Acoustic ejection tandem mass spectrometry for high-throughput screening of phencyclidine-type substances in urine, including authentic cases.

BACKGROUND: The abuse of the Phencyclidine-type substances, especially ketamine is a serious problem worldwide, and retrospective analysis are important for both the analysis and the identification of forms of drug abuse. The current major analytical methods, while all excellent in terms of accuracy, are time- and reagent-consuming. This depletion is made even more unfortunate by the fact that a large number of samples are negative in retrospective analyses. It is clear that a set of methods that can be analyzed both accurately and quickly need to be developed and applied to the screening and analysis of large quantities of samples. RESULTS: We described a urine test based on acoustic ejection mass spectrometry, which allows precise injection at very low volumes and near 1 ejection s-1 and data acquisition. The confidence in identification was increased by the characterization of the abundance ratio of the two pairs of ions. Urine samples could be diluted with water and loaded into a 384-well plate for sampling without complicated sample preparation. The sample in the transparent 384-well plate was pre-scanned by the laser, and then 20 nL droplets were ejected into the ion source for targeted analysis of 2 ion transitions per droplet totaling 9 targeted analytes in the sequence of acquisition methods. It took 90 min to screen 250 samples in this approach, yielding 10 ng mL-1 detection limits. Positive samples were further analyzed by UHPLC-MS/MS for confirmation and quantification of up to 36 analytes. SIGNIFICANCE: This was the first fast screening method for phencyclidine-type substances based on acoustic ejection mass spectrometry, which greatly reduces the analytical time, and can accomplish in 1.5 h what UHPLC-MS/MS needs 3 days to complete. And the samples can be analyzed without complicated sample preparation, and also can obtain good detectability. It was applied to a short-term retrospective analysis in Shanghai, and its accuracy was also extremely high.

Screening and effects of intestinal probiotics on growth performance, gut health, immunity, and disease resistance of Nile tilapia (Oreochromis niloticus) against Streptococcus agalactiae.

In the present study, 59 autochthonous bacteria were isolated from the intestine of tilapia. Following enzyme producing activity, antagonistic ability, hemolytic activity, drug sensitivity assessments, and in vivo safety evaluation, 7 potential probiotic strains were screened out: Bacillus tequilensis BT0825-2 (BT), Bacillus aryabhattai BA0829-3 (BA1), Bacillus megaterium BM0505-6 (BM), Bacillus velezensis BV0505-11 (BV), Bacillus licheniformis BL0505-18 (BL), B. aryabhattai BA0505-19 (BA2), and Lactococcus lactis LL0306-15 (LL). Subsequently, tilapia were fed basal diets (CT) and basal diets supplemented with 108 CFU/g of BT, BA1, BM, BV, BL, BA2 and LL, respectively. After 56 days of continuous feeding, the growth parameters (weight gain, final weight, and specific growth rate) showed significant improvement (p < 0.05) in both BM and BA2 groups. The total cholesterol and triglycerides of serum were significantly decreased in BV and LL groups (p < 0.05). The superoxide dismutase, glutathione reductase, and lysozyme of BV, BA2 and LL groups were increased, and the malondialdehyde of BV group was significantly decreased. The villous height and amylase of midgut were increased in BV, BA2 and LL groups. In addition, the expression levels of ZO-1 and occludin genes in the midgut of tilapia were enhanced in BM, BV, BA2 and LL groups. The supplementation of probiotics reduced the abundance of Cyanobacteria and increased the abundance of Actinobacteria at the phylum level. At the genus level, the addition of probiotics increased the abundance of Romboutsia. Furthermore, improvement in the expression of immune-related genes were observed, including interleukin 1ß, interleukin 10, tumor necrosis factor alpha, and transforming growth factor beta (p < 0.05). After challenging with S. agalactiae, the survival rates of BV, BA2 and LL groups were significantly higher than CT group (p < 0.05). Above results indicated that BM, BA2, BV and LL improved growth performance, gut health or immunity of tilapia, which can be applied in tilapia aquaculture.

Quantitative determination of nitrous oxide in human blood by HS-GC-MS: forensic application of two fatal poisoning cases.

Nitrous oxide (N2O), also known as laughing gas, has a euphoric effect and is becoming increasingly popular as a recreational inhalant drug. Deaths caused by recreational nitrous oxide abuse are rare, but may still occur. Although some methods for the quantification of N2O by GC-MS have been reported, elimination of carbon dioxide interference and the choice of a suitable internal standard remain current limitations to accurate N2O quantification. Here, a validated method using headspace-gas chromatography-mass spectrometry (HS-GC-MS) is described that allows the quantification of N2O in human blood samples: sodium hydroxide is used to remove carbon dioxide, and n-pentane is chosen as a suitable internal standard. Collectively, the validation results show a good linear relationship of N2O in blood within the concentration range of 0.02 ∼ 0.5 mL/mL and an LOD of 0.005 mL/mL. Subsequent application of the validated method to two real mortality cases due to N2O intoxication provided reference values for blood concentrations in forensic cases. Other biological specimens (gaseous samples and tissues) of the deceased were also analyzed to demonstrate that the deaths were caused by asphyxia due to the inhalation of N2O.

Application of atomic-scale mechanistic insights into carbon-catalyzed N2O reduction for kinetic modeling construction.

To achieve the collaborative elimination of N2O and carbon of potent greenhouse pollutants from automotive mobile sources, a chemical kinetic model is developed to accurately track the heterogeneous process of carbon-catalyzed N2O reduction based on density functional theory, with experimental data used to validate the model's reliability. The influence of carbon structure, site density, and surface chemical properties on N2O catalytic reduction can be analyzed within this system. Results reveal that the free-edge site of carbon accurately describes the catalytic reduction process of N2O. Adsorption of N2O to carbon edges in O-down, N-down, or parallel orientations exhibits an exothermic process with energy barriers. The N2O with O-down reduction pathway predominates due to the limitations imposed by the unitary carbon site. As the number of active carbon atoms at carbon edges increases, the N2O reaction mode tends towards parallel and N-down pathways, resulting in a significant enhancement of N2O conversion rates and a reduction in catalytic temperatures, with the lowest achievable temperature being 300 K. Furthermore, the triplet carbon structure exhibits higher efficiency in N2O catalytic reduction compared to the singlet carbon structure, achieving a remarkable N2O conversion rate of 93.8 % within the typical temperature exhaust window of diesel engines. This study supplies a breakthrough for carbon materials as catalysts for achieving high N2O conversion rates at low cost, which is important for the collaborative catalytic elimination of N2O and carbon black pollutants.

A sensitive fluorescence biosensor based on ligation-transcription and CRISPR/Cas13a-assisted cascade amplification strategies to detect the H1N1 virus.

We propose a sensitive H1N1 virus fluorescence biosensor based on ligation-transcription and CRISPR/Cas13a-assisted cascade amplification strategies. Products are generated via the hybridization of single-stranded DNA (ssDNA) probes containing T7 promoter and crRNA templates to a target RNA sequence using SplintR ligase. This generates large crRNA quantities in the presence of T7 RNA polymerase. At such crRNA quantities, ternary Cas13a, crRNA, and activator complexes are successfully constructed and activate Cas13a to enhance fluorescence signal outputs. The biosensor sensitively and specifically monitored H1N1 viral RNA levels down to 3.23 pM and showed good linearity when H1N1 RNA concentrations were 100 pM-1 µM. Biosensor specificity was also excellent. Importantly, our biosensor may be used to detect other viral RNAs by altering the sequences of the two probe junctions, with potential applications for the clinical diagnosis of viruses and other biomedical studies.

Fluorescence biosensor to detect microRNAs via integrating DNA hairpins transition mediated strand displacement amplification with primer exchange reaction.

Herein, we constructed a fluorescence biosensor for the ultra-sensitive analysis of microRNAs (miRNAs) by combining DNA hairpins transition triggered strand displacement amplification (DHT-SDA) with primer exchange reaction (PER). Target miRNA initiated DHT-SDA to facilitate the generation of multiple single-stranded DNA (ssDNA) as PER primer, which was extended into a long ssDNA. The biosensor is successfully utilized in detecting miRNAs with high sensitivity (limit of detection for miRNA-21 was 58 fM) and a good linear relationship between 100 nM and 100 fM. By simply changing the DNA hairpin sequence, the constructed biosensor can be extended to analyze another miRNAs. Moreover, the biosensor has the feasibility of detecting miRNAs in real samples with satisfactory accuracy and reliability. Therefore, the fluorescent biosensor has great application potential in clinical diagnosis.

Imidazolium-Functionalized Conjugated Polymer for On-Site Visual and Ultrasensitive Detection of Toxic Hexavalent Chromium.

Hexavalent chromium [Cr(VI)] is considered a serious environmental pollutant that possesses a hazardous effect on humans even at low concentrations. Thus, the development of a bifunctional material for ultratrace-selective detection and effective elimination of Cr(VI) from the environment remains highly desirable and scarcely reported. In this work, we explore an imidazolium-appended polyfluorene derivative PF-DBT-Im as a highly sensitive/selective optical probe and a smart adsorbent for Cr(VI) ions with an ultralow detection limit of 1.77 nM and removal efficiency up to 93.7%. In an aqueous medium, PF-DBT-Im displays obvious transformation in its emission color from blue to magenta on exclusively introducing Cr(VI), facilitating naked-eye colorimetric detection. Consequently, a portable sensory device integrated with a smartphone is fabricated for realizing real-time and on-site visual detection of Cr(VI). Besides, the imidazolium groups attached onto side chains of PF-DBT-Im are found to be highly beneficial for achieving selective and efficient elimination of Cr(VI) with capacity as high as 128.71 mg g-1. More interestingly, PF-DBT-Im could be easily regenerated following treatment with KBr and can be recycled at least five times in a row. The main factor behind ultrasensitive response and excellent removal efficiency is found to be anion-exchange-induced formation of a unique ground-state complex between PF-DBT-Im and Cr(VI), as evident by FT-IR, XPS, and simulation studies. Thus, taking advantage of the excellent signal amplification property and rich ion-exchange sites, a dual-functional-conjugated polymer PF-DBT-Im is presented for the concurrent recognition and elimination of Cr(VI) ions proficiently and promptly with great prospects in environmental monitoring and water decontamination.

Hair-based rapid UPLC-MS/MS analysis of 36 phencyclidine-type substances in forensic cases.

Phencyclidine (PCP) is a frequently abused dissociative agent. It causes confusion, increased tendencies toward violence, and concentration-dependent cytotoxicity after entry into the body. The parent nucleus of phencyclidine-type substances is arylcyclohexylamine, which is easy to modify; therefore, abusers and dealers can readily synthesize substitutes beyond the drug control catalog. An urgent need exists to establish screening methods for phencyclidine-type substances to provide technical support for abuse monitoring. In this study, 20 mg of hair was pulverized in 500 mL of methanol containing 0.5 ng/mL PCP-d5. After ultrasonication, centrifugation, and filtration, the supernatant was analyzed by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) operating in the multiple reaction monitoring mode. Phencyclidine-type substances were separated in 13 min on a biphenyl column using a mobile phase gradient composed of A (water, formic acid 0.1%, acetonitrile 5%, 20 mmol/L ammonium acetate) and B (acetonitrile). The developed and validated method showed good selectivity, sensitivity (limit of detection: 0.25-2 pg/mg and lower limit of quantitation: 0.5-4 pg/mg), linearity (R2 > 0.994), accuracy, and precision (< 20%), and a dilution effect. The method also showed good recovery and acceptable matrix effects for most of the targeted compounds. This analytical approach was successfully applied for the identification and quantification of phencyclidine-type substances in hair from 87 authentic forensic cases. Nine analytes were detected: ketamine (10.3-26211.3 pg/mg), 2-F-2-oxo-PCE (11.5-4034.9 pg/mg), 2-FDCK (14.0-43290.2 pg/mg), 2-BrDCK (10.6-21170.0 pg/mg), nor2-FDCK (10.1-16767.4 pg/mg), tiletamine (10.1-3250.8 pg/mg), O-PCE (43.3-166.1 pg/mg), DCK (10.2-90.4 pg/mg), and norDCK (24.9-103.0 pg/mg).

Evaluation of the suitability of elderly care in prefecture-level cities in China based on GIS.

The population of China is aging, and the demand for healthy elderly care is expanding. There is an urgent need to develop a market-oriented elderly care industry and cultivate a number of high-quality elderly care bases. The geographical environment is an important condition affecting the health of elderly individuals and the suitability of elderly care. Research on this topic has important guiding significance for the layout of elderly care bases and the choice of elderly care locations. In this study, a spatial fuzzy comprehensive evaluation was conducted to construct an evaluation index system based on the following standard layers: climatic conditions, topography, surface vegetation, atmospheric environment, traffic conditions, economy and population, elderly-friendly urban environments, elderly care service capabilities, and wellness and recreation resources. The index system analyzes the suitability of elderly care in 4 municipalities and 333 prefecture-level administrative regions in China, and development and layout suggestions are proposed. The results show the following: (1) The three concentrated areas with a highly suitable geographical environment for elderly care in China are the Yangtze River Delta, the Yunnan-Guizhou-Sichuan region and the Pearl River Delta. The areas with the most concentrated unsuitable areas are the southern Xinjiang and Qinghai-Tibet areas. (2) In areas with a geographical environment that is highly suitable for elderly care, high-end elderly care industries can be deployed, and national-level elderly care demonstration bases can be built. Areas with a highly suitable temperature in Central and Southwest China can develop characteristic elderly care bases for people with cardiovascular and cerebrovascular diseases. Scattered areas with a highly suitable temperature and humidity can develop characteristic elderly care bases for people with rheumatic and respiratory diseases.

Terrestrial transect study on pattern and driving mechanism of ecosystem services in the China-Mongolia-Russia Economic Corridor.

This study quantified the gradient characteristics, trade-off/synergy relationships, and spatiotemporal changes in five key ecosystem services on the meridional (east-west transect of the Siberian Railway (EWTSR) and zonal (north-south transect of Northeast Asia (NSTNEA)) transects of the China-Mongolia-Russia Economic Corridor from 1992 to 2020. The results showed significant regional differentiation of ecosystem services. The improvement in ecosystem services in the EWTSR was significantly greater than in the NSTNEA, and the synergy between water yield and food production in the EWTSR improved the most from 1992 to 2020. The relationship between ecosystem services and different levels of dominant factors was significant, and population expansion had the greatest impact on the trade-off between habitat quality and food production. The leading drivers of ecosystem services in the NSTNEA were normalized vegetation index, population density, and precipitation. This study helps reveal regional differentiation characteristics and drive factors of ecosystem services in Eurasia.

A kinetics mechanism of NOX formation and reduction based on density functional theory.

NOX are serious pollutants emitted during combustion, which are greatly harmful to human health and the environment. However, previous studies have not accurately elucidated the NOX conversion mechanism in complicated combustion reactions. To reveal the micro-chemical mechanism of NOX conversion and obtain accurate kinetics data, advanced quantum chemistry methods are employed in this study to systematically explore the pathways of NOX formation and reduction, and determine the new rate coefficients. An energy barrier analysis revealed that during NOX formation (N2 → N2O → NO→NO2), NO is primarily produced by a sequence of reactions (N2 + O → N2O → NO) rather than the traditional reaction (O + N2 → NO+N). Meanwhile, NO2 formation (NO→NO2) largely depends on the O and HO2 radicals, while the active O atom can promote both the formation and destruction of NO2. During NOX reduction (NO2 → NO→N2O → N2), NO2 reduction (NO2 → NO) is closely related to H, CO, and O, whereas CO plays a critical role in NO2 destruction. However, NO reduction (NO→N2O) is unfavourable because of a high energy barrier, while N2O reduction (N2O → N2) is strongly affected by the O atom instead of CO. HONO is mainly formed when NO2 reacts with the HO2 and H radicals, and when NO reacts with OH radicals; thus, HONO consumption largely depends on OH and H radicals. Based on the transition state theory, we obtained new kinetic parameters for NOX conversion, which supplement and correct critical kinetics data obtained from the current NOX model. Performance assessment of the proposed NOX kinetic mechanism reveals that it can improve the existing NOX kinetic mode, which is in good agreement with experimental data.

The impact of carbon emission reduction inputs of power generation enterprises on the cost of equity capital.

This paper aims to encourage power generation enterprises to actively carry out emission reduction activities by studying the impact of carbon emission reduction investment on long-term development. The existing literature mainly studies the interaction between national economic development and energy consumption at the macro level, and the impact of environmental protection investment by enterprises at the micro level on the short- and long-term economic consequences. However, there is still a large space for research on how specific emission reduction measures lead the long-term development of enterprises. Therefore, this paper chooses to introduce enterprise competitiveness, government subsidy, and management shareholding ratio respectively to conduct intermediary test and grouping regression, and empirically studies the mechanism of carbon emission reduction input of Chinese power generation enterprises on the cost of equity capital from 2013 to 2020. The results show that management shareholding can enhance the initiative of enterprises to reduce emissions. At the same time, carbon emission reduction investment can effectively reduce the cost of equity capital by improving the competitiveness of enterprises and forming a reputation mechanism faster. This study reveals the significance of carbon emission reduction investment as an important part of enterprise environmental protection investment, and emphasizes that the government and enterprises can effectively avoid the short-sighted tendency of enterprises and improve the long-term development power of enterprises by adjusting government subsidies, management shareholding ratio and other policies.

A database of water chemistry in eastern Siberian rivers.

Permafrost degradation leads to considerable changes in river ecosystems. The Eastern Siberian River Chemistry (ESRC) database was constructed to create a spatially extensive river chemistry database to assess climate warming-induced changes in freshwater systems in permafrost-dominated eastern Siberia. The database includes 9487 major ion (Na+, K+, Ca2+, Mg2+, Cl-, SO42- and HCO3-) data of chemical results from 1434 water samples collected mainly in six large river basins in eastern Siberia spanning 1940-2019. Data were obtained from public databases, scientific literature in English and Russian, and researchers and were formatted with a consistent table structure. The database is transparent and reproducible. Climate variable (air temperature and precipitation) data, discharge data, trace element concentration data, and isotope data at the basin and subbasin scales are also provided. This database enhances knowledge about the water chemistry of the permafrost region, especially in eastern Siberia, where data are scarce. The database will be useful to those assessing spatiotemporal changes in river water chemistry associated with permafrost degradation or other environmental stressors in a warmer climate.

Immunogenicity and safety of two novel human papillomavirus 4- and 9-valent vaccines in Chinese women aged 20-45 years: A randomized, blinded, controlled with Gardasil (type 6/11/16/18), phase III non-inferiority clinical trial.

BACKGROUND: Human papillomavirus (HPV) infections were the main cause of anogenital cancers and warts. HPV 6/11/16/18 vaccines provide protection against the high-risk types of HPV responsible for 70% of cervical cancers and 90% of genital warts. This randomized, blinded, non-inferiority phase III trial was to determine whether immunogenicity and tolerability would be non-inferior among women after receiving two novel 4- and 9-valent HPV vaccines (4vHPV, HPV 6/11/16/18; 9vHPV, HPV 6/11/16/18/31/33/45/52/58) compared with those receiving Gardasil 4 (4-valent). METHODS: 1680 females between 20 and 45 years were randomized in a 2:1:1 ratio to 20-26, 27-35, or 36-45 y groups. Subjects then equally assigned to receive 4vHPV, 9vHPV or Gardasil 4 (control) vaccine at months 0, 2, and 6. End points included non-inferiority of HPV-6/11/16/18 antibodies for 4vHPV versus control, and 9vHPV versus control and safety. The immunogenicity non-inferiority was pre-defined as the lower bound of 95% confidence interval (CI) of seroconversion rate (SCR) difference > -10% and the lower bound of 95% CI of geometric mean antibody titer (GMT) ratio > 0.5. RESULTS: Among the three vaccine groups, more than 99% of the participants seroconverted to all 4 HPV types. The pre-specified statistical non-inferiority criterion for the immunogenicity hypothesis was met: all the lower bounds of 95% CIs on SCR differences exceeded -10% for each vaccine HPV type and the corresponding lower bounds of 95% CIs for GMT ratios > 0.5. Across vaccination groups, the most common vaccination reaction were injection-site adverse events (AEs), including pain, swelling, and redness. General and serious AEs were similar in the three groups. There were no deaths. CONCLUSIONS: This study demonstrated that the novel 4- and 9-valent HPV vaccination was highly immunogenic and generally well tolerated, both of which were non-inferior to Gardasil 4 in immunogenicity and safety.

Impact of Education for Sustainable Development on Cognition, Emotion, and Behavior in Protected Areas.

Education for sustainable development (ESD) of protected areas is proposed to deal with global climate change and biodiversity conversation. It focuses on the "quality education" and "protection" of the United Nations' sustainable development goals (UN SDGs), not only taking protected areas as the education place, but also as the theme and content of education. Based on cognitive-behavior theory and social emotional learning theory, this study constructs a "cognitive-emotion-behavior" dimension framework of ESD in protected areas, selecting Potatso National Park in Yunnan as a case study. Based on 529 valid visitor questionnaires, this study uses structural equation modeling to verify theoretical hypotheses, and analyzes the impact of ESD in protected areas on public cognition, emotion, and behavior. The results show that: (1) Cognitive and emotional factors jointly drive the behavioral intentions of ESD in protected areas, and social-emotional factors are slightly higher than cognitive factors; (2) Environmental knowledge, personal norms, nature connectedness, and places attachment positively affects behavioral intentions; (3) Indigenous knowledge has an impact on behavioral intentions through emotional mediation, and personal norms have an impact on behavioral intentions through direct effects; (4) Gender and visit frequency are important moderating variables in the ESD of protected areas. These conclusions provide the following suggestions for further development of ESD. First, by forming environment-friendly social norms and focusing on the mining and presentation of indigenous knowledge, the behavioral intention can also be enhanced to a certain extent; second, improving people's emotion can also promote people's behavioral intention, especially referring to optimizing nature connectedness, strengthening place attachment, and creating emotional connections; Third, specific groups of people should be taught specifically, and improve the supporting services of ESD.

Comparison of Life Cycle Environmental Impact between Two Processes for Silver Separation from Copper Anode Slime.

The cost of silver separation is lowered when ammonia and hydrazine hydrate are replaced with sodium thiosulfate and sodium dithionite in the process of extracting of metallic silver from copper anode slime. The overall environmental impact of two types of copper silver separation processes from anode slime has been analyzed\using the LCA method. Through the subdivision analysis, we found the raw materials or emission items that should be improved first. The following conclusions are drawn: (1) The life cycle environmental impact of the sodium thiosulfate process is much lower than the existing process; (2) The resource and environmental impacts of the sodium thiosulfate method are mainly in the fields of climate change, photochemical smog, and ionizing radiation, exceeding two-thirds of the impact on all of the resources and environment; (3) In terms of input and output items, the main impact of the new process on the resources and the environment is concentrated on the use of sodium hydroxide, accounting for 33.98% of the total equivalent, followed by sodium thiosulfate and sodium carbonate, respectively. These input-output items are the key fields that need attention in future technology improvement.

Eco-Efficiency and Its Drivers in Tourism Sectors with Respect to Carbon Emissions from the Supply Chain: An Integrated EEIO and DEA Approach.

Eco-efficiency analysis can provide useful information about sustainability in the tourism industry, which has an important role in both global economy recovery and Sustainable Development Goals (SDGs), generating considerable indirect carbon emissions with respect to the supply chain due to its significant connections to other industries. This study, from the perspective of tourism sectors, including tourism hotels, travel agencies, and scenic spots, integrated the environmentally extended input-output analysis (EEIO) and data envelopment analysis (DEA) models to develop a research framework, analyzing the indirect carbon emissions of the tourism supply chain, evaluating eco-efficiency with respect to both direct carbon emissions and total carbon emissions (including direct and indirect parts), and exploring the driving factors of eco-efficiency of tourism sectors using Tobit regression models. This study took Gansu as a case, a province in China characterized by higher carbon intensity, an underdeveloped economy, and rapid tourism growth. The results demonstrate that (1) tourism hotels contribute the most carbon emissions in tourism sectors, especially indirectly due to the supply chain, with carbon emissions mainly resulting from the manufacturing of food and tobacco; (2) the eco-efficiency of tourism sectors in Gansu presents a U-shaped curve, which is consistent with Kuznets' theory; and (3) energy technology is key to improving the eco-efficiency of tourism sectors. The research results provide a clear path for the reduction of carbon emissions and the improvement of eco-efficiency in Gansu tourism sectors. Against the backdrop of global climate change and the post-COVID-19 era, our research framework and findings provide a reference for similar regions and countries who are in urgent need of rapid tourism development to effect economic recovery.

Mediator Engineering of Saccharomyces cerevisiae To Improve Multidimensional Stress Tolerance.

Saccharomyces cerevisiae is a well-performing workhorse in chemical production, which encounters complex environmental stresses during industrial processes. We constructed a multiple stress tolerance mutant, Med15V76R/R84K, that was obtained by engineering the KIX domain of Mediator tail subunit Med15. Med15V76R/R84K interacted with transcription factor Hap5 to improve ARV1 expression for sterol homeostasis for decreasing membrane fluidity and thereby enhancing acid tolerance. Med15V76R/R84K interacted with transcription factor Mga2 to improve GIT1 expression for phospholipid biosynthesis for increasing membrane integrity and thereby improving oxidative tolerance. Med15V76R/R84K interacted with transcription factor Aft1 to improve NFT1 expression for inorganic ion transport for reducing membrane permeability and thereby enhancing osmotic tolerance. Based on this Med15 mutation, Med15V76R/R84K, the engineered S. cerevisiae strain, showed a 28.1% increase in pyruvate production in a 1.0-L bioreactor compared to that of S. cerevisiae with its native Med15. These results indicated that Mediator engineering provides a potential alternative for improving multidimensional stress tolerance in S. cerevisiae. IMPORTANCE This study identified the role of the KIX domain of Mediator tail subunit Med15 in response to acetic acid, H2O2, and NaCl in S. cerevisiae. Engineered KIX domain by protein engineering, the mutant strain Med15V76R/R84K, increased multidimensional stress tolerance and pyruvate production compared with that of S. cerevisiae with its native Med15. The Med15V76R/R84K could increase membrane related genes expression possibly by enhancing interaction with transcription factor to improve membrane physiological functions under stress conditions.

Characterization of an intracellular humanized single-chain antibody to matrix protein (M1) of H5N1 virus.

We developed a human intracellular antibody based on the M1 protein from avian influenza virus H5N1 (A/meerkat/Shanghai/SH-1/2012) and then characterized the properties of this antibody. The M1 protein sequence was amplified by RT-PCR using the cDNA of the H5N1 virus as a template, expressed in bacterial expression system BL21 (DE3) and purified. A human strain, high affinity, and single chain antibody (HuScFv) against M1 protein was obtained by phage antibody library screening using M1 as an antigen. A recombinant TAT-HuScFv protein was expressed by fusion with the TAT protein transduction domain (PTD) gene of HIV to prepare a human intracellular antibody against avian influenza virus. Further analysis demonstrated that TAT-HuScFv could inhibit the hemagglutination activity of the 300 TCID50 H1N1 virus, thus providing preliminary validation of the universality of the antibody. After two rounds of M1 protein decomposition, the TAT-HuScFv antigen binding site was identified as Alanine (A) at position 239. Collectively, our data describe a recombinant antibody with high binding activity against the conserved sequences of avian influenza viruses. This intracellular recombinant antibody blocked the M1 protein that infected intracellular viruses, thus inhibiting the replication and reproduction of H5N1 viruses.