Maximizing antibody production in a targeted integration host by optimization of subunit gene dosage and position

Historically, therapeutic protein production in Chinese hamster ovary (CHO) cells has been accomplished by random integration (RI) of expression plasmids into the host cell genome. More recently, the development of targeted integration (TI) host cells has allowed for recombination of plasmid DNA into a predetermined genomic locus, eliminating one contributor to clone-to-clone variability. In this study, a TI host capable of simultaneously integrating two plasmids at the same genomic site was used to assess the effect of antibody heavy chain and light chain gene dosage on antibody productivity. Our results showed that increasing antibody gene copy number can increase specific productivity, but with diminishing returns as more antibody genes are added to the same TI locus. Random integration of additional antibody DNA copies in to a targeted integration cell line showed a further increase in specific productivity, suggesting that targeting additional genomic sites for gene integration may be beneficial. Additionally, the position of antibody genes in the two plasmids was observed to have a strong effect on antibody expression level. These findings shed light on vector design to maximize production of conventional antibodies or tune expression for proper assembly of complex or bispecific antibodies in a TI system.     

PTP1B inhibitory effects of tridepside and related metabolites isolated from the Antarctic lichen Umbilicaria antarctica

The selective inhibition of PTP1B has been widely recognized as a potential drug target for the treatment of type 2 diabetes and obesity. In the course of screening for PTP1B inhibitory natural products, the MeOH extract of the dried sample of the Antarctic lichen Umbilicaria antarctica was found to exhibit significant inhibitory effect, and the bioassay-guided fractionation and purification afforded three related lichen metabolites 1-3. Compounds 1-3 were identified as gyrophoric acid (1), lecanoric acid (2), and methyl orsellinate (3) mainly by analysis of NMR and MS data. These compounds inhibited PTP1B activity with 50% inhibitory concentration values of 3.6 ± 0.04 μM, 31 ± 2.7 μM, and 277 ± 8.6 μM, respectively. Furthermore, the kinetic analysis of PTP1B inhibition by compound 1 suggested that the compound inhibited PTP1B activity in a non-competitive manner.     

Network-Level Structural Abnormalities of Cerebral Cortex in Type 1 Diabetes Mellitus

Type 1 diabetes mellitus (T1DM) usually begins in childhood and adolescence and causes lifelong damage to several major organs including the brain. Despite increasing evidence of T1DM-induced structural deficits in cortical regions implicated in higher cognitive and emotional functions, little is known whether and how the structural connectivity between these regions is altered in the T1DM brain. Using inter-regional covariance of cortical thickness measurements from high-resolution T1-weighted magnetic resonance data, we examined the topological organizations of cortical structural networks in 81 T1DM patients and 38 healthy subjects. We found a relative absence of hierarchically high-level hubs in the prefrontal lobe of T1DM patients, which suggests ineffective top-down control of the prefrontal cortex in T1DM. Furthermore, inter-network connections between the strategic/executive control system and systems subserving other cortical functions including language and mnemonic/emotional processing were also less integrated in T1DM patients than in healthy individuals. The current results provide structural evidence for T1DM-related dysfunctional cortical organization, which specifically underlie the top-down cognitive control of language, memory, and emotion.     

Loss Mechanisms in Thick‐Film Low‐Bandgap Polymer Solar Cells

Polymer bulk heterojunction solar cells based on low bandgap polymer:fullerene blends are promising for next generation low‐cost photovoltaics. While these solution‐processed solar cells are compatible with large‐scale roll‐to‐roll processing, active layers used for typical laboratory‐scale devices are too thin to ensure high manufacturing yields. Furthermore, due to the limited light absorption and optical interference within the thin active layer, the external quantum efficiencies (EQEs) of bulk heterojunction polymer solar cells are severely limited. In order to produce polymer solar cells with high yields, efficient solar cells with a thick active layer must be demonstrated. In this work, the performance of thick‐film solar cells employing the low‐bandgap polymer poly(dithienogermole‐thienopyrrolodione) (PDTG‐TPD) was demonstrated. Power conversion efficiencies over 8.0% were obtained for devices with an active layer thickness of 200 nm, illustrating the potential of this polymer for large‐scale manufacturing. Although an average EQE > 65% was obtained for devices with active layer thicknesses > 200 nm, the cell performance could not be maintained due to a reduction in fill factor. By comparing our results for PDTG‐TPD solar cells with similar P3HT‐based devices, we investigated the loss mechanisms associated with the limited device performance observed for thick‐film low‐bandgap polymer solar cells.     

A new fused tetracyclic heterocyclic antioxidant from Serratia sp. PAMC 25557

A new fused tetracyclic heterocyclic compound, (4bR,10bR)-4b-hydroxy-10b,12-dihydrodibenzo[c,h][2,6]naphthyridine-5,11(4bH,6H)-dione (1), and a known compound, butyl 2-[(benzoyloxy)methyl]benzoate, spatozoate 2, were isolated from the broth culture of Serratia sp. PAMC 25557. The structure of 1 was determined by analyzing spectroscopic data. Compound 1 did not exhibit antimicrobial activity against Escherichia coli, Staphylococcus aureus, or Candida albicans. In addition, up to 100 μg/ml compound 1 did not show any toxicity against Artemia salina larvae. However, compound 1 showed DPPH free radical scavenging activity (IC₅₀ = 16.7 ± 0.34 μg/ml). This was the first report of spatozoate isolation from bacterial sources.     

Likely future extirpation of another Asian river dolphin: The critically endangered population of the Irrawaddy dolphin in the Mekong River is small and declining

The population of Irrawaddy dolphins that occupies the Mekong River in southern Lao People's Democratic Republic and Cambodia is classified as Critically Endangered by the IUCN. Based on capture‐recapture of photo‐identified individuals, we estimated that the total population numbered 93 ±  SE 3.90 individuals (95% CI 86–101), as of April 2007. The combined photo‐identification and carcass recovery program undertaken from 2001 to 2007 established that the Irrawaddy dolphin population inhabiting the Mekong River has reached a critical point with regards to its continued survival, where immediate research and management actions are required to greatly reduce adult mortality, and establish the cause of newborn mortality. In addition, community consultation is required to initiate, and evaluate, urgently required conservation measures. An ongoing well‐designed combined program of abundance estimation (i.e., photo‐identification) and carcass recovery is required to monitor total population size and mortality rates, to inform and evaluate management initiatives. The conclusions of this paper are likely generic to river dolphin populations, particularly where photo‐identification is possible.     

A Case of Human Pulmonary Dirofilariasis in a 48-Year-Old Korean Man

Dirofilariasis is a rare disease in humans. We report here a case of a 48-year-old male who was diagnosed with pulmonary dirofilariasis in Korea. On chest radiographs, a coin lesion of 1 cm in diameter was shown. Although it looked like a benign inflammatory nodule, malignancy could not be excluded. So, the nodule was resected by video-assisted thoracic surgery. Pathologically, chronic granulomatous inflammation composed of coagulation necrosis with rim of fibrous tissues and granulations was seen. In the center of the necrotic nodules, a degenerating parasitic organism was found. The parasite had prominent internal cuticular ridges and thick cuticle, a well-developed muscle layer, an intestinal tube, and uterine tubules. The parasite was diagnosed as an immature female worm of Dirofilaria immitis. This is the second reported case of human pulmonary dirofilariasis in Korea.     

Targeted delivery of a phosphopeptide prodrug inhibits the proliferation of a human glioma cell line

Peptides are ideal candidates for developing therapeutics. Polo-like kinase 1 is an important regulatory protein in the cell cycle and contains a C-terminal polo-box domain, which is the hallmark of this protein family. We developed a peptide inhibitor of polo-like kinase 1 that targets its polo-box domain. This new phosphopeptide, cRGDyK-S-S-CPLHSpT, preferentially penetrates the cancer cell membrane mediated by the integrin receptor, which is expressed at high levels by cancer cells. In the present study, using high performance liquid chromatography and mass spectroscopy, we determined the stability of cRGDyK-S-S-CPLHSpT and its cleavage by glutathione under typical conditions for cell culture. We further assessed the ability of the peptide to inhibit the proliferation of the U87MG glioma cell line. The phosphorylated peptide was stable, and the disulfide bond of cRGDyK-S-S-CPLHSpT was cleaved in 50 mM glutathione. This peptide inhibited the growth of cancer cells and changed their morphology. Therefore, we conclude that the phosphopeptide shows promise as a prodrug and has a high potential to act as an anticancer agent by inhibiting polo-like kinase 1 by binding its polo-box domain. These findings indicate the therapeutic potential of PLHSpT and peptides similarly targeted to surface receptors of cancer cells and to the functional domains of regulatory proteins.     

Distribution Dynamics of Recombinant Lactobacillus in the Gastrointestinal Tract of Neonatal Rats

One approach to deliver therapeutic agents, especially proteins, to the gastro-intestinal (GI) tract is to use commensal bacteria as a carrier. Genus Lactobacillus is an attractive candidate for use in this approach. However, a system for expressing exogenous proteins at a high level has been lacking in Lactobacillus. Moreover, it will be necessary to introduce the recombinant Lactobacillus into the GI tract, ideally by oral administration. Whether orally administered Lactobacillus can reach and reside in the GI tract has not been explored in neonates. In this study, we have examined these issues in neonatal rats. To achieve a high level of protein expression in Lactobacillus, we tested the impact of three promoters and two backbones on protein expression levels using mRFP1, a red fluorescent protein, as a reporter. We found that a combination of an L-lactate dehydrogenase (ldhL) promoter of Lactobacillus sakei with a backbone from pLEM415 yielded the highest level of reporter expression. When this construct was used to transform Lactobacillus casei, Lactobacillus delbrueckii and Lactobacillus acidophilus, high levels of mRFP1 were detected in all these species and colonies of transformed Lactobacillus appeared pink under visible light. To test whether orally administered Lactobacillus can be retained in the GI tract of neonates, we fed the recombinant Lactobacillus casei to neonatal rats. We found that about 3% of the bacteria were retained in the GI tract of the rats at 24 h after oral feeding with more recombinant Lactobacillus in the stomach and small intestine than in the cecum and colon. No mortality was observed throughout this study with Lactobacillus. In contrast, all neonatal rats died within 24 hours after fed with transformed E. coli. Taken together, our results indicate that Lactobacillus has the potential to be used as a vehicle for the delivery of therapeutic agents to neonates.     

Electrical Impedance Myography to Detect the Effects of Electrical Muscle Stimulation in Wild Type and Mdx Mice

Objective

Tools to better evaluate the impact of therapy on nerve and muscle disease are needed. Electrical impedance myography (EIM) is sensitive to neuromuscular disease progression as well as to therapeutic interventions including myostatin inhibition and antisense oligonucleotide-based treatments. Whether the technique identifies the impact of electrical muscle stimulation (EMS) is unknown.

Methods

Ten wild-type (wt) C57B6 mice and 10 dystrophin-deficient (mdx) mice underwent 2 weeks of 20 min/day EMS on left gastrocnemius and sham stimulation on the right gastrocnemius. Multifrequency EIM data and limb girth were obtained before and at the conclusion of the protocol. Muscle weight, in situ force measurements, and muscle fiber histology were also assessed at the conclusion of the study.

Results

At the time of sacrifice, muscle weight was greater on the EMS-treated side than on the sham-stimulated side (p = 0.018 for wt and p = 0.007 for mdx). Similarly, in wt animals, EIM parameters changed significantly compared to baseline (resistance (p = 0.009), reactance (p = 0.0003) and phase (p = 0.002); these changes were due in part to reductions in the EIM values on the EMS-treated side and elevations on the sham-simulated side. Mdx animals showed analogous but non-significant changes (p = 0.083, p = 0.064, and p = 0.57 for resistance, reactance and phase, respectively). Maximal isometric force trended higher on the stimulated side in wt animals only (p = 0.06). Myofiber sizes in wt animals were also larger on the stimulated side than on the sham-stimulated side (p = 0.034); no significant difference was found in the mdx mice (p = 0.79).

Conclusion

EIM is sensitive to stimulation-induced muscle alterations in wt animals; similar trends are also present in mdx mice. The mechanisms by which these EIM changes develop, however, remains uncertain. Possible explanations include longer-term trophic effects and shorter-term osmotic effects.