Compound effect and mechanism of oxidative damage induced by nanoplastics and benzo [a] pyrene.

Nanoplastics and polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in soil environments. In order to objectively evaluate the toxic interaction between polystyrene nanoplastics (PS NPs) and benzo [a] pyrene (BaP), oxidative damage at the level of earthworm cells and biomacromolecules was investigated by experiments combined with molecular dynamics simulation. Studies on cells reveal that PS NPs and BaP had synergistic toxicity when it came to causing oxidative stress. Cellular reactive oxygen species (ROS) levels under combined pollutant exposure were 24% and 19% higher, respectively than when PS NPs and BaP were exposed alone (compared to the blank group). In addition, BaP and PS NPs inhibited the ability of CAT to decompose H2O2 by affecting the structure of the proximal amino acid Tyr 357 in the active center of CAT, which exacerbated oxidative stress to a certain extent. Therefore, the synergistic toxic effect of BaP and PS NPs is due to the mutual complement of the two to the induction of protein structural looseness, and the strengthening of the stability of the conjugate (CAT-BaP-PS) under the weak interaction. This work provides a new perspective and approach on how to talk about the toxicity of combined pollutants.

Nitrogen and sulfur co-doped carbon quantum dots as "on-off-on" fluorescence probes to detect Hg2+ and MnO4- and improving the photostability of Rhodamine B.

BACKGROUND: Carbon quantum dot (CQDs) are zero-dimensional carbon nanomaterials with a size of less than 10 nm CQDs are widely used in the field of ion detection by virtue of their fluorescence characteristics such as strong fluorescence intensity, good optical stability and tunable emission wavelength. Although the traditional atomic absorption method, electrochemical method and other metal ion detection methods are highly sensitive, the operation is complex, expensive and limited by the site. Therefore, we prepared the N, S-CQDs capable of detecting Hg2+ and MnO4- in water with the advantages of simple operation, low cost, and direct visual signal. RESULTS: N, S-CQDs with high-quantum yield (77.68%), uniform particle size (0.4 nm-2.6 nm) and green fluorescence were created utilizing a one-pot hydrothermal process with the precursors ASDA-Na4 and m-phenylenediamine. N, S-CQDs has good optical properties such as high fluorescence intensity, wavelength independence, up-conversion luminescence and fluorescence stability. We examined 27 common ions in water and found that the fluorescence of N, S-CQDs could be selectively quenched by Hg2+ and MnO4-, and the detection limits are 0.41 µM and 1.2 µM, respectively. The mechanism of quenching is further investigated. The fluorescence of N, S-CQDs-Hg2+ system can be restored by halogen ions (Cl-, Br-, I-), while the fluorescence of N, S-CQDs-MnO4- system can be partially restored by Fe2+. This forms an "on-off-on" mode of fluorescent probes. In addition, we also studied that trace amounts of N, S-CQDs can improve the photostability of RhB. SIGNIFICANCE: The N, S-CQDs are fluorescent probes in an "on-off-on" mode. N, S-CQDs with green fluorescence (on) can be quenched by Hg2+ and MnO4- (off). The fluorescence quenched by Hg2+ can be restored by halogen ions again, while the fluorescence quenched by MnO4- can partially be restored (on). This ion detection method can be used to visually detect the two ions in the field, with the advantages of low cost, simple operation and visual intuition.

Novel highly selective fluorescence sensing strategy for Mercury(â ¡) in water based on nitrogen-doped carbon quantum dots.

In this work, a fluorescent signal-closing probe of nitrogen-doped carbon quantum dots (NCQDs) was developed for quantitative detection of mercury ions (Hg2+). In this detection system, the NCQDs with high quantum yield (QY, 63.80 %) were synthesized via simple hydrothermal method with Methyl Glycine Diacetic acid Trisodium Salt (MGDA) and m-phenylenediamine (MPD) as carbon and nitrogen sources. The NCQDs have a typical surface structure and exceptional fluorescence stability, and their fluorescence zones are centered on excitation wavelengths of 440 nm and emission wavelengths of 510 nm. Under optimal conditions, the NCQDs have outstanding anti-interference ability to various ions and high selectivity to mercury ions. The fluorescence intensity of the detection system is weakened due to the generation of non-fluorescent groups caused by the static quenching effect. The fluorescence quenching efficiency shows a fascinating linear relationship with Hg2+ ions at 0-100 µM (y = 0.0051x-0.015, R2 = 0.9943), and the detection limit is 0.9 µM. Acute toxicity test shows that NCQDs have low toxicity and little harm to environment. The detection system can be used for the quantification of mercury ions in environmental water samples, and the recovery rate is between 99.64 % and 103.43 %, indicating that it is a simple and economical fluorescence detection method.

Combined toxicity of micro/nanoplastics loaded with environmental pollutants to organisms and cells: Role, effects, and mechanism.

Micro/nanoplastics (MPs/NPs) are ubiquitous in the environment and living organisms have been exposed to these substances for a long time. When MPs/NPs enter different organisms, they transport various pollutants, including heavy metals, persistent organic pollutants, drugs, bacteria, and viruses, from the environment. On this basis, this paper summarizes the combined toxicity induced by MPs/NPs accumulating contaminants from the environment and entering organisms through a systematic review of 162 articles. Moreover, the factors influencing toxic interactions are critically discussed, thus highlighting the dominant role of the relative concentrations of contaminants in the combined toxic effects. Furthermore, for the first time, we describe the threats posed by MPs/NPs combined with other pollutants to human health, as well as their cytotoxic behavior and mechanism. We found that the "Trojan horse" effect of nanoplastics can increase the bioaccessibility of environmental pollutants, thus increasing the carcinogenic risk to humans. Simultaneously, the complex pollutants entering the cells are observed to be constantly dissociated due to the transport of lysosomes. However, current research on the intracellular release of MP/NP-loaded pollutants is relatively poor, which hinders the accurate in vivo toxicity assessment of combined pollutants. Based on the findings of our critical review, we recommend analyzing the toxic effects by clarifying the dose relationship of each component pollutant in cells, which is challenging yet crucial to exploring the toxic mechanism of combined pollution. In the future, our findings can contribute to establishing a system modeling the complete load-translocation toxicological mechanism of MP/NP-based composite pollutants.

Preparation of nitrogen-doped carbon quantum dots from chelating agent and used as fluorescent probes for accurate detection of ClO- and Cr(â ¥).

In this study, a novel ionic sensor based on nitrogen-doped carbon quantum dots (N-CQDs) was developed for sensitive detection of hypochlorite ions (ClO-) and dichromate ions (Cr(Ⅵ)) by fluorescence spectrometry. The N-CQDs was synthesized by hydrothermal method using Methyl Glycine Diacetic acid Trisodium Salt (MGDA) and Ethylenediamine (EDA) with bright blue fluorescence, high fluorescence quantum yield, abundant surface groups and good dispersion. The N-CQDs had a remarkable emission characteristic at 450 nm under the ultraviolet light of 350 nm, and the ClO- and Cr(Ⅵ) ions could quantificationally quench the fluorescence of this emission band. The results showed that N-CQDs had broad linear detection range and the detection limits of trace ClO- and Cr(Ⅵ) ions are 5.0 µM and 2.1 µM, respectively. Subsequently, further verify the reliability of this study and the N-CQDs played an excellent role in the ion detection of actual water samples. The quenching mechanism of ClO- is proved to be dominated by static quenching, while the quenching mechanism of Cr(Ⅵ) is mainly due to inner filter effect. This study is envisioned to efficiently prepare N-CQDs with novel raw materials, provide enlightening insights for enriching the detection of various trace ions by CQDs, and open up a new way to use fluorescence characteristics for water detection.

Degradation of a non-oxidizing biocide in circulating cooling water using UV/persulfate: Kinetics, pathways, and cytotoxicity.

In industry, isothiazolinone (a mixture containing 5-chloro-2-methyl-4-isothiazolin-3-one (CMIT) and 2-methyl-4-isothiazolin-3-one (MIT), CMIT-MIT) as a non-oxidizing biocide is extensively used to control the growth of microorganisms in the circulating cooling water system, which potentially threatens the ecological environment and human health. In this work, the oxidative degradation of CMIT-MIT by UV/persulfate (PS) technology on a laboratory-scale was systematically investigated. The degradation of CMIT-MIT was greatly improved by UV/PS compared with only UV or oxidant. During the photolysis of 60 mg/L PS, the degradation rate and TOC mineralization rate of CMIT-MIT were 91% and 34.7%, respectively. The contributions of .OH and SO4·- to CMIT-MIT degradation in the UV/PS system were estimated to be 0.93% and 32.12% respectively. The degradation rate of CMIT-MIT decreased slightly with the increase of pH. The presence of SO42- and NO3- had no significant effect on the degradation of CMIT-MIT, while the presence of Cl- and CO32- inhibited the CMIT-MIT removal rate. The degradation pathways and three possible intermediates of CMIT-MIT were obtained. After degradation of CMIT-MIT by UV/PS process, the cytotoxicity decreased within 20 min, effectively indicating that UV/PS could be as a potential technology to remove the CMIT-MIT in water treatment.

Probing the biological toxicity of pyrene to the earthworm Eisenia fetida and the toxicity pathways of oxidative damage: A systematic study at the animal and molecular levels.

Pyrene (Pyr), a widely used tetracyclic aromatic hydrocarbon, enters soil in large quantities and causes environmental pollution due to its production and mining. In order to systematically study the biotoxicity of pyrene to model organisms Eisenia fetida in soil, experiments were carried out from four dimensions: animal, tissue, cell and molecule. Experimental results proved that the mortality rate increased with increasing concentration and time of exposure to pyrene, while the mean body weight and spawning rate decreased. Meanwhile, when the pyrene concentration reached 900 mg/kg, the seminal vesicle and longitudinal muscle of the earthworm showed obvious atrophy. Experimental results at the cellular level showed that pyrene induced cell membrane damage and Ca2+ influx triggered mitochondrial membrane depolarization and a surge in ROS levels. Oxidative stress causes damage to proteins and lipids and DNA inside cells. When the mortality rate was 91.67 %, the Olive Tail Movement (OTM) of the comet experiment reached 15. The results of molecular level tests showed that pyrene inhibited the activity of Cu/Zn-superoxide dismutase (Cu/Zn-SOD) mainly by changing the microenvironment and secondary structure of amino acid Tyr 108. The weakened function of direct antioxidant enzymes may be the root cause of the excessive increase of reactive oxygen species (ROS) in cells. The systematic approach used in this study enriches the network of toxic pathways in toxicological studies, and basic data on the biological toxicity of pyrene can provide support for future soil contamination detection.

A Pyrene Fluorescent Probe for Rapid Detection of Ferric Ions.

The 1,3,6,8-pyrenetetrasulfonic acid tetrasodium salt (PTSA) is a pyrene derivative with high fluorescence characteristics and is widely used in fluorescence tracer. This study aims at investigating a simple and fast fluorescence detection method for determining the concentration of ferric ion by using PTSA, which the principle is that the fluorescence quenching of PTSA by ferric ions. Theoretical and experimental methods were adopted to deeply analyze its detection performance and characteristics. The fluorescence quenching phenomena under different pH conditions and the effect of the different interfering metal ions on PTSA/Fe3+ system was studied. The results showed that the PTSA was quite promising for the fluorescence detection of trace ferric ions, and the limit of detection is 9 µg/L. This study is envisioned to provide inspirational insights on trace detection of iron ions, opening new routes for water monitoring use fluorescence properties.

Toxic mechanism of pyrene to catalase and protective effects of vitamin C: Studies at the molecular and cell levels.

Polycyclic aromatic hydrocarbons, distributing extensively in the soil, would potentially threaten the soil organisms (Eisenia fetida) by triggering oxidative stress. As a ubiquitous antioxidant enzyme, catalase can protect organisms from oxidative damage. To reveal the potential impact of polycyclic aromatic hydrocarbon pyrene (Pyr) on catalase (CAT) and the possible protective effect of Ascorbic acid (vitamin C), multi-spectral and molecular docking techniques were used to investigate the influence of structure and function of catalase by pyrene. Fluorescence and circular dichroism analysis showed that pyrene would induce the microenvironmental changes of CAT amino acid residues and increase the α-helix in the secondary structure. Molecular simulation results indicated that the main binding force of pyrene around the active center of CAT is hydrogen bonding force. Furthermore, pyrene inhibited catalase activity to 69.9% compared with the blank group, but the degree of inhibition was significantly weakened after vitamin C added into the research group. Cell level experiments showed that pyrene can increase the level of ROS in the body cavity cell of earthworms, and put the cells under the threat of potential oxidative damage. Antioxidants-vitamin C has a protective effect on catalase and maintains the stability of intracellular ROS levels to a certain extent.

Degradation of fluorescent dye-Solvent Green 7 (HPTS) in wastewater by advanced oxidation process.

Solvent Green 7 (HPTS) is a widely used fluorescent dye. As a kind of polycyclic aromatic hydrocarbon (PAHs) derivative, HPTS would cause pollution when it is discharged into the environment. This study adopted advanced oxidation processes (UV/H2O2) to degrade the HPTS in aqueous solution and investigated the effects of various factors on the degradation. The results showed that: the initial concentration and the fluorescence characteristics of HPTS reduced the degradation efficiency. When the oxidant concentration of H2O2 was 3 mg/L, the degradation efficiency and cost of HPTS (20 mg/L) were the most appropriate; when there were various inorganic anions in the solution, the degradations were not affected, but when the solution was strong acid and there existed a lot of chloride ions, the degradation of HPTS was inhibited. The degradation pathways indicated HPTS degraded into naphthalene derivatives, benzene derivatives through oxidation and decarboxylation reactions, finally into water and carbon dioxide. Further research for substances similar to HPTS structure will make progress in understanding the degradation process of PAHs.

Probing the single and combined toxicity of PFOS and Cr(VI) to soil bacteria and the interaction mechanisms.

Many research focused on the removal of perfluorooctane sulfonic acid (PFOS) and hexavalent chromium (Cr(VI)) in some industrial wastewater (e.g. electroplating wastewater), but few research reported the combined toxicity of PFOS and Cr(VI) to soil bacteria. Therefore, the toxicity and mechanisms of the combined PFOS and Cr(VI) to bacteria (with Bacillus subtilis as a model) are explored. The results show that the combined PFOS and Cr(VI) exhibits much higher toxicity to the bacteria than that of Cr(VI) alone. The growth profile of Bacillus subtilis exposed by the combined pollution decreased by 18% and 56%, respectively, compared with that of single Cr(VI) and the control, indicating the combined toxicity to Bacillus subtilis is synergistic. Moreover, the changes of EPSs in Bacillus subtilis, such as decreased potential, increased extracellular polysaccharides, decreased extracellular proteins and irregular morphology, also confirmed that the combined PFOS and Cr(VI) caused greater toxicity. The increase of intracellular ROS and permeability of dye 4', 6-diamidino-2-phenylindoledihydrochloride (DAPI) suggest that oxidative damage and increased membrane permeability are the main mechanisms of toxicity induced by the combined PFOS and Cr(VI). This work could provide useful information for the risk assessment of co-exposure to PFOS and heavy metals in the natural environment.

Competition and enhancement effect in coremoval of atenolol and copper by an easily regenerative magnetic cation exchange resin.

This paper aimed to investigate the removal of combined Cu2+ and atenolol (ATL) in aqueous solution by using a newly synthesized magnetic cation exchange resin (MCER) as the adsorbent. The MCER exhibited efficient removal performance in sole, binary, pre-loading and saline systems. The adsorption kinetics of Cu2+ and ATL fitted both pseudo-first-order and pseudo-second order model, while better described by pseudo-second order model in binary system. In mixed Cu2+ and ATL solution, the adsorption of ATL was suppressed due to direct competition of carboxylic groups, while Cu2+ adsorption was enhanced because of the formation of surface complexes. This increasing in heterogeneity was demonstrated by adsorption isotherms, which were more suitable for Freundlich model in binary system, while better described by Langmuir model in sole system. As proved by FTIR and XPS spectra, both amino and hydroxyl groups of ATL could form complexes with Cu2+. Decomplexing-bridging interaction was elucidated as the leading mechanism in coremoval of Cu2+ and ATL, which involved [Cu-ATL] decomplexing and newly created Cu- or ATL sites for additional bridging. For saline system, the resulting competition and enhancement effects in mixed solution were amplified with the addition of co-existing cations. Moreover, the MCER could be effectively regenerated by 0.01 M HCl solution and maintain high stability over 5 adsorption-desorption cycles, which render it great potential for practical applications.

Probing the interactions between carboxylated multi-walled carbon nanotubes and copper-zinc superoxide dismutase at a molecular level.

In order to evaluate the toxicity of multi-walled carbon nanotubes (MWCNTs-COOH) at a molecular level, the effect of MWCNTs-COOH on antioxidant enzyme copper-zinc superoxide dismutase (Cu/ZnSOD) was investigated using fluorescence spectroscopy, UV/vis absorption spectroscopy, circular dichroism (CD) spectroscopy and isothermal titration calorimetry (ITC). By deducting the inner filter effect (IFE), the fluorescence emission spectra and synchronous fluorescence spectra indicated that there were interactions between MWCNTs-COOH and Cu/ZnSOD. Moreover, the microenvironment of the amino acid residues in the enzyme was changed slightly. The UV/vis absorption and CD spectroscopic results showed appreciable conformational changes in Cu/ZnSOD. However, the results of a Cu/ZnSOD activity determination did not show any significant difference. In other words, MWCNTs-COOH has no significant effect on enzyme activity. The ITC results showed that the binding of MWCNTs-COOH to Cu/ZnSOD was a weak endothermic process, indicating that the predominant force of the binding was hydrophobic interaction. Moreover, it was essential to consider the IFE in fluorescence assays, which might affect the accuracy and precision of the results. The above results are helpful in evaluating the oxidative stress induced by MWCNTs-COOH in vivo.

Interaction of Cu(2+), Pb (2+), Zn (2+) with trypsin: what is the key factor of their toxicity?

Heavy metals possess great endangerment to environment even human health because of their indissolubility and bioaccumulation. The toxicity of heavy metal ions (Cu(2+), Pb(2+), Zn(2+)) to trypsin was investigated by fluorescence, synchronous fluorescence, UV-vis absorption, circular dichroism (CD) spectroscopy, isothermal titration calorimetry (ITC), and enzyme activity assay. The experimental results showed that toxic effect of heavy metal ions was due to their own characteristic, rather than the electric charges of the ion. Zn(2+) could not show the obvious toxicity to trypsin, while the structure and function of trypsin was damaged when the enzyme explored to Cu(2+) and Pb(2+). From the spectra results, we found that Cu(2+) would bind with trypsin, which lead to the fluorescence quenched and hydrophobicity increased. Pb(2+) could also change the structure and reduce the activity of trypsin in high concentration. In vitro measurement, the toxicity order of heavy metal ions to trypsin is: Cu(2+) > Pb(2+) > Zn(2+). In addition, isothermal titration calorimetry analysis proved that the interactions between Cu(2+), Pb(2+), Zn(2+) and trypsin were all spontaneous and exothermic, which indicated the adverse effect of these heavy metal ions to trypsin.

Spectroscopic investigations on the interaction between carbon nanotubes and catalase on molecular level.

The interactions between well-dispersed multiwalled carbon nanotubes (MWCNTs) and catalase (CAT) were investigated. The activity of CAT was inhibited with the addition of MWCNTs. After deducting the inner filter effect, the fluorescence spectra revealed that the tryptophan (Trp) residues were exposed and the fluorescence intensities of CAT increased with the increase in the MWCNTs concentration. At the same time, the environment of the Trp residues became more hydrophobic. The results of UV-vis absorption spectroscopy and CD spectra indicated that the secondary structure of CAT had been changed, and the amino acid residues were located in a more hydrophobic environment. Meanwhile, the UV-vis spectra indicated that the conformation of the heme porphyrin rings was changed. The microenvironment of CAT activity sites may be interfered by MWCNTs. This research showed that MWCNTs could not only contribute to the conformational changes of protein but also change the enzyme function.

Interaction mechanism of Trp-Arg dipeptide with calf thymus DNA.

The interaction between Trp-Arg dipetide (WR) and calf thymus DNA (ctDNA) in pH 7.4 Tris-HCl buffer was investigated by multi-spectroscopic techniques and molecular modeling. The fluorescence spectroscopy and UV absorption spectroscopy indicated that WR interacted with ctDNA in a minor groove binding mode and the binding constant was 4.1 × 10(3). The ionic strength effect and single-stranded DNA (ssDNA) quenching effect further verified the minor groove binding mode. Circular dichroism spectroscopy (CD) was employed to measure the conformation change of ctDNA in the presence of WR. The molecular modeling results illustrated that electrostatic interaction and groove binding coexisted between them and the hydrogen bond and Van der Waals were main acting forces. All the above methods can be widely used to investigate the interaction of peptide with nucleic acids, which contributes to design the structure of new and efficient drugs.

Interactions of L-Arg with calf thymus DNA using neutral red dye as a fluorescence probe.

The interaction between l-Arg and calf thymus DNA (ctDNA) in sodium acetate-acetic acid buffer (pH=4) was investigated with the use of neutral red (NR) dye as a spectral probe coupled with UV-vis absorption, fluorescence, and circular dichroism (CD) spectroscopy technique. The UV absorption spectroscopy indicated that l-Arg interacted with ctDNA via electrostatic force and the fluorescence enhancing of the DNA-NR system verified the electrostatic interaction. In addition, detectable changes in the CD spectrum of ctDNA in the presence of l-Arg indicated conformational changes in the DNA double helix after interaction with the drug. Docking studies were found to corroborate the experimental results. All these results prove that this drug interacts with ctDNA via an electrostatic binding mode.

Toxic effects of different charged metal ions on the target--bovine serum albumin.

In this work, the toxic influence of metallic ions (Na+, Cu2+, Al3+) on the serum albumin were studied by fluorescence, resonance light scattering (RLS), synchronous fluorescence, UV-vis absorption and circular dichroism (CD) spectroscopy. The experimental results indicated that ion electric charge is not the main factor affecting the structure of bovine serum albumin (BSA). Na+ made the structure of BSA tighter and hydrophobicity enhanced, which improved fluorescence intensity, while Cu2+ could react with some functional groups of BSA, making the structure of BSA looser, so that the internal hydrophobic groups such as tryptophan (Trp) and other aromatic residues were gradually exposed. When we observed them with fluorescence spectra, we found fluorescence quenching with increasing Cu2+ dose. Al3+ is shown as little significant influence on the BSA, but BSA was found to aggregate with the dose of Al3+ by means of RLS because of the hydrolysis and ion strength effect of Al3+. The results also proved normal saline could keep lives healthy and good-working as a biological humour, however, heavy metals made harmful effects to the body when they exceeded the minimal effect level (MEL), such as Cu2+ chosen in our work.

Binding of oxytetracycline to bovine serum albumin: spectroscopic and molecular modeling investigations.

The residue of the widely used veterinary drug oxytetracycline (OTC) in the environment (e.g., animal food, soils, surface water, and groundwater) is potentially harmful. Knowledge of its binding to proteins contributes to the understanding of its toxicity in vivo. This work establishes the binding mode of OTC with bovine serum albumin (BSA) under physiological conditions by spectroscopic methods and molecular modeling techniques. The inner filter effect was eliminated to get accurate data (binding parameters). On the basis of the thermodynamic results and site marker competition experiments, it was considered that OTC binds to site II (subdomain IIIA) of BSA mainly by electrostatic interaction. Furthermore, using the BSA model established with CPHmodels, molecular docking and some other molecular modeling methods were applied to further define that OTC interacts with the Arg 433, Arg 436, Ala 429, and Pro 516 residues of BSA. In addition, UV-visible absorption, synchronous fluorescence, and circular dichroism (CD) results showed that the binding of OTC can cause conformational and some microenvironmental changes of BSA. The work provides accurate and full basic data for clarifying the binding mechanisms of OTC with BSA in vivo and is helpful for understanding its effect on protein function during its transportation and distribution in blood.

Investigation on the toxic interaction of toluidine blue with calf thymus DNA.

The gene toxic interactions of toluidine blue (TB) with calf thymus DNA (ct-DNA) were examined in vitro with UV-visible absorption spectroscopy, fluorescence spectroscopy, fluorescence polarization and circular dichroism techniques. The experimental results showed that TB interacted with ct-DNA by two binding modes. At low TB concentrations, TB intercalated into the DNA base pairs. At higher TB concentrations, TB was attached to the negatively charged phosphate groups. For the intercalation binding mode and electrostatic binding mode, the binding constants were 1.76 x1 0(6)L mol(-1) and 6.18 x 10(5)L mol(-1) and the number of binding sites was 0.48 and 0.79, respectively. Circular dichroism results showed that the two binding modes had different effects on the ct-DNA conformation. At high dye concentrations, Z-form DNA appears, while at low TB concentrations, a B to A form transition is observed.