Exogenous Poly Research Articles

Ultrasound-Activated Probiotics Vesicles Coating for Titanium Implant Infections Through Bacterial Cuproptosis-Like Death and Immunoregulation.

Implant-associated infections (IAIs) are the main cause of prosthetic implant failure. Bacterial biofilms prevent antibiotic penetration, and the unique metabolic conditions in hypoxic biofilm microenvironment may limit the efficacy of conventional antibiotic treatment. Escaping survival bacteria may not be continually eradicated, resulting in the recurrence of IAIs. Herein, a sonosensitive metal-organic framework of Cu-TCPP (tetrakis(4-carboxyphenyl) porphyrin) nanosheets and tinidazole doped probiotic-derived membrane vesicles (OMVs) with high-penetration sonodynamic therapy (SDT), bacterial metabolic state interference, and bacterial cuproptosis-like death to eradicate IAIs is proposed. The Cu-TCPP can convert O2 to toxic 1O2 through SDT in the normoxic conditions, enhancing the hypoxic microenvironment and activating the antibacterial activity of tinidazole. The released Cu(II) under ultrasound can be converted to Cu(I) by exogenous poly(tannic acid) (pTA) and endogenous glutathione. The disruption of the bacterial membrane by SDT can enhance the Cu(I) transporter activity. Transcriptomics indicate that the SDT-enhanced Cu(I) overload and hypoxia-activated therapy hinder the tricarboxylic acid cycle (TCA), leading to bacterial cuproptosis-like death. Moreover, the OMVs-activated therapy can polarize macrophages to a M2-like phenotype and facilitate bone repair. The sonodynamic biofilm microenvironment modulation strategy, whereby the hypoxia-enhanced microenvironment is potentiated to synergize SDT with OMVs-activated therapy, provides an effective strategy for antibacterial and osteogenesis performance.

Toll-like Receptor 3 in the Hybrid Yellow Catfish (Pelteobagrus fulvidraco ♀ × P. vachelli ♂): Protein Structure, Evolution and Immune Response to Exogenous Aeromonas hydrophila and Poly (I:C) Stimuli.

As a major mediator of cellular response to viral infection in mammals, Toll-like receptor 3 (TLR3) was proved to respond to double-stranded RNA (dsRNA). However, the molecular mechanism by which TLR3 functions in the viral infection response in teleosts remains to be investigated. In this study, the Toll-like receptor 3 gene of the hybrid yellow catfish was identified and characterized by comparative genomics. Furthermore, multiple sequence alignment, genomic synteny and phylogenetic analysis suggested that the homologous TLR3 genes were unique to teleosts. Gene structure analysis showed that five exons and four introns were common components of TLR3s in the 12 examined species, and interestingly the third exon in teleosts was the same length of 194 bp. Genomic synteny analysis indicated that TLR3s were highly conserved in various teleosts, with similar organizations of gene arrangement. De novo predictions showed that TLR3s were horseshoe-shaped in multiple taxa except for avian (with a round-shaped structure). Phylogenetic topology showed that the evolution of TLR3 was consistent with the evolution of the studied species. Selection analysis showed that the evolution rates of TLR3 proteins were usually higher than those of TLR3-TIR domains, indicating that the latter were more conserved. Tissue distribution analysis showed that TLR3s were widely distributed in the 12 tested tissues, with the highest transcriptions in liver and intestine. In addition, the transcription levels of TLR3 were significantly increased in immune-related tissues after infection of exogenous Aeromonas hydrophila and poly (I:C). Molecular docking showed that TLR3 in teleosts forms a complex with poly (I:C). In summary, our present results suggest that TLR3 is a pattern recognition receptor (PRR) gene in the immune response to pathogen infections in hybrid yellow catfish.

Combined PARP1-Targeted Nuclear Contrast and Reflectance Contrast Enhance Confocal Microscopic Detection of Basal Cell Carcinoma.

Reflectance confocal microscopy (RCM) with endogenous backscattered contrast can noninvasively image basal cell carcinomas (BCCs) in skin. However, BCCs present with high nuclear density, and the relatively weak backscattering from nuclei imposes a fundamental limit on contrast, detectability, and diagnostic accuracy. We investigated PARPi-FL, an exogenous nuclear poly(adenosine diphosphate ribose) polymerase (PARP1)-targeted fluorescent contrast agent, and fluorescence confocal microscopy toward improving BCC diagnosis. Methods: We tested PARP1 expression in 95 BCC tissues using immunohistochemistry, followed by PARPi-FL staining in 32 fresh surgical BCC specimens. The diagnostic accuracy of PARPi-FL contrast was evaluated in 83 surgical specimens. The optimal parameters for permeability of PARPi-FL through intact skin was tested ex vivo on 5 human skin specimens and invivo in 3 adult Yorkshire pigs. Results: We found significantly higher PARP1 expression and PARPi-FL binding in BCCs than in normal skin structures. Blinded reading of RCM-and-fluorescence confocal microscopy images by 2 experts demonstrated a higher diagnostic accuracy for BCCs with combined fluorescence and reflectance contrast than for RCM alone. Optimal parameters (time and concentration) for PARPi-FL transepidermal permeation through intact skin were successfully determined. Conclusion: Combined fluorescence and reflectance contrast may improve noninvasive BCC diagnosis with confocal microscopy.

Ultralong-Circulating and Self-Targeting "Watson-Crick A = T"-Inspired Supramolecular Nanotheranostics for NIR-II Imaging-Guided Photochemotherapy.

A carrier-free theranostic nanodrug directly coassembled using a NIR probe and a chemotherapeutic drug is a promising alternative for cancer theranostics. Nevertheless, this nanodrug still faces the limitations of short blood circulation and inefficient tumor accumulation/tumoral cellular uptake in vivo. Meanwhile, most exogenous targeting ligands and poly(ethylene glycol) have no therapeutic effect. Herein, we designed an ultralong-circulating and self-targeting nanodrug by an ordered supramolecular coassembly of indocyanine green (ICG), methotrexate (MTX, chemotherapeutic drug and cancer-cell-specific ligand), and clofarabine (CA). Notably, CA, as a surfactant-like chemotherapeutic drug, was introduced into the initial ICG-MTX coassembly by "Watson-Crick A = T-inspired" hydrogen-bond-driven sequential assembly with MTX. This carrier-free theranostic nanodrug with exceptionally high drug payload (100 wt %) not only showed superior serum stabilities but also displayed ultralong blood circulation (>7 days), enabling efficient accumulation at tumor sites. Moreover, our nanodrugs could be self-recognized by cancer cells and release the drugs on demand through lysosomal acidity and external laser stimulus. Under NIR-II imaging guidance, high-efficiency tumor ablation via synergistic photothermal-chemotherapy could be achieved in one treatment cycle while preventing the tumor recurrence. Our ultralong-circulating and self-recognizing carrier-free theranostic nanodrug based on the "drug-delivering-drug" strategy might have the potential for clinical theranostic application.

CGAS‐STING pathway activation in murine retina

AbstractPurposeThe intracellular DNA sensor, cGAS, initiates the host immune response by recognizing pathogen‐ or endogenous‐derived cytoplasmic DNA. After binding to DNA, activated cGAS‐STING pathway induces the transcription of the Type I interferon and other pro‐inflammatory genes. Mitochondrial dysfunction is known to be involved in various retinal degenerative and angiogenic diseases, including age‐related macular degeneration and diabetic retinopathy. As a consequence of mitochondrial stress, cytosolic escape of mitochondrial DNA has been reported to induce cGAS‐STING pathway activation. In this study, we characterized the expression of cGAS‐STING pathway in murine retina.MethodsThe expression of cGAS and STING in murine retina, microglia cells (BV2), primary RPE and B6‐RPE07, primary Müller glial and photoreceptor cells (661W) was examined by RT‐PCR and immunohistochemistry. The cells were transfected with exogenous dsDNA poly(dA:dT) (5ug/mL) for 8 hours. The expression of cGAS, STING, phosphorylated TBK1 (pTBK1) and IRF3 (pIRF3), type‐1 interferon, IL‐6 and TNFa was analysed by RT‐PCR and immunocytochemistry.ResultsBoth cGAS and STING were detected in the murine retina, microglia, Müller glial, photoreceptor and RPE cells. Following poly(dA:dT) treatment, the expression of cGAS and pTBK1 was increased in these cells. In addition, intranuclear translocation of pIRF3 was observed in poly(dA:dT)‐treated microglia, RPE, Müller glial and photoreceptor cells. In line with these observations, the mRNA expression of IFNa, IFNb, IL6 and TNFa was significantly increased (p < 0.05) in poly(dA:dT)‐treated cells.ConclusionsThe cGAS‐STING pathway is expressed in many cells within the retina, including microglia, Muller cells, photoreceptor and RPE cells. This pathway may play an important role in retinal innate immunity against endogenous or exogenous cytosolic DNA. Uncontrolled cGAS‐STING pathway may be involved in retinal degenerative diseases.

Establishment of Primary Transgenic Human Airway Epithelial Cell Cultures to Study Respiratory Virus-Host Interactions.

Primary human airway epithelial cell (hAEC) cultures represent a universal platform to propagate respiratory viruses and characterize their host interactions in authentic target cells. To further elucidate specific interactions between human respiratory viruses and important host factors in the airway epithelium, it is important to make hAEC cultures amenable to genetic modification. However, the short and finite lifespan of primary cells in cell culture creates a bottleneck for the genetic modification of these cultures. In the current study, we show that the incorporation of the Rho-associated protein kinase (ROCK) inhibitor (Y-27632) during cell propagation extends the life span of primary human cells in vitro and thereby facilitates the incorporation of lentivirus-based expression systems. Using fluorescent reporters for fluorescence-activated cell sorting (FACS)-based sorting, we generated homogenously fluorescent hAEC cultures that differentiate normally after lentiviral transduction. As a proof-of-principle, we demonstrate that host gene expression can be modulated post-differentiation via inducible short hairpin (sh)RNA-mediated knockdown. Importantly, functional characterization of these transgenic hAEC cultures with exogenous poly (I:C), as a proxy for virus infection, demonstrates that such modifications do not influence the host innate immune response. Moreover, the propagation kinetics of both human coronavirus 229E (HCoV-229E) and human respiratory syncytial virus (hRSV) were not affected. Combined, these results validate our newly established protocol for the genetic modification of hAEC cultures, thereby unlocking a unique potential for detailed molecular characterization of virus–host interactions in human respiratory epithelium.

Altered Nucleolar Phase Separation by NPM1 in ALS

Amyotrophic lateral sclerosis (ALS) is a lethal and incurable neurodegenerative disorder commonly associated with repeat expansion in the C9orf72 gene; termed C9-ALS. A major pathological feature of this is the accumulation of arginine-rich (R-rich) dipeptide repeat (DPR) polypeptides in neurons. R-rich DPRs interact with low complexity domains in proteins, accumulate in membraneless organelles (MLOs), alter the material properties of MLOs, and induce cell death. The molecular mechanisms underlying pathogenesis, though, remain unknown. R-rich DPRs infiltrate nucleoli, co-localize with nucleophosmin (NPM1), and alter NPM1 phase separation in vitro. NPM1 is crucial to the maintenance of nucleolar liquid-like properties through its ability to phase separate with proteins and nucleic acids. Elucidating the effects of DPR interactions with NPM1 on the liquid-like properties and overall architecture of nucleoli that ultimately lead to nucleolar dysfunction and cell death is critical to understanding C9-ALS. Here we employ poly(PR) as an archetypal R-rich DPR to (1) identify the interactions mediating phase separation with NPM1; (2) elucidate the mechanisms causing the DPR-dependent dissolution of in vitro NPM1/DPR droplets; and (3) confirm hypotheses stimulated by our biophysical results regarding the mechanisms of DPR-mediated nucleolar disruption in cells. Results from multiple, complementary biochemical and biophysical techniques show that (1) NPM1/DPR interactions are mediated by acidic tracts within the intrinsically disorder region of NPM1, (2) R-rich DPRs dissolve NPM1-containing droplets in vitro by sequestering NPM1 into large saturated complexes, and (3) exogenous poly(PR) induces NPM1 release from nucleoli, disrupting nucleolar organization and function. These results support the hypothesis that R-rich DPRs, mediate their toxic effects in part through saturation/sequestration of NPM1, perturbing NPM1 mediated phase separation in nucleoli, disrupting nucleolar function, and inducing cell death.

Effects of testosterone and 17β‑estradiol on angiotensin‑induced changes in tyrosine kinase activity in the androgen‑independent human prostate cancer cell line, DU145.

AngiotensinII (AngII), the main peptide of the renin‑angiotensin system(RAS), is involved in the proliferation of different types of cells, normal and pathological as well. The protein tyrosine kinases(PTKs) play an important role in the growth, differentiation and apoptosis of cells. AngII action depends on the hormonal milieu of the cell, and on sex steroid influence. Angiotensin1‑7 (Ang1‑7), metabolite of AngII, shows opposite action to AngII in cells. The present study aimed to examine the influence of 17β‑estradiol and testosterone on AngII and Ang1‑7 action on PTK activity in androgen‑independent humane prostate cancer cell line DU145. Cell cultures of human prostate cancer DU145 cells were used as a source of PTKs. Cultures were exposed to different concentrations of AngII(5x10‑11 to 5x10‑9M). The incubation with hormones lasted 15min to limit the genomic effects of steroids. In the phosphorylation reaction, we used γ32P‑ATP as a donor of phosphate and a synthetic peptide, Poly(Glu, Tyr) (4:1), as a substrate. The specific activities of PTKs were defined as pmol of 32P incorporated into 1mg of exogenous Poly(Glu, Tyr) per minute (pmol/mg/min). Our findings suggest that testosterone and 17β‑estradiol may change the effects of angiotensins in a rapid non‑genomic way, probably via membrane‑located receptors. The most significant change was caused by testosterone, whose effect was most significant on changes caused by Ang1‑7. AngII‑induced changes in phosphorylation appeared to be insensitive to the presence of testosterone, but were modified by 17β‑estradiol.

Manipulating the air-filled zebrafish swim bladder as a neutrophilic inflammation model for acute lung injury.

Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS), are life-threatening diseases that are associated with high mortality rates due to treatment limitations. Neutrophils play key roles in the pathogenesis of ALI/ARDS by promoting the inflammation and injury of the alveolar microenvironment. To date, in vivo functional approaches have been limited by the inaccessibility to the alveolar sacs, which are located at the anatomical terminal of the respiratory duct in mammals. We are the first to characterize the swim bladder of the zebrafish larva, which is similar to the mammalian lung, as a real-time in vivo model for examining pulmonary neutrophil infiltration during ALI. We observed that the delivery of exogenous materials, including lipopolysaccharide (LPS), Poly IC and silica nanoparticles, by microinjection triggered significant time- and dose-dependent neutrophil recruitment into the swim bladder. Neutrophils infiltrated the LPS-injected swim bladder through the blood capillaries around the pneumatic duct or a site near the pronephric duct. An increase in the post-LPS inflammatory cytokine mRNA levels coincided with the in vivo neutrophil aggregation in the swim bladder. Microscopic examinations of the LPS-injected swim bladders further revealed in situ injuries, including epithelial distortion, endoplasmic reticulum swelling and mitochondrial injuries. Inhibitor screening assays with this model showed a reduction in neutrophil migration into the LPS-injected swim bladder in response to Shp2 inhibition. Moreover, the pharmacological suppression and targeted disruption of Shp2 in myeloid cells alleviated pulmonary inflammation in the LPS-induced ALI mouse model. Additionally, we used this model to assess pneumonia-induced neutrophil recruitment by microinjecting bronchoalveolar lavage fluid from patients into swim bladders; this injection enhanced neutrophil aggregation relative to the control. In conclusion, our findings highlight the swim bladder as a promising and powerful model for mechanistic and drug screening studies of alveolar injuries.

Type I interferon responses are impaired in latently HIV infected cells.

Background The latent HIV-1 reservoir represents the primary barrier to the eradication of HIV-1 infection. The design of novel reservoir-clearance strategies, however, is impeded in part by the inability to distinguish latently HIV-infected cells from uninfected cells. Significant impairment of the type I interferon (IFN-I) response is observed during productive HIV-1 infection. Although this remains poorly described in the context of latent HIV-1 infection, presence of potential defects may serve as a novel therapeutic target. Therefore, IFN-I pathways were characterized using two latently HIV-1-infected cell lines, U1 and OM10.1, in comparison to their respective uninfected parental U937 and HL60 cell lines.FindingsConstitutive expression and induction of important mediators of IFN-I signaling including IFNα/β cytokines, IFNAR1, MHC-I, ISG15, and PKR were evaluated following exogenous IFNα or poly(I:C) treatment. Differences in basal expression of IFNAR1, MHC-I, and PKR were observed between the latently HIV-1 infected and uninfected cell lines. In parallel, significant impairments in the induction of MHC-I, ISG15 and PKR, as well as secretion of IFNα/β cytokines were observed in response to appropriate exogenous stimulation within the two latently HIV-infected U1 and OM10.1 cells, relative to their HIV-uninfected parental cells.ConclusionsIn comparison to the HIV-uninfected U937 and HL60 cell lines, widespread defects in IFN-I responsiveness were observed within the latently HIV-infected U1 and OM10.1 cells. These impairments represent novel therapeutic targets, which may be amenable to strategies currently employed in cancer therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-016-0302-9) contains supplementary material, which is available to authorized users.

Identification of poly‐ADP‐ribosylated mitochondrial proteins after traumatic brain injury

Poly-ADP-ribosylation is a post-translational modification performed by poly(ADP-ribose) polymerases (PARP), involved in many diverse cellular functions including DNA repair, transcription, and long-term potentiation. Paradoxically, PARP over-activation under pathologic conditions including traumatic brain injury (TBI) results in cell death. We previously demonstrated that intra-mitochondrial poly-ADP-ribosylation occurs following excitotoxic and oxidative injury in vitro. Here we sought to identify mitochondrial proteins modified by poly-ADP-ribosylation after TBI in vivo. Poly-ADP-ribosylation within mitochondria from injured brain after experimental TBI in rats was first verified using western blot and immuno-electron microscopy. Poly-ADP-ribosylated mitochondrial proteins identified using a targeted proteomic approach included voltage-dependent anion channel-1, mitofilin, mitochondrial stress proteins, and the electron transport chain components F1F0 ATPase, cytochrome c oxidase, and cytochrome c reductase. To examine the functional consequences of mitochondrial poly-ADP-ribosylation, isolated rat brain mitochondria were exposed to conditions of nitrosative stress known to activate PARP. PARP activation-induced reductions in State 3 respiration were prevented by the PARP-1 inhibitor 5-iodo-6-amino-1,2-benzopyrone or exogenous poly(ADP-ribose) glycohydrolase. As the effects of PARP activation on mitochondrial respiration appear regulated by poly(ADP-ribose) glycohydrolase, a direct effect of poly-ADP-ribosylation on electron transport chain function is suggested. These findings may be of relevance to TBI and other diseases where mitochondrial dysfunction occurs.

Effective delivery with enhanced translational activity synergistically accelerates mRNA-based transfection

mRNA instead of DNA provides a new and attractive approach for gene therapy and genetic vaccination. Current technologies for mRNA delivery are based on cationic lipids with DOTAP being the most efficient one. We previously reported on the synthesis of an inorganic–organic hybrid carrier by embedding inorganic nano-particles of carbonate apatite onto liposomal carrier DOTAP and demonstrated its high transfection potency of luciferase mRNA both in mitotic and non-mitotic cells. Here we show that in addition to the carrier design for effective endocytosis and release of mRNA to the cytoplasm, enhancement of mRNA translation efficiency is a prerequisite for maximum protein expression. We used the modified cap analog (ARCA) during in vitro transcription of luciferase DNA for proper cap orientation and demonstrated that transfection with ARCA–mRNA resulted in higher protein expression than the mRNA with usual cap structure for both DOTAP and DOTAP–apatite complex. Secondly, exogenous poly(A) was co-delivered with mRNA by the DOTAP–apatite, resulting in very significant expression compared to mRNA delivery only. Finally, when combined both of the effects of smart carrier and the modifications at mRNA translational level, a notable enhancement (100 times) was achieved as compared to the existing DOTAP-based liposome technology. Our findings, therefore, unveiled a novel approach that an effective delivery system can be developed by the improvement of the gene expression level in combination with the enhancement of the carrier potency.

Vaccinia virus infection attenuates innate immune responses and antigen presentation by epidermal dendritic cells.

Langerhans cells (LCs) are antigen-presenting cells in the skin that play sentinel roles in host immune defense by secreting proinflammatory molecules and activating T cells. Here we studied the interaction of vaccinia virus with XS52 cells, a murine epidermis-derived dendritic cell line that serves as a surrogate model for LCs. We found that vaccinia virus productively infects XS52 cells, yet this infection displays an atypical response to anti-poxvirus agents. Whereas adenosine N1-oxide blocked virus production and viral protein synthesis during a synchronous infection, cytosine arabinoside had no effect at concentrations sufficient to prevent virus replication in BSC40 monkey kidney cells. Vaccinia virus infection of XS52 cells not only failed to elicit the production of various cytokines, including tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, IL-10, IL-12 p40, alpha interferon (IFN-alpha), and IFN-gamma, it actively inhibited the production of proinflammatory cytokines TNF-alpha and IL-6 by XS52 cells in response to exogenous lipopolysaccharide (LPS) or poly(I:C). Infection with a vaccinia virus mutant lacking the E3L gene resulted in TNF-alpha secretion in the absence of applied stimuli. Infection of XS52 cells or BSC40 cells with the DeltaE3L virus, but not wild-type vaccinia virus, triggered proteolytic decay of IkappaBalpha. These results suggest a novel role for the E3L protein as an antagonist of the NF-kappaB signaling pathway. DeltaE3L-infected XS52 cells secreted higher levels of TNF-alpha and IL-6 in response to LPS and poly(I:C) than did cells infected with the wild-type virus. XS52 cells were productively infected by a vaccinia virus mutant lacking the K1L gene. DeltaK1L-infected cells secreted higher levels of TNF-alpha and IL-6 in response to LPS than wild-type virus-infected cells. Vaccinia virus infection of primary LCs harvested from mouse epidermis was nonpermissive, although a viral reporter protein was expressed in the infected LCs. Vaccinia virus infection of primary LCs strongly inhibited their capacity for antigen-specific activation of T cells. Our results highlight suppression of the skin immune response as a feature of orthopoxvirus infection.

Role of Corticosterone in Immunosuppressive Effects of Acute Ethanol Exposure on Toll-Like Receptor Mediated Cytokine Production

Acute ethanol (EtOH) exposure causes a stress response in humans, nonhuman primates, and rodents. Previous study results indicate that the suppression of some immunological parameters by EtOH is mediated in part or completely by elevated corticosterone concentrations induced by EtOH. However, initial results suggested that corticosterone is not involved in the modulation of cytokine production by macrophages in response to polyinosinic polycytidylic acid (poly I:C). New studies were conducted to further evaluate the role of corticosterone in EtOH-mediated changes in production of interleukin-6 (IL-6), IL-10, and IL-12 in serum and peritoneal fluid in mice treated with poly I:C or lipopolysaccharide (LPS). Suppression of IL-6, but not IL-12, production by EtOH was found to be mediated by corticosterone. However, poly I:C, LPS, and EtOH all caused similar elevations of corticosterone concentrations; thus, it is not clear if EtOH is required to induce levels or durations of corticosterone needed to mediate the observed effects. The situation with IL-10 was more complicated. Inhibition of corticosterone synthesis with aminoglutethimide prevented the increase in IL-10 production caused by EtOH plus poly I:C as compared to poly I:C only. This indicates that this increase is dependent on corticosterone, but exogenous corticosterone plus poly I:C did not increase IL-10 production. Thus, EtOH and corticosterone are required. However, with LPS inhibition of corticosterone synthesis (using aminoglutethimide) or inhibition of its action (using mifepristone) further increased, or did not affect IL-10 concentrations, suggesting fundamental differences in the signaling pathways leading from poly I:C and LPS to IL-10 production.

Influence of poly(ADP-ribose) polymerase-1 and its apoptotic 24-kD fragment on repair of DNA duplexes in bovine testis nuclear extract

Effects of exogenous proteins poly(ADP-ribose) polymerase-1 (PARP1) and its 24-kD proteolytic fragment (p24) on the repair of DNA duplexes containing a one nucleotide gap with furan phosphate or phosphate group at the 5'-end of the downstream primer were studied in bovine testis nuclear extract. These damaged DNAs are repaired by the long-patch or short-patch subpathways of base excision repair (BER), respectively. Exogenous PARP1 and p24 decreased the efficiency of gap filling DNA synthesis for both duplexes, but did not influence the ligation stage in the repair of DNA duplex by the short-patch subpathway. Under the same conditions, these proteins inhibited strand-displacement DNA synthesis and decreased the efficiency of the flap endonuclease 1 (FEN1)-catalyzed endonuclease reaction in the nuclear extract, blocking repair of DNA duplex by the long-patch subpathway. Addition of exogenous PARP1 and p24 also reduced the efficiency of UV light crosslinking of extract BER proteins to the photoreactive BER intermediates carrying a nick. Thus, PARP1 and p24 interact with DNA intermediates of BER and compete with nuclear extract proteins for binding to DNA. The interaction of PARP1 and p24 with DNA intermediates of the long-patch subpathway of BER resulted in inhibition of subsequent stages of the repair mediated by this mechanism. However, on recovery of the intact structure of DNA duplex by the short-patch subpathway, PARP1 and p24 suppressed the repair of the one nucleotide gap less efficiently and failed to influence the final stage of the repair, ligation.

Role of interferons in the control of Lassa virus replication in human dendritic cells and macrophages

Lassa fever is a hemorrhagic fever caused by Lassa virus (LV), which primarily targets human dendritic cells (DC) and macrophages (MP). Massive numbers of viral particles are released with no effect on the viability, activation or maturation of these cells. LV does not inhibit the activation of cells induced by sCD40L or LPS. We report here the consequences of exogenous activation of LV-infected human DC and MP for viral replication. The activation of cells with lipopolysaccharide or exogenous poly(I-C) and the transfection of cells with poly(I-C) strongly inhibited LV replication, at least partly by inducing type I interferon (IFN) synthesis. In contrast, cell stimulation with sCD40L did not induce type I IFN responses or inhibit LV release. Recombinant type I IFNs strongly inhibited LV replication in both cell types, whereas IFNγ and IFNλ did not. The modest type I IFN production observed in LV-infected MP, but not in DC, was involved in controlling LV replication in MP. These results provide an explanation for the slower replication of LV in MP than in DC, and suggest that type I IFNs are crucial in the control of LV.

Role of tight junction derangement in the endothelial dysfunction elicited by exogenous and endogenous peroxynitrite and poly(ADP-ribose) synthetase.

DNA single-strand breakage and activation of the nuclear enzyme poly(ADP-ribose) synthetase (PARS) triggers an energy consuming, inefficient repair cycle, which contributes to peroxynitrite-induced cellular injury. Here, we investigated whether peroxynitrite and PARS activation are involved in tight junctions (tight junction) derangement in the endothelial dysfunction in cells exposed to peroxynitrite and in vascular rings of animals subjected to zymosan non-septic shock. In human umbilical vein endothelial cells (HUVEC) in vitro, peroxynitrite caused a dose-dependent suppression of mitochondrial respiration, as measured by the mitochondrial-dependent conversion of the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan. Moreover, peroxynitrite caused activation of PARS. Inhibition of PARS by 3-aminobenzamide (3-AB; 1 mM) reduced the peroxynitrite-induced suppression of mitochondrial respiration in HUVECs. Vascular rings exposed to peroxynitrite exhibited reduced endothelium-dependent relaxant responses in response to acetylcholine. Peroxynitrite incubation also caused a significant derangement of zonula occludens (ZO)-1, which was significantly affected by pharmacological inhibition of PARS. 3-AB ameliorated the development of this peroxynitrite-induced endothelial dysfunction. In vascular rings obtained from the zymosan-treated rats, there was a marked suppression of the endothelium-dependent relaxation ex vivo, which was reduced by in vivo 3-AB treatment. A significant derangement of ZO-1 was observed in vascular rings from zymosan-treated rats. Tight junction alteration was significantly reduced by in vivo 3-AB treatment. Thus, activation of PARS by exogenous and endogenous peroxynitrite may be involved in the tight junction derangement associated with endothelial dysfunction. Inhibition of PARS may be a novel pharmacological approach to preserve endothelial tight junction function in shock and inflammation.

Free Poly(A) Stimulates Capped mRNA Translation in Vitro through the eIF4G-Poly(A)-binding Protein Interaction

The 5' cap and 3' poly(A) tail of classical eukaryotic mRNAs functionally communicate to synergistically enhance translation initiation. Synergy has been proposed to result in part from facilitated ribosome recapture on circularized mRNAs. Here, we demonstrate that this is not the case. In poly(A)-dependent, ribosome-depleted rabbit reticulocyte lysates, the addition of exogenous poly(A) chains of physiological length dramatically stimulated translation of a capped, nonpolyadenylated mRNA. When the poly(A):RNA ratio approached 1, exogenous poly(A) stimulated translation to the same extent as the presence of a poly(A) tail at the mRNA 3' end. In addition, exogenous poly(A) significantly improved translation of capped mRNAs carrying short poly(A(50)) tails. Trans stimulation of translation by poly(A) required the eIF4G-poly(A)-binding protein interaction and resulted in increased affinity of eIF4E for the mRNA cap, exactly as we recently described for cap-poly(A) synergy. These results formally demonstrate that mRNA circularization per se is not the cause of cap-poly(A) synergy at least in vitro.

Kinetic Proofreading Scanning Models for Eukaryotic Translational Initiation: the Cap and Poly(A) Tail Dependency of Translation

Two simplified kinetic proofreading scanning (KPS) models were proposed to describe the 5′ cap and 3′ poly(A) tail dependency of eukaryotic translation initiation. In Model I, the initiation factor complex starts scanning and unwinding the secondary structure of the 5′ untranslated region (UTR) from the 5′ terminus of mRNA. In Model II, the initiation factor complex starts scanning from any binding site in the 5′ UTR. In both models, following ATP hydrolysis, the initiation factor complex either dissociates from mRNA or continues to scan and unwind RNA secondary structure in the 5′ UTR. This step repeats n times until the AUG codon is reached. These two models show very different cap and/or poly(A) tail dependency of translation initiation. The models predict that both cap and poly(A) tail dependencies of translation, and translatability of mRNAs are coupled with the structure of 5′ UTR: the translation of mRNA with structured 5′ UTR is strongly cap- and poly(A) tail-dependent; while translation of mRNA with unstructured 5′ UTR is less cap- and poly(A) tail-dependent. We use these two models to explain: (1) the cap and poly(A) tail dependence of translation; (2) the effect of exogenous poly(A) on translation; (3) repression of host mRNA and translation of late adenovirus mRNA in the late phase of adenovirus infection; (4) repression of host mRNA and translation of Vaccinia virus mRNA in virus-infected cell; (5) heat shock repression of translation of normal mRNA and stimulation of translation of hsp mRNA; and (6) the synergistic effect of cap and poly(A) tail on stimulating translation. The kinetic proofreading scanning models provide a coherent interpretation of those phenomena.

Temporal patterns of poly(ADP-ribose) polymerase activation in the cortex following experimental brain injury in the rat.

The activation of poly(ADP-ribose) polymerase, a DNA base excision repair enzyme, is indicative of DNA damage. This enzyme also undergoes site-specific proteolysis during apoptosis. Because both DNA fragmentation and apoptosis are known to occur following experimental brain injury, we investigated the effect of lateral fluid percussion brain injury on poly(ADP-ribose) polymerase activity and cleavage. Male Sprague-Dawley rats (n = 52) were anesthetized, subjected to fluid percussion brain injury of moderate severity (2.5-2.8 atm), and killed at 30 min, 2 h, 6 h, 24 h, 3 days, or 7 days postinjury. Genomic DNA from injured cortex at 24 h, but not at 30 min, was both fragmented and able to stimulate exogenous poly(ADP-ribose) polymerase. Endogenous poly(ADP-ribose) polymerase activity, however, was enhanced in the injured cortex at 30 min but subsequently returned to baseline levels. Slight fragmentation of poly(ADP-ribose) polymerase was detected in the injured cortex in the first 3 days following injury, but significant cleavage was detected at 7 days postinjury. Taken together, these data suggest that poly(ADP-ribose) polymerase-mediated DNA repair is initiated in the acute posttraumatic period but that subsequent poly(ADP-ribose) polymerase activation does not occur, possibly owing to delayed apoptosis-associated proteolysis, which may impair the repair of damaged DNA.