Adenoviral vectors infect B lymphocytes in vivo.

B cells are the antibody-producing arm of the adaptive immune system and play a critical role in controlling pathogens. Several groups have now demonstrated the feasibility of using engineered B cells as a therapy, including infectious disease control and gene therapy of serum deficiencies. These studies have largely utilized ex vivo modification of the cells. Direct in vivo engineering would be of utility to the field, particularly in infectious disease control where the infrastructure needs of ex vivo cell modification would make a broad vaccination campaign highly challenging. In this study we demonstrate that engineered adenoviral vectors are capable of efficiently transducing murine and human primary B cells both ex vivo and in vivo. We found that unmodified human adenovirus C5 was capable of infecting B cells in vivo, likely due to interactions between the virus penton base protein and integrins. We further describe vector modification with B cell-specific gene promoters and successfully restrict transgene expression to B cells, resulting in a strong reduction in gene expression from the liver, the main site of human adenovirus C5 infection in vivo.

A New Gorilla Adenoviral Vector with Natural Lung Tropism Avoids Liver Toxicity and Is Amenable to Capsid Engineering and Vector Retargeting.

Human adenoviruses have many attractive features for gene therapy applications. However, the high prevalence of preexisting immunity against these viruses in general populations worldwide has greatly limited their clinical utility. In addition, the most commonly used human adenovirus, human adenovirus subgroup C serotype 5 (HAd5), when systemically administered, triggers systemic inflammation and toxicity, with the liver being the most severely affected organ. Here, we evaluated the utility and safety of a new low-seroprevalence gorilla adenovirus (GAd; GC46) as a gene transfer vector in mice. Biodistribution studies revealed that systemically administered GAd had a selective and robust lung endothelial cell (EC) tropism with minimal vector expression throughout many other organs and tissues. Administration of a high dose of GAd accomplished extensive transgene expression in the lung yet elicited no detectable inflammatory histopathology in this organ. Furthermore, GAd, unlike HAd5, did not exhibit hepatotropism or induce liver inflammatory toxicity in mice, demonstrating the exceptional safety profile of the vector vis-à-vis systemic utility. We further demonstrated that the GAd capsid fiber shared the flexibility of the HAd5 equivalent for permitting genetic modification; GAd with the pan-EC-targeting ligand myeloid cell-binding peptide (MBP) incorporated in the capsid displayed a reduced lung tropism and efficiently retargeted gene expression to vascular beds in other organs.IMPORTANCE In the aggregate, our mouse studies suggest that GAd is a promising gene therapy vector that utilizes lung ECs as a source of therapeutic payload production and a highly desirable toxicity profile. Further genetic engineering of the GAd capsid holds the promise of in vivo vector tropism modification and targeting.

Degradation of MCL-1 by bufalin reverses acquired resistance to osimertinib in EGFR-mutant lung cancer.

Although osimertinib, an EGFR tyrosine kinase inhibitor, has become the standard therapy for treating non-small cell lung cancer (NSCLC) patients with EGFR-activating mutation, upregulation of MCL-1 induces acquired resistance to osimertinib. Bufalin, a natural digoxin-like ingredient isolated from a traditional Chinese medicine Chan Su, has been shown to downregulate MCL-1 in NSCLC cells. However, whether bufalin reverses this acquired resistance to osimertinib in NSCLC cells remains unclear. In this study, bufalin reduced cell viability and promoted apoptosis in osimertinib-resistant cells. Moreover, co-treatment with bufalin and osimertinib restored the sensitivity of osimertinib-resistant cells to osimertinib-induced growth regression and apoptosis in vitro and in vivo. Mechanistically, MEK/ERK-dependent MCL-1 phosphorylation and Ku70-mediated MCL-1 overexpression confer osimertinib resistance in EGFR-mutant NSCLC cells. In osimertinib-resistant cells, bufalin modulates Ku70-mediated MCL-1 degradation, but not MEK/ERK/MCL-1 signaling. In conclusion, our study suggests that bufalin eliminates resistance to osimertinib by inhibiting Ku70-mediated MCL-1 overexpression, indicating that a combination of osimertinib and bufalin could be an effective additional treatment to overcome acquired resistance to osimertinib in NSCLC cells.

Pulmonary vasculature directed adenovirus increases epithelial lining fluid alpha-1 antitrypsin levels.

BACKGROUND: Gene therapy for inherited serum deficiency disorders has previously been limited by the balance between obtaining adequate expression and causing hepatic toxicity. Our group has previously described modifications of a replication deficient human adenovirus serotype 5 that increase pulmonary vasculature transgene expression. METHODS: In the present study, we use a modified pulmonary targeted adenovirus to express human alpha-1 antitrypsin (A1AT) in C57BL/6 J mice. RESULTS: Using the targeted adenovirus, we were able to achieve similar increases in serum A1AT levels with less liver viral uptake. We also increased pulmonary epithelial lining fluid A1AT levels by more than an order of magnitude compared to that of untargeted adenovirus expressing A1AT in a mouse model. These gains are achieved along with evidence of decreased systemic inflammation and no evidence for increased inflammation within the vector-targeted end organ. CONCLUSIONS: In addition to comprising a step towards clinically viable gene therapy for A1AT, maximization of protein production at the site of action represents a significant technical advancement in the field of systemically delivered pulmonary targeted gene therapy. It also provides an alternative to the previous limitations of hepatic viral transduction and associated toxicities.

The myeloid-binding peptide adenoviral vector enables multi-organ vascular endothelial gene targeting.

Vascular endothelial cells (ECs) are ideal gene therapy targets as they provide widespread tissue access and are the first contact surfaces following intravenous vector administration. Human recombinant adenovirus serotype 5 (Ad5) is the most frequently used gene transfer system because of its appreciable transgene payload capacity and lack of somatic mutation risk. However, standard Ad5 vectors predominantly transduce liver but not the vasculature following intravenous administration. We recently developed an Ad5 vector with a myeloid cell-binding peptide (MBP) incorporated into the knob-deleted, T4 fibritin chimeric fiber (Ad.MBP). This vector was shown to transduce pulmonary ECs presumably via a vector handoff mechanism. Here we tested the body-wide tropism of the Ad.MBP vector, its myeloid cell necessity, and vector-EC expression dose response. Using comprehensive multi-organ co-immunofluorescence analysis, we discovered that Ad.MBP produced widespread EC transduction in the lung, heart, kidney, skeletal muscle, pancreas, small bowel, and brain. Surprisingly, Ad.MBP retained hepatocyte tropism albeit at a reduced frequency compared with the standard Ad5. While binding specifically to myeloid cells ex vivo, multi-organ Ad.MBP expression was not dependent on circulating monocytes or macrophages. Ad.MBP dose de-escalation maintained full lung-targeting capacity but drastically reduced transgene expression in other organs. Swapping the EC-specific ROBO4 for the CMV promoter/enhancer abrogated hepatocyte expression but also reduced gene expression in other organs. Collectively, our multilevel targeting strategy could enable therapeutic biological production in previously inaccessible organs that pertain to the most debilitating or lethal human diseases.

Regioselective synthesis of cytarabine monopropionate by using a fungal whole-cell biocatalyst in nonaqueous medium.

The utilization of a dehydrated fungal biocatalyst of Aspergillus oryzae cells was successfully performed to achieve efficient acylation modification of a polar nucleoside cytarabine (ara-C). Organic solvents showed evident influence on the reaction catalyzed by the A. oryzae whole-cells. Except for hexane-pyridine, the catalytic activity and regioselectivity of the whole-cells clearly increased with increasing the polarity of the hydrophobic organic solvents used. The effects of some crucial factors on the reaction were further examined. The best reaction medium, hydrophobic solvent concentration, vinyl propionate/ara-C ratio, reaction temperature and shaking speed were confirmed as isopropyl ether (IPE)-pyridine, 30% (v/v), 90, 30 °C and 140-180 rpm, respectively. The cell biocatalyst also showed good thermal stabilities in both IPE-pyridine and hexane-pyridine systems. In addition, the desired 3'-O-propional derivative of ara-C was synthesized with the yields of 88.3% and regioselectivity (>70%). The resulting biocatalytic system appears to be an effective alternative, and can thus be employed for application in highly regioselective modification of nucleoside analogues.

Hydrogen-rich saline improves survival and neurological outcome after cardiac arrest and cardiopulmonary resuscitation in rats.

BACKGROUND: Sudden cardiac arrest is a leading cause of death worldwide. Three-fourths of cardiac arrest patients die before hospital discharge or experience significant neurological damage. Hydrogen-rich saline, a portable, easily administered, and safe means of delivering hydrogen gas, can exert organ-protective effects through regulating oxidative stress, inflammation, and apoptosis. We designed this study to investigate whether hydrogen-rich saline treatment could improve survival and neurological outcome after cardiac arrest and cardiopulmonary resuscitation, and the mechanism responsible for this effect. METHODS: Sprague-Dawley rats were subjected to 8 minutes of cardiac arrest by asphyxia. Different doses of hydrogen-rich saline or normal saline were administered IV at 1 minute before cardiopulmonary resuscitation, followed by injections at 6 and 12 hours after restoration of spontaneous circulation, respectively. We assessed survival, neurological outcome, oxidative stress, inflammation biomarkers, and apoptosis. RESULTS: Hydrogen-rich saline treatment dose dependently improved survival and neurological function after cardiac arrest/resuscitation. Moreover, hydrogen-rich saline treatment dose dependently ameliorated brain injury after cardiac arrest/resuscitation, which was characterized by the increase of survival neurons in hippocampus CA1, reduction of brain edema in cortex and hippocampus, preservation of blood-brain barrier integrity, as well as the decrease of serum S100ß and neuron-specific enolase. Furthermore, we found that the beneficial effects of hydrogen-rich saline treatment were associated with decreased levels of oxidative products (8-iso-prostaglandin F2α and malondialdehyde) and inflammatory cytokines (tumor necrosis factor-α, interleukin-1ß, and high-mobility group box protein 1), as well as the increased activity of antioxidant enzymes (superoxide dismutase and catalase) in serum and brain tissues. In addition, hydrogen-rich saline treatment reduced caspase-3 activity in cortex and hippocampus after cardiac arrest/resuscitation. CONCLUSIONS: Hydrogen-rich saline treatment improved survival and neurological outcome after cardiac arrest/resuscitation in rats, which was partially mediated by reducing oxidative stress, inflammation, and apoptosis.

Engineering a Novel Modular Adenoviral mRNA Delivery Platform Based on Tag/Catcher Bioconjugation.

mRNA vaccines have attracted widespread research attention with clear advantages in terms of molecular flexibility, rapid development, and potential for personalization. However, current mRNA vaccine platforms have not been optimized for induction of CD4/CD8 T cell responses. In addition, the mucosal administration of mRNA based on lipid nanoparticle technology faces challenges in clinical translation. In contrast, adenovirus-based vaccines induce strong T cell responses and have been approved for intranasal delivery. To leverage the inherent strengths of both the mRNA and adenovirus platforms, we developed a novel modular adenoviral mRNA delivery platform based on Tag/Catcher bioconjugation. Specifically, we engineered adenoviral vectors integrating Tag/Catcher proteins at specific locales on the Ad capsid proteins, allowing us to anchor mRNA to the surface of engineered Ad viruses. In proof-of-concept studies, the Ad-mRNA platform successfully mediated mRNA delivery and could be optimized via the highly flexible modular design of both the Ad-mRNA and protein bioconjugation systems.

In vivo editing of the pan-endothelium by immunity evading simian adenoviral vector.

Biological applications deriving from the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 site-specific nuclease system continue to impact and accelerate gene therapy strategies. Safe and effective in vivo co-delivery of the CRISPR/Cas9 system to target somatic cells is essential in the clinical therapeutic context. Both non-viral and viral vector systems have been applied for this delivery matter. Despite elegant proof-of-principle studies, available vector technologies still face challenges that restrict the application of CRISPR/Cas9-facilitated gene therapy. Of note, the mandated co-delivery of the gene-editing components must be accomplished in the potential presence of pre-formed anti-vector immunity. Additionally, methods must be sought to limit the potential of off-target editing. To this end, we have exploited the molecular promiscuities of adenovirus (Ad) to address the key requirements of CRISPR/Cas9-facilitated gene therapy. In this regard, we have endeavored capsid engineering of a simian (chimpanzee) adenovirus isolate 36 (SAd36) to achieve targeted modifications of vector tropism. The SAd36 vector with the myeloid cell-binding peptide (MBP) incorporated in the capsid has allowed selective in vivo modifications of the vascular endothelium. Importantly, vascular endothelium can serve as an effective non-hepatic cellular source of deficient serum factors relevant to several inherited genetic disorders. In addition to allowing for re-directed tropism, capsid engineering of nonhuman primate Ads provide the means to circumvent pre-formed vector immunity. Herein we have generated a SAd36. MBP vector that can serve as a single intravenously administered agent allowing effective and selective in vivo editing for endothelial target cells of the mouse spleen, brain and kidney. DATA AVAILABILITY: The data that support the findings of this study are available from the corresponding author upon reasonable request.

Efficient Genome Editing Achieved via Plug-and-Play Adenovirus Piggyback Transport of Cas9/gRNA Complex on Viral Capsid Surface.

The capacity to efficiently deliver the gene-editing enzyme complex to target cells is favored over other forms of gene delivery as it offers one-time hit-and-run gene editing, thus improving precision and safety and reducing potential immunogenicity against edited cells in clinical applications. Here we performed a proof-of-mechanism study and demonstrated that a simian adenoviral vector for DNA delivery can be repurposed as a robust intracellular delivery platform for a functional Cas9/guide RNA (gRNA) complex to recipient cells. In this system, the clinically relevant adenovirus was genetically engineered with a plug-and-display technology based on SpyTag003/SpyCatcher003 coupling chemistry. Under physiological conditions, an off-the-shelf mixture of viral vector with SpyTag003 incorporated into surface capsid proteins and Cas9 fused with SpyCatcher003 led to a rapid titration reaction yielding adenovirus carrying Cas9SpyCatcher003 on the virus surface. The Cas9 fusion protein-conjugated viruses in the presence of a reporter gRNA delivered gene-editing functions to cells with an efficiency comparable to that of a commercial CRISPR/Cas9 transfection reagent. Our data fully validate the adenoviral "piggyback" approach to deliver an intracellularly acting enzyme cargo and, thus, warrant the prospect of engineering tissue-targeted adenovirus carrying Cas9/gRNA for in vivo gene editing.

Advanced genetic engineering to achieve in vivo targeting of adenovirus utilizing camelid single domain antibody.

For the developing field of gene therapy the successful address of the basic requirement effective gene delivery has remained a critical barrier. In this regard, the "Holy Grail" vector envisioned by the field's pioneers embodied the ability to achieve efficient and specific in vivo gene delivery. Functional linkage of antibody selectivity with viral vector efficiency represented a logical strategy but has been elusive. Here we have addressed this key issue by developing the technical means to pair antibody-based targeting with adenoviral-mediated gene transfer. Our novel method allows efficient and specific gene delivery. Importantly, our studies validated the achievement of this key vectorology mandate in the context of in vivo gene delivery. Vectors capable of effective in vivo delivery embody the potential to dramatically expand the range of successful gene therapy cures.

Comparison of two vasopressor protocols for preventing hypotension post-spinal anesthesia during cesarean section: a randomized controlled trial.

BACKGROUND: Norepinephrine infusion decreases hypotension after spinal anesthesia during cesarean section. This study aimed to compare the efficacy of norepinephrine infusion and ephedrine bolus against post-spinal hypotension in parturients. METHODS: In this double-blinded, randomized controlled clinical trial, parturients scheduled for elective cesarean section were randomly allocated to receive norepinephrine infusion (0.05 µg·kg-1·min-1) just before spinal anesthesia continuing for 30 min or ephedrine bolus (0.15 mg/kg) just before spinal anesthesia. A rescue bolus (5 µg norepinephrine for the norepinephrine group, and 5 mg ephedrine for the ephedrine group) was administered whenever hypotension occurred. Our primary outcome was the incidence of hypotension within 30 min of spinal anesthesia administration. Secondary outcomes included maternal and neonatal outcomes 30 min after spinal block, and neonatal cerebral oxygenation 10 min after birth. RESULTS: In total, 190 patients were enrolled; of these patients, 177 were included in the final analysis. Fewer patients suffered hypotension in the norepinephrine group than in the ephedrine group (29.5% vs. 44.9%, odds ratio [OR]: 0.51, 95% confidence interval [CI]: 0.28-0.95, P = 0.034). Moreover, the tachycardia frequency was lower in the norepinephrine group than in the ephedrine group (OR: 0.22, 95% CI: 0.11-0.44, P < 0.001), and patients suffered less nausea and vomiting (OR: 0.28, 95% CI: 0.11-0.70, P = 0.004). There was no difference in Apgar scores and umbilical arterial blood gas analysis between the two groups. However, neonatal cerebral regional saturations were significantly higher after birth in the norepinephrine group than in the ephedrine group (mean difference: 2.0%, 95% CI: 0.55%-3.45%, P = 0.008). CONCLUSION: In patients undergoing elective cesarean section with spinal anesthesia, norepinephrine infusion compared to ephedrine bolus resulted in less hypotension and tachycardia, and exhibited potential neonatal benefits. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02542748; https://clinicaltrials.gov/ct2/show/record/NCT02542748.

Targeting Tumor Neoangiogenesis via Targeted Adenoviral Vector to Achieve Effective Cancer Gene Therapy for Disseminated Neoplastic Disease.

The application of cancer gene therapy has heretofore been restricted to local, or locoregional, neoplastic disease contexts. This is owing to the lack of gene transfer vectors, which embody the requisite target cell selectivity in vivo required for metastatic disease applications. To this end, we have explored novel vector engineering paradigms to adapt adenovirus for this purpose. Our novel strategy exploits three distinct targeting modalities that operate in functional synergy. Transcriptional targeting is achieved via the hROBO4 promoter, which restricts transgene expression to proliferative vascular endothelium. Viral binding is modified by incorporation of an RGD4C peptide in the HI loop of the fiber knob for recognition of cellular integrins. Liver sequestration is mitigated by ablation of factor X binding to the major capsid protein hexon by a serotype swap approach. The combination of these technologies into the context of a single-vector agent represents a highly original approach. Studies in a murine model of disseminated cancer validated the in vivo target cell selectivity of our vector agent. Of note, clear gains in therapeutic index accrued these vector modifications. Whereas there is universal recognition of the value of vector targeting, very few reports have validated its direct utility in the context of cancer gene therapy. In this regard, our article validates the direct gains that may accrue these methods in the stringent delivery context of disseminated neoplastic disease. Efforts to improve vector targeting thus represent a critical direction to fully realize the promise of cancer gene therapy.

Cold shock domain family members YB-1 and MSY4 share essential functions during murine embryogenesis.

Three cold shock domain (CSD) family members (YB-1, MSY2, and MSY4) exist in vertebrate species ranging from frogs to humans. YB-1 is expressed throughout embryogenesis and is ubiquitously expressed in adult animals; it protects cells from senescence during periods of proliferative stress. YB-1-deficient embryos die unexpectedly late in embryogenesis (embryonic day 18.5 [E18.5] to postnatal day 1) with a runting phenotype. We have now determined that MSY4, but not MSY2, is also expressed during embryogenesis; its abundance declines substantially from E9.5 to E17.5 and is undetectable on postnatal day 1(adult mice express MSY4 in testes only). Whole-mount analysis revealed similar patterns of YB-1 and MSY4 RNA expression in E11.5 embryos. To determine whether MSY4 delays the death of YB-1-deficient embryos, we created and analyzed MSY4-deficient mice and then generated YB-1 and MSY4 double-knockout embryos. MSY4 is dispensable for normal development and survival, but the testes of adult mice have excessive spermatocyte apoptosis and seminiferous tubule degeneration. Embryos doubly deficient for YB-1 and MSY4 are severely runted and die much earlier (E8.5 to E11.5) than YB-1-deficient embryos, suggesting that MSY4 indeed shares critical cellular functions with YB-1 in the embryonic tissues where they are coexpressed.

Long-term correction of hemophilia B using adenoviral delivery of CRISPR/Cas9.

Hemophilia B (HB) is a life-threatening inherited disease caused by mutations in the FIX gene, leading to reduced protein function and abnormal blood clotting. Due to its monogenic nature, HB is one of the primary targets for gene therapy. Indeed, successful correction of HB has been shown in clinical trials using gene therapy approaches. However, application of these strategies to non-adult patients is limited due to high cell turnover as young patients develop, resulting in vector dilution and subsequent loss of therapeutic expression. Gene editing can potentially overcome this issue by permanently inserting the corrective gene. Integration allows replication of the therapeutic transgene at every cell division and can avoid issues associated with vector dilution. In this study, we explored adenovirus as a platform for corrective CRISPR/Cas9-mediated gene knock-in. We determined as a proof-of-principle that adenoviral delivery of CRISPR/Cas9 is capable of corrective gene addition, leading to long-term augmentation of FIX activity and phenotypic correction in a murine model of juvenile HB. While we found on-target error-free integration in all examined samples, some mice also contained mutations at the integration target site. Additionally, we detected adaptive immune responses against the vector and Cas9 nuclease. Overall, our findings show that the adenovirus platform is suitable for gene insertion in juveniles with inherited disease, suggesting this approach may be applicable to other diseases.

Defining a murine ovarian cancer model for the evaluation of conditionally-replicative adenovirus (CRAd) virotherapy agents.

BACKGROUND: Virotherapy represents a promising approach for ovarian cancer. In this regard, conditionally replicative adenovirus (CRAd) has been translated to the context of human clinical trials. Advanced design of CRAds has sought to exploit their capacity to induce anti-tumor immunization by configuring immunoregulatory molecule within the CRAd genome. Unfortunately, employed murine xenograft models do not allow full analysis of the immunologic activity linked to CRAd replication. RESULTS: We developed CRAds based on the Ad5/3-Delta24 design encoding cytokines. Whereas the encoded cytokines did not impact adversely CRAd-induced oncolysis in vitro, no gain in anti-tumor activity was noted in immune-incompetent murine models with human ovarian cancer xenografts. On this basis, we explored the potential utility of the murine syngeneic immunocompetent ID8 ovarian cancer model. Of note, the ID8 murine ovarian cancer cell lines exhibited CRAd-mediated cytolysis. The use of this model now enables the rational design of oncolytic agents to achieve anti-tumor immunotherapy. CONCLUSIONS: Limits of widely employed murine xenograft models of ovarian cancer limit their utility for design and study of armed CRAd virotherapy agents. The ID8 model exhibited CRAd-induced oncolysis. This feature predicate its potential utility for the study of CRAd-based virotherapy agents.

Neurons can upregulate Cav-1 to increase intake of endothelial cells-derived extracellular vesicles that attenuate apoptosis via miR-1290.

Extracellular vesicles (EVs) including exosomes can serve as mediators of cell-cell communication under physiological and pathological conditions. However, cargo molecules carried by EVs to exert their functions, as well as mechanisms for their regulated release and intake, have been poorly understood. In this study, we examined the effects of endothelial cells-derived EVs on neurons suffering from oxygen-glucose deprivation (OGD), which mimics neuronal ischemia-reperfusion injury in human diseases. In a human umbilical endothelial cell (HUVEC)-neuron coculture assay, we found that HUVECs reduced apoptosis of neurons under OGD, and this effect was compromised by GW4869, a blocker of exosome release. Purified EVs could be internalized by neurons and alleviate neuronal apoptosis under OGD. A miRNA, miR-1290, was highly enriched in HUVECs-derived EVs and was responsible for EV-mediated neuronal protection under OGD. Interestingly, we found that OGD enhanced intake of EVs by neurons cultured in vitro. We examined the expression of several potential receptors for EV intake and found that caveolin-1 (Cav-1) was upregulated in OGD-treated neurons and mice suffering from middle cerebral artery occlusion (MCAO). Knock-down of Cav-1 in neurons reduced EV intake, and canceled EV-mediated neuronal protection under OGD. HUVEC-derived EVs alleviated MCAO-induced neuronal apoptosis in vivo. These findings suggested that ischemia likely upregulates Cav-1 expression in neurons to increase EV intake, which protects neurons by attenuating apoptosis via miR-1290.

YB-1 is important for late-stage embryonic development, optimal cellular stress responses, and the prevention of premature senescence.

Proteins containing "cold shock" domains belong to the most evolutionarily conserved family of nucleic acid-binding proteins known among bacteria, plants, and animals. One of these proteins, YB-1, is widely expressed throughout development and has been implicated as a cell survival factor that regulates the transcription and/or translation of many cellular growth and death-related genes. For these reasons, YB-1 deficiency has been predicted to be incompatible with cell survival. However, the majority of YB-1(-/-) embryos develop normally up to embryonic day 13.5 (E13.5). After E13.5, YB-1(-/-) embryos exhibit severe growth retardation and progressive mortality, revealing a nonredundant role of YB-1 in late embryonic development. Fibroblasts derived from YB-1(-/-) embryos displayed a normal rate of protein synthesis and minimal alterations in the transcriptome and proteome but demonstrated reduced abilities to respond to oxidative, genotoxic, and oncogene-induced stresses. YB-1(-/-) cells under oxidative stress expressed high levels of the G(1)-specific CDK inhibitors p16Ink4a and p21Cip1 and senesced prematurely; this defect was corrected by knocking down CDK inhibitor levels with specific small interfering RNAs. These data suggest that YB-1 normally represses the transcription of CDK inhibitors, making it an important component of the cellular stress response signaling pathway.

[Ischemic postconditioning induces neuroprotection via up-regulation of endogenous antioxidant enzyme activities: experiment with rabbits].

OBJECTIVE: To test the hypothesis that spinal cord protection induced by ischemic postconditioning is mediated by an increase of endogenous antioxidant enzyme activities during reperfusion phase in spinal cord. METHODS: Seventy-eight male New Zealand rabbits were randomly divided into 3 groups: Sham group (n = 18) undergoing sham operation without aortic occlusion; ischemia/reperfusion (I/R) group (n = 30) undergoing occlusion of the infrarenal abdominal aorta for 20 min, followed by reperfusion; and postconditioning (PostC) group (n = 30) undergoing occlusion of the infrarenal abdominal aorta for 20 min followed by 3 cycles of 30 s reperfusion/30 s ischemia just at the onset of reperfusion. 30 min, and 1, 3, 6, 24, and 48 h after reperfusion 5 rabbits from each group (and 3 from the Sham group) were killed with their spinal cords taken out, and spectrophotometric method was used to determine the antioxidant enzyme activity and malondialdehyde (MDA) content 6, 24, and 48 h after reperfusion motor function scoring of the hind limbs was conducted. RESULTS: (1) The motor function scores of the PostC group were significantly higher than those of the I/R group 6, 24, and 48 h after reperfusion (all P < 0.05). (2) The activities of superoxide dismutase (SOD) and catalase (CAT) in the spinal cord tissue of the PostC group 30 min-6 h after reperfusion were all significantly higher than those of the I/R group (all P < 0.05). There were no significant differences in the activity of glutathione peroxidase (GSH-px) at different time points between the PostC and I/R groups. (3) The MDA levels 24 h and 48 h after reperfusion of the PostC group were both significantly lower than that of the I/R group (both P < 0.01). CONCLUSION: Ischemic postconditioning shows effect against spinal cord ischemic-reperfusion injury mediated, at least partially, by up-regulating the activities of SOD and CAT in spinal cord during early reperfusion phase.

[Neuroprotective effects of combined application of JAK-STAT signal pathway inhibitor and free radical scavenger on focal cerebral ischemia/reperfusion injury in rats].

OBJECTIVE: To investigate the neuroprotective effects and dose-response relation by combining JAK-STAT signal pathway inhibitor (AG490) with free radical scavenger dimethylthiourea (DMTU) in rats subjected to focal cerebral ischemia/reperfusion (I/R) injury. METHODS: In all rats, the middle cerebral artery occlusion (MCAO) was produced by occlusion of right internal carotid artery with a nylon monofilament. One hundred male Sprague-Dawley (SD) rats were divided into ten groups according to random digits table, 10 rats were in each group. The first experiment involved I/R model control, dimethyl sulfoxide (DMSO) control, normal saline (NS) control, AG490, DMTU and combination of AG490 and DMTU (A+D) groups. The second experiment involved model group and three experimental groups in which various doses of DMTU and AG490 were administered. The neurological behavior scores (NBS) were assessed at 24, 48 and 72 hours after reperfusion respectively in both experiments, and all the animals were then decapitated to determine the brain infarct volume after 72 hours. RESULTS: The values of NBS in A+D group, AG490 group and DMTU group were higher than those in model group at 24, 48 and 72 hours after I/R, and their brain infarct volumes were obviously smaller than model group as well (all P<0.05). The brain infarct volume in A+D group was obviously smaller compared with AG490 and DMTU alone (all P<0.05). The values of NBS were higher and the brain infarct volumes were smaller in both high dose and medium dose combination groups than those in low dose combination and model groups respectively (all P<0.05). In addition, brain infarct volumes in high dose group were smaller than medium dose group (P<0.05), but there was no statistically significant difference between low dose and model groups. CONCLUSION: The combined application of AG490 and DMTU produces a dose-dependent synergistic neuroprotective effect.