Establishment of a human cell line with a surface display system for screening and optimizing Na+-taurocholate cotransporting polypeptide-binding peptides.

One of the most desirable targets for HBV medications is the sodium taurocholate cotransporting polypeptide (NTCP), an entry receptor for the hepatitis B virus (HBV). N-myristoylated preS1 2-48 (Myrcludex B or Hepcludex), an NTCP-binding peptide from the large surface protein of HBV, has been developed as the first-in-class entry inhibitor. However, its relatively large molecular weight contributes to increased immunogenicity and antibody production. As a result, it is preferable to look for an NTCP-binding peptide with a smaller size. To do this, we developed a human cell surface display strategy and screened peptides based on preS1-21. PreS1-21 (genotype D) was extended by 7 random amino acids and fused with mCherry and FasL transmembrane domain. The pooled constructs were transfected into HEK293 cells by using the transposon/transposase system to create a library displaying various peptides on the cell surface with red fluorescence. On the other hand, we expressed NTCP protein fused with EGFP on HEK293 and used the membrane lysate containing NTCP-GFP as the bait protein to select peptides with increased NTCP affinity. After 7 cycles of selection, the deep sequencing results revealed that some polypeptides were more than 1,000 times enriched. Further screening of the mostly enriched 10 peptides yields the peptide preS1-21-pep3. Replacing the preS1-21 sequence of preS1-21-pep3 with those from different genotypes demonstrated that the consensus sequence of genotype A-F had the best performance. The peptide (Myr-preS1-21-pep3) was synthesized and tested on the HepG2-NTCP cell model. The results showed that Myr-preS1-21-pep3 is approximately 10 times more potent than the initial peptide Myr-preS1-21 in preventing HBV infection. In conclusion, we developed a new strategy for screening peptides binding to membrane proteins and identified a new NTCP-binding peptide with a much smaller size than Hepcludex.

Caspase sensors based on NanoLuc.

Caspases are a family of evolutionary conserved cysteine proteases that play key roles in programmed cell death and inflammation. Among the methods for the detection of caspase activity, biosensors based on luciferases have advantages in genetical encoding and convenience in assay. In this study, we constructed a new set of caspase biosensors based on NanoLuc luciferase. This kind of sensors, named NanoLock, work in dark-to-bright model, with the help of a NanoLuc quencher peptide (HiBiT-R/D) mutated from HiBiT. Optimized NanoLock responded to proteases with high signal to noise ratio (S/N), 1233-fold activation by tobacco etch virus protease in HEK293 cells and > 500-fold induction to caspase 3 in vitro. We constructed NanoLocks for the detection of caspase 1, 3, 6, 7, 8, 9, and 10, and assays in HEK293 cells demonstrated that these sensors performed better than commercial kits in the aspect of S/N and convenience. We further established a cell line stably expressing NanoLock-casp 6 and provided a proof-of-concept for the usage of this cell line in the high throughput screening of caspase 6 modulator.

Angiographic Characteristics of Cerebral Perfusion and Hemodynamics of the Bridging Artery After Surgical Treatment of Unilateral Moyamoya Disease.

Purpose: To investigate the characteristics of cerebral perfusion and hemodynamics of bypass grafting in the treatment of moyamoya disease (MMD) using the iFlow color-coded flow map in comparison with magnetic resonance imaging-perfusion-weighted imaging (MRI-PWI) and computational fluid dynamic (CFD) analysis. Materials and Methods: Patients with MMD treated with bypass grafting who had undergone MRI PWI and digital subtraction angiography for iFlow color-coded map was retrospectively enrolled and CFD was performed for calculating the hemodynamic stresses around the bypass grafting. Results: Forty-five patients with unilateral MMD treated with bypass surgery were enrolled. The bypass surgery was successful in all patients, with no severe neurological complications during the periprocedural period. Followed up for 4-12 months (median 5.5), the neurological function was good in all patients. The cerebral blood flow (CBF), cerebral blood volume (CBV), mean transit time (MTT), and time to peak (TTP) were significantly (p < 0.05) improved in the middle cerebral artery distribution area on the surgical side before and after vascular bypass, and the difference of TTP (s) measured from the proximal bifurcation of common carotid artery to the confluence of sinus was also significant (p < 0.05). A significant (p < 0.05) positive correlation existed in the perfusion parameters between the iFlow blood perfusion and the MRI-PWI perfusion, with r-value for TTP of 0.765 (p < 0.01). The iFlow color-coded blood flow map showed warm color changes on the diseased side, similar to those on the contralateral side. In CFD analysis, the hemodynamic stresses were all improved, in and around the bypass grafting and distal vessels, which were beneficial to blood flow entering distal arterial branches. Conclusion: The iFlow color-coded flow map can be used to analyze cerebral perfusion after bypass grafting for MMD, similar to MRI-PWI, and CFD can be used to analyze the hemodynamics after bypass grafting, revealing improved hemodynamics to promote blood flow entering distal arteries.

Identification of STAU1 as a regulator of HBV replication by TurboID-based proximity labeling.

The core promoter (CP) of hepatitis B virus (HBV) is critical for HBV replication by controlling the transcription of pregenomic RNA (pgRNA). Host factors regulating the activity of the CP can be identified by different methods. Biotin-based proximity labeling, a powerful method with the capability to capture weak or dynamic interactions, has not yet been used to map proteins interacting with the CP. Here, we established a strategy, based on the newly evolved promiscuous enzyme TurboID, for interrogating host factors regulating the activity of HBV CP. Using this strategy, we identified STAU1 as an important factor involved in the regulation of HBV CP. Mechanistically, STAU1 indirectly binds to CP mediated by TARDBP, and recruits the SAGA transcription coactivator complex to the CP to upregulate its activity. Moreover, STAU1 binds to HBx and enhances the level of HBx by stabilizing it in a ubiquitin-independent manner.

Early use of omeprazole benefits patients with acute myocardial infarction.

BACKGROUND: Bleeding complications are not uncommon in patients with acute myocardial infarction (AMI) during treatments. How to prevent the occurrence of upper gastrointestinal bleeding in AMI patients has become one of the most intractable problems. And there are conflicting data on the efficacy and complication rate of omeprazole treatment. We conducted an intervention study to determine whether using omeprazole could benefit AMI patients. METHODS: A total of 237 patients with AMI were divided into two groups at random: omeprazole group including 114 patients and control group including 123 patients. Omeprazole 40 mg by intravenous drip was given to the patients in omeprazole group when they were admitted to the hospitals. From the second day they were given omeprazole 20 mg per day by oral administration for 7 days. In contrast, no gastric acid inhibitor was given to the patients in control group. The incidence of upper gastrointestinal bleeding, the recanalization rate and overall mortality in both groups were observed. RESULTS: The incidence of upper gastrointestinal bleeding in omeprazole group was 5.3% (6/114) which was much lower than 14.6% (18/123) in control group (P = 0.017), but the recanalization rate had no significant difference between the two groups (P = 0.681). The overall mortality in omeprazole group was lower than that of control group (3.5% vs. 10.6%, P = 0.035). CONCLUSIONS: Our findings suggest that early use of omeprazole in AMI patients could decrease the incidence of upper gastrointestinal bleeding and the overall mortality, without influencing the recanalization rate. Early use of omeprazole might benefit AMI patients.