A Recombinant System and Reporter Viruses for Papiine Alphaherpesvirus 2

Primate simplex viruses, including Herpes simplex viruses 1 and 2, form a group of closely related herpesviruses, which establish latent infections in neurons of their respective host species. While neuropathogenic infections in their natural hosts are rare, zoonotic transmission of Macacine alphaherpesvirus 1 (McHV1) from macaques to humans is associated with severe disease. Human infections with baboon-derived Papiine alphaherpesvirus 2 (PaHV2) have not been reported, although PaHV2 and McHV1 share several biological properties, including neuropathogenicity in mice. The reasons for potential differences in PaHV2 and McHV1 pathogenicity are presently not understood, and answering these questions will require mutagenic analysis. Here, we report the development of a recombinant system, which allows rescue of recombinant PaHV2. In addition, we used recombineering to generate viruses carrying reporter genes (Gaussia luciferase or enhanced green fluorescent protein), which replicate with similar efficiency as wild-type PaHV2. We demonstrate that these viruses can be used to analyze susceptibility of cells to infection and inhibition of infection by neutralizing antibodies and antiviral compounds. In summary, we created a recombinant system for PaHV2, which in the future will be invaluable for molecular analyses of neuropathogenicity of PaHV2.

A simple secretion assay for assessing new and existing myocilin variants

With the increasing use of molecular genetics approaches for determination of potential disease-causing mutations, it is becoming more important to be able to interpret and act upon the provided results. As an example of such an instance, nearly 300 mutations have been identified in the myocilin (MYOC) gene, which is the most commonly mutated gene causing primary open angle glaucoma. Yet a lack of sufficient information exists for many of these variants, hindering their definitive classification. While the function of MYOC is unclear, biochemically, the vast majority of glaucoma-causing MYOC mutations result in protein non-secretion and intracellular insoluble aggregate formation in cultured cells. Previously we generated a Gaussia luciferase-based MYOC fusion protein to sensitively track secretion of the protein. Herein we applied this same assay to fourteen clinically-derived MYOC variants with varying degrees of predicted pathogenicity and compared the luciferase secretion results with the better established MYOC assay of western blotting. Eight of the variants (G12R, V53A, T204T, P254L, T325T, D380H, D395_E396insDP, and P481S) had not been biochemically assessed previously. Of these, P254L and D395_E396insDP demonstrated significant secretion defects from human embryonic kidney (HEK-293A) cells reminiscent of glaucoma-causing mutations. Overall, we found that the luciferase assay results agreed with western blotting for thirteen of the fourteen variants (93%), suggesting a strong concordance. These results suggest that the Gaussia luciferase assay may be used as a complementary or standalone assay for quickly assessing MYOC variant behavior, and anticipate that these results will be useful in MYOC variant curation and reclassification.

A High-Content Screen for the Identification of Plant Extracts with Insulin Secretion-Modulating Activity

Bioactive plant compounds and extracts are of special interest for the development of pharmaceuticals. Here, we describe the screening of more than 1100 aqueous plant extracts and synthetic reference compounds for their ability to stimulate or inhibit insulin secretion. To quantify insulin secretion in living MIN6 β cells, an insulin–Gaussia luciferase (Ins-GLuc) biosensor was used. Positive hits included extracts from Quillaja saponaria, Anagallis arvensis, Sapindus mukorossi, Gleditsia sinensis and Albizia julibrissin, which were identified as insulin secretion stimulators, whereas extracts of Acacia catechu, Myrtus communis, Actaea spicata L., Vaccinium vitis-idaea and Calendula officinalis were found to exhibit insulin secretion inhibitory properties. Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) were used to characterize several bioactive compounds in the selected plant extracts, and these bioactives were retested for their insulin-modulating properties. Overall, we identified several plant extracts and some of their bioactive compounds that may be used to manipulate pancreatic insulin secretion.

Glucose-Responsive Gene Delivery at Physiological pH through Tertiary-Amine Stabilized Boronate-PVA Particles Synthesized by One-Pot Reaction

We report a physiologically stable and cytocompatible glucose-responsive nonviral gene delivery system made up of boronate functionalized polymeric material. Herein, we utilize boronate cis-diol interactions to develop a glucose-responsive submicron particle (SMP) system. The stability of the boronate interaction at a physiological pH was achieved by copolymerization of dimethyl aminoethyl methacrylate (DMAEMA) with acrylamidophenylboronic acid (AAPBA) and the formation of a complex with polyvinylalcohol (PVA) which is governed by cis-diol interactions. The shift in hydrodynamic diameter of SMPs was observed and correlated with increasing glucose concentrations at a physiological pH. Optimal transfection was observed for a 5 µg dose of the gaussia luciferase reporter gene in NIH3T3 cells without any adverse effect on cellular viability. The destabilization of the AAPBA–PVA complex by interacting with glucose allowed the release of encapsulated bovine serum albumin (BSA) in a glucose-responsive manner. In total, 95% of BSA was released from SMPs at a 50 mM glucose concentration after 72 h. A two-fold increase in transfection was observed in 50 mM glucose compared to that of 10 mM glucose.

Solution structure of Gaussia Luciferase with five disulfide bonds and identification of a putative coelenterazine binding cavity by heteronuclear NMR

Gaussia luciferase (GLuc) is a small luciferase (18.2 kDa; 168 residues) and is thus attracting much attention as a reporter protein, but the lack of structural information is hampering further application. Here, we report the first solution structure of a fully active, recombinant GLuc determined by heteronuclear multidimensional NMR. We obtained a natively folded GLuc by bacterial expression and efficient refolding using a Solubility Enhancement Petide (SEP) tag. Almost perfect assignments of GLuc’s 1H, 13C and 15N backbone signals were obtained. GLuc structure was determined using CYANA, which automatically identified over 2500 NOEs of which > 570 were long-range. GLuc is an all-alpha-helix protein made of nine helices. The region spanning residues 10–18, 36–81, 96–145 and containing eight out of the nine helices was determined with a Cα-atom RMSD of 1.39 Å ± 0.39 Å. The structure of GLuc is novel and unique. Two homologous sequential repeats form two anti-parallel bundles made by 4 helices and tied together by three disulfide bonds. The N-terminal helix 1 is grabbed by these 4 helices. Further, we found a hydrophobic cavity where several residues responsible for bioluminescence were identified in previous mutational studies, and we thus hypothesize that this is a catalytic cavity, where the hydrophobic coelenterazine binds and the bioluminescence reaction takes place.

Screening for Anti-Influenza Actives of Prefractionated Traditional Chinese Medicines

Traditional Chinese medicines (TCMs) have proven to possess advantages in counteracting virus infections according to clinical practices. It’s therefore of great value to discover novel antivirals from TCMs. In this paper, One hundred medicinal plants which have been included in TCM prescriptions for antiviral treatment were selected and prefractionated into 5 fractions each by sequentially using cyclohexane, dichloromethane, ethyl acetate, n-butanol, and water. 500 TCM-simplified extracts were then subjected to a phenotypic screening using a recombinant IAV expressing Gaussia luciferase. Ten TCM fractions were identified to possess antiviral activities against influenza virus. The IC50’s of the hit fractions range from 1.08 to 6.45 μg/mL, while the SIs, from 7.52 to 98.40. Furthermore, all the ten hit fractions inhibited the propagation of progeny influenza virus significantly at 20 μg/mL. The hit TCM fractions deserve further isolation for responsible constituents leading towards anti-influenza drugs. Moreover, a library consisting of 500 simplified TCM extracts was established, facilitating antiviral screening in quick response to emerging and re-emerging viruses such as Ebola virus and current SARS-CoV-2 pandemic.