Swarms of chemically modified antiviral siRNA targeting herpes simplex virus infection in human corneal epithelial cells

Herpes simplex virus type 1 (HSV-1) is a common virus of mankind and HSV-1 infections are a significant cause of blindness. The current antiviral treatment of herpes infection relies on acyclovir and related compounds. However, acyclovir resistance emerges especially in the long term prophylactic treatment that is required for prevention of recurrent herpes keratitis. Earlier we have established antiviral siRNA swarms, targeting sequences of essential genes of HSV, as effective means of silencing the replication of HSV in vitro or in vivo. In this study, we show the antiviral efficacy of 2´-fluoro modified antiviral siRNA swarms against HSV-1 in human corneal epithelial cells (HCE). We studied HCE for innate immunity responses to HSV-1, to immunostimulatory cytotoxic double stranded RNA, and to the antiviral siRNA swarms, with or without a viral challenge. The panel of studied innate responses included interferon beta, lambda 1, interferon stimulated gene 54, human myxovirus resistance protein A, human myxovirus resistance protein B, toll-like receptor 3 and interferon kappa. Our results demonstrated that HCE cells are a suitable model to study antiviral RNAi efficacy and safety in vitro. In HCE cells, the antiviral siRNA swarms targeting the HSV UL29 gene and harboring 2´-fluoro modifications, were well tolerated, induced only modest innate immunity responses, and were highly antiviral with more than 99% inhibition of viral release. The antiviral effect of the 2’-fluoro modified swarm was more apparent than that of the unmodified antiviral siRNA swarm. Our results encourage further research in vitro and in vivo on antiviral siRNA swarm therapy of corneal HSV infection, especially with modified siRNA swarms.     

A Histochemical Method for the Simultaneous Demonstration of Capillaries and Fiber Type in Skeletal Muscle

A modified ATPasc method for the simultaneous demonstration of capillaries and fiber types in skeletal muscle is presented. Muscle biopsies were obtained front mice, hamsters, rats, cats, and dogs, quick frozen, and sectioned at 8 μm in a cryostat. The frozen slides were fixed in a neutral formalin solution at 4 C for 5 min, and then incubated at 37 C for 1 hr in a medium containing ATP, Pb²⁺, and Ca²⁺ in a tris-maleate buffer (pH 7.2). Dilute (NH₄)₂S was used as a developer. To test the reliability of the proposed method, serial sections of each biopsy were stained separately for capillaries (amylase-PAS method) and for fiber types by a standard myosin ATPase (m-ATPase) method. Fiber type percent and capillary parameters were determined for each biopsy. No difference in results was observed for parameters determined using the modified ATPase method compared to the standard capillary and fiber type staining methods. This modified technique is therefore suitable for the simultaneous demonstration of capillaries and fiber types in skeletal muscle.     

A histochemical method for the simultaneous demonstration of capillaries and fiber type in skeletal muscle

A modified ATPase method for the simultaneous demonstration of capillaries and fiber types in skeletal muscle is presented. Muscle biopsies were obtained from mice, hamsters, rats, cats, and dogs, quick frozen, and sectioned at 8 microns in a cryostat. The frozen slides were fixed in a neutral formalin solution at 4 C for 5 min, and then incubated at 37 C for 1 hr in a medium containing ATP, Pb2+, and Ca2+ in a tris-maleate buffer (pH 7.2). Dilute (NH4)2S was used as a developer. To test the reliability of the proposed method, serial sections of each biopsy were stained separately for capillaries (amylase-PAS method) and for fiber types by a standard myosin ATPase (m-ATPase) method. Fiber type percent and capillary parameters were determined for each biopsy. No difference in results was observed for parameters determined using the modified ATPase method compared to the standard capillary and fiber type staining methods. This modified technique is therefore suitable for the simultaneous demonstration of capillaries and fiber types in skeletal muscle.     

Comparison of three differently shaped ROIs in free breathing breast radiotherapy setup using surface guidance with AlignRT®

BackgroundSetup accuracy within adjuvant radiotherapy of breast cancer treated in free breathing is well studied, but a comparison of the typical regions of interest (ROI) used in surface guided radiation therapy (SGRT) does not exist. The aim of this study was to estimate the setup accuracy obtained with differently shaped ROIs in SGRT.Materials and methodsA total of 573 orthogonal image pairs were analyzed from free breathing breast patients in two groups: positioning using AlignRT^® surface guidance system (Group A, n = 20), and setup using conventional laser and tattoo setup (Group L, n = 20). For SGRT, three different setup ROIs were used: a Breast-shaped, O-shaped and T-shaped (B-O and T-ROI). We evaluated the isocenter-, rotation-, pitch and arm position accuracy and residual errors for the chest wall and shoulder joint in kV orthogonal and tangential setup images with laser- or SGRT-based setup.ResultsLess isocenter variance was found in Group A than in Group L. Rotations and posture errors were larger in group L than in Group A (p ≤ 0.05). Rotation error was smaller with T-shaped ROI than with O- or B-shape (p = 0.01–0.04).ConclusionSetup with AlignRT^® improves reproducibility compared to laser setup. Between the different ROI shapes only small differences were found in the patient posture or the isocenter position in the images. The T-ROI is recommended to set up the chest wall bony structure and an additional B-ROI may be used to fine-tune the soft tissue accuracy.     

Specificity and rate of human and mouse liver and plasma phosphatidylcholine synthesis analyzed in vivo

Phosphatidylcholine (PC) synthesis by the direct cytidine diphosphate choline (CDP-choline) pathway in rat liver generates predominantly mono- and di-unsaturated molecular species, while polyunsaturated PC species are synthesized largely by the phosphatidylethanolamine-N-methyltransferase (PEMT) pathway. Although altered PC synthesis has been suggested to contribute to development of hepatocarcinoma and nonalcoholic steatohepatitis, analysis of the specificity of hepatic PC metabolism in human patients has been limited by the lack of sensitive and safe methodologies. Here we incorporated a deuterated methyl-D(9)-labled choline chloride, to quantify biosynthesis fluxes through both of the PC synthetic pathways in vivo in human volunteers and compared these fluxes with those in mice. Rates and molecular specificities of label incorporated into mouse liver and plasma PC were very similar and strongly suggest that label incorporation into human plasma PC can provide a direct measure of hepatic PC synthesis in human subjects. Importantly, we demonstrate for the first time that the PEMT pathway in human liver is selective for polyunsaturated PC species, especially those containing docosahexaenoic acid. Finally, we present a multiple isotopomer distribution analysis approach, based on transfer of deuterated methyl groups to S-adenosylmethionine and subsequent sequential methylations of PE, to quantify absolute flux rates through the PEMT pathway that are applicable to studies of liver dysfunction in clinical studies.     

A strategy to accelerate protein production from a pool of clones in Chinese hamster ovary cells for toxicology studies

In the biopharmaceutical industry, a clonally derived cell line is typically used to generate material for investigational new drug (IND)‐enabling toxicology studies. The same cell line is then used to generate material for clinical studies. If a pool of clones can be used to produce material for IND‐enabling toxicology studies (Pool for Tox (PFT) strategy) during the time a lead clone is being selected for clinical material production, the toxicology studies can be accelerated significantly (approximately 4 months at Genentech), leading to a potential acceleration of 4 months for the IND submission. We explored the feasibility of the PFT strategy with three antibodies—mAb1, mAb2, and mAb3—at the 2 L scale. For each antibody, two lead cell lines were identified that generated material with similar product quality to the material generated from the associated pool. For two antibody molecules, mAb1 and mAb2, the material generated by the lead cell lines from 2 L bioreactors was tested in an accelerated stability study and was shown to have stability comparable to the material generated by the associated pool. Additionally, we used this approach for two antibody molecules, mAb4 and mAb5, at Tox and GMP production. The materials from the Tox batch at 400 L scale and three GMP batches at 2000 L scale have comparable product quality attributes for both molecules. Our results demonstrate the feasibility of using a pool of clonally derived cell lines to generate material of similar product quality and stability for use in IND‐enabling toxicology studies as was derived from the final production clone, which enabled significant acceleration of timelines into clinical development. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1449–1455, 2017     

Correlating Behavioral Responses to fMRI Signals from Human Prefrontal Cortex: Examining Cognitive Processes Using Task Analysis

The aim of this methods paper is to describe how to implement a neuroimaging technique to examine complementary brain processes engaged by two similar tasks. Participants'' behavior during task performance in an fMRI scanner can then be correlated to the brain activity using the blood-oxygen-level-dependent signal. We measure behavior to be able to sort correct trials, where the subject performed the task correctly and then be able to examine the brain signals related to correct performance. Conversely, if subjects do not perform the task correctly, and these trials are included in the same analysis with the correct trials we would introduce trials that were not only for correct performance. Thus, in many cases these errors can be used themselves to then correlate brain activity to them. We describe two complementary tasks that are used in our lab to examine the brain during suppression of an automatic responses: the stroop¹ and anti-saccade tasks. The emotional stroop paradigm instructs participants to either report the superimposed emotional ''word'' across the affective faces or the facial ''expressions'' of the face stimuli^1,2. When the word and the facial expression refer to different emotions, a conflict between what must be said and what is automatically read occurs. The participant has to resolve the conflict between two simultaneously competing processes of word reading and facial expression. Our urge to read out a word leads to strong ''stimulus-response (SR)'' associations; hence inhibiting these strong SR''s is difficult and participants are prone to making errors. Overcoming this conflict and directing attention away from the face or the word requires the subject to inhibit bottom up processes which typically directs attention to the more salient stimulus. Similarly, in the anti-saccade task^3,4,5,6, where an instruction cue is used to direct only attention to a peripheral stimulus location but then the eye movement is made to the mirror opposite position. Yet again we measure behavior by recording the eye movements of participants which allows for the sorting of the behavioral responses into correct and error trials⁷ which then can be correlated to brain activity. Neuroimaging now allows researchers to measure different behaviors of correct and error trials that are indicative of different cognitive processes and pinpoint the different neural networks involved.     

Structure-Based and Random Mutagenesis Approaches Increase the Organophosphate-Degrading Activity of a Phosphotriesterase Homologue from Deinococcus radiodurans

An enzyme from the amidohydrolase family from Deinococcus radiodurans (Dr-OPH) with homology to phosphotriesterase has been shown to exhibit activity against both organophosphate (OP) and lactone compounds. We have characterized the physical properties of Dr-OPH and have found it to be a highly thermostable enzyme, remaining active after 3 h of incubation at 60 °C and withstanding incubation at temperatures up to 70 °C. In addition, it can withstand concentrations of at least 200 mg/mL. These properties make Dr-OPH a promising candidate for development in commercial applications. However, compared to the most widely studied OP-degrading enzyme, that from Pseudomonas diminuta, Dr-OPH has low hydrolytic activity against certain OP substrates. Therefore, we sought to improve the OP-degrading activity of Dr-OPH, specifically toward the pesticides ethyl and methyl paraoxon, using structure-based and random approaches. Site-directed mutagenesis, random mutagenesis, and site-saturation mutagenesis were utilized to increase the OP-degrading activity of Dr-OPH. Out of a screen of more than 30,000 potential mutants, a total of 26 mutant enzymes were purified and characterized kinetically. Crystal structures of w.t. Dr-OPH, of Dr-OPH in complex with a product analog, and of 7 mutant enzymes were determined to resolutions between 1.7 and 2.4 Å. Information from these structures directed the design and production of 4 additional mutants for analysis. In total, our mutagenesis efforts improved the catalytic activity of Dr-OPH toward ethyl and methyl paraoxon by 126- and 322-fold and raised the specificity for these two substrates by 557- and 183-fold, respectively. Our work highlights the importance of an iterative approach to mutagenesis, proving that large rate enhancements are achieved when mutations are made in already active mutants. In addition, the relationship between the kinetic parameters and the introduced mutations has allowed us to hypothesize on those factors most important for maintaining the structure and function of the enzyme.