In vitro corrosion resistance,antibacterial activity and cytocompatibility of a layer-by-layer assembled DNA coating on magnesium alloy

A chitosan/deoxyribonucleic acid(CHI/DNA)_(5)coating was constructed by layer-by-layer(LbL)assembly dip coating method with Mg(OH)_(2)coating as an inner protective layer on AZ31 alloy.X-ray diffractometry,X-ray photoelectron spectrometry,Fourier transform infrared spectroscopy and field-emission scanning electron microscopy were utilized to represent the chemical compositions and surface morphologies of the coatings.Electrochemical tests and hydrogen evolution measurements were implemented to confirm the good corrosion resistance of the composite coating in artificial body fluid.Antimicrobial activity of the composite coatings was tested via the plate-counting method,and the cytotoxicity of the samples was appraised by MTT assay and Live/dead staining.A double action was put into effect for the composite coating,which the inner Mg(OH)2 coating plays the part of physical barrier,and the outer(CHI/DNA)5 coating is employed as an inducer to fabricate a biocompatible Ca-P corrosion product coating during immersion,making up for its thin thickness.Otherwise,the composite coating is also beneficial for the growth of bone,resulting from the biomineralization effect of the outer polyelectrolyte multilayer.The good antibacterial property of the(CHI/DNA)5/Mg(OH)2 coating is ascribed to the contact-killing strength of CHI.Thus,the obtained(CHI/DNA)5/Mg(OH)2 coating has a wide application prospect in the field of Mg-based bone implantation.

Acute and joint toxicity of three agrochemicals to Chinese tiger frog （Hoplobatrachus chinensis） tadpoles

We studied acute and joint toxicity of three different agrochemicals （chlorantraniliprole, flubendiamide-abamectin and penoxsulam） to Chinese tiger frog （Hoplobatrachus chinensis） tadpoles with the method of stability water tests. Results showed that the three agrochemicals increased tadpole mortality. For acute toxicity, the LC50 values after 24, 48 and 72 h of chlorantraniliprole, flubendiamide-abamectin and penoxsulam exposure were 5.37, 4.90 and 4.68 mg/L; 0.035, 0.025 and 0.021 rag/L; 1.74, 1.45 and 1.29 mg/L, respectively. The safety concentrations （SC） of chlorantraniliprole, fiubendiamide-abamectin and penoxsulam to the tadpoles were 1.23, 0.30 and 0.003 mg/L, respectively. Based on these findings, chlorantraniliprole and penoxsulam were moderately toxic, while flubendiamide-abamectin was highly toxic. All pairwise joint toxicity tests showed moderate toxicity. The LCs0 values after 24, 48 and 72 h of exposure were 7.08, 6.61 and 6.03 mg/L for chlorantra- niliprole＋penoxsulam, with corresponding values of 2.455, 2.328 and 2.183 mg/L for chlorantraniliprole＋flubendiamide-abamectin, and 1.132, 1.084 and 1.050 mg/L for penoxsulam＋flubendiamide-abamectin, with safe concentrations of 1.73, 0.63 and 0.30 mg/L, respectively. For toxic evaluations of pairwise combinations of the three agrochemicals, only the joint toxicity of chlorantraniliprole and flubendiamide-abamectin after 24 h was found to be synergistic, whereas all other tests were antagonistic. Our findings provide valuable information on the toxic effects of agrochemicals on amphibians and how various types of agrochemicals can be reasonably used in agricultural areas.

Effects of hydroperiod duration on developmental plasticity in tiger frog （Hoplobatrachus chinensis） tadpoles

While developmental plasticity can facilitate evolutionary diversification of organisms, the effects of water levels as an environmental pressure on tiger frogs remains unclear. This study clarifies the relationship by studying the responses of tiger frog （Hoplobatrachus chinensis） tadpoles to simulated hydroperiods （i.e., constant low water levels, constant high water levels, increasing water levels, decreasing water levels, rapid changes in water levels and gradual fluctuations in water levels） in a laboratory setting. ANOVA analysis showed that none of the water level treatments had any significant effect on the total length, body mass, or developmental stages of H. chinensis tadpoles half way through development （11 days old）. Tadpoles raised in rapidly fluctuating water levels had protracted metamorphosis, whereas tadpoles raised under low and gradually fluctuating water levels had shortened metamorphosis. None of the water level treatments had a significant effect on the snout-vent length （SVL） or body mass of H. chinensis tadpoles at Gosner stage 42, or on the body mass of tadpoles at Gosner stage 45. However, the＇ tadpoles raised in high levels and rapidly fluctuating water levels, significantly larger SVL at Gosner stage 45, while ones under gradually fluctuating water levels had smaller SVL than the other groups. Time to metamorphosis was positively correlated with body size （SVL） at metamorphosis in H. chinensis tadpoles. H. chinensis tadpoles under constant low water level had the highest mortality rate among all the treatments （G-test）. Moreover, ANOVA and ACNOVA （with body length as the covariate） indicated that water levels had no significant effect on either the morphology （i.e. head length, head width, forelimb length, hindlimb length and body width） or the jumping ability of juvenile H. chinensis. These results suggest that the observed accelerated metamorphosis and high mortality of H. chinensis tadpoles under decreasing water level treatment was driven by density-induced physical interaetions among increasing conspeeifics.

Effects of light intensity on activity in four sympatric anuran tadpoles

Though light conditions are known to affect the development and anti-predation strategies of several aquatic species, relatively little is known about how different species react to light, or how light can affect these species during different points in their life-cycle. In this study, we used four sympatric anuran tadpoles （Bufo gargarizans, B. melanostictus, Pelophylax nigromaculatus and Microhyla fissipes） as animal system to examine species-specific activities of the underdoing different light intensity treatments, so as to better understand how they respond to light. We exposed four different species of tadpoles to 1660 and 14 lux light intensity treatments and then measured several parameters including development stage, body length and tail length, and as well as their basic activities. The results of this observation and analysis showed that the activities of tadpoles were significantly greater in B. gargarizans and B. melanostictus than in P. nigromaculatus and M. fissipes; and were also significantly greater during times of high light intensity as compared to during low light intensity. Moreover, the observed relationship between species and light intensity was significant. The activities ofB. gargarizans and B. melanostictus tadpoles were greater in high light, while the activity of P. nigromaculatus tadpoles was greater in low light intensity, while M. fissipes tadpoles showed no differences in either low or high intensity light. Furthermore, the activities orB. gargarizans, B. melanostictus and M. fissipes tadpoles in terms of developmental stage, body size or tail length did not seem to differ with light intensity, but during early larval developmental period of P. nigromaculatus, the activity of tadpoles was negatively correlated with development stage, but irrelevant to either body size or tail length in different light intensities. These results lead us to conclude the observed activities of the four sympatric anuran tadpoles are closely correlated with their specific anti-predation strategies.

In vitro degradation resistance of glucose and L-cysteine-bioinspired Schiff-base anodic Ca−P coating on AZ31 magnesium alloy

A Schiff base(a compound containing a C=N bond)induced anodic Ca−P coating was prepared on AZ31 Mg alloy in a mixed solution of CaCl_(2) and KH_(2)PO_(4) at 60℃ in the presence of glucose and L-cysteine.The microstructure and chemical composition of the coatings were characterized using FE-SEM,FT-IR,XRD,and XPS.The in vitro degradation resistance of the coated samples was evaluated via potentiodynamic polarization(PDP),electrochemical impedance spectroscopy(EIS),and hydrogen evolution test.The experimental results show that the Ca−PSchiff base coating is composed of CaHPO_(4)(DCPA)and hydroxyapatite(HA),whereas HA is not present in the Ca−P coating.The Ca−P_(Schiff base) coating thickness is about 2 times that of Ca−P coating(Ca−P coating:(9.13±4.20)μm and Ca−P_(Schiff base):(18.13±5.78)μm).The corrosion current density of the Ca−P_(Schiff base) coating is two orders of magnitude lower than that of the Ca−P coating.The formation mechanism of the Ca−P_(Schiff base) is proposed.

Study on effect of imbalance of TRAF-6, IRAK-1 and NALP3 inflammatory factors in patients with gouty arthritis

Objective:To explore the effect of imbalance of tumor necrosis factor receptor related factor-6(TRAF-6),interleukin 1 receptor associated kinase-1(IRAK-1)and neutrophil alkaline phosphatase-3(NALP3)in patients with gouty arthritis.Methods:The retrospective experiment was conducted on 105 patients with gouty arthritis admitted to our hospital(47 patients with acute onset and 58 patients with remission,namely group A and group B);meanwhile,another 61 healthy volunteers were selected for control,namely group C.The enrolling of the three groups was dated from May 2017 to May 2018,and TRAF-6,IRAK-1 and NALP3 of all subjects were tested through real-time fluorescence quantification(RT-PCR),and the correlation between the three inflammatory factors and gouty arthritis was compared.Results:1)Through treatment,ESR,BUA and total addiment in group A and B were higher than those in group C,among which the three indicators in group A were higher than those in group B(P<0.05),while CRP was lower than that of group C,and the two indicators in group A were lower than those in group B(P<0.05).2)There was no significant difference in the relative expression of TRAF-6 mRNA between group A and group B before treatment(P>0.05),significantly lower than group C(P<0.05);the above indicators of group A and group B were improved to some extent after treatment,but group A was still lower than group B(P<0.05),and the degree of improvement of group A was also lower than that of group C(P<0.05),while the degree of improvement of group B was not significantly different from that of group C(P>0.05).3)The relative expression level of IRAK-1mRNA in group A and group B before treatment showed no significant difference(P>0.05),but was also lower than that in group C(P<0.05).The relative expression level of IRAK-1mRNA in group A and group B increased to some extent after treatment,with group A significantly lower than group C(P<0.05),and group B showed no significant difference compared with group C(P>0.05).4)The relative expression level of NALP-3 mRNA in group A and group B showed no significant difference(P>0.05)before treatment,significantly higher than that in group C(P<0.05);the relative expression of NALP-3 mRNA in group A was not significantly decreased(P>0.05)after treatment,while that in group B was significantly decreased after treatment(P<0.05),indicating significant different compared with group A and group C.5)There was no correlation between)TRAF-6,ESR,CRP and total addiment(P>0.05);IRAK-1 was negatively correlated with CRP,BUA and total addiment(P<0.05);NALP-3 was negatively correlated with ESR and CRP(P<0.05).Conclusion:TRAF-6,IRAK-1 and NALP-3 are all under abnormal expression in the developing of new gouty arthritis,acting as important participants in promoting the occurrence,development and outcome of illness states,so the intervening measures should be taken.

Corrosion resistance of in-situ growth of nano-sized Mg（OH）2 on micro-arc oxidized magnesium alloy AZ31—Influence of EDTA

One of the major obstacles for the clinical use of biodegradable magnesium(Mg)-based materials is their high corrosion rate. Micro-arc oxidation(MAO) coatings on Mg alloys provide mild corrosion protection owing to their porous structure. Hence, in this study a dense Mg(OH)2 film was fabricated on MAO-coated Mg alloy AZ31 in an alkaline electrolyte containing ethylenediamine tetraacetic acid disodium(EDTA-2 Na) to reinforce the protection. Surface morphology, chemical composition and growth process of the MAO/Mg(OH)2 hybrid coating were examined using field-emission scanning electron microscopy, energy dispersive X-ray spectrometer, X-ray diffraction, X-ray photoelectron spectroscopy and Fourier transform infrared spectrophotometer. Corrosion resistance of the coatings was evaluated via potentiodynamic polarization curves and hydrogen evolution tests. Results manifested that the Mg(OH)2 coating possesses a porous nano-sized structure and completely seals the micro-pores and micro-cracks of the MAO coating.The intermetallic compound of AlMn phase in the substrate plays a key role in the growth of Mg(OH)2 film. The current density of Mg(OH)2-MAO composite coating decreases three orders of magnitude in comparison with that of bare substrate, indicating excellent corrosion resistance. The Mg(OH)2-MAO composite coating is beneficial to the formation of calcium phosphate corrosion products on the surface of Mg alloy AZ31, demonstrating a great promise for orthopaedic applications.

Corrosion resistance of nanostructured magnesium hydroxide coating on magnesium alloy AZ31: influence of EDTA

A hexagonal nanosheet Mg(OH)2 coating was prepared through a one-step hydrothermal method using LiOH solution as mineralizer and then modified by ethylenediaminetetraacetic acid(EDTA) to minimize the rapid corrosion of AZ31 Mg alloy.The performance of the coating was evaluated using electrochemical technique,hydrogen evolution measurements, nanoscratch test,Fourier-transform infrared spectroscopy(FTIR), X-ray diffraction(XRD) patterns and field-emission scanning electron microscopy(FESEM).The results suggested that the corrosion rate of bare AZ31 Mg alloys was significantly reduced by one and two orders of magnitude through the protection from Mg(OH)2 coating and modification with EDTA(i.e., EDTA-Mg(OH)2 coating), respectively.FESEM micrographs indicated that the modification in EDTA elicits to the formation of an EDTA-Mg(OH)2 composite with a thickness as twice as that of as-prepared Mg(OH)2 coating.Nanoscratch tests revealed strong adhesion between the composite or Mg(OH)2 coating and the substrate.The study of formation and corrosion mechanisms of the coatings manifested that Mg(OH)2 was first formed near the intermetallic compound AlMn particles and gradually covered the entire surface, wherein the AlMn particles played an important role in the coating growth process.And it also proved that EDTA accelerated the formation of Mg(OH)2.

Corrosion resistance of an amino acid-bioinspired calcium phosphate coating on magnesium alloy AZ31

An L-cysteine-bioinspired calcium phosphate(Ca-P)coating is prepared upon magnesium alloy AZ31 in a water bath at 60℃.FE–SEM,FTIR,XRD,electrochemical characterization,hydrogen evolution tests and XPS were used to evaluate the microstructure,chemistry and corrosion performance of the samples.Results indicate that L-cysteine promotes the nucleation process of the coating and significantly increases its thickness.This can be attributed to the complexation of the carboxyl group and mercapto group of L-cysteine with calcium ions.Indeed,the obtained Ca-P coating possesses higher corrosion resistance than that prepared in L-cysteine-free bath.

In vitro degradation and cytocompatibility of a low temperature in-situ grown self-healing Mg-Al LDH coating on MAO-coated magnesium alloy AZ31

Basically,Mg–Al layered double hydroxide(LDH)coatings are prepared on the surface of micro-arc oxidation(MAO)coated magnesium(Mg)alloys at a high temperature or a low pH value.This scenario leads to the growth rate of LDH coating inferior to the dissolution rate of the MAO coating.This in turn results in limited corrosion resistance of the composite coating.In this study,a Mg–Al LDH coating on MAO-coated Mg alloy AZ31 is prepared through a water bath with a higher pH(13.76)at a lower temperature(60℃).FE-SEM,EDS,XRD,XPS,and FT-IR are applied to analyze the surface morphology,chemical compositions,and growth process.Electrochemical polarization,electrochemical impedance spectroscopy(EIS)and hydrogen evolution tests are employed to evaluate the corrosion resistance of the samples.The results disclose that the MAO coating is completely covered by the nanosheet-structured LDH coating with a thickness of approximately 3.8μm.The corrosion current density of the MAO-LDH composite coating is decreased four orders of magnitude in comparison to its substrate;the presence of a wide passivation region in anodic polarization branch demonstrates its strong self-healing ability,indicating the hybrid coating possesses excellent corrosion resistance.The formation mechanism of the LDH coating on the MAO-coated Mg alloy is proposed.Furthermore,the cytocompatibility is assessed via an indirect extraction test for MC3T3-E1 pre-osteoblasts,which indicates an acceptable cytocompatibility of osteoblasts for the composite coating.

Male size does not correlate with fertilization success in two bufonid toads that show size-assortative mating

We examined sexual size dimorphism （SSD）, mating pattem, fertilization efficiency and female reproductive traits in two bufonid toads （Bufo gargarizans and Duttaphrynus melanostictus） to test the idea that importance of male body size for egg fertilization success depends on the mating pattern. Female-biased SSD was evident only in D. melanostictus. Female B. gar- garizans laid fewer larger eggs nearly three months earlier than did female D. melanostictus. Fertilization efficieneies on average were higher in B. gargarizans （95%） than in D. melanostictus （91%）. Though differing in the degree of SSD, body size, breeding season, clutch size, egg size and fertilization efficiency, the two toads were similar in four aspects： （1） both showed size-assortative mating; （2） females did not tradeoff egg size against egg number; （3） male size, clutch size and clutch dry mass were greater in male-larger than in female-larger pairs after accounting for female snout-vent length （SVL）; and （4） the ratio of male to female SVL did not affect fertilization efficiency. Our data show that： （1） a female preference for large males is likely not important in terms of egg fertilization success; （2） a male preference for large females is likely important because larger females are more fecund; and （3） size-assortative mating arises from a male preference for large females. Our study demonstrates that male size is not always important for egg fertilization success in anurans that show size-assortative mating.