection correlated with antibody responses to the envelope variable 1 and 2 regions in the RV144 vaccine trial. To understand the relationship between antibody responses, V2 sequence, and structure, plasma samples (n = 16) from an early acute HIV-1 infection cohort from Thailand infected with CRF01_AE strain were analyzed for binding to V2 peptides by surface plasmon resonance. Five participants with a range of V2 binding responses at week 24 post-infection were further analyzed against a set of four overlapping V2 peptides that were designed based on envelope single-genome amplification. Antibody responses that were relatively consistent over the four segments of the V2 region or a focused response to the C-strand (residues 165–186) of the V2 region were observed. Viral escape in the V2 region resulted in significantly reduced antibody binding. Structural modeling indicated that the C-strand and the sites of viral variation were highly accessible in the open conformation of the HIV-1 Env trimer. V2 residues, 165–186 are preferentially targeted during acute infection. Residues 169–184 were also preferentially targeted by the protective immune response in the RV144 trial, thus emphasizing the importance of these residues for vaccine design. Full article

Parkinson’s disease (PD) is the second most common degenerative disorder. Although the disease was described more than 200 years ago, its pathogenetic mechanisms have not yet been fully described. In recent years, the discovery of the association between mutations of the GBA gene (encoding for the lysosomal enzyme glucocerebrosidase) and PD facilitated a better understating of this disorder. GBA mutations are the most common genetic risk factor of the disease. However, mutations of this gene can be found in different phenotypes, such as Gaucher’s disease (GD), PD, dementia with Lewy bodies (DLB) and rapid eye movements (REM) sleep behavior disorders (RBDs). Understanding the pathogenic role of this mutation and its different manifestations is crucial for geneticists and scientists to guide their research and to select proper cohorts of patients. Moreover, knowing the implications of the GBA mutation in the context of PD and the other associated phenotypes is also important for clinicians to properly counsel their patients and to implement their care. With the present review we aim to describe the genetic, clinical, and therapeutic features related to the mutation of the GBA gene. Full article

ATP-binding cassette subfamily G member 2 (ABCG2) is a physiologically important urate transporter. Accumulating evidence demonstrates that congenital dysfunction of ABCG2 is an important genetic risk factor in gout and hyperuricemia; recent studies suggest the clinical significance of both common and rare variants of ABCG2. However, the effects of rare variants of ABCG2 on the risk of such diseases are not fully understood. Here, using a cohort of 250 Czech individuals of European descent (68 primary hyperuricemia patients and 182 primary gout patients), we examined exonic non-synonymous variants of ABCG2. Based on the results of direct sequencing and database information, we experimentally characterized nine rare variants of ABCG2: R147W (rs372192400), T153M (rs753759474), F373C (rs752626614), T421A (rs199854112), T434M (rs769734146), S476P (not annotated), S572R (rs200894058), D620N (rs34783571), and a three-base deletion K360del (rs750972998). Functional analyses of these rare variants revealed a deficiency in the plasma membrane localization of R147W and S572R, lower levels of cellular proteins of T153M and F373C, and null urate uptake function of T434M and S476P. Accordingly, we newly identified six rare variants of ABCG2 that showed lower or null function. Our findings contribute to deepening the understanding of ABCG2-related gout/hyperuricemia risk and the biochemical characteristics of the ABCG2 protein. Full article

The cytoskeleton of animal cells is one of the most complicated and functionally versatile structures, involved in processes such as endocytosis, cell division, intra-cellular transport, motility, force transmission, reaction to external forces, adhesion and preservation, and adaptation of cell shape. These functions are mediated by three classical cytoskeletal filament types, as follows: Actin, microtubules, and intermediate filaments. The named filaments form a network that is highly structured and dynamic, responding to external and internal cues with a quick reorganization that is orchestrated on the time scale of minutes and has to be tightly regulated. Especially in brain tumors, the cytoskeleton plays an important role in spreading and migration of tumor cells. As the cytoskeletal organization and regulation is complex and many-faceted, this review aims to summarize the findings about cytoskeletal filament types, including substructures formed by them, such as lamellipodia, stress fibers, and interactions between intermediate filaments, microtubules and actin. Additionally, crucial regulatory aspects of the cytoskeletal filaments and the formed substructures are discussed and integrated into the concepts of cell motility. Even though little is known about the impact of cytoskeletal alterations on the progress of glioma, a final point discussed will be the impact of established cytoskeletal alterations in the cellular behavior and invasion of glioma. Full article

The nuclear lamina consists of a dense fibrous meshwork of nuclear lamins, Type V intermediate filaments, and is ~14 nm thick according to recent cryo-electron tomography studies. Recent advances in light microscopy have extended the resolution to a scale allowing for the fine structure of the lamina to be imaged in the context of the whole nucleus. We review quantitative approaches to analyze the imaging data of the nuclear lamina as acquired by structured illumination microscopy (SIM) and single molecule localization microscopy (SMLM), as well as the requisite cell preparation techniques. In particular, we discuss the application of steerable filters and graph-based methods to segment the structure of the four mammalian lamin isoforms (A, C, B1, and B2) and extract quantitative information. Full article

As key post-transcriptional regulators, microRNAs (miRNAs) play an indispensable role in skeletal muscle development. Our previous study suggested that miR-34b-5p and IGFBP2 could have a potential role in skeletal muscle growth. Our goal in this study is to explore the function and regulatory mechanism of miR-34b-5p and IGFBP2 in myogenesis. In this study, the dual-luciferase reporter assay and Western blot analysis showed that IGFBP2 is a direct target of miR-34b-5p. Flow cytometric analysis and EdU assay showed that miR-34b-5p could repress the cell cycle progression of myoblasts, and miR-34b-5p could promote the formation of myotubes by promoting the expression of MyHC. On the contrary, the overexpression of IGFBP2 significantly facilitated the proliferation of myoblasts and hampered the formation of myotubes. Together, our results indicate that miR-34b-5p could mediate the proliferation and differentiation of myoblasts by targeting IGFBP2. Full article

Xanthohumol (XN), a prenylated chalcone from hops, has been reported to exhibit a variety of health-beneficial effects. However, poor bioavailability may limit its application in the prevention and therapy of diseases. The objective of this study was to determine whether a micellar solubilization of xanthohumol could enhance the bioavailability and biological efficacy of xanthohumol in a Western-type diet (WTD) induced model of obesity, diabetes and non-alcoholic fatty liver disease (NAFLD). After 3 weeks feeding with WTD, XN was additionally applied per oral gavage as micellar solubilizate (s-XN) or native extract (n-XN) at a daily dose of 2.5 mg/kg body weight for a further 8 weeks. Control mice received vehicle only in addition to the WTD. WTD-induced body weight-gain and glucose intolerance were significantly inhibited by s-XN application. Furthermore, WTD-induced hepatic steatosis, pro-inflammatory gene expression (MCP-1 and CXCL1) and immune cell infiltration as well as activation of hepatic stellate cells (HSC) and expression of collagen alpha I were significantly reduced in the livers of s-XN-treated mice compared to WTD controls. In contrast, application of n-XN had no or only slight effects on the WTD-induced pathological effects. In line with this, plasma XN concentration ranged between 100–330 nmol/L in the s-XN group while XN was not detectable in the serum samples of n-XN-treated mice. In conclusion, micellar solubilization enhanced the bioavailability and beneficial effects of xanthohumol on different components of the metabolic syndrome including all pathological steps of NAFLD. Notably, this was achieved in a dose more than 10-fold lower than effective beneficial doses of native xanthohumol reported in previous in vivo studies. Full article

Myricetin is a naturally occurring flavonoid with protective effects against a variety of cancers. However, the molecular mechanism of myricetin against hepatocellular carcinoma (HCC) has still not been fully elucidated. Previous studies have indicated that YAP is essential for cancer initiation and progression. However, whether YAP contributes to the anti-cancer effects of myricetin remains unclear. Herein, we aimed to investigate the effect of myricetin on HCC, and identify the underlying mechanisms. We report that myricetin induced apoptosis and proliferation inhibition in HepG2 and Huh-7 cells. Myricetin inhibited expression of YAP by promoting its phosphorylation and subsequent degradation. Myricetin inhibited YAP expression by stimulating kinase activation of LATS1/2. Knockdown expression of LATS1/2 by shRNA attenuated myricetin-induced phosphorylation and degradation of YAP. Furthermore, myricetin sensitized HCC cells to cisplatin treatment through inhibiting YAP and its target genes, both in vitro and in vivo. The identification of the LATS1/2-YAP pathway as a target of myricetin may help with the design of novel strategies for human HCC prevention and therapy. Full article

The beta-3 adrenergic receptor (β₃-AR) is by far the least studied isotype of the beta-adrenergic sub-family. Despite its study being long hampered by the lack of suitable animal and cellular models and inter-species differences, a substantial body of literature on the subject has built up in the last three decades and the physiology of β₃-AR is unraveling quickly. As will become evident in this work, β₃-AR is emerging as an appealing target for novel pharmacological approaches in several clinical areas involving metabolic, cardiovascular, urinary, and ocular disease. In this review, we will discuss the most recent advances regarding β₃-AR signaling and function and summarize how these findings translate, or may do so, into current clinical practice highlighting β₃-AR’s great potential as a novel therapeutic target in a wide range of human conditions. Full article

Reactive oxygen species (ROS) induce nuclear factor erythroid 2–related factor 2 (Nrf2) activation as an adaptive defense mechanism, determining the synthesis of antioxidant molecules, including heme-oxygenase-1 (HO-1). HO-1 protects cells against oxidative injury, degrading free heme and inhibiting ROS production. HO-1 is highly expressed in macrophages during plaque growth. Macrophages are morpho-functionally heterogeneous, and the prevalence of a specific phenotype may influence the plaque fate. This heterogeneity has also been observed in monocyte-derived macrophages (MDMs), a model of macrophages infiltrating tissue. The study aims to assess oxidative stress status and Nrf2/HO-1 axis in MDM morphotypes obtained from healthy subjects and coronary artery disease (CAD) patients, in relation to coronary plaque features evaluated in vivo by optical coherence tomography (OCT). We found that MDMs of healthy subjects exhibited a lower oxidative stress status, lower Nrf2 and HO-1 levels as compared to CAD patients. High HO-1 levels in MDMs were associated with the presence of a higher macrophage content, a thinner fibrous cap, and a ruptured plaque with thrombus formation, detected by OCT analysis. These findings suggest the presence of a relationship between in vivo plaque characteristics and in vitro MDM profile, and may help to identify patients with rupture-prone coronary plaque. Full article

Fusion proteins incorporating the TLR5-ligand flagellin are currently undergoing clinical trials as vaccine candidates for many diseases. We recently reported a flagellin:allergen fusion protein containing the TLR5-ligand flagellin A (FlaA) from Listeria monocytogenes and the major birch pollen allergen Bet v 1 (rFlaA:Betv1) to prevent allergic sensitization in an experimental mouse model. This study analyzes the signaling pathways contributing to rFlaA:Betv1-mediated pro- and anti-inflammatory cytokine secretion and cell metabolism in myeloid dendritic cells (mDCs) in vitro. The influence of mammalian target of rapamycin (mTOR)-, NFκB-, and MAP kinase (MAPK)-signaling on cytokine secretion and metabolic activity of bone marrow (BM)-derived mDCs stimulated with rFlaA:Betv1 were investigated by pre-treatment with either mTOR- (rapamycin), NFκB- (dexamethason, BMS-345541, TPCA-1, triptolide, or BAY-11) or MAPK- (SP600125, U0126, or SB202190) inhibitors, respectively. rFlaA:Betv1-mediated IL-10 secretion as well as activation of mDC metabolism, rather than pro-inflammatory cytokine secretion, were inhibited by rapamycin. Inhibition of NFκB-signaling suppressed rFlaA:Betv1-induced IL-12, while inhibition of MAPK-signaling dose-dependently suppressed rFlaA:Betv1-induced IL-10 as well as pro-inflammatory IL-6 and TNF-α production. Notably, with the exception of a partial JNK-dependency, rFlaA:Betv1-mediated effects on mDC metabolism were mostly NFκB- and MAPK-independent. Therefore, MAPK-mediated activation of both NFκB- and mTOR-signaling likely is a key pathway for the production of pro- and anti-inflammatory cytokines by flagellin fusion protein vaccines. Full article

Lipid droplets (LDs) are cytosolic lipid storage organelles that are important for cellular lipid metabolism, energy homeostasis, cell signaling, and inflammation. Several bacterial, viral and protozoal pathogens exploit host LDs to promote infection, thus emphasizing the importance of LDs at the host–pathogen interface. In this review, we discuss the thus far reported relation between host LDs and bacterial pathogens including obligate and facultative intracellular bacteria, and extracellular bacteria. Although there is less evidence for a LD–extracellular bacterial interaction compared to interactions with intracellular bacteria, in this review, we attempt to compare the bacterial mechanisms that target LDs, the host signaling pathways involved and the utilization of LDs by these bacteria. Many intracellular bacteria employ unique mechanisms to target host LDs and potentially obtain nutrients and lipids for vacuolar biogenesis and/or immune evasion. However, extracellular bacteria utilize LDs to either promote host tissue damage or induce host death. We also identify several areas that require further investigation. Along with identifying LD interactions with bacteria besides the ones reported, the precise mechanisms of LD targeting and how LDs benefit pathogens should be explored for the bacteria discussed in the review. Elucidating LD–bacterial interactions promises critical insight into a novel host–pathogen interaction. Full article

The most common cystic fibrosis-causing mutation (F508del, present in ~85% of CF patients) leads to CFTR misfolding, which is recognized by the endoplasmic reticulum (ER) quality control (ERQC), resulting in ER retention and early degradation. It is known that CFTR exit from the ER is mediated by specific retention/sorting signals that include four arginine-framed tripeptide (AFT) retention motifs and a diacidic (DAD) exit code that controls the interaction with the COPII machinery. Here, we aim at obtaining a global view of the protein interactors that regulate CFTR exit from the ER. We used mass spectrometry-based interaction proteomics and bioinformatics analyses to identify and characterize proteins interacting with selected CFTR peptide motifs or full-length CFTR variants retained or bypassing these ERQC checkpoints. We conclude that these ERQC trafficking checkpoints rely on fundamental players in the secretory pathway, detecting key components of the protein folding machinery associated with the AFT recognition and of the trafficking machinery recognizing the diacidic code. Furthermore, a greater similarity in terms of interacting proteins is observed for variants sharing the same folding defect over those reaching the same cellular location, evidencing that folding status is dominant over ER escape in shaping the CFTR interactome. Full article

Functional studies of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hCMs) under different gravity conditions contribute to aerospace medical research. To study the effects of altered gravity on hCMs, we exposed them to acute hypergravity and microgravity phases in the presence and absence of the β-adrenoceptor isoprenalin (ISO), L-type Ca²⁺ channel (LTCC) agonist Bay-K8644, or LTCC blocker nifedipine, and monitored their beating rate (BR). These logistically demanding experiments were executed during the 66th Parabolic Flight Campaign of the European Space Agency. The hCM cultures were exposed to 31 alternating hypergravity, microgravity, and hypergravity phases, each lasting 20–22 s. During the parabolic flight experiment, BR and cell viability were monitored using the xCELLigence real-time cell analyzer Cardio Instrument^®. Corresponding experiments were performed on the ground (1 g), using an identical set-up. Our results showed that BR continuously increased during the parabolic flight, reaching a 40% maximal increase after 15 parabolas, compared with the pre-parabolic (1 g) phase. However, in the presence of the LTCC blocker nifedipine, no change in BR was observed, even after 31 parabolas. We surmise that the parabola-mediated increase in BR was induced by the LTCC blocker. Moreover, the increase in BR induced by ISO and Bay-K8644 during the pre-parabola phase was further elevated by 20% after 25 parabolas. This additional effect reflects the positive impact of the parabolas in the absence of both agonists. Our study suggests that acute alterations of gravity significantly increase the BR of hCMs via the LTCC. Full article

Small guanosine triphosphatases (GTPases) gathered in the Rat sarcoma (Ras) superfamily represent a large family of proteins involved in several key cellular mechanisms. Within the Ras superfamily, the Ras homolog (Rho) family is specialized in the regulation of actin cytoskeleton-based mechanisms. These proteins switch between an active and an inactive state, resulting in subsequent inhibiting or activating downstream signals, leading finally to regulation of actin-based processes. The On/Off status of Rho GTPases implicates two subsets of regulators: GEFs (guanine nucleotide exchange factors), which favor the active GTP (guanosine triphosphate) status of the GTPase and GAPs (GTPase activating proteins), which inhibit the GTPase by enhancing the GTP hydrolysis. In humans, the 20 identified Rho GTPases are regulated by over 70 GAP proteins suggesting a complex, but well-defined, spatio-temporal implication of these GAPs. Among the quite large number of RhoGAPs, we focus on p190RhoGAP, which is known as the main negative regulator of RhoA, but not exclusively. Two isoforms, p190A and p190B, are encoded by ARHGAP35 and ARHGAP5 genes, respectively. We describe here the function of each of these isoforms in physiological processes and sum up findings on their role in pathological conditions such as neurological disorders and cancers. Full article