Screening and detection of multivalent human papillomavirus antibodies using a high-throughput liquid chip fluoroimmunoassay system.

Immunoassays are commonly used in disease diagnosis and vaccine evaluation but can be costly and time-consuming when confronted with multivalent targets, such as antisera containing antibodies to human papillomavirus (HPV), because of their limited ability to discriminate between multiple analytes in a single reaction well. This study describes the development of a high-throughput liquid chip system that combines immunoassay techniques and magnetic beads to allow the simultaneous screening and quantitative detection of antibodies to four types of HPV using the Luminex fluoroimmunoassay system. Groups of beads embedded with fluorescent dyes at various ratios were coated with optimized HPV capture antigens and demonstrated excellent dose-dependent response to four monoclonal antibodies used as reference standards. This assay is sensitive, accurate, repeatable, and simple to perform, enabling multiplex antibody detection with a high degree of orthogonality. The performance of the Luminex system was compared with conventional immunoassays for quantitative detection of quadrivalent HPV antibodies in antisera of mice immunized with five lots of HPV vaccines, verifying the accuracy and detection efficiency of the assay. This strategy is a promising approach to characterizing antibodies present in polyclonal antisera and has promising applications in research, clinical, and industrial settings, for example, streamlining vaccine efficacy trials and vaccine lot inspection and release procedures.

Immunogenicity and protective effects of recombinant bivalent COVID-19 vaccine in mice and rhesus macaques.

Coronavirus disease (COVID-19) is still spreading rapidly worldwide, and a safe, effective, and cheap vaccine is still required to combat the COVID-19 pandemic. Here, we report a recombinant bivalent COVID-19 vaccine containing the RBD proteins of the prototype strain and beta variant. Immunization studies in mice demonstrated that this bivalent vaccine had far greater immunogenicity than the ZF2001, a marketed monovalent recombinant protein COVID-19 vaccine, and exhibited good immunization effects against the original COVID-19 strain and various variants. Rhesus macaque challenge experiments showed that this bivalent vaccine drastically decreased the lung viral load and reduced lung lesions in SARS-CoV-2 (the causative virus of COVID-19)-infected rhesus macaques. In summary, this bivalent vaccine showed immunogenicity and protective efficacy that was far superior to the monovalent recombinant protein vaccine against the prototype strain and provided an important basis for developing broad-spectrum COVID-19 vaccines.

Seismically imaged lithospheric delamination and its controls on the Mesozoic Magmatic Province in South China.

The current lithospheric root of the South China Block has been partly removed, yet what mechanisms modified the lithospheric structure remain highly controversial. Here we use a new joint seismic inversion algorithm to image tabular high-velocity anomalies at depths of ~90-150 km in the asthenosphere beneath the convergent belt between the Yangtze and Cathaysia blocks that remain weakly connected with the stable Yangtze lithosphere. Based on obtained seismic images and available geochemical data, we interpret these detached fast anomalies as partially destabilized lower lithosphere that initially delaminated at 180-170 Ma and has relaminated to their original position after warming up in the mantle by now. We conclude that delamination is the most plausible mechanism for the lithospheric modification and the formation of a Mesozoic Basin and Range-style magmatic province in South China by triggering adiabatic upwelling of the asthenosphere and consequent lithospheric extension and extensive melting of the overlying crust.

Lithosphere architecture characterized by crust-mantle decoupling controls the formation of orogenic gold deposits.

This study, via combined analysis of geophysical and geochemical data, reveals a lithospheric architecture characterized by crust-mantle decoupling and vertical heat-flow conduits that control orogenic gold mineralization in the Ailaoshan gold belt on the southeastern margin of Tibet. The mantle seismic tomography indicates that the crust-mantle decoupled deformation, defined from previous seismic anisotropy analysis, was formed by upwelling and lateral flow of the asthenosphere, driven by deep subduction of the Indian continent. Our magnetotelluric and seismic images show both a vertical conductor across the Moho and high Vp/Vs anomalies both in the uppermost mantle and lowest crust, suggesting that crust-mantle decoupling promotes ponding of mantle-derived basic melts at the base of the crust via a heat-flow conduit. Noble gas isotope and halogen ratios of gold-related ore minerals indicate a mantle source of ore fluid. A rapid decrease in Cl/F ratios of lamprophyres under conditions of 1.2 GPa and 1050°C suggests that the ore fluid was derived from degassing of the basic melts. Similar lithospheric architecture is recognized in other orogenic gold provinces, implying analogous formational controls.

Acoustic Source Localization in CFRP Composite Plate Based on Wave Velocity-Direction Function Fitting.

Composite materials are widely used, but they are often subjected to impacts from foreign objects, causing structural damage. To ensure the safety of use, it is necessary to locate the impact point. This paper investigates impact sensing and localization technology for composite plates and proposes a method of acoustic source localization for CFRP composite plates based on wave velocity-direction function fitting. This method divides the grid of composite plates, constructs the theoretical time difference matrix of the grid points, and compares it with the actual time difference to form an error matching matrix to localize the impact source. In this paper, finite element simulation combined with a lead-break experiment is used to explore the wave velocity-angle function relationship of Lamb waves in composite materials. The simulation experiment is used to verify the feasibility of the localization method, and the lead-break experimental system is built to locate the actual impact source. The results show that the acoustic emission time-difference approximation method can effectively solve the problem of impact source localization in composite structures, and the average localization error is 1.44 cm and the maximum localization error is 3.35 cm in 49 experimental points with good stability and accuracy.

Development and characterization of anti-HPV16 monoclonal antibodies for assembly of an HPV16 detection kit.

Quality control is very important during the development of 3-valent (16/18/58), 9-valent (6/11/16/18/31/33/45/52/58), and 15-valent human papillomavirus (HPV) vaccines (6/11/16/18/31/33/35/39/45/52/56/58/59/68). All 3-valent, 9-valent, and 15-valent HPV vaccines contain the HPV16 antigen; therefore, a detection method that can specifically identify HPV16 in vaccines is urgently required. This study aimed to develop and characterize monoclonal antibodies to assemble a highly specific HPV16 detection kit. The HPV16 L1 pentameric protein developed as an immunogen was used to prepare monoclonal antibodies. From the pool of prepared monoclonal antibodies, we selected 4G12 and 5A6 to screen and evaluate their subtypes, specificity, neutralizing activity, serum competition, binding affinity, and gene sequencing. After these characterizations, an enzyme-linked immunosorbent assay kit for these monoclonal antibodies was developed, and excellent quality was demonstrated in the assessment of linearity, repeatability, and specificity. The developed detection kit has great potential for wide use in clinical testing and quality control in vaccine production processes.

Development of HPV58 type-specific antibodies and detection kit.

Currently, human papillomavirus (HPV) vaccines are in short supply, so the development of HPV vaccines has a broad market prospect. The 3-, 9-, and 15-valent HPV vaccines developed by ourselves all contain HPV58-derived antigen components. It is important to detect HPV 58 during vaccine production. Here, we introduced a development process of HPV58 type-specific antibodies and a detection kit. Briefly, HPV58 L1-Virus Like Particles (VLPs) were used as antigens to immunize mice, followed by extraction of the ascites to prepare hybridoma cells. After culturing, the supernatants containing secreted antibodies were harvested, purified, and screened to obtain monoclonal antibodies (mAbs). In the pool of attained monoclonal antibodies, we selected 2F7 and 2G7 to evaluate their subtypes, specificity, neutralizing activity, serum competition, binding affinity and gene sequencing. Finally, an enzyme-linked immunosorbent assay (ELISA) detection kit was assembled with 2F7 and 2G7 mAbs which possessed high specificity to HPV58 L1-VLPs. The detection kit developed by 2F7 and 2G7 could be adopted to specifically detect HPV58 L1 protein with good linearity and detection range, which could be widely used in clinical testing and quality control in the production of HPV vaccines.Abbreviations: BSA: Bovine serum albumin; CDRs: Complementarity-determining regions; CV: Coefficient of variation; DTT: Dithiothreitol; ELISA: Enzyme-linked immunosorbent assay; HAT: Hypoxanthine-aminopterin-thymidine; HPV: Human Papillomavirus; IC50: 50% inhibition rate; IC90: 90% inhibition rate; mAbs: Monoclonal antibodies; VLP: Virus-like particle.

Polysaccharides from Vaccaria segetalis seeds reduce urinary tract infections by inhibiting the adhesion and invasion abilities of uropathogenic Escherichia coli.

The seeds of Vaccaria segetalis (Neck.) are from a traditional medicinal plant Garcke, also called Wang-Bu-Liu-Xing in China. According to the Chinese Pharmacopoeia, the seeds of V. segetalis can be used for treating urinary system diseases. This study was designed to investigate the underlying mechanism of VSP (polysaccharides from Vaccaria segetalis) against urinary tract infections caused by uropathogenic Escherichia coli (UPEC). Here, both in vitro and in vivo infection models were established with the UPEC strain CFT073. Bacterial adhesion and invasion into bladder epithelial cells were analyzed. We found that VSP reduced the adhesion of UPEC to the host by inhibiting the expression of bacterial hair follicle adhesion genes. VSP also reduced the invasion of UPEC by regulating the uroplakins and Toll-like receptors of host epithelial cells. In addition, the swarming motility and flagella-mediated motility genes flhC, flhD and Flic of UPEC were diminished after VSP intervention. Taken together, our findings reveal a possible mechanism by which VSP interferes with the adhesion and invasion of UPEC.

Novel combretastatin A-4 derivative containing aminophosphonates as dual inhibitors of tubulin and matrix metalloproteinases for lung cancer treatment.

Here, sixteen novel conjugates containing tubulin inhibitor and matrix metalloproteinase inhibitor was synthesized together with their activity evaluated. Among them, 9e exhibited the most potent activity against various human cancer cells (IC50 values was 0.19-0.42 µM) as well as multidrug-resistant tumor cells (A549/CDDP and MCF-7/DOX) and also showed significantly lower cytotoxic activity toward human normal liver cells LO2 in comparison with that of CA-4. Interestingly, 9e not only strongly inhibited tubulin polymerization, and induced cell apoptosis and cell cycle arrest in G2/M stage, but also remarkably displayed inhibition of cell migration against A549 cells in vitro, and exhibited a moderate activity toward MMP-2, MMP-3 and MMP-9, respectively. Moreover, the significant down-regulation in the levels of Bcl-2 protein and up-regulated levels of proteins, such as Bax, p53 and caspase-3, indicated that 9e can induce apoptosis via mitochondria-dependent apoptosis pathway. Additionally, 9e can also cause ER stress demonstrating as up-regulation express of proteins (CHOP, p-eIF2a, and p-PERK). Importantly, 9e displayed significant in vivo antitumor efficacy in A549 xenograft models without inducing apparent systemic toxicity. Collectively, this work indicated that compound 9e, a dual MMPs and tubulin inhibitor, is a novel and promising agent for cancer therapy.

Associations Between Fundus Types and Clinical Manifestations in Patients with RDH12 Gene Mutations.

To study the associations between RDH12 gene mutations, fundus types, and clinical manifestations. In total, 46 patients with inherited eye diseases caused by RDH12 gene mutations were included in this study. High-throughput chip capture sequencing, Sanger sequencing, and gene panel detection were used to determine that RDH12 was the pathogenic gene. All patients underwent the following detailed ophthalmic examinations: visual acuity, visual field, intraocular pressure, fundus photography, electroretinography, and optical coherence tomography (OCT). Statistical analysis was used to evaluate the clinical phenotype. A total of 32 mutations were identified in 46 patients. The most common mutations were c.437T > A, c.184C > T, and c.524C > T; the corresponding amino acid changes were p.Val146Asp, p.Arg62Ter, and p.Ser175Leu. Of the 46 patients, retinitis pigmentosa (RP) was found in 31 (68.9%); leber congenital amaurosis (LVA) was found in 11 (24.4%); early onset of severe retinal dystrophy (EOSRD) was found in one (2.2%); cone rod dystrophy (CORD) was found in one (2.2%); and Stargardt disease was found in one (2.2%). There was a significant difference in best-corrected visual acuity among patients based on fundus type (p = 0.0124). Linear trend analysis showed that best-corrected visual acuity gradually decreased as the fundus type increased in severity. In addition, there was a significant difference in the incidence of night blindness among patients with different fundus types (p = 0.0429): types I and IV fundi were associated with the highest incidences of night blindness. RDH12 gene mutation can cause serious inherited retinal diseases, which primarily include RP and LCA. Combined with clinical symptoms and fundus types, the progression of the disease can be characterized and used to guide genetic diagnosis and gene therapy.

[Effect of polysaccharides from seeds of Vaccaria segetalis in alleviating urinary tract infection induced bladder injury by inhibiting NLRP3 inflammasome].

To study the mechanism of polysaccharides from seeds of Vaccaria segetalis( PSV) in the treatment of bacterial cystitis through the NLRP3 inflammasome pathway. The rat model of urinary tract infection was used and treated with PSV,and the urine and bladders were collected. The level of interleukin-10( IL-10) in rat urine was detected by enzyme linked immunosorbent assay( ELISA). Western blot and immunofluorescence staining were used to detect the expressions of sonic hedgehog( SHH) and NLRP3 inflammasome [NOD-like receptor thermoprotein domain 3( NLRP3),apoptosis associated speck like protein( ASC) and pro-caspase-1]. The expression of Toll-like receptor pathway was detected by RT-PCR. The death of 5637 cells induced by uropathogenic Escherichia coli( UPEC) and lactate dehydrogenase( LDH) release were evaluated using live/dead staining. The results showed that in the rat bladder,the expressions of SHH,NLRP3 inflammasomes and Toll-like receptors were significantly up-regulated,and NLRP3 inflammasomes were significantly activated by UPEC infection. The administration with PSV could significantly increase the concentration of IL-10 in urine,inhibit the expressions of SHH,NLRP3 inflammasomes and Toll-like receptors in bladder,and inhibit the activation of NLRP3 inflammasomes. A large number of 5637 cells were dead after UPEC infection and caused LDH production. PSV could significantly inhibit the death of 5637 cells and the release of LDH. In conclusion,PSV could inhibit the expression and activation of NLRP3 inflammasomes by inhibiting the Toll-like receptor pathway,thereby mitigating the bladder injury.

A novel cytotoxic flavonoid C-glycoside from premna fulva.

Premna fulva Craib, locally known as "Zhangu" in China, is a kind of traditional medicinal plant. A phytochemical investigation on this plant led to the isolation of a novel flavonoid glycoside along with three known analogues. The chemical structure of the new compound was determined by spectral and chemical analysis as apigenin 8-C-ß-D-xylopyranoside (1). Compound 1 showed weak inhibitory activities in vitro against to four tumor cell lines (HL-60, Bcap37, SMMC7721, and P388) with IC50 values of 12.58, 19.31, 31.02, and 48.19 µg/mL, respectively. The result might be helpful to explain the application of P. fulva in Traditional Chinese Medicine.[Figure: see text].

Polysaccharides extract from Vaccaria segetalis seeds inhibits kidney infection by regulating cathelicidin expression.

ETHNOPHARMACOLOGICAL RELEVANCE: According to the Chinese Pharmacopoeia, the seeds of Vaccaria segetalis, a traditional medicinal herb, can be used for treating urinary diseases. The polysaccharides extract from V. segetalis seeds (VSP) has been shown to prevent urinary tract infections (UTIs). AIM OF THE STUDY: Investigate the effects of VSP on treating kidney infection induced by uropathogenic Escherichia coli (UPEC) and the underlying mechanisms. MATERIALS AND METHODS: Both in vivo and in vitro infection models were established with the UPEC strain CFT073. After oral administration of VSP, the levels of bacterial load, cathelicidin (CRAMP), Toll-like receptors (TLRs) in the kidney were evaluated. The expression of cathelicidin (LL-37) in human renal cell carcinoma cell line (A498) was tested after the treatment of VSP. RESULTS: In the kidneys of infection models, high-titer bacteria was detected. In the kidney of rat model, the expression of CRAMP was down-regulated, no significant change was observed in the levels of TLRs. After oral administration of VSP, the bacterial load was significantly decreased in rat and mouse models, and the levels of CRAMP and TLRs were significantly up-regulated in rat model. In vitro, the expression of LL-37 was significantly inhibited by CFT073. VSP up-regulated the expression of LL-37 in A498 cells. CONCLUSIONS: The up-regulation of cathelicidin expression may contribute to the therapeutic effects of VSP on kidney infection.

Cross-neutralizing antibody titres against non-vaccine types induced by a recombinant trivalent HPV vaccine (16/18/58) in rhesus macaques.

Human papillomavirus (HPV) causes not only most cervical cancers but also cancers of the vagina, vulva, penis, anus, rectum, and oropharynx. Every year, 200,000 women die of cervical cancer in the world, and China accounts for about 10%. HPV vaccines are effective in preventing HPV infections thus HPV-related cancers worldwide. Studies on the clinical trials of the 2v Cervarix™ and the 4v Gardasil® have suggested that immunization with either of these vaccines provided some level of protection against other HPV types that are closely related to the types contained in the vaccines. Here we conducted a preliminary evaluation on the ability to induce cross-neutralizing antibodies in rhesus monkeys by a 3v HPV vaccine that targets HPV16, 18, and 58 and it is specifically designed for Chinese women. We found that this vaccine is no less than Gardasil® in terms of the ability to induce NAbs against non-vaccine types of HPV in rhesus macaques. These results provided evidence from the immunogenicity point of view that the KLWS 3v HPV vaccine is a strong competitor to the imported 2v and 4v HPV vaccines currently available on the market.

Comparison of inflammatory cytokines levels in the aqueous humor with diabetic retinopathy.

PURPOSE: Evidence has accumulated suggesting that various inflammatory cytokines are involved in the progress of diabetic retinopathy (DR), but there are few studies concerning the relationship between individual cytokines levels in the aqueous humor (AH) and the severity of DR. This study aimed to explore the differences of interleukin (IL)-23, IL-17, IL-10 and transforming growth factor (TGF)-ß in AH form patients with different proliferative stages of DR. METHODS: From June 2016 to June 2019, patients for senile cataract surgery were enrolled with the informed consent. All cases were graded into 4 groups: the control group (patients without diabetes), non-retinopathy (NDR) group (diabetic patients without retinopathy), non-proliferative diabetic retinopathy (NPDR) group, and proliferative diabetic retinopathy (PDR) group. The concentrations of IL-23, IL-17, IL-10, and TGF-ß in AH were measured using ELISA and compared them within four groups by ANOVA. RESULTS: In this study, 20 (28.57%), 18 (25.71%), 17 (24.29%), and 15 (21.43%) patients were included in the control, NDR, NPDR, and PDR groups, respectively. There had no significant differences in demographic characteristics (P > 0.05). Comparison of these cytokines within four groups revealed: the IL-23 level was increased in NDR group initially and raised along with the progression of DR (P < 0.01). The IL-17 level was significantly higher in NPDR and PDR groups compared to NDR and the control groups, and positively correlated with more-severe DR (P < 0.01). By contrast, The IL-10 level was significantly lower in diabetic patients than in non-diabetic controls, and decreased as the severity of DR increased (P < 0.05). In addition, TGF-ß was also elevated in diabetic patients, but showed no differences based on the presence or severity of DR (P > 0.05). CONCLUSIONS: The over-expression of IL-23 and IL-17 in AH might have a synergistic effect on the pathogenesis well before the proliferative stage, and was typical positively correlated with the severity of DR. These results offer a novel early therapeutic target for DR.

Crude polysaccharides from the seeds of Vaccaria segetalis prevent the urinary tract infection through the stimulation of kidney innate immunity.

ETHNOPHARMACOLOGICAL RELEVANCE: The seeds of Vaccaria segetalis (Neck.) Garcke is used for the treatment of urinary diseases in Traditional Chinese Medicine according to the Chinese Pharmacopoeia. Crude polysaccharides and the aqueous extract from the seeds of V. segetalis (SVCP) were proved to be effective on treating benign prostatic hyperplasia. AIM OF THE STUDY: The aim of this study was to test the effects of SVCP on urinary tract infection (UTI) induced by uropathogenic Escherichia coli (UPEC) strain CFT073 in the rat model and to investigate the underlying mechanisms. MATERIALS AND METHODS: A rat UTI model was established with the infection of UPEC strain CFT073. After oral administration of SVCP, the urinalysis and histological examination were evaluated. The levels of pro-inflammatory cytokines, procalcitonin (PCT) and polymeric Ig receptor (PIGR) were used to test the effects of SVCP on host immunity. The mRNA level of PapG in CFT073 was used to test the influence of SVCP on virulence factor. The effects of SVCP on the inhibition of bacterial adhesion were evaluated with mice UTI model. RESULTS: In the rat UTI model, the levels of bacterial load, white blood cells (WBC) and red blood cells (RBC) in urine and the pathological injury in the bladder were significantly up-regulated, the expression of PIGR in kidney was down-regulated, no significant change was observed on the pro-inflammatory cytokines in urine. After oral administration of SVCP for 3 days, the levels of bacterial load, WBC and RBC in urine were significantly decreased, the pathological injury in the bladder were remarkably inhibited. The expression of IL-6, IL-8 in urine and PIGR in kidney were significantly up-regulated by SVCP (200 mg/kg). SVCP showed no effect on the concentration of PCT in serum. SVCP failed to down-regulate the mRNA level of PapG in CFT073. In the mice UTI model, pre-treatment of SVCP failed to inhibit the intracellular bacterial load in the bladder. CONCLUSIONS: The therapeutic effects of SVCP on treating UTIs might result from the up-regulation of innate immunity in the kidney. SVCP can be used as an alternative therapeutic agent for UTIs.

Synthesis, mechanisms of action, and toxicity of novel aminophosphonates derivatives conjugated irinotecan in vitro and in vivo as potent antitumor agents.

Twenty novel aminophosphonates derivatives (5a-5j and 6a-6j) conjugated irinotecan were synthesized through esterification reaction, and evaluated their anticancer activities using MTT assay. In vitro evaluation revealed that they displayed similar or superior cytotoxicity compared to the positive drug irinotecan against A549, MCF-7, SK-OV-3, MG-63, U2OS and multidrug-resistant (MDR) SK-OV-3/CDDP cancer cell lines. Among them, 9b displayed the most potent activity, with IC50 values of 0.92-3.23 µM against five human cancer cells, which exhibited a 5.4-19.1-fold increase in activity compared to the reference drug irinotecan, respectively. Moreover, cellular mechanism studies suggested that 9b arrested cell cycle at S stage and induced cell apoptosis along with the decrease of mitochondrial membrane potential (MMP). Interestingly, 9b significantly inhibited tumor growth in SK-OV-3 xenograft models in vivo without apparent toxicity, which was better than the positive drug irinotecan. Taken together, 9b possessed potent antitumor activity and may be a promising candidate for the potential treatment of human ovarian cancer cells.

Quantitative evaluation of in-situ bioremediation of compound pollution of oil and heavy metal in sediments from the Bohai Sea, China.

Owing to the semi-enclosed environment of the Bohai Sea, the ecological effects caused by an oil spill would be significant. A typical in- situ bioremediation engineering project for of oil-spilled marine sediments was performed in the Bohai Sea and a quantitative assessment of the ecological restoration was performed. The bioremediation efficiencies of n-alkane and PAHs in the sediment are 32.84 ± 21.66% and 50.42 ± 17.49% after 70 days of bioremediation, and 60.99 ± 10.14% and 68.01 ± 18.60% after 210 days, respectively. After 210 days of bioremediation, the degradation rates of two- to three ring PAHs and four-ring PAHs are 84.44 ± 23.03% and 26.62 ± 43.76%, respectively. In addition, the concentrations of the heavy metals first increased by 6.00% due to oil spill degradation and release, and then decreased by 72.60% with the degradation of oil caused by bioremediation or vertical migration. According to the continuous tracking monitoring, the composition of the microbial community in the restored area was similar to that in the control area and the clean area in Bohai Sea after 210 days of bioremediation. These results may provide some theoretical and scientific data to understand the degradation mechanism and assessing the ecological remediation efficiency for oil spills in open sea areas.