Anti-Epstein-Barr Virus BNLF2b for Mass Screening for Nasopharyngeal Cancer.

BACKGROUND: Population screening of asymptomatic persons with Epstein-Barr virus (EBV) DNA or antibodies has improved the diagnosis of nasopharyngeal carcinoma and survival among affected persons. However, the positive predictive value of current screening strategies is unsatisfactory even in areas where nasopharyngeal carcinoma is endemic. METHODS: We designed a peptide library representing highly ranked B-cell epitopes of EBV coding sequences to identify novel serologic biomarkers for nasopharyngeal carcinoma. After a retrospective case-control study, the performance of the novel biomarker anti-BNLF2b total antibody (P85-Ab) was validated through a large-scale prospective screening program and compared with that of the standard two-antibody-based screening method (EBV nuclear antigen 1 [EBNA1]-IgA and EBV-specific viral capsid antigen [VCA]-IgA). RESULTS: P85-Ab was the most promising biomarker for nasopharyngeal carcinoma screening, with high sensitivity (94.4%; 95% confidence interval [CI], 86.4 to 97.8) and specificity (99.6%; 95% CI, 97.8 to 99.9) in the retrospective case-control study. Among the 24,852 eligible participants in the prospective cohort, 47 cases of nasopharyngeal carcinoma (38 at an early stage) were identified. P85-Ab showed higher sensitivity than the two-antibody method (97.9% vs. 72.3%; ratio, 1.4 [95% CI, 1.1 to 1.6]), higher specificity (98.3% vs. 97.0%; ratio, 1.01 [95% CI, 1.01 to 1.02]), and a higher positive predictive value (10.0% vs. 4.3%; ratio, 2.3 [95% CI, 1.8 to 2.8]). The combination of P85-Ab and the two-antibody method markedly increased the positive predictive value to 44.6% (95% CI, 33.8 to 55.9), with sensitivity of 70.2% (95% CI, 56.0 to 81.4). CONCLUSIONS: Our results suggest that P85-Ab is a promising novel biomarker for nasopharyngeal carcinoma screening, with higher sensitivity, specificity, and positive predictive value than the standard two-antibody method. (Funded by the National Key Research and Development Program of China and others; ClinicalTrials.gov number, NCT04085900.).

The neutralizing breadth of antibodies targeting diverse conserved epitopes between SARS-CoV and SARS-CoV-2.

Antibody therapeutics for the treatment of COVID-19 have been highly successful. However, the recent emergence of the Omicron variant has posed a challenge, as it evades detection by most 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 the potential candidates, we found that nAbs X01, X10, and X17 offer broad neutralizing potential against most variants of concern, with X17 further identified as a Class 5 nAb with undiminished neutralization against the Omicron variant. Cryo-electron microscopy structures of the three antibodies together in complex with each of the spike proteins of the prototypical SARS-CoV, SARS-CoV-2, and Delta and Omicron variants of SARS-CoV-2 defined three nonoverlapping conserved epitopes on the receptor-binding domain. The triple-antibody mixture exhibited enhanced resistance to viral evasion and effective protection against infection of the Beta variant in hamsters. Our findings will aid the development of antibody therapeutics and broad vaccines against SARS-CoV-2 and its emerging variants.

Therapeutic vaccine-induced plasma cell differentiation is defective in the presence of persistently high HBsAg levels.

BACKGROUND & AIMS: Mechanisms behind the impaired response of antigen-specific B cells to therapeutic vaccination in chronic hepatitis B virus (HBV) infection remain unclear. The development of vaccines or strategies to overcome this obstacle is vital for advancing the management of chronic hepatitis B. METHODS: A mouse model, denominated as E6F6-B, was engineered to feature a knock-in of a B-cell receptor (BCR) that specifically recognizes HBsAg. This model served as a valuable tool for investigating the temporal and spatial dynamics of humoral responses following therapeutic vaccination under continuous antigen exposure. Using a suite of immunological techniques, we elucidated the differentiation trajectory of HBsAg-specific B cells post-therapeutic vaccination in HBV carrier mice. RESULTS: Utilizing the E6F6-B transfer model, we observed a marked decline in antibody-secreting cells 2 weeks after vaccination. A dysfunctional and atypical pre-plasma cell population (BLIMP-1+ IRF4+ CD40- CD138- BCMA-) emerged, manifested by sustained BCR signaling. By deploying an antibody to purge persistent HBsAg, we effectively prompted the therapeutic vaccine to provoke conventional plasma cell differentiation. This resulted in an enhanced anti-HBs antibody response and facilitated HBsAg clearance. CONCLUSIONS: Sustained high levels of HBsAg limit the ability of therapeutic hepatitis B vaccines to induce the canonical plasma cell differentiation necessary for anti-HBs antibody production. Employing a strategy combining antibodies with vaccines can surmount this altered humoral response associated with atypical pre-plasma cells, leading to improved therapeutic efficacy in HBV carrier mice. IMPACT AND IMPLICATIONS: Therapeutic vaccines aimed at combatting HBV encounter suboptimal humoral responses in clinical settings, and the mechanisms impeding their effectiveness have remained obscure. Our research, utilizing the innovative E6F6-B mouse transfer model, reveals that the persistence of HBsAg can lead to the emergence of an atypical pre-plasma cell population, which proves to be relevant to the potency of therapeutic HBV vaccines. Targeting the aberrant differentiation process of these atypical pre-plasma cells stands out as a critical strategy to amplify the humoral response elicited by HBV therapeutic vaccines in carrier mouse models. This discovery suggests a compelling avenue for further study in the context of human chronic hepatitis B. Encouragingly, our findings indicate that synergistic therapy combining HBV-specific antibodies with vaccines offers a promising approach that could significantly advance the pursuit of a functional cure for HBV.

Critical Residues Involved in the Coassembly of L1 and L2 Capsid Proteins of Human Papillomavirus 16.

Human papillomaviruses (HPV) are small DNA viruses associated with cervical cancer, warts, and other epithelial tumors. Structural studies have shown that the HPV capsid consists of 360 copies of the major capsid protein, L1, arranged as 72 pentamers in a T=7 icosahedral lattice, coassembling with substoichiometric amounts of the minor capsid protein, L2. However, the residues involved in the coassembly of L1 and L2 remain undefined due to the lack of structure information. Here, we investigated the solvent accessibility surfaces (SASs) of the central cavity residues of the HPV16 L1 pentamer in the crystal structure because those internal exposed residues might mediate the association with L2. Twenty residues in L1 protein were selected to be analyzed, with four residues in the lumen of the L1 pentamer identified as important: F256, R315, Q317, and T340. Mutations to these four residues reduced the PsV (pseudovirus) infection capacity in 293FT cells, and mutations to R315, Q317, and T340 substantially perturb L2 from coassembling into L1 capsid. Compared with wild-type (WT) PsVs, these mutant PsVs also have a reduced ability to become internalized into host cells. Finally, we identified a stretch of negatively charged residues on L2 (amino acids [aa] 337 to 340 [EEIE]), mutations to which completely abrogate L2 assembly into L1 capsid and subsequently impair the endocytosis and infectivity of HPV16 PsVs. These findings shed light on the elusive coassembly between HPV L1 and L2. IMPORTANCE Over 200 types of HPV have been isolated, with several high-risk types correlated with the occurrence of cervical cancer. The HPV major capsid protein, L1, assembles into a T=7 icosahedral viral shell, and associates with the minor capsid protein, L2, which plays a critical role in the HPV life cycle. Despite the important role of the L2 protein, its structure and coassembly with L1 remain elusive. In this study, we analyzed the amino acid residues at the proposed interface between L1 and L2. Certain mutations at these sites decreased the amount of L2 protein assembled into the capsid, which, in turn, led to a decrease in viral infectivity. Knowledge about these residues and the coassembly of L1 and L2 could help to expand our understanding of HPV biology and aid in the development of countermeasures against a wide range of HPV types by targeting the L2 protein.

Redeveloping antigen detection kits for the diagnosis of rat hepatitis E virus.

The emergence of Rocahepevirus ratti [species HEV ratti (r HEV)] as a causative agent of hepatitis E in humans presents a new potential threat to global public health. The R. ratti genotype 1 (r-1 HEV) variant only shares 50%-60% genomic identity with Paslahepevirus balayani [species HEV balayani (b HEV)] variants, which are the main causes of hepatitis E infection in humans. Here, we report antigen diagnoses for r-1 HEV and b HEV using an enzymatic immunoassay (EIA) method. We detected recombinant virus-like particles protein (HEV 239) of r HEV and b HEV using a collection of hepatitis E virus (HEV)-specific monoclonal antibodies. Two optimal candidates, the capture antibody P#1-H4 and the detection antibodies C145 (P#1-H4*/C145#) and C158 (P#1-H4*/C158#), were selected to detect antigen in infected rat samples and r-1 HEV- or b HEV-infected human clinical samples. The two candidates showed similar diagnostic efficacy to the Wantai HEV antigen kit in b HEV-infected clinical samples. Genomic divergence resulted in low diagnostic efficacy of the Wantai HEV antigen kit (0%, 0 of 10) for detecting r-1 HEV infection. Compared with the P#1-H4*/C145# candidate (80%, 8 of 10), the P#1-H4*/C158# candidate had excellent diagnostic efficacy in r-1 HEV-infected clinical samples (100%, 10 of 10). The two candidates bind to a discrete antigenic site that is highly conserved across r HEV and b HEV. P#1-H4*/C145# and P#1-H4*/C158# are efficacious candidate antibody combinations for rat HEV antigen detection.

Omics-guided bacterial engineering of Escherichia coli ER2566 for recombinant protein expression.

The goal of bacterial engineering is to rewire metabolic pathways to generate high-value molecules for various applications. However, the production of recombinant proteins is constrained by the complexity of the connections between cellular physiology and recombinant protein synthesis. Here, we used a rational and highly efficient approach to improve bacterial engineering. Based on the complete genome and annotation information of the Escherichia coli ER2566 strain, we compared the transcriptomic profiles of the strain under leaky expression and low temperature-induced stress. Combining the gene ontology (GO) enrichment terms and differentially expressed genes (DEGs) with higher expression, we selected and knocked out 36 genes to determine the potential impact of these genes on protein production. Deletion of bluF, cydA, mngR, and udp led to a significant decrease in soluble recombinant protein production. Moreover, at low-temperature induction, 4 DEGs (gntK, flgH, flgK, flgL) were associated with enhanced expression of the recombinant protein. Knocking out several motility-related DEGs (ER2666-ΔflgH-ΔflgL-ΔflgK) simultaneously improved the protein yield by 1.5-fold at 24 °C induction, and the recombinant strain had the potential to be applied in the expression studies of different exogenous proteins, aiming to improve the yields of soluble form to varying degrees in comparison to the ER2566 strain. Totally, this study focused on the anabolic and stress-responsive hub genes of the adaptation of E. coli to recombinant protein overexpression on the transcriptome level and constructs a series of engineering strains increasing the soluble protein yield of recombinant proteins which lays a solid foundation for the engineering of bacterial strains for recombinant technological advances. KEY POINTS: • Comparative transcriptome analysis shows host responses with altered induction stress. • Deletion of bluF, cydA, mngR, and udp genes was identified to significantly decrease the soluble recombinant protein productions. • Synchronal knockout of flagellar genes in E. coli can enhance recombinant protein yield up to ~ 1.5-fold at 24 °C induction. • Non-model bacterial strains can be re-engineered for recombinant protein expression.

Effect of Steam Flash-Explosion on Physicochemical Properties and Structure of High-Temperature Denatured Defatted Rice Bran Protein Isolate.

The effects of Steam Flash-Explosion (SFE) on the physicochemical properties and molecular structure of high-temperature denatured defatted rice bran protein isolate (RBPI) were investigated. The mechanism of SFE treatment on high-temperature denatured defatted RBPI was revealed. The analysis of the physical and chemical properties of RBPI showed that the surface hydrophobicity, characteristic viscosity, and thermal stability of rice bran protein isolate were significantly affected by the pressure of saturated steam and pressure holding time. Under the conditions of 2.1 MPa and 210 s, the surface hydrophobicity index decreased significantly from 137.5 to 17.5, and the characteristic viscosity increased significantly. The peak temperature of denaturation decreases from 114.2 to 106.7 °C, and the enthalpy of denaturation decreases from 356.3 to 231.4 J/g. The higher structure (circular dichroic spectrum and endogenous fluorescence spectrum) of rice bran protein isolate was analyzed by volume rejection chromatography (SEC). The results showed that steam flash treatment could depolymerize and aggregate RBPI, and the relative molecular weight distribution changed greatly. The decrease in small molecules with poor solubility was accompanied by the increase in macromolecules (>550 kDa) soluble aggregates, which were the products of a Maillard reaction. The contents of free sulfhydryl and disulfide bonds in high-temperature rice bran meal protein isolate were significantly increased, which resulted in the increase in soluble aggregates containing disulfide bonds. Circular dichroism (CD) analysis showed that the α-helix content of the isolated protein was significantly decreased, the random curl content was increased, and the secondary structure of the isolated protein changed from order to disorder. The results of endogenous fluorescence spectroscopy showed that the high-temperature rice bran meal protein isolate was more extended, tryptophan was in a more hydrophilic microenvironment, the fluorescence intensity was reduced, and the tertiary structure was changed. In addition, the mean particle size and net surface charge of protein isolate increased in the aqueous solution, which was conducive to the development of the functional properties of the protein.

Influence of Pasteurization on Maillard Reaction in Lactose-Free Milk.

In order to improve the safety and quality of lactose-free milk (LFM) Maillard reaction products (MRPs), this study used raw cow's milk as raw material and lactase hydrolysis to prepare LFM, which was heat-treated using pasteurization and then placed in storage temperatures of 4 °C, 25 °C and 37 °C to investigate the changes in the Maillard reaction (MR). The results of the orthogonal test showed that the optimal conditions for the hydrolysis of LFM are as follows: the hydrolysis temperature was 38 °C, the addition of lactase was 0.03%, and the hydrolysis time was 2.5 h. Under these conditions, the lactose hydrolysis rate reached 97.08%, and the lactose residue was only 0.15 g/100 g as determined by high-performance liquid chromatography (HPLC), complying with the standard of LFM in GB 28050-2011. The contents of furoamic acid and 5-hydroxymethylfurfural were determined by high-performance liquid chromatography, the color difference was determined by CR-400 color difference meter, and the internal fluorescence spectrum was determined by F-320 fluorescence spectrophotometer. The test results showed that the variation range of furosine in lactose-free milk after pasteurization was 44.56~136.45 mg/100g protein, the range of 5-hydroxymethylfurfural (HMF) was 12.51~16.83 mg/kg, the color difference ranges from 88.11 to 102.53 in L*, from -0.83 to -0.10 in a*, and from 1.88 to 5.47 in b*. The furosine content of LFM during storage at 4, 25, and 37 °C ranged from 44.56 to 167.85, 44.56 to 287.13, and 44.56 to 283.72 mg/100 g protein, respectively. The average daily increase in protein content was 1.18-3.93, 6.46-18.73, and 15.7-37.66 mg/100 g, respectively. The variation range of HMF was 12.51~17.61, 12.51~23.38, and 12.51~21.1 mg/kg, and the average daily increase content was 0.03~0.07, 0.47~0.68, and 0.51~0.97 mg/kg, respectively. During storage at 4 °C, the color difference of LFM ranged from 86.82 to 103.82, a* ranged from -1.17 to -0.04, and b* ranged from 1.47 to 5.70. At 25 °C, color difference L* ranges from 72.09 to 102.35, a* ranges from -1.60 to -0.03, b* ranges from 1.27 to 6.13, and at 37 °C, color difference L* ranges from 58.84 to 102.35, a* ranges from -2.65 to 1.66, and b* ranges from 0.54 to 5.99. The maximum fluorescence intensity (FI) of LFM varies from 131.13 to 173.97, 59.46 to 173.97, and 29.83 to 173.97 at 4, 25, and 37 °C. In order to reduce the effect of the Maillard reaction on LFM, it is recommended to pasteurize it at 70 °C-15 s and drink it as soon as possible during the shelf life within 4 °C.

A novel ZsGreen knock-in melanoma cell line reveals the function of CD11b in tumor phagocytosis.

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) is an efficient tool for establishing genetic models including cellular models, and has facilitated unprecedented advancements in biomedical research. In both patients and cancer animal models, immune cells infiltrate the tumor microenvironment and some of them migrate to draining lymph nodes to exert antitumor effects. Among these immune cells, phagocytes such as macrophages and dendritic cells engulf tumor antigens prior to their crosstalk with T cells and elicit adaptive immune response against tumors. Melanoma cells are frequently used as a tumor model because of their relatively high level of somatic mutations and antigenicity. However, few genetic models have been developed using melanoma cell lines to track tumor cell phagocytosis, which is essential for understanding protective immune response in vivo. In this study, we used CRISPR/Cas9-mediated DNA cleavage and homologous recombination to develop a novel knock-in tool which expresses the ultra-bright fluorescent probe ZsGreen in YUMM1.7 melanoma cells. Using this novel tool, we measured the macrophagic engulfment of melanoma cells inside the tumor microenvironment. We also found that in tumor-grafted mice, a subset of dendritic cells efficiently engulfed YUMM1.7 cells and was preferentially trafficking tumor antigens to draining lymph nodes. In addition, we used this knock-in tool to assess the impact of a point mutation of CD11b on phagocytosis in the tumor microenvironment. Our results demonstrate that the ZsGreen-expressing YUMM1.7 melanoma model provides a valuable tool for the study of phagocytosis in vivo.

Structural basis for the shared neutralization mechanism of three classes of human papillomavirus type 58 antibodies with disparate modes of binding.

Human papillomavirus type 58 (HPV58) is associated with cervical cancer and poses a significant health burden worldwide. Although the commercial 9-valent HPV vaccine covers HPV58, the structural and molecular-level neutralization sites of the HPV58 complete virion are not fully understood. Here, we report the high-resolution (∼3.5 Å) structure of the complete HPV58 pseudovirus (PsV58) using cryo-electron microscopy (cryo-EM). Three representative neutralizing monoclonal antibodies (nAbs 5G9, 2H3 and A4B4) were selected through clustering from a nAb panel against HPV58. Bypassing the steric hindrance and symmetry-mismatch in the HPV Fab-capsid immune-complex, we present three different neutralizing epitopes in the PsV58, and show that, despite differences in binding, these nAbs share a common neutralization mechanism. These results offer insight into HPV58 genotype specificity and broaden our understanding of HPV58 neutralization sites for antiviral research.IMPORTANCE Cervical cancer primarily results from persistent infection with high-risk types of human papillomavirus (HPV). HPV type 58 (HPV58) is an important causative agent, especially within Asia. Despite this, we still have limited data pertaining to the structural and neutralizing epitopes of HPV58, and this encumbers our in-depth understanding of the virus mode of infection. Here, we show that representative nAbs (5G9, 10B11, 2H3, 5H2 and A4B4) from three different groups share a common neutralization mechanism that appears to prohibit the virus from associating with the extracellular matrix and cell surface. Furthermore, we identify that the nAbs engage via three different binding patterns: top-center binding (5G9 and 10B11), top-fringe binding (2H3 and 5H2), and fringe binding (A4B4). Our work shows that, despite differences in the pattern in binding, nAbs against HPV58 share a common neutralization mechanism. These results provide new insight into the understanding of HPV58 infection.

Risk of hepatocellular carcinoma in antiviral treatment-naïve chronic hepatitis B patients treated with entecavir or tenofovir disoproxil fumarate: a network meta-analysis.

BACKGROUND: Long-term antiviral treatments are associated with a significantly lower hepatocellular carcinoma (HCC) incidence in chronic hepatitis B (CHB) patients by reducing HBV DNA concentrations. However, it is still controversial whether antiviral strategies affect HCC development in antiviral treatment-naïve CHB patients. This study aimed to estimate the incidence of HCC in antiviral treatment-naïve CHB patients who were treated with Entecavir (ETV) and Tenofovir Disoproxil Fumarate (TDF) and compare the efficacy of two treatment regimens in HCC reduction. METHODS: The PubMed, Embase, China National Knowledge Infrastructure, and Wanfang databases were systematically searched until June 24, 2021. The pooled incidence and 95% confidence interval of HCC were calculated by the Freeman-Tukey double arcsine transformation method. The efficacies of ETV and TDF treatments in HCC reduction were compared through a network meta-analysis. RESULTS: A total of 27 studies were identified as eligible for this systematic review. The incidence densities in the ETV and TDF treatment groups were 2.78 (95% CI: 2.21-3.40) and 2.59 (95% CI: 1.51-3.96) per 100 persons-year among patients with preexisting cirrhosis and 0.49 (95% CI: 0.32-0.68) and 0.30 (95% CI: 0.06-0.70) per 100 persons-year among patients without preexisting cirrhosis. As the proportion of CHB patients with preexisting cirrhosis increased, the incidence density of HCC also increased gradually. Compared with other Nucleos(t)ide analogs (NAs) treatments, ETV and TDF treatments significantly lowered the risk of HCC, with hazard ratios (HRs) of 0.60 (95% CI: 0.40-0.90) and 0.56 (95% CI: 0.35-0.89), respectively. However, there was no difference in the incidence density of HCC between ETV and TDF treatments (HR = 0.92, 95% CI: 0.71-1.20) regardless of preexisting cirrhosis. CONCLUSION: ETV and TDF treatments were associated with significantly lower risks of HCC than other NAs treatments. However, no difference was observed between ETV and TDF treatments in the risk of HCC development regardless of preexisting cirrhosis among treatment-naïve CHB patients.

Whole blood GBP5 protein levels in patients with and without active tuberculosis.

BACKGROUND: The host blood transcriptional levels of several genes, such as guanylate binding protein 5 (GBP5), have been reported as potential biomarkers for active tuberculosis (aTB) diagnosis. The aim of this study was to investigate whole blood GBP5 protein levels in aTB and non-tuberculosis patients. METHODS: An in-house immunoassay for testing GBP5 protein levels in whole blood was developed, and suspected aTB patients were recruited. Whole blood samples were collected and tested at enrolment using interferon-gamma release assay (IGRA) and the GBP5 assay. RESULTS: A total of 470 participants were enrolled, and 232 and 238 patients were finally diagnosed with aTB and non-TB, respectively. The GBP5 protein levels of aTB patients were significantly higher than those of non-tuberculosis patients (p < 0.001), and the area under the ROC curve of the GBP5 assay for aTB diagnosis was 0.76. The reactivity of the GBP5 assay between pulmonary and extrapulmonary tuberculosis patients was comparable (p = 0.661). With the optimal cut-off value, the sensitivity and specificity of the GBP5 assay for diagnosing aTB were 78.02 and 66.81%, respectively, while those of IGRA were 77.59 and 76.47%. The combination of the GBP5 assay and IGRA results in 88.52% accuracy for diagnosing aTB in 63.83% of suspected patients with a positive predictive value of 89.57% and a negative predictive value of 87.59%. CONCLUSIONS: Whole blood GBP5 protein is a valuable biomarker for diagnosing of aTB. This study provides an important idea for realizing the clinical application of whole blood transcriptomics findings by immunological methods.

Coadministration of Curcumin and Hydromorphone Hydrochloride Alleviates Postoperative Pain in Rats.

This study aimed to explore the effect of curcumin and hydromorphone hydrochloride (HH) cotreatment on postoperative pain in rats. An incision + formaldehyde-induced pain rat model was established. Rats were treated with vehicle, curcumin, HH, or curcumin + HH. Paw mechanical withdrawal threshold and thermal withdrawal latency were measured at 1 d before surgery as well as 1 , 2 h, 1 , 3 , and 7 d after surgery to assess pain sensitivity. The L4-6 region of the spinal cord was collected from each rat at 2 h, 1 , 3 , and 7 d after surgery. Western blot analysis and immunohistochemical staining were carried out to detect the protein expression of pain-related genes. Quantitative real-time PCR and enzyme-linked immunosorbent assay were conducted to measure the expression and production of proinflammatory mediators. Compared with other groups, Curcumin + HH significantly reduced pain sensitivity in the model rats. Mechanistically, curcumin + HH suppressed protein expression of stromal cell-derived factor-1 (SDF-1), CXC chemokine receptor 4 (CXCR4), p-Akt, and c-fos while enhancing protein expression of nerve growth factor (NGF) in the dorsal root ganglia (DRG) of model rats. Curcumin + HH inhibited the expression and production of interleukin 1ß (IL-1ß), cyclooxygenase-2 (COX-2), tumor necrosis factor α (TNF-α), and p65 nuclear factor kappa B (NF-κB) in the DRG. Coadministration of curcumin and HH alleviates incision + formaldehyde-induced pain in rats, possibly by suppressing the SDF-1/CXCR4 pathway and the production of proinflammatory mediators. Our results provide curcumin and HH cotreatment as a promising therapeutic strategy in the management of postoperative pain.

Viral neutralization by antibody-imposed physical disruption.

In adaptive immunity, organisms produce neutralizing antibodies (nAbs) to eliminate invading pathogens. Here, we explored whether viral neutralization could be attained through the physical disruption of a virus upon nAb binding. We report the neutralization mechanism of a potent nAb 8C11 against the hepatitis E virus (HEV), a nonenveloped positive-sense single-stranded RNA virus associated with abundant acute hepatitis. The 8C11 binding flanks the protrusion spike of the HEV viruslike particles (VLPs) and leads to tremendous physical collision between the antibody and the capsid, dissociating the VLPs into homodimer species within 2 h. Cryo-electron microscopy reconstruction of the dissociation intermediates at an earlier (15-min) stage revealed smeared protrusion spikes and a loss of icosahedral symmetry with the capsid core remaining unchanged. This structural disruption leads to the presence of only a few native HEV virions in the ultracentrifugation pellet and exposes the viral genome. Conceptually, we propose a strategy to raise collision-inducing nAbs against single spike moieties that feature in the context of the entire pathogen at positions where the neighboring space cannot afford to accommodate an antibody. This rationale may facilitate unique vaccine development and antimicrobial antibody design.

Anterior segment structures in dark iris Chinese patients with unilateral Fuchs' uveitis syndrome.

PURPOSE: To compare binocular anterior segment structures in Chinese patients with dark iris and unilateral Fuchs' uveitis syndrome (FUS). METHODS: This was a cross-sectional study including 34 phakic eyes (17 patients) with unilateral FUS. Anterior segment parameters were measured by rotating Scheimpflug imaging camera, noncontact specular microscopy, and anterior segment optical coherence tomography. RESULTS: Corneal volume was higher in FUS eyes compared to unaffected eyes (p < 0.05). The iridocorneal angles were larger in FUS eyes compared to contralateral eyes (p < 0.05). Mean endothelial cell density (ECD) was lower, and the coefficient of variation in endothelial cell size and average cell area of endothelial cells (ACA) were higher, in FUS eyes (p < 0.05). Mean densitometry values of the midstromal cornea (zones with a diameter of 0-2, 2-6, or 10-12 mm), posterior (0-2, 2-6, 10-12, or 0-12 mm), or total thickness (0-2 or 2-6 mm) were higher in FUS eyes compared with unaffected eyes (p < 0.05). ECD, percentage of hexagonal cells, and ACA were strongly related to densitometry values of the midstromal and posterior cornea in the FUS eyes (p < 0.05). Smoothness index of iris was lager in affected eyes (p < 0.05). CONCLUSION: In Chinese patients with unilateral FUS, loss of endothelial cells, wider iridocorneal angle, thicker cornea, higher corneal densitometry of midstromal and posterior layer, and smoother iris were observed in affected eyes compared to contralateral eyes. These data can help to elucidate anterior segment characteristics of unilateral FUS in this population.

Engineering for an HPV 9-valent vaccine candidate using genomic constitutive over-expression and low lipopolysaccharide levels in Escherichia coli cells.

BACKGROUND: The various advantages associated with the growth properties of Escherichia coli have justified their use in the production of genetically engineered vaccines. However, endotoxin contamination, plasmid vector instability, and the requirement for antibiotic supplementation are frequent bottlenecks in the successful production of recombinant proteins that are safe for industrial-scaled applications. To overcome these drawbacks, we focused on interrupting the expression of several key genes involved in the synthesis of lipopolysaccharide (LPS), an endotoxin frequently responsible for toxicity in recombinant proteins, to eliminate endotoxin contamination and produce better recombinant proteins with E. coli. RESULTS: Of 8 potential target genes associated with LPS synthesis, we successfully constructed 7 LPS biosynthesis-defective recombinant strains to reduce the production of LPS. The endotoxin residue in the protein products from these modified E. coli strains were about two orders of magnitude lower than that produced by the wild-type strain. Further, we found that 6 loci-lpxM, lpxP, lpxL, eptA, gutQ and kdsD-were suitable for chromosomal integrated expression of HPV L1 protein. We found that a single copy of the expression cassette conferred stable expression during long-term antibiotic-free cultivation as compared with the more variable protein production from plasmid-based expression. In large-scale fermentation, we found that recombinant strains bearing 3 to 5 copies of the expression cassette had 1.5- to 2-fold higher overall expression along with lower endotoxin levels as compared with the parental ER2566 strain. Finally, we engineered and constructed 9 recombinant E. coli strains for the later production of an HPV 9-valent capsid protein with desirable purity, VLP morphology, and antigenicity. CONCLUSIONS: Reengineering the LPS synthesis loci in the E. coli ER2566 strain through chromosomal integration of expression cassettes has potential uses for the production of a 9-valent HPV vaccine candidate, with markedly reduced residual endotoxin levels. Our results offer a new strategy for recombinant E. coli strain construction, engineering, and the development of suitable recombinant protein drugs.

Exploring the effect of virtual reality relaxation environment on white coat hypertension in blood pressure measurement.

A phenomenon called White Coat Hypertension (WCH) often occurs when measuring blood pressure (BP) in a real medical environment. Utilizing virtual reality (VR) technology to present appropriate relaxation scenes can isolate the real medical environments and may provide a new method to avoid WCH and improve the accuracy of BP measurement. In this study, we designed four immersive VR relaxation scenes and conducted an experiment to explore the role of VR scenes in eliminating/detecting WCH in BP measurement. Results from the current sample showed that both systolic BP and diastolic BP measured in the simulated medical environment were significantly higher than the baseline level and the VR scene condition, while there were no significant differences between the BPs measured in VR scenes and the baseline level. It can be concluded that VR provides an effective approach to avoid WCH in BP measurement by visually and aurally isolating the real environment and assisting relaxation and provides a new approach to detect the occurrence of WCH by the comparison of BPs measured in the VR scene condition and real medical environments.

Quantitative analysis of retinal vessel density and thickness changes in diabetes mellitus evaluated using optical coherence tomography angiography: a cross-sectional study.

BACKGROUND: Diabetic retinopathy is the most common microvascular complication of diabetes; however, early changes in retinal microvessels are difficult to detect clinically, and a patient's vision may have begun to deteriorate by the time a problem is identified. Optical coherence tomography angiography (OCTA) is an innovative tool for observing capillaries in vivo. The aim of this study was to analyze retinal vessel density and thickness changes in patients with diabetes. METHODS: This was a retrospective, observational cross-sectional study. Between August 2018 and February 2019, we collected OCTA data from healthy participants and diabetics from the First Affiliated Hospital of Harbin Medical University. Analyzed their retinal vessel density and thickness changes. RESULTS: A total of 97 diabetic patients with diabetes at different severity stages of diabetic retinopathy and 85 controls were involved in the experiment. Diabetic patients exhibited significantly lower retinal VD (particularly in the deep vascular complexes), thickening of the neurosensory retina, and thinning of the retinal pigment epithelium compared with controls. In the control group, nondiabetic retinopathy group and mild diabetic retinopathy group, superficial VD was significantly correlated with retinal thickness (r = 0.3886, P < 0.0001; r = 0.3276, P = 0.0019; r = 0.4614, P = 0.0024, respectively). CONCLUSIONS: Patients with diabetes exhibit ischemia of the retinal capillaries and morphologic changes in vivo prior to vision loss. Therefore, OCTA may be useful as a quantitative method for the early detection of diabetic retinopathy.

A unique B cell epitope-based particulate vaccine shows effective suppression of hepatitis B surface antigen in mice.

OBJECTIVE: This study aimed to develop a novel therapeutic vaccine based on a unique B cell epitope and investigate its therapeutic potential against chronic hepatitis B (CHB) in animal models. METHODS: A series of peptides and carrier proteins were evaluated in HBV-tolerant mice to obtain an optimised therapeutic molecule. The immunogenicity, therapeutic efficacy and mechanism of the candidate were investigated systematically. RESULTS: Among the HBsAg-aa119-125-containing peptides evaluated in this study, HBsAg-aa113-135 (SEQ13) exhibited the most striking therapeutic effects. A novel immunoenhanced virus-like particle carrier (CR-T3) derived from the roundleaf bat HBV core antigen (RBHBcAg) was created and used to display SEQ13, forming candidate molecule CR-T3-SEQ13. Multiple copies of SEQ13 displayed on the surface of this particulate antigen promote the induction of a potent anti-HBs antibody response in mice, rabbits and cynomolgus monkeys. Sera and purified polyclonal IgG from the immunised animals neutralised HBV infection in vitro and mediated efficient HBV/hepatitis B virus surface antigen (HBsAg) clearance in the mice. CR-T3-SEQ13-based vaccination induced long-term suppression of HBsAg and HBV DNA in HBV transgenic mice and eradicated the virus completely in hydrodynamic-based HBV carrier mice. The suppressive effects on HBsAg were strongly correlated with the anti-HBs level after vaccination, suggesting that the main mechanism of CR-T3-SEQ13 vaccination therapy was the induction of a SEQ13-specific antibody response that mediated HBV/HBsAg clearance. CONCLUSIONS: The novel particulate protein CR-T3-SEQ13 suppressed HBsAg effectively through induction of a humoural immune response in HBV-tolerant mice. This B cell epitope-based therapeutic vaccine may provide a novel immunotherapeutic agent against chronic HBV infection in humans.

Mesenchymal stem cell-secreted extracellular vesicles carrying TGF-ß1 up-regulate miR-132 and promote mouse M2 macrophage polarization.

The effects of mesenchymal stem cells (MSCs) on different types of diseases are controversial, and the inner mechanisms remain unknown, which retards the utilization of MSCs in disease therapy. In this study, we aimed to elucidate the mechanisms of MSCs-extracellular vesicles (EVs) carrying transforming growth factor-beta 1 (TGF-ß1) in M2 polarization in mouse macrophages via the microRNA-132 (miR-132)/E3 ubiquitin ligase myc binding protein 2 (Mycbp2)/tuberous sclerosis complex 2 (TSC2) axis. Mouse MSCs were isolated for adipogenic and osteogenic induction, followed by co-culture with mouse macrophages RAW264.7. Besides, mouse macrophages RAW264.7 were co-cultured with MSCs-EVs in vitro, where the proportion of macrophages and inflammation were detected by flow cytometry and ELISA. The experimental data revealed that MSCs-EVs promoted M2 polarization of macrophages, and elevated interleukin (IL)-10 expression and inhibited levels of IL-1ß, tumour necrosis factor (TNF)-α and IL-6. MSC-EV-treated macrophages RAW264.7 increased TGF-ß1 expression, thus elevating miR-132 expression. MiR-132 directly bound to Mycbp2, as confirmed by luciferase activity assay. Meanwhile, E3 ubiquitin ligase Mycbp2 could ubiquitinate TSC2 protein. Furthermore, silencing TGF-ß1 inhibited M2 polarization of MSC-EV-treated macrophages. Taken conjointly, this study provides evidence reporting that MSC-secreted EVs carry TGF-ß1 to promote M2 polarization of macrophages via modulation of the miR-132/Mycbp2/TSC2 axis.