Ultrasensitive Electrochemiluminescence Biosensor to Detect Ampicillin Resistance Gene (ARGAMP) Based on a Novel Near-Infrared Ruthenium Carbene Complex/TPrA/PEI Ternary ECL System.

The establishment of rapid target identification and analysis methods for antibiotic resistance genes (ARGs) is urgently needed. In this study, we unprecedently designed a target-catalyzed hairpin assembly (CHA) electrochemiluminescent (ECL) biosensor for the ultrasensitive detection of ampicillin resistance genes (ARGAMP) based on a novel, efficient near-infrared ruthenium carbene complex/TPrA/PEI ternary ECL system with low oxidation potential. The ternary NIR-ECL system illustrated in this work displayed double ECL intensity in comparison with their corresponding traditional binary ECL system. The as-prepared ECL biosensor illustrated in this work demonstrates highly selective and sensitive determination of ARGAMP from 1 fM to 1 nM and a low detection limit of 0.23 fM. Importantly, it also exhibits good accuracy and stabilities to identify ARGAMP in plasmid and bacterial genome DNA, which demonstrates its excellent reliability and great potential in detecting ARGAMP in real environmental samples.

Bis-tridentate Iridium(III) Complex with the N-Heterocyclic Carbene Ligand as a Novel Efficient Electrochemiluminescence Emitter for the Sandwich Immunoassay of the HHV-6A Virus.

Human herpesvirus type 6A (HHV-6A) can cause a series of immune and neurological diseases, and the establishment of a sensitive biosensor for the rapid detection of HHV-6A is of great significance for public health and safety. Herein, a bis-tridentate iridium complex (BisLT-Ir-NHC) comprising the N-heterocyclic carbene (NHC) ligand as a novel kind of efficient ECL luminophore has been unprecedently reported. Based on its excellent ECL properties, a new sensitive ECL-based sandwich immunosensor to detect the HHV-6A virus was successfully constructed by encapsulating BisLT-Ir-NHC into silica nanoparticles and embellishing ECL sensing interface with MXene@Au-CS. Notably, the immunosensor illustrated in this work not only had a wide linear range of 102 to 107 cps/µL but also showed outstanding recoveries (98.33-105.11%) in real human serum with an RSD of 0.85-3.56%. Undoubtedly, these results demonstrated the significant potential of the bis-tridentate iridium(III) complex containing an NHC ligand in developing ECL-based sensitive analytical methods for virus detection and exploring novel kinds of efficient iridium-based ECL luminophores in the future.

Generation of SARS-CoV-2 spike receptor binding domain mutants and functional screening for immune evaders using a novel lentivirus-based system.

The emergence of rapid and continuous mutations of severe acute respiratory syndrome 2 (SARS-CoV-2) spike glycoprotein that increased with the Omicron variant points out the necessity to anticipate such mutations for conceiving specific and adaptable therapies to avoid another pandemic. The crucial target for the antibody treatment and vaccine design is the receptor binding domain (RBD) of the SARS-CoV-2 spike. It is also the site where the virus has shown its high ability to mutate and consequently escape immune response. We developed a robust and simple method for generating a large number of functional SARS-CoV-2 spike RBD mutants by error-prone PCR and a novel nonreplicative lentivirus-based system. We prepared anti-RBD wild type (WT) polyclonal antibodies and used them to screen and select for mutant libraries that escape inhibition of virion entry into recipient cells expressing human angiotensin-converting enzyme 2 and transmembrane serine protease 2. We isolated, cloned, and sequenced six mutants totally bearing nine mutation sites. Eight mutations were found in successive WT variants, including Omicron and other recombinants, whereas one is novel. These results, together with the detailed functional analyses of two mutants provided the proof of concept for our approach.

Novel Electrochemiluminescent Biosensor to Ultrasensitively Detect U94 Gene in Human Herpesvirus 6 Using Metal-Organic Framework-Based Nanoemitters Comprising Iridium(III) Complexes via One-Pot Coordination Reaction Strategy.

The detection of the U94 gene in human herpesvirus 6 is crucial for early diagnosis of HHV-6 infections, which could induce acute febrile illness in infants. In this work, the first ultrasensitive electrochemiluminescence (ECL) biosensor for detecting U94 gene in Human Herpesvirus 6 was successfully designed by utilizing efficient novel metal-organic framework (MOF)-based ECL nanoemitters comprising iridium(III) complexes (Ir-ZIF-8-NH2) synthesized via one-pot coordination reaction strategy as an ECL indicator and a target-catalyzed hairpin assembly (CHA) signal amplification strategy. The as-prepared ECL indicator Ir-ZIF-8-NH2 exhibited an approximately 2.7-fold ECL intensity compared with its small molecular analogue of emissive iridium(III) complex named IrppymIM formed by in situ coordination reaction between iridium(III) solvent complex and imidazole ligands. In addition, a target-catalyzed hairpin assembly (CHA) strategy was employed to further improve the sensitivity of the proposed ECL biosensor, which demonstrated a wide linear range from 1 fM to 1 µM and the limit of detection as low as 0.113 fM (S/N = 3). Significantly, this biosensor was successfully applied to detect U94 gene in plasmids and real virus samples. The recoveries were in the range of 97.0-109.0% for plasmids and 95.7-107.5% for real virus samples with a relative standard deviation (RSD) of 1.87-2.53%. These satisfactory experimental results from the proposed ECL biosensor in this work would inevitably promote the development of new time/cost-effective and sensitive methods to detect HHV-6 with a major global health threat and substantial burden on healthcare in the future.

Human Herpesvirus 6A U4 Inhibits Proteasomal Degradation of the Amyloid Precursor Protein.

Human herpesvirus 6 (HHV-6) belongs to the betaherpesvirus subfamily and is divided into two distinct species, HHV-6A and HHV-6B. HHV-6 can infect nerve cells and is associated with a variety of nervous system diseases. Recently, the association of HHV-6A infection with Alzheimer's disease (AD) has been suggested. The main pathological phenomena of AD are the accumulation of ß-amyloid (Aß), neurofibrillary tangles, and neuroinflammation; however, the specific molecular mechanism of pathogenesis of AD is not completely clear. In this study, we focused on the effect of HHV-6A U4 gene function on Aß expression. Coexpression of HHV-6A U4 with amyloid precursor protein (APP) resulted in inhibition of ubiquitin-mediated proteasomal degradation of APP. Consequently, accumulation of ß-amyloid peptide (Aß), insoluble neurofibrillary tangles, and loss of neural cells may occur. Immunoprecipitation coupled with mass spectrometry (IP-MS) showed that HHV-6A U4 protein interacts with E3 ubiquitin ligase composed of DDB1 and cullin 4B, which is also responsible for APP degradation. We hypothesize that HHV-6A U4 protein competes with APP for binding to E3 ubiquitin ligase, resulting in the inhibition of APP ubiquitin modification and clearance. Finally, this leads to an increase in APP expression and Aß deposition, which are the hallmarks of AD. These findings provide novel evidence for the etiological hypothesis of AD, which can contribute to the further analysis of the role of HHV-6A in AD. IMPORTANCE The association of HHV-6A infection with Alzheimer's disease has attracted increasing attention, although its role and molecular mechanism remain to be established. Our results here indicate that HHV-6A U4 inhibits amyloid precursor protein (APP) degradation. U4 protein interacts with CRLs (cullin-RING E3 ubiquitin-protein ligases), which is also responsible for APP degradation. We propose a model in which U4 competitively binds to CRLs with APP, resulting in APP accumulation and Aß generation. Our findings provide new insights into the etiological hypothesis of HHV-6A in AD that can help further analyses.

Human herpesvirus 6A promotes glycolysis in infected T cells by activation of mTOR signaling.

Human herpesvirus 6 (HHV-6) is an important immunosuppressive and immunomodulatory virus worldwide. However, whether and how HHV-6 infection influences the metabolic machinery of the host cell to provide the energy and biosynthetic resources for virus propagation remains unknown. In this study, we identified that HHV-6A infection promotes glucose metabolism in infected T cells, resulting in elevated glycolytic activity with an increase of glucose uptake, glucose consumption and lactate secretion. Furthermore, we explored the mechanisms involved in HHV-6A-mediated glycolytic activation in the infected T cells. We found increased expressions of the key glucose transporters and glycolytic enzymes in HHV-6A-infected T cells. In addition, HHV-6A infection dramatically activated AKT-mTORC1 signaling in the infected T cells and pharmacological inhibition of mTORC1 blocked HHV-6A-mediated glycolytic activation. We also found that direct inhibition of glycolysis by 2-Deoxy-D-glucose (2-DG) or inhibition of mTORC1 activity in HHV-6A-infected T cells effectively reduced HHV-6 DNA replication, protein synthesis and virion production. These results not only reveal the mechanism of how HHV-6 infection affects host cell metabolism, but also suggest that targeting the metabolic pathway could be a new avenue for HHV-6 therapy.

gp96 Is Critical for both Human Herpesvirus 6A (HHV-6A) and HHV-6B Infections.

Human herpesviruses 6A and 6B (HHV-6A and HHV-6B, respectively) are two virus species in the betaherpesvirus subfamily that exhibit T cell tropism. CD46 and CD134 are the cellular receptors for HHV-6A and HHV-6B, respectively. Interestingly, the efficiency of HHV-6A/6B entry is different among different types of target cells despite similar receptor expression levels on these cells. Here, we found that the cellular factor gp96 (also known as glucose-regulated protein 94 [GRP94]) is expressed on the cell surface and interacts with viral glycoprotein Q1 (gQ1) during virus entry. gp96 cell surface expression levels are associated with the efficiency of HHV-6A and HHV-6B entry into target cells. Both loss-of-function and gain-of-function experiments indicated that gp96 plays an important role in HHV-6 infection. Our findings provide new insight into the HHV-6 entry process and might suggest novel therapeutic targets for HHV-6 infection.IMPORTANCE Although new clinical importance has been revealed for human herpesviruses 6A (HHV-6A) and 6B, much is still unknown about the life cycles of these viruses in target cells. We identified a novel cellular factor, gp96, that is critical for both HHV-6A and -6B entry into host cells. As gp96 can function as an adjuvant in vaccine development for both infectious agents and cancers, it can be a potential therapeutic target for infection by these two viruses.

Disrupted architecture and fast evolution of the mitochondrial genome of Argeia pugettensis (Isopoda): implications for speciation and fitness.

BACKGROUND: Argeia pugettensis is an isopod species that parasitizes other crustaceans. Its huge native geographic range spans the Pacific from China to California, but molecular data are available only for a handful of specimens from North-American populations. We sequenced and characterised the complete mitogenome of a specimen collected in the Yellow Sea. RESULTS: It exhibited a barcode (cox1) similarity level of only 87-89% with North-American populations, which is unusually low for conspecifics. Its mitogenome is among the largest in isopods (≈16.5 Kbp), mostly due to a large duplicated palindromic genomic segment (2 Kbp) comprising three genes. However, it lost a segment comprising three genes, nad4L-trnP-nad6, and many genes exhibited highly divergent sequences in comparison to isopod orthologues, including numerous mutations, deletions and insertions. Phylogenetic and selection analyses corroborated that this is one of the handful of most rapidly evolving available isopod mitogenomes, and that it evolves under highly relaxed selection constraints (as opposed to positive selection). However, its nuclear 18S gene is highly conserved, which suggests that rapid evolution is limited to its mitochondrial genome. The cox1 sequence analysis indicates that elevated mitogenomic evolutionary rates are not shared by North-American conspecifics, which suggests a breakdown of cox1 barcoding in this species. CONCLUSIONS: A highly architecturally disrupted mitogenome and decoupling of mitochondrial and nuclear rates would normally be expected to have strong negative impacts on the fitness of the organism, so the existence of this lineage is a puzzling evolutionary question. Additional studies are needed to assess the phylogenetic breadth of this disrupted mitochondrial architecture and its impact on fitness.

CD137 agonist induces gastric cancer cell apoptosis by enhancing the functions of CD8+ T cells via NF-κB signaling.

BACKGROUND: CD137 is a target for tumor immunotherapy. However, the role of CD137 in gastric cancer (GC), especially in inducing GC cell apoptosis, has not been studied. METHODS: Foxp3+ and CD8+ T cells in GCs were investigated using immunohistochemistry (IHC). CD137 expression in GCs was detected using flow cytometry, IHC and immunofluorescence (IF). Peripheral blood mononuclear cells (PBMCs) and CD8+ T cells isolated from peripheral blood were stimulated with a CD137 agonist in vitro. CD8+ T cell proliferation and p65 expression was examined using flow cytometry. P65 nuclear translocation was analyzed using IF. IL-10, TGF-ß, IFN-γ, perforin and granzyme B were detected using real-time quantitative PCR (real-time PCR). PBMCs and primary GC cells were cocultured and stimulated with a CD137 agonist in vitro. Apoptosis of primary GC cells was detected using flow cytometry. RESULTS: Our data demonstrated that GC tumors showed characteristics of an immunosuppressive microenvironment. CD137 was predominantly expressed in CD8+ T cells in GCs and had a positive correlation with tumor cell differentiation. The CD137 agonist promoted CD8+ T cell proliferation and increased the secretion of IFN-γ, perforin and granzyme B, which induced primary GC cell apoptosis. Mechanistically, this study found that the CD137 agonist induced NF-κB nuclear translocation in CD8+ T cells. CONCLUSION: Our results demonstrated that a CD137 agonist induced primary GC cell apoptosis by enhancing CD8+ T cells via activation of NF-κB signaling.

Protein Staining Agents from Cationic and Neutral Luminescent Iridium(III) Complexes.

Seven luminescent iridium(III) complexes were prepared to investigate the relationships between chemical structures and properties of protein staining. For the first time, the effect of the main ligand, the π conjugation effect of the ancillary ligand, and the charge effect of organometallic complexes on protein staining has been revealed. Most importantly, this study gives the first experimental evidence of the potential applications of charge-neutral organometallic complexes in protein staining, which could open an avenue of exploiting novel protein staining agents in the future.

Polyethylene glycol and divalent salt-induced DNA reentrant condensation revealed by single molecule measurements.

In this study, we investigated the DNA condensation induced by polyethylene glycol (PEG) with different molecular weights (PEG 600 and PEG 6000) in the presence of NaCl or MgCl2 by using magnetic tweezers (MT) and atomic force microscopy (AFM). The MT measurements show that with increasing NaCl concentration, the critical condensation force in the PEG 600-DNA or PEG 6000-DNA system increased approximately linearly. PEG 6000 solution has a larger critical force than PEG 600 solution at a given NaCl concentration. In comparison, a parabolic trend of the critical condensation force was observed with increasing MgCl2 concentration, indicating that DNA undergoes a reentrant condensation. The AFM results show that the morphologies of the compacted DNA-PEG complexes depended on the salt concentration and were consistent with the MT results.

Histidine-iridium(III) coordination-based peptide luminogenic cyclization and cyclo-RGD peptides for cancer-cell targeting.

In the field of peptide drug discovery, structural constraining and fluorescent labeling are two sought-after techniques important for both basic research and pharmaceutical development. In this work, we describe an easy-to-use approach for simultaneous peptide cyclization and luminescent labeling based on iridium(III)-histidine coordination (Ir-HH cyclization). Using a series of model peptides with histidine flanking each terminus, the binding activity and reaction kinetics of Ir-HH cyclization of different ring sizes were characterized. In the series, Ir-HAnH (n = 2, 3) with moderate ring sizes provides appropriate flexibility and proper distance between histidines for cyclic formation, which leads to the best binding affinity and structural stability in physiological conditions, as compared to other Ir-HH-cyclized peptides with smaller (n = 0, 1) or larger (n = 4, 5) ring sizes. Ir-HRGDH, an Ir-HH-cyclized peptide containing integrin targeting motif Arg-Gly-Asp (RGD), showed better targeting affinity than its linear form and enhanced membrane permeability in comparison with fluorescein-labeled cyclic RGDyK peptide. Cell death inducing peptide KLA-linked Ir-HRGDH (Ir-HRGDH-KLA) showed dramatically enhanced cytotoxicity and high selectivity for cancer cells versus noncancer cells. These data demonstrate that the method conveniently combines structural constraining of peptides with luminescent imaging capabilities, which facilitates functional and intracellular characterization of potential peptide-based drug leads, thus introducing a new tool to meet emerging needs in medicinal research.

AMPK restricts HHV-6A replication by inhibiting glycolysis and mTOR signaling.

AMP-activated protein kinase (AMPK) is a cellular energy sensor regulating metabolic homeostasis. In this study, we investigated the role of AMPK in response to human herpesvirus 6A (HHV-6A) infection. We show that HHV-6A infection significantly downregulates the active phosphorylated state of AMPK in infected T cells. Pharmacological activation of AMPK highly attenuated HHV-6A propagation. Mechanistically, we found that the activation of AMPK by AICAR blocked HHV-6-induced glycolysis by inhibiting glucose metabolism and lactate secretion, as well as decreasing expressions of key glucose transporters and glycolytic enzymes. In addition, mTOR signaling has been inactivated in HHV-6A infected T cells by AICAR treatment. We also showed that HHV-6A infection of human umbilical cord blood mononuclear cells (CBMCs) reduced AMPK activity whereas the activation of AMPK by metformin drastically reduced HHV-6A DNA replication and virions production. Taken together, this study demonstrates that AMPK is a promising antiviral therapeutic target against HHV-6A infection.

Genomic characterization of two carbapenem-resistant Serratia marcescens isolates causing bacteremia: Emergence of KPC-2-encoding IncR plasmids.

The occurrence and transmission of carbapenemase-producing-Enterobacterales (CPE) on a global scale has become a major issue. Clinical reports are rarely providing information on the genomic and plasmid features of carbapenem-resistant Serratia marcescens. Our objective was to investigate the resistance and transmission dynamics of two carbapenem-resistant S. marcescens that are resistant to carbapenem and have caused bacteremia in China. Blood specimens were taken from two individuals with bacteremia. Multiplex PCR was employed to identify genes that code for carbapenemase. Antimicrobial susceptibility tests and plasmid analysis were conducted on S. marcescens isolates SM768 and SM4145. The genome of SM768 and SM4145 were completely sequenced using NovaSeq 6000-PE150 and PacBio RS II platforms. Antimicrobial resistance genes (ARGs) were predicted using the ResFinder tool. S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and southern blotting were employed to analyze plasmids. Two S. marcescens that produced KPC-2 were identified from bloodstream infections. The antimicrobial susceptibility testing demonstrated that both of the isolates had a resistance to various antibiotics. The whole-genome sequence (WGS) and plasmid analysis revealed the presence of bla KPC-2-bearing IncR plasmids and multiple plasmid-borne antimicrobial resistance genes in the isolates. Our comparative plasmid analysis suggested that the two IncR plasmids identified in this study could be derived from a common ancestor. Our findings revealed the emergence of bla KPC-2-bearing IncR plasmid in China, which could be a hindrance to the transmission of KPC-2-producing S. marcescens in clinical settings.

New species of the genus Pseudocuneopsis Huang, Dai, Chen & Wu, 2022 (Bivalvia, Unionidae) from Guangxi Province, China.

A new species of freshwater mussel belonging to the genus Pseudocuneopsis, namely Pseudocuneopsisyangshuoensissp. nov., is diagnosed and described from Guangxi Province, China. This paper provides a detailed morphological description, photograph of the type specimen, and anatomical characteristics along with partial sequences of mitochondrial COI as DNA barcode data for this novel species. The new species can be distinguished from its congeners (Pseudocuneopsissichuanensis and Pseudocuneopsiscapitata) by shell shape, beak position and surface sculpture. The interspecies genetic distance based on the COI barcode between P.yangshuoensissp. nov. and P.sichuanensis is 8%, while it reaches 9% with P.capitata. Therefore, we provide robust morphological and molecular evidence to support the validity of this new species.

Luminescent iridium(III) complexes as novel protein staining agents.

This article reports a new class of luminescent metal complexes, biscyclometalated iridium(III) complexes with an ancillary bathophenanthroline disulfonate ligand, for staining protein bands that are separated by electrophoresis. The performances of these novel staining agents have been studied in comparison with tris(bathophenanthroline disulfonate) ruthenium(II) tetrasodium salt (i.e. RuBPS) using a commercially available imaging system. The staining agents showed different limits of detection, linear dynamic ranges, and protein-to-protein variations. The overall performances of all three stains were found to be better than or equivalent to RuBPS under the experimental conditions.

Iridium tetrazolato complexes as efficient protein staining agents.

In this work, three iridium(III) tetrazolato complexes have been designed and successfully synthesized. Beside photophysical properties, their performances in protein staining have been comprehensively investigated in this work for the first time. Notably, these iridium(III) tetrazolato complexes with high quantum efficiency exhibited much better protein staining properties than the commercial agent Coomassie Brilliant Blue (CBB) under the same experimental conditions, which may pave the way to explore new efficient iridium-based protein staining agents both for commercial markets and academic research in the future.

LINC00467 facilitates the proliferation, migration and invasion of glioma via promoting the expression of inositol hexakisphosphate kinase 2 by binding to miR-339-3p.

Our previous studies indicate that long noncoding RNA (lncRNA) LINC00467 can act as an oncogene to participate in the malignant progression of glioma, but the underlying molecular mechanism remains to be studied further. This study aimed to explore the biological role of the LINC00467/miR-339-3p/ inositol hexakisphosphate kinase 2 (IP6K2) regulatory axis in glioma. The Cancer Genome Atlas (TCGA), Oncomine databases and reverse transcription­quantitative PCR (RT­qPCR) were used to analyze IP6K2 expression in glioma. RT-PCR, EdU and transwell assays were conducted to observe the effect of IP6K2 on glioma cell proliferation, migration and invasion. Using bioinformatics analysis, RT-PCR, and dual luciferase reporter gene assay, the potential role of the LINC00467/miR-339-3p/IP6K2 regulatory axis in glioma was verified. The results showed that IP6K2 was up-regulated in glioma tissues and cell lines. Moreover, the expression level of IP6K2 was correlated with the clinical features of glioma patients. In vitro and in vivo experiments indicated that IP6K2 overexpression could promote the proliferation, migration, and invasion of glioma cells. Further bioinformatics analysis and in vitro assays revealed that LINC00467 could promote IP6K2 expression by binding to miR-339-3p and promote the malignant progression of glioma. Overall, LINC00467 could upregulate IP6K2 by binding to miR-339-3p and promote the proliferation, migration, and invasion of glioma cells. The LINC00467/miR-339-3p/IP6K2 regulatory axis might be a potential therapeutic target for glioma.

Activation and Evasion of RLR Signaling by DNA Virus Infection.

Antiviral innate immune response triggered by nucleic acid recognition plays an extremely important role in controlling viral infections. The initiation of antiviral immune response against RNA viruses through ligand recognition of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) was extensively studied. RLR's role in DNA virus infection, which is less known, is increasing attention. Here, we review the research progress of the ligand recognition of RLRs during the DNA virus infection process and the viral evasion mechanism from host immune responses.

Evaluation of the curative effects of Bailing capsules for treating chronic obstructive pulmonary disease: A protocol for systematic review and meta-analysis.

BACKGROUND: The goal of the present study is to evaluate the efficacy and safety of Bailing capsules, which is a traditional Chinese drug that can improve lung functionality when used to treat chronic obstructive pulmonary disease (COPD) patients. METHODS: A comprehensive search will be performed on the following primary electronic databases: PubMed, EMBASE, Cochrane Library, Chinese National Knowledge Infrastructure, and WanFang database. A search of secondary sources includes reference lists of included studies. Two pairs of review authors will screen and scrutinize selected articles. This study will analyze continuous data as mean differences and dichotomous data as odds ratios, both with 95% confidence intervals. A sensitivity analysis will also be conducted to evaluate the stableness of the outcomes. RevMan 5.3 software was adopted to accomplish all the statistical analysis. RESULTS: The results obtained in this research shall be published in a peer-reviewed journal. CONCLUSION: Based on the interpretations of the results, useful conclusions will be presented. These conclusions will offer additional insights with useful evidence to assess whether it is viable to use Bailing capsules as an effective and safety treatment option for COPD. ETHICS AND DISSEMINATION: The present work does not involve any humans or animals; therefore, ethical approval is not needed. SYSTEMATIC REVIEW REGISTRATION: March 26, 2021.osf.io/kvgbu. (https://osf.io/kvgbu/).