Rapidly progressing central-type primitive neuroectodermal tumor of the ovary: a case report.

Primitive neuroectodermal tumors (PNET) belong to a group of highly malignant tumors comprised of small round cells of neu- roectodermal origin. These tumors can be either of peripheral-type (Ewing family tumors/PNET) or central-type. A number of case re- ports have described PNET involving the gynecologic organs and the prognosis is generally poor. The authors describe the case of a 53-year-old woman with a rapidly progressing central-type PNET of the ovary.

Urinary neutrophil gelatinase-associated lipocalin for diagnosis and estimating activity in lupus nephritis: a meta-analysis.

Urinary neutrophil gelatinase-associated lipocalin (uNGAL) is relatively specific in lupus nephritis (LN) patients. However, its diagnostic value has not been evaluated. The aim of this review was to determine the value of uNGAL for diagnosis and estimating activity in LN. A comprehensive search was performed on PubMed, EMBASE, Web of Knowledge, Cochrane electronic databases through December 2014. Meta-analysis of sensitivity and specificity was performed with a random-effects model. Additionally, summary receiver operating characteristic (SROC) curves and area under the curve (AUC) values were calculated. Fourteen studies were selected for this review. With respect to diagnosing LN, the pooled sensitivity and specificity were 73.6% (95% confidence interval (CI), 61.9-83.3) and 78.1% (95% CI, 69.0-85.6), respectively. The SROC-AUC value was 0.8632. Regarding estimating LN activity, the pooled sensitivity and specificity were 66.2% (95% CI, 60.4-71.7) and 62.1% (95% CI, 57.9-66.3), respectively. The SROC-AUC value was 0.7583. In predicting renal flares, the pooled sensitivity and specificity were 77.5% (95% CI, 68.1-85.1) and 65.3% (95% CI, 60.0-70.3), respectively. The SROC-AUC value was 0.7756. In conclusion, this meta-analysis indicates that uNGAL has relatively fair sensitivity and specificity in diagnosing LN, estimating LN activity and predicting renal flares, suggesting that uNGAL is a potential biomarker in diagnosing LN and monitoring LN activity.

[Scoping review on prevention of central venous catheter-related bloodstream infection in burn patients].

Objective: To investigate the research trend and scope of prevention of central venous catheter-related bloodstream infection (CRBSI) in burn patients. Methods: The scoping review method was adopted. Pre-retrieval was carried out with search terms of ", , " and "central venous catheter, infection, catheter-related bloodstream infection, burn". On the basis of pre-retrieval, different retrieval formulas were formulated to retrieve researches related to central venous CRBSI in burn patients in China National Knowledge Internet, Wanfang Database, VIP Database, PubMed, Embase, CINAHL, and Cochrane Library from the establishment of each database to August 2020. Data were extracted from the included literature, including the first author, research publication time, research country, research type, diagnosis basis and intervention measures of central venous CRBSI, research sample selection, incidence related to infection, and research conclusion. Results: A total of 20 randomized controlled trials, quasi-experimental studies, case-control studies, cohort studies, and implementation researches published in 1990-2020 were included in this study with the first authors from China, the United States of America, or Argentina. The diagnostic bases for central venous CRBSI in burn patients were not uniform in the included literature, including adopting the Guidelines of American Centers for Disease Control and Prevention, Diagnostic Criteria for Nosocomial Infection, and other diagnostic criteria without specifying the source. The intervention measures included the use of new materials such as antibiotics coated catheter and ethanol impregnated port protectors, multidisciplinary cooperation, and comprehensive preventive measures. The sample size in the included literature was small, and the sample selection was different, including the number of patients and the the number of placement of central venous catheter. The outcome indicators for infection in the included literature were diversified. The incidence per 1 000 days of central venous CRBSI was 20.41‰-29.1‰ of patients in control group in China, the incidence per 1 000 days of central venous CRBSI was mostly <16.6‰ in control group in foreign countries, and the incidence of central venous CRBSI was decreased to varying degrees after implementing the corresponding intervention measures. Related research conclusions showed that new materials, multidisciplinary cooperation, and comprehensive preventive measures had good effects on prevention of central venous CRBSI in burn patients. Conclusions: The researches on prevention of central venous CRBSI in burn patients in China start early and the research types are diversified. The diagnostic criteria of central venous CRBSI in burn patients are not uniform, intervention measures have shifted from standardizing relevant operational measures to exploring the prevention effects of new materials, multidisciplinary cooperation, and multiple measures, and the latter has good effects on preventing central venous CRBSI in burn patients.

Safety evaluation of zinc methionine in laying hens: Effects on laying performance, clinical blood parameters, organ development, and histopathology.

The study was conducted to investigate whether high-dose zinc methionine (Zn-Met) affected the safety of laying hens, including laying performance, hematological parameters, serum chemical parameters, organ index, and histopathology. A total of 540 20-week-old Hy-Line White laying hens was randomly allocated to 6 groups with 6 replicates of 15 birds each. Birds were fed diets supplemented with 0 (control), 70, 140, 350, 700, or 1,400 mg Zn/kg diet as Zn-Met. The experiment lasted for 8 wk after a 2-week acclimation period. Results showed that dietary supplementation with 70 or 140 mg Zn/kg diet as Zn-Met significantly increased average daily egg mass (ADEM), laying rate (LR), and feed conversion ratio (FCR) (P < 0.05) and lowered broken and soft-shelled egg ratio (BSER) (P < 0.05) in comparison with the control group; no significant differences were detected among hens fed with 0, 350, or 700 mg Zn/kg as Zn-Met (P > 0.05); hens administered 1,400 mg Zn/kg showed a significant increase in BSER and remarkable decreases in ADEM, LR, and FCR (P < 0.001). There were no significant differences among hens receiving 0, 70, 140, 350, or 700 mg Zn/kg as Zn-Met in serum chemical parameters (P > 0.05); supplementation with 1,400 mg Zn/kg as Zn-Met remarkably elevated the concentrations of serum total bilirubin (TBILI), glucose (GLU), uric acid (UA), and creatinine (CRE) (P < 0.001), and enhanced activities of serum glutamic oxalacetic transaminase (GOP) and alkaline phosphatase (AKP) (P < 0.001) compared with the control group. No significant histopathological changes were found in hens administered 0, 70, 140, 350, or 700 mg Zn/kg as Zn-Met, while significant histological lesions were observed in the heart, liver, lung, and kidney tissues of hens receiving 1,400 mg Zn/kg as Zn-Met. No significant differences were detected in hematological parameters or organ index (P > 0.05). In conclusion, a nominal Zn concentration of 700 mg/kg as Zn-Met is considered to be no-observed-adverse-effect level following daily administration to hens for 56 days.

The bone marrow mononuclear cells reduce the oxidative stress of cerebral infarction through PI3K/AKT/NRF2 signaling pathway.

OBJECTIVE: To evaluate the mechanism of bone marrow mononuclear cells (BMMNCs) in reducing the oxidative stress after cerebral infarction through PI3K/AKT/NRF2 signaling pathway. MATERIALS AND METHODS: 96 healthy SD rats, which were 6-8-week old, weighting about 250-280 g, were selected for the study. The middle cerebral artery occlusion model (MCAO) was established in SD rats using the suture method. The rats were randomly divided into sham operation group, model group, BMMNCs group and PI3K inhibitor group. 24 rats in each group were selected. 200 µl phosphate-buffered saline (PBS) solution was injected into the caudal vein of the rats in the model group, 200 µl PBS solution containing 5×106 BMMNCs that obtained by gradient centrifugation was injected into the rats in the BMMNCs group, meanwhile, in the PI3K inhibitor group, LY294002 (10 mmol/L/kg) was injected into the lateral ventricle of the brain. After the 3d, 7d and 14d, the modified neurological severity scores (mNSS) were used to evaluate the neurological function. The volume of cerebral infarction was assessed by TTC staining, the VEGF, BDNF, TNF-α, IL-1ß, malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels were detected by ELISA. RESULTS: The mNSS and the volume of cerebral infarction of the model group were significantly higher than those of the sham operation group (p<0.05), while the mNSS and the volume of cerebral infarction of the BMMNCs group were lower than those of the model group, higher than those of the sham operation group (p<0.05). The VEGF, BDNF, TNF-α, IL-1ß, MDA, SOD and GSH-Px levels of the model group were significantly higher than those of the sham operation group (p<0.05). CONCLUSIONS: BMMNCs can reduce the oxidative stress, apoptosis, and inflammatory reaction through PI3K/AKT/NRF2 signaling pathway, thus promoting the secretion of nerve and vascular cytokines, improving the neurological function and reducing the infarct scope.

[Role of sleep apnea monitoring management platform in the treatment of patients with obstructive sleep apnea hypopnea syndrome].

Objective:To investigate the clinical significance and value of the sleep apnea monitoring management platform in the treatment of patients with obstructive sleep apnea hypopnea syndrome (OSAHS) by comparing with the traditional continuous positive airway pressure (CPAP) card reader mode.Method:A total of 48 severe adult OSAHS patients from Department of Otorhinolaryngology Head and Neck Surgery-Sleep Medical Center of the Third People's Hospital of Honghe during the period of Nov. 2015 to Aug. 2016 were collected in this prospective study. All of them were diagnosed by PSG and treated with nasal continuous positive airway pressure. They were randomly divided into group A (n=24) and group B (n=24). Group A and B were treated with the traditional CPAP card reader mode and the sleep apnea monitoring management platform respectively. During the follow-up, the compliance, mean blood oxygen saturation, titration pressure, Epworth sleepiness scale after 1, 3, 6 and 12 month treatment were compared between two groups. Ttest was used to analyze the difference. Statistical significance was set at P <0.05.Result:Statistical analysis showed that there were significant difference in the compliance of using CPAP, mean blood oxygen saturation and Epworth sleepiness scale score between the two groups (P < 0.05), but the titration pressure had no difference between the two groups (P>0.05).Conclusion:Compared the sleep apnea monitoring management platform with traditional CPAP card reader mode in the treatment of OSAHS patients, the former could solve the problems during the CPAP use in time, improve the compliance of using CPAP, and which could increase the efficacy of CPAP. Thus, the sleep apnea monitoring management platform is of more clinical value and deserve promotion.

Evidence for regulation of transcription and replication of the human neurotropic virus JCV genome by the human S(mu)bp-2 protein in glial cells.

Glial factor 1 (GF-1) is a partial cDNA isolated from a human brain cDNA library which encodes a truncated protein with binding ability to the B-regulatory domain of the human neurotropic virus, JCV. GF-1 exhibits sequence homology to the central region of the newly identified human DNA-binding protein S(mu)bp-2. GF-1 appears to be a partial cDNA for human S(mu)bp-2 based on its sequence homology to S(mu)bp-2 and their chromosomal co-localization. In this report, we have employed transfection assay and have compared the ability of GF-1 and its full-length form, S(mu)bp-2, on regulating the activity of JCV promoters in glial cells. Our results demonstrate that, unlike GF-1 which stimulates JCV early promoter in glial cells, overexpression of S(mu)bp-2 exhibits no drastic effect on the transcription of the viral early promoter. The activity of the viral late promoter was noticeably increased by both GF-1 and S(mu)bp-2, although the level of induction by GF-1 was consistently higher than that detected by S(mu)bp-2. Use of deletion constructs in co-transfection assay revealed that the B-domain of the JCV promoter is required for transcriptional activation by GF-1 and S(mu)bp-2. Expression of GF-1 and S(mu)bp-2 in glial cells increased the induced level of JCV late gene transcription by the viral early protein, T-antigen. Examination of the viral DNA replication by DpnI assay indicated that, unlike GF-1, S(mu)bp-2 has the ability to decrease the level of JCV DNA replication in glial cells. These observations suggest that the N-terminal portion of S(mu)bp-2 which encompasses several helicase motifs and/or its C-terminus, both of which are missing in GF-1, may confer differential effects on viral gene transcription and replication. The biological importance of our findings in regulation of the JCV lytic cycle in glial cells is discussed.

Chairside sensor for rapid monitoring of Enterococcus faecalis activity.

In this study, optical spectroscopy was used to monitor a chromogenic, enzyme-substrate reaction for the rapid identification of Enterococcus faecalis. The detection system, comprising a miniature spectrophotometer and an accompanying data acquisition system, was placed in an incubator. During testing, a 3-ml test sample was placed in a cuvette within the spectrophotometer. This permitted online, real-time, and remote analysis of spectral signature needed to monitor the bacteria. It was observed that the absorption peak intensity increased conspicuously 3.5 h after inoculation and through the entire period of testing. A linear-regression analysis demonstrated a significant correlation between the increase in absorption peak intensity at 610 nm (r = 0.9389) and 653 nm (r = 0.9387) with the formation of colony-forming units. Optical spectroscopy-based sensing systems can pave the way for rapid, nonlaboratory-based approaches to monitor microbial status quantitatively and qualitatively from clinical samples.

Prophylactic treatment of dens evaginatus using mineral trioxide aggregate.

Two case reports with dens evaginatus are presented. Each patient had one tooth affected. There was a prominent tubercle on the occlusal surface of the mandibular second premolar. Under local anesthesia and rubber dam isolation a partial pulpotomy was conducted and mineral trioxide aggregate was placed. After 6 months the teeth were removed as part of planned orthodontic treatment. Histological examination of these teeth showed an apparent continuous dentin bridge formation in both teeth, and the pulps were free of inflammation. These cases show that mineral trioxide aggregate can be used as an alternative to existing materials in the proplylactic treatment of dens evaginatus.

Risk factors of adverse drug reaction caused by nimesulide in Shanghai patients with osteoarthropathy.

The aim of this study was to investigate the risk factors for adverse drug reactions (ADRs) to nimesulide in patients from Shanghai with osteoarthropathy. A retrospective epidemiological study was performed to obtain information (observational variables) on demographics, primary disease, family history of disease, quality of life, dietary habits, lifestyle, use of nonsteroidal anti-inflammatory drugs (NSAIDs) and ADR history of NSAIDs. Univariate and multivariate analyses were performed to establish the relationship between these observational variables and the occurrence of ADRs caused by nimesulide. Among the 726 variables, five risk factors for ADRs to nimesulide were identified. The study showed an increased risk for ADR occurrence with increased scoring of the following four factors: (i) "Concomitant drug therapy" (odds ratio [OR]: 4.66, 95% confidence intervals [CI]: 1.26-17.26, p < 0.05); (ii) "Compared with six months ago, how would you rate your health in general now?" (OR: 1.38, 95% CI: 1.03-1.84, p < 0.05); (iii) "General feeling of health status" (OR: 1.27, 95% CI: 1.03-1.56, p < 0.05) and (iv) "1 expect my health to get worse" (OR: 2.05, 95% CI: 1.22-3.44, p < 0.01). There was a decreased risk for ADR occurrence with increased scoring of the factor "Have you ever suffered from depression that impacted on your life?" (OR: 0.15, 95% CI: 0.03-0.66, p < 0.05). The predictive model for the overall incidence rate of ADRs caused by nimesulide was then established. In conclusion, the predictive model helps to indicate the risk of ADRs to nimesulide and provides clinicians with an alternative method for decision making when prescribing this drug.

[Effect of recombinant human interleukin-1 beta on K+ channels of mice bone marrow stromal cells].

In the present study, both cell-attached and inside-out mode of patch clamp technique were applied to detect the effect of recombinant human interleukin-1 beta (IL-1 beta) on K+ channels of mice bone marrow stromal cells. A 16.7 pS voltage-dependent K+ channel resembling the delayed rectifier K+ channel in excitable cells was identified and characterized. Under stimulation of IL-1 beta (1000 U/ml), the single channel conductance increased to 26.1 +/- 3.6 pS (P < 0.01). IL-1 beta also markedly increased the open time constant and the open probability, but decreased the closed time constant as compared to the same patches without IL-1 beta. In addition, IL-1 beta induced multi-channel activities in about 80 percent patches by inducing more K+ channels of the same type open. These results suggest that activation of K+ channels plays a role in signal transduction of interleukin-1.

Combination therapy targeting ectopic ATP synthase and 26S proteasome induces ER stress in breast cancer cells.

F1Fo ATP synthase is present in all organisms and is predominantly located on the inner membrane of mitochondria in eukaryotic cells. The present study demonstrated that ATP synthase and electron transport chain complexes were ectopically expressed on the surface of breast cancer cells and could serve as a potent anticancer target. We investigated the anticancer effects of the ATP synthase inhibitor citreoviridin on breast cancer cells through proteomic approaches and revealed that differentially expressed proteins in cell cycle regulation and in the unfolded protein response were functionally enriched. We showed that citreoviridin triggered PERK-mediated eIF2α phosphorylation, which in turn attenuated general protein synthesis and led to cell cycle arrest in the G0/G1 phase. We further showed that the combination of citreoviridin and the 26S proteasome inhibitor bortezomib could improve the anticancer activity by enhancing ER stress, by ameliorating citreoviridin-caused cyclin D3 compensation, and by contributing to CDK1 deactivation and PCNA downregulation. More interestingly, the combined treatment triggered lethality through unusual non-apoptotic caspase- and autophagy-independent cell death with a cytoplasmic vacuolization phenotype. The results imply that by boosting ER stress, the combination of ATP synthase inhibitor citreoviridin and 26S proteasome inhibitor bortezomib could potentially be an effective therapeutic strategy against breast cancer.

Survival of root filled cracked teeth in a tertiary institution.

AIM: To assess the survival rate of root filled cracked teeth over a 2-year period in a tertiary institute. METHODOLOGY: Forty-nine patients who had root canal treatment completed on their cracked teeth at the National Dental Centre (Singapore) were recalled for a 2-year review. Collected review data included presence of periodontal pocketing, sinus tract and swelling associated with the teeth. The date of extraction was noted if a tooth was missing at review. Pre-treatment data collected were number, extent and location of crack, presence of periodontal pocketing, patients' age and gender, location of cracked teeth, type of teeth and presence of terminal cracked tooth. RESULTS: Fifty teeth in 49 patients were included. The Kaplan-Meier estimate of 2-year survival rate was 85.5% (95% confidence interval: 75.5-95.5). Cracked teeth which were the terminal teeth in the dental arch (RR = 4.9, 95% CI: 1.2-2.0, P = 0.04), teeth with pre-root filling periodontal pocketing (RR = 4.9, 95% CI: 1.2-2.0, P = 0.04) and teeth with multiple cracks (RR = infinity, 95% CI: 1.9-infinity, P = 0.01) were more likely to be extracted. CONCLUSIONS: Within the limitations of this study, multiple cracks, terminal teeth and pre-root filling pocketing were significant prognostic factors for the survival of root filled cracked teeth.

Regulatory effects of D2 receptors in the ventral tegmental area on the mesocorticolimbic dopaminergic pathway.

To investigate the regulatory effects of somatodendritic D2 receptors on the terminal's extracellular dopamine (DA) concentration, a D2 antagonist (eticlopride) was infused directly into the ventral tegmental area via a microdialysis probe in chloral hydrate-anesthetized rats. Extracellular DA changes in both the nucleus accumbens (N ACC) and the medial prefrontal cortex (mPFC) were monitored. Infusion of 10.0 fM eticlopride had no effect on DA in the mPFC (110.2 +/- 10.0% of baseline) but significantly increased DA in the N ACC (150.1 +/- 11.7%). Infusion of a higher dose of eticlopride (100.0 or 1,000.0 fM) significantly augmented the DA in the mPFC (121.1 +/- 7.6 and 180.7 +/- 25.8%, respectively) but surprisingly had no effect on DA in the N ACC (111.5 +/- 7.3 and 104.1 +/- 8.7%, respectively). To further investigate whether the bluntness of DA increase in the N ACC was due to DA receptor activation in the mPFC, eticlopride or SCH23390 was infused into the mPFC prior to and during intrategmental eticlopride infusion, and the change of DA in the N ACC was simultaneously monitored. During intra-mPFC 1.0 nM eticlopride infusion but not during 10.0 nM SCH23390 administration (95.5 +/- 6.1%), intrategmental 1,000.0 fM eticlopride infusion could further elevate DA in the N ACC (130.0 +/- 4.6%). Our results indicated that (1) the mesolimbic and the mesocortical pathways were under tonic inhibition by somatodendritic D2 receptors; (2) the DA concentration in the N ACC first increased and then returned to baseline while the intrategmental infusion dose of eticlopride increased; and (3) the bluntness of DA increase in the N ACC resulted from the D2 receptor activation in the mPFC.

Transcriptional regulation of human JC polyomavirus promoters by cellular proteins YB-1 and Pur alpha in glial cells.

Transcription of the human polyomavirus (JCV) genome is regulated by host cell proteins and the viral early protein, T antigen. A region called the lytic control element (LCE), located within the enhancer of JCV, is important for transcription of JCV early and late promoters. Earlier studies have led to the identification of two single-stranded DNA-binding proteins, YB-1 and Pur alpha, with the ability to interact with nucleotides on the early and late strands of LCE, respectively. Of particular interest is the notion that the unique interplay between these two cellular proteins and JCVT antigen determines their binding activities with the LCE. In this study, we employed a series of cotransfection experiments to evaluate the levels of transcription from JCV early and late promoters in the presence of YB-1, Pur alpha, and T antigen. Results from these studies indicated that Pur alpha stimulates JCV early and has little effect on the late promoter. Moreover, T antigen was able to decrease the induced level of early gene transcription by Pur alpha. On the other hand, the extent of transactivation of the viral late promoter by T antigen was reduced upon overexpression of Pur alpha in the transfected cells. These observations suggest that Pur alpha and T antigen exert an antagonistic effect on each other's regulatory action upon their responsive promoters. Of particular interest was the observation that YB-1 liberated T-antigen-induced late promoter activity from repression imposed by overexpression of pur alpha. Under similar conditions, overexpression of YB-1 showed no effect on the transcriptional activity of the early promoter in cells transfected with T-antigen- and Pur alpha-producing plasmids. On the basis of the data presented here and the previous binding results, a model is proposed which describes the potential role of Pur alpha, YB-1, and T antigen in differential expression of the viral genome during the lytic cycle.

In vitro antimicrobial effect of chlorhexidine-impregnated gutta percha points on Enterococcus faecalis.

AIM: To evaluate the in vitro antimicrobial effect of chlorhexidine-impregnated gutta percha points, Roeko activ point (Roeko, Langenau, Germany) on Enterococcus faecalis. METHODOLOGY: Human maxillary premolar roots were prepared with.04 rotary ProFile instruments to a master apical file size 40, autoclave-sterilized and then infected with E. faecalis (ATCC 29212) for 3 weeks. Baseline controls were carried out verifying negligible effects of plain gutta percha cones on E. faecalis. Subsequent to intracanal placement of calcium hydroxide, 'activ points' or saline (positive control) and the 2-week incubation in 54 root specimens, dentine sampling at depths of 100 and 250 micro m was carried out using.04 rotary ProFile instruments at sizes 60 and 90 to assess the quantity of bacteria present. Inactivating agents were used prior to sampling and the colony-forming units (CFU) of E. faecalis were then plate-counted after culturing. Statistical analysis was completed using the paired t-test. RESULTS: In comparison to the positive control, treatment with calcium hydroxide (P = 0.000 and 0.000) or activ points (P = 0.000 and 0.002) produced significantly lower colony counts of E. faecalis at dentine depths of 100 and 250 micro m, respectively. Calcium hydroxide (2.10 x 102 CFU mL-1) was significantly more effective than activ points (1.58 x 103 CFU mL-1) at 100 micro m (P = 0.013), but not at 250 micro m (P = 0.353). Neither of these two medications was able to eliminate E. faecalis completely. CONCLUSIONS: Chlorhexidine-impregnated activ points did not possess an in vitro inhibitory activity strong enough to eliminate E. faecalis completely from infected dentinal tubules.

Evidence that replication of human neurotropic JC virus DNA in glial cells is regulated by the sequence-specific single-stranded DNA-binding protein Pur alpha.

Initiation of polyomavirus DNA replication in eukaryotic cells requires the participation of the viral early protein T antigen, cellular replication factors, and DNA polymerases. The human polyomavirus JC virus (JCV) is the etiologic agent of the fatal demyelinating disease progressive multifocal leukoencephalopathy in immunocompromised individuals. This virus exhibits a narrow host range and a tissue specificity that restricts its replication to glial cells of the central nervous system. Restriction of viral DNA replication due to species specificity of the DNA polymerase, coupled with glial cell-specific transcription of the viral early promoter, is thought to account for the brain-specific replication of JCV. In this report we demonstrate that overexpression of Pur alpha, a protein which binds to single-stranded DNA in a sequence-specific manner, suppresses replication of JCV DNA in glial cells. Results from footprinting studies indicate that Pur alpha and T antigen share a common binding region spanning the single-stranded ori sequence of JCV. Further, T antigen was capable of stimulating the association of Pur alpha with the ori sequence in a band shift assay. Whereas no evidence for simultaneous binding of Pur alpha and T antigen to single-stranded DNA has been observed, results from coimmunoprecipitation and Western blot (immunoblot) analyses of proteins derived from cells producing JCV T antigen indicate a molecular association of JCV T antigen and Pur alpha. The binding of Pur alpha to the single-stranded ori sequence and its association with T antigen suggest that Pur alpha interferes with the activity of T antigen and/or other regulatory proteins to exert its negative effect on JCV DNA replication. The importance of these findings in the reactivation of JCV in the latently infected individual under immunosuppressed conditions is discussed.

Overexpression of cyclin A inhibits augmentation of recombinant adeno-associated virus transduction by the adenovirus E4orf6 protein.

The 34-kDa product of adenovirus E4 region open reading frame 6 (E4orf6) dramatically enhances transduction by recombinant adeno-associated virus vectors (rAAV). This is achieved by promoting the conversion of incoming single-stranded viral genomes into transcriptionally competent duplex molecules. The molecular mechanism for enhancing second-strand synthesis is not fully understood. In this study, we analyzed the cellular consequences of E4orf6 expression and the requirements for efficient rAAV transduction mediated by E4orf6. Expression of E4orf6 in 293 cells led to an inhibition of cell cycle progression and an accumulation of cells in S phase. This was preceded by specific degradation of cyclin A and p53, while the levels of other proteins involved in cell cycle control remained unchanged. In addition, the kinase activity of cdc2 was inhibited. We further showed that p53 expression is not necessary or inhibitory for augmentation of rAAV transduction by E4orf6. However, overexpression of cyclin A inhibited E4orf6-mediated enhancement of rAAV transduction. A cyclin A mutant incapable of recruiting protein substrates for cdk2 was unable to inhibit E4orf6-mediated augmentation. In addition, we created an E4orf6 mutant that is selectively defective in rAAV augmentation of transduction. Based on these findings, we suggest that cyclin A degradation represents a viral mechanism to disrupt cell cycle progression, resulting in enhanced viral transduction. Understanding the cellular pathways used during transduction will increase the utility of rAAV vectors in a wide range of gene therapy applications.