Peptide Stapling by Crosslinking Two Amines with α-Ketoaldehydes through Diverse Modified Glyoxal-Lysine Dimer Linkers.

α-Ketoaldehydes play versatile roles in the ubiquitous natural processes of protein glycation. However, leveraging the reactivity of α-ketoaldehydes for biomedical applications has been challenging. Previously, the reactivity of α-ketoaldehydes with guanidine has been harnessed to design probes for labeling Arg residues on proteins in an aqueous medium. Herein, a highly effective, broadly applicable, and operationally simple protocol for stapling native peptides by crosslinking two amino groups through diverse imidazolium linkers with various α-ketoaldehyde reagents is described. The use of hexafluoroisopropanol as a solvent facilitates rapid and clean reactions under mild conditions and enables unique selectivity for Lys over Arg. The naturally occurring GOLD/MOLD linkers have been expanded to encompass a wide range of modified glyoxal-lysine dimer (OLD) linkers. In a proof-of-concept trial, these modular stapling reactions enabled a convenient two-round strategy to streamline the structure-activity relationship (SAR) study of the wasp venom peptide anoplin, leading to enhanced biological activities.

2',3'-Cyclic GMP-AMP Dinucleotides for STING-Mediated Immune Modulation: Principles, Immunotherapeutic Potential, and Synthesis.

The cGAS-STING pathway discovered ten years ago is an important component of the innate immune system. Activation of cGAS-STING triggers downstream signalling, such as TBK1-IRF3, NF-κB and autophagy, which in turn leads to antipathogen responses, durable antitumour immunity or autoimmune diseases. 2',3'-Cyclic GMP-AMP dinucleotides (2',3'-cGAMP), the key second messengers produced by cGAS, play a pivotal role in cGAS-STING signalling by binding and activating STING. Thus, 2',3'-cGAMP has immunotherapeutic potential, which in turn has stimulated research on the design and synthesis of 2',3'-cGAMP analogues for clinical applications over the past ten years. This review presents the discovery, metabolism, and function of 2',3'-cGAMP in the cGAS-STING innate immune signalling axis. The enzymatic and chemical syntheses of 2',3'-cGAMP analogues as STING-targeting therapeutics are also summarized.

N-(2-Aminoethyl)-2-(hexylthio) Acetamide-Functionalized Pillar[5]arene for the Selective Detection of l-Trp through Guest-Adaptive Multisupramolecular Interactions.

Tryptophan (Trp) is very necessary for biosystems; therefore, high-efficient detection of Trp is an important subject. Hereof, based on our early research works on fluorescent sensors, we rationally designed and synthesized a fluorescent sensor (SNP5) based on N-(2-aminoethyl)-2-(hexylthio) acetamide-functionalized pillar[5]arene, which showed high selectivity and sensitive recognition for l-Trp (LOD = 2.19 × 10-8 M). Moreover, SNP5 exhibited aggregation-induced emission enhancement fluorescence. Within SNP5, the pillar[5]arene group could act as N-H···π- and C-H···π-interaction sites, as well as a H-bond-interaction site; meanwhile, the N-(2-aminoethyl)-2-(hexylthio) acetamide group also served as a multihydrogen-bonding site. As a result, SNP5 could selectively detect l-Trp through the synergy of the pillar[5]arene group and the N-(2-aminoethyl)-2-(hexylthio) acetamide group. Compared with previous work, the results of this work support the strategy that changing the functionalized group of the pillar[5]arene can adjust the selectivity of the pillar[5]arene-based sensor and achieve the detection of different amino acids. The detection mechanism was specifically researched through experiments and theoretical calculations including frontier orbitals, electrostatic potential, and the independent gradient model approach. Interestingly, these theoretical calculations not only supported the experimental results but also provided a visualized understanding of guest-adaptive multisupramolecular interactions between SNP5 and l-Trp.

A pillar[5]arene-based fluorescent sensor for sensitive detection of L-Met through a dual-site collaborative mechanism.

L-Methionine (L-Met) is one of the essential amino acids in human health, efficiently detect L-Met is a significant issue. Herein, a concept "dual-site collaborative recognition" had been successfully introduced into the design and achieved high selective and sensitive recognition of L-Met. In order to realize the "dual-site collaborative recognition", we rationally designed and synthesized an ester functionalized pillar[5]arene-based fluorescent sensor (SP5). And it shows blue Aggregation-induced emission (AIE) fluorescence. In the SP5, the pillar[5]arene group act as C-H···π interactions site, and ester group serve as multi hydrogen bonding acceptor. Interestingly, the SP5 exhibited high selectivity and sensitivity (2.84 × 10-8 M) towards L-Met based on the collaboration of electron-rich cavernous pillar[5]arene group and ester group through C-H···π and H-bond interactions, respectively. This "dual-site collaborative recognition" mechanism has been investigated by 1H NMR, ESI-MS and theoretical calculation including frontier orbital (HOMO and LUMO), electrostatic potential (ESP) and the noncovalent interaction (NCI). These theoretical calculations not only support the proposed host-guest recognition mechanism, but also provided visualized information on the "dual-site collaborative recognition" mode. Furthermore, the concept "dual-site collaborative recognition" is an effective strategy for easily detecting biological molecules.

Microarray analysis of preterm preeclampsia.

Premature preeclampsia is the second cause of maternal mortalities around the world. To investigate its potential driving mechanism(s), we constructed a multi-regulatory-mediated preeclampsia dysfunction module. Through combining differential expression analysis, co-expression analysis, and enrichment analysis, we obtained 23 sets of preeclampsia expression disorder modules in the disease, which involve the modular aggregations of 3016 genes. The modules were subjected to be analyzed for GO and KEGG paths for enrichment analysis. Based on these pivotal regulators, it is possible to manipulate the essential parts of the modular subnetwork and study their cooperative acts to mediate the driving mechanism of the preeclampsia. Simultaneously, they mainly cause the onset of the disease through the regulation of the apoptotic signaling pathway, down-regulation of an inflammatory response and retinol metabolism. This may present a potential driving mechanism for the disease. The predictor analysis of the regulators showed a series of non-coding RNAs that have potentially significant regulatory effects on the disease, including miR-182-5p, miR-200b-3p, miR-23a-3p, miR -429, miR-590-3p, and transcription factors. These pivotal regulators might mediate the potential driving processes. Based on a comprehensive multivariate analysis, we found a possible driving mechanism in which significant pivotal regulators were used as distinct functional segments in the preterm preeclampsia-driven process.

Hepatitis C Virus Genotype Analyses in Chronic Hepatitis C Patients and Individuals With Spontaneous Virus Clearance Using a Newly Developed Serotyping Assay.

BACKGROUND: We developed a novel HCV serotyping assay and detected the genotypes in chronic hepatitis C (CHC) patients and individuals with spontaneous viral clearance (SVC). METHODS: Nine hundred and ninety-seven patients were enrolled in a previous study; their samples were genotyped originally using the molecular assays. Among them, 190 patients achieved sustained virological response; the post-treatment samples were also serotyped. Moreover, 326 samples from follow-up cohorts were serotyped, among whom 66 were from SVC individuals, and 260 from CHC patients. RESULTS: Nine hundred and fifty-eight out of 997 samples were available for serotyping, among which 29 samples generated indeterminate serotyping results. The consistency between the genotyping and serotyping assays was 91.50% (850/929). The specificity and sensitivity were 98.45% and 88.77% for genotype 1, 96.42% and 93.97% for genotype 2, and 94.15% and 80.52% for non-genotype 1 or 2. However, only 41 of 60 genotype-6 samples were correctly serotyped. Little difference was found in the 190 paired serotyping results. No difference existed in the genotype distribution between the SVC and CHC groups (P = 0.08). CONCLUSIONS: The assay provides an accurate alternative for determining HCV genotypes, whereas it is not recommended for detecting genotype 6. Furthermore, it facilitates identifying the genotypes in SVC individuals. HCV genotype has little impact on SVC.

The Lumipulse G HBsAg-Quant assay for screening and quantification of the hepatitis B surface antigen.

Qualitative HBsAg assay is used to screen HBV infection for decades. The utility of quantitative assay is also rejuvenated recently. We aimed to evaluate and compare the performance of a novel ultra-sensitive and quantitative assay, the Lumipulse assay, with the Architect and Elecsys assays. As screening methods, specificity was compared using 2043 consecutive clinical routine samples. As quantitative assays, precision and accuracy were assessed. Sera from 112 treatment-naïve chronic hepatitis B patients, four patients undergoing antiviral therapy and one patient with acute infection were tested to compare the correlations. Samples with concurrent HBsAg/anti-HBs were also quantified. The Lumipulse assay precisely quantified ultra-low level of HBsAg (0.004 IU/mL). It identified additional 0.98% (20/2043) clinical samples with trance amount of HBsAg. Three assays displayed excellent linear correlations irrespective of genotypes and S-gene mutations (R(2)>0.95, P<0.0001), while minor quantitative biases existed. The Lumipulse assay did not yield higher HBsAg concentrations in samples with concomitant anti-HBs. Compared with other assays, the Lumipulse assay is sensitive and specific for detecting HBsAg. The interpretation of the extremely low-level results, however, is challenging. Quantitative HBsAg results by different assays are highly correlated, but they should be interpreted interchangeably only after conversion to eliminate the biases.

2',3'-cyclic nucleotide 3'-phosphodiesterases inhibit hepatitis B virus replication.

2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) is a member of the interferon-stimulated genes, which includes isoforms CNP1 and CNP2. CNP1 is locally expressed in the myelin sheath but CNP2 is additionally expressed at low levels outside the nervous system. CNPs regulate multiple cellular functions and suppress protein production by association with polyadenylation of mRNA. Polyadenylation of Hepatitis B virus (HBV) RNAs is crucial for HBV replication. Whether CNPs interact with polyadenylation signal of HBV RNAs and interfere HBV replication is unknown. In this study, we evaluated expressions of CNP isoforms in hepatoma cell lines and their effects on HBV replication. We found that CNP2 is moderately expressed and gently responded to interferon treatment in HepG2, but not in Huh7 cells. The CNP1 and CNP2 potently inhibited HBV production by blocking viral proteins synthesis and reducing viral RNAs, respectively. In chronic hepatitis B patients, CNP was expressed in most of HBV-infected hepatocytes of liver specimens. Knockdown of CNP expression moderately improved viral production in the HepG2.2.15 cells treated with IFN-α. In conclusion, CNP might be a mediator of interferon-induced response against HBV.

Performance evaluation and comparison of the newly developed Elecsys anti-HCV II assay with other widely used assays.

BACKGROUND: Anti-HCV assays remain the first choice for screening HCV infection in most clinical laboratories. The Elecsys anti-HCV II assay has been recently launched and we aimed to evaluate its performance compared with other widely used methods. METHODS: Four seroconversion panels, 861 consecutive sera under routine clinical conditions, 100 preselected sera with low positive anti-HCV results and 178 samples from patients infected with HIV were tested using Elecsys anti-HCV II, Architect anti-HCV and Vitros anti-HCV assays. Confirmatory testing was performed using RIBA and HCV RNA tests. Moreover, 203 samples with different HCV genotypes were assessed using Elecsys anti-HCV II. RESULTS: Elecsys anti-HCV II detected seroconversion 7-14 days earlier than the Architect and Vitros assays. Furthermore, it had 100% sensitivity and superior specificity in screening routine clinical samples, including those with low positive anti-HCV, and detected 97.6% (122/125) anti-HCV-positive samples from HIV-infected patients with HCV viremia. However, the anti-HCV levels in the genotype 3b samples were slightly underestimated. CONCLUSIONS: Elecsys anti-HCV II shortens the seroconversion window. It is suitable for screening HCV infection in clinical samples, including those from immunocompromised patients, due to the excellent sensitivity and specificity. Further investigation of the subtype inclusivity in a larger sample number might be warranted.

Evaluation of the performance of the EIAgen HCV test for detection of hepatitis C virus infection.

The EIAgen HCV test (Adaltis Inc., Montreal, Canada) is an enzyme immunoassay (EIA) for the detection of anti-hepatitis C virus (HCV) antibodies. This study compared the performance of this test side-by-side with the current Ortho HCV 3.0 Anti-HCV assay (Ortho-Clinical Diagnostics Inc., Johnson & Johnson Company, Raritan, NY, USA). Among 2559 specimens examined, 178 were true positives, 2376 were true negatives and 5 were indeterminate. The sensitivity of the EIAgen HCV test was 100%, versus 98.3% for the Ortho HCV test, while their respective specificities were 98.1% and 98.2%. The EIAgen HCV test gave a positive predictive value of 79.8% and a negative predictive value of 100%. Overall, the concordance of this test with the Ortho HCV test was 98.2%. Specimens from potentially interfering substances, such as sera from pregnant women, sera from patients with acute non-C hepatitis, autoimmune diseases, lipidemia, or from patients undergoing hemolysis, showed no interference with either EIA. An EIAgen HCV test signal-to-cut-off ratio of >5.9 would be highly predictive of a true-positive finding in these specimens. The EIAgen HCV test is well suited for screening blood and blood products in antibodies to HCV.