Routine evaluation of HBV-specific T cell reactivity in chronic hepatitis B using a broad-spectrum T-cell epitope peptide library and ELISpot assay.

BACKGROUND: The clinical routine test of HBV-specific T cell reactivity is still limited due to the high polymorphisms of human leukocyte antigens (HLA) in patient cohort and the lack of universal detection kit, thus the clinical implication remains disputed. METHODS: A broad-spectrum peptide library, which consists of 103 functionally validated CD8+ T-cell epitopes spanning overall HBsAg, HBeAg, HBx and HBpol proteins and fits to the HLA polymorphisms of Chinese and Northeast Asian populations, was grouped into eight peptide pools and was used to establish an ELISpot assay for enumerating the reactive HBV-specific T cells in PBMCs. Totally 294 HBV-infected patients including 203 ones with chronic hepatitis B (CHB), 13 ones in acute resolved stage (R), 52 ones with liver cirrhosis (LC) and 26 ones with hepatocellular carcinoma (HCC) were detected, and 33 CHB patients were longitudinally monitored for 3 times with an interval of 3-5 months. RESULTS: The numbers of reactive HBV-specific T cells were significantly correlated with ALT level, HBsAg level, and disease stage (R, CHB, LC and HCC), and R patients displayed the strongest HBV-specific T cell reactivity while CHB patients showed the weakest one. For 203 CHB patients, the numbers of reactive HBV-specific T cells presented a significantly declined trend when the serum viral DNA load, HBsAg, HBeAg or ALT level gradually increased, but only a very low negative correlation coefficient was defined (r = - 0.21, - 0.21, - 0.27, - 0.079, respectively). Different Nucleotide Analogs (NUCs) did not bring difference on HBV-specific T cell reactivity in the same duration of treatment. NUCs/pegIFN-α combination led to much more reactive HBV-specific T cells than NUCs monotherapy. The dynamic numbers of reactive HBV-specific T cells were obviously increasing in most CHB patients undergoing routine treatment, and the longitudinal trend possess a high predictive power for the hepatitis progression 6 or 12 months later. CONCLUSION: The presented method could be developed into an efficient reference method for the clinical evaluation of cellular immunity. The CHB patients presenting low reactivity of HBV-specific T cells have a worse prognosis for hepatitis progression and should be treated using pegIFN-α to improve host T-cell immunity.

Bioinformatics analysis of GPS1 expression and biological function in breast cancer.

G protein pathway suppressor 1 (GPS1) is involved in the development of many diseases including tumors, but its specific regulatory mechanism in breast cancer is not clear. The goal of the present study was to explore the biological effects and underlying mechanism of GPS1 in breast cancer. Public databases were used to analyze GPS1 expression and the relationship with clinicopathological characteristics and prognosis of breast cancer patients, combined with in vitro experiments to analyze the mechanism of action and immune relevance of GPS1 in breast cancer. Data analysis showed that the expression of GPS1 in breast cancer tissues was significantly higher than that in paracancerous tissues (p < 0.001), and the receiver operating curve (ROC) revealed a higher diagnostic efficiency (AUC = 0.832). Survival analyses indicated that patients with high GPS1 expression made the prognosis worse in Luminal B, low to intermediate-grade breast cancers. Enrichment analysis showed that GPS1 was involved in the formation of ribonucleoprotein complexes, which dynamically altered the fate of RNA; it could also enhance the responsiveness of the Wnt pathway by interacting with WBP2. In addition, GPS1 expression was closely related to the immune microenvironment. GPS1 knockdown inhibits the proliferation, invasion and migration of MCF7 and MDA-MB-231 cells in vitro. This study suggests that the upregulation of GPS1 is associated with the malignant biological behavior and prognosis of breast cancer and may promote cancer progression. The correlation between GPS1 and the immune microenvironment suggests that it may be a potential target for immunotherapy.

[Determination of four fatty acid ethyl esters in olive oil by solid phase extraction-gas chromatography].

The fatty acid ethyl ester (FAEE) content of olive oil is an important indicator of its quality. At present, the international standard method used to detect FAEEs in olive oil is silica gel (Si) column chromatography-gas chromatography (GC); however, this technique presents a number of disadvantages, including complex operation, long analysis times, and high reagent consumption. In this study, a method based on Si solid phase extraction (SPE)-GC was established to determine four FAEEs in olive oil, namely, ethyl palmitate, ethyl linoleate, ethyl oleate, and ethyl stearate. First, the effects of the carrier gas were investigated, and He gas was ultimately selected as the carrier gas. Next, several internal standards were screened, and ethyl heptadecenoate (cis-10) was determined as the optimal internal standard. The SPE conditions were also optimized, and the effects of different brands of Si SPE columns on the recoveries of analytes were compared. Finally, a pretreatment method in which 0.05 g of olive oil was extracted with n-hexane and purified through a Si SPE column (1 g/6 mL) was developed. A sample could be processed within approximately 2 h using a total reagent volume of about 23 mL. Validation of the optimized method revealed that the four FAEEs have good linearities within the range of 0.1-5.0 mg/L (coefficients of determination (R2)>0.999). The limits of detection (LODs) of the method were within 0.78-1.11 mg/kg, and its limits of quantification (LOQs) were in the range of 2.35-3.33 mg/kg. The recoveries ranged from 93.8% to 104.0% at all spiked levels tested (4, 8, and 20 mg/kg), and the relative standard deviations were 2.2%-7.6%. Fifteen olive oil samples were tested using the established method, and the total FAEEs of three extra-virgin olive oil samples were found to exceed 35 mg/kg. Compared with the international standard method, the proposed method has the advantages of simpler pretreatment process, shorter operation time, lower reagent consumption and detection cost, high precision, and good accuracy. The findings provide an effective theoretical and practical reference for improving olive oil detection standards.

Screening of Efficient Adjuvants for the RBD-Based Subunit Vaccine of SARS-CoV-2.

The variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are more transmissible, with a reduced sensitivity to vaccines targeting the original virus strain. Therefore, developing an effective vaccine against both the original SARS-CoV-2 strain and its variants is an urgent need. It is known that the receptor-binding domain (RBD) in the S protein of SARS-CoV-2 is an important vaccine target, but subunit vaccines usually have lower immunogenicity and efficacy. Thus, selecting appropriate adjuvants to enhance the immunogenicity of protein-based subunit vaccine antigens is necessary. Here, an RBD-Fc subunit vaccine of SARS-CoV-2 has been generated, followed by vaccination in B6 mice, and four adjuvant regimens were investigated, including aluminum salts (Alum) + 3-O-desacyl-4'-monophosphoryl lipid A (MPL), AddaVax, QS21 + MPL, and Imiquimod. The adjuvant potency was evaluated by comparing the elicited polyclonal antibodies titers with measuring binding to RBD and S protein in ELISA and Western blot analysis, and also the cross-neutralizing antibodies titers using a pseudovirus infection assay of hACE2-expressing 293T cells, with pseudoviruses expressing the S protein of the SARS-CoV-2 original strain and Delta strain. The presence of QS21 + MPL adjuvant induced stronger polyclonal antibody response and neutralization potency blocking the original strain and Delta strain, as compared with the non-adjuvant RBD-Fc group and other adjuvant groups. Meanwhile, Imiquimod even had a negative effect in inducing specific antibodies and cross-neutralizing antibody production as an adjuvant.

A Non-Hydrolysis Reaction-Based Imine for Fluorescence Response toward Al3+ Ions with Extremely High Selectivity.

Designing an imine-based fluorescent probe capable of greatly suppressing the tendency of intrinsic hydrolysis reaction is an attractive topic in the field of chemo-/biosensing. In this work, hydrophobic 1,1'-binaphthyl-2,2'-diamine containing two amine groups was introduced to synthesize probe R-1 bearing two imine bonds linked by two salicylaldehyde (SAs). The hydrophobicity of binaphthyl moiety and the unique clamp-like structure formed from double imine bonds and from ortho-OH on SA part make probe R-1 is able to function as an ideal receptor to coordinate with Al3+ ions, leading to the fluorescence originated from the complex rather than from the assumed hydrolyzed fluorescent amine is turned on. Further study revealed that, when Al3+ ions were introduced, both the hydrophobic binaphthyl moiety and the clamp-like double imine structure in the designed imine-based probe showed important contributions to suppress the intrinsic hydrolysis reaction, resulting in generating a stable coordination complex with an extremely high selectivity in fluorescence response.

Modulating Dual Fluorescence Emissions in Imine-Based Probes to Distinguish D2O and H2O.

How to distinguish D2O and H2O and determine the trace H2O content in D2O solvent, by using molecule-based spectral probes, is an intriguing topic in analytical chemistry, yet considerably few examples remain up to now, likely due to the very similar physical/chemical properties between D2O and H2O. In this work, we found that both the hydrolysis reactions to release fluorescent amines and aggregation-induced emission (AIE) of imines, functioning as dual fluorescence signals to distinguish D2O and H2O, could be modulated by changing the imine structures. The hydrophobicity of imines showed an important contribution to the ability of modulating the hydrolysis reactions and AIE, demonstrating a significant difference on fluorescence signals in D2O and H2O solvents. Among all tested imines, probe 3, condensed from 2-naphthylamine and salicylaldehyde, was found to have the potential ability to act as an ideal candidate for probing the H2O content in D2O solvent, particularly in a low H2O content range, using the ratiomeric emission signals.

Enantioselective Dynamic Exchange Reactions of Imines.

Although dynamic reactions of imines have been extensively studied, the dynamic behaviors manipulated by chirality remain nearly unexplored. In this work, enantioselective amine exchange reactions were demonstrated as a first example via the reaction of enantiomeric chiral amines such as natural amino acids with a series of innovative axially chiral 1,1'-binaphthyl-2,2'-diamine (BNDA)-based imines that were prepared from the condensation reactions between BNDA and salicylaldehyde (SA) or its derivatives. This enantioselective dynamic behavior can be directly indicated by the degree of the fluorescence response of the R-configuration of imines to the d-enantiomer of chiral amine, because the released BNDA can serve as the fluorescence signal output when the amine exchange reaction occurs, which is far higher than the response to its l-enantiomer under identical experimental conditions. For the S-configuration of chiral imines, the fluorescence response is the opposite. The enantioselective exchange reaction can be tuned by altering the electron-withdrawing or electron-donating capability of the substituent at position 4 or 5 of the SA part of chiral imines. Not only o-OH groups in SA-based imines but also protic solvents used as reaction media were found to be important to the dynamic behavior at high rates.

Self-Assembled Multivalent Ag-SR Coordination Polymers with Phosphatase-Like Activity.

We show herein the phosphatase-like catalytic activity of coordination polymers obtained after adding Ag+ -ions to thiols bearing hydrophobic alkyl chains terminated with a 1,4,7-triazacyclononane (TACN) group. The subsequent addition of Zn2+ -ions to the self-assembled polymers resulted in the formation of multivalent metal coordination polymers capable of catalysing the transphosphorylation of an RNA-model compound (2-hydroxypropyl-4-nitrophenyl phosphate, HPNPP) with high reactivity. Analysis of a series of metal ions showed that the highest catalytic activity was obtained when Ag+ -ions were used as the first metal ions to construct the backbone of the coordination polymer through interaction with the -SH group followed by Zn2+ -ions as the second metal ions complexed by the TACN-macrocycle. Furthermore, it was demonstrated that the catalytic activity could be modulated by changing the length of the hydrophobic alkyl chain.

Purification, characterization and immunostimulatory effects of polysaccharides from Anemarrhena asphodeloides rhizomes.

The crude polysaccharide was extracted from A. asphodeloides rhizomes and further purified to produce two fractions F1 (50.0%) and F2 (19.6%). The chemical constitutions of the polysaccharides were neutral sugars (51.4%-89.7%), uronic acids (1.0%-30.2%) and sulfate esters (3.4%-8.1%), with various ratios of monosaccharides including rhamnose (1.4%-6.1%), arabinose (7.1%-21.2%), xylose (0.2%-4.8%), mannose (39.9%-79.0%), glucose (6.0%-11.1%) and galactose (2.6%-22.0%). The molecular properties of the polysaccharides were investigated by the HPSEC-UV-MALLS-RI system, revealing the Mw 130.0 × 103-576.5 × 103 g/moL, Rg 87.6-382.6 nm and SVg 0.3-54.3 cm3/g. The polysaccharides stimulated RAW264.7 cells to produce considerable amounts of NO and up-regulate the expression of TNF-α, IL-1 and COX-2 genes. Polysaccharides exhibited the growth inhibitory effects on cancer cells lines of AGS, MKN-28 and MKN-45, in which F2 fraction exhibited prominent bioactivities. The AGS cells treated with F2 experienced condensed cytoplasm, shrinkage of nucleus and chromatin marginalization with the highest number of cells at early-stage apoptosis reaching 54.6%. The inhibitory effect of F2 polysaccharide on AGS cells was through MAPKs and STAT3 signaling pathways. The backbone of the F2 was mainly linked by (1 â†’ 4)-linked mannopyranosyl and (1 â†’ 3)-linked galactopyranosyl. Taken together, the polysaccharide from A. asphodeloides rhizomes could be utilized as medicinal, pharmacological and functional food ingredients.

Extraction, structural elucidation and immunostimulating properties of water-soluble polysaccharides from wheat bran.

A water-soluble polysaccharide was extracted from wheat bran (WBP) and investigate their structural characteristics and immunostimulatory activities. The chemical composition of WBP and purified fraction (WBP-F) mainly consists of neutral sugars (91.2 ± 1.2 and 98.7 ± 1.2%), proteins (8.6 ± 0.3 and 0.2 ± 0.1%) and uronic acids (0.7 ± 0.1 and 0.6 ± 0.1%). The molecular weight (Mw ) of WBP and WBP-F was calculated as 911.7 and 510.2 × 103  g/mol, respectively. The WBP-F stimulates the RAW264.7 cells through the production of nitric oxide and various cytokines. The treatment of WBP-F facilitated the phosphorylation of P38, JNK, ERK, and NF-ƘB in RAW264.7 cells suggesting that they might stimulate RAW264.7 cells through the activation of NF-ƙB and MAPKs pathways. Furthermore, the structural details of WBP-F were studied by GC-MS and NMR spectrum, which confirms that the main backbone consists of 4-α-D-linked glucopyranosyl residues with branching points at C-6. PRACTICAL APPLICATIONS: Wheat bran is a potential source of health-promoting compounds. It has been reported that polysaccharides of wheat bran containing numerous beneficial activities. In this study, the wheat bran polysaccharide was extracted, fractionated and investigated their immunostimulatory activities. The results found in this study revealed that the purified polysaccharide from wheat bran potentially enhanced the RAW264.7 cells activation. Hence, these polysaccharides could be utilized as a potent immunity-enhancing agent in food and pharmaceutical industries.

A pyrazolo[1,5-a]pyridine-based ratiometric fluorescent probe for sensing Cu2+ in cell.

Ratiometric fluorescent probes based on FRET mechanism have attracted great attention due to their large pseudo-Stokes shifts and built-in correction for environmental effects. However, most donors failed to meet the requirement that the emission of the donor must overlap well with the absorption of the acceptor. Therefore, searching for new fluorophore to construct FRET system is in great need. In this paper, a new fluorescent dye pyrazolo[1,5-a]pyridine was synthesized and used as a donor in the FRET system for ratiometric sensing of Cu2+. The probe is based on FRET and PET mechanism. It shows high selectivity and sensitivity toward Cu2+ (detection limit 30 nM). Furthermore, it was successfully used to detect Cu2+ in Glioma cells.

A FRET-based ratiometric fluorescent probe for detection of intrinsically generated SO2 derivatives in mitochondria.

A mitochondria-targeted ratiometric fluorescent probe based on hemicyanine and pyrido[1,2-a]benzimidazole was presented. It shows high sensitivity and selectivity toward SO2 in pure water. The limit of detection (LOD) was as low as 26.7 nM, which is superior to most reported probes. Most importantly, the probe was successfully used for fluorescence imaging of endogenous bisulfite in mitochondria in Glioma cells.

A mitochondria-targeted and FRET-based ratiometric fluorescent probe for detection of SO2 derivatives in water.

A mitochondria-targeted ratiometric fluorescent probe based on the fluorophore pyrido[1,2-a]benzimidazole was developed. It showed high sensitivity and selectivity toward SO2 derivatives in water. The probe was also successfully applied to detect intrinsically generated SO2 derivatives in living glioma cells, which may be helpful in ascertaining the biological roles of SO2 in mitochondria.

A FRET based ratiometric fluorescent probe for detection of sulfite in food.

A new fluorophore pyrido[1,2-a]benzimidazole based ratiometric fluorescent probe for the selective detection of sulfite ions in water was investigated. It shows large (pseudo) Stokes shifts (260 nm), high FRET efficiency, high selectivity and sensitivity. A distinct color change from red to colorless was observed and importantly, it proves to be a convenient and efficient tool to detect the sulfite levels in sugar samples.

A ratiometric fluorescent probe for detection of exogenous mitochondrial SO2 based on a FRET mechanism.

A novel imidazo[1,5-a]pyridine-hemicyanine based ratiometric fluorescent probe for detection of mitochondrial SO2 was designed and synthesized. The probe is based on a fluorescence resonance energy transfer (FRET) mechanism. It exhibits high selectivity and sensitivity towards SO3 2- with a fast response time (3 min) and detection limit of 0.13 µM. Further, it showed low cytotoxicity and was successfully applied to image exogenous mitochondrial SO2 in cells.

FRET-based ratiometric fluorescent probe for Cu2+ with a new indolizine fluorophore.

A new fluorophore indolizine was synthesized and used as a donor to construct FRET system. The chemodosimeter shows a large Stokes shifts (193 nm), high sensitivity, and high selectivity. The probe can quickly (10 min) detect Cu2+ effectively in a wide pH range from 5 to 12 and with a detection limit as low as 9.1 × 10-9 M. Furthermore, it could be used for imaging Cu2+ in living Glioma cells which demonstrated its value of practical applications in biological systems.

A new pyrido[1,2-a]benzimidazole-rhodamine FRET system as an efficient ratiometric fluorescent probe for Cu2+ in living cells.

A novel fluorophore pyrido[1,2-a]benzimidazole was synthesized and used as a donor to construct FRET system. The probe shows a large Stokes shifts (190 nm), high selectivity, fast response (within 2 min), and high sensitivity toward Cu2+ (detection limit 42 nM). A significant colour change from colourless to pink could be observed, enabling naked-eye detection of Cu2+. Furthermore, it could be used for imaging Cu2+ in living cells which demonstrated its value of practical applications in biological systems.