Natural M-segment reassortment in Potosi and Main Drain viruses: implications for the evolution of orthobunyaviruses

Summary Recently, we identified Batai virus as the M-segment reassortment partner of Ngari virus. Extension of genetic analyses to other orthobunyaviruses related to the Bunyamwera serogroup indicates additional natural genome reassortments. Whereas the relative phylogenetic positions of all three genome segment sequences were similar for Northway and Kairi viruses, the relative positions of Potosi and Main Drain virus M-segment sequences diverged from those of their S- and L-segments. Our findings indicate M-segment reassortment in Potosi and Main Drain viruses and demonstrate natural genome reassortment as a driving force in the evolution of viruses of the Bunyamwera serogroup.     

Rapid Delivery of Diazepam from Supersaturated Solutions Prepared Using Prodrug/Enzyme Mixtures: Toward Intranasal Treatment of Seizure Emergencies

Current treatments for seizure emergencies, such as status epilepticus, include intravenous or rectal administration of benzodiazepines. While intranasal delivery of these drugs is desirable, the small volume of the nasal cavity and low drug solubility pose significant difficulties. Here, we prepared supersaturated diazepam solutions under physiological conditions and without precipitation, using a prodrug/enzyme system. Avizafone, a peptide prodrug of diazepam, was delivered with—Aspergillus oryzae (A.O.) protease, an enzyme identified from a pool of hydrolytic enzymes in assay buffer, pH 7.4 at 32°C. This enzyme converted avizafone to diazepam at supersaturated concentrations. In vitro permeability studies were performed at various prodrug/enzyme ratios using Madin-Darby canine kidney II-wild type (MDCKII-wt) monolayers, a representative model of the nasal epithelium. Monolayer integrity was examined using TEER measurement and the lucifer yellow permeability assay. Prodrug/drug concentrations were measured using HPLC. Enzyme kinetics with avizafone-protease mixtures revealed K_M = 1,501 ± 232 μM and V_max = 1,369 ± 94 μM/s. Prodrug-protease mixtures, when co-delivered apically onto MDCKII-wt monolayers, showed 2–17.6-fold greater diazepam flux (S = 1.3–15.3) compared to near-saturated diazepam (S = 0.7). Data for prodrug conversion upstream (apical side) and drug permeability downstream (basolateral side) fitted reasonably well to a previously developed in vitro two compartment pharmacokinetic model. Avizafone-protease mixtures resulted in supersaturated diazepam in less than 5 min, with the rate and extent of supersaturation determined by the prodrug/enzyme ratio. Together, these results suggest that an intranasal avizafone-protease system may provide a rapid and alternative means of diazepam delivery.

Electronic supplementary material

The online version of this article (doi:10.1208/s12248-014-9596-5) contains supplementary material, which is available to authorized users.KEY WORDS: avizafone enzyme activation, diazepam delivery, hydrophobic drugs, MDCK monolayers, rapid absorption, seizure emergencies, supersaturation     

Asymptomatic prostatic inflammation in men with clinical BPH and erectile dysfunction affects the positive predictive value of prostate-specific antigen

ObjectiveTo test the hypothesis that sexual dysfunction in elderly men with benign prostatic hyperplasia leads to prostatic inflammation, diagnosed by prostatic fluid interleukin-8 (IL-8), which lowers the positive predictive value of prostate-specific antigen (PSA).MethodsOverall, 160 men with lower urinary tract symptoms between 50 and 75 years of age with an elevated PSA level of more than 4 ng/ml with normal digital rectal examination and 50 age-matched controls with normal PSA level were prospectively evaluated for prostatic fluid IL-8 levels. Erectile dysfunction was measured by self-administered questionnaire of the Sexual Health Inventory for Men. Total and free serum PSA levels and IL-8 in prostatic fluid were measured 6 to 8 weeks after a course of 400 mg of ofloxacin and 20 mg of piroxicam given daily for 2 weeks. Transrectal ultrasonography–guided biopsy was done only when PSA level did not decrease less than 4 ng/ml.ResultsMean ages of patients and controls were 63.18 (standard deviation [SD]±7.10) and 60.18 (SD+6.02) years, respectively. Mean concentration of IL-8 in prostatic fluid of the patients was significantly higher, i.e., 6678 pg/ml (SD±1985.7) than in control, i.e., 1543 pg/ml (SD±375.7) (P<0.001). Following anti-inflammatory treatment, there was a significant decrease in the mean level of IL-8 from baseline to 5622 pg/ml (SD±1870.66) (P<0.001). Corresponding to this, a significant decrease was noted in total PSA levels to less than 4 ng/ml in 105 (65.62%) patients. Men with the highest levels of IL-8 had a greater degree of erectile dysfunction.ConclusionMen with symptomatic benign prostatic hyperplasia and erectile dysfunction had significant inflammation of the prostate to cause spurious rise in PSA level resulting in an unnecessary biopsy.     

Effect of body mass index on the outcomes of patients with upper and lower urinary tract cancers treated by radical surgery: Results from a Canadian multicenter collaboration

ObjectiveTo evaluate the effect of body mass index (BMI) on the outcomes of patients with urinary tract carcinoma treated with radical surgery.Materials and methodsData were collected from 10 Canadian centers on patients who underwent radical cystectomy (RC) (1998–2008) or radical nephroureterectomy (RNU) (1990–2010). Various parameters among subsets of patients (BMI<25, 25≤BMI<30, and BMI≥30 kg/m²) were analyzed. Kaplan-Meier and multivariate analyses were performed to assess the effect of BMI on overall survival, disease-specific survival, and recurrence-free survival (RFS).ResultsAmong the 847 RC and 664 RNU patients, there was no difference in histology, stage, grade, and margin status among the 3 patient subsets undergoing either surgery. However, RC patients with lower BMIs (<25 kg/m²) were significantly older (P = 0.004), had more nodal metastasis (P = 0.03), and trended toward higher stage (P = 0.052). RNU patients with lower BMIs (<25 kg/m²) were significantly older (P = 0.0004) and fewer received adjuvant chemotherapy (P = 0.04) compared with those with BMI≥30 kg/m²; however, there was no difference in tumor location (P = 0.20), stage (P = 0.48), and management of distal ureter among the groups (P = 0.30). On multivariate analysis, BMI was not prognostic for overall survival, disease-specific survival, and RFS in the RC group. However, BMI≥30 kg/m² was associated with more bladder cancer recurrences and worse RFS in the RNU group (HR = 1.588; 95% CI: 1.148–2.196; P = 0.0052).ConclusionsIncreased BMI did not influence survival among RC patients. BMI≥30 kg/m² is associated with worse bladder cancer recurrences among RNU patients; whether this is related to difficulty in obtaining adequate bladder cuff in patients with obesity requires further evaluation.     

Characterization of the GBoV1 Capsid and Its Antibody Interactions

Human bocavirus 1 (HBoV1) has gained attention as a gene delivery vector with its ability to infect polarized human airway epithelia and 5.5 kb genome packaging capacity. Gorilla bocavirus 1 (GBoV1) VP3 shares 86% amino acid sequence identity with HBoV1 but has better transduction efficiency in several human cell types. Here, we report the capsid structure of GBoV1 determined to 2.76 Å resolution using cryo-electron microscopy (cryo-EM) and its interaction with mouse monoclonal antibodies (mAbs) and human sera. GBoV1 shares capsid surface morphologies with other parvoviruses, with a channel at the 5-fold symmetry axis, protrusions surrounding the 3-fold axis and a depression at the 2-fold axis. A 2/5-fold wall separates the 2-fold and 5-fold axes. Compared to HBoV1, differences are localized to the 3-fold protrusions. Consistently, native dot immunoblots and cryo-EM showed cross-reactivity and binding, respectively, by a 5-fold targeted HBoV1 mAb, 15C6. Surprisingly, recognition was observed for one out of three 3-fold targeted mAbs, 12C1, indicating some structural similarity at this region. In addition, GBoV1, tested against 40 human sera, showed the similar rates of seropositivity as HBoV1. Immunogenic reactivity against parvoviral vectors is a significant barrier to efficient gene delivery. This study is a step towards optimizing bocaparvovirus vectors with antibody escape properties.     

Normal Weight Obesity: An Underrecognized Problem in Individuals of South Asian Descent

PurposeObesity has attained pandemic proportions across the world, and its prevalence in developing countries is also on the rise. Nevertheless, there is still a large gap in understanding the reasons behind a disproportionately high prevalence of diabetes as opposed to a lesser degree of obesity seen in individuals of South Asian origin. This research letter highlights the importance of identifying individuals with normal weight obesity, which may partially bridge this knowledge gap.MethodsWe reviewed recently published evidence on normal weight obesity.FindingsNormal weight obesity is a common public health problem and may be prevalent in up to one-third of individuals of certain Asian ethnicities. Literature is emerging on its pathophysiology and association with metabolic diseases, such as type 2 diabetes mellitus, hypertension, and dyslipidemia. More recently, normal weight obesity was also identified as an independent strong predictor of cardiovascular mortality. However, evidence is particularly lacking on its appropriate management.ImplicationsNormal weight obesity is an underrecognized yet widely prevalent problem in individuals of Asian descent. Further research on pathogenic mechanisms, diagnostic modalities, and therapeutic options in individuals with normal weight obesity is needed to appropriately manage this condition.