[Effects of Different Topdressing Nitrogen Rates on Soil Fungal Community Structure and Ecological Network in Wheat Field Under Crop Residue Retention].

Crop residue retention and fertilizer application are the main sources of soil nutrient input in fields. Crop residue retention combined with appropriate fertilizer application rates could provide necessary nutrients for crop production under the premise of environmentally friendly conditions. The aim of this study was to clarify the influence of different topdressing nitrogen rates on the soil fungal community in a wheat field under crop residue retention and to evaluate the rationality of nitrogen fertilizer management in winter wheat from the perspective of soil ecological function. On the basis of full straw retention and 150 kg·hm-2 basal nitrogen, treatments with five topdressing nitrogen rates (0, 37.5, 75, 112.5, and 150 kg·hm-2) were set up. The abundance, diversity, structure, and ecological network of soil fungal communities were analyzed using real-time fluorescence quantitative PCR and high-throughput sequencing, and the main soil physical and chemical factors driving the change in soil fungal communities were explored. The results showed that, compared with the no topdressing nitrogen and low topdressing nitrogen rate treatments, high topdressing nitrogen rate treatments increased soil total nitrogen and mineral nitrogen and decreased soil pH, total phosphorus, available phosphorus, and available potassium. Compared with the no topdressing nitrogen treatments, the 37.5-150 kg·hm-2 topdressing nitrogen treatments significantly increased soil fungal community abundance (P<0.05), whereas there was no significant difference among different topdressing nitrogen treatments (P>0.05). The Heip index and Shannon index of soil fungal communities decreased gradually with the increase in topdressing nitrogen rate, and the Sobs index, Heip index, and Shannon index of soil fungal communities in the treatment with 150 kg·hm-2 topdressing nitrogen were significantly lower than those of 0-75 kg·hm-2 topdressing nitrogen treatments (P<0.05). Principal component analysis and similarity analysis showed that there were significant differences in soil fungal community structure under different topdressing nitrogen rate treatments (P<0.05). With the increase in topdressing nitrogen rate, the number of network edges and average number of neighbors of soil fungal ecological network increased first and then decreased, and the network complexity of 37.5 kg·hm-2 topdressing nitrogen treatments was the highest. Compared with 0-75 kg·hm-2 topdressing nitrogen treatments, 112.5 kg·hm-2 and 150 kg·hm-2 topdressing nitrogen treatments increased the characteristic path length of the soil fungal ecological network, whereas it decreased the network density. With the increase in topdressing nitrogen rate, the relative abundance of soil saprotrophs gradually increased, and the pathotroph-saprotroph-symbiotroph relative abundance gradually decreased. Redundancy analysis showed that soil pH, total phosphorus, mineral nitrogen, available phosphorus, and available potassium were the main soil physicochemical factors affecting the soil fungal community structure in the wheat field under different topdressing nitrogen rate treatments. In conclusion, on the basis of straw retention and basal nitrogen, topdressing nitrogen at the wheat jointing stage could change the diversity, structure, and species composition of the soil fungal community, in turn affecting the soil fungal ecological network and function, and high topdressing nitrogen rates could reduce soil fungal community diversity, ecological network complexity, and network density.

[Soil bacterial communities of different crop rotations and yield of succeeding wheat.] / 不同轮作茬口土壤细菌群落及后茬小麦产量.

Evaluating ecological sustainability and crop productivity of different crop rotation patterns could provide theoretical support for adjusting and optimizing crop planting structure. We set seven treatments with different rotation crops and periods. We used real-time quantitative PCR to determine the abundance of soil bacterial community and 16S rRNA gene amplicon high-throughput sequencing technology to analyze diversity and taxa composition of soil bacterial community. Both soil available nutrients and succeeding wheat yield were measured. The results showed that, compared with the rotation with summer maize, the rotations with summer peanut or soybean in diffe-rent periods reduced soil organic carbon, mineral nitrogen, and available potassium, but significantly increased soil available phosphorus. The 16S rRNA gene copy numbers of soil bacteria in the treatments of rotations with summer peanut or soybean in different periods were significantly decreased, while community richness and diversity were increased. Different rotation crops significantly changed the structure and taxonomic composition of soil bacterial community. Compared with the rotation with summer maize, the rotations with summer soybean in different periods significantly increased the 1000-grain weight and grain yield of succeeding winter wheat. In conclusion, rotations with summer peanut or soybean in different periods could increase soil available phosphorus content and bacterial diversity, and significantly change soil bacterial community structure. In particular, rotation with summer soybean performed best in promoting yield formation of succeeding winter wheat.

Inhalable antibiotic resistomes emitted from hospitals: metagenomic insights into bacterial hosts, clinical relevance, and environmental risks.

BACKGROUND: Threats of antimicrobial resistance (AMR) to human health are on the rise worldwide. Airborne fine particulate matter (PM2.5), especially those emitted from hospitals, could serve as a substantial yet lesser-known environmental medium of inhalable antibiotic resistomes. A genome-centric understanding of the hosting bacterial taxa, mobility potential, and consequent risks of the resistomes is needed to reveal the health relevance of PM2.5-associated AMR from clinical settings. RESULTS: Compared to urban ambient air PM2.5, the hospital samples harbored nearly twice the abundance of antibiotic resistantance genes (ARGs, ~ 0.2 log10(ARGs/16S rRNA gene)) in the summer and winter sampled. The profiled resistome was closely correlated with the human-source-influenced (~ 30% of the contribution) bacterial community (Procrustes test, P < 0.001), reflecting the potential antibiotic-resistant bacteria (PARB), such as the human commensals Staphylococcus spp. and Corynebacterium spp. Despite the reduced abundance and diversity of the assembled metagenomes from summer to winter, the high horizontal transfer potential of ARGs, such as the clinically relevant blaOXA and bacA, in the human virulent PARB remained unaffected in the hospital air PM samples. The occurring patterns of ß-lactam resistance genes and their hosting genomes in the studied hospital-emitting PM2.5 were closely related to the in-ward ß-lactam-resistant infections (SEM, std = 0.62, P < 0.01). Featured with more abundant potentially virulent PARB (2.89 genome copies/m3-air), the hospital samples had significantly higher resistome risk index scores than the urban ambient air samples, indicating that daily human exposure to virulent PARB via the inhalation of PM2.5 was ten times greater than from the ingestion of drinking water. CONCLUSIONS: The significance of AMR in the studied hospital-emitting PM2.5 was highlighted by the greater abundance of ARGs, the prevalence of potentially virulent PARB, and the close association with hospital in-ward ß-lactam infections. A larger-scale multi-source comparison of genome-resolved antibiotic resistomes is needed to provide a more holistic understanding to evaluate the importance of airborne AMR from the "One-Health" perspective. Video Abstract.

Complete chloroplast genome sequencing of Job's tears (Coix L.): genome structure, comparative analysis, and phylogenetic relationships.

Job's tears, also known as adlay, is a valuable plant that has commonly been used in traditional Chinese medicine, as well as an edible food. Due to the lack of knowledge of its genetics and gaps in its evolutionary analysis, breeding of adlay has been hindered. Here, we report five complete chloroplast genomes of various species and varieties in the genus by Illumina sequencing, while their genome structure, comparative analysis, and phylogenetic relationships were conducted. Genome sizes ranged from 140,860 to 140,864 bp in length, GC contents were 38.43%, and genome architecture was of a typical quadripartite structure. We annotated 82～83 protein-coding genes and 46～47 non-coding RNA genes in each genome and they functionally associated with self-replication, photosynthesis, cytochrome synthesis and other unknown functions. Three codons that encoded tryptophan, arginine and leucine were used frequently at rates of 41.42, 37.98, and 32.28% respectively. The preferred codons consistently ended with A or T. A total of 146 simple sequence repeats (SSR), 9 insertions and deletions (InDels) and 143 single nucleotide polymorphisms (SNPs) were observed among genomes. The InDel and SNP variations were mostly distributed in intergenic regions. It confirmed that Coix, Sorghum, Saccharum, Zea, Tripsacum and Saccharum were closely genera and the genetic distance of Sorghum to Coix was closer than Zea to Coix. These results give us more insight into the evolution of Coix in a wide range of evolutionary studies.

Development and Evaluation of Stable Sugarcane Mosaic Virus Mild Mutants for Cross-Protection Against Infection by Severe Strain.

Sugarcane mosaic virus (SCMV; genus Potyvirus) induces maize dwarf mosaic disease that has caused serious yield losses of maize in China. Cross-protection is one of the efficient strategies to fight against severe virus strains. Although many mild strains have been identified, the spontaneous mutation is one of the challenging problems affecting their application in cross-protection. In this study, we found that the substitution of cysteine (C) at positions 57 or 60 in the zinc finger-like motif of HC-Pro with alanine (A; C57A or C60A) significantly reduced its RNA silencing suppression activity and SCMV virulence. To reduce the risk of mild strains mutating to virulent ones by reverse or complementary mutations, we obtained attenuated SCMV mutants with double-mutations in the zinc finger-like and FRNK motifs of HC-Pro and evaluated their potential application in cross-protection. The results showed that the maize plants infected with FKNK/C60A double-mutant showed symptomless until 95 days post-inoculation and FKNK/C60A cross-protected plants displayed high resistance to severe SCMV strain. This study provides theoretical and material bases for the control of SCMV through cross-protection.

Development and application of a full-length infectious clone of potato virus Y isolate belonging to SYR-I strain.

Potato virus Y (PVY) causes huge damage to potato and tobacco production worldwide. The complete genome sequence of GZ, a PVY isolate (strain SYR-I) from Guizhou province, China, was cloned into the binary vector pCambia0390. Three introns were individually inserted into the P3 and CI ORFs to produce plasmid pCamPVY-GZ. The plasmid could infect plants of Nicotiana benthamiana, N. tabacum via agroinfiltration and plants of pepper and potato by mechanical inoculation. The green fluorescence protein gene of Aequoria victoriae was cloned into the encoding regions between nuclear inclusion body 'b' and coat protein genes in pCamPVY-GZ to produce pCamPVY-GZ-GFP, which could infect plants of N. benthamiana, N. tabacum, potato and tomato, and produce green fluorescence in the systemic leaves of inoculated plants. Mutations were introduced to pCamPVY-GZ to make the lysine (K) 391 and glutamic acid (E)410 of helper component-proteinase to arginine (R) and asparagic acid (E), respectively. Unlike wild type PVY-GZ, the mutant PVY-K391R/E410D could not induce veinal necrosis in N. tabacum plants. With an interval of 14 days, mutant PVY-K391R/E410D could protect N. tabacum plants from the infection of severe PVY strain. The results presented here provide a promising alternate for the prevention of diseases caused by PVY.

Phosphorus Removal and Recovery from Wastewater using Fe-Dosing Bioreactor and Cofermentation: Investigation by X-ray Absorption Near-Edge Structure Spectroscopy.

A new phosphorus (P) removal and recovery process that integrates an FeCl3-dosing, membrane bioreactor (MBR), and side-stream cofermentation was developed for wastewater treatment. The Fe and P species and their transformation mechanisms via aerobic and anaerobic conditions were investigated with X-ray absorption near edge structure (XANES) spectroscopy. In the new treatment system, 98.4% of the total P in domestic wastewater was removed and retained in activated sludge in the MBR. During the subsequent acidogenic cofermentation with food waste, P in the MBR sludge was released and eventually recovered as vivianite, achieving an overall P recovery efficiency of 61.9% from wastewater. The main pathways for P removal and recovery with iron dosing and acidogenic fermentation were determined by XANES analysis. The results showed that Fe-enhanced P removal with the MBR was mainly achieved by precipitation as ferric phosphate (24.2%) and adsorption onto hydrous iron oxides (60.3%). During anaerobic fermentation, transition from Fe(III)-P to Fe(II)-P complex occurred in the sludge, leading to Fe(II) dissolution and P release. The pH decrease and microbial Fe reduction were crucial conditions for effective P extraction from the MBR sludge. The efficiency of P recovery increased with an increase in the fermentation time and organic load and a decrease of pH in the solution.

Chelant-enhanced washing of CCA-contaminated soil: Coupled with selective dissolution or soil stabilization.

Remediation of CCA-contaminated soil (Cr, Cu, and As) by biodegradable chelant-enhanced washing (EDDS, S,S-ethylene-diamine-disuccinic-acid) needs further enhancement. This study investigated the effectiveness of coupling with pre-treatment by selective dissolution and post-treatment by soil amendments, respectively. Three groups of reagents (reductants, alkaline solvents, and organic ligands) were adopted in the pre-treatment to dissolve the oxide minerals before EDDS extraction. In the post-treatment, soil amendments (coal fly ash (CFA), acid mine drainage sludge (AMDS), green waste compost (GWC)), and their mixtures) were used for a 2-month stabilization after 2-h EDDS washing. Multi-endpoint evaluation was performed by assessing the chemical state, leachability, mobility, bioaccessibility, and plant-availability of residual metal(loid)s as well as the cytotoxicity, enzyme activities, and available nutrients of the treated soils. Pre-treatment by dithionite-citrate-bicarbonate significantly enhanced extraction efficiency, but also increased the leachability of As and Cr and bioaccessibility of Cr in the treated soils. While sodium hydroxide removed the majority of As without increasing its leachability and bioaccessibility, it increased the cytotoxicity and inhibited the acid phosphatase activity. Post-treatment with AMDS and CFA effectively controlled the mobility and leachability of residual As and Cr after EDDS washing. However, destabilized Cu was only marginally immobilized by GWC due to strong Cu-EDDS complexation. The bioaccessibility and phytoavailability of Cu was primarily reduced by EDDS washing, while those of As and Cr could be attenuated by AMDS and CFA. This study indicates that coupling chemical extraction with subsequent soil amendment plays complementary roles in mitigating effects of residual metal(loid)s and improving environmental quality.

Inhibition of miR-128-3p by Tongxinluo Protects Human Cardiomyocytes from Ischemia/reperfusion Injury via Upregulation of p70s6k1/p-p70s6k1.

Background and Aims: Tongxinluo (TXL) is a multifunctional traditional Chinese medicine that has been widely used to treat cardiovascular and cerebrovascular diseases. However, no studies have explored whether TXL can protect human cardiomyocytes (HCMs) from ischemia/reperfusion (I/R) injury. Reperfusion Injury Salvage Kinase (RISK) pathway activation was previously demonstrated to protect the hearts against I/R injury and it is generally activated via Akt or (and) Erk 1/2, and their common downstream protein, ribosomal protein S6 kinase (p70s6k). In addition, prior studies proved that TXL treatment of cells promoted secretion of VEGF, which could be stimulated by the increased phosphorylation of one p70s6k subtype, p70s6k1. Consequently, we hypothesized TXL could protect HCMs from I/R injury by activating p70s6k1 and investigated the underlying mechanism. Methods and Results: HCMs were exposed to hypoxia (18 h) and reoxygenation (2 h) (H/R), with or without TXL pretreatment. H/R reduced mitochondrial membrane potential, increased bax/bcl-2 ratios and cytochrome C levels and induced HCM apoptosis. TXL preconditioning reversed these H/R-induced changes in a dose-dependent manner and was most effective at 400 µg/mL. The anti-apoptotic effect of TXL was abrogated by rapamycin, an inhibitor of p70s6k. However, inhibitors of Erk1/2 (U0126) or Akt (LY294002) failed to inhibit the protective effect of TXL. TXL increased p70s6k1 expression and, thus, enhanced its phosphorylation. Furthermore, transfection of cardiomyocytes with siRNA to p70s6k1 abolished the protective effects of TXL. Among the micro-RNAs (miR-145-5p, miR-128-3p and miR-497-5p) previously reported to target p70s6k1, TXL downregulated miR-128-3p in HCMs during H/R, but had no effects on miR-145-5p and miR-497-5p. An in vivo study confirmed the role of the p70s6k1 pathway in the infarct-sparing effect of TXL, demonstrating that TXL decreased miR-128-3p levels in the rat myocardium during I/R. Transfection of HCMs with a hsa-miR-128-3p mimic eliminated the protective effects of TXL. Conclusions: The miR-128-3p/p70s6k1 signaling pathway is involved in protection by TXL against HCM apoptosis during H/R. Overexpression of p70s6k1 is, therefore, a potential new strategy for alleviating myocardial reperfusion injury.

Arsenic-containing soil from geogenic source in Hong Kong: Leaching characteristics and stabilization/solidification.

Geogenic sources of arsenic (As) have aroused extensive environmental concerns in many countries. This study evaluated the vertical profiles, leaching characteristics, and surface characteristics of As-containing soils in Hong Kong. The results indicated that elevated levels of As (486-1985 mg kg-1) were mostly encountered in deeper layer (15-20 m below ground). Despite high concentrations, geogenic As displayed a high degree of chemical stability in the natural geochemical conditions, and there was minimal leaching of As in various leaching tests representing leachability, mobility, phytoavailability, and bioaccessibility. Microscopic/spectroscopic investigations suggested that As in the soils was predominantly present as As(V) in a coordination environment with Fe oxides. Sequential extraction indicated that the majority of As were strongly bound with crystalline Fe/Al oxides and residual phase. Yet, uncertainties may remain with potential As exposure through accidental ingestion and abiotic/biotic transformation due to changes in geochemical conditions. Hence, the effectiveness of stabilization/solidification (S/S) treatment was evaluated. Although the leached concentrations of As from the S/S treated soils increased to varying extent in different batch leaching tests due to the increase in alkalinity, the mobility of As was considered very low based on semi-dynamic leaching test. This suggested that As immobilization in the S/S treated soils was predominantly dependent on physical encapsulation by interlocking framework of hydration products, which could also prevent potential exposure and allow controlled utilization of S/S treated soils as monolithic materials. These results illustrate the importance of holistic assessment and treatment/management of As-containing soils for enabling flexible future land use.

Quantitative Proteomics Analysis of Ischemia/Reperfusion Injury-Modulated Proteins in Cardiac Microvascular Endothelial Cells and the Protective Role of Tongxinluo.

BACKGROUND: The protection of endothelial cells (ECs) against reperfusion injury has received little attention. In this study, we used Tandem Mass Tag (TMT) labeling proteomics to investigate the modulated proteins in an in vitro model of cardiac microvascular endothelial cells (CMECs) subjected to ischemia/reperfusion (I/R) injury and their alteration by traditional Chinese medicine Tongxinluo (TXL). METHODS: Human CMECs were subjected to 2 h of hypoxia followed by 2 h of reoxygenation with different concentrations of TXL Protein expression profiles of CMECs were determined using tandem mass spectrometry. We evaluated several proteins with altered expression in I/R injury and summarized some reported proteins related to I/R injury. RESULTS: TXL dose-dependently decreased CMEC apoptosis, and the optimal concentration was 800 µg/mL. I/R significantly altered proteins in CMECs, and 30 different proteins were detected between a normal group and a hypoxia and serum deprivation group. In I/R injury, TXL treatment up-regulated 6 types of proteins including acyl-coenzyme A synthetase ACSM2B mitochondrial (ACSM2B), cyclin-dependent kinase inhibitor 1B (CDKN1B), heme oxygenase 1 (HMOX1), transcription factor SOX-17 (SOX17), sequestosome-1 isoform 1 (SQSTM1), and TBC1 domain family member 10B (TBC1D10B). Also, TXL down-regulated 5 proteins including angiopoietin-2 isoform c precursor (ANGPT2), cytochrome c oxidase assembly factor 5 (COA5), connective tissue growth factor precursor (CTGF), cathepsin L1 isoform 2 (CTSL), and eukaryotic elongation factor 2 kinase (LOC101930123). These types of proteins mainly had vital functions, including cell proliferation, stress response, and regulation of metabolic process. CONCLUSIONS: The study presented differential proteins upon I/R injury through a proteomic analysis. TXL modulated the expression of proteins in CMECs and has a protective role in response to I/R.

Insights into the subsurface transport of As(V) and Se(VI) in produced water from hydraulic fracturing using soil samples from Qingshankou Formation, Songliao Basin, China.

Produced water is a type of wastewater generated from hydraulic fracturing, which may pose a risk to the environment and humans due to its high ionic strength and the presence of elevated concentrations of metals/metalloids that exceed maximum contamination levels. The mobilization of As(V) and Se(VI) in produced water and selected soils from Qingshankou Formation in the Songliao Basin in China were investigated using column experiments and synthetic produced water whose quality was representative of waters arising at different times after well creation. Temporal effects of produced water on metal/metalloid transport and sorption/desorption were investigated by using HYDRUS-1D transport modelling. Rapid breakthrough and long tailings of As(V) and Se(VI) transport were observed in Day 1 and Day 14 solutions, but were reduced in Day 90 solution probably due to the elevated ionic strength. The influence of produced water on the hydrogeological conditions (i.e., change between equilibrium and non-equilibrium transport) was evidenced by the change of tracer breakthrough curves before and after the leaching of produced water. This possibly resulted from the sorption of polyacrylamide (PAM (-CH2CHCONH2-)n) onto soil surfaces, through its use as a friction reducer in fracturing solutions. The sorption was found to be reversible in this study. Minimal amounts of sorbed As(V) were desorbed whereas the majority of sorbed Se(VI) was readily leached out, to an extent which varied with the composition of the produced water. These results showed that the mobilization of As(V) and Se(VI) in soil largely depended on the solution pH and ionic strength. Understanding the differences in metal/metalloid transport in produced water is important for proper risk management.

Potential impact of flowback water from hydraulic fracturing on agricultural soil quality: Metal/metalloid bioaccessibility, Microtox bioassay, and enzyme activities.

Hydraulic fracturing has advanced the development of shale gas extraction, while inadvertent spills of flowback water may pose a risk to the surrounding environment due to its high salt content, metals/metalloids (As, Se, Fe and Sr), and organic additives. This study investigated the potential impact of flowback water on four representative soils from shale gas regions in Northeast China using synthetic flowback solutions. The compositions of the solutions were representative of flowback water arising at different stages after fracturing well establishment. The effects of solution composition of flowback water on soil ecosystem were assessed in terms of metal mobility and bioaccessibility, as well as biological endpoints using Microtox bioassay (Vibrio fischeri) and enzyme activity tests. After one-month artificial aging of the soils with various flowback solutions, the mobility and bioaccessibility of As(V) and Se(VI) decreased as the ionic strength of the flowback solutions increased. The results inferred a stronger binding affinity of As(V) and Se(VI) with the soils. Nevertheless, the soil toxicity to Vibrio fischeri only presented a moderate increase after aging, while dehydrogenase and phosphomonoesterase activities were significantly suppressed with increasing ionic strength of flowback solutions. On the contrary, polyacrylamide in the flowback solutions led to higher dehydrogenase activity. These results indicated that soil enzyme activities were sensitive to the composition of flowback solutions. A preliminary human health risk assessment related to As(V) suggested a low level of cancer risk through exposure via ingestion, while holistic assessment of environmental implications is required.

Identification of the Isoform-specific Interactions between the Tail and the Head of Class V Myosin.

Vertebrates have three isoforms of class V myosin (Myo5), Myo5a, Myo5b, and Myo5c, which are involved in transport of multiple cargoes. It is well established that the motor functions of Myo5a and Myo5b are regulated by a tail inhibition mechanism. Here we found that the motor function of Myo5c was also inhibited by its globular tail domain (GTD), and this inhibition was abolished by high Ca(2+), indicating that the tail inhibition mechanism is conserved in vertebrate Myo5. Interestingly, we found that Myo5a-GTD and Myo5c-GTD were not interchangeable in terms of inhibition of motor function, indicating isoform-specific interactions between the GTD and the head of Myo5. To identify the isoform-specific interactions, we produced a number of Myo5 chimeras by swapping the corresponding regions of Myo5a and Myo5c. We found that Myo5a-GTD, with its H11-H12 loop being substituted with that of Myo5c, was able to inhibit the ATPase activity of Myo5c and that Myo5a-GTD was able to inhibit the ATPase activity of Myo5c-S1 and Myo5c-HMM only when their IQ1 motif was substituted with that of Myo5a. Those results indicate that the H11-H12 loop in the GTD and the IQ1 motif in the head dictate the isoform-specific interactions between the GTD and head of Myo5. Because the IQ1 motif is wrapped by calmodulin, whose conformation is influenced by the sequence of the IQ1 motif, we proposed that the calmodulin bound to the IQ1 motif interacts with the H11-H12 loop of the GTD in the inhibited state of Myo5.

Mouse myosin-19 is a plus-end-directed, high-duty ratio molecular motor.

Class XIX myosin (Myo19) is a vertebrate-specific unconventional myosin, responsible for the transport of mitochondria. To characterize biochemical properties of Myo19, we prepared recombinant mouse Myo19-truncated constructs containing the motor domain and the IQ motifs using the baculovirus/Sf9 expression system. We identified regulatory light chain (RLC) of smooth muscle/non-muscle myosin-2 as the light chain of Myo19. The actin-activated ATPase activity and the actin-gliding velocity of Myo19-truncated constructs were about one-third and one-sixth as those of myosin-5a, respectively. The apparent affinity of Myo19 to actin was about the same as that of myosin-5a. The RLCs bound to Myo19 could be phosphorylated by myosin light chain kinase, but this phosphorylation had little effect on the actin-activated ATPase activity and the actin-gliding activity of Myo19-truncated constructs. Using dual fluorescence-labeled actin filaments, we determined that Myo19 is a plus-end-directed molecular motor. We found that, similar to that of the high-duty ratio myosin, such as myosin-5a, ADP release rate was comparable with the maximal actin-activated ATPase activity of Myo19, indicating that ADP release is a rate-limiting step for the ATPase cycle of acto-Myo19. ADP strongly inhibited the actin-activated ATPase activity and actin-gliding activity of Myo19-truncated constructs. Based on the above results, we concluded that Myo19 is a high-duty ratio molecular motor moving to the plus-end of the actin filament.

Salvianolic acid B attenuates spinal cord ischemia-reperfusion-induced neuronal injury and oxidative stress by activating the extracellular signal-regulated kinase pathway in rats.

BACKGROUND: Salvianolic acid B (SalB), the main bioactive compound isolated from the traditional Chinese medicinal herb broad Radix Salviae Miltiorrhizae exerts a spectrum of pharmacologic activities. We investigated the effects of SalB treatment in a rat model of spinal cord ischemia and reperfusion (I/R) injury and the underlying mechanism. MATERIALS AND METHODS: SalB was administered at 1, 10, or 50 mg/kg after spinal cord ischemia. The potential protective effects on spinal cord injury were determined by spinal cord edema, infarct volume, and motor function assessment of the hind limbs. RESULTS: SalB treatment significantly decreased spinal cord edema and infarct volume and preserved motor function of the hind limbs in a dose-dependent manner. SalB administration ameliorated the generation of oxidative products and preserved antioxidant defense activities in the injured spinal cord at both 4 and 24 h after I/R injury. Moreover, SalB prolonged the I/R injury-induced activation of extracellular signal-regulated kinase (ERK), and blocking ERK activation with PD98059 partially prevented the neuroprotective effects of SalB. CONCLUSIONS: These findings demonstrate the neuroprotective effects of SalB in a spinal cord I/R injury model and suggest that SalB-induced neuroprotection was mediated by ERK activation.

Protein kinase A-mediated cardioprotection of Tongxinluo relates to the inhibition of myocardial inflammation, apoptosis, and edema in reperfused swine hearts.

BACKGROUND: Our previous studies have demonstrated that Tongxinluo (TXL), a traditional Chinese medicine, can protect hearts against no-reflow and reperfusion injury in a protein kinase A (PKA)-dependent manner. The present study was to investigate whether the PKA-mediated cardioprotection of TXL against no-reflow and reperfusion injury relates to the inhibition of myocardial inflammation, edema, and apoptosis. METHODS: In a 90-minute ischemia and 3-hour reperfusion model, minipigs were randomly assigned to sham, control, TXL (0.05 g/kg, gavaged one hour prior to ischemia), and TXL + H-89 (a PKA inhibitor, intravenously and continuously infused at 1.0 µg/kg per minute) groups. Myocardial no-reflow, necrosis, edema, and apoptosis were determined by pathological and histological studies. Myocardial activity of PKA and myeloperoxidase was measured by colorimetric method. The expression of PKA, phosphorylated cAMP response element-binding protein (p-CREB) (Ser(133)), tumor necrosis factor α (TNF-α), P-selectin, apoptotic proteins, and aquaporins was detected by Western blotting analysis. RESULTS: TXL decreased the no-reflow area by 37.4% and reduced the infarct size by 27.0% (P < 0.05). TXL pretreatment increased the PKA activity and the expression of Ser(133) p-CREB in the reflow and no-reflow myocardium (P < 0.05). TXL inhibited the ischemia-reperfusion-induced elevation of myeloperoxidase activities and the expression of TNF-α and P-selectin, reduced myocardial edema in the left ventricle and the reflow and no-reflow areas and the expression of aquaporin-4, -8, and -9, and decreased myocytes apoptosis by regulation of apoptotic protein expression in the reflow and no-reflow myocardium. However, addition of the PKA inhibitor H-89 counteracted these beneficial effects of TXL. CONCLUSION: PKA-mediated cardioprotection of TXL against no-reflow and reperfusion injury relates to the inhibition of myocardial inflammation, edema, and apoptosis in the reflow and no-reflow myocardium.

Calmodulin bound to the first IQ motif is responsible for calcium-dependent regulation of myosin 5a.

Myosin 5a is as yet the best-characterized unconventional myosin motor involved in transport of organelles along actin filaments. It is well-established that myosin 5a is regulated by its tail in a Ca(2+)-dependent manner. The fact that the actin-activated ATPase activity of myosin 5a is stimulated by micromolar concentrations of Ca(2+) and that calmodulin (CaM) binds to IQ motifs of the myosin 5a heavy chain indicates that Ca(2+) regulates myosin 5a function via bound CaM. However, it is not known which IQ motif and bound CaM are responsible for the Ca(2+)-dependent regulation and how the head-tail interaction is affected by Ca(2+). Here, we found that the CaM in the first IQ motif (IQ1) is responsible for Ca(2+) regulation of myosin 5a. In addition, we demonstrate that the C-lobe fragment of CaM in IQ1 is necessary for mediating Ca(2+) regulation of myosin 5a, suggesting that the C-lobe fragment of CaM in IQ1 participates in the interaction between the head and the tail. We propose that Ca(2+) induces a conformational change of the C-lobe of CaM in IQ1 and prevents interaction between the head and the tail, thus activating motor function.

Action mechanisms of complementary and alternative medicine therapies for rheumatoid arthritis.

Rheumatoid arthritis (RA) is characterized as a chronic inflammatory disease in joints and concomitant destruction of cartilage and bone. Cartilage extracellular matrix components, such as type II collagen and aggrecan are enzymatically degraded by matrix metalloproteinases (MMPs) and aggrecanases in RA. Currently, treatments targeting cytokines, including anti-tumor necrosis factor (TNF) α antibodies, soluble TNF receptor, anti-interleukin (IL)-6 receptor antibody, and IL-1 receptor antagonist, are widely used for treating RA in addition to antiantiinflammatory agents and disease-modifying antirheumatic drugs (DMARDs), such as inflmethotrexate, but these treatments have some problems, especially in terms of cost and the increased susceptibility of patients to infection in addition to the existence of low-responders to these treatments. Therefore, therapeutics that can be safely used for an extended period of time would be preferable. Complementary and alternative medicines including traditional Chinese medicines (TCM) have been used for the arthritic diseases through the ages. Recently, there are many reports concerning the anti-arthritic action mechanisms of TCM-based herbal formulas and crude herbal extracts or isolated ingredients. These natural herbal medicines are thought to moderately improve RA, but they exert various actions for the treatment of RA. In this review, the current status of the mechanism exploration of natural compounds and TCM-based herbal formulas are summarized, focusing on the protection of cartilage destruction in arthritic diseases including RA and osteoarthritis.

Tongxinluo reduces myocardial no-reflow and ischemia-reperfusion injury by stimulating the phosphorylation of eNOS via the PKA pathway.

The objective of the present study was to investigate whether pretreatment with single low loading dose of tongxinluo (TXL), a traditional Chinese medicine, 1 h before myocardial ischemia could attenuate no-reflow and ischemia-reperfusion injury by regulating endothelial nitric oxide synthase (eNOS) via the PKA pathway. In a 90-min ischemia and 3-h reperfusion model, minipigs were randomly assigned to the following groups: sham, control, TXL (0.05 g/kg, gavaged 1 h before ischemia), TXL + H-89 (a PKA inhibitor, intravenously infused at a dose of 1.0 µg·kg(-1)·min(-1) 30 min before ischemia), and TXL + N(ω)-nitro-L-arginine (L-NNA; an eNOS inhibitor, intravenously administered at a dose of 10 mg/kg 30 min before ischemia). TXL decreased creatine kinase (CK) activity (P < 0.05) and reduced the no-reflow area from 48.6% to 9.5% and infarct size from 78.5% to 59.2% (P < 0.05), whereas these effects of TXL were partially abolished by H-89 and completely reversed by L-NNA. TXL elevated PKA activity and the expression of PKA, Thr(198) phosphorylated PKA, Ser(1179) phosphorylated eNOS, and Ser(635) phosphorylated eNOS in the ischemic myocardium. H-89 repressed the TXL-induced enhancement of PKA activity and phosphorylation of eNOS at Ser(635), and L-NNA counteracted the phosphorylation of eNOS at Ser(1179) and Ser(635) without an apparent influence on PKA activity. In conclusion, pretreatment with a single low loading dose of TXL 1 h before ischemia reduces myocardial no-reflow and ischemia-reperfusion injury by upregulating the phosphorylation of eNOS at Ser(1179) and Ser(635), and this effect is partially mediated by the PKA pathway.