Characterization of a novel carboxylesterase from Streptomyces lividans TK24 and site-directed mutagenesis for its thermostability.

As an industrial enzyme that catalyzes the formation and cleavage of ester bonds, carboxylesterase has attracted attention in fine chemistry, pharmaceutical, biological energy and bioremediation fields. However, the weak thermostability limits their further developments in industrial applications. In this work, a novel carboxylesterase (EstF) from Streptomyces lividans TK24, belonging to family XVII, was acquired by successfully heterologous expressed and biochemically identified. The EstF exhibited optimal activity at 55 °C, pH 9.0 and excellent catalytic performances (Km = 0.263 mM, kcat/Km = 562.3 s-1 mM-1 for p-nitrophenyl acetate (pNPA2) hydrolysis). Besides, the EstF presented exceptionally high thermostability with a half-life of 387.23 h at 55 °C and 2.86 h at 100 °C. Furthermore, the EstF was modified to obtain EstFP144G using the site-directed mutation technique to investigate the effect of single glycine on thermostability. Remarkably, the mutant EstFP144G displayed a 5.10-fold increase of half-life at 100 °C versus wild-type without affecting catalytic performance. Structural analysis implied that the glycine introduction could release a steric strain and induce cooperative effects between distal residues to increase the thermostability. Therefore, the thermostable EstF and EstFP144G with prominently catalytic characteristics have potential industrial applications and the introduction of a single glycine strategy opens up alternative avenues for the thermostability engineering of other enzymes.

Correction: A poly(thymine)-templated fluorescent copper nanoparticle hydrogel-based visual and portable strategy for an organophosphorus pesticide assay.

Correction for 'A poly(thymine)-templated fluorescent copper nanoparticle hydrogel-based visual and portable strategy for an organophosphorus pesticide assay' by Jihua Chen et al., Analyst, 2019, 144, 2423-2429, https://doi.org/10.1039/C9AN00017H.

Dual-functional cellulase-mediated gold nanoclusters for ascorbic acid detection and fluorescence bacterial imaging.

Protein-protected metal nanomaterials are becoming the most promising fluorescent nanomaterials for biosensing, bioimaging, and therapeutic applications due to their obvious fluorescent molecular properties, favorable biocompatibility and excellent physicochemical properties. Herein, we pioneeringly prepared a cellulase protected fluorescent gold nanoclusters (Cel-Au NCs) exhibiting red fluorescence under the excitation wavelength of 560 nm via a facile and green one-step method. Based on the fluorescence turn-off mechanism, the Cel-Au NCs were used as a biosensor for specificity determination of ascorbic acid (AA) at the emission of 680 nm, which exhibited satisfactory linearity over the range of 10-400 µM and the detection limit of 2.5 µM. Further, the actual sample application of the Au NCs was successfully established by evaluating AA in serum with good recoveries of 98.76%-104.83%. Additionally, the bacteria, including gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus) and gram-negative bacteria (Escherichia coli), were obviously stained by Cel-Au NCs with strong red emission. Thereby, as dual-functional nanoclusters, the prepared Cel-Au NCs have been proven to be an excellent fluorescent bioprobe for the detection of AA and bacterial labeling in medical diagnosis and human health maintenance.

Reusable carboxylesterase immobilized in ZIF for efficient degradation of chlorpyrifos in enviromental water.

The past few decades have witnessed biodegradation of pesticides as a significant method in remediation of the environment for its specificity, efficiency and biocompatibility. However, the tolerability and recyclability of the enzymes in pesticide degradation and the development of enzymes that biodegrad pesticides are still urgent problems to be solved so far. Herein, a novel hyper-thermostable and chlorpyrifos-hydrolyzing carboxylesterase EstC was immobilized by biomineralization using zeolitic imidazolate framework (ZIF), one of the metal-organic frameworks (MOFs) with highly diverse structure and porosity. Compared with free enzyme, EstC@ZIF with a cruciate flower-like morphology presented scarcely variation in catalytic efficiency and generally improved the tolerance to organic solvents or detergents. Furthermore, there was scarcely decrease in the catalytic efficiency of EstC@ZIF and it also showed good reusability with about 50% residual activity after 12 continuous uses. Notably, EstC@ZIF could be used in actual water environment with an excellent value of degradation rate of 90.27% in 120 min, and the degradation efficiency remained about 50% after 9 repetitions. The present strategy of immobilizing carboxylesterase to treat pesticide-contaminated water broadens the method of immobilized enzymes on MOFs, and envisions its recyclable applicability in globe environmental remediation.

Recent progress of Bioinspired Hydrogel-based delivery system for endometrial repair.

Endometrial injury is the main fact leading to infertility. Current treatments of endometrial injury present many problems, such as unable to achieve desired effects due to low retention and the inherent potential risk of injury. Besides, it is important to the development of bioinspired material that can mimic the natural tissue and possess native tissue topography. Hydrogel is a kind of bioinspired superhydrophilic materials with unique characteristics, such as excellent biocompatibility, biodegradability, porosity, swelling, and cross-linkage. These unique physiochemical properties of bioinspired hydrogels enable their promising application as novel delivery platform and alternative therapies for endometrial injury. In this mini review, we summarize the recent advances in bioinispred hydrogel-based delivery system for endometrial repair, including as a post-operative physical barrier and therapeutic delivery system. In addition, present status, limitations, and future perspectives are also discussed.

Acupuncture for Female Infertility: Discussion on Action Mechanism and Application.

A higher incidence of female infertility has been reported with an unexpectedly early appearance in recent years. The female infertility treatment and application of assisted reproductive technology have recently gained immense interest from scientists. Many studies have discussed the beneficial effects of acupuncture on female infertility. With advancements in science and medical technology, acupuncture-related research has increased in investigating its effectiveness in treating female infertility. This review focuses on a compilation of research in recent years on acupuncture for female infertility treatment and the exploration of the underlying mechanism. For this purpose, literature was searched using various search engines like PubMed, Web of Science, and Google Scholar. The search was refined by only focusing on recent studies on acupuncture effectiveness and mechanism in female infertility and evaluating pregnancy outcomes.

Excellent catalytic properties of luminescent Cu@Cu2S nanozymes and their antibacterial applications.

Luminescent Cu@Cu2S nanozymes have been prepared by a one-pot method, displaying high peroxidase-like and oxidase-like activity. Fluorescence images show that these nanozymes adhere to the surface of the bacterial cell and scanning electron microscopy reveals that the nanomaterials cause folding and collapse of the bacterial surface, resulting in bacterial death.

Transcriptomic study of the mechanism by which the Kai Yu Zhong Yu recipe improves oocyte quality in a stressed mouse model.

ETHNOPHARMACOLOGICAL RELEVANCE: The Kai Yu Zhong Yu recipe (KYZY) is a classic herbal formula in traditional Chinese medicine (TCM) that has been used to treat infertility associated with psychological stress for more than three hundred years. AIM OF THE STUDY: Psychological stress has major impacts on fertility, with variable outcomes depending on the nature, strength, and duration of the stress. Stress can directly disturb ovulation, oocyte quality, maturation, and embryo development. The aim of this study is to investigate the molecular mechanism by which KYZY improves oocyte developmental potential under psychological stress. MATERIALS AND METHODS: ICR female mice aged 4-5 weeks were randomly divided into five groups: control, stressed in the chronic unpredictable stress model (CUSM), and stressed plus KYZY treatment at 38.2 g/kg (KYZYH), 19.1 g/kg (KYZYM), or 9.6 g/kg (KYZYL). Ovary function was assessed by measuring serum levels of estradiol (E2), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and anti-Müllerian hormone (AMH). Oocyte quality was evaluated in terms of reactive oxygen species (ROS) levels, apoptotic DNA fragmentation, and mitochondria distribution. We used RNA sequencing (RNAseq) to identify differentially expressed genes (DEGs) between groups and then further analyzed the DEGs for gene ontology (GO) term enrichment and protein-protein interactions. RESULTS: Mice in the stressed group had reduced serum E2, LH, and FSH as well as increased ROS levels, increased apoptosis, and disturbed mitochondria distribution in oocytes. Treatment with KYZY at all three doses reversed or ameliorated these negative effects of stress. DEG analysis identified 187 common genes between the two comparisons (stressed vs. control and KYZYM vs. stressed), 33 of which were annotated with six gene ontology (GO)'s biological process (BP) terms: cell differentiation, apoptosis, ATP synthesis, protein homo-oligomerization, neuron migration, and negative regulation of peptidase activity. Protein-protein interaction network analysis of DEGs identified key hub genes. Notably, the genes Atp5o and Cyc1 were both involved in the ATP synthesis and among the top three hub genes, suggesting that regulation of oocyte mitochondrial electron transport and ATP synthesis is important in the response to stress and also is a possible mechanism of action for KYZY. CONCLUSIONS: KYZY was effective in ameliorating the adverse effects of stress on oocyte competence, possibly by targeting the mitochondrial respiratory chain and ATP synthase.

Autonomous operation of 3D DNA walkers in living cells for microRNA imaging.

Three dimensional (3D) DNA walkers hold great potential in serving as an ideal candidate for signal transduction and amplification in bio-assays. However, the autonomous operation of 3D DNA walkers inside living cells is still few and far between, which could be attributed to the lack of suitable driving forces and moderate efficiency in terms of the cellular uptake of such complex 3D DNA components. Herein, a newly updated autonomously operated and highly integrated 3D DNA walker on Au nanoparticles (Au NPs)/zeolitic imidazolate framework-8 (ZIF-8) was activated in a tumor microenviroment and its signal amplified assay capability in living cells was demonstrated using miRNA as a sensing model biomolecule. Specifically, we assembled a 3D DNA motor, including Zn2+-dependent DNAzyme and substrates on the AuNPs grafted on ZIF-8. After being delivered into a living cell, ZIF-8 was efficiently degraded in the tumor microenvironment (low pH value), locally releasing the Zn2+ and DNA motor. Then, a self-sufficient DNA motor autonomously performed the bio-analytical task of imaging miRNA-10b, with a low detection limit of 34 pM. Also, such self-sufficient 3D walkers allowed real-time imaging of MDA-MB-231 cells by intracellular operation. This method demonstrates the self-sufficient 3D DNA motor's bioanalytical application in living cells which may inspire various other biological applications including gene delivery, therapy, etc.

Characterization of a carboxylesterase with hyper-thermostability and alkali-stability from Streptomyces lividans TK24.

A gene (estA', 804 bp) from Streptomyces lividans TK24 was artificially synthesized and successfully overexpressed as a 6His-tagged fusion protein in Escherichia coli. It encoded a carboxylesterase (EstA) that composed of 267 amino acids with a predicted molecular weight of 28.56 kDa. Multiple sequence alignment indicated that EstA has typical characteristics of esterases, including a catalytic triad (Ser93-Asp194-His224) and a conserved pentapeptide motif (Gly91-Leu92-Ser93-Met94-Gly95). Simultaneously, phylogenetic analysis indicated that EstA belongs to family VI. Biochemical characterization displayed its optimum enzyme activity was at 55 â„ƒ and pH 8.5. Additionally, EstA exhibited higher activity towards short carbon substrates and showed the outstanding catalytic efficiency for pNPA2 with kcat/Km of 2296.14 ± 10.35 s-1 mM-1. Notably, EstA has hyper-thermostability and good alkali stability. The activity of EstA did not change obviously when incubated at 50 and 100 â„ƒ for 337 and 1 h, independently. Besides, by incubating at 100 â„ƒ for 6 h, EstA remained about half of its initial activity. Moreover, EstA showed stability at pH ranging from 8.0 to 11.0, and about 90% residual enzyme activity was reserved by being treated at pH 8.0 or 9.0 for 80 h, especially. Such multiple features prepare EstA for a potential candidate in the field of biological catalysis of some industrial applications under harsh conditions.

Characterization of a novel hyper-thermostable and chlorpyrifos-hydrolyzing carboxylesterase EstC: A representative of the new esterase family XIX.

Carboxylesterases have widely been used in a series of industrial applications, especially, the detoxification of pesticide residues. In the present study, EstC, a novel carboxylesterase from Streptomyces lividans TK24, was successfully heterogeneously expressed, purified and characterized. Phylogenetic analysis showed that EstC can be assigned as the first member of a novel family XIX. Multiple sequence alignment indicated that EstC has highly conserved structural features, including a catalytic triad formed by Ser155, Asp248 and His278, as well as a canonical Gly-His-Ser-Ala-Gly pentapeptide. Biochemical characterization indicated that EstC exhibited maximal activity at pH 9.0 (Tris-HCl buffer) and 55 °C. It also showed higher activity towards short-chain substrates, with the highest activity for p-nitrophenyl acetate (pNPA2) (Km = 0.31 ± 0.02 mM, kcat/Km = 1923.35 ± 9.62 s-1 mM-1) compared to other pNP esters used in this experiment. Notably, EstC showed hyper-thermostability and good alkali stability. The activity of EstC had no significant changes when it was incubated under 55 °C for 100 h and reached half-life after incubation at 100 °C for 8 h. Beyond that, EstC also showed stability at pH ranging from 6.0 to 11.0 and about 90% residual activity still reserved after treatment at pH 8.0 or 9.0 for 26 h, especially. Furthermore, EstC had outstanding potential for bioremediation of chlorpyrifos-contaminated environment. The recombinant enzyme (0.5 U mL-1) could hydrolyze 79.89% chlorpyrifos (5 mg L-1) at 37 °C within 80 min. These properties will make EstC have a potential application value in various industrial productions and detoxification of chlorpyrifos residues.

Flash nanoprecipitation of ultra-small semiconducting polymer dots with size tunability.

Small-sized semiconducting polymer dots (Pdots) provide better tissue and subcellular penetration while minimizing unspecific interactions, and make the fast clearance of Pdots from human bodies possible by urinary excretion. We employ a powerful and scalable technology, flash nanoprecipitation, to prepare Pdots with small sizes (hydrodynamic diameters ∼10 nm).

Intracellular Imaging of Glutathione with MnO2 Nanosheet@Ru(bpy)32+-UiO-66 Nanocomposites.

Fluorescent detection of glutathione (GSH) in the living system has attracted much attention, but current fluorescent probes are usually exposed to the exterior environment, leading to photobleaching and premature leakage and subsequently limiting the sensitivity and photostability. Herein, luminescent metal-organic frameworks [Ru(bpy)32+ encapsulated in UiO-66] coated with manganese dioxide nanosheets [MnO2 NS@Ru(bpy)32+-UiO-66] were prepared by an in situ growth method and further explored to construct a GSH-switched fluorescent sensing platform. Because of the splendid fluorescence quenching ability, special probe leakage blocking role and distinguished recognition of the MnO2 NS, and the improved fluorescence of Ru(bpy)32+ by UiO-66, a low background, highly sensitive and selective detection of GSH with a low limit of detection as 0.28 µM was realized. At the same time, the preparation of MnO2 NS@Ru(bpy)32+-UiO-66 nanocomposites is simple and less toxic, and there was no notable loss of cell survivability after being exposed to MnO2 NS@Ru(bpy)32+-UiO-66 below the concentrations of 120 µg mL-1 for 24 h. Consequently, the results coming from this effort suggest that the new sensing platform will have a great potential in the detection of GSH in living cells.

Fluorometric methods for determination of H2O2, glucose and cholesterol by using MnO2 nanosheets modified with 5-carboxyfluorescein.

A fluorescent probe for H2O2 is described. It is composed of MnO2 nanosheets and 5-carboxyfluorescein and was characterized by fluorescence, transmission electron microscopy, ultraviolet-visible absorption spectra, energy dispersive X-ray and Fourier transform infrared spectroscopy. The probe, with fluorescence excitation/emission maxima at 490/518 nm, responds to H2O2 in the 1 to 200 µM concentration range and has a 0.33 µM detection limit. The probe was used in enzymatic assays for glucose and cholesterol by using the respective oxidases which produce H2O2. Responses are linear in the concentration range from 0.5 to 200 µM in case of glucose, and from 1 to 300 µM in case of cholesterol. The method was applied to quantify glucose and cholesterol in (spiked) serum samples. Graphical abstract Schematic presentation of the principle based on hydrogen peroxide-induced degradation of MnO2 nanosheet-FAM complex for detection of H2O2 (a), glucose and cholesterol (b).

A poly(thymine)-templated fluorescent copper nanoparticle hydrogel-based visual and portable strategy for an organophosphorus pesticide assay.

Since fluorescence assays with high sensitivity for organophosphorus pesticides (OPs) are urgently required to protect the ecosystem and prevent disease, an environmentally friendly and label-free fluorescent probe is desirable. Herein, a poly-thymine30 DNA-templated copper nanoparticle (poly T30-Cu NPs) hydrogel fluorescent probe was explored for the construction of an OPs sensing platform via tyrosinase (TYR) enzyme-controlled quenching. Initially, TYR can efficiently quench the fluorescence of poly T30-Cu NPs; however, when OPs are mixed with a certain amount of TYR, the fluorescence of poly T30-Cu NPs can be recovered. Based on this phenomenon, we designed a functionalized hydrogel based on poly T30-Cu NPs for portable and visible detection of OPs with high sensitivity and selectivity. This proposed fluorescent platform was demonstrated to enable rapid detection of OPs (paraoxon as the model analyte) and provide excellent sensitivity with a detection limit of 3.33 × 10-5 ng µL-1 and a linear range of 1.0 × 10-4-1.0 ng µL-1. The fluorescent probe does not require a sophisticated synthesis and labeling process; in addition, it is environmentally friendly because of the presence of a biotemplate of DNA and biocompatible copper. Moreover, the functional hydrogel combines the features of portability, visualization, fast signal response and environmental anti-interference that make the proposed strategy more feasible in complex practical detection.

PABPC1L depletion inhibits proliferation and migration via blockage of AKT pathway in human colorectal cancer cells.

Numerous studies have demonstrated that PABPC1 participates in the process of carcinogenesis and its function is inconsistent in different types of cancers. PABPC1-like (PABPC1L) is an important paralog of PABPC1 and few studies are available on the roles of PABPC1L in colorectal cancer (CRC) development. Hence, we explored the biological function and prognostic impact of PABPC1L in CRC. The mRNA expression of PABPC1L in CRC was determined based on the data obtained from The Cancer Genome Atlas (TCGA) database. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was utilized to determine the PABPC1L mRNA expression level in CRC HT-29 and LS-174T cell lines. Kaplan-Meier method and Cox proportional-hazards model were utilized to conduct the survival and prognosis analyses. HT-29 cells with silenced PABPC1L were constructed to explore the effect of PABPC1L on cell proliferation, invasion and migration capacities using cell counting kit-8 (CCK-8), clone formation, wound-healing and Transwell assays, respectively. To uncover the potential mechanisms of how PABPC1L influences CRC proliferation and migration, we analyzed the expression of AKT, p-AKT, PI3K, and p-PI3K in HT-29 cells using western blotting. Our results revealed that PABPC1L was overexpressed in CRC tissues compared with normal tissues based on the data obtained from TCGA database. Similarly, the mRNA expression of PABPC1L was higher in HT-29 and LS-174T cells than that in CCD-18Co cells. The expression of PABPC1L in CRC was found to be significantly related to age, pathologic stage, pathologic-node, pathologic-metastasis, and death. In univariate and multivariate analyses, pathologic-tumor and pathologic-metastasis were identified as independent prognostic factors for CRC. After PABPC1L depletion, cell proliferation rate, colony numbers, and the invasive and migratory capacity of HT-29 cells were all reduced. Western blot analysis showed that reduction of PABPC1L significantly inhibited p-AKT, and p-PI3K expression level in HT-29 cells. Collectively, our results suggested that PABPC1L is a potential novel candidate oncogene in CRC, and targeting PABPC1L may provide clinical utility in CRC.

Fe Foil-Guided Fabrication of Uniform Ag@AgX Nanowires for Sensitive Detection of Leukemia DNA.

Herein, we report a novel Fe foil-guided, in situ etching strategy for the preparation of highly uniform Ag@AgX (X = Cl, Br) nanowires (NWs) and applied the photoelectric-responsive materials for sensitive photoelectrochemical (PEC) detection of leukemia DNA. The Ag@AgX NW formation process was discussed from the redox potential and Ksp value. The fabricated PEC platform for sensing leukemia DNA showed good assay performance with a wide linear range (0.1 pM to 50 nM) and low detection limit of 0.033 pM. We envision that our Fe foil-guided synthetic method could be applied to synthesize more photoactive materials for sensitive PEC detections.

Aprotinin Encapsulated Gold Nanoclusters: A Fluorescent Bioprobe with Dynamic Nuclear Targeting and Selective Detection of Trypsin and Heavy Metal.

Fluorescence imaging has currently emerged as one of the most frequently used noninvasive imaging technologies to selectively monitor biological processes in living systems. In past decades, gold nanoclusters (Au NCs) has received increasing attraction because of their intrinsic fluorescence and their inherent biocompatibility. As a stabilizing and reducing agent, an abundant, sustainable, and widely used polypeptide derived drug molecule, aprotinin (Ap), is selected for the synthesis of Au nanoclusters (Ap-Au NCs) due to characteristic bioactivity, excellent biocompatibility, biodegradability, and non-allergenic character. Herein, Ap encapsulated Au NCs with desirable red fluorescence was facilely produced for the first time, which were subsequently used for cell imaging and detection of various analytes. Much interestingly, dynamically subcellular targeting  from the cytoplasm to the nucleus in HeLa cells was observed. Besides, it has shown that, the selective and quantitative detection of trypsin has been established by using Ap-Au NCs. Finally, Ap-Au NCs were readily used for quantitative detection of mercury and copper. The photoluminescence of the Ap-Au NCs was quenched with the addition of the aforementioned analytes. This study not only  discusses a multifunctional nanomaterial  for cell imaging, dynamically nuclear targeting and biosensing, but also opens crucial insights on the integration of funtional biomolecule with metal nanoclusters intended for extensively biomedical applications.

Portable Aptasensor of Aflatoxin B1 in Bread Based on a Personal Glucose Meter and DNA Walking Machine.

Despite some recent developments on the portable on-site sensor of Aflatoxin B1 (AFB1), the complex and expensive preparation of recognition elements have still limited their wide applications. In this paper, using the fast, low-cost, and stable recognition of aptamer DNA-AFB1, a portable aptasensor was constructed for the on-site detection of AFB1 in food matrixes, with the readout of personal glucose meter (PGM) and DNA walking machine for signal probe separation. In such an assay protocol, the target could trigger the DNA walker to autonomously move on the electrode surface, propelled by unidirectional Pb2+-specific DNAzyme digestion, which could amplify the signal and separate the signal probe as well for further quantification by the PGM. Under optimized conditions, the increase of PGM signal was relative with the concentration of AFB1 ranging from 0.02 to 10 nM and the low limit of detection (LOD) was 10 pM (S/N = 3). With the features of portability, and cheapness, the presented user-friendly method could be extended to various other analytes for wide point-of-care applications.

Long Noncoding RNA PVT1 Promotes Melanoma Progression via Endogenous Sponging miR-26b.

Melanoma is an extremely aggressive malignant skin tumor with a high mortality. Various long noncoding RNAs (lncRNAs) have been reported to be associated with the oncogenesis of melanoma. The purposes of this study were to investigate the potential role of lncRNA PVT1 in melanoma progression and to explore its possible mechanisms. A total of 35 patients who were diagnosed with malignant melanoma were enrolled in this study. Expression of PVT1 was significantly upregulated in melanoma tissue and was associated with a poor prognosis. Loss-of-function experiments showed that PVT1 knockdown markedly suppressed the proliferation activity, induced cell cycle arrest at the G0/G1 phase, and enhanced the apoptosis of melanoma cell lines. Bioinformatics analysis and dual-luciferase reporter assay revealed that PVT1 directly bound to miR-26b, which had been verified to be a tumor suppressor in melanoma. Moreover, further functional rescue experiments revealed that PVT1 knockdown could observably reverse the tumor-promoting role of the miR-26b inhibitor. Overall, our study demonstrates the oncogenic role of PVT1 as a miR-26b sponge, possibly providing a novel therapeutic target for melanoma.