Search

Antibody therapeutics for the treatment of COVID-19 has been highly successful while faces a challenge of the recent emergence of the Omicron variant which escapes the majority of existing SARS-CoV-2 neutralizing antibodies (nAbs). Here, we successfully generated a panel of SARS-CoV-2/SARS-CoV cross-neutralizing antibodies by sequential immunization of the two pseudoviruses. Of which, nAbs X01, X10 and X17 showed broadly neutralizing breadths against most variants of concern (VOCs) and X17 was further identified as a Class 5 nAb with undiminished neutralization against the Omicron variant. Cryo-EM structures of three-antibody in complex with the spike proteins of prototyped SARS-CoV-2, Delta, Omicron and SARS-CoV defined three non-overlapping conserved epitopes on the receptor-binding domain (RBD). The triple antibody cocktail exhibited enhanced resistance to viral escape and effective protection against the infection of Beta variant in hamsters. Our finding will aid the development of both antibody therapeutics and broad vaccines against SARS-CoV-2 and emerging variants.

The classification networks have already existed for a long time and achieve great success. However, in biomedical image processing, classifying normal and abnormal ones only is not enough clinically, the desired output should include localization, i.e., where the lesion is. In this paper, we present a method for detecting protrusion lesion in digestive tract. We use a deep learning-based model to build a computer-aided diagnosis system to help doctors examine the intestinal diseases. Learn from existing detection method, one-stage and two-stage detection algorithm, a new network suitable for protrusion lesion detection is proposed. We inherit the method of anchor generation in SSD, a fast single-stage object detector outperform R-CNN series in terms of speed. Multi-scale feature layers are assigned to generate different sizes of default anchor boxes. Different from the previous work, our method doesnt require additional preprocessing because the network can learn features autonomously. For the 256*256 input, our method achieves 73% AP, perform a novel way to detect protrusion lesions.

SummaryAlthough vaccines have been successfully developed and approved against SARS-CoV-2, it is still valuable to perform studies on conserved antigenic sites for preventing possible pandemic-risk of other SARS-like coronavirus in the future and prevalent SARS-CoV-2 variants. By antibodies obtained from convalescent COVID-19 individuals, receptor binding domain (RBD) were identified as immunodominant neutralizing domain that efficiently elicits neutralizing antibody response with on-going affinity mature. Moreover, we succeeded to define a quantitative antigenic map of neutralizing sites within SARS-CoV-2 RBD, and found that sites S2, S3 and S4 (new-found site) are conserved sites and determined as subimmunodominant sites, putatively due to their less accessibility than SARS-CoV-2 unique sites. P10-6G3, P07-4D10 and P05-6H7, respectively targeting S2, S3 and S4, are relatively rare antibodies that also potently neutralizes SARS-CoV, and the last mAbs performing neutralization without blocking S protein binding to receptor. Further, we have tried to design some RBDs to improve the immunogenicity of conserved sites. Our studies, focusing on conserved antigenic sites of SARS-CoV-2 and SARS-CoV, provide insights for promoting development of universal SARS-like coronavirus vaccines therefore enhancing our pandemic preparedness.

Background Hepatitis E virus (HEV)-caused acute viral hepatitis is a major threat to public health worldwide. Recently, an enzyme linked immunosorbent assay (ELISA) kit detecting the HEV antigen was reported to have good concordance with the HEV RNA load and showed good clinical performance. But the ELISA kits can barely satisfy the needs of community clinics. In this study, a fluorescent microbead-based immunoassay (FMIA) for detecting the HEV antigen was developed and evaluated. Methods A mouse anti-HEV monoclonal antibody (mAb) conjugated with fluorescent microbeads was used as capturing antibody and another mouse mAb was used as detection antibody. Overall, 150 serum samples were collected from HEV-infected patients (n = 50) and non-HEV cases (n = 100) to evaluate the performance of the FMIA. Results The FMIA results showed a strong linear correlation with the viral RNA load. The diagnostic sensitivity and specificity of the HEV antigen FMIA were 92.0% (46/50) and 100.0% (100/100), respectively, and the test was consistent (kappa = 0.937, p = 0.627) with the commercial HEV antigen ELISA. The FMIA also showed good consistency with the PCR results (kappa = 0.939, p = 0.134). Conclusions As a rapid point-of-care (POC) test, a FMIA that is developed with acceptable performance is suitable for acute hepatitis E diagnosis, especially in developing countries and regions, because of its reduced time and simplified operation.

Hepatitis E virus (HEV) is one of the major pathogens that cause acute viral hepatitis. The human (genotypes 1 and 2) and zoonotic (genotypes 3 and 4) groups of HEV present different epidemiology and clinical features. In this study, we developed a classification method for rapidly classifying HEV into human or zoonotic groups that combines a general antigen test with a zoonotic group-specific antigen test. Evaluation of serial samples from HEV-infected rhesus monkeys indicated that HEV antigen-positive samples can be classified using the antigen-based classification method. The antigen-based classification method was evaluated further on 55 genotyped samples from acute hepatitis E patients, including 9 human and 46 zoonotic groups. The novel method was completely consistent with the sequencing results: 9/9 for the human groups (100%, 95% confidence interval [CI] 66.4-100%) and 46/46 for the zoonotic groups (100%, 95% CI 92.3-100%). This method was also successfully used for the clustering of some samples that could not be clustered by sequencing. Compared with the sequencing-based method, this method is less time-consuming, less expensive, and less technically complex and is therefore ideal for large numbers of samples. In conclusion, this study provides a convenient and sensitive method for classifying different groups of HEV, and it has potentially important public health applications, especially in underdeveloped areas that cannot afford the high cost of nucleic acid testing.

Background Nepal is an endemic area for hepatitis E virus (HEV) epidemics. The research on viral hepatitis in Nepal is limited. Methods Serum samples from 170 patients presenting with symptoms of hepatitis were collected from April to May 2014 in Biratnagar, Nepal, and then transported to Xiamen, China, for further evaluation. All samples were tested for HEV RNA, HEV antigen, anti-HEV IgM, anti-HEV IgG and anti-HBc IgM, anti-HCV IgG, and anti-HAV IgM. Results Sixteen patients were identified as acute hepatitis E with the presence of ≥ 2 HEV acute phase markers (antigen, RNA, and anti-HEV IgM). HEV infection was the major cause of potential active viral hepatitis (59.2%, 16 of 27), followed by HBV (25.9%, 7 of 27, anti-HBc IgM positive), HAV (18.5%, 5 of 27, anti-HAV IgM positive), and HCV (3.7%, 1 of 27, anti-HCV antibodies). All 16 confirmed HE cases were positive for HEV antigen, while 5 cases were HEV RNA positive, as well as 15 anti-HEV IgM positive. The low positive rate of RNA might be related to the collection and/or the transportation of these samples. Conclusions This study showed that HEV is a major cause of acute hepatitis in developing countries and regions. Application of immunoassay diagnostic kits, especially the HEV antigen tests, showed great potential for HE detection in these countries and regions.

With the aim of sending electric power to autonomous underwater vehicles (AUVs), an automatic wireless power supply system by means of electromagnetic coupler was proposed. An equivalent circuit of the electromagnetic coupler with compensating capacitors was presented to analyze the performance of the system. The magnetizing inductance and leakage inductance, which are important for choosing compensating capacitors, were calculated by finite element analysis (FEA) methods. Then the voltage gain, power loss and system efficiency were analyzed to optimize the winding turns. Finally, a phase-shift full bridge zero-voltage-switching converter and an electromagnetic coupler were produced. The experimental results demonstrated that the proposed system has a high voltage gain with small variation and sufficient power feeding.

The hepatitis E virus (HEV) is one of the main causes of enterically transmitted hepatitis worldwide. Although the mortality rates associated with HEV are generally low, they can be up to 28% in HEV-infected pregnant women, and the elderly are more susceptible. The reasons for this selective severity are unclear, partially because there is no suitable, easy-to-use model in which to study HEV infection. Non-human primates and standard swine have been identified as being sensitive to infection with HEV and have been used for HEV infection studies. However, studies in these animals have been limited by high housing costs and the difficulty of manipulating these animals. In the current study, we established a model of HEV infection using Bama miniature swine. The model is easy to use and is sensitive to infections with HEV genotypes 3 and 4, which are classified as zoonotic HEVs. In this model, infection of Bama miniature swine with HEV genotypes 3 and 4 caused the typical features. All Bama miniature swine that were infected with HEV genotypes 3 and 4 exhibited significant HEV viremia, shedding, anti-HEV antibody responses and partial liver inflammation. Bama miniature swine may serve as an alternative to standard swine models for the study of zoonotic HEV infection and HEV genotype specificity research.

Hepatitis E virus (HEV) is the aetiological agent of enterically transmitted hepatitis. The traditional methods for evaluating neutralizing antibody titres against HEV are real-time PCR and the immunofluorescence foci assay (IFA), which are poorly repeatable and operationally complicated, factors that limit their applicability to high-throughput assays. In this study, we developed a novel high-throughput neutralizing assay based on biotin-conjugated p239 (HEV recombinant capsid proteins, a.a. 368–606) and staining with allophycocyanin-conjugated streptavidin (streptavidin APC) to amplify the fluorescence signal. A linear regression analysis indicated that there was a high degree of correlation between IFA and the novel assay. Using this method, we quantitatively evaluated the neutralization of sera from HEV-infected and vaccinated macaques. The anti-HEV IgG level had good concordance with the neutralizing titres of macaque sera. However, the neutralization titres of the sera were also influenced by anti-HEV IgM responses. Further analysis also indicated that, although vaccination with HEV vaccine stimulated higher anti-HEV IgG and neutralization titres than infection with HEV in macaques, the proportions of neutralizing antibodies in the infected macaques’ sera were higher than in the vaccinated macaques with the same anti-HEV IgG levels. Thus, the infection more efficiently stimulated neutralizing antibody responses.

In intracerebral hemorrhage, microglia become rapidly activated and remove the deposited blood and cellular debris. To survive in a harmful hemorrhagic or posthemorrhagic condition, activated microglia must be equipped with appropriate self-defensive mechanism(s) to resist the toxicity of hemin, a component released from damaged RBCs. In the current study, we found that activation of microglia by pretreatment with LPS markedly reduced their vulnerability to hemin toxicity in vitro. Similarly, intracorpus callosum microinjection of LPS prior to hemin treatment reduced the brain tissue damage caused by hemin and increased microglial density in the penumbra in rats. LPS induced the expressions of inducible NO synthase (iNOS) and heme oxygenase (HO)-1, the rate-limiting enzyme in heme degradation in microglia. The preventive effect by LPS was significantly diminished by an iNOS inhibitor, L-N6-(1-iminoethyl)lysine, whereas it was mimicked by a NO donor, diethylamine-NONOate, both suggesting the crucial role of NO in the modulation of hemin-induced toxicity in activated microglia. We further found that NO reduced hemin toxicity via inhibition of hemin-induced activation of JNK and p38 MAPK pathways in microglia. Whereas HO-1 expression in LPS-stimulated microglia was markedly blocked by L-N6-(1-iminoethyl)lysine, the HO-1 inhibitor, tin protoporphyrin, increased iNOS expression and decreased the susceptibility of LPS-activated microglia to hemin toxicity. The data indicate that the mutual interaction between NO and HO-1 plays a critical role in modulating the adaptive response of activated microglia to hemin toxicity. Better understanding of the survival mechanism of activated microglia may provide a therapeutic strategy to attenuate the devastating intracerebral hemorrhagic injury.

Huperzine A (HupA) is a reversible and selective inhibitor of acetylcholinesterase (AChE), and it has multiple targets when used for Alzheimer's disease (AD) therapy. In this study, we searched for new mechanisms by which HupA could activate Wnt signaling and reduce amyloidosis in AD brain. A nasal gel containing HupA was prepared. No obvious toxicity of intranasal administration of HupA was found in mice. HupA was administered intranasally to β-amyloid (Aβ) precursor protein and presenilin-1 double-transgenic mice for 4 months. We observed an increase in ADAM10 and a decrease in BACE1 and APP695 protein levels and, subsequently, a reduction in Aβ levels and Aβ burden were present in HupA-treated mouse brain, suggesting that HupA enhances the nonamyloidogenic APP cleavage pathway. Importantly, our results further showed that HupA inhibited GSK3α/β activity, and enhanced the β-catenin level in the transgenic mouse brain and in SH-SY5Y cells overexpressing Swedish mutation APP, suggesting that the neuroprotective effect of HupA is not related simply to its AChE inhibition and antioxidation, but also involves other mechanisms, including targeting of the Wnt/β-catenin signaling pathway in AD brain.

The hepatitis E virus (HEV) ORF2 encodes a single structural capsid protein. The E2s domain (amino acids 459–606) of the capsid protein has been identified as the major immune target. All identified neutralizing epitopes are located on this domain; however, a comprehensive characterization of antigenic sites on the domain is lacking due to its high degree of conformation dependence. Here, we used the statistical software SPSS to analyze cELISA (competitive ELISA) data to classify monoclonal antibodies (mAbs), which recognized conformational epitopes on E2s domain. Using this novel analysis method, we identified various conformational mAbs that recognized the E2s domain. These mAbs were distributed into 6 independent groups, suggesting the presence of at least 6 epitopes. Twelve representative mAbs covering the six groups were selected as a tool box to further map functional antigenic sites on the E2s domain. By combining functional and location information of the 12 representative mAbs, this study provided a complete picture of potential neutralizing epitope regions and immune-dominant determinants on E2s domain. One epitope region is located on top of the E2s domain close to the monomer interface; the other is located on the monomer side of the E2s dimer around the groove zone. Besides, two non-neutralizing epitopes were also identified on E2s domain that did not stimulate neutralizing antibodies. Our results help further the understanding of protective mechanisms induced by the HEV vaccine. Furthermore, the tool box with 12 representative mAbs will be useful for studying the HEV infection process.

Hepatitis E virus (HEV) is a serious public health problem that causes acute hepatitis in humans and is primarily transmitted through fecal and oral routes. The major anti-HEV antibody responses are against conformational epitopes located in a.a. 459–606 of HEV pORF2. All reported neutralization epitopes are present on the dimer domain constructed by this peptide. While looking for a neutralizing monoclonal antibody (MAb)-recognized linear epitope, we found a novel neutralizing linear epitope (L2) located in a.a. 423–437 of pORF2. Moreover, epitope L2 is proved non-immunodominant in the HEV-infection process. Using the hepatitis B virus core protein (HBc) as a carrier to display this novel linear epitope, we show herein that this epitope could induce a neutralizing antibody response against HEV in mice and could protect rhesus monkeys from HEV infection. Collectively, our results showed a novel non-immunodominant linear neutralizing epitope of hepatitis E virus, which provided additional insight of HEV vaccine.

He Lian1, Jin Sun1, Yan Ping Yu1, Yan Hua Liu2, Wen Cao1, Yong Jun Wang1, Ying Hua Sun1, Si Ling Wang1, Zhong Gui He11School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, 2Department of Pharmaceutics, School of Pharmacy, Ningxia Medical University, Ningxia, People's Republic of ChinaBackground: Nowadays, many cytotoxic anticancer drugs exhibit low solubility and poor tumor selectivity, which means that the drug formulation is very important. For example, in the case of paclitaxel (PTX), Cremophor EL® (BASF, Ludwigshafen, Germany) needs to be used as a solubilizer in its clinical formulation (Taxol®, Bristol-Myers Squibb, New York, NY), although it can cause serious side effects. Nanomicellar systems are promising carriers to resolve the above problems, and the polymer chosen is the key element.Methods: In this study, a novel amphiphilic chitosan/vitamin E succinate (CS-VES) copolymer was successfully synthesized for self-assembling polymeric micelles. Proton nuclear magnetic resonance spectroscopy and infrared were used to characterize the molecular structure of the copolymer. The PTX-loaded CS-VES polymeric micelles (PTX-micelles) were characterized by dynamic light scattering, transmission electron microscopy, X-ray diffraction, and differential scanning calorimetry.Results: The critical micelle concentration of CS-VES was about 12.6 µg/mL, with the degree of amino group substitution being 20.4%. PTX-micelles were prepared by a nanoprecipitation/dispersion technique without any surfactant being involved. PTX-micelles exhibited a drug loading as high as 21.37% and an encapsulation efficiency of 81.12%, with a particle size ranging from 326.3 to 380.8 nm and a zeta potential of +20 mV. In vitro release study showed a near zero-order sustained release, with 51.06%, 50.88%, and 44.35% of the PTX in the micelles being released up to 168 hours at three drug loadings of 7.52%, 14.09%, and 21.37%, respectively. The cellular uptake experiments, conducted by confocal laser scanning microscopy, showed an enhanced cellular uptake efficiency of the CS-VES micelles in MCF-7 cells compared with Taxol. The PTX-micelles exhibited a comparable but delayed cytotoxic effect compared with Taxol against MCF-7 cells, due to the sustained-release characteristics of the nanomicelles. More interestingly, blank nanomicelles based on CS-VES copolymer demonstrated significant cytotoxicity against MCF-7 cells.Conclusion: The supramolecular micellar aggregates based on CS-VES copolymer is a promising nanocarrier and efficacy enhancer when used as an anticancer drug-delivery system.Keywords: nanomicelles, cellular uptake, cytotoxicity

Valproate (VPA) is a widely used anticonvulsant and mood-stabilizing drug. Recent studies have shown that VPA could reduce amyloid-β generation, and improve memory deficits in transgenic mouse models of Alzheimer's disease (AD). However, whether VPA affects tau phosphorylation and the underlying mechanism has not been established. Here, we showed that systemic treatment of APP and presenilin 1 double transgenic mice with VPA (50mg/kg, once a day for 12 weeks), significantly reduced the levels of tau phosphorylation at the sites of Thr205, Ser396 and Thr231. Meanwhile, VPA treatment markedly reduced the activities of cyclin-dependent kinase 5 (CDK5) and glycogen synthase kinase 3β (GSK3β), two protein kinases involved in abnormal hyperphosphorylation of tau. In an okadaic acid-induced tau hyperphosphorylation SH-SY5Y cell model, the anti-tau-phosphorylation effect of VPA was further confirmed, accompanied by a marked decrease in the activities of CDK5 and GSK3β. Our present data suggest that the inhibitory effects of VPA on tau hyperphosphorylation might be mediated through both CDK5 and GSK3β signaling pathways.

Polymeric micelles which are self-assembled from amphiphilic copolymers are thermodynamically stable, and they can solubilize hydrophobic drugs by the hydrophilic core. Many excellent active compounds are confined because of general low oral bioavailability due to poor solubility. Take into account from the two points above, polymeric micelles may be used as proper oral carrier to improve the dissolubility of hydrophobic drugs, and enhance the permeation though gastrointestinal tract, therefore, the pharmacodynamics is elevated. Meanwhile, the segments in copolymers are multivariate, so many kinds of micelles can be obtained, such as, pH- or thermo- sensitive as well as mucoadhesive ones. The modified micelles can alter drug release profiles while solubilizing them, that is why the oral bioavailability increase further. In this review, recent progress of polymeric micelles used in oral administration is summarized, and the prospect of polymeric micelles' application in this field is also evaluated.

The 18-mer oligodeoxynucleotides (ODNs) that can inhibit survivin gene expression were selected as a model gene drug to study hepatic-targeting drug delivery system. Novel galactosylated polymers (cholesteryloxycarbonylamino) ethylamine-alpha,beta-polyasparthydrazied (CHE-PAHy-Lacs), which target asialoglycoprotein receptor on hepatic parenchymal cells (PC), were designed and synthesized as non-toxic, non-antigenic and non-teratogenic ligands for liposomes. The liposomes incorporating different CHE-PAHy-Lacs were prepared and characterized by zeta potential and particle size analyzer. The drug encapsulation efficiency was measured by gel filtration method. 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate was used as a marker for all the liposome preparations in the in vivo experiments. The CHE-PAHy-Lac liposomes produced a significant improvement in the encapsulation efficiency of ODNs (28.73-51.37%) compared with conventional liposomes (9.88%). The in vivo results showed that the liposomes incorporating CHE-PAHy-Lac, which contained about 30% (w/w) galactosyl residues, exhibited marked accumulation in the liver and hepatic PC. These results suggest that the novel galactosylated polymers used for liposomes have a great potential as a gene delivery system for hepatic targeting.

Background Although increasing evidence has indicated that brain insulin dysfunction is a risk factor for Alzheimer disease (AD), the underlying mechanisms by which insulin deficiency may impact the development of AD are still obscure. Using a streptozotocin (STZ)-induced insulin deficient diabetic AD transgenic mouse model, we evaluated the effect of insulin deficiency on AD-like behavior and neuropathology. Results Our data showed that administration of STZ increased the level of blood glucose and reduced the level of serum insulin, and further decreased the phosphorylation levels of insulin receptors, and increased the activities of glycogen synthase kinase-3α/β and c-Jun N-terminal kinase in the APP/PS1 mouse brain. We further showed that STZ treatment promoted the processing of amyloid-β (Aβ) precursor protein resulting in increased Aβ generation, neuritic plaque formation, and spatial memory deficits in transgenic mice. Conclusions Our present data indicate that there is a close link between insulin deficient diabetes and cerebral amyloidosis in the pathogenesis of AD.