Biomimetic, pH-Responsive Nanoplatforms for Cancer Multimodal Imaging and Photothermal Immunotherapy.

Photothermal therapy (PTT), by converting light to thermal energy, has become a novel and noninvasive technique for tumor thermal ablation in clinical practice. However, as a result of phagocytosis of reticuloendothelial cells, current photothermal agents (PTAs) derived from exogenous materials suffer from incompetent tumor targeting and brief internal circulation time. The resulting poor accumulation of PTAs in the target area severely reduces the efficacy of PTT. In addition, the potential toxicity of PTAs, excessive laser exposure, and possibilities of tumor recurrence and metastasis following PTT are still intractable problems that severely influence patients' quality of life. Herein, a biomimetic pH-responsive nanoprobe was prepared via cancer cell membrane coating polydopamine (PDA)-CaCO3 nanoparticles (CPCaNPs) for photoacoustic (PA)/ultrasonic (US)/thermal imaging-guided PTT. When CPCaNPs targeted and infiltrated into the tumor's acidic microenvironment, the decomposed CO2 bubbles from homologous targeting CPCaNPs enhanced ultrasonic (US) signals obviously. At the same time, the PDA of CPCaNPs not only performed efficient PTT of primary tumors but also generated photoacoustic (PA) signals. In addition, an immune checkpoint pathway blockade was combined, which inhibited tumor recurrence and metastasis significantly and improved the immunosuppressive microenvironment after PTT to a large extent. Thus, these proposed biomimetic pH-responsive CPCaNPs provide a promising strategy for precise PTT immunotherapy under the intelligent guidance of PA/US/thermal imaging and show great potential for clinical translation.

Thermochromic Tissue-Mimicking Phantoms for Thermal Ablation Based on Polyacrylamide Gel.

In recent years, thermal ablation has played an increasingly important role in treating various tumors in the clinic. A practical thermochromic phantom model can provide a favorable platform for clinical thermotherapy training of young physicians or calibration and optimization of thermal devices without risk to animals or human participants. To date, many tissue-mimicking thermal phantoms have been developed and are well liked, especially the polyacrylamide gel (PAG)-based phantoms. This review summarizes the PAG-based phantoms in the field of thermotherapy, details their advantages and disadvantages and provides a direction for further optimization. The relevant physical parameters (such as electrical, acoustic, and thermal properties) of these phantoms are also presented in this review, which can assist operators in a deeper understanding of these phantoms and selection of the proper recipes for phantom fabrication.

Structure of an Alkaline Pectate Lyase and Rational Engineering with Improved Thermo-Alkaline Stability for Efficient Ramie Degumming.

Alkaline pectate lyases have biotechnological applications in plant fiber processing, such as ramie degumming. Previously, we characterized an alkaline pectate lyase from Bacillus clausii S10, named BacPelA, which showed potential for enzymatic ramie degumming because of its high cleavage activity toward methylated pectins in alkaline conditions. However, BacPelA displayed poor thermo-alkaline stability. Here, we report the 1.78 Å resolution crystal structure of BacPelA in apo form. The enzyme has the characteristic right-handed ß-helix fold of members of the polysaccharide lyase 1 family and shows overall structural similarity to them, but it displays some differences in the details of the secondary structure and Ca2+-binding site. On the basis of the structure, 10 sites located in flexible regions and showing high B-factor and positive ΔTm values were selected for mutation, aiming to improve the thermo-alkaline stability of the enzyme. Following site-directed saturation mutagenesis and screening, mutants A238C, R150G, and R216H showed an increase in the T5015 value at pH 10.0 of 3.0 °C, 6.5 °C, and 7.0 °C, respectively, compared with the wild-type enzyme, interestingly accompanied by a 24.5%, 46.6%, and 61.9% increase in activity. The combined mutant R150G/R216H/A238C showed an 8.5 °C increase in the T5015 value at pH 10.0, and an 86.1% increase in the specific activity at 60 °C, with approximately doubled catalytic efficiency, compared with the wild-type enzyme. Moreover, this mutant retained 86.2% activity after incubation in ramie degumming conditions (4 h, 60 °C, pH 10.0), compared with only 3.4% for wild-type BacPelA. The combined mutant increased the weight loss of ramie fibers in degumming by 30.2% compared with wild-type BacPelA. This work provides a thermo-alkaline stable, highly active pectate lyase with great potential for application in the textile industry, and also illustrates an effective strategy for rational design and improvement of pectate lyases.

Exogenous melatonin improves cotton (Gossypium hirsutum L.) pollen fertility under drought by regulating carbohydrate metabolism in male tissues.

Although exogenous melatonin can enhance the drought tolerance of plants, reports on the role of melatonin in drought tolerance in male reproductive organs are limited. To explore this, a pot experiment was conducted with cotton cultivar Yuzaomian 9110 to study the effects of exogenous melatonin (100, 200, and 1000 µM) on male fertility and related carbohydrate metabolism in anther under drought. Results showed that drought inhibited the translocation of carbon assimilates to anthers, however, melatonin application (100 and 200 µM) significantly improved the translocation of carbon assimilates to drought-stressed anthers. Drought reduced the deposition of starch, the hydrolysis of sucrose into hexoses, the generation of adenosine triphosphate (ATP) in anthers, restricting pollen viability and germination. Nevertheless, the appropriate melatonin concentrations (100 and 200 µM) increased the starch accumulation by enhancing ADP-glucose pyrophosphorylase and soluble starch synthases activities and accelerated the hydrolysis of sucrose by increasing sucrose synthase, acid and alkaline invertases activities in drought-stressed anthers. Appropriate melatonin concentrations (100 and 200 µM) also could help to generate more ATP for reproductive activities of drought-stressed anthers, finally increasing the pollen viability and germination of drought-stressed plants. These findings suggest that drought inhibited male fertility of cotton, but a precise melatonin application could regulate the carbohydrate balance of drought-stressed anthers to improve male fertility. This is the first report demonstrating the important role of exogenous melatonin in improving male fertility under drought conditions by regulating the carbohydrate metabolism in the male part of cotton.

A Multicolor Fluorescence Nanoprobe Platform Using Two-Dimensional Metal Organic Framework Nanosheets and Double Stirring Bar Assisted Target Replacement for Multiple Bioanalytical Applications.

Multicolor fluorescence probes can show fluorescence of different colors when detecting different targets, and the excellent feature can create a highly differentiated multicolor sensing platform. However, most of the previously reported multicolor luminescent materials usually suffer from high toxicity and photobleaching, complex preparation procedures, and poor water solubility, which may not be conducive to bioanalytical applications. Two-dimensional metal organic frameworks (2D MOFs), which have large specific surface areas with long-range fluorescence quenching coupled with biomolecular recognition events, have encouraged innovation in biomolecular probing. Here, we propose a 2D-MOF-based multicolor fluorescent aptamer nanoprobe using a double stirring bar assisted target replacement system for enzyme-free signal amplification. It utilizes the interaction between 2D MOFs and DNA molecules to detect multiple antibiotics quickly, sensitively, and selectively. Since 2D MOFs have excellent quenching efficiency for luminescence of fluorescent-dye-labeled single-strand DNA (ssDNA), the background fluorescence can be largely reduced and the signal-to-noise ratio can be improved. When the adsorbed ssDNA formed double helix double-stranded DNA with its complementary ssDNA, its fluorescence can be almost fully recovered. The assay was tested by detecting chloramphenicol (CAP), oxytocin (OTC), and kanamycin (KANA) in biological samples. The developed aptasensor was sufficiently sensitive to detect the antibiotic residues as low as 1.5 pM CAP, 2.4 pM OTC, and 1 pM KANA (S/N = 3). It has been preliminarily used for multicolor imaging of three different antibiotics in fish tissue slices with satisfactory results.

Novel label-free and high-throughput microchip electrophoresis platform for multiplex antibiotic residues detection based on aptamer probes and target catalyzed hairpin assembly for signal amplification.

Novel label-free and multiplex aptasensors have been developed for simultaneous detection of several antibiotics based on a microchip electrophoresis (MCE) platform and target catalyzed hairpin assembly (CHA) for signal amplification. Kanamycin (Kana) and oxytetracycline (OTC) were employed as models for testing the system. These aptasensors contained six DNA strands termed as Kana aptamer-catalysis strand (Kana apt-C), Kana inhibit strand (Kana inh), OTC aptamer-catalysis strand (OTC apt-C), OTC inhibit strand (OTC inh), hairpin structures H1 and H2 which were partially complementary. Upon the addition of Kana or OTC, the binding event of aptamer and target triggered the self-assembly between H1 and H2, resulting in the formation of many H1-H2 complexes. They could show strong signals which represented the concentration of Kana or OTC respectively in the MCE system. With the help of the well-designed and high-quality CHA amplification, the assay could yield 300-fold amplified signal comparing that from non-amplified system. Under optimal conditions, this assay exhibited a linear correlation in the ranges from 0.001ngmL-1 to 10ngmL-1, with the detection limits of 0.7pgmL-1 and 0.9pgmL-1 (S/N=3) toward Kana and OTC, respectively. The platform has the following advantages: firstly, the aptamer probes can be fabricated easily without labeling signal tags for MCE detection; Secondly, the targets can just react with probes and produce the amplified signal in one-pot. Finally, the targets can be simultaneously detected within 10min in different channels, thus high-throughput measurement can be achieved. Based on this work, it is estimated that this detection platform will be universally served as a simple, sensitive and portable platform for antibiotic contaminants detection in biological and environmental samples.

Isolation of CHS Gene from Brunfelsia acuminata Flowers and Its Regulation in Anthocyanin Biosysthesis.

Chalcone synthase gene (BaCHS) from Brunfelsia acuminata flowers was isolated using RT-PCR and RACE. The coding region of the gene is 1425-bp with an open reading frame of 1170-bp, 73-bp 5'UTR, and 172-bp 3'UTR. Its deduced protein does not have a signal peptide but does contain a cond_enzyme superfamily domain, and consists of 389 amino acids with a predicted molecular mass of 42,699 Da and a pI of 6.57. The deduced amino acid sequence of BaCHS shares 90%, 88%, 85%, 84% and 79% identity with CHS from Petunia hybrida, Nicotiana tabacum, Solanum lycopersicum, Capsicum annuum and Camellia sinensis, respectively. The striking color change from dark purple to light purple and ultimately lead to pure white resulted from a decline in anthocyanin content of the petals and was preceded by a decrease in the expression of BaCHS. Its gene expression was positively correlated with the contents of anthocyanin (p ≤ 0.01).

A triple-amplification colorimetric assay for antibiotics based on magnetic aptamer-enzyme co-immobilized platinum nanoprobes and exonuclease-assisted target recycling.

Herein, an ultrasensitive and selective colorimetric assay for antibiotics, using chloramphenicol (CAP) as the model analyte, was developed based on magnetic aptamer-HRP-platinum composite probes and exonuclease-assisted target recycling. The composite probes were prepared through immunoreactions between the double stranded DNA antibody (anti-DNA) labeled on core-shell Fe3O4@Au nanoparticles (AuMNP-anti-DNA) as the capture probe, and the double stranded aptamer (aptamer hybrid with its complementary oligonucleotides) labeled on Pt@HRP nanoparticles as the nanotracer (ds-Apt-HRP-PtNPs). When the CAP samples were incubated with the probes for 30 min at room temperature, they could be captured by the aptamer to form a nanotracer-CAP complex, which was then released into the supernatant after magnetic separation. This is because the anti-DNA on the capture probes cannot recognize the single strand aptamer-CAP complex. The exonuclease I (Exo I) added into the supernatant can further digest the aptamer-CAP from the 3'-end of the aptamer and the CAP in the aptamer-CAP complex can be released again, which can further participate in a new cycling process to react with the probes. Pt and HRP in the nanotracer could both catalyze and dual amplify the absorbance at 650 nm ascribed to the 3,3',5,5'-tetramethylbenzidine (TMB)-H2O2 system. Moreover, Exo I can assist the target recycling, which can further amplify the signal. Thus, the triple amplified signal can be quantified by ultraviolet-visible spectroscopy. The experimental results showed that the CAP detection possessed a linear range of 0.001-10 ng mL(-1) and a detection limit of 0.0003 ng mL(-1) (S/N = 3). The assay was successfully employed to detect CAP in milk, which is much more facile, time saving, and sensitive than the commercial ELISA kits.

A "signal-on'' aptasensor for simultaneous detection of chloramphenicol and polychlorinated biphenyls using multi-metal ions encoded nanospherical brushes as tracers.

A "signal-on'' aptasensor was developed for simultaneous detection of chloramphenicols (CAP) and polychlorinated biphenyl-72 (PCB72) with a novel multi-metal ions encoded nanospherical brushes as nanotracers. To construct the assay, the respective aptamer of CAP and PCB72 labeled magnetic gold nanoparticles as capture probes (aptamer-MGPs), and their complementary single strand DNA (s-DNA) encoded metal ions (Cd(2+) and Pb(2+)) on nanospherical branched polyethylene imine brushes as tracers (s-DNA-MSPEIs), were simultaneously synthesized. After that, the capture probe and tracers were connected through a hybridization reaction between s-DNA and aptamers. In the presence of CAP and PCB72, the analytes could react with the aptamers on capture probes and release the tracers into supernatant after magnetic separation. The released tracers with metal ions (Cd(2+) and Pb(2+)) could be simultaneously detected through the square wave voltammetry (SWV) without acid dissolution, which can switch the signals of the biosensor to "on'' state. Under optimal conditions, the assay could detect CAP and PCB72 as low as 0.3 pg mL(-1) with the dynamitic range from 0.001 to 100 ng mL(-1) and exhibited excellent selectivity. More importantly, the strategy can be extended easily to other targets after changing the corresponding aptamers and other metal ions tracers, which provides a promising and facile approach in multiplex detection of ultra-trace level of pollutants in food safety without more complex separation and washing steps.

A novel "dual-potential" electrochemiluminescence aptasensor array using CdS quantum dots and luminol-gold nanoparticles as labels for simultaneous detection of malachite green and chloramphenicol.

A novel type of "dual-potential" electrochemiluminescence (ECL) aptasensor array was fabricated on a homemade screen-printed carbon electrode (SPCE) for simultaneous detection of malachite green (MG) and chloramphenicol (CAP) in one single assay. The SPCE substrate consisted of a common Ag/AgCl reference electrode, carbon counter electrode and two carbon working electrodes (WE1 and WE2). In the system, CdS quantum dots (QDs) were modified on WE1 as cathode ECL emitters and luminol-gold nanoparticles (L-Au NPs) were modified on WE2 as anode ECL emitters. Then the MG aptamer complementary strand (MG cDNA) and CAP aptamer complementary strand (CAP cDNA) were attached on CdS QDs and L-Au NPs, respectively. The cDNA would hybridize with corresponding aptamer that was respectively tagged with cyanine dye (Cy5) (as quenchers of CdS QDs) and chlorogenic acid (CA) (as quenchers of l-Au NPs) using poly(ethylenimine) (PEI) as a bridging agent. PEI could lead to a large number of quenchers on the aptamer, which increased the quenching efficiency. Upon MG and CAP adding, the targets could induce strand release due to the highly affinity of analytes toward aptamers. Meanwhile, it could release the Cy5 and CA, which recovered cathode ECL of CdS QDs and anode ECL of L-Au NPs simultaneously. This "dual-potential" ECL strategy could be used to detect MG and CAP with the linear ranges of 0.1-100 nM and 0.2-150 nM, with detection limits of 0.03 nM and 0.07 nM (at 3sB), respectively. More importantly, this designed method was successfully applied to determine MG and CAP in real fish samples and held great potential in the food analysis.