Replicon vectors derived from Sindbis virus and Semliki forest virus that establish persistent replication in host cells

Alphavirus replicon vectors are well suited for applications where transient, high-level expression of a heterologous gene is required. Replicon vector expression in cells leads to inhibition of host macromolecular synthesis, culminating in eventual cell death by an apoptotic mechanism. For many applications, including gene expression studies in cultured cells, a longer duration of transgene expression without resulting cytopathic effects is useful. Recently, noncytopathic Sindbis virus (SIN) variants were isolated in BHK cells, and the mutations responsible were mapped to the protease domain of nonstructural protein 2 (nsP2). We report here the isolation of additional variants of both SIN and Semliki Forest virus (SFV) replicons encoding the neomycin resistance gene that can establish persistent replication in BHK cells. The SIN and SFV variant replicons resulted from previously undescribed mutations within one of three discrete regions of the nsP2 gene. Differences among the panel of variants were observed in processing of the nonstructural polyprotein and in the ratios of subgenomic to genomic RNAs. Importantly, high-level expression of a heterologous gene was retained with most replicons. Finally, in contrast to previous studies, efficient packaging was obtained with several of the variant replicons. This work expands the utility of noncytopathic replicons and the understanding of how alphavirus replicons establish persistent replication in cultured cells.     

Automated assay optimization with integrated statistics and smart robotics

The transition from manual to robotic high throughput screening (HTS) in the last few years has made it feasible to screen hundreds of thousands of chemical entities against a biological target in less than a month. This rate of HTS has increased the visibility of bottlenecks, one of which is assay optimization. In many organizations, experimental methods are generated by therapeutic teams associated with specific targets and passed on to the HTS group. The resulting assays frequently need to be further optimized to withstand the rigors and time frames inherent in robotic handling. Issues such as protein aggregation, ligand instability, and cellular viability are common variables in the optimization process. The availability of robotics capable of performing rapid random access tasks has made it possible to design optimization experiments that would be either very difficult or impossible for a person to carry out. Our approach to reducing the assay optimization bottleneck has been to unify the highly specific fields of statistics, biochemistry, and robotics. The product of these endeavors is a process we have named automated assay optimization (AAO). This has enabled us to determine final optimized assay conditions, which are often a composite of variables that we would not have arrived at by examining each variable independently. We have applied this approach to both radioligand binding and enzymatic assays and have realized benefits in both time and performance that we would not have predicted a priori. The fully developed AAO process encompasses the ability to download information to a robot and have liquid handling methods automatically created. This evolution in smart robotics has proven to be an invaluable tool for maintaining high-quality data in the context of increasing HTS demands.     

Differential pathogenesis of respiratory syncytial virus clinical isolates in BALB/c mice

Airway mucus is a hallmark of respiratory syncytial virus (RSV) lower respiratory tract illness. Laboratory RSV strains differentially induce airway mucus production in mice. Here, we tested the hypothesis that RSV strains differ in pathogenesis by screening six low-passage RSV clinical isolates for mucogenicity and virulence in BALB/cJ mice. The RSV clinical isolates induced variable disease severity, lung interleukin-13 (IL-13) levels, and gob-5 levels in BALB/cJ mice. We chose two of these clinical isolates for further study. Infection of BALB/cJ mice with RSV A2001/2-20 (2-20) resulted in greater disease severity, higher lung IL-13 levels, and higher lung gob-5 levels than infection with RSV strains A2, line 19, Long, and A2001/3-12 (3-12). Like the line 19 RSV strain, the 2-20 clinical isolate induced airway mucin expression in BALB/cJ mice. The 2-20 and 3-12 RSV clinical isolates had higher lung viral loads than laboratory RSV strains at 1 day postinfection (p.i.). This increased viral load correlated with higher viral antigen levels in the bronchiolar epithelium and greater histopathologic changes at 1 day p.i. The A2 RSV strain had the highest peak viral load at day 4 p.i. RSV 2-20 infection caused epithelial desquamation, bronchiolitis, airway hyperresponsiveness, and increased breathing effort in BALB/cJ mice. We found that RSV clinical isolates induce variable pathogenesis in mice, and we established a mouse model of clinical isolate strain-dependent RSV pathogenesis that recapitulates key features of RSV disease.     

Extracorporeal bicarbonate space after bicarbonate or a bicarbonate-carbonate mixture in acidotic dogs

The effects of sodium bicarbonate and a bicarbonate-carbonate mixture on expired CO2 and the volume of distribution of bicarbonate were studied in eight anesthetized, paralyzed, and ventilated dogs made acidotic with HCl (5 mmol/kg) infused over 90 min. Both sodium bicarbonate and Carbicarb resulted in systemic alkalinization and comparable increases in the serum bicarbonate at 50 min (7.07 +/- 0.91 vs. 7.99 +/- 0.77, respectively; P = NS). Sodium bicarbonate infusion resulted in an increase in CO2 excretion that accounted for a fractional CO2 excretion of 0.20 +/- 0.09, whereas infusion of a bicarbonate-carbonate mixture resulted in a fractional CO2 excretion of -0.06 +/- 0.09 (P less than 0.01). The uncorrected volume of distribution of bicarbonate after sodium bicarbonate infusion was higher than that seen with the bicarbonate-carbonate mixture (0.60 +/- 0.07 vs. 0.34 +/- 0.03 l/kg; P less than 0.01). However, when the volume of bicarbonate distribution was corrected for expired CO2, there was no difference between treatment with sodium bicarbonate and the bicarbonate-carbonate mixture (0.44 +/- 0.07 vs. 0.38 +/- 0.04 l/kg; P = NS). These data demonstrate that, in this animal model of acidosis, sodium bicarbonate treatment of systemic acidosis is accompanied by a generation of a considerable amount of CO2, whereas treatment with a bicarbonate-carbonate mixture is not. This suggests that in states of impaired ventilation, a bicarbonate-carbonate mixture may offer more efficient systemic alkalinization and may be associated with less CO2 generation than sodium bicarbonate.     

A Multifunctional Mesothelin Antibody-tagged Microparticle Targets Human Mesotheliomas

Pleural and peritoneal mesotheliomas (MMs) are chemoresistant tumors with no effective therapeutic strategies. The authors first injected multifunctional, acid-prepared mesoporous spheres (APMS), microparticles functionalized with tetraethylene glycol oligomers, intraperitoneally into rodents. Biodistribution of APMS was observed in major organs, peritoneal lavage fluid (PLF), and urine of normal mice and rats. After verification of increased mesothelin in human mesotheliomas injected into severe combined immunodeficient (SCID) mice, APMS were then functionalized with an antibody to mesothelin (APMS-MB) or bovine serum albumin (BSA), a nonspecific protein control, and tumor targeting was evaluated by inductively coupled plasma mass spectrometry and multifluorescence confocal microscopy. Some APMS were initially cleared via the urine over a 24 hr period, and small amounts were observed in liver, spleen, and kidneys at 24 hr and 6 days. Targeting with APMS-MB increased APMS uptake in mesenteric tumors at 6 days. Approximately 10% to 12% of the initially injected amount was observed in both spheroid and mesenteric MM at this time point. The data suggest that localized delivery of APMS-MB into the peritoneal cavity after encapsulation of drugs, DNA, or macromolecules is a novel therapeutic approach for MM and other tumors (ovarian and pancreatic) that overexpress mesothelin.     

Partial information decomposition reveals that synergistic neural integration is greater downstream of recurrent information flow in organotypic cortical cultures

The directionality of network information flow dictates how networks process information. A central component of information processing in both biological and artificial neural networks is their ability to perform synergistic integration–a type of computation. We established previously that synergistic integration varies directly with the strength of feedforward information flow. However, the relationships between both recurrent and feedback information flow and synergistic integration remain unknown. To address this, we analyzed the spiking activity of hundreds of neurons in organotypic cultures of mouse cortex. We asked how empirically observed synergistic integration–determined from partial information decomposition–varied with local functional network structure that was categorized into motifs with varying recurrent and feedback information flow. We found that synergistic integration was elevated in motifs with greater recurrent information flow beyond that expected from the local feedforward information flow. Feedback information flow was interrelated with feedforward information flow and was associated with decreased synergistic integration. Our results indicate that synergistic integration is distinctly influenced by the directionality of local information flow.     

Effects of concentration and sniff flow rate on the rat electroolfactogram

Previous reports using the electroolfactogram (EOG) to study the spatial and temporal aspects of response in the rodent olfactory epithelium had focused on high odorant concentrations that gave large responses. This investigation has used lower concentrations to test the difference between responses in the rat dorsomedial and lateral recesses with a range of nasal flow rates and a range of chemical properties. The responses to a highly polar, more hydrophilic odorant changed more steeply with flow rate than responses to a very nonpolar, hydrophobic odorant. With low flow rates there was a response delay in the lateral recess, which is consistent with the models indicating lower flow rates in that region. We observed significant volume conduction effects in which large responses in the dorsomedial region obscured smaller initial portions of the lateral responses. These effects could be removed by destroying the dorsomedial response with a high concentration of a low molecular weight ester. We caution that investigators of EOG recordings from the intact epithelium must attend to the possible presence of volume conduction, which can be assessed by attention to the selectivity of odorant response, response waveform, and response latency.