Modular Peroxidase-Based Reporters for Detecting Protease Activity and Protein Interactions with Temporal Gating.

Enzymatic reporters have been widely applied to study various biological processes because they can amplify signal through enzymatic reactions and provide good sensitivity. However, there is still a need for modular motifs for designing a series of enzymatic reporters. Here, we report a modular peroxidase-based motif, named CLAPon, that features acid-base coil-caged enhanced ascorbate peroxidase (APEX). We demonstrate the modularity of CLAPon by designing a series of reporters for detecting protease activity and protein-protein interactions (PPIs). CLAPon for protease activity showed a 390-fold fluorescent signal increase upon tobacco etch virus protease cleavage. CLAPon for PPI detection (PPI-CLAPon) has two variants, PPI-CLAPon1.0 and 1.1. PPI-CLAPon1.0 showed a signal-to-noise ratio (SNR) of up to 107 for high-affinity PPI pairs and enabled imaging with sub-cellular spatial resolution. However, the more sensitive PPI-CLAPon1.1 is required for detecting low-affinity PPI pairs. PPI-CLAPon1.0 was further engineered to a reporter with light-dependent temporal gating, called LiPPI-CLAPon1.0, which can detect a 3-min calcium-dependent PPI with an SNR of 17. LiPPI-CLAPon enables PPI detection within a specific time window with rapid APEX activation and diverse readout. Lastly, PPI-CLAPon1.0 was designed to have chemical gating, providing more versatility to complement the LiPPI-CLAPon. These CLAPon-based reporter designs can be broadly applied to study various signaling processes that involve protease activity and PPIs and provide a versatile platform to design various genetically encoded reporters.

Plasma melatonin levels in patients with diabetic retinopathy secondary to type 2 diabetes.

BACKGROUND: Melatonin is reported to be related to diabetes mellitus (DM) risk; however, the effect of melatonin on diabetic retinopathy (DR) risk remains unclear. AIM: The aim of this study was to determine the effect of melatonin on DR risk. METHODS: A hospital-based case-control study was conducted from January 2020 to June 2020. DR was assessed using the Diabetic Retinopathy preferred practice pattern (PPP)-updated 2019 criteria. The participants were divided into the DM cases without DR (NDR) group, non-proliferative DR (NPDR) group and proliferative DR (PDR) group. Plasma melatonin concentration was detected with the enzyme-linked immunosorbent assay kit. The relationship between plasma melatonin concentration and DR risk as well as severity was assessed. RESULTS: It was found that plasma melatonin was 72.83 ± 16.25, 60.38 ± 13.43, 44.48 ± 10.30 and 44.69 ± 8.95 pg/mL in healthy controls, NDR group, NPDR and PDR group, respectively. In addition, it was found that plasma melatonin could be used as a potential diagnostic biomarker for DR (AUC = 0.893, P < 0.001). There was a significant positive relationship between total bilirubin and melatonin content (P < 0.001) based on the correlation assay. Significant associations between total bilirubin and melatonin content were also detected in the NPDR (R 2 = 0.360, P < 0.001) and PDR (R 2 = 0.183, P < 0.001) groups. CONCLUSION: The data obtained in this study demonstrated that plasma melatonin concen-tration was decreased in DR cases and could be used as a sensitive and specific marker for the diagnosis of DR. A significant positive relationship between total bilirubin and melatonin was detected. More related studies are required to understand the role of melatonin in DR.

Calcium channel blockers reduce severe fever with thrombocytopenia syndrome virus (SFTSV) related fatality.

Severe fever with thrombocytopenia syndrome (SFTS), an emerging tick-borne infectious disease caused by a novel phlebovirus (SFTS virus, SFTSV), was listed among the top 10 priority infectious diseases by the World Health Organization due to its high fatality of 12%-50% and possibility of pandemic transmission. Currently, effective anti-SFTSV intervention remains unavailable. Here, by screening a library of FDA-approved drugs, we found that benidipine hydrochloride, a calcium channel blocker (CCB), inhibited SFTSV replication in vitro. Benidipine hydrochloride was revealed to inhibit virus infection through impairing virus internalization and genome replication. Further experiments showed that a broad panel of CCBs, including nifedipine, inhibited SFTSV infection. The anti-SFTSV effect of these two CCBs was further analyzed in a humanized mouse model in which CCB treatment resulted in reduced viral load and decreased fatality rate. Importantly, by performing a retrospective clinical investigation on a large cohort of 2087 SFTS patients, we revealed that nifedipine administration enhanced virus clearance, improved clinical recovery, and remarkably reduced the case fatality rate by >5-fold. These findings are highly valuable for developing potential host-oriented therapeutics for SFTS and other lethal acute viral infections known to be inhibited by CCBs in vitro.

HBV induces different responses of the hepatocytes and oval cells during HBV-related hepatic cirrhosis.

Although hepatitis B virus (HBV)-related cirrhosis and hepatocellular carcinoma (HCC) cause a sever health problem worldwide, the underlying mechanisms are still elusive. This study aimed to investigate the responses of different cell types isolated from HBV transgenic mice. A cross-sectional set of hepatocytes and oval cells were obtained from HBV transgenic and control mice. Flow cytometry, immunohistochemistry and microarray were applied to investigate the cell biology of the hepatocytes and oval cells. Our results showed that HBV induced the proliferation of both cell oval cells and hepatocytes, and induced cell death of HBV hepatocytes while had minimal effects on oval cells. Further molecular and pathways analysis identified some genes and signaling pathways may be responsible for the different responses between oval cells and hepatocytes. In addition, analyses of selectively ten genes by IHC staining in human samples were consistent with microarray data. In summary, HBV transgenic mice is a useful model for studying the biological behaviors of oval cells affected by HBV and HBV-cirrhosis. Also, our results help better understand the mechanisms of HBV induced cirrhosis, and provide novel progenitor markers or prognostic/therapeutic markers.

Global quantitative proteomic analysis profiles host protein expression in response to Sendai virus infection.

Sendai virus (SeV) is an enveloped nonsegmented negative-strand RNA virus that belongs to the genus Respirovirus of the Paramyxoviridae family. As a model pathogen, SeV has been extensively studied to define the basic biochemical and molecular biologic properties of the paramyxoviruses. In addition, SeV-infected host cells were widely employed to uncover the mechanism of innate immune response. To identify proteins involved in the SeV infection process or the SeV-induced innate immune response process, system-wide evaluations of SeV-host interactions have been performed. cDNA microarray, siRNA screening and phosphoproteomic analysis suggested that multiple signaling pathways are involved in SeV infection process. Here, to study SeV-host interaction, a global quantitative proteomic analysis was performed on SeV-infected HEK 293T cells. A total of 4699 host proteins were quantified, with 742 proteins being differentially regulated. Bioinformatics analysis indicated that regulated proteins were mainly involved in "interferon type I (IFN-I) signaling pathway" and "defense response to virus," suggesting that these processes play roles in SeV infection. Further RNAi-based functional studies indicated that the regulated proteins, tripartite motif (TRIM24) and TRIM27, affect SeV-induced IFN-I production. Our data provided a comprehensive view of host cell response to SeV and identified host proteins involved in the SeV infection process or the SeV-induced innate immune response process.

Activation of the RLR/MAVS Signaling Pathway by the L Protein of Mopeia Virus.

The family Arenaviridae includes several important human pathogens that can cause severe hemorrhagic fever and greatly threaten public health. As a major component of the innate immune system, the RLR/MAVS signaling pathway is involved in recognizing viral components and initiating antiviral activity. It has been reported that arenavirus infection can suppress the innate immune response, and NP and Z proteins of pathogenic arenaviruses can disrupt RLR/MAVS signaling, thus inhibiting production of type I interferon (IFN-I). However, recent studies have shown elevated IFN-I levels in certain arenavirus-infected cells. The mechanism by which arenavirus infection induces IFN-I responses remains unclear. In this study, we determined that the L polymerase (Lp) of Mopeia virus (MOPV), an Old World (OW) arenavirus, can activate the RLR/MAVS pathway and thus induce the production of IFN-I. This activation is associated with the RNA-dependent RNA polymerase activity of Lp. This study provides a foundation for further studies of interactions between arenaviruses and the innate immune system and for the elucidation of arenavirus pathogenesis. IMPORTANCE: Distinct innate immune responses are observed when hosts are infected with different arenaviruses. It has been widely accepted that NP and certain Z proteins of arenaviruses inhibit the RLR/MAVS signaling pathway. The viral components responsible for the activation of the RLR/MAVS signaling pathway remain to be determined. In the current study, we demonstrate for the first time that the Lp of MOPV, an OW arenavirus, can activate the RLR/MAVS signaling pathway and thus induce the production of IFN-I. Based on our results, we proposed that dynamic interactions exist among Lp-produced RNA, NP, and the RLR/MAVS signaling pathway, and the outcome of these interactions may determine the final IFN-I response pattern: elevated or reduced. Our study provides a possible explanation for how IFN-I can become activated during arenavirus infection and may help us gain insights into the interactions that form between different arenavirus components and the innate immune system.

[Synthesis and identification of artificial complete antigen 25-hydroxyvitamin D(3);].

AIM: To synthesize the 25-hydroxyvitamin D(3); artificial complete antigen and to prepare the specific antibody against 25-hydroxyvitamin D(3);. METHODS: The active group carboxyl was introduced into 25-hydroxyvitamin D(3); and formed 25-hydroxyvitamin D(3);-hemisuccinate which possessed the structure of the hapten by chemical modification. The EDC method was applied to conjugate 25-hydroxyvitamin D(3);-hemisuccinate to bovine serum albumin as an artificial immunogen. The coating antigen 25-hydroxyvitamin D(3);-hemisuccinate-OVA was obtained in the same way. Ultraviolet, SDS-PAGE and MALDI-TOF were used to identify 25-hydroxyvitamin D(3);-hemisuccinate-BSA. RESULTS: BALB/c mice were immunized with 25-hydroxyvitamin D(3);-hemisuccinate-BSA to generate the polyclonal antibody of the 25-hydroxyvitamin D(3); worth high titer and the immunogen, 25-hydroxyvitamin D(3);-hemisuccinate-BSA, was successfully prepared with coupling ratio (12±0.16):1(N=3) coupling. CONCLUSION: The high titer and good sensitivity of anti-25-hydroxyvitamin D(3); antibody are produced in sera immunized BALB/c mice, which made it possible to develop a clinical diagnostics for illness.