PAICS ubiquitination recruits UBAP2 to trigger phase separation for purinosome assembly.

Purinosomes serve as metabolons to enhance de novo purine synthesis (DNPS) efficiency through compartmentalizing DNPS enzymes during stressed conditions. However, the mechanism underpinning purinosome assembly and its pathophysiological functions remains elusive. Here, we show that K6-polyubiquitination of the DNPS enzyme phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthetase (PAICS) by cullin-5/ankyrin repeat and SOCS box containing 11 (Cul5/ASB11)-based ubiquitin ligase plays a driving role in purinosome assembly. Upon several purinosome-inducing cues, ASB11 is upregulated by relieving the H3K9me3/HP1α-mediated transcriptional silencing, thus stimulating PAICS polyubiquitination. The polyubiquitinated PAICS recruits ubiquitin-associated protein 2 (UBAP2), a ubiquitin-binding protein with multiple stretches of intrinsically disordered regions, thereby inducing phase separation to trigger purinosome assembly for enhancing DNPS pathway flux. In human melanoma, ASB11 is highly expressed to facilitate a constitutive purinosome formation to which melanoma cells are addicted for supporting their proliferation, viability, and tumorigenesis in a xenograft model. Our study identifies a driving mechanism for purinosome assembly in response to cellular stresses and uncovers the impact of purinosome formation on human malignancies.

CRM1-spike-mediated nuclear export of hepatitis B virus encapsidated viral RNA.

Hepatitis B virus (HBV) is a global pathogen. We report here that the cellular CRM1 machinery can mediate nuclear export of entire HBV core (HBc) particles containing encapsidated viral RNAs. Two CRM1-mediated nuclear export signals (NESCRM1) cluster at the conformationally flexible spike tips of HBc particles. Mutant NESCRM1 capsids exhibit strongly reduced associations with CRM1 and nucleoporin358 in vivo. CRM1 and NXF1 machineries mediate nuclear export of HBc particles independently. Inhibition of nuclear export has pleiotropic consequences, including nuclear accumulation of HBc particles, a significant reduction of encapsidated viral RNAs in the cytoplasm but not in the nucleus, and barely detectable viral DNA. We hypothesize an HBV life cycle where encapsidation of the RNA pregenome can initiate early in the nucleus, whereas DNA genome maturation occurs mainly in the cytoplasm. We identified a druggable target for HBV by blocking its intracellular trafficking.

Peptide-guided JC polyomavirus-like particles specifically target bladder cancer cells for gene therapy.

The ultimate goal of gene delivery vectors is to establish specific and effective treatments for human diseases. We previously demonstrated that human JC polyomavirus (JCPyV) virus-like particles (VLPs) can package and deliver exogenous DNA into susceptible cells for gene expression. For tissue-specific targeting in this study, JCPyV VLPs were conjugated with a specific peptide for bladder cancer (SPB) that specifically binds to bladder cancer cells. The suicide gene thymidine kinase was packaged and delivered by SPB-conjugated VLPs (VLP-SPBs). Expression of the suicide gene was detected only in human bladder cancer cells and not in lung cancer or neuroblastoma cells susceptible to JCPyV VLP infection in vitro and in vivo, demonstrating the target specificity of VLP-SPBs. The gene transduction efficiency of VLP-SPBs was approximately 100 times greater than that of VLPs without the conjugated peptide. JCPyV VLPs can be specifically guided to target particular cell types when tagged with a ligand molecule that binds to a cell surface marker, thereby improving gene therapy.

Gene therapy for castration-resistant prostate cancer cells using JC polyomavirus-like particles packaged with a PSA promoter driven-suicide gene.

Prostate cancer is the second most common cancer in men globally. Prostate cancer patients at advanced stages are usually treated with androgen deprivation therapy (ADT). However, with disease progression, it often becomes the incurable castration-resistant prostate cancer (CRPC). JC polyomavirus (JCPyV) is a human DNA virus. Its virus-like particles (VLPs) exhibit similar tropism to native virions and they are capable of delivering exogenous genes to the target cells for expression. JCPyV has been detected in prostate cells; therefore, prostate cancer cells may be susceptible to JCPyV infection and JCPyV VLPs may be used as a vector for gene therapy against prostate cancer. Here we constructed a plasmid (pPSAtk) that allows expression of the thymidine kinase suicide gene only in androgen receptor (AR) positive prostate cancer cells using the prostate-specific antigen (PSA) promoter, and used JCPyV VLPs as a vector to carry pPSAtk (PSAtk-VLPs) for transcriptional targeting in prostate cancer cells. In this study, we found that PSAtk-VLPs could only kill AR-positive CRPC 22Rv1 cells in vitro and inhibit the growth of tumor nodules in the xenograft mouse model. Our results reveal that PSAtk-VLPs could potentially be used as a new option for treating CRPC patients in the future.

Gene therapy for human glioblastoma using neurotropic JC virus-like particles as a gene delivery vector.

Glioblastoma multiforme (GBM), the most common malignant brain tumor, has a short period of survival even with recent multimodality treatment. The neurotropic JC polyomavirus (JCPyV) infects glial cells and oligodendrocytes and causes fatal progressive multifocal leukoencephalopathy in patients with AIDS. In this study, a possible gene therapy strategy for GBM using JCPyV virus-like particles (VLPs) as a gene delivery vector was investigated. We found that JCPyV VLPs were able to deliver the GFP reporter gene into tumor cells (U87-MG) for expression. In an orthotopic xenograft model, nude mice implanted with U87 cells expressing the near-infrared fluorescent protein and then treated by intratumoral injection of JCPyV VLPs carrying the thymidine kinase suicide gene, combined with ganciclovir administration, exhibited significantly prolonged survival and less tumor fluorescence during the experiment compared with controls. Furthermore, JCPyV VLPs were able to protect and deliver a suicide gene to distal subcutaneously implanted U87 cells in nude mice via blood circulation and inhibit tumor growth. These findings show that metastatic brain tumors can be targeted by JCPyV VLPs carrying a therapeutic gene, thus demonstrating the potential of JCPyV VLPs to serve as a gene therapy vector for the far highly treatment-refractory GBM.

In situ gelation of PEG-PLGA-PEG hydrogels containing high loading of hydroxyapatite: in vitro and in vivo characteristics.

Thermosensitive hydrogels are renowned carriers that are used to deliver a variety of drugs with the aim of combating diseases. In this study, the injectability of thermosensitive hydrogels comprised of poly(ethylene glycol)-poly(lactic acid-co-glycolic acid)-poly(ethylene glycol) (PEG-PLGA-PEG, PELGE) and hydroxyapatite (HA) were examined for their ability to deliver bone morphological protein 2 (BMP-2). The physicochemical characteristics of PELGE, HA, and PELGE/HA hydrogel composites were investigated by (1)H NMR, GPC, FTIR, XRD, SEM, and TEM. The rheological properties, injectability, in vitro degradation, and in vivo biocompatibility were investigated. The hydrogel with a weight ratio of 4:6 of polymer to HA was found to be resistant to auto-catalyzed degradation of acidic monomers (LA, GA) for a period of 70 days owing to the presence of alkaline HA. Injectability was quantitatively determined by the ejected weight of the hydrogel composite at room temperature and was a close match to the weight amount predetermined by the syringe pump. The results not only revealed that the PELGE/HA hydrogel composite presented a minor tissue response in the subcutis of ICR mice at eight weeks, but they also indicated an acceptable tolerance of the hydrogel composite in animals. Thus, PELGE/HA hydrogel composite is expected to be a promising injectable orthopedic substitute because of its desirable thermosensitivity and injectability.

Health-care utilization and costs in Taiwanese pediatric patients with asthma.

BACKGROUND: In many countries, the burden of asthma is sufficient to warrant recognition as a high-priority disorder in governmental health strategies. However, the components of the total health-care costs for pediatric patients with asthma have not been well studied, and an overall understanding of health-care utilization patterns in this population is lacking in Taiwan. METHODS: A total of 33 461 patients aged 3-17 years who were enrolled in the National Health Insurance Research database from 1 January to 31 December 2002 were evaluated. Health-care utilization and costs, including those related to office, outpatient hospital, emergency department, and inpatient hospital visits were compared between pediatric patients with and without asthma. RESULTS: In 2002, the period prevalence of treated asthma was 6.0%. Pediatric patients with asthma used substantially more services than did those without asthma in all categories. Hospital outpatient visits and overall health-care expenditure for patients with asthma were 2.2-fold higher than those of patients without asthma. Asthma care represented 20% of all health-care services that patients with asthma received, while the remaining 80% were for non-asthma care. Almost three-fourths of all asthma-related costs were attributable to office and hospital outpatient visits; one-fourth was attributable to urgent care and hospitalizations. CONCLUSIONS: These findings may serve as baseline data for future evaluation of changes in health-care utilization and expenditure among pediatric patients with asthma.

The relationship of air pollution to ED visits for asthma differ between children and adults.

The purpose of this study was to evaluate the relationship between air pollution and asthma exacerbation in children and adults. Pearson analysis was used to establish correlations between air pollutants-sulfur dioxide, nitrogen dioxide, ozone, carbon monoxide, and particles with an aerodynamic diameter of 10 microm or less (PM(10))--and ED visits for asthma in 2004. Among children, there were significant positive correlations between nitrogen dioxide (r = 0.72), carbon monoxide (r = 0.65), and PM(10) (r = 0.63) and ED visits for asthma. Among adults, only weakly positive, non significant correlations between all air pollution measures and ED visits for asthma were found. This study suggests that air pollution plays a role in acute exacerbation of asthma in children but not in adults.

Differences in the prescription patterns of anti-asthmatic medications for children by pediatricians, family physicians and physicians of other specialties.

BACKGROUND: Prescription patterns of anti-asthma medications in children vary among doctors in different disciplines and settings, and may reflect differences in treatment outcome. The purpose of this study was to analyze the prescribing patterns of anti-asthma drugs by pediatricians, family physicians and other practitioners. METHODS: Data for a total of 225,537 anti-asthma prescriptions were collected from the National Health Insurance Research Database for the period from January 1, 2002 to March 31, 2002. These medications included inhaled and oral adrenergics, inhaled and oral corticosteroids, xanthine derivatives, and leukotriene receptor antagonists prescribed by general pediatricians, family physicians and physicians in other disciplines. RESULTS: Oral beta2-agonist was the most commonly prescribed drug used as monotherapy, with prescription rates of 70.4%, 46.9% and 58.0% by pediatricians, family physicians and other physicians, respectively. A xanthine derivative was the next most commonly prescribed monotherapy. Oral corticosteroid combined with oral beta2-agonist, followed by oral beta2-agonist combined with a xanthine derivative were the two most commonly prescribed dual-agent combined therapies by all three physician categories. The prescription rate for inhaled corticosteroid monotherapy was 7.8% by pediatricians, 5.6% by family physicians, and 8.0% by other physicians. The prescription rate for inhaled adrenergic was the highest in family physicians (14.9%), followed by the other physicians (7.2%), and was lowest in pediatricians (3.1%). CONCLUSION: Pediatricians and family physicians appeared to share similar opinions on the medical management of children with asthma in that both most commonly prescribed oral beta2-agonists and xanthine derivatives, either alone or in combination. Family physicians were least likely to prescribe an inhaled corticosteroid and most likely to prescribe an inhaled adrenergic agent.