Fate and Transport of Mercury through Waterflows in a Tropical Rainforest.

Knowledge gaps of mercury (Hg) biogeochemical processes in the tropical rainforest limit our understanding of the global Hg mass budget. In this study, we applied Hg stable isotope tracing techniques to quantitatively understand the Hg fate and transport during the waterflows in a tropical rainforest including open-field precipitation, throughfall, and runoff. Hg concentrations in throughfall are 1.5-2 times of the levels in open-field rainfall. However, Hg deposition contributed by throughfall and open-field rainfall is comparable due to the water interception by vegetative biomasses. Runoff from the forest shows nearly one order of magnitude lower Hg concentration than those in throughfall. In contrast to the positive Δ199Hg and Δ200Hg signatures in open-field rainfall, throughfall water exhibits nearly zero signals of Δ199Hg and Δ200Hg, while runoff shows negative Δ199Hg and Δ200Hg signals. Using a binary mixing model, Hg in throughfall and runoff is primarily derived from atmospheric Hg0 inputs, with average contributions of 65 ± 18 and 91 ± 6%, respectively. The combination of flux and isotopic modeling suggests that two-thirds of atmospheric Hg2+ input is intercepted by vegetative biomass, with the remaining atmospheric Hg2+ input captured by the forest floor. Overall, these findings shed light on simulation of Hg cycle in tropical forests.

Vertical variation in leaf functional traits of Parashorea chinensis with different canopy layers.

Introduction: Canopy species need to shift their ecological adaptation to improve light and water resources utilization, and the study of intraspecific variations in plant leaf functional traits based at individual scale is of great significance for evaluating plant adaptability to climate change. Methods: In this study, we evaluate how leaf functional traits of giant trees relate to spatial niche specialization along a vertical gradient. We sampled the tropical flagship species of Parashorea chinensis around 60 meters tall and divided their crowns into three vertical layers. Fourteen key leaf functional traits including leaf morphology, photosynthetic, hydraulic and chemical physiology were measured at each canopy layer to investigate the intraspecific variation of leaf traits and the interrelationships between different functional traits. Additionally, due to the potential impact of different measurement methods (in-situ and ex-situ branch) on photosynthetic physiological parameters, we also compared the effects of these two gas exchange measurements. Results and discussion: In-situ measurements revealed that most leaf functional traits of individual-to-individual P. chinensis varied significantly at different canopy heights. Leaf hydraulic traits such as midday leaf water potential (MWP) and leaf osmotic potential (OP) were insignificantly correlated with leaf photosynthetic physiological traits such as maximal net assimilation rate per mass (A mass). In addition, great discrepancies were found between in-situ and ex-situ measurements of photosynthetic parameters. The ex-situ measurements caused a decrease by 53.63%, 27.86%, and 38.05% in A mass, and a decrease of 50.00%, 19.21%, and 27.90% in light saturation point compared to the in-situ measurements. These findings provided insights into our understanding of the response mechanisms of P. chinensis to micro-habitat in Xishuangbanna tropical seasonal rainforests and the fine scale adaption of different resultant of decoupled traits, which have implications for understanding ecological adaption strategies of P. chinensis under environmental changes.

Facile Synthesis of Hollow Fe3O4-rGO Nanocomposites for the Electrochemical Detection of Acetaminophen.

Acetaminophen (AC) is one of the most popular pharmacologically active substances used as an analgesic and antipyretic drug. Herein, a new type of hollow Fe3O4-rGO/GCE electrode was prepared for electrochemical detection of AC through a three-step approach involving a solvothermal method for the synthesis of hollow Fe3O4 and the chemical reduction of graphene oxide (GO) for reduced graphene oxide (rGO) and Fe3O4-rGO nanocomposites modified on the glassy carbon electrode (GCE) surface. The as-prepared Fe3O4-rGO nanocomposites were characterized using a transmission electron microscope (TEM), X-ray diffraction (XRD), and a magnetic measurement system (SQUID-VSM). The magnetic Fe3O4-rGO/GCE electrodes were employed for the electrochemical detection of AC using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and square wave voltammetry (SWV) and exhibited an ultra-high selectivity and accuracy, a low detection limit of 0.11 µmol/L with a wider linear range from 5 × 10-7 to 10-4 mol/L, and high recovery between 100.52% and 101.43%. The obtained Fe3O4-rGO-modified GCE displays great practical significance for the detection of AC in drug analysis.

China Requires a Sustainable Transition of Vegetable Supply from Area-Dependent to Yield-Dependent and Decreased Vegetable Loss and Waste.

China, the largest country in vegetable supply, faces a transition to sustainable vegetable production to counteract resource waste and environmental pollution. However, there are knowledge gaps on the main constraints and how to achieve sustainable vegetable supply. Herein, we integrated the vegetable production and supply data in China, compared its current status with five horticulture-developed countries US, the Netherlands, Greece, Japan and South Korea, using data from the Food and Agriculture Organization (FAO) and National Bureau of Statistics of China, and predicted the vegetable supply in 2030 and 2050 by a model prediction. The vegetable supply in China increased from 592 g capita-1 d-1 in 1995 to 1262 g capita-1 d-1 in 2018 and will keep constant in 2030 and 2050. Compared to the five countries, the greater vegetable supply is primarily achieved by higher harvested areas rather than higher yield. However, it is predicted that the harvested areas will decrease by 13.6% and 24.7% in 2030 and 2050. Instead, steady increases in vegetable yield by 11.8% and 28.3% are predicted for this period. The high vegetable supply and greater vegetable preference indicated by the high vegetable-to-meat production ratio cannot guarantee recommended vegetable intake, potentially due to the high rate of vegetable loss and waste. Under the scenarios of decreased vegetable loss and waste, the harvested area will decrease by 37.3-67.2% in 2030 and 2050. This study points out that the sustainable transition of Chinese vegetable supply can be realized by enhancing yield and limiting vegetable loss and waste instead of expanding the harvested area.

Elevated CO2 improves phosphorus nutrition and growth of citrate-secreting wheat when grown under adequate phosphorus supply on an Al3+ -toxic soil.

BACKGROUND: Understanding how climate change affects the phosphorus (P) nutrition of crops grown on acid soils is important in optimizing the management of P, and to secure future food production on these soils. This study assessed the impact of elevated CO2 (eCO2 ) on the P nutrition of wheat (Triticum aestivum) grown on Al3+ -toxic and P-deficient soils or in hydroponics. The aluminium-resistant near-isogenic wheat lines EGA-Burke (malate efflux only) and EGA-Burke TaMATE1B (malate and citrate efflux) were grown under ambient (400 µmol mol-1 ) and elevated CO2 (800 µmol mol-1 ) in growth chambers for 4-6 weeks. RESULTS: Elevated CO2 enhanced shoot growth and total P uptake of both lines at P rates >250 mg kg-1 , which was associated with improved root biomass allocation and thus increased root growth, but these effects were not apparent at lower P rates. Elevated CO2 decreased specific P uptake (P uptake per unit root length) at P supply >250 mg kg-1 , but did not significantly affect external or internal P requirements. This effect on the specific P uptake was less for EGA-Burke TaMATE1B than for EGA-Burke, possibly due to the increased citrate efflux and decreased Al concentration in root tips of EGA-Burke TaMATE1B. Compared to EGA-Burke, citrate-exuding EGA-Burke TaMATE1B had greater shoot P concentration and greater specific P uptake. CONCLUSION: Elevated CO2 improved root growth, and thus total P uptake and plant production of both lines when high P alleviated Al3+ toxicity and improved P nutrition in acid soils. The decreased P uptake efficiency under eCO2 was less for EGA-Burke TaMATE1B than EGA-Burke. © 2022 Society of Chemical Industry.

Dose-Dependent Application of Straw-Derived Fulvic Acid on Yield and Quality of Tomato Plants Grown in a Greenhouse.

Fulvic acids are organic compounds widely distributed in soils, and the application of fulvic acids is thought to increase crop yield and quality. However, the effects vary among various sources and doses of fulvic acids and environmental and growth conditions of crops. Here, we investigated the effects of bioresource-derived (corn straw) fulvic acids on plant production and quality of tomato plants and soil chemical properties in soil cultures while experiments on seed germination and hydroponics were conducted to explore the underlying mechanism. Base dressing with 2.7 g kg-1 increased the yield of tomato by 35.0% at most as increased fruit number. Fulvic acids increased the concentrations of minerals, such as Ca, Fe, and Zn and the concentrations of citric, malic, and some amino acids in berries of tomato but did not affect the concentrations of soluble sugars and aromatic substances in tomato fruits. Similarly, fulvic acids at 80-160 mg L-1 increased germination rate, growth vigor, and radicle elongation of tomato seeds while it increased plant biomass, concentrations of nutrients, and root length of tomato plants in hydroponics to the greatest extent in general. The increases in yield and quality can be attributed to the improvement in root growth and, thus, increased nutrient uptake. In addition, the base application of fulvic acids improved soil cation exchange capacity and soil organic matter to an extent. In conclusion, base dressing and the addition into solution of fulvic acids at moderate doses facilitate root growth and nutrient uptake and, thus, vegetable production and quality; therefore, fulvic acids can be an effective component for designing new biofertilizers for sustainable agricultural production.

Heavy metals and health risk of rice sampled in Yangtze River Delta, China.

Dietary exposure to heavy metals is threatening human health worldwide. In this study, the concentration of cadmium, mercury, arsenic and chromium in 258 samples of brown rice, grown in Yangtze River Delta where the soils were low-level contaminated, were investigated. In 12 (4.6%) and 10 (3.9%) rice samples the concentrations of Cd and Hg, respectively, exceeded the limit for food. ANOVA showed that Cd and Hg concentrations in rice grains collected from Nanjing and Jiaxing were higher than in the less developed city Yancheng. Students' t-test showed Cd and Hg were accumulated in hybrid rice higher than in conventional rice. The hazard quotients (HQs) showed a low risk from rice consumption. Conventional rice was recommended to cultivate to reduce the current risk in the soil defined as safe use level in Yangtze River Delta.

Impacts of elevated CO2 on plant resistance to nutrient deficiency and toxic ions via root exudates: A review.

Elevated atmospheric CO2 (eCO2) concentration can increase root exudation into soils, which improves plant tolerance to abiotic stresses. This review used a meta-analysis to assess effect sizes of eCO2 on both efflux rates and total amounts of some specific root exudates, and dissected whether eCO2 enhances plant's resistance to nutrient deficiency and ion toxicity via root exudates. Elevated CO2 did not affect efflux rates of total dissolved organic carbon, a measure of combined root exudates per unit of root biomass or length, but increased the efflux amount of root systems per plant by 31% which is likely attributed to increased root biomass (29%). Elevated CO2 increased efflux rates of soluble-sugars, carboxylates, and citrate by 47%, 111%, and 16%, respectively, but did not affect those of amino acids and malate. The increased carbon allocation to roots, increased plant requirements of mineral nutrients, and heightened detoxification responses to toxic ions under eCO2 collectively contribute to the increased efflux rates despite lacking molecular evidence. The increased efflux rates of root exudates under eCO2 were closely associated with improved nutrient uptake whilst less studies have validated the associations between root exudates and resistance to toxic ions of plants when grown under eCO2. Future studies are required to reveal how climate change (eCO2) affect the efflux of specific root exudates, particularly organic anions, the corresponding nutrient uptake and toxic ion resistance from plant molecular biology and soil microbial ecology perspectives.

Elevated CO2 (free-air CO2 enrichment) increases grain yield of aluminium-resistant but not aluminium-sensitive wheat (Triticum aestivum) grown in an acid soil.

BACKGROUND AND AIMS: Soil acidity currently limits root growth and crop production in many regions, and climate change is leading to uncertainties regarding future food supply. However, it is unknown how elevated CO2 (eCO2) affects the performance of wheat crops in acid soils under field conditions. We investigated the effects of eCO2 on plant growth and yield of three pairs of near-isogenic hexaploid wheat lines differing in alleles of aluminium-resistant genes TaALMT1 (conferring root malate efflux) and TaMATE1B (conferring citrate efflux). METHODS: Plants were grown until maturity in an acid soil under ambient CO2 (aCO2; 400 µmol mol-1) and eCO2 (550 µmol mol-1) in a soil free-air CO2 enrichment facility (SoilFACE). Growth parameters and grain yields were measured. KEY RESULTS: Elevated CO2 increased grain yield of lines carrying TaMATE1B by 22 % and lines carrying only TaALMT1 by 31 %, but did not increase the grain yield of Al3+-sensitive lines. Although eCO2 promoted tiller formation, coarse root length and root biomass of lines carrying TaMATE1B, it did not affect ear number, and it therefore limited yield potential. By contrast, eCO2 decreased or did not change these parameters for lines carrying only TaALMT1, and enhanced biomass allocation to grains thereby resulting in increased grain yield. Despite TaMATE1B being less effective than TaALMT1 at conferring Al3+ resistance based on root growth, the gene promoted grain yield to a similar level to TaALMT1 when the plants were grown in acid soil. Furthermore, TaALMT1 and TaMATE1B were not additive in their effects. CONCLUSIONS: As atmospheric CO2 increases, it is critical that both Al3+-resistance genes (particularly TaALMT1) should be maintained in hexaploid wheat germplasm in order for yield increases from CO2 fertilization to be realized in acid soils.

Small Molecules as PD-1/PD-L1 Pathway Modulators for Cancer Immunotherapy.

Blockade of PD-1/PD-L1 interactions using PD-1/PD-L1 pathway modulators has shown unprecedented clinical efficacy in various cancer models. Current PD-1/PD-L1 modulators approved by FDA are exclusively dominated by therapeutic antibodies. Nevertheless, therapeutic antibodies also exhibit several disadvantages such as low tumor penetration, difficulty in crossing physiological barriers, lacking oral bioavailability, high manufacturing costs, inaccessible to intracellular targets, immunogenicity, immune-related adverse events (irAEs). Modulation of PD-1/PD-L1 pathway using small molecules may be an alternative approach to mobilize immune system to fight against cancers. In this review, we focus on summarizing the recently disclosed chemical structures and preliminary structure-activity relationships (SARs) of small molecules as PD-1/PD-L1 modulators for cancer immunotherapy.

Effects of Elevated CO2 on Nutritional Quality of Vegetables: A Review.

Elevated atmospheric CO2 (eCO2) enhances the yield of vegetables and could also affect their nutritional quality. We conducted a meta-analysis using 57 articles consisting of 1,015 observations and found that eCO2 increased the concentrations of fructose, glucose, total soluble sugar, total antioxidant capacity, total phenols, total flavonoids, ascorbic acid, and calcium in the edible part of vegetables by 14.2%, 13.2%, 17.5%, 59.0%, 8.9%, 45.5%, 9.5%, and 8.2%, respectively, but decreased the concentrations of protein, nitrate, magnesium, iron, and zinc by 9.5%, 18.0%, 9.2%, 16.0%, and 9.4%. The concentrations of titratable acidity, total chlorophyll, carotenoids, lycopene, anthocyanins, phosphorus, potassium, sulfur, copper, and manganese were not affected by eCO2. Furthermore, we propose several approaches to improving vegetable quality based on the interaction of eCO2 with various factors, including species, cultivars, CO2 levels, growth stages, light, O3 stress, nutrient, and salinity. Finally, we present a summary of the eCO2 impact on the quality of three widely cultivated crops, namely, lettuce, tomato, and potato.

Increased DHRS12 expression independently predicts poor survival in patients with high-grade serous ovarian cancer.

AIM: To explore the expression profile of some DHRS genes in high-grade serous ovarian cancer (SOVC) and to study their prognostic values. PATIENTS & METHODS: A retrospective bioinformatic analysis was performed using data in the Gene Expression Omnibus, the Human Protein Atlas and the Cancer Genome Atlas-Ovarian Cancer. RESULTS: Increased DHRS12 expression was an independent indicator of poor overall survival (hazard ratio [HR]: 1.265, 95% CI: 1.075-1.488; p = 0.005) and recurrence-free survival (RFS; HR: 2.242, 95%CI: 1.464-3.432; p < 0.001) in patients with high-grade SOVC. DNA deletion was associated with decreased DHRS12 expression, as well as the best overall survival and RFS among the three copy number alteration groups. CONCLUSION: DHRS12 might serve as a valuable prognostic biomarker in high-grade SOVC.

Cost-effective three dimensional Ag/polymer dyes/graphene-carbon spheres hybrids for high performance nonenzymatic sensor and its application in living cell H2O2 detection.

We describe a facile method to synthesize a new type of catalyst by electrodepositing Ag nanocrystals (AgNCs) on the different polymer dyes, Poly (methylene blue) (PMB) or Poly (4-(2-Pyridylazo)-Resorcinol) (PAR) modified graphene­carbon spheres (GS) hybrids. The self-assembled GS take dual advantages of carbon spheres and graphene. Carbon spheres acts as nano-spacers prevent the aggregation of graphene and guarantee the fast electron transfer of GS. Secondly, polymerized dyes used here are beneficial for AgNCs growing as a linker. The effects of dyes on the growth habits, morphologies and catalytic properties for AgNCs were investigated. A novel electrochemical nonenzymatic sensor for hydrogen peroxide (H2O2) detection is fabricated based on the Ag/Polymer dyes/GS ternary composites modified glass carbon electrode (GCE) for the first time. It was found that the proposed electrodes, especially for Ag/PMB/GS/GCE, displayed a peculiar electrocatalytic activity towards H2O2 reduction synergistically as compared to Ag/PAR/GS/GCE or Ag/GS/GCE alone. Ag/PMB/GS/GCE showed a linear response over the H2O2 concentration range of 0.5 to 1112 µM. The detection limit and sensitivity is 0.15 µM and 400 µA mM-1 cm-2, respectively. These outstanding results enable the practical application of Ag/PMB/GS/GCE for the H2O2 tracking released from MCF-7 (human breast cancer cells) with satisfactory results.

Elevated and super-elevated CO2 differ in their interactive effects with nitrogen availability on fruit yield and quality of cucumber.

BACKGROUND: Elevated carbon dioxide (CO2 ) and nitrogen (N) availability can interactively promote cucumber yield, but how the yield increase is realized remains unclear, whilst the interactive effects on fruit quality are unknown. In this study, cucumber plants (Cucumis sativus L. cv. Jinmei No. 3) were grown in a paddy soil under three CO2 concentrations - 400 (ambient CO2 ), 800 (elevated CO2 , eCO2 ) and 1200 µmol mol-1 (super-elevated CO2 ) - and two N applications - 0.06 (low N) and 0.24 g N kg-1 soil (high N). RESULTS: Compared with ambient CO2 , eCO2 increased yield by 106% in high N but the increase in total biomass was only 33%. This can result from greater carbon translocation to fruits from other organs, indicated by the increased biomass allocation from stems and leaves, particularly source leaves, to fruits and the decreased concentrations of fructose and glucose in source leaves. Super-elevated CO2 reduced the carbon allocation to fruits thus yield increase (71%). Additionally, eCO2 also increased the concentrations of fructose and glucose in fruits, maintained the concentrations of dietary fiber, phosphorus, potassium, calcium, magnesium, sulfur, manganese, copper, molybdenum and sodium, whilst it decreased the concentrations of nitrate, protein, iron, and zinc in high N. Compared with eCO2 , super-elevated CO2 can still improve the fruit quality to some extent in low N availability. CONCLUSIONS: Elevated CO2 promotes cucumber yield largely by carbon allocation from source leaves to fruits in high N availability. Besides a dilution effect, carbon allocation to fruits, carbohydrate transformation, and nutrient uptake and assimilation can affect the fruit quality. © 2018 Society of Chemical Industry.

PD-1/PD-L1 Inhibitors for Immuno-oncology: From Antibodies to Small Molecules.

BACKGROUND: The recent regulatory approvals of immune checkpoint protein inhibitors, such as ipilimumab, pembrolizumab, nivolumab, atezolizumab, durvalumab, and avelumab ushered a new era in cancer therapy. These inhibitors do not attack tumor cells directly but instead mobilize the immune system to re-recognize and eradicate tumors, which endows them with unique advantages including durable clinical responses and substantial clinical benefits. PD-1/PD-L1 inhibitors, a pillar of immune checkpoint protein inhibitors, have demonstrated unprecedented clinical efficacy in more than 20 cancer types. Besides monoclonal antibodies, diverse PD- 1/PD-L1 inhibiting candidates, such as peptides, small molecules have formed a powerful collection of weapons to fight cancer. METHODS: The goal of this review is to summarize and discuss the current PD-1/PD-L1 inhibitors including candidates under clinical development, their molecular interactions with PD-1 or PD-L1, the disclosed structureactivity relationships of peptides and small molecules as inhibitors. RESULTS: Current PD-1/PD-L1 inhibitors under clinical development are exclusively dominated by antibodies. The molecular interactions of therapeutic antibodies with PD-1 or PD-L1 have been gradually elucidated for the design of novel inhibitors. Various peptides and traditional small molecules have been investigated in preclinical model to discover novel PD-1/PD-L1 inhibitors. CONCLUSION: Peptides and small molecules may play an important role in immuno-oncology because they may bind to multiple immune checkpoint proteins via rational design, opening opportunity for a new generation of novel PD-1/PD-L1 inhibitors.

Juglanin induces apoptosis and autophagy in human breast cancer progression via ROS/JNK promotion.

Breast cancer is one of the most common primary malignant tumors of among women, the long-term survival of which has stagnated in the past decades. Juglanin as a natural production mainly extracted from green walnut husks of Juglans mandshurica has been defined as the functional composition among a series of compounds. It showed powerful protective effect in various diseases by inhibiting inflammation and tumor cells growth. However, the effect of juglanin on human breast cancer and the underlying mechanisms remains to be elucidated. We reported here that juglanin could inhibit cell proliferation by leading to G2/M phase arrest. Exposure to juglanin resulted in the activation of cleaved caspase -3, -8, and -9, indicating that juglanin induced apoptosis. Autophagy occurred in juglanin-treated cells as evidenced by formation of autophagosome and up-regulation of LC3B-II. The juglanin-induced cell death was significantly restored by the combination of autophagy and apoptosis. Further, juglanin also induced JNK activation and ROS production. The JNK inhibitor attenuated juglanin-caused apoptosis and autophagy significantly while ROS scavenger could reverse them. In addition, the ROS scavenger also inhibited G2/M phase arrest and phosphorylated JNK. Of note, we found that juglanin had the similar effects on breast cancer cells. Finally, juglanin inhibited tumor growth in the mouse xenograft model in vivo. Together, our results suggested that juglanin led to G2/M phase arrest, induced apoptosis as well as autophagy through the ROS/JNK signaling pathway in human breast cancer cells. Hence, juglanin might be a promising candidate for development of anti-tumor drugs targeting breast cancer.

Design, synthesis and in vitro evaluation of amidoximes as histone deacetylase inhibitors for cancer therapy.

Amindoximes are geometric isomers of N-hydroxyamidines which are bioisosteres of hydroxamates. Since amindoxime group is capable of chelating transition metal ions including zinc ion, amindoximes should possess histone deacetylases (HDACs) inhibitory activity. In this work, we designed and synthesized a series of amindoximes, examined their inhibitory activities against HDACs, and investigated their cytotoxicity to human cancer cells. Preliminary results demonstrated that amindoximes possessed submicromolar HDACs inhibitory activity, with noteworthy enhancement compared with hydroxamates. Furthermore, the amindoximes arrested HCT116 and A549 cells in G2/M phase and showed good efficacy in inducing cells death. We provided a proof-of-concept that amindoximes could be used as HDACs inhibitors and hold great promise as epigenetic drugs.

The stability and the metal ions binding properties of mutant A85M of CopC.

In this work, the mutant A85M of CopC was obtained. The stability of mutant A85M of CopC and the binding properties of metal ions were clarified through various spectroscopic techniques. The binding capacity of A85M to metal ions was measured by fluorescence spectroscopy and UV differential absorbance. The results suggested that Cu(2+) can bind with A85M in 1:1 form, and the constant of A85M was nearly the same as that of CopC. Ag(+) can occupy the Cu(+) binding site located at C-terminal, and the binding constant was (2.64±0.48)×10(6)L/mol. Hg(2+) not only can occupy the Cu(+) binding site located at C-terminal, but also can occupy the Cu(2+) binding site located at N-terminal. The stability of A85M was measured by chemical unfolding experiment. The intermediate was observed in the unfolding pathway of A85M-Cu(2+) induced by urea. In addition, the interaction of SDS with A85M also can result in the formation of the intermediate. The effect of metal ions on the stability of intermediate suggested that the C terminal region of intermediate was unfolded and the N terminal region suffered few effects. Compared with CopC, the stability of A85M was decreased. The main reason was the lower stability of N terminal region. The results of molecular dynamic simulation suggested that when the alanine at 85 site was mutated to methionine, the hydrophobic almost unchanged, but the distance between the phenylalanine at 25 site and tryptophan at 83 site increased because of the spatial effect. And it made the stacking interaction of aromatic rings decreased, which was the main reason for the decreasing stability of N terminal region for A85M.