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BackgroundAdverse childhood experiences (ACEs) are strongly linked to many mental health problems, and play important role in the intergenerational transmission of psychopathology. Additionally, the developmental timing may also be critical in ACEs' impact on these problems. The Adverse Life Experiences Scale (ALES), as a recently developed measure, has demonstrated good reliability and validity in indexing cumulative risk, developmental timing, and intergenerational transmission. This scale has not been used in China. The purpose of present study was to revise the Chinese version of the ALES and examine its psychometric properties.MethodsA total of 527 parents (fathers n = 246, mothers n = 281) from families with at least one child (12–18 years) completed this online survey. Internal consistency, test–retest reliability, correlations, regression models were examined for assessing the psychometric properties of the Chinese version of the ALES.ResultsThe Chinese version of the ALES showed acceptable internal consistency (children: α = .72, parents: α = .74) and test–retest reliability (children: r = .86, parents: r = .84). In terms of validity, both parents and children's ACEs scores (total score and most age intervals scores) were significantly correlated with their current symptoms; ACEs scores of some age intervals in early childhood and adolescence significantly predicted symptoms in regression models; and parents' ACEs total score significantly correlated with children's ACEs total score and symptoms (all, girls, boys) except boys' Strengths and Difficulties Questionnaire total score.ConclusionThe Chinese version of the ALES showed good psychometric properties for assessing ACEs cumulative risk, developmental timing, and intergenerational transmission, and can serve as a reliable tool to evaluate ACEs in Chinese samples.

Fibromyalgia is characterised by widespread chronic pain and is often accompanied by comorbidities such as sleep disorders, anxiety, and depression. Because it is often accompanied by many adverse symptoms and lack of effective treatment, it is important to search for the pathogenesis and treatment of fibromyalgia. Astaxanthin, a carotenoid pigment known for its anti-inflammatory and antioxidant properties, has demonstrated effective analgesic effects in neuropathic pain. However, its impact on fibromyalgia remains unclear. Therefore, in this study, we constructed a mouse model of fibromyalgia and investigated the effect of astaxanthin on chronic pain and associated symptoms through multiple intragastrical injections. We conducted behavioural assessments to detect pain and depression-like states in mice, recorded electroencephalograms to monitor sleep stages, examined c-Fos activation in the anterior cingulate cortex, measured activation of spinal glial cells, and assessed levels of inflammatory factors in the brain and spinal cord, including interleukin (IL)-1β, IL-6, and tumour necrosis factor- α(TNF-α).Additionally, we analysed the expression levels of IL-6, IL-10, NOD-like receptor thermal protein domain associated protein 3 (NLRP3), Apoptosis-associated speck-like protein containing CARD, and Caspase-1 proteins. The findings revealed that astaxanthin significantly ameliorated mechanical and thermal pain in mice with fibromyalgia and mitigated sleep disorders and depressive-like symptoms induced by pain. A potential mechanism underlying these effects is the anti-inflammatory action of astaxanthin, likely mediated through the inhibition of the NLRP3 inflammasome, which could be one of the pathways through which astaxanthin alleviates fibromyalgia. In conclusion, our study suggests that astaxanthin holds promise as a potential analgesic medication for managing fibromyalgia and its associated symptoms.

Inhalation anesthesia stands as a pivotal modality within clinical anesthesia practices. Beyond its primary anesthetic effects, inhaled anesthetics have non-anesthetic effects, exerting bidirectional influences on the physiological state of the body and disease progression. These effects encompass impaired cognitive function, inhibition of embryonic development, influence on tumor progression, and so forth. For many years, inhaled anesthetics were viewed as inhibitors of stem cell fate regulation. However, there is now a growing appreciation that inhaled anesthetics promote stem cell biological functions and thus are now regarded as a double-edged sword affecting stem cell fate. In this review, the effects of inhaled anesthetics on self-renewal and differentiation of neural stem cells (NSCs), embryonic stem cells (ESCs), and cancer stem cells (CSCs) were summarized. The mechanisms of inhaled anesthetics involving cell cycle, metabolism, stemness, and niche of stem cells were also discussed. A comprehensive understanding of these effects will enhance our comprehension of how inhaled anesthetics impact the human body, thus promising breakthroughs in the development of novel strategies for innovative stem cell therapy approaches.

When studying an unfamiliar system, we first look for the symmetry that the system has so that we can make many predictions about the possible properties of the system. The symmetry in transport network security needs to maintain a stable state and maintain a constant state of transport network security. With the development of China–Europe freight trains, the transport between Asia and Europe has gradually formed the Belt and Road (B&R) land–sea transport network. In order to analyze the cascading failure mechanism of the B&R land–sea transport network, a network cascading failure model is constructed. Then, the quantitative analysis of the connectivity indicators of the land–sea transport network is conducted from the node attack strategy, and it is compared with the Maritime Silk Road (MSR) shipping network. Finally, the robustness of the land–sea transport network under emergencies is analyzed. From the results of deliberate attacks, the attack threshold of the B&R land–sea transport network is the same as that of the MSR shipping network, and the maximum number of attacks is slightly less than that of the MSR shipping network. The Russia–Ukraine conflict has a minimal impact on the robustness of cascading failure in the land–sea transport network. The Red Sea crisis may have a significant impact on the robustness of cascading failure in the land–sea transport network. The research results can provide suggestions for improving the robustness of the B&R land–sea transport network.

Current conceptualizations of dental caries etiology center primarily on the local role of sugar, starch, or other fermentable carbohydrates on tooth enamel demineralization—a well-established and empirically supported mechanism. However, in addition to this mechanism, studies dating back to the early 1900s point to an important systemic role of diet and nutrition, particularly from pasture-raised animal-source foods (ASF), in dental caries etiology and arrest. Findings from animal and human studies suggest that adherence to a diet high in calcium, phosphorus, fat-soluble vitamins A and D, and antioxidant vitamin C, as well as low in phytates, may contribute to arrest and reversal of dental caries, particularly among children. Furthermore, findings from observational and experimental studies of humans across the life-course suggest that fat-soluble vitamins A, D, and K2 may interact to protect against dental caries progression, even within a diet that regularly contains sugar. While these historic studies have not been revisited in decades, we emphasize the need for them to be reinvestigated and contextualized in the 21st century. Specifically, methodologically rigorous studies are needed to reinvestigate whether historical knowledge of systemic impacts of nutrition on dental health can help to inform current conceptualizations of dental caries etiology, prevention, and arrest.

This paper investigates the effect of Nb and B microalloying on the wear and corrosion resistance of 20MnCr5 steel after high-temperature carburization at 950 °C. The abrasion and corrosion performance of the steel was compared using electrochemical experiments and dry sliding wear tests. The results show that Nb microalloying reduces the size of residual austenite, thereby improving the wear resistance of 20MnCr5 steel. Furthermore, the combination of Nb and B microalloying reduces the amount of residual austenite, leading to further improvements in wear resistance. Both microalloying methods reduce the wear scar depth and debris accumulation. The corrosion potentials of 20MnCr5, 20MnCr5Nb, and 20MnCr5NbB are 0.645 V, 0.627 V, and 0.675 V, respectively. The addition of Nb to 20MnCr5 steel results in higher corrosion resistance after carburization due to the refinement of needle-like martensite. However, 20MnCr5NbB steel exhibits low corrosion resistance due to the coarsening of needle-like martensite caused by B microalloying. In summary, this study provides new insights into the effects of microalloying elements on the properties of steel. The findings contribute to a deeper understanding of how to design and manufacture gears and other components with superior wear and corrosion resistance.

Abstract Background Decomposition of plant litter is a key driver of carbon and nutrient cycling in terrestrial ecosystems. Mixing litters of different plant species may alter the decomposition rate, but its effect on the microbial decomposer community in plant litter is not fully understood. Here, we tested the effects of mixing with maize (Zea mays L.) and soybean [Glycine max (Linn.) Merr.] stalk litters on the decomposition and microbial decomposer communities of common bean (Phaseolus vulgaris L.) root litter at the early decomposition stage in a litterbag experiment. Results Mixing with maize stalk litter, soybean stalk litter, and both of these litters increased the decomposition rate of common bean root litter at 56 day but not 14 day after incubation. Litter mixing also increased the decomposition rate of the whole liter mixture at 56 day after incubation. Amplicon sequencing found that litter mixing altered the composition of bacterial (at 56 day after incubation) and fungal communities (at both 14 and 56 day after incubation) in common bean root litter. Litter mixing increased the abundance and alpha diversity of fungal communities in common bean root litter at 56 day after incubation. Particularly, litter mixing stimulated certain microbial taxa, such as Fusarium, Aspergillus and Stachybotrys spp. In addition, a pot experiment with adding litters in the soil showed that litter mixing promoted growth of common bean seedlings and increased soil nitrogen and phosphorus contents. Conclusions This study showed that litter mixing can promote the decomposition rate and cause shifts in microbial decomposer communities, which may positively affect crop growth.

Antibiotic abuse and environmental pollution caused by animal husbandry has aroused extensive concern from the government and the industry. As a kind of efficient alternative to agricultural chemicals like hormones and antibiotics, functional microorganisms can enhance the comprehensive growth performance of animals and thus play a crucial role in ecological and healthy breeding. The use of functional microorganisms in animal breeding for eco-agriculture has been a key development pathway for achieving the objective of sustainable development. Functional microorganisms are used at the beginning, middle, and end of the entire breeding process. Functional microorganisms can effectively eliminate anti-nutritional factors from feed sources, increasing their utilization rate and nutritional value. Animals fed with functional microorganisms not only have balanced gut microbiota and intestinal homeostasis, but their immune system is also well regulated. Functional microorganisms can also control odor emissions and pathogens in the environment. Finally, functional microorganisms also play an important role in the bio-safety treatment and circular utilization of breeding wastes. This study provides references for the continued application and promotion of functional microorganisms in animal breeding by summarizing the progress of their application and related mechanisms in such areas as feed processing, intestinal health, breeding environmental control and waste treatment.

【Objective】A strain of Lactobacillus fermentum was isolated from the feces of healthy piglets, and its probiotic properties were tested in order to provide references for its applications in livestock and poultry breeding.【Method】106 strains of lactic acid bacteria were isolated by using Rogosa SL medium, and one strain which was potential for feeding was preliminarily screened by gram staining, acid and bile salt tolerance tests. The probiotic ability of strain LF1, which was identified as L. fermentum LF1 by 16S rDNA sequencing, was evaluated, including its antagonistic activity (Escherichia coli K88, Staphylococcus aureus, Streptococcus suis, Salmonella, and Vibrio, etc.), tolerance to the stimulated acidic environment in stomach and bile acid, and adhesion to small intestinal epithelial cells.【Result】L. fermentum LF1 had strong tolerance to acid and bile salt, showing its strong probiotic potential. However, the LF1 strain showed low adhesion to pig intestinal epithelial cells. In terms of antagonism, the culture supernatant of L. fermentum LF1 inhibited the growth of many animal pathogens significantly.【Conclusion】L. fermentum LF1 had a good probiotics ability. After further experimental evaluation on animals, it can be used as a probiotic candidate strain for livestock and poultry breeding industry.

Abstract Background Lung cancers arising in never smokers have been suggested to be substantially different from lung cancers in smokers at an epidemiological, genetic and molecular level. Focusing on non-small cell lung cancer (NSCLC), we characterized lung cancer patients in China looking for demographic and clinical differences between the smoking and never-smoking subgroups. Methods In total, 891 patients with NSCLC, including 841 with adenocarcinoma and 50 with squamous cell carcinoma, were recruited in this study. Association of smoking status with demographic and clinical features of NSCLC was determined, and risk factors for lymph node metastasis and TNM stage were evaluated using Multivariate logistic regression analysis. Results In patients with adenocarcinoma, never smokers showed a younger age at diagnosis (54.2 ± 12.7vs. 59.3 ± 9.4, p adjusted

Reciprocating piezoelectric micropumps enable miniaturization in microfluidics for lab-on-a-chip applications such as organs-on-chips (OoC). However, achieving a steady flow when using these micropumps is a significant challenge because of flow ripples in the displaced liquid, especially at low frequencies or low flow rates (RFfl.), which is the ratio of the root mean square (RMS) value of the fluctuations present in the rectified output to the average fluctuation-free value of the discharge flow, as a metric to express the quality of the flow. The fluidic ripple factor was reduced by more than 90% by using three-phase rectification when compared to one-phase rectification in the 2–60 μL/min flow rate range. Analytical equations to estimate the fluidic ripple factor for a chosen number of pumps connected in parallel are presented, and we experimentally confirmed up to four pumps. The analysis shown can be used to design a frequency-independent multi-phase fluid rectifier to the desired ripple level in a flow for reciprocating pumps.

PurposeAging is a process associated with degeneration and dysfunction of peripheral vestibular system or apparatus. This study aimed to investigate the influence of aging on ocular vestibular-evoked myogenic potential (oVEMP) response rates and recording parameters using the B81 bone vibrator and compare them with air conduction stimuli (ACS) oVEMP response characteristics.MethodsIn 60 healthy participants aged 10–71 years (mean age 39.9; 29 male participants), the oVEMP response was elicited using a B81 bone vibrator and an ER-3A insert earphone. The effects of age and stimulus on oVEMP response rates and recording parameters were evaluated.ResultsResponse rates and amplitudes declined with aging using either ACS or bone-conducted vibration (BCV) stimulation, particularly in individuals over 60 years of age, whereas thresholds increased and N1 latencies were prolonged. BCV showed fewer risks of absent oVEMP response than ACS (p = 0.002). BCV acquired higher amplitudes (p < 0.001), lower thresholds, and shorter N1 and P1 latencies (all p < 0.001) than ACS.ConclusionsThe absence of an oVEMP response may be attributed to aging rather than a concurrent vestibular disorder. B81-BCV likely produces higher mechanical drives to the vestibular hair cells at safer and non-traumatic levels compared with ACS and therefore may be more likely to evoke a response in the elderly cohort, whose vestibular function and mechanical sensitivity have declined. Thus, B81-BCV stimulation is more effective and safer to elicit oVEMPs, and it should be recommended when ACS fails in the clinic, particularly in the elderly population.

Purpose: The aim of the study was to evaluate the status of psychological resilience among women in their second pregnancy and to investigate the possible influencing factors. Methods: A total of 275 women in their second pregnancy and who met the criteria were surveyed from two public hospitals in China from July 2018 to January 2019. The instruments included the General Self-designed Questionnaire, Connor–Davidson Resilience Scale, Social Support Rate Scale, and 36-item Pregnancy Stress Rating Scale. Results: The total psychological resilience score of second-pregnancy women was relatively low. Multivariate regression analysis identified five factors associated with psychological resilience: intimacy with husbands, social support utilization, gender of the first child, high-risk pregnancy of the first child, and the stress caused by worrying about the health and safety of the mother and fetus. Conclusion: Women in their second pregnancy represent a unique population, and their low psychological resilience score deserves attention. Identification of factors contributing to decreased psychological resilience may enable us to design prevention and intervention strategies and to deliver specific psychological supports to pregnant women at high risk of developing negative psychology.

Outbreaks of influenza D virus (IDV) continue to be reported in many countries. On the basis of the hemagglutinin-esterase fusion (HEF) gene, five IDV genetic lineages have been identified: D/OK, D/660, D/Yama2016, D/Yama2019 and D/CA2019 lineages. Previously reported IDV strains in China all form a sub-clade (D/China sub-lineage) within D/OK lineage. From October 2021 to February 2022, nasal swab samples (n = 250) were collected from apparently healthy cattle in slaughterhouses around the city of Guangzhou, China, and screened for IDV by RT-PCR. Ten samples were positive for IDV. An IDV strain with nearly complete genome sequences was identified and designated as D/bovine/CHN/JY3001/2021. Importantly, sequence alignments and phylogenetic analyses revealed that this IDV strain is genetically close to the strains (>98% homology) in the D/Yama2019 lineage that has been found only in Japan, but distant from the previously reported Chinese IDV strains (~95% similarity). These results demonstrate the emergence of D/Yama2019 lineage IDV in Chinese cattle herds, highlighting a need for future surveillance of D/Yama2019-like viruses toward better understanding both epidemiology and diversity of IDV in China.

ABSTRACT Mixing plant litters of multiple species can alter litter decomposition, a key driver of carbon and nutrient cycling in terrestrial ecosystems. Changes in microbial decomposer communities is proposed as one of the mechanisms explaining this litter-mixture effect, but the underlying mechanism is unclear. In a microcosm litterbag experiment, we found that, at the early stage of decomposition, litter mixing promoted tomato root litter decomposition, thus generating a synergistic nonadditive litter-mixture effect. The transplanting decomposer community experiment showed that changes in microbial decomposer communities contributed to the nonadditive litter-mixture effect on tomato root litter decomposition. Moreover, litter mixing altered the abundance and diversity of bacterial and fungal communities on tomato root litter. Litter mixing also stimulated several putative keystone operational taxonomic units (OTUs) in the microbial correlation network, such as Fusarium sp. fOTU761 and Microbacterium sp. bOTU6632. Then, we isolated and cultured representative isolates of these two taxa, named Fusarium sp. F13 and Microbacterium sp. B26. Subsequent in vitro tests found that F13, but not B26, had strong decomposing ability; moreover, these two isolates developed synergistic interaction, thus promoted litter decomposition in coculture. Addition of F13 or B26 both promoted the decomposing activity of the resident decomposer community on tomato root litter, confirming their importance for litter decomposition. Overall, litter mixing promoted tomato root litter decomposition through altering microbial decomposers, especially through stimulating certain putative keystone taxa. IMPORTANCE Microbial decomposer community plays a key role in litter decomposition, which is an important regulator of soil carbon and nutrient cycling. Though changes in decomposer communities has been proposed as one of the potential underlying mechanisms driving the litter-mixture effects, direct evidence is still lacking. Here, we demonstrated that litter mixing stimulated litter decomposition through altering microbial decomposers at the early stage of decomposition. Moreover, certain putative keystone taxa stimulated by litter mixing contributed to the nonadditive litter-mixture effect. In vitro culturing validated the role of these taxa in litter decomposition. This study also highlights the possibility of regulating litter decomposition through manipulating certain microbial taxa.

The importance of transaction fees in maintaining blockchain security and sustainability has been confirmed by extensive research, although they are not mandatory in most current blockchain systems. To enhance blockchain in the long term, it is crucial to design effective transaction pricing mechanisms. Different from the existing schemes based on auctions with more consideration about the profit of miners, we resort to game theory and propose a correlated equilibrium based transaction pricing mechanism through solving a pricing game among users with transactions, which can achieve both the individual and global optimum. To avoid the computational complexity exponentially increasing with the number of transactions, we further improve the game-theoretic solution with an approximate algorithm, which can derive almost the same results as the original one but costs significantly reduced time. We also propose a truthful assessment model for pricing mechanism to collect the feedback of users regarding the price suggestion. Extensive experimental results demonstrate the effectiveness and efficiency of our proposed mechanism.

Abstract Background Porcine epidemic diarrhea virus (PEDV), an intestinal coronavirus that causes acute diarrhea and high mortality in suckling piglets, can result in high economic losses in the swine industry. In recent years, despite the use of China’s current vaccine immunization strategy, multiple types of PEDV strains were still found in immunized swine herds. Our research aims to explore a new rapid differentiation method to distinguish the different types of PEDV strains and assess the safety evaluation of classical attenuated vaccine strains in swine herds. Results In the study, a differential one-step quantitative real-time fluorescent reverse transcription recombinase polymerase amplification (real-time RT-RPA) method based on the PEDV universal real-time RT-RPA assay was established according to the ORF1 deletion sequences of three classical attenuated vaccine strains (PEDV attenuated vaccine KC189944, attenuated CV777 and DR13) and five Vero cell-adapted isolates (JS2008, SDM, SQ2014, SC1402, HLJBY), which could effectively differentiate PEDV classical attenuated vaccine strains from wild-type strains (PEDV classical wild strains and variant strains). The detection limits of PEDV RNA in the both PEDV real-time RT-RPA assays were 300 copies within 20 min at 39 °C, and the detection limits of classical attenuated vaccine strain CV777, Vero-cell-adapted isolate JS2008, and PEDV wild-type strain DX were 100.5 TCID50/100 μL, 101.1 TCID50/100 μL, and 101.2 TCID50/100 μL, respectively. Both assays were highly specific for PEDV, showing no cross-reactivity with other enteral viruses. Conclusion This RPA method we developed is simple, time-effective, and safe and provides a reliable technical tool for the differential diagnosis and clinical epidemic surveillance of PEDV classical attenuated vaccine strains and wild-type strains.

This paper is a study on the elongational deformation induced by pins in pin-barrel cold-feed extruders. The mathematical model is established by using simple flow field, plate model, and Newton constitutive relation. It is proved that there is elongational deformation when the fluid bypasses the pin, and the elongational deformation is quantitatively calculated. The finite element method is used to simulate the flow field of the screw mixing section of the pin-barrel cold-feed extruder with different specifications. The velocity vector nephogram in the simulation results is analyzed by numerical analysis of elongational deformation and compared with the theoretical elongational deformation value. The results show that the simulated and theoretical values of elongational deformation are approximately consistent. The numerical analysis of the key factors affecting the elongational deformation shows that the elongational deformation will gradually increase with the increase of screw diameter. For the screw of the same specification, the increasing helical angle will reduce the elongational deformation, and the increasing rotational speed will linearly increase the elongational deformation in unit time. This study can be used to roughly estimate the elongational deformation induced by pins and establish the mixing theoretical model of the pin-barrel cold-feed extruder.

Quickly and accurately identifying water leakage is one of the important components of the health monitoring of subway tunnels. A mobile vision measurement system consisting of several high-resolution, industrial, charge-coupled device (CCD) cameras is placed on trains to implement structural health monitoring in tunnels. Through the image processing technology proposed in this paper, water leakage areas in subway tunnels can be found and repaired in real time. A lightweight automatic segmentation approach to water leakage using hybrid-supervised-deep-learning technology is proposed. This approach consists of the weakly supervised learning Water Leakage-CAM and fully supervised learning WRDeepLabV3+. The Water Leakage-CAM is used for the automatic labeling of data. The WRDeepLabV3+ is used for the accurate identification of water leakage areas in subway tunnels. Compared with other end-to-end semantic segmentation networks, the hybrid-supervised learning approach can more completely segment the water leakage region when dealing with water leakage in complex environments. The hybrid-supervised-deep-learning approach proposed in this paper achieves the highest MIoU of 82.8% on the experimental dataset, which is 6.4% higher than the second. The efficiency is also 25% higher than the second and significantly outperforms other end-to-end deep learning approaches.

The variation of crustal thickness is a critical index to reveal how the continental crust evolved over its four billion years. Generally, ratios of whole-rock trace elements, such as Sr/Y, (La/Yb)n and Ce/Y, are used to characterize crustal thicknesses. However, sometimes confusing results are obtained since there is no enough filtered data. Here, a state-of-the-art approach, based on a machine-learning algorithm, is proposed to predict crustal thickness using global major- and trace-element geochemical data of intermediate arc rocks and intraplate basalts, and their corresponding crustal thicknesses. After the validation processes, the root-mean-square error (RMSE) and the coefficient of determination (R2) score were used to evaluate the performance of the machine learning algorithm based on the learning dataset which has never been used during the training phase. The results demonstrate that the machine learning algorithm is more reliable in predicting crustal thickness than the conventional methods. The trained model predicts that the crustal thickness of the eastern North China Craton (ENCC) was ~45 km from the Late Triassic to the Early Cretaceous, but ~35 km from the Early Cretaceous, which corresponds to the paleo-elevation of 3.0 ± 1.5 km at Early Mesozoic, and decease to the present-day elevation in the ENCC. The estimates are generally consistent with the previous studies on xenoliths from the lower crust and on the paleoenvironment of the coastal mountain of the ENCC, which indicates that the lower crust of the ENCC was delaminated abruptly at the Early Cretaceous.

Heavy metal pollution in water environments poses a great threat to public health and to the ecological environment due to its high toxicity and non-degradability. However, many existing detection methods require laboratory-based bulky instruments and time-consuming manual operations. Although some on-site systems exist, they are difficult to deploy on a large scale owing to their large size and high cost. Here, we report a sensing node featuring low power consumption and low cost, achieved by integrating microsensor, microfluidic, and electronic modules into a compact size for automatic and scalable heavy metal pollution monitoring. Digital microfluidic and electrochemical sensing modules are integrated on a chip, thereby combining the procedures of sample pretreatment, electrochemical sensing, and waste removal for automatic and continuous monitoring. The feasibility of the platform is demonstrated by Pb2+ detection in tap water. With a 3500 mA·h battery, the compact sensing node could work for several years in principle. There is scope for further improvements to the system in terms of wider functionality and reductions in size, power consumption, and cost. The sensing node presented here is a strong candidate for distributed monitoring of water quality as an Internet-of-Things application.

Persimmon fruits accumulate a large amount of proanthocyanidins (PAs), which makes an astringent sensation. Proanthocyanidins (PAs) are the polymers of flavan-3-ols stored in plant vacuoles under laccase activation. A laccase gene, DkLAC2, is putatively involved in PAs biosynthesis and regulated by microRNA (DkmiR397) in persimmon. However, the polymerization of PAs in association with miRNA397 still needs to be explored in persimmon. Here, we identified pre-DkmiR397 and its target gene DkLAC2 in ‘Eshi 1’ persimmon. Histochemical staining with GUS and dual luciferase assay both confirmed DkmiR397-DkLAC2 binding after co-transformation in tobacco leaves. Diverse expression patterns of DkLAC2 and DkmiR397 were exhibited during persimmon fruit development stages. Moreover, a contrasting expression pattern was also observed after the combined DkLAC2-miR397 transformation in persimmon leaves, suggesting that DkmiR397 might be a negative regulator of DkLAC2. Similarly, the transient transformation of DkmiR397 in persimmon fruit discs in vitro also reduced PA accumulation by repressing DkLAC2, whereas the up-regulation of DkLAC2 increased the accumulation of PAs by short tandem target mimic STTM-miR397. A similar expression pattern was observed when overexpressing of DkLAC2 in Arabidopsis wild type (WT) and overexpression of DkLAC2, DkmiR397 in persimmon leaf callus. Our results revealed that the role of DkmiR397 repressed the expression of DkLAC2 concerning PA biosynthesis, providing a potential target for the manipulation of PAs metabolism in persimmon.

In the cracking process of waste tires, pyrolysis carbon black (CBp), as a solid product, accounts for about 35% of the total tire rubber content. Here, the treated CBp has been gradually applied to the tire formula to improve the recycling efficiency of waste tires. This study elucidated the influence of adding CBp during the tire mixing process on soft friction and metal wear. Compared with industrial carbon black (I-CB), the friction coefficient of CBp was smaller at different mixing stages, and the ripple caused by adhesion friction was not evident. After the modified CBp (M-CBp) was obtained by implementing the surface activation of common CBp (C-CBp), the friction coefficient between M-CBp and metal increased by 10%, while the filler dispersion and comprehensive mechanical properties showed an upward trend. The wear rate of metal was higher than that observed after adding I-CB during the same mixing mode; thus, it was necessary to strengthen the wear resistance of the inner-wall surface of the mixing chamber. The –OH group on the M-CBp surface can also participate in the silane coupling reaction and aggravate the metal wear of the mixer chamber wall. Through a comparison of results, the mixing friction coefficient can reflect the strength of filler–rubber interaction, which in turn can preliminarily represent the dispersion effect and comprehensive properties, reveal the reason behind the poor performance of CBp, and highlight the need for modification from the perspective of tribology.

The dam foundation rock mass, at the Baihetan hydropower station on the Jinsha River, is mainly columnar jointed basalt, with faults and fissures developed. Considering adverse factors such as the unloading relaxation or the opening of the fissures due to excavation blasting, consolidation grouting is needed to improve the integrity of the dam foundation rock mass. According to the physical and mechanical properties of columnar jointed basalt and the continuity of construction, the effectiveness of overburden consolidation grouting is experimentally studied. The results show that this grouting technology can obviously improve the integrity and uniformity of a dam foundation rock mass and reduce the permeability of the rock mass. After grouting, the average increase in the wave velocity of the rock mass is 7.3%. The average improvement in the deformation modulus after grouting is 13.5%. After grouting, the permeability of 99% of the inspection holes in the Lugeon test section had Lugeon values of no more than 3 LU. This improvement is considerable and provides a case to engineering application.

To obtain critical rainfall (CR) estimates similar to the rainfall value that causes minor basin outlet flooding, and to reduce the flash flood warning missed/false alarm rate, the effect of unit hydrographs (UHs) and rainfall hyetographs on computed threshold rainfall (TR) values was investigated. The Tanjia River basin which is a headwater subbasin of the Greater Huai River basin in China was selected as study basin. Xin’anjiang Model, with subbasins as computation units, was constructed, and time-variant distributed unit hydrographs (TVUHs) were used to route the channel network concentration. Calibrated Xin’anjiang Model was employed to derive the TVUHs and to obtain the maximum critical rainfall duration (Dmax) of the study basin. Initial soil moisture condition was represented by the antecedent precipitation index (Pa). Rainfall hyetographs characterized by linearly increasing, linearly decreasing, and uniform hyetographs were used. Different combinations of the three hyetographs and UHs including TVUHs and time-invariant unit hydrographs (TIVUHs) were utilized as input to the calibrated Xin’anjiang Model to compute the relationships between TR and Pa (TR-Pa curves) by using trial and error methodology. The computed TR-Pa curves reveal that, for given Pa and UH, the TR corresponding to linearly increasing hyetograph is the minimum one. So, the linearly increasing hyetograph is the optimum hyetograph type for estimating CR. In the linearly increasing hyetograph context, a comparison was performed between TR-Pa curves computed from different UHs. The results show that TR values for different TIVUHs are significantly different and the TR-Pa curve gradient of TVUHs is lower than that of TIVUHs. It is observed that CR corresponds to the combination of linearly increasing hyetograph and TVUHs. The relationship between CR and Pa (CR-Pa curves) and that between CR and duration (D) (CR-D curves) were computed. Warnings for 12 historical flood events were performed. Warning results show that the success rate was 91.67% and that the critical success index (CSI) was 0.91. It is concluded that the combination of linearly increasing hyetograph and TVUHs can provide the CR estimate similar to the minimum rainfall value necessary to cause flash flooding.

(1) Background: Residue degradation plays a very important role in terrestrial ecosystems and residue mixing is the main factor affecting the degradation rates. However, in the agricultural systems, the effect of residue mixing on the degradation of pepper residues and the microbial community in pepper root residues is not clear. (2) Methods: In this study, we added different residues into soil by using double-layered nylon litterbags in culture bottles. The treatments including pepper root (P: Capsicum annuum L.), soybean [S: Glycine max (L.) Merr.] and maize (M: Zea mays L.) residue, as well as mixtures of soybean + pepper (SP), maize + pepper (MP), maize + soybean + pepper (MSP) mixtures. Litterbags were harvested after 7, 14, 28, and 56 days, respectively. Mass loss and nitrogen and phosphorus contents in pepper residue were quantified and bacterial and fungal community levels in pepper residues were analyzed using quantitative PCR and high throughput amplicon sequencing; (3) Results: The study showed that the mass loss and fungal community abundance of pepper root residue in mixtures were higher than P, except day 7. The phosphorus contents in MSP-P and MP-P were significantly lower than that for P at day 28 and day 56. Illumina MiSeq sequencing showed that the presence of maize residue significantly altered the microbial community composition of pepper root pepper. Day 56. (4) Conclusions: Our results suggest that residue mixing changed the microbial community abundance in pepper residue and promoted the degradation of pepper residues compared to pepper residue decomposition alone, especially for mixtures with soybean.

Abstract Background Iron is a kind of essential trace mineral in the human body, while the studies on its physiological requirement are very limited recently, especially in China. And most studies were performed with the radioisotope tracer technique, which was harmful to health. This study aimed to first get the value of iron physiological requirements in Chinese adults assessed by the stable isotope labeling technique. Methods Forty-four eligible young Chinese healthy adults were randomly recruited from the Bethune Military Medical College (Shijiazhuang, Hebei, China) between January 2010 and March 2011, and 19 subjects were included in the final data analysis. After adaptive diets and observation, subjects received 58Fe intravenously. The baseline venous blood sample and general information were collected on day 0. Venous blood samples were also collected on day 14, 30, 60, 100, 120, 150, 240, 330, 425, 515, 605, 767, 1155, respectively. The blood samples were acid digested by a Microwave Digestion System and then analyzed by the MC-ICP-MS and Atomic Absorption Spectroscopy to get the abundance of Fe isotopes and the total iron concentration respectively. The circulation rate (the proportion of blood iron to whole body iron) could be calculated by the intake amount, background content and the peak isotope content. When the abundance changed stably, the iron physiological requirement could be calculated by the iron loss in a period of time. Results The abundance of 58Fe reached its peak on day 14, and changed stably from day 425. The average circulation rate was 84%, with no significance difference between the 2 genders. The mean iron requirement in females was 1101.68 μg/d, and the mean requirement adjusted by body weight was 20.69 μg/kg.d. For males, the mean iron requirement was 959.9 μg/d, and the requirement adjusted by body weight was 14.04 μg/kg.d. Conclusion Our study has obtained the data about the iron physiological requirements of Chinese adults using stable isotope labeling technique, which could provide the basis for adjusting iron DRIs of Chinese people in the future. Trial registration The trial was registered at the Chinese Clinical Trial Registry (No: ChiCTR-TRC-09000581).

Summary: To maintain proper locomotive patterns, animals constantly monitor body posture with their proprioceptive receptors. In Drosophila, the chordotonal organs (Cho) are especially important in the regulation of locomotion pattern. However, how Cho neurons that are normally activated with sound (vibration) transduce static displacement caused by body position change remains unclear. Here, we report that piezo-like (pzl), a homolog for mammalian piezo1 and 2, is essential for Cho’s function in locomotion. The mutant allele of pzl showed severe defects in crawling pattern and body gesture control, which were rescued by expressing Pzl specifically in Cho neurons. The ability of Cho neurons to respond to micrometer-scale body wall displacement requires pzl. Intriguingly, human or mouse Piezo1 can rescue pzl-mutant phenotypes, suggesting a conserved role of the Piezo-family proteins in locomotion. : Proprioception is critical for animals to maintain body postures and locomotive patterns. Hu et al. find that Drosophila piezo-like (pzl), a putative mechanosensitive channel gene and a homolog for mammalian piezo1 and 2, functions in the stretch-sensing proprioceptive chordotonal neurons to regulate Drosophila larval locomotion. Keywords: mechanotransduction, piezo family, proprioception, chordotonal neurons, locomotion, Drosophila

[This corrects the article DOI: 10.1371/journal.pone.0224296.].

Dehydration-responsive element-binding (DREB) transcription factors (TFs) are key regulators of stress-inducible gene expression in plants. Anthocyanins, an important class of flavonoids, protect plants from reactive oxygen species produced under abiotic stresses. However, regulation of DREBs on anthocyanin accumulation is largely unknown. Here, an A-5 subgroup DREB gene (AmDREB3) isolated from Ammopiptanthus mongolicus, a desert broadleaf shrub with very high tolerance to harsh environments, was characterized in terms of both abiotic stress tolerance and anthocyanin accumulation. AmDREB3 does not contain the transcriptional repression motif EAR, and the protein was located in the nucleus and has transcriptional activation capacity. The transcription of AmDREB3 was differentially induced in the shoots and roots of A. mongolicus seedlings under drought, salt, heat, cold, ultraviolet B, and abscisic acid treatments. Moreover, the transcript levels in twigs, young leaves, and roots were higher than in other organs of A. mongolicus shrubs. Constitutively expressing AmDREB3 improved the tolerance of transgenic Arabidopsis to drought, high salinity and heat, likely by inducing the expression of certain stress-inducible genes. The transgenic Arabidopsis seedlings also exhibited an obvious purple coloration and significant increases in anthocyanin accumulation and/or oxidative stress tolerance under drought, salt, and heat stresses. These results suggest that the AmDREB3 TF may be an important positive regulator of both stress tolerance and anthocyanin accumulation.

Soil microbial communities have profound effects on the growth, nutrition and health of plants in agroecosystems. Understanding soil microbial dynamics in cropping systems can assist in determining how agricultural practices influence soil processes mediated by microorganisms. In this study, soil bacterial communities were monitored in a continuously monocropped Jerusalem artichoke (JA) system, in which JA was successively monocropped for 3 years in a wheat field. Soil bacterial community compositions were estimated by amplicon sequencing of the 16S rRNA gene. Abundances of ammonia-oxidizing and denitrifying bacteria were estimated by quantitative PCR analysis of the amoA, nirS, and nirK genes. Results showed that 1–2 years of monocropping of JA did not significantly impact the microbial alpha diversity, and the third cropping of JA decreased the microbial alpha diversity (P < 0.05). Principal coordinates analysis and permutational multivariate analysis of variance analyses revealed that continuous monocropping of JA changed soil bacterial community structure and function profile (P < 0.001). At the phylum level, the wheat field was characterized with higher relative abundances of Latescibacteria, Planctomycetes, and Cyanobacteria, the first cropping of JA with Actinobacteria, the second cropping of JA with Acidobacteria, Armatimonadetes, Gemmatimonadetes, and Proteobacteria. At the genus level, the first cropping of JA was enriched with bacterial species with pathogen-antagonistic and/or plant growth promoting potentials, while members of genera that included potential denitrifiers increased in the second and third cropping of JA. The first cropping of JA had higher relative abundances of KO terms related to lignocellulose degradation and phosphorus cycling, the second cropping of JA had higher relative abundances of KO terms nitrous-oxide reductase and nitric-oxide reductase, and the third cropping of JA had higher relative abundances of KO terms nitrate reductase and nitrite reductase. The abundances of amoA genes decreased while nirK increased in the third cropping of JA, nirS continuously increased in the second and third cropping of JA (P < 0.05). Redundancy analysis and Mantel test found that soil organic carbon and Olsen phosphorus contents played important roles in shaping soil bacterial communities. Overall, our results revealed that continuous monocropping of JA changed soil bacterial community composition and its functional potentials.

Intestinal epithelial barrier damage disrupts immune homeostasis and leads to many intestinal disorders. Lactobacillus reuteri strains have probiotic functions in their modulation of the microbiota and immune system in intestines. In this study, the effects of L. reuteri LR1, a new strain isolated from the feces of weaning piglets, on intestinal epithelial barrier damage in IPEC-1 cells caused by challenge with enterotoxigenic Escherichia coli (ETEC) K88 were examined. It was found that L. reuteri LR1, in large part, offset the ETEC K88-induced increase in permeability of IPEC-1 cell monolayers and decreased the adhesion and invasion of the coliform in IPEC-1 cells. In addition, L. reuteri LR1 increased transcript abundance and protein contents of tight junction (TJ) proteins zonula occluden-1 (ZO-1) and occludin in ETEC K88-infected IPEC-1 cells, whereas it had no effects on claudin-1 and F-actin expression. Using colloidal gold immunoelectron microscopy, these effects of L. reuteri LR1 on ZO-1 and occludin content in IPEC-1 cells were confirmed. By using ML-7, a selective inhibitor of myosin light-chain kinase (MLCK), the beneficial effect of L. reuteri LR1 on contents of ZO-1 and occludin was shown to be dependent on the MLCK pathway. In conclusion, L. reuteri LR1 had beneficial effects on epithelial barrier function consistent with increasing ZO-1 and occludin expression via a MLCK-dependent manner in IPEC-1 cells during challenge with ETEC K88.

Phytotoxic effects of phenolic compounds have been extensively studied, but less attention has been given to the effects of these compounds on soil microbial communities, which are crucial to the productivity of agricultural systems. Responses of cucumber rhizosphere bacterial and fungal communities to syringic acid (SA), a phenolic compound with autotoxicity to cucumber, were analyzed by high-throughput sequencing of 16S rRNA gene and internal transcribed spacer amplicons. SA at the concentration of 0.1 μmol g-1 soil changed rhizosphere bacterial and fungal community compositions, decreased bacterial community diversity but increased fungal community richness and diversity (P

In gas gathering pipeline system, 90° elbow suffers constant and severe collision of gas-carrying particles, which causes serious erosion on the inner wall. In this study, models of standard elbows (carbon steel with 210 GPa in elasticity modulus and 8300 kg/m 3 in density) whose diameter varies from 65 to 200 mm with curvature ratio 1.5 are established to predict erosion rate causes by silica sand (100 μm in diameter, 1550 kg/m 3 in bulk density) under gas gathering operating conditions (5.0 MPa, 10 m/s). Unlike most of the previous studies which only focus on erosion result on the wall, in this article, author extracts sample particles as research objects and studies the motion of every single particle. It has been found that there are three typical types of particle motion patterns in the elbow, and a part of sand particles released from lower area of incident plane will not even cause erosion.

Gut microbiomes in various fish species were widely investigated with the rapid development of next-generation sequencing technologies. However, little is known about gastrointestinal (GI) microbial communities in mudskippers, a representative group of marine amphibious fishes, and their comparisons with other vertebrate animals from different habitats. Here, we performed a comprehensive analysis on microbial composition in five representative vertebrate groups (including amphibious mudskippers, marine and freshwater aquatic fishes, amphibians, and terrestrial animals) via operational taxonomic unit (OTU) survey and obtained a microbial gene catalog of five common fish species by metagenome sequencing. We observed that Cyanobacteria, Proteobacteria, Firmicutes, Bacteroidetes, and Fusobacteria were the most substantial bacteria in mudskippers. Differential variances in composition patterns of GI microbiota among the vertebrate groups were determined, although Proteobacteria and Firmicutes were the shared phyla with high abundance. In addition, Cetobacterium and Photobacterium were the most abundant genera in core OTUs of these examined omnivores, carnivores, and herbivores. Our metagenomic analysis also showed significant differences between the representative blue-spotted mudskipper and grass carp (both are herbivorous fishes) in microbes at the phylum and class levels and functional gene terms. Moreover, several bacteriocin-related genes were identified in the five common fishes, suggesting their potential contributions to pathogen resistance. In summary, our present work not only sheds new light on the correlation of GI microbiota composition with living habitats and feeding habits of the hosts, but also provides valuable bacterial genetic resources for healthy growth of aquaculture fishes.

BACKGROUND:This study was to observe the non-heme iron absorption and biological utilization from typical whole Chinese diets in young Chinese healthy urban men, and to observe if the iron absorption and utilization could be affected by the staple food patterns of Southern and Northern China. MATERIALS AND METHODS:Twenty-two young urban men aged 18-24 years were recruited and randomly assigned to two groups in which the staple food was rice and steamed buns, respectively. Each subject received 3 meals containing approximately 3.25 mg stable 57FeSO4 (the ratio of 57Fe content in breakfast, lunch and dinner was 1:2:2) daily for 2 consecutive days. In addition, approximately 2.4 mg 58FeSO4 was administered intravenously to each subject at 30-60 min after dinner each day. Blood samples were collected from each subject to measure the enrichment of the 57Fe and 58Fe. Fourteen days after the experimental diet, non-heme iron absorption was assessed by measuring 57Fe incorporation into red blood cells, and absorbed iron utilization was determined according to the red blood cell incorporation of intravenously infused 58Fe SO4. RESULTS:Non-heme iron intake values overall, and in the rice and steamed buns groups were 12.8 ±2.1, 11.3±1.3 and 14.3±1.5 mg, respectively; the mean 57Fe absorption rates were 11±7%, 13±7%, and 8±4%, respectively; and the mean infused 58Fe utilization rates were 85±8%, 84±6%, and 85±10%, respectively. There was no significantly difference in the iron intakes, and 57Fe absorption and infused 58Fe utilization rates between rice and steamed buns groups (all P>0.05). CONCLUSION:We present the non-heme iron absorption and utilization rates from typical whole Chinese diets among young Chinese healthy urban men, which was not affected by the representative staple food patterns of Southern and Northern China. This study will provide a basis for the setting of Chinese iron DRIs.

Since arsenic trioxide (As3+) has been successfully used in the treatment of acute promyelocytic leukemia (APL), its adverse effects on patients have been problematic and required a solution. Considering the good therapeutic potency and low toxicity of tetraarsenictetrasulfide (As4S4) in the treatment of APL, we investigated the effects of combining As4S4 and As3+ on the apoptosis and differentiation of NB4 and primary APL cells. As4S4, acting similarly to As3+, arrested the G1/S transition, induced the accumulation of cellular reactive oxygen species, and promoted apoptosis. Additionally, low concentrations of As4S4 (0.1-0.4 μM) induced differentiation of NB4 and primary APL cells. Compared with the As4S4- or As3+-treated groups, the combination of As4S4 and As3+ obviously promoted apoptosis and differentiation of NB4 and primary APL cells. Mechanistic studies suggested that As4S4 acted synergistically with As3+ to down-regulate Bcl-2 and nuclear factor-κB expression, up-regulate Bax and p53 expression, and induce activation of caspase-12 and caspase-3. Moreover, the combination of low concentrations of As4S4 and As3+ enhanced degradation of the promyelocytic leukemia-retinoic acid receptor α oncoprotein. In summary, As4S4 and As3+ synergistically induce the apoptosis and differentiation of NB4 and primary APL cells.

Hypoxia inducible factor 1 (HIF-1) plays a pivotal role in cellular responses to hypoxia. Prolyl hydroxylase 3 (PHD3) degrades HIF-1α under normoxic conditions through the hydroxylation of HIF-1α for proteolysis. Inhibiting PHD3 activity is crucial for up-regulating HIF-1α, thereby acting as a potential target for treating hypoxia-related diseases. In this study, two proline analogues (PA1 and PA2) were screened as PHD3 inhibitors with apparent EC50 values of 1.53 and 3.17 µM respectively, indicating good inhibition potency. Nine proteins, significantly regulated by PA1, were identified using 2-DE coupled with MALDI-TOF/TOF MS. Pyruvate kinase isozymes M1/M2 (PKM) and alpha-enolase 1 (ENO1), which are key modulators of glycolysis, are directly regulated by HIF-1α. Moreover, VEGF, a signal protein stimulating angiogenesis, was strongly promoted by PA1. Our findings suggest that PA1 stabilized HIF-1α as well as up-regulated glycolysis and angiogenesis proteins. Herein, for the first time, we systematically studied proline analogue PA1 as a PHD3 inhibitor, which provides innovative evidence for the treatment of HIF-related diseases.

Arsenic (III) methyltransferase (AS3MT) is a cysteine (Cys)-rich enzyme that catalyzes the biomethylation of arsenic. To investigate how these crucial Cys residues promote catalysis, we used matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) to analyze Cys residues in recombinant human arsenic (III) methyltransferase (hAS3MT). We detected two disulfide bonds, Cys250-Cys32 and Cys368-Cys369, in hAS3MT. The Cys250-Cys32 disulfide bond was reduced by glutathione (GSH) or other disulfide bond reductants before the enzymatic methylation of arsenite (iAs3+). In addition to exposing residues around the active sites, cleavage of the Cys250-Cys32 pair modulated the conformation of hAS3MT. This adjustment may stabilize the binding of S-Adenosyl-L-methionine (AdoMet) and favor iAs3+ binding to hAS3MT. Additionally, we observed the intermediate of Cys250-S-adenosylhomocysteine (AdoHcy), suggesting that Cys250 is involved in the transmethylation. In recovery experiments, we confirmed that trivalent arsenicals were substrates for hAS3MT, methylation of arsenic occurred on the enzyme, and an intramolecular disulfide bond might be formed after iAs3+ was methylated to dimethylarsinous acid (DMA3+). In this work, we clarified both the functional roles of GSH and the crucial Cys residues in iAs3+ methylation catalyzed by hAS3MT.

Residues Tyr59, Gly78, Ser79, Met103, Gln107, Ile136 and Glu137 in human arsenic (+3 oxidation state) methyltransferase (hAS3MT) were deduced to form a potential hydrogen bond network around S-adenosylmethionine (SAM) from the sequence alignment between Cyanidioschyzon merolae arsenite S-adenosylmethyltransferase (CmArsM) and hAS3MT. Herein, seven mutants Y59A, G78A, S79A, M103A, Q107A, I136A and E137A were obtained. Their catalytic activities and conformations were characterized and models were built. Y59A and G78A were completely inactive. Only 7.0%, 10.6% and 13.8% inorganic arsenic (iAs) was transformed to monomethylated arsenicals (MMA) when M103A, Q107A and I136A were used as the enzyme. The Vmax (the maximal velocity of the reaction) values of M103A, Q107A, I136A and E137A were decreased to 8%, 22%, 15% and 50% of that of WT-hAS3MT, respectively. The KM(SAM) (the Michaelis constant for SAM) values of mutants M103A, I136A and E137A were 15.7, 8.9 and 5.1 fold higher than that of WT-hAS3MT, respectively, indicating that their affinities for SAM were weakened. The altered microenvironment of SAM and the reduced capacity of binding arsenic deduced from KM(As) (the Michaelis constant for iAs) value probably synergetically reduced the catalytic activity of Q107A. The catalytic activity of S79A was higher than that of WT despite of the higher KM(SAM) , suggesting that Ser79 did not impact the catalytic activity of hAS3MT. In short, residues Tyr59 and Gly78 significantly influenced the catalytic activity of hAS3MT as well as Met103, Ile136 and Glu137 because they were closely associated with SAM-binding, while residue Gln107 did not affect SAM-binding regardless of affecting the catalytic activity of hAS3MT. Modeling and our experimental results suggest that the adenine ring of SAM is sandwiched between Ile136 and Met103, the amide group of SAM is hydrogen bonded to Gly78 in hAS3MT and SAM is bonded to Tyr59 with van der Waals, cation-π and hydrogen bonding contacts.

Arsenic (III) methyltransferase (AS3MT) catalyzes the process of arsenic methylation. Each arsenite (iAs(3+)) binds to three cysteine residues, methylarsenite (MMA(3+)) binds to two, and dimethylarsenite (DMA(3+)) binds to one. However, only two As-binding sites (Cys156 and Cys206) have been confirmed on human AS3MT (hAS3MT). The third As-binding site is still undefined. Residue Cys72 in Cyanidioschyzon merolae arsenite S-adenosylmethyltransferase (CmArsM) may be the third As-binding site. The corresponding residue in hAS3MT is Cys61. Functions of Cys32, Cys61, and Cys85 in hAS3MT are unclear though Cys32, Cys61, and Cys85 in rat AS3MT have no effect on the enzyme activity. This is why the functions of Cys32, Cys61, and Cys85 in hAS3MT merit investigation. Here, three mutants were designed, C32S, C61S, and C85S. Their catalytic activities and conformations were determined, and the catalytic capacities of C156S and C206S were studied. Unlike C85S, mutants C32S and C61S were completely inactive in the methylation of iAs(3+) and active in the methylation of MMA(3+). The catalytic activity of C85S was also less pronounced than that of WT-hAS3MT. All these findings suggest that Cys32 and Cys61 markedly influence the catalytic activity of hAS3MT. Cys32 and Cys61 are necessary to the first step of methylation but not to the second. Cys156 and Cys206 are required for both the first and second steps of methylation. The S(C32) is located far from arsenic in the WT-hAS3MT-SAM-As model. The distances between S(C61) and arsenic in WT-hAS3MT-As and WT-hAS3MT-SAM-As models are 7.5 Å and 4.1 Å, respectively. This indicates that SAM-binding to hAS3MT shortens the distance between S(C61) and arsenic and promotes As-binding to hAS3MT. This is consistent with the fact that SAM is the first substrate to bind to hAS3MT and iAs is the second. Model of WT-hAS3MT-SAM-As and the experimental results indicate that Cys61 is the third As-binding site.