Chinese hamster ovary (CHO) cells, first isolated in 1957, are the preferred production host for many therapeutic proteins. Although genetic heterogeneity among CHO cell lines has been well documented, a systematic, nucleotide-resolution characterization of their genotypic differences has been stymied by the lack of a unifying genomic resource for CHO cells. Here we report a 2.4-Gb draft genome sequence of a female Chinese hamster, Cricetulus griseus, harboring 24,044 genes. We also resequenced and analyzed the genomes of six CHO cell lines from the CHO-K1, DG44 and CHO-S lineages. This analysis identified hamster genes missing in different CHO cell lines, and detected >3.7 million single-nucleotide polymorphisms (SNPs), 551,240 indels and 7,063 copy number variations. Many mutations are located in genes with functions relevant to bioprocessing, such as apoptosis. The details of this genetic diversity highlight the value of the hamster genome as the reference upon which CHO cells can be studied and engineered for protein production.

Mutational bias is a potentially important agent of evolution, but it is difficult to disentangle the effects of mutation from those of natural selection. Mutation-accumulation experiments, in which mutations are allowed to accumulate at very small population size, thus minimizing the efficiency of natural selection, are the best way to separate the effects of mutation from those of selection. Body size varies greatly among species of nematode in the family rhabditidae; mutational biases are both a potential cause and a consequence of that variation. We report data on the cumulative effects of mutations that affect body size in three species of rhabditid nematode that vary fivefold in adult size. Results are very consistent with previous studies of mutations underlying fitness in the same strains: two strains of Caenorhabditis briggsae decline in body size about twice as fast as two strains of C. elegans, with a concomitant higher point estimate of the genomic mutation rate; the confamilial Oscheius myriophila is intermediate. There is an overall mutational bias, such that mutations reduce size on average, but the bias appears consistent between species. The genetic correlation between mutations that affect size and those underlying fitness is large and positive, on average.T HE importance of mutation to the evolutionary process is universally appreciated by biologists, both in terms of the deleterious effects on fitness (Morgan 1903;Fisher 1930;Haldane 1937;Sturtevant 1937) and as the ultimate source of potentially adaptive genetic variation. It has been recognized for a long time that there is substantial variation in the mutational process at a variety of taxonomic levels, even among genotypes within species (Sturtevant 1937 and references therein;Woodruff et al. 1984;Fry 2004b;Baer et al. 2005;Á vila et al. 2006;Haag-Liautard et al. 2007). The factors responsible for that variation are poorly understood, but there are two classes of potential explanations. First, the mutation rate may be primarily a by-product of intrinsic or extrinsic environmental factors, e.g., temperature, metabolic rate, UV exposure, etc. (Martin and Palumbi 1993;Hebert et al. 2002;Gillooly et al. 2005). Alternatively, the mutation rate may be an evolutionarily optimized property, with either the optimum or the deviation from the optimum varying among taxa (Kimura 1967;Leigh 1973;Kondrashov 1995;Dawson 1998). Elucidating the taxonomic distribution of variation in mutational properties may provide important insights into several disparate areas of evolutionary biology, among them the causes of adaptive radiation (Bjedov et al. 2003;Sikorski and Nevo 2005) and cladogenesis (Shpak 2005), the rate of molecular evolution (Martin and Palumbi 1993;Gillooly et al. 2005), the nature of selection on modifier loci (Kondrashov 1995), the evolution of genetic architecture underlying the phenotype ( Jones et al. 2003), and the evolution of mating system and sexual reproduction (Kondrashov 1988(Kondrashov , 1995Keightley and Otto 2006).Of part...

Objectives: There are no approved pharmacologic therapies for chronic sensorineural hearing loss (SNHL). The combination of CHIR99021+valproic acid (CV, FX-322) has been shown to regenerate mammalian cochlear hair cells ex vivo . The objectives were to characterize the cochlear pharmacokinetic profile of CV in guinea pigs, then measure FX-322 in human perilymph samples, and finally assess safety and audiometric effects of FX-322 in humans with chronic SNHL. Study Designs: Middle ear residence, cochlear distribution, and elimination profiles of FX-322 were assessed in guinea pigs. Human perilymph sampling following intratympanic FX-322 dosing was performed in an open-label study in cochlear implant subjects. Unilateral intratympanic FX-322 was assessed in a Phase 1b prospective, randomized, double-blinded, placebo-controlled clinical trial. Setting: Three private otolaryngology practices in the US. Patients: Individuals diagnosed with mild to moderately severe chronic SNHL (≤70 dB standard pure-tone average) in one or both ears that was stable for ≥6 months, medical histories consistent with noise-induced or idiopathic sudden SNHL, and no significant vestibular symptoms. Interventions: Intratympanic FX-322. Main Outcome Measures: Pharmacokinetics of FX-322 in perilymph and safety and audiometric effects. Results: After intratympanic delivery in guinea pigs and humans, FX-322 levels in the cochlear extended high-frequency region were observed and projected to be pharmacologically active in humans. A single dose of FX-322 in SNHL subjects was well tolerated with mild, transient treatment-related adverse events (n = 15 FX-322 vs 8 placebo). Of the six patients treated with FX-322 who had baseline word recognition in quiet scores below 90%, four showed clinically meaningful improvements (absolute word recognition improved 18–42%, exceeding the 95% confidence interval determined by previously published criteria). No significant changes in placebo-injected ears were observed. At the group level, FX-322 subjects outperformed placebo group in word recognition in quiet when averaged across all time points, with a mean improvement from baseline of 18.9% ( p = 0.029). For words in noise, the treated group showed a mean 1.3 dB signal-to-noise ratio improvement ( p = 0.012) relative to their baseline scores while placebo-treated subjects did not (−0.21 dB, p = 0.71). Conclusions: Delivery of FX-322 to the extended high-frequency region of the cochlea is well tolerated and enhances speech recognition performance in multiple subjects with stable chronic hearing loss.

BackgroundNasal airway obstruction (NAO) is a highly prevalent disorder. Septal swell body (SSB) hypertrophy is an often overlooked contributor to NAO. SSB treatment may relieve symptoms of NAO. The objective of this study was to assess the clinical use of a temperature‐controlled radiofrequency (TCRF) device to treat SSBs to improve symptoms in adults with NAO.MethodsIn this prospective, multicenter, open‐label, single arm study, patients with severe or extreme NAO related to SSB hypertrophy received bilateral TCRF treatment in the SSB area. The primary endpoint was improvement in Nasal Obstruction Symptom Evaluation (NOSE) Scale scores from baseline to 3 months postprocedure. A subset of study patients underwent computed tomography (CT) imaging to evaluate posttreatment changes in SSB size.ResultsMean NOSE Scale scores significantly improved from 73.5 (SD 14.2) at baseline to 27.9 (SD 17.2) at 3 months postprocedure, a reduction of −45.3 (SD 21.4, 95% confidence interval [CI]: −50.4 to −40.1; p < 0.0001); the responder rate was 95.7% (95% CI: 0.88 to 0.99; p < 0.0001). CT evaluation at 3 months showed statistically significant reductions in the SSB with the greatest reduction in the middle thickness (mean change −3.4 [SD 1.8] mL, 95% CI: −4.0 to −2.8; p < 0.0001). Minimal adverse events with any relationship to the device or procedure were reported; none were serious in nature and no septal perforations occurred.ConclusionsThis study demonstrates that TCRF treatment of SSB hypertrophy is well tolerated and effective at reducing both SSB size and symptoms of NAO at 3 months posttreatment.

Cell Linking Moieties (CLMs) are bi-specific antibody fragments that are capable of recognizing epitopes on tumor cells and specific effector cells via unique cell surface receptors. They are comprised of 2 scFv elements recognizing unique epitopes, bound by a linking element. CLMs offer a distinct advantage in that they do not rely on the expression of MHC class I molecules, generation of specific antigenic peptide, or ex vivo manipulation of cells to exert their tu mor cell killing effect. To identify the proper orientation of scFv elements and to define the most potent and selective CLM, we have established an imagebased screening platform capable of interrogating a large number of CLMs based on their ability to activate effectors and specifically kill tumor cells. ee demonstrate the ability to screen a number of CLMs targeting wild type EGFR and HER2 receptors on fluorescently-tagged tumor cell lines for their ability to recruit CD3+ T-cells from unstimulated PBMC pools and mediate selective cell killing. CLMs were created by transient transfection of HEK293 cells with PCR-generated DNA fragments in 96 well plates. Supernatant was harvested 3 days post transfection. Dilutions of crude supernatant were applied to A549, MDA-MB-468, and Se620 (negative) target cells expressing EGFR or HER2 receptors in the presence of unstimulated PBMCs at 10:1 PBMC:Target ratio in 384 well plates. PBMC to target ratios were pre-optimized by running a titration from 2:1 through 1:30 Target:PBMCs in a kill assay. Resulting target cell loss (cell death) was compared to control wells (mock transfected supernatant). In the screening campaign, a primary screen first selects CLM hits based on killing ability, and then a secondary screen is employed to provide dose-responses, IC50s, and additional information, such as non-specific killing of nega tive lines. Using this image-based screen, consistent Z-scores above 0.5 were obtained, which corresponds to 12 standard deviations between the positive and negative controls, indicating an excellent dynamic range and low variability in the assay. In a proof of concept study using this platform, two αEGFR-αCD3 CLMs were selected from a larger list and characterized further in the secondary screen. In the top candidate, secondary screening resulted in IC50 average values of 1632 and 12,971-fold dilution of CLM-containing HEK293 supernatant in A549 cells, and MDA-MB-468 cells, respectively. At high doses of top-candidate CLMs, no target cells remained in PBMC co-cultures, representing a near 100% killing effect. No killing was observed in the negative con trol line, Se620. Consistent ranking of CLM candidates was observed among 4 donors of PBMCs evaluated. In conclusion, this image-based screening platform allows for well-and cell-level killing information to be obtained with specific loss of target cells in complex mixtures to be accurately calculated without the presence of radioactive compounds like 51 Cr. Furthermore, this method allows for kinetic analysis where the same well can be scanned...