Unravelling chilling-stress resistance mechanisms in endangered Mangrove plant Lumnitzera littorea (Jack) Voigt.

Lumnitzera littorea (Jack) Voigt is one of the most endangered mangrove species in China. Previous studies have showed the impact of chilling stress on L. littorea and the repsonses at physiological and biochemical levels, but few attentions have been paid at molecular level. In this study, we conducted genome-wide investigation of transcriptional and post-transcriptional dynamics in L. littorea in response to chilling stress (8 °C day/5 °C night). In the seedlings of L. littorea, chilling sensing and signal transducing, photosystem II regeneration and peroxidase-mediated reactive oxygen species (ROS) scavenging were substantially enhanced to combat the adverse impact induced by chilling exposure. We further revealed that alternative polyadenylation (APA) events participated in chilling stress-responsive processes, including energy metabolism and steroid biosynthesis. Furthermore, APA-mediated miRNA regulations downregulated the expression of the genes involved in fatty acid biosynthesis and elongation, and protein phosphorylation, reflecting the important role of post-transcriptional regulation in modulating chilling tolerance in L. littorea. Our findings present a molecular view to the adaptive characteristics of L. littorea and shed light on the conservation genomic approaches of endangered mangrove species.

Effects of 5-HT on the cold resistance of mangrove Kandelia obovata seedlings.

5-hydroxytryptamine (5-HT) participates in plant growth and development, and can also delay senescence and cope with abiotic stress. To explore the role of 5-HT in regulating the abilities of mangrove in cold resis-tance, we examined the effects of cold acclimation and the spraying of p-chlorophenylalanine (p-CPA, 5-HT synthesis inhibitor) on leaf gas exchange parameters and CO2 response curves (A/Ca), as well as the endogenous phytohormone content levels in the mangrove species Kandelia obovata seedlings under low temperature stress. The results showed that low temperature stress significantly reduced the contents of 5-HT, chlorophyll, endogenous auxin (IAA), gibberellin (GA), and abscisic acid (ABA). It weakened the CO2 utilization abilities of plants and reduced net photosynthetic rate, which ultimately reduced carboxylation efficiency (CE). Under low temperature stress, exogenous p-CPA reduced the contents of photosynthetic pigments, endogenous hormones, and 5-HT in the leaves, which aggravated the damages caused by low temperature stress on photosynthesis. By enhancing cold acclimation abilities, the endogenous IAA content in the leaves could was reduced under low temperature stress, promoted the production of 5-HT, improved the contents of photosynthetic pigments, GA, and ABA, as well as enhanced photosynthetic carbon assimilation abilities, which would increase photosynthesis in the K. obovata seedlings. Under cold acclimation conditions, the spraying of p-CPA could significantly inhibit the synthesis of 5-HT, promote the production of IAA, and reduce the contents of photosynthetic pigments, GA, ABA, and CE, which would weaken the effects of cold acclimation by improving the cold resistance of mangroves. In conclusion, cold acclimation could improve the cold resistance abilities of K. obovata seedlings by regulating photosynthetic carbon assimilation capacity and the contents of endogenous phytohormone. 5-HT synthesis is one of the necessary conditions for improving the cold resistance abilities of mangroves.

Inhibitory Effect of Two Closely Related Phages on Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a foodborne pathogenic bacterium commonly found in seafood. The emergence of drug-resistant strains poses a threat to human public health and economic development. Therefore, there are increasing needs to develop new technologies in controlling multidrug-resistant V. parahaemolyticus strains and to evaluate their practical efficiency in seafood or mariculture. In this study, we screened two genetically related V. parahaemolyticus phages, F23s2 and H256D1, which belonged to the siphoviridae family and podoviridae family, respectively. They showed 97.13% and 96.13% identity with Vibrio phage vB_Vpap_MGD1, respectively. Both phages were stable at pH 4-11 and displayed temperature tolerance (<70°C). Meanwhile they showed a broad host spectrum for multidrug-resistant V. parahaemolyticus, and Phage F23s2 lysed 16 of all 23 V. parahaemolyticus strains, while phage H256D1 lysed 10 strains. Phage F23s2 and H256D1 had a good inhibitory effect on V. parahaemolyticus in shrimp meat. Compared with the negative group, the bacterial amount of experimental group with phage F23s2 decreased by 1.60 log colony-forming unit (CFU)/mL at 12 h. For phage H256D1, the bacterial concentration of shrimp meat contaminated with V. parahaemolyticus H256 increased to 5.65 log CFU/mL at 72 h, while the concentration of the experimental group in presence of phage H256D1 was 3.58 log CFU/mL. All live clams infected with V. parahaemolyticus died after 96 h in the absence of phage, whereas clams with phage F23s2 and H256D1 still had a survival rate of 12% and 4%, respectively. Understanding the gene function and biology of phages facilitates its application for control of V. parahaemolyticus contamination worldwide.

Composition and Leaching Toxicity of Hydrochloric Acid Pickling Sludge Generated from the Hot-Dip Galvanized Steel Industry.

Steel hydrochloric acid pickling sludge (SHPS), containing the heavy metals Fe, Zn, and Ni and a high chloride salt content, is considered a type of hazardous solid waste because of its potential harm to human health and the environment. In addition, the SHPS yield is large, but the main treatment currently used is only safe for landfills. Although studying the composition and leaching toxicity of SHPS is of great importance, only a small amount of related literature is available. This paper can help compensate for this deficiency. SHPS is analyzed from the aspects of its formation mechanism, pH, moisture content, elemental concentration, phase composition, microstructure, and leaching toxicity. The results show that its pH ranges from 2.25 to 11.11, and the moisture content ranges from 45.47% to 83.34%. Additionally, the concentration of Fe is the highest, with values from 29.80% to 50.65%, while other alkali metal elements, namely, Ca, K, and Na, have values of 0.36% to 23.07%, 0.02% to 19.82%, and 0.38% to 3.31%, respectively. Heavy metal elements, namely, Zn, Ni, Mn, Cr, and Pb, have values of 0.02% to 14.88%, 0.001% to 0.05%, 0.03% to 0.38%, 0.01% to 0.09%, and 0.02% to 0.19%, respectively. Anions, namely, SO4 2-, Cl-, F-, and NO3 -, have contents of 0.09% to 0.34%, 0.54% to 5.73%, 0.001% to 0.04%, and 0.01% to 0.15%, respectively. X-ray diffraction (XRD) analysis shows that Fe and Zn are mainly present in oxides, Ca is present as CaO and CaCO3, and chlorine is present in NaCl. Moreover, scanning electron microscopy (SEM) analysis shows that the microscopic structure consists mainly of bright and fluffy irregular spheres; stripes; flakes; and dark, very small irregular particles. The leaching toxicity test based on HJ/T 299-2007 (China) was performed, where SHPS samples were treated with a mixed solution of sulfuric acid, nitric acid, and pure water (pH = 3.20 ± 0.05) at a liquid-to-solid ratio of 10:1 for a period of 18 h. The leachate was filtered and analyzed for Cr, Ni, Mn, Zn, etc. The leaching results indicate that Zn and Ni are the main elements that cause SHPS to be hazardous to the environment. These research results can provide a reference for later researchers studying the effective treatment of SHPS, such as more effective treatments for reducing toxicity and resource utilization.

Grafting of polymers via ring-opening polymerization for electrochemical assay of alkaline phosphatase activity.

As an important hydrolytic enzyme, abnormal activity of alkaline phosphatase (ALP) is closely associated with a variety of diseases. It has been identified as an important diagnostic indicator for clinical hepatobiliary and bone diseases. Herein, a novel electrochemical sensor based on signal amplification strategy through ring-opening polymerization (ROP) has been developed to assay of ALP activity. First of all, 3-mercaptopropanoic acid (MPA) was employed as a cross-linking agent to attach O-phosphoethanolamine to the electrode surface via amide bond. Then, ALP catalyzed the hydrolysis of phosphate monoester structures to hydroxyl groups, which could initiate ROP reaction. The polymer grafted on the electrode surface contains a large number of ferrocene electroactive molecules, which effectively increased the signal output of the electrochemical sensor and improved the sensitivity of ALP activity detection. Under optimum conditions, this electrochemical sensor rendered a satisfactory linear dependence over the range from 20 to 120 mU mL-1, with a low detection limit of 0.66 mU mL-1. Furthermore, this strategy presented satisfactory selectivity and interference resistance in human serum sample, and compared with clinical data, the relative error of the results obtained by this method was less than 5%. Thus, this method showed considerable potential for the detection of ALP activity in clinical application.

Ultrasensitive electrochemical detection of CYFRA 21-1 via in-situ initiated ROP signal amplification strategy.

The cytokeratin19 fragment (CYFRA 21-1) is an essential biomarker for non-small cell lung cancer (NSCLC). This work proposed a novel electrochemical immunosensor with a high selective and sensitive detection of CYFRA 21-1via the ring-opening polymerization (ROP) signal amplification strategy. Specifically, 3-mercaptopropionic (MPA) was employed as a cross-linking agent to immobilize cAb on the electrode surface for subsequent specific capture of CYFRA 21-1. After CYFRA 21-1 bound to cAb, the amino groups of them were blocked with acrolein. Then, the sandwich-type compositions were formed via the specific recognition between detection antibody (dAb) and CYFRA 21-1. Finally, the ROP was triggered by the amino group on dAb and the polymers containing a large number of ferrocene electroactive molecules were in situ grown on the electrode surface, thereby outputting a high sensing signal. Under optimal conditions, the fabricated immunosensor showed an ultrasensitive and highly selective with a linear range of 1 pg/mL ∼1 µg/mL, and the detection limit down to 9.08 fg/mL. Furthermore, a bright correlation was obtained for CYFRA 21-1 detection in the clinical serum samples. By merits of its ease of operation, environmental friendliness and low cost, this method had considerable potential application in bioanalytical for the ultrasensitive quantitation of biological molecules.

Konjac Oligosaccharides Modulate the Gut Environment and Promote Bone Health in Calcium-Deficient Mice.

This study aimed to investigate the beneficial effect of konjac oligosaccharides (KOS) on bone health in calcium-deficient mice. During the experimental period, low-calcium diet-fed mice were administered with calcium chloride to simulate daily calcium supplementation. Meanwhile, different levels of KOS intervened by adding them into the diet. After 8 weeks, the calcium balance status, bone mass parameters, and gut environment modulation were evaluated. The results showed that dietary KOS intervention alleviated the negative calcium balance, significantly promoted the trabecular number and cortical thickness, and remarkably enhanced the skeletal mechanical strength. Moreover, Pearson's correlation analysis among significantly changed gut microbiota, gut metabolites, and relevant physiological indexes showed that the microbial genera of Lactobacillus, Bifidobacterium, Mucispirillum, Alistipes, and unidentified Clostridia and gut metabolites of kynurenine and testosterone were significantly associated with increased bone mass. These findings provided a new insight into the effect of prebiotics on bone health.

Electrochemiluminescence immunosensor for cytokeratin fragment antigen 21-1 detection using electrochemically mediated atom transfer radical polymerization.

The cytokeratin fragment antigen 21-1 (CYFRA 21-1) protein is a critical tumor biomarker tightly related to non-small cell lung cancer (NSCLC). Herein, we prepared an effective electrochemiluminescence (ECL) immunosensor for CYFRA 21-1 detection using electrochemically mediated atom transfer radical polymerization (eATRP). The CYFRA 21-1 antigen was fixed on the electrode surface by constructing a sandwich type antibody-antigen-antibody immune system. The sensitivity of ECL was improved by using the eATRP reaction. In this method, eATRP was applied to CYFRA 21-1 detection antibody with N-acryloyloxysuccinimide as functional monomer. This is the first time that ECL and eATRP signal amplification technology had been combined. Under the optimized testing conditions, the immunosensor showed a good linear relation in the range from 1 fg mL-1 to 1 µg mL-1 at a limit of detection of 0.8 fg mL-1 (equivalent to ~ 134 molecules in a 10 µL sample). The ECL immunosensing system based on eATRP signal amplification technology provided a new way for rapid diagnosis of lung cancer by detecting CYFRA 21-1. The paper prepared an electrochemiluminescence biosensor for ultrasensitive detection of CYFRA 21-1 via eATRP signal amplification strategy, which had the advantages of high sensitivity, reproducibility, and held potential prospect for analysis of low-abundance.

Functionalized graphene oxide in situ initiated ring-opening polymerization for highly sensitive sensing of cytokeratin-19 fragment.

Improving the sensitivity of detection is crucial to monitor biomarker, assess toxicity, and track therapeutic agent. Herein, a sensitivity-improved immunosensor is reported for the first time via functionalized graphene oxide (GO) and a "grafting-to" ring-opening polymerization (ROP) dual signal amplification strategy. Through the ROP reaction using 2-[(4-ferrocenylbutoxy)methyl] oxirane (FcEpo) as the monomer, lots of electroactive tags are linked in situ from multiple initiation sites on the GO surface modified with ethanol amine (GO-ETA), thereby achieving high sensitivity even in the case of trace amounts of tumor markers. The utmost important factor for achieving this high sensitivity is to select functionalized GO as the initiator that contains a large number of repeated hydroxyl functional groups so as to trigger additional ROP reaction. Under the optimal conditions, the high sensitivity and applicability is demonstrated by the use of GO-ETA-mediated ROP-based immunosensor to detect non-small cell lung cancer (NSCLC)-specific biomarker down to 72.58 ag/mL (equivalent to ~6 molecules in a 5 µL sample). Furthermore, the satisfactory results for the determination of biomarkers in clinical serum samples highlighted that this immunosensor holds a huge potential in practical clinical application. This work described an electrochemical immunosensor for ultrasensitive detection of CYFRA 21-1 via the functionalized graphene oxide (GO) and a "grafting-to" ring-opening polymerization (ROP) dual signal amplification strategy, which hold the merits of high sensitivity, applicability, selectivity, efficiency, easy operation and environmental friendliness.

Electrochemical detection of alkaline phosphatase activity through enzyme-catalyzed reaction using aminoferrocene as an electroactive probe.

As a nonspecific phosphomonoesterase, alkaline phosphatase (ALP) plays a pivotal role in tissue mineralization and osteogenesis which is an important biomarker for the clinical diagnosis of bone and hepatobiliary diseases. Herein, we described a novel electrochemical method that used aminoferrocene (AFC) as an electroactive probe for the ALP activity detection. In the condition with imidazole and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), the AFC probe could be directly labeled on single-stranded DNA (ssDNA) by one-step conjugation. Specifically, thiolated ssDNA at 3'-terminals was modified to the electrode surface through Au-S bond. In the condition without ALP, AFC could be labeled on ssDNA by conjugating with phosphate groups. In the presence of ALP, phosphate groups were catalyzed to be removed from the 5'-terminal of ssDNA. The AFC probe cannot be labeled on ssDNA. Thus, the electrochemical detection of ALP activity was achieved. Under optimal conditions, the strategy presented a good linear relationship between current intensity and ALP concentration in the range of 20 to 100 mU/mL with the limit of detection (LOD) of 1.48 mU/mL. More importantly, the approach rendered high selectivity and satisfactory applicability for ALP activity detection. In addition, this method has merits of ease of operation, low cost, and environmental friendliness. Thus, this strategy presents great potential for ALP activity detection in practical applications. An easy, sensitive and reliable strategy was developed for the detection of alkaline phosphatase activity via electrochemical "Signal off".

Ultrasensitive label-free detection for lung cancer CYFRA 21-1 DNA based on ring-opening polymerization.

Cytokeratin fragment antigen 21-1 (CYFRA 21-1) DNA is perceived as sensitive tumor marker for the diagnosis of non-small cell lung cancer and other tumor. Herein, linear chain poly(ε-caprolactone) (PCL) synthesized by ring-opening polymerization is applied to ultrasensitive label-free electrochemical impedance detection system for CYFRA 21-1 DNA. First, thiolated peptide nucleic acid (PNA) is self-assembled into the Au electrode surface through the formation of Au-S bonds, allowing the PNA to act as biomolecular probe and form PNA/DNA heteroduplex with the target DNA via specific hybridization. Then, PCL is conjugated to the immobilized DNA on the electrode via "carboxylate-Zr4+-phosphate" bridges. Finally, the electrochemical response of modified PNA/DNA/Zr4+/PCL electrode is determined by electrochemical impedance method to quantify of CYFRA 21-1 DNA. Under optimal conditions, this method exhibits highly sensitivity with a broad linear range (0.1 fM - 1 nM) (R2 = 0.995) and the limit of detection (LOD) is as low as 10.73 aM, which is equivalent to just 64 molecules in a 10 µL sample. What's more, the high selectivity, good anti-interference, label-free operation, and real-time monitoring in complex samples of the proposed strategy demonstrate its broad application for the early diagnosis and clinical monitoring.