Germ cell-specific eIF4E1b regulates maternal mRNA translation to ensure zygotic genome activation.

Translation of maternal mRNAs is detected before transcription of zygotic genes and is essential for mammalian embryo development. How certain maternal mRNAs are selected for translation instead of degradation and how this burst of translation affects zygotic genome activation remain unknown. Using gene-edited mice, we document that the oocyte-specific eukaryotic translation initiation factor 4E family member 1b (eIF4E1b) is the regulator of maternal mRNA expression that ensures subsequent reprogramming of the zygotic genome. In oocytes, eIF4E1b binds to transcripts encoding translation machinery proteins, chromatin remodelers, and reprogramming factors to promote their translation in zygotes and protect them from degradation. The protein products are thought to establish an open chromatin landscape in one-cell zygotes to enable transcription of genes required for cleavage stage development. Our results define a program for rapid resetting of the zygotic epigenome that is regulated by maternal mRNA expression and provide new insights into the mammalian maternal-to-zygotic transition.

eIF4E1b is a non-canonical eIF4E protecting maternal dormant mRNAs.

Maternal mRNAs are essential for protein synthesis during oogenesis and early embryogenesis. To adapt translation to specific needs during development, maternal mRNAs are translationally repressed by shortening the polyA tails. While mRNA deadenylation is associated with decapping and degradation in somatic cells, maternal mRNAs with short polyA tails are stable. Here we report that the germline-specific eIF4E paralog, eIF4E1b, is essential for zebrafish oogenesis. eIF4E1b localizes to P-bodies in zebrafish embryos and binds to mRNAs with reported short or no polyA tails, including histone mRNAs. Loss of eIF4E1b results in reduced histone mRNA levels in early gonads, consistent with a role in mRNA storage. Using mouse and human eIF4E1Bs (in vitro) and zebrafish eIF4E1b (in vivo), we show that unlike canonical eIF4Es, eIF4E1b does not interact with eIF4G to initiate translation. Instead, eIF4E1b interacts with the translational repressor eIF4ENIF1, which is required for eIF4E1b localization to P-bodies. Our study is consistent with an important role of eIF4E1b in regulating mRNA dormancy and provides new insights into fundamental post-transcriptional regulatory principles governing early vertebrate development.

Adenovirus E1B-55K controls SUMO-dependent degradation of antiviral cellular restriction factors.

IMPORTANCE: Human adenoviruses (HAdVs) generally cause mild and self-limiting diseases of the upper respiratory and gastrointestinal tracts but pose a serious risk to immunocompromised patients and children. Moreover, they are widely used as vectors for vaccines and vector-based gene therapy approaches. It is therefore vital to thoroughly characterize HAdV gene products and especially HAdV virulence factors. Early region 1B 55 kDa protein (E1B-55K) is a multifunctional HAdV-encoded oncoprotein involved in various viral and cellular pathways that promote viral replication and cell transformation. We analyzed the E1B-55K dependency of SUMOylation, a post-translational protein modification, in infected cells using quantitative proteomics. We found that HAdV increases overall cellular SUMOylation and that this increased SUMOylation can target antiviral cellular pathways that impact HAdV replication. Moreover, we showed that E1B-55K orchestrates the SUMO-dependent degradation of certain cellular antiviral factors. These results once more emphasize the key role of E1B-55K in the regulation of viral and cellular proteins in productive HAdV infections.

[Expression and significance of hypoxia-inducible factor 1α and Bcl-2/adenovirus E1B19kDa-interacting protein 3 in children with traumatic brain injury]. / ç¼ºæ°§è¯±å¯¼å› å­1α、Bcl-2/è ºç— æ¯’E1B19kDa相互作用蛋白3在儿童创伤性脑损伤中的表达及意义.

OBJECTIVES: To dynamically observe the changes in hypoxia-inducible factor 1α (HIF-1α) and Bcl-2/adenovirus E1B19kDa-interacting protein 3 (BNIP3) in children with traumatic brain injury (TBI) and evaluate their clinical value in predicting the severity and prognosis of pediatric TBI. METHODS: A prospective study included 47 children with moderate to severe TBI from January 2021 to July 2023, categorized into moderate (scores 9-12) and severe (scores 3-8) subgroups based on the Glasgow Coma Scale. A control group consisted of 30 children diagnosed and treated for inguinal hernia during the same period, with no underlying diseases. The levels of HIF-1α, BNIP3, autophagy-related protein Beclin-1, and S100B were compared among groups. The predictive value of HIF-1α, BNIP3, Beclin-1, and S100B for the severity and prognosis of TBI was assessed using receiver operating characteristic (ROC) curves. RESULTS: Serum levels of HIF-1α, BNIP3, Beclin-1, and S100B in the TBI group were higher than those in the control group (P<0.05). Among the TBI patients, the severe subgroup had higher levels of HIF-1α, BNIP3, Beclin-1, and S100B than the moderate subgroup (P<0.05). Correlation analysis showed that the serum levels of HIF-1α, BNIP3, Beclin-1, and S100B were negatively correlated with the Glasgow Coma Scale scores (P<0.05). After 7 days of treatment, serum levels of HIF-1α, BNIP3, Beclin-1, and S100B in both non-surgical and surgical TBI patients decreased compared to before treatment (P<0.05). ROC curve analysis indicated that the areas under the curve for predicting severe TBI based on serum levels of HIF-1α, BNIP3, Beclin-1, and S100B were 0.782, 0.835, 0.872, and 0.880, respectively (P<0.05), and for predicting poor prognosis of TBI were 0.749, 0.775, 0.814, and 0.751, respectively (P<0.05). CONCLUSIONS: Serum levels of HIF-1α, BNIP3, and Beclin-1 are significantly elevated in children with TBI, and their measurement can aid in the clinical assessment of the severity and prognosis of pediatric TBI.

Conserved E1B-55K SUMOylation in Different Human Adenovirus Species Is a Potent Regulator of Intracellular Localization.

Over the past decades, studies on the biology of human adenoviruses (HAdVs) mainly focused on the HAdV prototype species C type 5 (HAdV-C5) and revealed fundamental molecular insights into mechanisms of viral replication and viral cell transformation. Recently, other HAdV species are gaining more and more attention in the field. Reports on large E1B proteins (E1B-55K) from different HAdV species showed that these multifactorial proteins possess strikingly different features along with highly conserved functions. In this work, we identified potential SUMO-conjugation motifs (SCMs) in E1B-55K proteins from HAdV species A to F. Mutational inactivation of these SCMs demonstrated that HAdV E1B-55K proteins are SUMOylated at a single lysine residue that is highly conserved among HAdV species B to E. Moreover, we provide evidence that E1B-55K SUMOylation is a potent regulator of intracellular localization and p53-mediated transcription in most HAdV species. We also identified a lysine residue at position 101 (K101), which is unique to HAdV-C5 E1B-55K and specifically regulates its SUMOylation and nucleo-cytoplasmic shuttling. Our findings reveal important new aspects on HAdV E1B-55K proteins and suggest that different E1B-55K species possess conserved SCMs while their SUMOylation has divergent cellular effects during infection. IMPORTANCE E1B-55K is a multifunctional adenoviral protein and its functions are highly regulated by SUMOylation. Although functional consequences of SUMOylated HAdV-C5 E1B-55K are well studied, we lack information on the effects of SUMOylation on homologous E1B-55K proteins from other HAdV species. Here, we show that SUMOylation is a conserved posttranslational modification in most of the E1B-55K proteins, similar to what we know about HAdV-C5 E1B-55K. Moreover, we identify subcellular localization and regulation of p53-dependent transcription as highly conserved SUMOylation-regulated E1B-55K functions. Thus, our results highlight how HAdV proteins might have evolved in different HAdV species with conserved domains involved in virus replication and differing alternative functions and interactions with the host cell machinery. Future research will link these differences and similarities to the diverse pathogenicity and organ tropism of the different HAdV species.

E1B-55K Is a Phosphorylation-Dependent Transcriptional and Posttranscriptional Regulator of Viral Gene Expression in Human Adenovirus C5 Infection.

The multifunctional adenoviral E1B-55K phosphoprotein is a major regulator of viral replication and plays key roles in virus-mediated cell transformation. While much is known about its function in oncogenic cell transformation, the underlying features and exact mechanisms that implicate E1B-55K in the regulation of viral gene expression are less well understood. Therefore, this work aimed to unravel basic intranuclear principles of E1B-55K-regulated viral mRNA biogenesis using wild-type human adenovirus C5 (HAdV-C5) E1B-55K, a virus mutant with abrogated E1B-55K expression, and a mutant that expresses a phosphomimetic E1B-55K. By subnuclear fractionation, mRNA, DNA, and protein analyses as well as luciferase reporter assays, we show that (i) E1B-55K promotes the efficient release of viral late mRNAs from their site of synthesis in viral replication compartments (RCs) to the surrounding nucleoplasm, (ii) E1B-55K modulates the rate of viral gene transcription and splicing in RCs, (iii) E1B-55K participates in the temporal regulation of viral gene expression, (iv) E1B-55K can enhance or repress the expression of viral early and late promoters, and (v) the phosphorylation of E1B-55K regulates the temporal effect of the protein on each of these activities. Together, these data demonstrate that E1B-55K is a phosphorylation-dependent transcriptional and posttranscriptional regulator of viral genes during HAdV-C5 infection. IMPORTANCE Human adenoviruses are useful models to study basic aspects of gene expression and splicing. Moreover, they are one of the most commonly used viral vectors for clinical applications. However, key aspects of the activities of essential viral proteins that are commonly modified in adenoviral vectors have not been fully described. A prominent example is the multifunctional adenoviral oncoprotein E1B-55K that is known to promote efficient viral genome replication and expression while simultaneously repressing host gene expression and antiviral host responses. Our study combined different quantitative methods to study how E1B-55K promotes viral mRNA biogenesis. The data presented here propose a novel role for E1B-55K as a phosphorylation-dependent transcriptional and posttranscriptional regulator of viral genes.

LncRNA HOX transcript antisense RNA mitigates cardiac function injury in chronic heart failure via regulating microRNA-30a-5p to target KDM3A.

Long noncoding RNA HOX transcript antisense RNA (HOTAIR) has been studied in multiple diseases, but the role of HOTAIR on chronic heart failure (CHF) through the regulation of microRNA (miR)-30a-5p and lysine-specific demethylase 3A (KDM3A) remains unexplored. This research aims to probe the effects of HOTAIR on CHF progression via modulating miR-30a-5p to target KDM3A. CHF mouse model was established by intraperitoneal injection of doxorubicin. The CHF mice were then injected with high-expressed HOTAIR, miR-30a-5p or KDM3A adenovirus vectors to determine the cardiac function, oxidative stress, inflammatory response, pathological change and cardiomyocyte apoptosis. HOTAIR, miR-30a-5p, KDM3A and Bcl-2/adenovirus E1B 19kDa interacting protein 3 (BNIP3) expression in CHF mice was detected. The binding relations among HOTAIR, miR-30a-5p and KDM3A were validated. HOTAIR and KDM3A were depleted, while miR-30a-5p was augmented in CHF mice. The elevated HOTAIR or KDM3A or could improve cardiac function, mitigate oxidative stress and pathological change, reduce inflammatory factor levels and cardiomyocyte apoptosis, while the increased miR-30a-5p exerted opposite effects. The miR-30a-5p elevation could reverse the effects of enriched HOTAIR, while BNIP3 reduction abrogated the effects of KDM3A on CHF. HOTAIR sponged miR-30a-5p that targeted KDM3A. HOTAIR improves cardiac injury in CHF via modulating miR-30a-5p to target KDM3A. This study provides novel therapeutic strategies for CHF treatment.

E1B-55K-Mediated Regulation of RNF4 SUMO-Targeted Ubiquitin Ligase Promotes Human Adenovirus Gene Expression.

Human adenovirus (HAdV) E1B-55K is a multifunctional regulator of productive viral replication and oncogenic transformation in nonpermissive mammalian cells. These functions depend on E1B-55K's posttranslational modification with the SUMO protein and its binding to HAdV E4orf6. Both early viral proteins recruit specific host factors to form an E3 ubiquitin ligase complex that targets antiviral host substrates for proteasomal degradation. Recently, we reported that the PML-NB-associated factor Daxx represses efficient HAdV productive infection and is proteasomally degraded via a SUMO-E1B-55K-dependent, E4orf6-independent pathway, the details of which remained to be established. RNF4, a cellular SUMO-targeted ubiquitin ligase (STUbL), induces ubiquitinylation of specific SUMOylated proteins and plays an essential role during DNA repair. Here, we show that E1B-55K recruits RNF4 to the insoluble nuclear matrix fraction of the infected cell to support RNF4/Daxx association, promoting Daxx PTM and thus inhibiting this antiviral factor. Removing RNF4 from infected cells using RNA interference resulted in blocking the proper establishment of viral replication centers and significantly diminished viral gene expression. These results provide a model for how HAdV antagonize the antiviral host responses by exploiting the functional capacity of cellular STUbLs. Thus, RNF4 and its STUbL function represent a positive factor during lytic infection and a novel candidate for future therapeutic antiviral intervention strategies.IMPORTANCE Daxx is a PML-NB-associated transcription factor that was recently shown to repress efficient HAdV productive infection. To counteract this antiviral measurement during infection, Daxx is degraded via a novel pathway including viral E1B-55K and host proteasomes. This virus-mediated degradation is independent of the classical HAdV E3 ubiquitin ligase complex, which is essential during viral infection to target other host antiviral substrates. To maintain a productive viral life cycle, HAdV E1B-55K early viral protein inhibits the chromatin-remodeling factor Daxx in a SUMO-dependent manner. In addition, viral E1B-55K protein recruits the STUbL RNF4 and sequesters it into the insoluble fraction of the infected cell. E1B-55K promotes complex formation between RNF4- and E1B-55K-targeted Daxx protein, supporting Daxx posttranslational modification prior to functional inhibition. Hence, RNF4 represents a novel host factor that is beneficial for HAdV gene expression by supporting Daxx counteraction. In this regard, RNF4 and other STUbL proteins might represent novel targets for therapeutic intervention.

Degradation of a Novel DNA Damage Response Protein, Tankyrase 1 Binding Protein 1, following Adenovirus Infection.

Infection by most DNA viruses activates a cellular DNA damage response (DDR), which may be to the detriment or advantage of the virus. In the case of adenoviruses, they neutralize antiviral effects of DDR activation by targeting a number of proteins for rapid proteasome-mediated degradation. We have now identified a novel DDR protein, tankyrase 1 binding protein 1 (TNKS1BP1) (also known as Tab182), which is degraded during infection by adenovirus serotype 5 and adenovirus serotype 12. In both cases, degradation requires the action of the early region 1B55K (E1B55K) and early region 4 open reading frame 6 (E4orf6) viral proteins and is mediated through the proteasome by the action of cullin-based cellular E3 ligases. The degradation of Tab182 appears to be serotype specific, as the protein remains relatively stable following infection with adenovirus serotypes 4, 7, 9, and 11. We have gone on to confirm that Tab182 is an integral component of the CNOT complex, which has transcriptional regulatory, deadenylation, and E3 ligase activities. The levels of at least 2 other members of the complex (CNOT3 and CNOT7) are also reduced during adenovirus infection, whereas the levels of CNOT4 and CNOT1 remain stable. The depletion of Tab182 with small interfering RNA (siRNA) enhances the expression of early region 1A proteins (E1As) to a limited extent during adenovirus infection, but the depletion of CNOT1 is particularly advantageous to the virus and results in a marked increase in the expression of adenovirus early proteins. In addition, the depletion of Tab182 and CNOT1 results in a limited increase in the viral DNA level during infection. We conclude that the cellular CNOT complex is a previously unidentified major target for adenoviruses during infection.IMPORTANCE Adenoviruses target a number of cellular proteins involved in the DNA damage response for rapid degradation. We have now shown that Tab182, which we have confirmed to be an integral component of the mammalian CNOT complex, is degraded following infection by adenovirus serotypes 5 and 12. This requires the viral E1B55K and E4orf6 proteins and is mediated by cullin-based E3 ligases and the proteasome. In addition to Tab182, the levels of other CNOT proteins are also reduced during adenovirus infection. Thus, CNOT3 and CNOT7, for example, are degraded, whereas CNOT4 and CNOT1 are not. The siRNA-mediated depletion of components of the complex enhances the expression of adenovirus early proteins and increases the concentration of viral DNA produced during infection. This study highlights a novel protein complex, CNOT, which is targeted for adenovirus-mediated protein degradation. To our knowledge, this is the first time that the CNOT complex has been identified as an adenoviral target.

Synergistic effect of HIF-1α and FoxO3a trigger cardiomyocyte apoptosis under hyperglycemic ischemia condition.

Cardiomyocyte death is an important pathogenic feature of ischemia and heart failure. Through this study, we showed the synergistic role of HIF-1α and FoxO3a in cardiomyocyte apoptosis subjected to hypoxia plus elevated glucose levels. Using gene specific small interfering RNAs (siRNA), semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR), Western blot, immunofluorescence, nuclear and cytosolic localization and TUNEL assay techniques, we determined that combined function of HIF-1α and FoxO3a under high glucose plus hypoxia condition lead to enhanced expression of BNIP3 inducing cardiomyocyte death. Our results highlighted the importance of the synergistic role of HIF-1α and FoxO3a in cardiomyocyte death which may add insight into therapeutic approaches to pathophysiology associated with ischemic diabetic cardiomyopathies.

Efficient Transformation of Primary Human Mesenchymal Stromal Cells by Adenovirus Early Region 1 Oncogenes.

Previous observations that human amniotic fluid cells (AFC) can be transformed by human adenovirus type 5 (HAdV-5) E1A/E1B oncogenes prompted us to identify the target cells in the AFC population that are susceptible to transformation. Our results demonstrate that one cell type corresponding to mesenchymal stem/stroma cells (hMSCs) can be reproducibly transformed by HAdV-5 E1A/E1B oncogenes as efficiently as primary rodent cultures. HAdV-5 E1-transformed hMSCs exhibit all properties commonly associated with a high grade of oncogenic transformation, including enhanced cell proliferation, anchorage-independent growth, increased growth rate, and high telomerase activity as well as numerical and structural chromosomal aberrations. These data confirm previous work showing that HAdV preferentially transforms cells of mesenchymal origin in rodents. More importantly, they demonstrate for the first time that human cells with stem cell characteristics can be completely transformed by HAdV oncogenes in tissue culture with high efficiency. Our findings strongly support the hypothesis that undifferentiated progenitor cells or cells with stem cell-like properties are highly susceptible targets for HAdV-mediated cell transformation and suggest that virus-associated tumors in humans may originate, at least in part, from infections of these cell types. We expect that primary hMSCs will replace the primary rodent cultures in HAdV viral transformation studies and are confident that these investigations will continue to uncover general principles of viral oncogenesis that can be extended to human DNA tumor viruses as well. IMPORTANCE: It is generally believed that transformation of primary human cells with HAdV-5 E1 oncogenes is very inefficient. However, a few cell lines have been successfully transformed with HAdV-5 E1A and E1B, indicating that there is a certain cell type which is susceptible to HAdV-mediated transformation. Interestingly, all those cell lines have been derived from human embryonic tissue, albeit the exact cell type is not known yet. We show for the first time the successful transformation of primary human mesenchymal stromal cells (hMSCs) by HAdV-5 E1A and E1B. Further, we show upon HAdV-5 E1A and E1B expression that these primary progenitor cells exhibit features of tumor cells and can no longer be differentiated into the adipogenic, chondrogenic, or osteogenic lineage. Hence, primary hMSCs represent a robust and novel model system to elucidate the underlying molecular mechanisms of adenovirus-mediated transformation of multipotent human progenitor cells.

Modelling heterogeneity in viral-tumour dynamics: The effects of gene-attenuation on viral characteristics.

The use of viruses as a cancer treatment is becoming increasingly more robust; however, there is still a long way to go before a completely successful treatment is formulated. One major challenge in the field is to select which virus, out of a burgeoning number of oncolytic viruses and engineered derivatives, can maximise both treatment spread and anticancer cytotoxicity. To assist in solving this problem, an in-depth understanding of the virus-tumour interaction is crucial. In this article, we present a novel integro-differential system with distributed delays embodying the dynamics of an oncolytic adenovirus with a fixed population of tumour cells in vitro, allowing for heterogeneity to exist in the virus and cell populations. The parameters of the model are optimised in a hierarchical manner, the purpose of which is not to obtain a perfect representation of the data. Instead, we place our parameter values in the correct region of the parameter space. Due to the sparse nature of the data it is not possible to obtain the parameter values with any certainty, but rather we demonstrate the suitability of the model. Using our model we quantify how modifications to the viral genome alter the viral characteristics, specifically how the attenuation of the E1B 19 and E1B 55 gene affect the system performance, and identify the dominant processes altered by the mutations. From our analysis, we conclude that the deletion of the E1B 55 gene significantly reduces the replication rate of the virus in comparison to the deletion of the E1B 19 gene. We also found that the deletion of both the E1B 19 and E1B 55 genes resulted in a long delay in the average replication start time of the virus. This leads us to propose the use of E1B 19 gene-attenuated adenovirus for cancer therapy, as opposed to E1B 55 gene-attenuated adenoviruses.

Oncolytic E1B 55KDa-deleted adenovirus replication is independent of p53 levels in cancer cells.

Oncolytic adenoviruses represent a new approach for cancer therapy due to its tumor specificity. E1B 55kDa-deleted adenovirus type 5 (Ad5dlE1B 55kDa) is a promising therapeutic agent that can selectively replicate in and lyse p53 defective cancer cells. However, the overall efficacy has shown varying degrees of success with raised doubts about the correlation between p53 status and E1B-deleted adenovirus replication ability. In this study, we investigated the relationship between the efficiency of Ad5dlE1B 55kDa replication and p53 levels in cancer cells. Five transient p53 expression vectors were engineered to expresses different p53 levels in transfected cells. Then, the effect of the variable p53 levels and cellular backgrounds on the replication efficiency of oncolytic Ad5dlE1B 55kDa was evaluated in H1299 and HeLa cell lines. We found that the replication efficiency of these oncolytic viruses is dependent on the status, but not the expression levels, of p53. Ad5dlE1B 55kDa was shown to have selective replication activity in H1299 cells (p53-null) and decreased viral replication in HeLa cells (p53-positive), relative to the wild-type adenovirus in both cell lines. Our findings suggest that there is a relation between the E1B-deleted adenovirus replication and the presence as well as the activity of p53, independent of its quantity.

Strategies to optimize capsid protein expression and single-stranded DNA formation of adeno-associated virus in Saccharomyces cerevisiae.

AIMS: Adeno-associated virus type 2 (AAV) is a nonpathogenic parvovirus that is a promising tool for gene therapy. We aimed to construct plasmids for optimal expression and assembly of capsid proteins and evaluate adenovirus (Ad) protein effect on AAV single-stranded DNA (ssDNA) formation in Saccharomyces cerevisiae. METHODS AND RESULTS: Yeast expression plasmids have been developed in which the transcription of AAV capsid proteins (VP1,2,3) is driven by the constitutive ADH1 promoter or galactose-inducible promoters. Optimal VP1,2,3 expression was obtained from GAL1/10 bidirectional promoter. Moreover, we demonstrated that AAP is expressed in yeast and virus-like particles (VLPs) assembled inside the cell. Finally, the expression of two Ad proteins, E4orf6 and E1b55k, had no effect on AAV ssDNA formation. CONCLUSIONS: This study confirms that yeast is able to form AAV VLPs; however, capsid assembly and ssDNA formation are less efficient in yeast than in human cells. Moreover, the expression of Ad proteins did not affect AAV ssDNA formation. SIGNIFICANCE AND IMPACT OF THE STUDY: New manufacturing strategies for AAV-based gene therapy vectors (rAAV) are needed to reduce costs and time of production. Our study explores the feasibility of yeast as alternative system for rAAV production.

MicroRNA-210 alleviates oxidative stress-associated cardiomyocyte apoptosis by regulating BNIP3.

Oxidative stress-induced myocardial apoptosis and necrosis are involved in ischemia/reperfusion (I/R) injury. This study was performed to investigate microRNA (miR)-210's role in oxidative stress-related myocardial damage. The expression of miR-210 was upregulated in myocardial tissues of I/R rats, while that of Bcl-2 adenovirus E1B 19kDa-interacting protein 3 (BNIP3) was downregulated. To simulate in vivo oxidative stress, H9c2 cells were treated with H2O2 for 48 h. MiR-210 level was increased upon H2O2 stimulation, peaked at 8 h, and then decreased. An opposite expression pattern of BNIP3 was observed. BNIP3 was demonstrated as a direct target of miR-210 via luciferase reporter assay. H2O2-induced cell apoptosis was attenuated by miR-210 mimics, whereas aggravated by miR-210 inhibitor. MiR-210 knockdown-induced cell apoptosis in presence of H2O2 was attenuated by BNIP3 siRNA. Our work demonstrates that miR-210 plays a protective role in H2O2-induced cardiomyocyte apoptosis at least by regulating the pro-apoptotic BNIP3.

Regulation of autophagy by stress-responsive transcription factors.

Autophagy is an evolutionarily conserved process that promotes the lysosomal degradation of intracellular components including organelles and portions of the cytoplasm. Besides operating as a quality control mechanism in steady-state conditions, autophagy is upregulated in response to a variety of homeostatic perturbations. In this setting, autophagy mediates prominent cytoprotective effects as it sustains energetic homeostasis and contributes to the removal of cytotoxic stimuli, thus orchestrating a cell-wide, multipronged adaptive response to stress. In line with the critical role of autophagy in health and disease, defects in the autophagic machinery as well as in autophagy-regulatory signaling pathways have been associated with multiple human pathologies, including neurodegenerative disorders, autoimmune conditions and cancer. Accumulating evidence indicates that the autophagic response to stress may proceed in two phases. Thus, a rapid increase in the autophagic flux, which occurs within minutes or hours of exposure to stressful conditions and is entirely mediated by post-translational protein modifications, is generally followed by a delayed and protracted autophagic response that relies on the activation of specific transcriptional programs. Stress-responsive transcription factors including p53, NF-κB and STAT3 have recently been shown to play a major role in the regulation of both these phases of the autophagic response. Here, we will discuss the molecular mechanisms whereby autophagy is orchestrated by stress-responsive transcription factors.

Chronic hypoxia alters mitochondrial composition in human macrophages.

Hypoxia inducible factors (HIFs) are important mediators of the cellular adaptive response during acute hypoxia. The role of HIF-1 and HIF-2 during prolonged periods of hypoxia, i.e. chronic hypoxia is less defined. Therefore, we used human THP-1 macrophages with a knockdown of either HIF-1α, HIF-2α, or both HIFα-subunits, incubated them for several days under hypoxia (1% O2), and analyzed responses to hypoxia using 2D-DIGE coupled to MS/MS-analysis. Chronic hypoxia was defined as a time point when the early but transient accumulation of HIFα-subunits and mRNA expression of classical HIF target genes returned towards basal levels, with a new steady state that was constant from 72h onwards. From roughly 800 spots, that were regulated comparing normoxia to chronic hypoxia, about 100 proteins were unambiguously assigned during MS/MS-analysis. Interestingly, a number of glycolytic proteins were up-regulated, while a number of inner mitochondrial membrane proteins were down-regulated independently of HIF-1α or HIF-2α. Chronic hypoxic conditions depleted the mitochondrial mass by autophagy, which occurred independently of HIF proteins. Macrophages tolerate periods of chronic hypoxia very well and adaptive responses occur, at least in part, independently of HIF-1α and/or HIF-2α and comprise mitophagy as a pathway of particular importance.

Establishment of a mouse melanoma model system for the efficient infection and replication of human adenovirus type 5-based oncolytic virus.

Due to poor adenoviral infectivity and replication in mouse tumor cell types compared with human tumor cell types, use of human-type adenoviral vectors in mouse animal model systems was limited. Here, we demonstrate enhanced infectivity and productive replication of adenovirus in mouse melanoma cells following introduction of both the Coxsackievirus and adenovirus receptor (CAR) and E1B-55K genes. Introduction of CAR into B16BL6 or B16F10 cells increased the infectivity of GFP-expressing adenovirus; however, viral replication was unaffected. We demonstrated a dramatic increase of adenoviral replication (up to 100-fold) in mouse cells via E1B-55K expression and subsequent viral spreading in mouse tissue. These results reveal for the first time that human adenovirus type 5 (Ad5)-based oncolytic virus can be applied to immunocompetent mouse with the introduction of CAR and E1B-55K to syngenic mouse cell line.

Genetic tools for multicolor imaging in zebrafish larvae.

Zebrafish gain increasing popularity as animal model for the study of various aspects of modern cell biology as well as model organism for human diseases. This is owed to the fact that zebrafish represent a cost effective and versatile in vivo alternative to in vitro cell culture systems and to invertebrate- and classic rodent models as they combine many strengths of each of these systems. Zebrafish with their small size and rapid embryonic development can be maintained at relatively low costs with females giving rise to more than hundred eggs per week, thus allowing for the efficient analysis of cellular and subcellular processes. Moreover, such analysis can be performed using sophisticated imaging techniques, and transgenic zebrafish lines that express any gene of interest can be generated relatively easily. Among other advantages, the powerful genetic tractability of this vertebrate model organism combined with the in vivo multicolor imaging options make zebrafish unique for addressing questions of in vivo cell biology in vertebrates. In this article we outline these options by reviewing recent advances in zebrafish genetics with focus on the molecular tools and methods that are currently established for the use of zebrafish for multicolor imaging.

Benzo[a]pyrene exposure causes exonal switch resulting in reduced surface CD5 expression in an AHR-dependent manner.

The function of CD5 protein in T cells is well documented, but regulation of its surface-level expression has yet to be fully understood. However, variation in its surface expression is associated with various immunopathological conditions and haematological malignancies. Briefly, expression of an alternate exon E1B of a human endogenous retroviruses (HERV) origin directly downregulates the conventional transcript variant (E1A), as its expression leads to the retention of the resultant protein at the intracellular level (cCD5). A separate promoter governs the expression of E1B and may be influenced by different transcription factors. Hence, we performed in silico transcription factor binding site (TFBS) analysis of the 3 kb upstream region from TSS of exon E1B and found five putative DREs (Dioxin Response elements) with good similarity scores. Further, we observed the upregulation in E1B expression after the exposure of BaP (a dioxin) and the reduction of E1A expression and their respective protein, i.e. sCD5 and cCD5. The binding of AHR at the predicted DRE sites was confirmed by ChIP qPCR and AHR specific inhibitor and gene silencing studies suggested the involvement of AHR in exonal switch. This study indicates that the polycyclic aromatic hydrocarbon decreases the sCD5 expression by upregulating alternative exon expression, which may adversely affect the overall T cell functions.