ACS Omega
·
2022
·
IF 4.132
Xianjing Zhang,Jun Guo,Bo Song,Feng Zhang
The polymerase chain reaction (PCR) has been widely used in medical diagnosis and forensic identification due to its ultrahigh sensitivity and signal amplification. Metal ions (i.e., Cu2+, Zn2+) have been considered PCR inhibitors and rarely shown their positive roles in PCR amplification until our report, in which we discovered that metal ions can significantly improve the PCR specificity and the yield of target DNA sequences. For an in-depth investigation with taking copper ions as a typical model, here we found an interesting spatiotemporal regulation mechanism of metal ions in PCR. The ionic concentration window for improving PCR specificity not only was independent of annealing temperature but also can be well regulated by both the annealing time and extension time. Using the ionic concentration window as a measure, the time affects either the amount or the sequence length of nonspecific amplicons in the space. The mechanism proposed in this work will deepen our understanding of the unneglectable roles of metal ions in DNA replication and meanwhile provide a new strategy for designing regulation kits for PCR-based biomedical applications.
研究领域:
分子生物学
·
生物化学
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DOI
PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2022
·
IF 5.895
Yawen Jiang,Xiaoxiao Li,Tjaard Pijning,Yuxiang Bai,Lubbert Dijkhuizen
Limosilactobacillus reuteri 121 4,6-α-glucanotransferase (Lr121 4,6-α-GTase), belonging to the glycosyl hydrolase (GH) 70 GtfB subfamily, converts starch and maltodextrins into linear isomalto/malto polysaccharides (IMMPs) with consecutive (α1 → 6) linkages. The recent elucidation of its crystal structure allowed identification and analysis of further structural features that determine its reaction and product specificity. Herein, sequence alignments between GtfB enzymes with different product linkage specificities (4,6-α-GTase and 4,3-α-GTase) identified amino acid residues in GH70 homology motifs, which may be critical for reaction and product specificity. Based on these alignments, four Lr121 GtfB-ΔN mutants (I1020M, S1057P, H1056G, and Q1126I) were constructed. Compared to wild-type Lr121 GtfB-ΔN, mutants S1057P and Q1126I had considerably improved catalytic efficiencies. Mutants H1056G and Q1126I showed a 9% decrease and an 11% increase, respectively, in the ratio of (α1 → 6) over (α1 → 4) linkages in maltodextrin-derived products. A change in linkage type (e.g., (α1 → 6) linkages to (α1 → 3) linkages) was not observed. The possible functional roles of these Lr121 GtfB-ΔN residues located around the acceptor substrate-binding subsites are discussed. The results provide new insights into structural determinants of the reaction and product specificity of Lr121 GtfB 4,6-α-GTase.
研究领域:
酶学
·
分子生物学
·
微生物学
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DOI
PubMed
高影响因子
CELL RESEARCH
·
2025
·
IF 25.9
Qin Rui,Zhang Yong,Shi Jiawei,Wu Peng,An Chenyi,Li Zhenhai,Liu Nuo,Wan Ziyan,Hua Ting,Li Xiaolong,Lou Jizhong,Yin Weiwei,Chen Wei
Naturally evolved T-cell receptors (TCRs) exhibit remarkably high specificity in discriminating non-self antigens from self-antigens under dynamic biomechanical modulation. In contrast, engineered high-affinity TCRs often lose this specificity, leading to cross-reactivity with self-antigens and off-target toxicity. The underlying mechanism for this difference remains unclear. Our study reveals that natural TCRs exploit mechanical force to form optimal catch bonds with their cognate antigens. This process relies on a mechanically flexible TCR–pMHC binding interface, which enables force-enhanced CD8 coreceptor binding to MHC-α 1 α 2 domains through sequential conformational changes induced by force in both the MHC and CD8. Conversely, engineered high-affinity TCRs create rigid, tightly bound interfaces with cognate pMHCs of their parental TCRs. This rigidity prevents the force-induced conformational changes necessary for optimal catch-bond formation. Paradoxically, these high-affinity TCRs can form moderate catch bonds with non-stimulatory pMHCs of their parental TCRs, leading to off-target cross-reactivity and reduced specificity. We have also developed comprehensive force-dependent TCR–pMHC kinetics-function maps capable of distinguishing functional and non-functional TCR–pMHC pairs and identifying toxic, cross-reactive TCRs. These findings elucidate the mechano-chemical basis of the specificity of natural TCRs and highlight the critical role of CD8 in targeting cognate antigens. This work provides valuable insights for engineering TCRs with enhanced specificity and potency against non-self antigens, particularly for applications in cancer immunotherapy and infectious disease treatment, while minimizing the risk of self-antigen cross-reactivity.
研究领域:
免疫学
·
细胞生物学
·
生物力学
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产品关联
DOI
PubMed
高影响因子
Analytical Methods
·
2015
·Vol. 7
·期 16
·6712-6717
·
IF 1.821
Weiwen Hu,Yong Ning,Lianzhi Li,Jinming Kong,Xueji Zhang
In this work, a highly effective biosensor supported on morpholino-functionalized magnetic microspheres was investigated for the sequence-specific detection of DNA. Briefly, morpholino was first modified on the surface of magnetic microspheres through amido bonds, and then hybridized with target DNA in the ensuing step. Biotin-labeled signal probe DNA was designed to hybridize with the DNA nearby the 5′ end of sequence, and the biotin was used to bind with streptavidin-alkaline phosphatase (SA-ALP). L-Ascorbic acid-2-phosphate (AAP) was hydrolyzed by ALP to generate L-ascorbic acid, which could reduce resazurin to resorufin, resulting in a turn-on fluorescence signal. UV-vis absorbance spectroscopy was applied to prove the conjugation of ALP with signal probe DNA. Under optimal conditions, this biosensor displayed a good linear relationship between the fluorescence intensity and logarithm of single-stranded DNA concentrations in the range of 0.1 pM to 0.1 nM with a low detection limit of 9.32 fM. Moreover, fully complementary versus single-base mismatched, three-base mismatched and non-complementary DNA could be effectively distinguished. In addition, this novel approach rendered satisfactory analytical results for the determination of DNA in serum, thus exhibiting practical significance. These results demonstrated that this assay method displays excellent sensitivity and specificity for DNA detection and has great potential for practical applications.
研究领域:
生物化学
·
分子生物学
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产品关联
DOI
高影响因子
Jove-Journal of Visualized Experiments
·
2018
·
IF 1.184
Yanlan Wang,Jiayu Liu,Haitao Pan,Jieyu Xing,Xiaoqiong Wu,Qing Li,Zhong Wang
This protocol describes the construction and functional studies of a bispecific antibody (bsAb), GPC3-S-Fab. bsAbs can recognize two different epitopes through their two different arms. bsAbs have been actively studied for their ability to directly recruit immune cells to kill tumor cells. Currently, the majority of bsAbs are produced in the form of recombinant proteins, either as Fc-containing bsAbs or as smaller bsAb derivatives without the Fc region. In this study, GPC3-S-Fab, an antibody fragment (Fab) based bispecific antibody, was designed by linking the Fab of anti-GPC3 antibody GC33 with an anti-CD16 single domain antibody. The GPC3-S-Fab can be expressed in Escherichia coli and purified by two affinity chromatographies. The purified GPC3-S-Fab can specifically bind to and kill GPC3 positive liver cancer cells by recruiting natural killer cells, suggesting a potential application of GPC3-S-Fab in liver cancer therapy.
研究领域:
肿瘤学
·
免疫学
·
分子生物学
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产品关联
DOI
PubMed
高影响因子
FOOD AND AGRICULTURAL IMMUNOLOGY
·
2015
·
IF 0.986
Naifeng Xu,Liguang Xu,Wei Ma,Hua Kuang,Chuanlai Xu
In this study, a highly sensitive monoclonal antibody (McAb) against chloramphenicol (CAP) was successfully raised and characterised by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). Three antigens coupled with different proteins were synthesised and determined by the UV-vis method. After five rounds of immunisation, antisera were collected from six Balb/C mice and screened by ic-ELISA. The standard inhibition curve of one CAP McAb (labelled 6F11) showed the best sensitivity to CAP after optimisation of the principal working conditions. The half-maximal inhibition concentration (IC50) was calculated to be 0.01 ng/mL and the linear working range was from 0.0028 ng/mL to 0.040 ng/mL with a correlation coefficient of 0.996. This McAb showed high specificity, with negligible cross-reactivities detected towards CAP analogues (florfinicol, florfinicol amine, thiamphenicol). The recovery rate was 98.9–106.8% while the coefficient of variation was 4.45–6.05%. The IC50 value and other parameters obtained for this CAP McAb suggest that it is likely to have a promising future in further applications in combination with different analytical techniques.
研究领域:
免疫学
·
分析化学
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产品关联
DOI
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2022
·
IF 9.221
Shuang Li,Jiawei Zhang,Cherie S. Tan,Chong Chen,Chang Hu,Yongchang Bai,Dong Ming
As important biomarkers for prostate cancer, total prostate specific antigen (tPSA) and free prostate specific antigen (fPSA) levels in serum play essential roles in early diagnosis. In this study, hybrid molybdenum disulfide (MoS 2 )/Pt@Au-nanoprism/polydopamine (PDA)-modified screen-printed electrodes were utilized to detect both fPSA and tPSA in serum simultaneously. MoS 2 generated by the hydrothermal method assembled on the electrode to provide binding sites for further modification. Au nanoprisms were obtained by reducing chloroauric acid with sodium thiosulfate to achieve uniformity. Pt nanoparticles covered on Au nanoprisms by the displacement method, and the generated Pt@Au-nanoprism structure was then deposited onto the electrode to enhance the electrochemical signals. An electropolymerization-synthesized PDA film further modified the electrode to increase antibody binding sites. The results demonstrated that the proposed electrochemical immunosensor for fPSA and tPSA analysis in serum using differential pulse voltammetry (DPV) had a wide linear range from 0.0001 ng/mL to 100 ng/mL, with limits of detection (3 SD/slope) at 0.1 pg/mL and 1.1 fg/mL, respectively. For clinical patient samples, the proposed biosensor demonstrated good accuracy according to the clinical electrochemiluminescence immunoassay and improved the diagnostic specificity of tPSA concentrations in the “gray zone", suggesting promising prospects for clinical applications of our multiplex biosensors.
研究领域:
生物医学工程
·
临床诊断学
·
材料科学
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产品关联
DOI
高影响因子
MOLECULAR BIOTECHNOLOGY
·
2016
·Vol. 58
·期 6
·428-440
·
IF 1.752
Yang Yuanyuan,Lin Jinping,Wei Dongzhi
A NADPH-dependent and FMN-containing nitroreductase (Gox0834) from Gluconobacter oxydans was cloned and heterogeneously expressed in Escherichia coli . The purified enzyme existed as a dimer with an apparent molecular mass of about 31.4 kDa. The enzyme displayed broad substrate specificity and reduced a variety of mononitrated, polynitrated, and polycyclic nitroaromatic compounds to the corresponding amino products. The highest activity was observed for the reduction of CB1954 (5-(1-aziridinyl)-2,4-dinitrobenzamide). The enzyme kinetics analysis showed that Gox0834 had relatively low K
m
(54 ± 11 μM) but high k
cat / K
m
value (0.020 s −1 /μM) for CB1954 when compared with known nitroreductases. Nitrobenzene and 2,4,6-trinitrotoluene (TNT) were preferred substrates for this enzyme with specific activity of 11.0 and 8.9 μmol/min/mg, respectively. Gox0834 exhibited a broad temperature optimum of 40–60 °C for the reduction of CB1954 with a pH optimum between 7.5 and 8.5. The purified enzyme was very stable below 37 °C over a broad pH range of 6.0–10.0. These characteristics suggest that the nitroreductase Gox0834 may be a possible candidate for catalyzing prodrug activation, bioremediation, or biocatalytic processes.
研究领域:
微生物学
·
酶学
·
环境科学
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产品关联
DOI
PubMed
高影响因子
MOLECULAR PLANT-MICROBE INTERACTIONS
·
2018
·Vol. 32
·期 2
·
IF 3.588
Dandan Zhang,Caijuan Tian,Kangquan Yin,Wenyi Wang,Jin-Long Qiu
Stomata are leaf pores that regulate gas exchange and water transpiration in response to environmental cues. They also function in innate immunity by limiting pathogen entry through actively closing in so-called stomatal defense. However, roles of stomata in plant disease resistance are not fully elucidated, especially in monocots. Here, we report that non–race specific resistance of the rice abscisic acid-deficient mutant Osaba1 to Xanthomonas oryzae pv. oryzae is due to increased stomatal conductance. Reducing stomatal conductance in the Osaba1 mutant increases its susceptibility to X. oryzae pv. oryzae . Artificial opening of stomata in wild-type plants leads to enhanced resistance to X. oryzae pv. oryzae . The rice mutant es1-1 with constitutively higher stomatal conductance exhibits strong resistance to X. oryzae pv. oryzae . Additionally, Osaba1 and es1-1 are resistant to X. oryzae pv. oryzicola . The data support that open stomata confer postinvasive resistance against bacterial pathogens in rice, and such resistance probably results from decreased leaf water potential. Our findings reveal a novel role of stomata in plant immunity through modulation of leaf water status, which provides physiological insight into the interactions between plant, pathogen, and environment.
研究领域:
植物学
·
分子生物学
·
植物病理学
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产品关联
DOI
PubMed
高影响因子
APPLIED CATALYSIS B-ENVIRONMENTAL
·
2023
·
IF 22.1
Xuewu Deng,Xiaobing Zheng,Feifei Jia,Cuiyao Cao,Haolei Song,Yanjun Jiang,Yunting Liu,Guanhua Liu,Shijie Li,Lihui Wang
The selective oxyfunctionalization of C-H and C C bonds is highly important and catalyzed by unspecific peroxygenases (UPOs) which suffer from inactivation by hydrogen peroxide (H 2 O 2 ). Herein, photo-enzyme integrated catalysts were constructed by immobilizing Aae UPO as cross-linked enzyme aggregates (CLEAs) in Pd-loaded three-dimensional ordered macroporous titania (3DOM TiO 2 ) with in situ photocatalytic H 2 O 2 production. The prepared Aae UPO@3DOM TiO 2 -Pd obtained higher catalytic efficiency compared with CLEAs- Aae UPO. The conversion of ethylbenzene to (R) -1-phenethyl alcohol was significantly improved, reaching 99 % compared with free enzyme (38 %) and CLEAs- Aae UPO (49 %), with ee value of > 99 %. Aae UPO@3DOM TiO 2 -Pd exhibited good stability and reusability attributed to the protection of macropores and in situ H 2 O 2 production. By using the designed 3DOM platform for photocatalysis and enzyme immobilization, the in situ photocatalytic generation of H 2 O 2 was highly efficient, and the monodisperse CLEAs- Aae UPO in the macropores facilitated the contact of substrate and enzyme, thus obtaining excellent photo-enzyme coupled catalysis.
研究领域:
生物化学
·
环境科学
·
材料科学
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产品关联
DOI
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2025
·
IF 7.7
Ya-Xue Jia,Na Wang,Juan Zhang,Cheng-Peng Sun
Thioredoxin reductase (TrxR) is a pivotal biomarker and therapeutic target, monitoring its expression levels with good accuracy and sensitivity in cells or tissues is of great significance for clinical disease diagnosis and prognosis studies. Herin, we first develop a novel TrxR fluorescence sensing strategy based on the nucleic acid site-specific cleavage (NASC) mechanism, which highlights the benefits of prominent selectivity and high sensitivity. Notably, our NASC-based sensing strategy shows the limit of detection (LOD) is over two orders of magnitude lower than other site-specific cleavage-based approaches. Our proposed method has demonstrated the successful application in determining the TrxR level of in living cell and tissue, highlighting its potential for analyzing TrxR in clinical samples. The results convincingly illustrated that the NASC strategy stands as a highly promising molecular instrument for unraveling the physiological roles of TrxR within living organisms.
研究领域:
生物化学
·
分子生物学
·
细胞生物学
·
分析化学
·
生物医学工程
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产品关联
DOI
高影响因子
MOLECULES
·
2024
·Vol. 29
·期 23
·5722
·
IF 4.2
Cuimei Chen,Ping Ding
Purposes: To explore the optimization method and application of Au-NP-enhanced luminol––H2O2luminescence system in TORCH (TOX, RV, CMV, HSVI, and HSVII) detection. Method: 4.5 × 10−5mmol/L gold nano solution was prepared with chloroauric acid as the reducing agent and trisodium citrate as the stabilizer. After curing for 3 days, Au NPs participate in the luminal–H2O2luminescence system to detect TORCH antibodies and establish the cut off value. SPSS 18.0 software was used to analyze the TORCH antibodies detected by the nano-gold-enhanced luminol luminescence method and TORCH kit. Additionally, its detection performance is studied. Results: The results of a pairedt-test for the absorbance values of samples with and without gold nanoparticles showed that there were statistically significant differences (p< 0.001) between the two methods in the detection of TOX, RV, CMV, HSVI, and HSVII. The luminescence values with the addition of gold nanoparticles were significantly higher than those without gold nanoparticles. Using the Au NP–luminol–H2O2chemiluminescence method, 127 serum samples were tested for TORCH antibodies. The sensitivities were 84.6%, 83.3%, 90.9%, 85.7%, and 84.6%, while the specificities were 94.7%, 96.5%, 96.6%, 97.3%, and 95.6%, respectively. The sensitivity and specificity of the chemiluminescence method enhanced by gold nanoparticles are significantly improved compared to the chemiluminescence method without enhancers. Conclusions: Au NPs participate in the luminal–H2O2luminescent system. The absorbance, sensitivity, and specificity of TORCH antibodies show that Au NPs can enhance the luminol–H2O2luminescent system. Au NP–luminol–H2O2luminescence system has broad application prospects in the detection of eugenics.
研究领域:
生物医学
·
免疫学
·
纳米技术
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产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）