Analysis of binding and authentic virus-neutralizing activities of immune sera induced by various monkeypox virus antigens.

Monkeypox cases continue to increase globally, and there is an urgent need to develop a highly effective vaccine against monkeypox. This study investigated the binding and authentic-virus neutralizing activities of sera from mice immunized with EEV (extracellularly enveloped viruses) antigens B6R and A35R, and IMV (intrinsic material viruses) antigens M1R, A29L, E8L, and H3L against monkeypox virus. The results showed that immunizations of A35R and E8L could only induce lower titers of binding antibodies, in contrast, immunization of M1R induced the highest titers of binding antibodies, while immunization of B6R, H3L, and A29L induced moderate titers of binding antibodies. For the live monkeypox virus neutralization assay, the results showed that immunization with two doses of EEV antigen B6R did not effectively induce humoral immune responses to neutralize monkeypox live virus, immunization with EEV-A35R only induced weak monkeypox-neutralizing antibodies. In contrast, the immunization of the four types of monkeypox virus IMV antigens can all induce neutralizing antibodies against authentic monkeypox virus, among them, A29L and H3L induced the highest neutralizing antibody titers. The results of this study provide important references for the selection of antigens in the development of the next generation of monkeypox vaccines.

Thrombectomy for delayed thromboembolism in a recurrent cerebral aneurysm previously treated with coiling: A case report.

RATIONALE: Giant intracranial aneurysms pose a significant threat due to high mortality rates upon rupture, prompting interventions such as neurosurgical clipping or endovascular coiling. PATIENT CONCERNS: We present a rare case involving a 47-year-old female with a history of successfully treated ruptured giant intracranial aneurysms. Six months post-surgical clipping, she developed symptoms of acute ischemic stroke, prompting the decision for neurosurgical coiling and stent-assisted aneurysm coil embolization due to recurrent intracranial aneurysms. DIAGNOSES: Subsequently, occlusion occurred at the previously implanted stent site during embolization, necessitating exploration of alternative therapeutic options. Digital subtraction angiography confirmed stent occlusion in the right middle cerebral artery. INTERVENTIONS: Despite an initial unsuccessful attempt using a direct aspiration first-pass technique, the patient underwent successful mechanical thrombectomy with a retrievable stent, leading to successful reperfusion. This study aims to highlight the challenges and therapeutic strategies in managing delayed cerebral vascular occlusion following stent-assisted coil embolization, emphasizing the significance of exploring alternative interventions to enhance patient outcomes. OUTCOMES: The patient achieved successful reperfusion, and the study underscores the importance of recognizing and addressing delayed cerebral vascular occlusion after stent-assisted coil embolization for recurrent cerebral aneurysms. LESSONS: Our findings suggest that retrievable stent mechanical thrombectomy may serve as a viable therapeutic option in challenging scenarios, emphasizing the need for further exploration of alternative interventions to enhance patient care.

Development of monoclonal antibodies targeting the conserved fragment of hexon protein to detect different serotypes of human adenovirus.

Human adenovirus (HAdV) infects the respiratory system, thus posing a threat to health. However, immunodiagnostic reagents for human adenovirus are limited. This study aimed to develop efficient diagnostic reagents based on monoclonal antibodies for diagnosing various human adenovirus infections. Evolutionary and homology analyses of various human adenoviral antigen genes revealed highly conserved antigenic fragments. The prokaryotic expression system was applied to recombinant penton, hexon, and IVa2 conserved fragments of adenovirus, which were injected into BALB/c mice to prepare human adenovirus-specific monoclonal antibodies. Enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), and Western blotting were used to determine the immune specificity of the monoclonal antibodies. Indirect ELISA showed that monoclonal antibodies 1F10, 8D3, 4A1, and 9B2 were specifically bound to HAdV-3 and HAdV-55 and revealed high sensitivity and low detection limits for various human adenoviruses. Western blotting showed that 1F10 and 8D3 specifically recognized various human adenovirus types, including HAdV-1, HAdV-2, HAdV-3, HAdV-4, HAdV-5, HAdV-7, HAdV-21, and HAdV-55, and 4A1 specifically recognized HAdV-1, HAdV-2, HAdV-3, HAdV-5, HAdV-7, HAdV-21, and HAdV-55. IFAs showed that 1F10, 8D3, and 4A1 exhibited highly selective localization to A549 cells infected with HAdV-3 and HAdV-55. Finally, two antibody pairs that could detect hexon antigens HAdV-3 and HAdV-55 at low concentrations were developed. The monoclonal antibodies developed in this study show potential for detecting human adenoviruses. IMPORTANCE: In this study, we selected the three most conserved antigenic fragments of human adenovirus to prepare a murine monoclonal antibody for the first time, and human adenovirus antigenic fragments with heretofore unheard of degrees of conservatism were isolated. The three monoclonal antibodies with the ability to recognize human respiratory adenovirus over a broad spectrum were screened by hybridoma and monoclonal antibody preparation. Human adenovirus infections are serious; however, therapeutic drugs and diagnostic reagents are scarce. Thus, to reduce the serious consequences of human viral infections and adenovirus pneumonitis, early diagnosis of infection is required. The present study provides three monoclonal antibodies capable of recognizing a wide range of human adenoviruses, thereby offering guidance for subsequent research and development.

Development of a quadruple qRT-PCR assay for simultaneous identification of hypervirulent and carbapenem-resistant Klebsiella pneumoniae.

IMPORTANCE: Globally, the increasing number of hypervirulent Klebsiella pneumoniae (hvKp) and carbapenem-resistant Kp (CR-Kp) infections poses a huge public health challenge with high morbidity and mortality. Worrisomely, due to the mobility of elements carrying virulence and drug-resistance genes, the increasing prevalence of CR-hvKp has also been found with an overwhelming mortality rate in recent years. However, the current detection methods for hvKp and CR-Kp have many disadvantages, such as long turnaround time, complex operation, low sensitivity, and specificity. Herein, a more sensitive, rapid, single-reaction, and multiplex quantitative real-time PCR was developed and validated to differentiate the circulating lineages of Kp with excellent performance in sensitivity and specificity, providing a useful tool for the differential diagnosis and the surveillance of the circulating Kp.

Construction and application of the conditionally essential gene knockdown library in Klebsiella pneumoniae to screen potential antimicrobial targets and virulence genes via Mobile-CRISPRi-seq.

Klebsiella pneumoniae is a ubiquitous human pathogen, and its clinical treatment faces two major challenges: multidrug resistance and the pathogenesis of hypervirulent K. pneumoniae. The discovery and study of conditionally essential (CE) genes that can function as potential antimicrobial targets has always been a research concern due to their restriction in the development of novel antibiotics. However, the lack of essential functional genomic data has hampered the study of the mechanisms of essential genes related to antimicrobial susceptibility. In this study, we developed a pooled CE genes mobile clustered regularly interspaced short palindromic repeat (CRISPR) interference screening method (Mobile-CRISPRi-seq) for K. pneumoniae to identify genes that play critical roles in antimicrobial fitness in vitro and host immunity in vivo. Targeting 870 predicted CE genes in K. pneumoniae, Mobile-CRISPRi-seq uncovered the depletion of tetrahydrofolate synthesis pathway genes folB and folP under trimethoprim pressure. Our screening also identified genes waaE and fldA related to polymyxin and ß-lactam susceptibility by applying a screening strategy based on Mobile-CRISPRi-seq and comparative genomics. Furthermore, using a mouse infection model and Mobile-CRISPRi-seq, multiple virulence genes were identified, and among these genes, pal, yciS, and ribB were demonstrated to contribute to the pathogenesis of K. pneumoniae. This study provides a simple, rapid, and effective platform for screening potential antimicrobial targets and virulence genes in K. pneumoniae, and this broadly applicable system can be expanded for high-throughput functional gene study in multiple pathogenic bacteria, especially in gram-negative bacteria. IMPORTANCE The discovery and investigation of conditionally essential (CE) genes that can function as potential antimicrobial targets has always been a research concern because of the restriction of antimicrobial targets in the development of novel antibiotics. In this study, we developed a pooled CE gene-wide mobile clustered regularly interspaced short palindromic repeat (CRISPR) interference sequencing (Mobile-CRISPRi-seq) strategy in Klebsiella pneumoniae to identify genes that play critical roles in the fitness of antimicrobials in vitro and host immunity in vivo. The data suggest a robust tool to screen for loss-of-function phenotypes in a pooled gene knockdown library in K. pneumoniae, and Mobile-CRISPRi-seq may be expanded to multiple bacteria for screening and identification of genes with crucial roles in the fitness of antimicrobials and hosts.

Loss of PWT7, Located on a Supernumerary Chromosome, Is Associated with Parasitic Specialization of Pyricularia oryzae on Wheat.

Pyricularia oryzae, a blast fungus of gramineous plants, is composed of various host genus-specific pathotypes. The avirulence of an Avena isolate on wheat is conditioned by PWT3 and PWT4. We isolated the third avirulence gene from the Avena isolate and designated it as PWT7. PWT7 was effective as an avirulence gene only at the seedling stage or on leaves. PWT7 homologs were widely distributed in a subpopulation of the Eleusine pathotype and the Lolium pathotype but completely absent in the Triticum pathotype (the wheat blast fungus). The PWT7 homolog found in the Eleusine pathotype was one of the five genes involved in its avirulence on wheat. A comparative analysis of distribution of PWT7 and the other two genes previously identified in the Eleusine pathotype suggested that, in the course of parasitic specialization toward the wheat blast fungus, a common ancestor of the Eleusine, Lolium, Avena, and Triticum pathotypes first lost PWT6, secondly PWT7, and, finally, the function of PWT3. PWT7 or its homologs were located on core chromosomes in Setaria and Eleusine isolates but on supernumerary chromosomes in Lolium and Avena isolates. This is an example of interchromosomal translocations of effector genes between core and supernumerary chromosomes. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Three neutralizing mAbs induced by MPXV A29L protein recognizing different epitopes act synergistically against orthopoxvirus.

The worldwide outbreak of the monkeypox virus (MPXV) has become a "Public Health Emergency of International Concern" (PHEIC). Severe monkeypox virus infection can be fatal, however, effective therapeutic methods are yet to be developed. Mice were immunized with A35R protein and A29L protein of MPXV, and the binding and neutralizing activities of the immune sera against poxvirus-associated antigens and viruses were identified. A29L protein and A35R protein-specific monoclonal antibodies (mAbs) were generated and their antiviral activities of these mAbs were characterized in vitro and in vivo. Immunization with the MPXV A29L protein and A35R protein induced neutralizing antibodies against the orthopoxvirus in mice. None of the mAbs screened in this study against A35R could effectively neutralize the vaccinia virus (VACV), while three mAbs against A29L protein, 9F8, 3A1 and 2D1 were confirmed to have strong broad binding and neutralizing activities against orthopoxvirus, among which 9F8 showed the best neutralizing activity. 9F8, 3A1, and 2D1 recognized different epitopes on MPXV A29L protein, showing synergistic antiviral activity in vitro against the VACV Tian Tan and WR strains; the best activity was observed when the three antibodies were combined. In the vivo antiviral prophylactic and therapeutic experiments, 9F8 showed complete protective activity, whereas 3A1 and 2D1 showed partial protective activity. Similarly, the three antibodies showed synergistic antiviral protective activity against the two VACVs. In conclusion, three mAbs recognized different epitopes on MPXV A29L protein were developed and showed synergistic effects against orthopoxvirus.

Broadly neutralizing antibodies recognizing different antigenic epitopes act synergistically against the influenza B virus.

The discovery of broadly neutralizing monoclonal antibodies against influenza viruses has raised hope for the successful development of new antiviral drugs. However, due to the speed and variety of mutations in influenza viruses, single-component antibodies that recognize specific epitopes are susceptible to viral escape and have limited efficacy when administration is delayed. Hence, it is necessary to develop alternative strategies with better antiviral activity. Influenza B virus infection can cause severe illness in children and the elderly. Commonly used anti-influenza drugs have low clinical efficacy against influenza B virus. In this study, we investigated the antiviral efficacy of combinations of representative monoclonal antibodies targeting different antigenic epitopes against the influenza B virus. We found that combinations of antibodies recognizing the hemagglutinin (HA) head and stem regions showed a stronger neutralizing activity than single antibodies and other antibody combinations in vitro. In addition, we found that pair-wise combinations of antibodies recognizing the HA head region, HA stem region, and neuraminidase enzyme-activated region showed superior antiviral activity than single antibodies in both mouse and ferret in vivo protection assays. Notably, these antibody combinations still displayed good antiviral efficacy when treatment was delayed. Mechanistic studies further revealed that combining antibodies recognizing different epitope regions resulted in extremely strong antibody-dependent cell-mediated cytotoxicity, which may partly explain their superior antiviral effects. Together, the findings of this study provide new avenues for the development of better antiviral drugs and vaccines against influenza viruses.

Rational Design and Characterization of Trispecific Antibodies Targeting the HIV-1 Receptor and Envelope Glycoprotein.

Multitudinous broadly neutralizing antibodies (bNAbs) against HIV-1 have been developed as novel antiviral prophylactic and therapeutic agents. Combinations of bNAbs are generally even more effective than when they are applied individually, showing excellent neutralization coverage and limiting the emergence of escape mutants. In this study, we investigated the design and characterization of three trispecific antibodies that allow a single molecule to interact with independent HIV-1 envelope determinants-(1) the host receptor CD4, (2) the host co-receptor CCR5 and (3) distinct domains in the envelope glycoprotein of HIV-1-using an ELISA, an HIV-1 pseudovirus neutralization assay and in vivo antiviral experiments in humanized mice. We found that trispecific bNAbs and monovalent ones all had satisfactory binding activities against the corresponding antigens in the ELISA, exhibited higher potency and breadth than any previously described single bnAb in the HIV-1 pseudovirus neutralization assay and showed an excellent antiviral effect in vivo. The trispecific antibodies simultaneously recognize the host receptor CD4, host co-receptor CCR5 and HIV-1 envelope glycoprotein, which could mean they have promise as prophylactic and therapeutic agents against HIV-1.

Assessment of antibody responses against SARS-CoV-2 in unvaccinated individuals and vaccinees from Omicron-BA.2 infection in Zhaoqing, Guangdong Province, China.

Currently, the majority of the global population has been vaccinated with the COVID-19 vaccine, and characterization studies of antibodies in vivo from Omicron breakthrough infection and naive infection populations are urgently needed to provide pivotal clues about accurate diagnosis, treatment, and next-generation vaccine design against SARS-CoV-2 infection. We showed that after infection with Omicron-BA.2, the antibody levels of specific IgM against the Wuhan strain and specific IgG against Omicron were not significantly elevated within 27 days of onset. Interestingly, in this study, the levels of humoral immunity against Omicron-specific IgM were significantly increased after breakthrough infection, suggesting that the detection of Omicron-specific IgM antibodies can be used as a test criterion of Omicron breakthrough infection. In addition, we observed that serums from unvaccinated individuals and the majority of vaccinated infections possessed only low or no neutralizing activity against Omicron at the onset of Omicron breakthrough infections, and at the later stage of Omicron-BA.2 breakthrough infection, levels of neutralization antibody against the Wuhan and Omicron strains were elevated in infected individuals. The findings of this study provide important clues for the diagnosis of Omicron breakthrough infections, antibody characterization studies and vaccine design against COVID-19.

Serosurvey in SARS-CoV-2 inactivated vaccine-elicited neutralizing antibodies against authentic SARS-CoV-2 and its viral variants.

Various variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been emerging and circulating in different parts of the world. Millions of vaccine doses have been administered globally, which reduces the morbidity and mortality of coronavirus disease-2019 efficiently. Here, we assess the immune responses of individuals after two shots of BBIBP-CorV or CoronaVac inactivated vaccine. We measured neutralizing antibody responses after the second vaccination by using authentic SARS-CoV-2 and its viral variants. All the serum samples efficiently neutralized SARS-CoV-2 wild-type lineage, in contrast, a part of serum samples failed to neutralize Alpha, Beta, Gamma, Delta, or Eta lineages, and only several serum samples were able to neutralize Omicron lineage virus strains (BA.1 and BA.2) with low neutralization titer. As compared with the neutralization of SARS-CoV-2 wild-type lineage, the neutralization of all other SARS-CoV-2 variant lineages was significantly lower. Considering that all the SARS-CoV-2 mutation viruses challenged the antibody neutralization induced by BBIBP-CorV and CoronaVac, it is necessary to carry out a third booster vaccination to increase the humoral immune response against the SARS-CoV-2 mutation viruses.

Humoral immune response to authentic circulating severe acute respiratory syndrome coronavirus 2 variants elicited by booster vaccination with distinct receptor-binding domain subunits in mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants could induce immune escape by mutations of the spike protein which are threatening to weaken vaccine efficacy. A booster vaccination is expected to increase the humoral immune response against SARS-CoV-2 variants in the population. We showed that immunization with two doses of wild type receptor-binding domain (RBD) protein, and booster vaccination with wild type or variant RBD protein all significantly increased binding and neutralizing antibody titers against wild type SARS-CoV-2 and its variants in mice. Only the booster immunization by Omicron (BA.1)RBD induced a strong antibody titer against the omicron virus strain and comparable antibody titers against all the other virus strains. These findings might shed the light on coronavirus disease 2019 booster immunogens.

Physical Properties of Peanut and Soy Protein-Based Emulsion Gels Induced by Various Coagulants.

Emulsions of peanut and soy proteins, including their major components (arachin, conarachin, glycinin and ß-conglycinin), were prepared by ultrasonication (300 W, 20 min) at a constant protein concentration (4%, w/v) and oil fraction (30%, v/v). These emulsions were then induced by CaCl2, transglutaminase (TGase) and glucono-δ-lactone (GDL) to form emulsion gels. The optimum coagulant concentrations were obtained for peanut and soy protein-stabilized emulsion gels, such as CaCl2 (0.15 and 0.25 g/dL, respectively), TGase (25 U/mL) and GDL (0.3% and 0.5%, w/v, respectively). For the CaCl2-induced emulsion gels, the hardness of the ß-conglycinin gel was the highest, whereas that of the conarachin gel was the lowest. However, when TGase and GDL were used as coagulants, the strength of the conarachin emulsion gel was the best. For the GDL-induced emulsion gels, microstructural analysis indicated that the conarachin gel showed more homogeneous and compact structures. The gelation kinetics showed that the storage modulus (G') of all the GDL-induced emulsions increased sharply except for the arachin-stabilized emulsion. The interactive force nature varied between conarachin and arachin emulsion gels. This work reveals that peanut conarachin could be used as a good protein source to produce emulsion gels when suitable coagulants are selected.

Effect of nicotinamide riboside on lipid metabolism and gut microflora-bile acid axis in alcohol-exposed mice.

Alcoholic liver disease (ALD) is the most common complication of alcohol abuse, while we lack safe and effective treatment for ALD. This study aimed to explore the effects of nicotinamide riboside (NR) on lipid metabolism and gut microflora-bile acid axis in alcohol-exposed mice. NR significantly improved liver histopathological damage and abnormal liver function. NR as a provider of nicotinamide adenine dinucleotide (NAD+) increased the NAD+/NADH ratio. Meanwhile, NR inhibited the activation of the protein phosphatase 1 signaling pathway, decreased the liver triglyceride and total bile acid levels, and reduced lipid accumulation. According to the results of gut microflora species analysis, NR intervention changed the microbial community structure at the phylum, family and genus levels, and the species abundances returned to a level similar to these of the normal control group. Besides, the results of high-performance liquid chromatograph-mass spectrometry showed that NR intervention resulted in fecal bile acid levels tending to be normal with decreased chenodeoxycholic acid level and increased deoxycholic acid and hyocholic acid levels. Spearman's correlation analysis showed a correlation between gut microflora and bile acids. Therefore, NR supplementation has the potential to prevent ALD, and its mechanism may be related to regulating lipid metabolism disorders and the gut microflora-bile acid axis.

Pricing Constraint and the Complexity of IPO Timing in the Stock Market: A Dynamic Game Analysis.

The timing of an initial public offering (IPO) is a complex dynamic game in the stock market. Based on a dynamic game model with the real option, this paper investigates the relationship between pricing constraint and the complexity of IPO timing in the stock market, and further discusses its mechanism. The model shows that the IPO pricing constraint reduced the exercise value of the real option of IPO timing, thus restricting the enterprise's independent timing and promoting an earlier listing. The IPO price limit has a stronger effect on high-trait enterprises, such as technology enterprises. Lowering the upper limit of the pricing constraint increases the probability that enterprises are bound by this restriction during IPO. A high discount cost and stock-market volatility are also reasons for early listing. This paper suggests a theoretical explanation for the mechanism of the pricing constraint on IPO timing in the complex market environment, which is an extension of IPO timing theory, itself an interpretation of the IPO behavior of Chinese enterprises. These findings provide new insights in understanding the complexity of IPOs in relation to the Chinese stock market.

Aplysin Retards Pancreatic Necrosis and Inflammatory Responses in NOD Mice by Stabilizing Intestinal Barriers and Regulating Gut Microbial Composition.

Aplysin is a brominated sesquiterpene with an isoprene skeleton and has biological activities. The purpose of this study is to investigate the inhibitory effect of aplysin on spontaneous pancreatic necrosis in nonobese diabetic (NOD) mice and its potential mechanisms. Results showed that NOD mice at 12 weeks of age showed obvious spontaneous pancreatic necrosis, damaged tight junctions of intestinal epithelia, and widened gaps in tight and adherens junctions. Aplysin intervention was able to alleviate spontaneous pancreatic necrosis in NOD mice, accompanied with decreased serum endotoxin levels and downregulated expressions of Toll-like receptor 4 and its related molecules MyD88, TRAF-6, NF-κB p65, TRIF, TRAM, and IRF-3, as well as protein levels of interleukin-1ß and interferon-ß in pancreatic tissues. In addition, we observed obvious improvements of intestinal mucosal barrier function and changes of gut microbiota in the relative abundance at the phylum level and the genus level in aplysin-treated mice compared with control mice. Together, these data suggested that aplysin could retard spontaneous pancreatic necrosis and inflammatory responses in NOD mice through the stabilization of intestinal barriers and regulation of gut microbial composition.

An interlayer composed of a porous carbon sheet embedded with TiO2 nanoparticles for stable and high rate lithium-sulfur batteries.

The shuttling of lithium polysulfides (LiPSs) in lithium-sulfur (Li-S) batteries results in low sulfur utilization and fast capacity decay, hindering their practical applications. Constructing an interlayer is an efficient way to block the LiPS shuttling, but maintaining a low Li ion diffusion resistance with such an interlayer is hard to achieve. Herein, a thin porous carbon nanosheet embedded with TiO2 nanoparticles (denoted PCNS-TiO2) was used to fabricate an interlayer on the separator, which effectively solves the above problem. The PCNS-TiO2 was prepared by using the Ti3C2Tx MXene as the two-dimensional (2D) template directing the porous carbon sheet formation, and the Ti3C2Tx transformed into TiO2 nanoparticles embedded in the PCNS. The decomposition of the MXene eliminates the ion blocking effect by the 2D nanosheet structure. The thin and hierarchical porous structure allows fast Li ion diffusion across the interlayer, and at the same time, the porous structure and the strong adsorption ability of TiO2 effectively block the polysulfide diffusion. Thus, the Li-S battery with this interlayer shows good rate performance with a high capacity of 627 mA h g-1 at 2 C. Meanwhile, stable cycling performance is also achieved, showing a low capacity decay of 0.063% per cycle after 300 cycles at 0.5 C.