Ultralow RuO2 Doped NiS2 Heterojunction as a Multifunctional Electrocatalyst for Hydrogen Evolution linking to Biomass Organics Oxidation.

Hydrogen energy and biomass energy are green and sustainable forms that can solve the energy crisis all over the world. Electrocatalytic water splitting is a marvelous way to produce hydrogen and biomass platform molecules can be added into the electrolyte to reduce the overpotential and meanwhile are converted into some useful organics, but the key point is the design of electrocatalyst. Herein, ultralow noble metal Ru is doped into NiS2 to form RuO2@NiS2 heterojunction. Amongst them, the 0.06 RuO2@NiS2 has low overpotentials of 363 mV for OER and 71 mV for HER in 1 m KOH, which are superior to the RuO2 and Pt/C. Besides, the 0.06 RuO2@NiS2 shows a low overpotential of 173 mV in 1 m KOH+0.1 m glycerol, and the glycerol is oxidized to glyceraldehyde and formic acid via the high Faraday efficiency GlyOR process, and the splitting voltage is only 1.17 V. In addition, the 0.06 RuO2@NiS2 has a low overpotential of 206 mV in 1 m KOH+0.1 m glucose, and the glucose is converted to glucaric acid, lactic acid, and formic acid. This work has a "one stone three birds" effect for the production of hydrogen, low splitting voltage, and high-value-added biomass chemicals.

Oil-film extinction coefficient inversion based on thickness difference: erratum.

An erratum is presented to correct errors in Eqs. (2) and (3) in our previously published paper [Opt. Express30(17), 30368 (2022).10.1364/OE.461162].

Mesothelin CAR-T cells expressing tumor-targeted immunocytokine IL-12 yield durable efficacy and fewer side effects.

Chimeric antigen receptor (CAR)-modified T cell therapy has achieved remarkable efficacy in treating hematological malignancies, but it confronts many challenges in treating solid tumors, such as the immunosuppressive microenvironment of the solid tumors. These factors reduce the antitumor activity of CAR-T cells in clinical trials. Therefore, we used the immunocytokine interleukin-12 (IL-12) to enhance the efficacy of CAR-T cell therapy. In this study, we engineered CAR-IL12R54 T cells that targeted mesothelin (MSLN) and secreted a single-chain IL-12 fused to a scFv fragment R54 that recognized a different epitope on mesothelin. The evaluation of the anti-tumor activity of the CAR-IL12R54 T cells alone or in combination with anti-PD-1 antibody in vitro and in vivo was followed by the exploration of the functional mechanism by which the immunocytokine IL-12 enhanced the antitumor activity. CAR-IL12R54 T cells had potency to lyse mesothelin positive tumor cells in vitro. In vivo studies demonstrated that CAR-IL12R54 T cells were effective in controlling the growth of established tumors in a xenograft mouse model with fewer side effects than CAR-T cells that secreted naked IL-12. Furthermore, combination of PD-1 blockade antibody with CAR-IL12R54 T cells elicited durable anti-tumor responses. Mechanistic studies showed that IL12R54 enhanced Interferon-γ (IFN-γ) production and dampened the activity of regulatory T cells (Tregs). IL12R54 also upregulated CXCR6 expression in the T cells through the NF-κB pathway, which facilitated T cell infiltration and persistence in the tumor tissues. In summary, the studies provide a good therapeutic option for the clinical treatment of solid tumors.

Impact of CD40 gene polymorphisms on the risk of cervical squamous cell carcinoma: a case-control study.

BACKGROUND: Cervical cancer is the fourth most common cancer among women worldwide. Genome-wide association studies have revealed multiple susceptible genes and their polymorphisms for cervical cancer risk. Therefore, we aimed to investigate the correlation between single nucleotide polymorphisms (SNPs) of the CD40 gene and susceptibility to cervical squamous cell carcinoma (CSCC) in a population from the northeastern Han Chinese population. METHODS: The three SNPs (rs1800686, rs3765459, and rs4810485) of the CD40 gene were analyzed by multiplex polymerase chain reaction (PCR) combined with next-generation sequencing methods in 421 patients with CSCC, 594 patients with high-grade squamous intraepithelial lesions (HSIL), and 504 healthy females. Multivariate logistic regression analysis was used to analyze the potential relationship between CD40 gene polymorphisms and CSCC, or HSIL. RESULTS: Our research results showed the AA genotype of rs1800686 had a protective effect on CSCC in comparison to the GG genotype and AG+GG genotypes (AA vs. GG: p = 0.0389 and AA vs. AG+GG: p = 0.0280, respectively). After FDR correction, the results were still statistically significant (p = 0.0389 and p = 0.0389, respectively). Similarly, rs3765459 showed a reduced risk association for CSCC in the codominant and recessive models (AA vs. GG: p = 0.0286 and AA vs. AG+GG: p = 0.0222, respectively). Significant differences remained after FDR correction (p = 0.0286 and p = 0.0286, respectively). However, these differences were no longer significant after the Bonferroni correction. In addition, the genotypes for the rs4810485 polymorphisms were associated with parity of the patients with CSCC. The genotypes for the rs3765459 polymorphisms were significantly correlated with the D-dimer of the patients with CSCC. The 3 SNPs genotypes of the CD40 gene were closely related to the squamous cell carcinoma antigen (SCC) of the patients with HSIL. CONCLUSIONS: The CD40 gene may play a role in the occurrence and development of CSCC.

Patterns of lymph node metastasis in level IIB and contralateral level VI for papillary thyroid carcinoma with pN1b and safety of low collar extended incision for neck dissection in level II.

OBJECTIVE: To explore relevant clinical factors of level IIB and contralateral level VI lymph node metastasis and evaluate the safety of low-collar extended incision (LCEI) for lymph node dissection in level II for papillary thyroid carcinoma (PTC) with pN1b. METHOD: A retrospective analysis was performed on 218 patients with PTC with pN1b who were treated surgically in the Head and Neck Surgery Center of Sichuan Cancer Hospital from September 2021 to May 2022. Data on age, sex, body mass index (BMI), tumor location, maximum tumor diameter, multifocality, Braf gene, T staging, surgical incision style, and lymph node metastasis in each cervical subregion were collected. The chi-square test was used for comparative analysis of relevant factors. All statistical analyses were completed by SPSS 24 software. RESULT: Each subgroup on sex, age, BMI, multifocality, tumor location, extrathyroidal extension, Braf gene, and lymphatic metastasis in level III, level IV, and level V had no significant difference in the positive rate of lymph node metastasis in level IIB (P > 0.05). In contrast, patients with bilateral lateral cervical lymphatic metastasis were more likely to have level IIB lymphatic metastasis than those with unilateral lateral cervical lymphatic metastasis, with a statistically significant difference (P = 0.000). In addition, lymph node metastasis in level IIA was significantly associated with lymph node metastasis in level IIB (P = 0.001). After multivariate analysis, lymph node metastasis in level IIA was independently associated with lymph node metastasis in level IIB (P = 0.010). The LCEI group had a similar lymphatic metastasis number and lymphatic metastasis rate in both level IIA and level IIB as the L-shaped incision group (P > 0.05). There were 86 patients with ipsilateral central lymphatic metastasis (78.2%). Patients with contralateral central lymphatic metastasis accounted for 56.4%. The contralateral central lymphatic metastasis rate was not correlated with age, BMI, multifocality, tumor invasion, or ipsilateral central lymphatic metastasis, and there was no significant difference (P > 0.05). The contralateral central lymphatic metastasis in males was slightly higher than that in females, and the difference was statistically significant (68.2% vs. 48.5%, P = 0.041). CONCLUSION: Lymphatic metastasis in level IIA was an independent predictor of lymphatic metastasis in level IIB. When bilateral lateral cervical lymphatic metastasis or lymph node metastasis of level IIA is found, lymph node dissection in level IIB is strongly recommended. When unilateral lateral cervical lymphatic metastasis and lymphatic metastasis in level IIA are negative, lymph node dissection in level IIB may be performed as appropriate on the premise of no damage to the accessory nerve. LCEI is safe and effective for lymph node dissection in level II. When the tumor is located in the unilateral lobe, attention should be given to contralateral central lymph node dissection because of the high lymphatic metastasis rate.

An odorant receptor tuned to an attractive plant volatile vanillin in Spodoptera litura.

The insect olfaction plays crucial roles in many important behaviors, in which ORs are key determinants for signal transduction and the olfactory specificity. Spodoptera litura is a typical polyphagous pest, possessing a large repertoire of ORs tuning to broad range of plant odorants. However, the specific functions of those ORs remain mostly unknown. In this study, we functionally characterized one S. litura OR (OR51) that was highly expressed in the adult antennae. First, by using Xenopus oocyte expression and two-electrode voltage clamp recording system (XOE-TEVC), OR51 was found to be strongly and specifically responsive to vanillin (a volatile of S. litura host plants) among 77 tested odorants. Second, electroantennogram (EAG) and Y-tube behavioral experiment showed that vanillin elicited significant EAG response and attraction behavior especially of female adults. This female attraction was further confirmed by the oviposition experiment, in which the soybean plants treated with vanillin were significantly preferred by females for egg-laying. Third, 3D structural modelling and molecular docking were conducted to explore the interaction between OR51 and vanillin, which showed a high affinity (-4.46 kcal/mol) and three residues (Gln163, Phe164 and Ala305) forming hydrogen bonds with vanillin, supporting the specific binding of OR51 to vanillin. In addition, OR51 and its homologs from other seven noctuid species shared high amino acid identities (78-97%) and the same three hydrogen bond forming residues, suggesting a conserved function of the OR in these insects. Taken together, our study provides some new insights into the olfactory mechanisms of host plant finding and suggests potential applications of vanillin in S. litura control.

Release of moth pheromone compounds from Nicotiana benthamiana upon transient expression of heterologous biosynthetic genes.

BACKGROUND: Using genetically modified plants as natural dispensers of insect pheromones may eventually become part of a novel strategy for integrated pest management. RESULTS: In the present study, we first characterized essential functional genes for sex pheromone biosynthesis in the rice stem borer Chilo suppressalis (Walker) by heterologous expression in Saccharomyces cerevisiae and Nicotiana benthamiana, including two desaturase genes CsupYPAQ and CsupKPSE and a reductase gene CsupFAR2. Subsequently, we co-expressed CsupYPAQ and CsupFAR2 together with the previously characterized moth desaturase Atr∆11 in N. benthamiana. This resulted in the production of (Z)-11-hexadecenol together with (Z)-11-hexadecenal, the major pheromone component of C. suppressalis. Both compounds were collected from the transformed N. benthamiana headspace volatiles using solid-phase microextraction. We finally added the expression of a yeast acetyltransferase gene ATF1 and could then confirm also (Z)-11-hexadecenyl acetate release from the plant. CONCLUSIONS: Our results pave the way for stable transformation of plants to be used as biological pheromone sources in different pest control strategies.

Action recognition based on multimode fusion for VR online platform.

The current popular online communication platforms can convey information only in the form of text, voice, pictures, and other electronic means. The richness and reliability of information is not comparable to traditional face-to-face communication. The use of virtual reality (VR) technology for online communication is a viable alternative to face-to-face communication. In the current VR online communication platform, users are in a virtual world in the form of avatars, which can achieve "face-to-face" communication to a certain extent. However, the actions of the avatar do not follow the user, which makes the communication process less realistic. Decision-makers need to make decisions based on the behavior of VR users, but there are no effective methods for action data collection in VR environments. In our work, three modalities of nine actions from VR users are collected using a virtual reality head-mounted display (VR HMD) built-in sensors, RGB cameras and human pose estimation. Using these data and advanced multimodal fusion action recognition networks, we obtained a high accuracy action recognition model. In addition, we take advantage of the VR HMD to collect 3D position data and design a 2D key point augmentation scheme for VR users. Using the augmented 2D key point data and VR HMD sensor data, we can train action recognition models with high accuracy and strong stability. In data collection and experimental work, we focus our research on classroom scenes, and the results can be extended to other scenes.

Oil-film extinction coefficient inversion based on thickness difference.

The extinction coefficient of oil films on the sea surface was inversion using a physical model based on two-beam interference and the equal-thickness difference method. The coefficient is simplified to a quadratic equation in one variable related to oil-film thickness and incident angle and wavelength of light. Through a laboratory-simulated oil spill experiment, the reflectivities of oil films of different thicknesses were obtained. The extinction coefficients of the oil film under visible light were inversion. The model considered the light beam on the oil-film surface and effects of scattering properties and photon attenuation of the oil film on spectral reflectance.

SYT-SSX1 enhances the invasiveness and maintains stem-like cell properties in synovial sarcoma via induction of TGF-ß1/Smad signaling.

BACKGROUND: Synovial sarcoma (SS) is a type of soft tissue sarcoma (STS) of undetermined tissue origin, which is characterized by the recurrent pathognomonic chromosomal translocation t (X;18)(p11.2; q11.2). Studies have shown that SS is a malignant tumor originating from cancer stem cells or pluripotent mesenchymal stem cells and may be related to fusion genes. In addition, some studies have indicated that the induction of epithelial-mesenchymal transition (EMT) via the TGF-ß1/Smad signaling pathway leads to SS metastasis. METHODS: We analyzed the effects of SYT-SSX1 on the stemness of SS cells via TGF-ß1/Smad signaling in vitro. The SYT-SSX1 fusion gene high expression cell was constructed by lentiviral stable transfer technology. SYT-SSX1 and SW982 cells were cultured and tested for sphere-forming ability. The transwell migration assay and flow cytometry were used to assess the migration ability of the sphere cells as well as the expression of CSC-related markers. We treated SYT-SSX1 cells with rhTGF-ß1 (a recombinant agent of the TGF-ß1 signaling pathway) and SB431542 and observed morphological changes. A CCK-8 experiment and a western blot (WB) experiment were conducted to detect the expression of TGF-ß1 signaling pathway- and EMT-related proteins after treatment. The SYT-SSX1 cells were then cultured and their ability to form spheres was tested. Flow cytometry, WB, and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the expression of CSC surface markers on SYT-SSX1 sphere cells. RESULTS: It was found that SYT-SSX1 has stronger sphere-forming ability, migration ability, and higher expression of CSC-related molecules than SW982 cells. Through treating SYT-SSX1 and SW982 cells with rhTGF-ß1 and SB431542, we found that TGF-ß1 enhanced the proliferation of cells, induced EMT, and that TGF-ß1 enhanced the characteristics of tumor stem cells. CONCLUSIONS: Our results suggest that SYT-SSX1 enhances invasiveness and maintains stemness in SS cells via TGF-ß1/Smad signaling. These findings reveal an effective way to potentially improve the prognosis of patients with SS by eliminating the characteristics of cancer stem cells (CSCs) during treatment.

A novel cocktail therapy based on quintuplet combination of oncolytic herpes simplex virus-2 vectors armed with interleukin-12, interleukin-15, GM-CSF, PD1v, and IL-7 × CCL19 results in enhanced antitumor efficacy.

BACKGROUND: Selectively replicating herpes simplex virus-2 (HSV-2) vector is a promising treatment for cancer therapy. The insertion of multiple transgenes into the viral genome has been performed to improve its oncolytic activity. METHODS: Herein, we simultaneously constructed five "armed" oncolytic viruses (OVs), designated oHSV2-IL12, -IL15, GM-CSF, -PD1v, and IL7 × CCL19. These OVs delete the ICP34.5 and ICP47 genes with the insertion of transgenes into the deleted ICP34.5 locus. The anti-tumor efficacy in vivo was tested in the syngeneic 4T1 and CT26 tumor-bearing mice model. RESULTS: The OVs showed comparable oncolytic capability in vitro. The combination therapy of oHSV2-IL12, -IL15, GM-CSF, -PD1v, and IL7 × CCL19 exhibited the highest tumor inhibition efficacy compared with the treatment of single OV or two OVs combination. CONCLUSIONS: The OVs armed with different transgenes combination therapy also named 5-valent oHSV2 (also called cocktail therapy) might be an effective therapeutic strategy for solid tumors.

Enantioselectivity and key residue of Herbaspirillum huttiense monooxygenase in asymmetric epoxidation of styrenes.

Styrene monooxygenases (SMOs) are powerful enzymes for the synthesis of enantiopure epoxides, but these SMOs have narrow substrate spectra, and the residues in controlling enantioselectivity of SMOs remains unclear. A monooxygenase from Herbaspirillum huttiense (HhMO) was found to have excellent enantioselectivities and diastereoselectivities in the epoxidation of unconjugated terminal alkenes. Here we found that HhMO could also transfer styrene into styrene epoxide with 75% ee, and it could also catalyze the epoxidation of styrene derivatives into the corresponding epoxides with enantioselectivities up to 99% ee. Meanwhile, site 199 in the substrate access channel of HhMO was found to play an important role in the controlling enantioselectivity of the epoxidation. The E199L variant catalyzed the epoxidation of styrene with > 99% ee. The identification of critical residue that affects the enantioselectivity of SMOs would thus be valuable for creating efficient monooxygenases for the preparation of essential enantiopure epoxides. KEY POINTS: • Bioexpoxidation of both conjugated and unconjugated alkenes by HhMO with excellent enantioselectivities. • Gating residue 199 played an essential role in controlling the enantioselectivity of SMO. • HhMO E199L catalyzed the epoxidation of styrenes with up to > 99% ee.

Improved spectral reflectance model of oil films on seawater based on two-beam interference theory.

The theoretical properties of electromagnetic transmission and radiation of light are combined with the propagation law of light in a uniform medium and the Fresnel formula to provide an improved two-beam interference model for simulating oil spills. This model enables the study of spectral reflectance under different thicknesses and different illumination angles. Through observation and analysis of simulation results, it is found that light waves reflected at the interface of water and oil films and of oil films and air oscillate due to interference. In order to overcome this problem, solar irradiance is simulated and multiplied with the reflection model to obtain the outgoing illuminance values at different wavelengths, which are convoluted with the slit function of the hyperspectral camera. The processed results are compared with incoming illuminance values to obtain new simulation results. In addition, based on portable hyperspectral imagery and imported xenon lamp light sources and other experimental equipment, indoor simulated oil spill experiments were conducted. By comparing and analyzing the results of these simulations, the accuracy of the improved model was further improved, demonstrating the feasibility and utility of the model in studying oil spills at water surfaces.

Functional differentiation of three pheromone binding proteins in Orthaga achatina using mixed-type sex pheromones.

Pheromone-binding proteins (PBPs) play important roles in perception of insect sex pheromones, functioning to recognize and transport pheromone components onto the olfactory receptors of the odorant sensing neurons. Orthaga achatina, a serious pest of camphor trees, uses a mixture of three Type I (Z11-16:OAc, Z11-16:OH and Z11-16:Ald) and one Type II (Z3,Z6,Z9,Z12,Z15-23:H) sex pheromone components in its sex communication, in which Z11-16:OAc is the major component and others are minor components. In this study, we for the first time demonstrated that the three PBPs differentiated in recognition among pheromone components in a moth using mixed-type sex pheromones. First, tissue expression study showed that all three PBPs of O. achatina were expressed only in antennae and highly male-biased, suggesting their involvement in perception of the sex pheromones. Second, the three PBPs were expressed in Escherichia coli and the binding affinities of PBPs to four sex pheromone components and some pheromone analogs were determined by the fluorescence competition binding assays. The results showed that OachPBP1 bound all four sex pheromone components with high binding affinity, while OachPBP2 had high or moderate binding affinity only to three Type I components, and OachPBP3 had high binding affinity only to three minor pheromone components. Furthermore, key amino acid residues that bind to sex pheromone components were identified in three PBPs by 3-D structure modeling and ligand molecular docking, predicting the interactions between PBPs and pheromone components. Our study provides a fundamental insight into the olfactory mechanism in moths that use mixed-type sex pheromones.

Tumor radiomics signature for artificial neural network-assisted detection of neck metastasis in patient with tongue cancer.

BACKGROUND AND PURPOSE: To determine the neck management of tongue cancer, this study attempted to construct an artificial neural network (ANN)-assisted model based on computed tomography (CT) radiomics of primary tumors to predict neck lymph node (LN) status in patients with tongue squamous cell carcinoma (SCC). MATERIALS AND METHODS: Three hundred thirteen patients with tongue SCC were retrospectively included and randomly divided into training (60%), validation (20%) and internally independent test (20%) sets. In total, 1673 feature values were extracted after the semiautomatic segmentation of primary tumors and set as input layers of a classical 3-layer ANN incorporated with or without clinical LN (cN) status after dimension reduction. The receiver operating characteristic (ROC) curve, accuracy (ACC), sensitivity (SEN), specificity (SPE), area under curve (AUC) and Net Reclassification Index (NRI), were used to evaluate and compare the models. RESULTS: Four models with different settings were constructed. The ACC, SEN, SPE and AUC reached 84.1%, 93.1%, 76.5% and 0.943 (95% confidence interval: 0.891-0.996, p<.001), respectively, in the test set. The NRI of models compared with radiologists reached 40% (p<.001). The occult nodal metastasis rate was reduced from 30.9% to a minimum of 12.7% in the T1-2 group. CONCLUSION: ANN-based models that incorporated CT radiomics of primary tumors with traditional LN evaluation were constructed and validated to more precisely predict neck LN metastasis in patients with tongue SCC than with naked eyes, especially in early-stage cancer.

Enzymatic Enantioselective anti-Markovnikov Hydration of Aryl Alkenes.

The addition of water to alkenes is an important method for the synthesis of alcohols, but the regioselectivity of acid-catalyzed hydration of terminal alkenes yields secondary alcohols according to Markovnikov's rule, making it difficult to obtain primary alcohols. Here we report a styrene monooxygenase that catalyzes the anti-Markovnikov hydration of the terminal aryl alkenes under anaerobic conditions. This hydration provides primary alcohols in good yields (up to 100 %), excellent anti-Markovnikov regioselectivity (>99 : 1), and good enantiomeric purity (60-83 % ee). Residues Asn46, Asp100, and Asn309 are essential for catalysis suggesting an acid-base mechanism with a carbanion-like intermediate that could account for the anti-Markovnikov regioselectivity. Our work reveals a new enzymatic tool with unusual regioselectivity based on the promiscuous catalytic activity of a monooxygenase.

Numerically modelling the reflectance of a rough surface covered with diesel fuel based on bidirectional reflectance distribution function.

Oil spills have become a problem that negatively affects the oceanic environment and maritime transportation. Optical remote sensing technology is a potential method to monitor oil spills by analyzing the reflectance spectra of oil-polluted and clean water surface. In this paper, a numerical model for the reflectance of a rough oil surface is constructed by combining Fresnel reflection and bidirectional reflectance distribution function (BRDF). The way that visible light is reflected from the rough diesel fuel surface is quantitatively described and discussed based on the reflection theory of electromagnetic waves. The simulation result of the proposed model shows reasonable agreement with experimental measurements. With reliable prediction and a low computational complexity, the proposed model is expected to provide a theorical basis for rapid detection of oil spills on rough sea surfaces using optical remote sensing technology.

Serum interleukin-6 is an indicator for severity in 901 patients with SARS-CoV-2 infection: a cohort study.

BACKGROUND: Interleukin-6 (IL-6) was proposed to be associated with the severity of coronavirus disease 2019 (COVID-19). The present study aimed to explore the kinetics of IL-6 levels, validate this association in COVID-19 patients, and report preliminary data on the efficacy of IL-6 receptor blockade. METHODS: We conducted a retrospective single-institutional study of 901 consecutive confirmed cases. Serum IL-6 concentrations were tested on admission and/or during hospital stay. Tocilizumab was given to 16 patients with elevated IL-6 concentration. RESULTS: 366 patients were defined as common cases, 411 patients as severe, and 124 patients as critical according to the Chinese guideline on diagnosis and treatment of COVID-19. The median concentration of IL-6 was < 1.5 pg/ml (IQR < 1.50-2.15), 1.85 pg/ml (IQR < 1.50-5.21), and 21.55 pg/ml (IQR 6.47-94.66) for the common, severe, and critical groups respectively (P < 0.001). The follow-up kinetics revealed serum IL-6 remained high in critical patients even when cured. An IL-6 concentration higher than 37.65 pg/ml was predictive of in-hospital death (AUC 0.97 [95% CI 0.95-0.99], P < 0.001) with a sensitivity of 91.7% and a specificity of 95.7%. In the 16 patients who received tocilizumab, IL-6 concentrations were significantly increased after administration, and survival outcome was not significantly different from that of propensity-score matched counterparts (n = 53, P = 0.12). CONCLUSION: Serum IL-6 should be included in diagnostic work-up to stratify disease severity, but the benefit of tocilizumab needs further confirmation. Trial registration retrospectively registered.

Purification of low-abundance lysozyme in egg white via free-flow electrophoresis with gel-filtration chromatography.

As an effective separation tool, free-flow electrophoresis has not been used for purification of low-abundance protein in complex sample matrix. Herein, lysozyme in complex egg white matrix was chosen as the model protein for demonstrating the purification of low-content peptide via an FFE coupled with gel fitration chromatography (GFC). The crude lysozyme in egg while was first separated via free-flow zone electrophoresis (FFZE). After that, the fractions with lysozyme activity were condensed via lyophilization. Thereafter, the condensed fractions were further purified via a GFC of Sephadex G50. In all of the experiments, a special poly(acrylamide- co-acrylic acid) (P(AM-co-AA)) gel electrophoresis and a mass spectrometry were used for identification of lysozyme. The conditions of FFZE were optimized as follows: 130 µL/min sample flow rate, 4.9 mL/min background buffer of 20 mM pH 5.5 Tris-Acetic acid, 350 V, and 14 °C as well as 2 mg/mL protein content of crude sample. It was found that the purified lysozyme had the purity of 80% and high activity as compared with its crude sample with only 1.4% content and undetectable activity. The recoveries in the first and second separative steps were 65% and 82%, respectively, and the total recovery was about 53.3%. The reasons of low recovery might be induced by diffusion of lysozyme out off P(AM-co-AA) gel and co-removing of high-abundance egg ovalbumin. All these results indicated FFE could be used as alternative tool for purification of target solute with low abundance.

Functional characterization of two spliced variants of fructose gustatory receptor in the diamondback moth, Plutella xylostella.

Insect gustatory system plays important roles in multiple behaviors including feeding, mating, and oviposition. Gustatory receptors (GRs), located on the dendritic membrane of gustatory sensory neurons (GSNs), are crucial in peripheral coding of non-volatile compounds. However, GRs and their detailed functions remain poorly understood in lepidopteran pests. In the present work, focusing on GR genes of Plutella xylostella, an important worldwide crop pest, we cloned a candidate fructose GR gene that has two spliced variants (PxylGR43a-1 and PxylGR43a-2), and determined the tissue expression profiles by semi-quantitative reverse transcription PCR (RT-PCR). It revealed that both GR variants were highly expressed in antennae and less highly in heads of adults, while PxylGR43a-2 was also weakly expressed in other tested tissues. Functional analyses were further conducted using the Xenopus oocyte system. PxylGR43a-1 and PxylGR43a-2 both responded specifically to the d-fructose among the 12 tested sugar compounds, but PxylGR43a-2 showed much higher current response than PxylGR43a-1. In addition, the proboscis extension reflex (PER) assay was conducted, demonstrating that female moths could respond to d-fructose following stimulation of antenna. Taken together, our study contributes to elucidation of the molecular mechanisms of fructose reception and provides a potential target for development of GR based pest control techniques.