Development of FRET and Stress Granule Dual-Based System to Screen for Viral 3C Protease Inhibitors.

3C proteases (3Cpros) of picornaviruses and 3C-like proteases (3CLpros) of coronaviruses and caliciviruses represent a group of structurally and functionally related viral proteases that play pleiotropic roles in supporting the viral life cycle and subverting host antiviral responses. The design and screening for 3C/3CLpro inhibitors may contribute to the development broad-spectrum antiviral therapeutics against viral diseases related to these three families. However, current screening strategies cannot simultaneously assess a compound's cytotoxicity and its impact on enzymatic activity and protease-mediated physiological processes. The viral induction of stress granules (SGs) in host cells acts as an important antiviral stress response by blocking viral translation and stimulating the host immune response. Most of these viruses have evolved 3C/3CLpro-mediated cleavage of SG core protein G3BP1 to counteract SG formation and disrupt the host defense. Yet, there are no SG-based strategies screening for 3C/3CLpro inhibitors. Here, we developed a fluorescence resonance energy transfer (FRET) and SG dual-based system to screen for 3C/3CLpro inhibitors in living cells. We took advantage of FRET to evaluate the protease activity of poliovirus (PV) 3Cpro and live-monitor cellular SG dynamics to cross-verify its effect on the host antiviral response. Our drug screen uncovered a novel role of Telaprevir and Trifluridine as inhibitors of PV 3Cpro. Moreover, Telaprevir and Trifluridine also modulated 3Cpro-mediated physiological processes, including the cleavage of host proteins, inhibition of the innate immune response, and consequent facilitation of viral replication. Taken together, the FRET and SG dual-based system exhibits a promising potential in the screening for inhibitors of viral proteases that cleave G3BP1.

A laboratory exercise for visible gel filtration chromatography using fluorescent proteins.

Gel filtration chromatography (GFC) separates molecules according to size and is one of the most widely used methods for protein purification. Here, red fluorescent protein (RFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), and/or their fusion proteins were prokaryotically expressed, purified, and used in a laboratory exercise to intuitively demonstrate GFC. Different bands, corresponding to RFP, RFP-CFP (RC), YFP-RFP-YFP (YRY), and pyruvate kinase II-GFP (PKG) were well separated on a Superdex 200 column from a 0.5-mL sample. Increasing the sample volume and changing the chromatographic resin to Sephadex G-100 resulted in lower resolution separation. Students enjoyed identifying combinations of colored proteins and found this exercise helpful for understanding the factors that affect GFC resolution.

[Change of unsaturated fatty acids in hippocampus of mice exposed to lead].

OBJECTIVE: To study possible impairment mechanisms of learning and memory abilities from unsaturated fatty acids in hippocampus of mice exposed to lead. METHODS: Forty-eight healthy mice were divided into 4 groups: low dose (0.625 g/L), middle dose (1.250 g/L) and high dose (2.500 g/L) of lead solution in diet and control group (distilled water). The mice in treatment groups were fed with lead solution every day while the mice in control group were fed with distilled water for 50 days. After learning and memory abilities were measured, the mice were killed and contents of oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3), arachidonic acid (AA,C20:4), eicosapentaenoic acid (EPA,C20:5) and docosahexaenoic acid (DHA, C22:6 ) in hippocampus of mice were determined by high performance liquid chromatography (HPLC). RESULTS: (1) In the four training days, the mice treated with lead in the middle dose group and high dose group significantly increased the escape latencies compared with the mice treated with distilled water (P<0.05), and on the 4th day, the low dosage mice's escape latencies were delayed (P<0.05). The escape latencies of the 1st, 2nd, 3rd and 4th day had significantly positive linear relation with lead dose. Their relative coefficient in turn is r=0.973, 0.985, 0.929 and 0.936, indicating that lead harmed spatial memory of mice in Morris water maze (MWM). (2) The contents of C18:2 and AA were obviously enhanced in hippocampus of middle and high dosage (P<0.05); while there was evident decrease in the contents of C18:3, EPA and DHA (P<0.05); the content of C18:1 was decreased significantly in high dosage group (P<0.01). The mice's escape latencies had significantly negative linear relation with contents of C18:1, C18:3, EPA and DHA, while there was positive linear relation significantly with contents of C18:2 and AA. Their relative coefficient in turn was r=-0.901, -0.914, -0.893, -0.855, 0.936, 0.727. CONCLUSION: Lead interferes with the metabolism of hippocampus fatty acids and affects membrane function in hippocampus of mice, which might contribute to change of the synthesis, metabolism and release of central neurotransmitter and decrease of the learning and memory abilities.

[High-level transcription of human mitochondrial tRNA(Trp) based on ribozyme and characterization on its aminoacylation activity].

In vitro transcription systems with T7 RNA polymerase (T7 RNAP) were widely used in preparation of RNA because of their simplicity and high efficiency. The transcripts would have additional 5' sequence since T7 promoter spans the transcription start site, while deletion of the transcription start site would severely reduce the T7 RNAP transcriptional activity. We successfully developed an in vitro transcription by combining of T7 RNAP high efficient transcription system and highly specific self-splicing technology of ribozymes, in this system, ribozyme self-splices at the designed specific site and releases the aim RNA without affecting transcription efficiency of T7 RNAP, the aminoacylation activity of human mitochondrial tRNA(Trp) (HmtRNA(Trp) (UCA)) is 113.6 pmol/microg. This method with its high efficiency on transcription and good repeatability is very suitable for preparation of accurate RNA in large scale.

[Effects of low temperature at 10 degrees C on some antioxidant enzyme activities and ultrastructures of hypocotylar cells in mung bean and garden pea].

Mung bean (Phaseolus radiatus Linn.) and garden pea (Pisum satium Linn.), which were stressed 4 days under a low temperature of 10 degrees C, were used as materials to study the cold tolerance of plant with different resistance. On the 2nd and 3rd day under 10 degrees C stress, both the malondialdehyde (MDA) content and the superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activities increased significantly in hypocotylar cells of mung bean, so did SOD activity in garden pea, but other physiological indexes in garden pea were not different from the non-treatment groups (Figs. 1-5). In hypocotylar cells of mung bean, SOD activity always maintain at the highest level in a period of time,and so does POD activity (Figs. 3, 4). Ultrastructural results after stress indicated as follows: (1) Plastids in hypocotylar cells of mung bean accumulated much starch (Plate I-6), whereas the form of plastids in hypocotylar cells of garden pea changed maskedly to become dumb-bell-shaped, round or irregular, with the last one being the most common form (Plate I-8, 12); (2) In both mung bean and garden pea, central vacuole was divided into small vacuoles (Plate I-4, 10), and the number of mitochondria increased and became aggregated (Plate I-3, 11, 12). Judging from the activities of protective enzymes and ultrastructures, 10 degrees C low temperature caused non-lethal, temporary injuries to hypocotyls ultrastructures in mung bean, but no visible injury at all, and even improved its cold tolerance to a certain degree in garden pea.

[Effects of soybean phospholipids on learning and memory ability and contents of lipids in mice's brain].

OBJECTIVE: To study the effects of soybean phospholipids on learning and memory abilities and contents of protein and lipids in brain of mice. METHODS: Mice were randomly divided into four groups: low dose (2.5 g/kg), middle dose(5.0 g/kg) and high dose (10.0 g/kg) of soybean Phospholipids in diet and control group (distilled water). The mice were fed with soybean phospholipids everyday while control groups were fed with distilled water for 30 days. After learning and memory abilities were measured, the mice were killed and contents of protein, fatty acids and phospholipids in brain of mice were determined. RESULTS: The learning and memory abilities of mice fed with middle and high dose of soybean Phospholipids were improved obviously(P < 0.05), while memory abilities of low dose's mice were stronger than that of control(P < 0.05). (2) The brain weight v.8 body weight of those in three doses' group was obviously increased, and the protein content in brain was significantly higher compared with control(P < 0.05). (3) The contents of linoleic acid(C18: 2), linolenic acid(C18: 3) and ratio of polyunsaturated fatty acids(PUFA) to total fatty acids (TFA) obviously increased (P < 0.05). In brain of high dose mice, there was obvious change in the content of linolenic acid(C18: 3) in middle's (P < 0.05). (4) The contents of phosphatidylcholine (PS) phosphatidylethanolamine (PE), Phosphatidylcholine (PC) and phospholipids (PL) obviously increase (P < 0.05), while ratio of SM to PC was decreased significantly in brain of high dose mice(P < 0.05). The contents of PS, PC and PL were increased obviously(P < 0.05). CONCLUSION: Soybean phospholipids may increase contents of protein, PUFA and PL in brain of mice and improve learning and memory abilities of mice effectively in a dose-response manner.