Porous Flow Field for Next-Generation Proton Exchange Membrane Fuel Cells: Materials, Characterization, Design, and Challenges.

Porous flow fields distribute fuel and oxygen for the electrochemical reactions of proton exchange membrane (PEM) fuel cells through their pore network instead of conventional flow channels. This type of flow fields has showed great promises in enhancing reactant supply, heat removal, and electrical conduction, reducing the concentration performance loss and improving operational stability for fuel cells. This review presents the research and development progress of porous flow fields with insights for next-generation PEM fuel cells of high power density (e.g., ∼9.0 kW L-1). Materials, fabrication methods, fundamentals, and fuel cell performance associated with porous flow fields are discussed in depth. Major challenges are described and explained, along with several future directions, including separated gas/liquid flow configurations, integrated porous structure, full morphology modeling, data-driven methods, and artificial intelligence-assisted design/optimization.

Lactococcus lactis HkyuLL 10 suppresses colorectal tumourigenesis and restores gut microbiota through its generated alpha-mannosidase.

OBJECTIVE: Probiotic Lactococcus lactis is known to confer health benefits to humans. Here, we aimed to investigate the role of L. lactis in colorectal cancer (CRC). DESIGN: L. lactis abundance was evaluated in patients with CRC (n=489) and healthy individuals (n=536). L. lactis was isolated from healthy human stools with verification by whole genome sequencing. The effect of L. lactis on CRC tumourigenesis was assessed in transgenic Apc Min/+ mice and carcinogen-induced CRC mice. Faecal microbiota was profiled by metagenomic sequencing. Candidate proteins were characterised by nano liquid chromatography-mass spectrometry. Biological function of L. lactis conditioned medium (HkyuLL 10-CM) and functional protein was studied in human CRC cells, patient-derived organoids and xenograft mice. RESULTS: Faecal L. lactis was depleted in patients with CRC. A new L. lactis strain was isolated from human stools and nomenclated as HkyuLL 10. HkyuLL 10 supplementation suppressed CRC tumourigenesis in Apc Min/+ mice, and this tumour-suppressing effect was confirmed in mice with carcinogen-induced CRC. Microbiota profiling revealed probiotic enrichment including Lactobacillus johnsonii in HkyuLL 10-treated mice. HkyuLL 10-CM significantly abrogated the growth of human CRC cells and patient-derived organoids. Such protective effect was attributed to HkyuLL 10-secreted proteins, and we identified that α-mannosidase was the functional protein. The antitumourigenic effect of α-mannosidase was demonstrated in human CRC cells and organoids, and its supplementation significantly reduced tumour growth in xenograft mice. CONCLUSION: HkyuLL 10 suppresses CRC tumourigenesis in mice through restoring gut microbiota and secreting functional protein α-mannosidase. HkyuLL 10 administration may serve as a prophylactic measure against CRC.

Advancing MicroRNA Detection: Enhanced Biotin-Streptavidin Dual-Mode Phase Imaging Surface Plasmon Resonance Aptasensor.

MicroRNAs (miRNAs) are novel tumor biomarkers owing to their important physiological functions in cell communication and the progression of multiple diseases. Due to the small molecular weight, short sequence length, and low concentration levels of miRNA, miRNA detection presents substantial challenges, requiring the advancement of more refined and sensitive techniques. There is an urgent demand for the development of a rapid, user-friendly, and sensitive miRNA analysis method. Here, we developed an enhanced biotin-streptavidin dual-mode phase imaging surface plasmon resonance (PI-SPR) aptasensor for sensitive and rapid detection of miRNA. Initially, we evaluated the linear sensing range for miRNA detection across two distinct sensing modalities and investigated the physical factors that influence the sensing signal in the aptamer-miRNA interaction within the PI-SPR aptasensor. Then, an enhanced biotin-streptavidin amplification strategy was introduced in the PI-SPR aptasensor, which effectively reduced the nonspecific adsorption by 20% and improved the limit of detection by 548 times. Furthermore, we have produced three types of tumor marker chips, which utilize the rapid sensing mode (less than 2 min) of PI-SPR aptasensor to achieve simultaneous detection of multiple miRNA markers in the serum from clinical cancer patients. This work not only developed a new approach to detect miRNA in different application scenarios but also provided a new reference for the application of the biotin-streptavidin amplification system in the detection of other small biomolecules.

Repurposing sodium stibogluconate as an uracil DNA glycosylase inhibitor against prostate cancer using a time-resolved oligonucleotide-based drug screening platform.

Repurposing drugs can significantly reduce the time and costs associated with drug discovery and development. However, many drug compounds possess intrinsic fluorescence, resulting in aberrations such as auto-fluorescence, scattering and quenching, in fluorescent high-throughput screening assays. To overcome these drawbacks, time-resolved technologies have received increasing attention. In this study, we have developed a rapid and efficient screening platform based on time-resolved emission spectroscopy in order to screen for inhibitors of the DNA repair enzyme, uracil-DNA glycosylase (UDG). From a database of 1456 FDA/EMA-approved drugs, sodium stibogluconate was discovered as a potent UDG inhibitor. This compound showed synergistic cytotoxicity against 5-fluorouracil-resistant cancer cells. This work provides a promising future for time-resolved technologies for high-throughput screening (HTS), allowing for the swift identification of bioactive compounds from previously overlooked scaffolds due to their inherent fluorescence properties.

New association between splicing factor-coding gene polymorphisms and the risk of acute lymphoblastic leukemia in southern Chinese children: A five-center case-control study.

BACKGROUND: The role of splicing factor-coding gene polymorphisms in pediatric acute lymphoblastic leukemia (ALL) susceptibility is still unclear. METHODS: A case-control designed model was used to estimate the overall risk of pediatric ALL and five single nucleotide polymorphisms (SNPs) of splicing factor-coding genes in 808 cases and 1,340 controls, which were genotyped using a TaqMan assay. Stratified analysis was performed to explore the association of rs2233911 genotype and pediatric ALL susceptibility. The influence of splicing factor arginine/serine-rich 1 (SFRS1) polymorphisms on the sensitivity to different chemotherapeutic regimens based on minimal residual disease (MRD) levels was analyzed. The haplotype analysis was adopted to evaluate the association between inferred haplotypes of the splicing factor-coding genes and pediatric ALL risk. RESULTS: Among the five analyzed SNPs, SFRS1 rs2233911 AG/GG exhibited a significant association with increased pediatric ALL risk. The stratified analysis further identified the harmful effect of SFRS1 rs2233911 AG/GG in specific subgroups. Moreover, rs2233911 AG/GG had a protective effect on MRD in marrow of ≥0.01%  12 weeks of Chinese Children Cancer Group chemotherapeutics, but provided a harmful effect on MRD in the marrow of ≥0.01% at days 15-19 of the South China Children Leukemia Group chemotherapeutics. Haplotype analysis of these five SNPs yielded haplotypes ACGCC and ACGTC significantly correlating with increased pediatric ALL susceptibility. On the contrary, haplotypes GCATG and GTACC were linked with remarkably decreased pediatric ALL risk. CONCLUSION: SFRS1 gene polymorphism was associated with increased pediatric ALL risk and indicated that rs2233911 AG/GG might be a potential biomarker for choosing chemotherapeutics.

ADAR1 polymorphisms contribute to increased susceptibility in pediatric acute lymphoblastic leukemia.

Adenosine deaminase acting on RNA1 (ADAR1), catalyzing post-transcriptional adenosine-to-inosine RNA editing, promotes cancer progression and therapeutic resistance. However, very little is known about the association of ADAR1 variants with acute lymphoblastic leukemia (ALL). Here we first explored the potential association of three polymorphisms (rs9616, rs2229857, and rs1127313) of ADAR1 with susceptibility in Chinese children ALL, then functionally characterized ADAR1 in ALL. Our results demonstrated that rs9616 T and rs2229857 T were associated with increased expression of ADAR1 mRNA and higher risk to ALL. Of note, a stronger risk effect of rs2229857 T genotypes was found among relapse children. Furthermore, ADAR1 knockdown specifically inhibited proliferation and promoted apoptosis in ALL cells. These findings give insights into a mechanism by which the risk variant at rs9616 and rs2229857 modulate ADAR1 expression and confers a predisposition and relapse risk to ALL, and representing a potential novel biomarker for pediatric ALL.

Porous Carbon with Alumina Coating Nanolayer Derived from Biomass and the Enhanced Electrochemical Performance as Stable Anode Materials.

With the ever-increasing world population, the energy produced from green, environmentally friendly approaches is in high demand. In this work, we proposed a green and cost-effective strategy for synthesizing a porous carbon electrode decorated with alumina oxide (Al2O3) from cherry blossom leaves using the pyrolysis method followed by a sol-gel method. An Al2O3-coating nano-layer (4-6 nm) is formed on the porous carbon during the composition fabrication, which further adversely affects battery performance. The development of a simple rich-shell-structured C@Al2O3 nanocomposite anode is expected to achieve stable electrochemical performances as lithium storage. A significant contributing factor to enhanced performance is the structure of the rich-shell material, which greatly enhances conductivity and stabilizes the solid-electrolyte interface (SEI) film. In the battery test assembled with composite C@Al2O3 electrode, the specific capacity is 516.1 mAh g-1 at a current density of 0.1 A g-1 after 200 cycles. The average discharge capacity of carbon is 290 mAh g-1 at a current density of 1.0 A g-1. The present study proposes bioinspired porous carbon electrode materials for improving the performance of next-generation lithium-ion batteries.

Porphyran-derived oligosaccharides alleviate NAFLD and related cecal microbiota dysbiosis in mice.

Porphyran and its derivatives possess a variety of biological activities, such as ameliorations of oxidative stress, inflammation, hyperlipemia, and immune deficiencies. In this study, we evaluated the potential efficacy of porphyran-derived oligosaccharides from Porphyra yezoensis (PYOs) in alleviating nonalcoholic fatty liver disease (NAFLD) and preliminarily clarified the underlying mechanism. NAFLD was induced by a high-fat diet for six months in C57BL/6J mice, followed by treatment with PYOs (100 or 300 mg/kg/d) for another six weeks. We found that PYOs reduced hepatic oxidative stress in mice with NAFLD, which plays a critical role in the occurrence and development of NAFLD. In addition, PYOs could markedly decrease lipid accumulation in liver by activating the IRS-1/AKT/GSK-3ß signaling pathway and the AMPK signaling pathway in mice with NAFLD. PYOs also apparently relieved the hepatic fibrosis induced by oxidative stress via downregulation of TGF-ß and its related proteins, so that liver injury was markedly alleviated. Furthermore, PYOs treatment relieved cecal microbiota dysbiosis (such as increasing the relative abundance of Akkermansia, while decreasing the Helicobacter abundance), which could alleviate oxidative stress, inflammation, and lipid metabolism, and protect the liver to a certain degree. In summary, PYOs treatment remarkably improved NAFLD via a specific molecular mechanism and reshaped the cecal microbiota.

External quality assessment for detection of methylated Syndecan 2 (SDC2) in China.

OBJECTIVES: Detection of Syndecan 2 (SDC2) methylation in stool DNA is a novel method for the auxiliary diagnosis of early colorectal cancer (CRC). Currently, this method has been widely applied; however, its accuracy and reliability have not been determined. The objective of this pioneering study was to evaluate the performance of clinical laboratories in China for their ability to detect SDC2 methylation from stool DNA. METHODS: We generated a sample panel consisting of clinical and cell samples. The clinical samples were stool specimens from patients with or without CRC, including four positives (prepared by serial dilution from one stool specimen), one negative and one interferential sample. Two cell samples, with positive or negative methylated SDC2, were used as controls. The panel was distributed to 32 clinical laboratories for analysis of SDC2 methylation, and the results were compared and scored. RESULTS: The sample panel was compatible with commercially available assays and it showed appropriate stability to be an external quality assessment material. There were four false results; one hospital laboratory and one commercial diagnostic laboratory had a false-positive and a false-negative result, respectively, and one commercial diagnostic laboratory had both a false-positive and false-negative result. Among the 32 participating laboratories, 29 (90.62%) obtained an acceptable or better performance score, while 3 (9.38%) laboratories required improvement. CONCLUSIONS: Our results demonstrate that the detection of SDC2 methylation from stool DNA was satisfactory in China. Additionally, the importance of external quality assessment was highlighted for monitoring the performance of clinical laboratories.

Effects of ambient PM2.5 on development of psoriasiform inflammation through KRT17-dependent activation of AKT/mTOR/HIF-1α pathway.

Exposure to fine particulate matter (PM2.5) has significant effects on human skin health, mainly disrupting skin homeostasis and accelerating aging. To date, the effects of PM2.5 on psoriasis (PSO) have not been elucidated. An ambient particulate matter exposed and well characterized imiquimod (IMQ)-induced psoriasis mouse model was established. Thirty male C57BL/6 mice aged 8 weeks were randomly divided into three groups: filtered air (FA) group (Control group), PSO+ FA group and PSO + PM2.5 group. A KRT17 knockdown (KRT17-KD) mouse model was simultaneously established by subcutaneously injecting KRT17-KD lentivirus. Forty male C57BL/6 mice were randomly divided into four groups: PSO + FA + KRT17-RNAi negative control lentivirus (KRT17-NC) group, PSO+ FA+ KRT17-KD group, PSO + PM2.5 + KRT17-NC group and PSO + PM2.5 + KRT17-KD group. PM2.5 exposure continued for 8 weeks. Psoriasis was induced by topically applying IMQ on the dorsal skin of the mice for 6 days during week 8. Morphometric and histological analyses were performed to investigate the changes in psoriatic lesions. Differentially expressed genes and enriched pathways were explored using bioinformatics analysis and showed that KRT17 gene and the vascular endothelial growth factor receptor signaling pathway were associated with psoriasis. HaCaT cells were stimulated with interleukin-17A and infected with KRT17-KD lentivirus to establish an in vitro KRT17 knockdown psoriasis cell model. Notably, PM2.5 exposure increased the expression of KRT17 protein and activated AKT/mTOR/HIF-1α signaling pathway in vivo. Moreover, specific agonist of AKT (740Y-P) reversed the decreased neovascularization induced by KRT17 knockdown through AKT/mTOR/HIF-1α signaling pathway in vitro. Consequently, PM2.5 exposure could promote the development and progression of psoriasis through KRT17-dependent activation of AKT/mTOR/HIF-1α signaling pathway.

Ultrafast Surface Plasmon Resonance Imaging Sensor via the High-Precision Four-Parameter-Based Spectral Curve Readjusting Method.

A variety of surface plasmon resonance (SPR) sensing devices have been extensively used in biochemical detection for their characteristics of label-free, highly sensitive, and faster detecting. Among them, the spectrum-based SPR sensing devices have offered us great advantages in high-throughput sensing due to their large dynamic range and the possibility of detection resolution similar to that offered by angle interrogation. This paper demonstrates a spectrum-based SPR imaging sensing system with fast wavelength scanning capability achieved by an acousto-optic tunable filter (AOTF) and a low-cost and speckle-free halogen lamp implemented as the SPR excitation source. Especially, we developed a novel four-parameter-based spectral curve readjusting (4-PSCR) method for data processing, which offered us a faster and more accurate spectral data curve fitting process than the traditional polynomial fitting method. With the configuration, we have also conducted an SPR high-throughput detection of the novel coronavirus (COVID-19) spike protein, proving its application possibility in the screening of COVID-19 with high accuracy. We believe that the higher sensitivity and accuracy of the system have made it readily used in biochemical imaging and detecting applications.

Phase interrogation surface plasmon resonance hyperspectral imaging sensor for multi-channel high-throughput detection.

Phase interrogation surface plasmon resonance (SPR) imaging is, in principle, suitable in multiple samples and high-throughput detection, but the refractive index difference of various samples can be largely varied, while the dynamic range of phase interrogation SPR is narrow. So it is difficult to perform multi-sample detection in phase interrogation mode. In this paper, we successfully designed a multi-channel phase interrogation detection SPR imaging sensing scheme based on a common optical interference path between p- and s-polarized light without using any mechanical moving components. The fixed optical path difference between p- and s-polarized light is introduced by a birefringence crystal to produce sinusoidal spectral interference fringes. We adopted a time-division-multiplexing peak-finding algorithm to track the resonance wavelength so that the detection range can cover every channel. The phase values which carry the high sensitivity signal of the corresponding samples are calculated by the iterative parameter scanning cross-correlation algorithm.

COVID-19-another influential event impacts on laboratory medicine management.

BACKGROUND: Before public health emergencies became a major challenge worldwide, the scope of laboratory management was only related to developing, maintaining, improving, and sustaining the quality of accurate laboratory results for improved clinical outcomes. Indeed, quality management is an especially important aspect and has achieved great milestones during the development of clinical laboratories. CURRENT STATUS: However, since the coronavirus disease 2019 (COVID-19) pandemic continues to be a threat worldwide, previous management mode inside the separate laboratory could not cater to the demand of the COVID-19 public health emergency. Among emerging new issues, the prominent challenges during the period of COVID-19 pandemic are rapid-launched laboratory-developed tests (LDTs) for urgent clinical application, rapid expansion of testing capabilities, laboratory medicine resources, and personnel shortages. These related issues are now impacting on clinical laboratory and need to be effectively addressed. CONCLUSION: Different from traditional views of laboratory medicine management that focus on separate laboratories, present clinical laboratory management must be multidimensional mode which should consider consolidation of the efficient network of regional clinical laboratories and reasonable planning of laboratories resources from the view of overall strategy. Based on relevant research and our experience, in this review, we retrospect the history trajectory of laboratory medicine management, and also, we provide existing and other feasible recommended management strategies for laboratory medicine in future.

Associations of long-term exposure to traffic-related air pollution with risk of valvular heart disease based on a cross-sectional study.

Emerging evidence demonstrated that traffic-related air pollution induced adverse effects on cardiovascular system. We designed a population-based cross-sectional study to explore the association between residential proximity to major roadways, traffic density and the prevalence of valvular heart disease (VHD). A total of 34040 subjects from a Rural Health Project between 2013 and 2018 were collected. According to the inclusion and exclusion criteria, 4158 participants were enrolled in the final analysis. And we calculated the subjects' proximity to major roadways and collected the traffic density on the major roadways. Transthoracic echocardiography (TTE) was performed to diagnose the VHD, according to the current AHA/ACC (the American Heart Association and the American College of Cardiology) guidelines. Differences between groups were examined by the one-way ANOVAs for continuous variables and the chi-square tests for categorical variables. A logistic regression models were used to assess the associations. The stratified analysis by age and sex were conducted to further analyze the association. The restricted cubic spline analysis was performed to further evaluate the association between road way distance and VHD. Bonferroni test was used to adjust the significance level. The subjects closer to the major roads had the higher risk of tricuspid regurgitation (TR) (odds risk, OR = 1.519, 95% confidence intervals, 95%CI: 1.058-2.181), especially in female. The risk of VHD was positive (high traffic density VS low traffic density, OR = 1.799, 95%CI: 1.221-2.651), especially in female. In addition, the high traffic density was associated with the risk of mitral regurgitation (MR) (OR = 1.758, 95%CI: 1.085-2.848). The restricted cubic spline analysis found a threshold distance of about 300 m, where had the lowest risk of VHD, aortic regurgitation (AR), MR, TR. Our results found a positive association between traffic-related air pollution and VHD especially in female.

High-throughput imaging surface plasmon resonance biosensing based on ultrafast two-point spectral-dip tracking scheme.

Wavelength interrogation surface plasmon resonance imaging (λSPRi) has potential in detecting 2-dimensional (2D) sensor array sites, but the resonance wavelength imaging rate limits the application of detecting biomolecular binding process in real time. In this paper, we have successfully demonstrated an ultrafast λSPRi biosensor system. The key feature is a two-point tracking algorithm that drives the liquid crystal tunable filter (LCTF) to achieve fast-tracking of the resonance wavelength movement caused by the binding of target molecules with the probe molecules on the sensing surface. The resonance wavelength measurement time is within 0.25s. To date, this is the fastest speed ever reported in λSPRi. Experiment results show that the sensitivity and dynamic are 2.4 × 10-6 RIU and 4.6 × 10-2 RIU, respectively. In addition, we have also demonstrated that the system has the capability of performing fast high-throughput detection of biomolecular interactions, which confirms that this fast real-time detecting approach is most suitable for high-throughput and label-free biosensing applications.

Phase interrogation SPR sensing based on white light polarized interference for wide dynamic detection range.

A phase surface plasmon resonance (SPR) sensing technology based on white light polarized interference in common-path geometry is reported. A halogen lamp is used as the excitation source of the SPR sensor. The fixed optical path difference (OPD) between p- and s-polarized light is introduced by a birefringence crystal to produce sinusoidal spectral interference fringes. The SPR phase is accurately extracted from the interference fringes using a novel iterative parameter-scanning cross-correlation algorithm. The dynamic detection range is expanded by tracking the best SPR wavelength, which is identified using a window Fourier algorithm. The experimental results show that the sensitivity of this SPR system was 1.3 × 10-7 RIU, and the dynamic detection range was 0.029 RIU. This sensor, not only simple to implement and cost efficient, requires no modulators.

Detection of genes mutations in cerebrospinal fluid circulating tumor DNA from neoplastic meningitis patients using next generation sequencing.

BACKGROUND: This study profiled the somatic genes mutations and the copy number variations (CNVs) in cerebrospinal fluid (CSF)-circulating tumor DNA (ctDNA) from patients with neoplastic meningitis (NM). METHODS: A total of 62 CSF ctDNA samples were collected from 58 NM patients for the next generation sequencing. The data were bioinformatically analyzed by (Database for Annotation, Visualization and Integrated Discovery) DAVID software. RESULTS: The most common mutated gene was TP53 (54/62; 87.10%), followed by EGFR (44/62; 70.97%), PTEN (39/62; 62.90%), CDKN2A (32/62; 51.61%), APC (27/62: 43.55%), TET2 (27/62; 43.55%), GNAQ (18/62; 29.03%), NOTCH1 (17/62; 27.42%), VHL (17/62; 27.42%), FLT3 (16/62; 25.81%), PTCH1 (15/62; 24.19%), BRCA2 (13/62; 20.97%), KDR (10/62; 16.13%), KIT (9/62; 14.52%), MLH1 (9/62; 14.52%), ATM (8/62; 12.90%), CBL (8/62; 12.90%), and DNMT3A (7/62; 11.29%). The mutated genes were enriched in the PI3K-Akt signaling pathway by the KEGG pathway analysis. Furthermore, the CNVs of these genes were also identified in these 62 samples. The mutated genes in CSF samples receiving intrathecal chemotherapy and systemic therapy were enriched in the ERK1/2 signaling pathway. CONCLUSIONS: This study identified genes mutations in all CSF ctDNA samples, indicating that these mutated genes may be acted as a kind of biomarker for diagnosis of NM, and these mutated genes may affect meningeal metastasis through PI3K-Akt signaling pathway.

Fucoidan from Ascophyllum nodosum Suppresses Postprandial Hyperglycemia by Inhibiting Na+/Glucose Cotransporter 1 Activity.

We previously demonstrated that fucoidan with a type II structure inhibited postprandial hyperglycemia by suppressing glucose uptake, but the mechanism remains elusive. Here, we aimed to assess whether the effect of glucose absorption inhibition was related to the basic structure of fucoidans and preliminarily clarified the underlying mechanism. Fucoidans with type II structure and type I structure were prepared from Ascophyllumnodosum (AnF) or Laminariajaponica (LjF) and Kjellmaniellacrassifolia (KcF), respectively. The effects of various fucoidans on suppressing postprandial hyperglycemia were investigated using in vitro (Caco-2 monolayer model), semi-in vivo (everted gut sac model), and in vivo (oral glucose tolerance test, OGTT) assays. The results showed that only AnF with a type II structure, but not LjF or KcF with type I structure, could inhibit the glucose transport in the Caco-2 monolayer and everted gut sac models. A similar result was seen in the OGTT of Kunming mice and leptin receptor-deficient (db/db) mice, where only AnF could effectively inhibit glucose transport into the bloodstream. Furthermore, AnF (400 mg/kg/d) treatment decreased the fasting blood glucose, HbA1c, and fasting insulin levels, while increasing the serum glucagon-like peptide-1 (GLP-1) level in obese leptin receptor-deficient (db/db) mice. Furthermore, surface plasmon resonance (SPR) analysis revealed the specific binding of AnF to Na+/glucose cotransporter 1 (SGLT1), which indicated the effect of AnF on postprandial hyperglycemia could be due to its suppression on SGLT1 activity. Taken together, this study suggests that AnF with a type II structure can be a promising candidate for hyperglycemia treatment.

Different binding properties of two general-odorant binding proteins in Athetis lepigone with sex pheromones, host plant volatiles and insecticides.

Athetis lepigone (Alep) is a polyphagous pest native to Europe and Asia that has experienced major outbreaks in the summer maize area of China since 2011 and has shown evidence of resistance to some insecticides. Insect olfaction is crucial for recognition of sex pheromones, host plant volatiles and even insecticides, in which two general-odorant binding proteins (GOBPs) play important roles. To elucidate the functions of GOBPs in A. lepigone, we first expressed the two AlepGOBP proteins in the E. coli expression system. Then, the results of fluorescence competitive binding assays demonstrated that the high binding affinity of AlepGOBP2 with sex pheromones [(Z)-7-dodecenyl acetate (Z7-12:Ac), Ki = 0.65 µM; (Z)-9-tetradecenyl acetate (Z9-14:Ac), Ki = 0.83 µM], two maize plant volatiles [Ocimene, Ki = 9.63 µM; (E)-ß-Farnesene, Ki = 4.76 µM] and two insecticides (Chlorpyrifos Ki =5.61 µM; Phoxim, Ki = 4.38 µM). However, AlepGOBP1 could only bind Ocimene (Ki = 13.0 µM) and two insecticides (Chlorpyrifos Ki =4.46 µM; Phoxim, Ki = 3.27 µM). These results clearly suggest that AlepGOBP1 and AlepGOBP2 differentiate among odorants and other ligands. The molecular docking results further revealed different key residues involved in the ligand binding of AlepGOBPs. In summary, this study provides a foundation for exploring the olfactory mechanism of A. lepigone and identified two potential target genes for the development of highly effective insecticides in the future.

Simultaneous enhancement of barley ß-amylase thermostability and catalytic activity by R115 and T387 residue sites mutation.

OBJECTIVE: To simultaneously increase the thermostability and catalytic activity of barley ß-amylase. METHODS: The amino acid sequences of various barley ß-amylases with different enzyme properties were aligned, two amino acid residues R115 and T387 were identified to be important for barley ß-amylase properties. R115C and T387V were then generated using site-directed and saturation mutagenesis. RESULTS: R115C and T387V mutants increased the enzyme catalytic activity and thermostability, respectively. After combinational mutagenesis, the T50 value and t(1/2,60oC) value of R115C/T387V mutant reached 59.4 °C and 48.8 min, which were 3.6 °C higher and 29.5 min longer than those of wild-type. The kcat/Km value of mutant R115C/T387V were 59.82/s·mM, which were 54.7% higher than that of wild-type. The increased surface hydrophobicity and newly formed strong hydrogen bonds and salt bridges might be responsible for the enzyme thermostability improvement while the two additional hydrogen bonds formed in the active center may lead to the catalytic property enhancement. CONCLUSIONS: The mutant R115C/T387V showed high catalytic activity and thermostability indicating great potential for application in industry.