Isolation and characterization of distinctive pyrene-degrading bacteria from an uncontaminated soil.

Considerable efforts that isolate and characterize degrading bacteria for polycyclic aromatic hydrocarbons (PAHs) have focused on contaminated environments so far. Here we isolated three distinctive pyrene (PYR)-degrading bacteria from a paddy soil that was not contaminated with PAHs. These included a novel Bacillus sp. PyB-9 and efficient degraders, Shigella sp. PyB-6 and Agromyces sp. PyB-10. All three strains could utilize naphthalene, phenanthrene, anthracene, fluoranthene and PYR as sole carbon sources, and degraded PYR in a range of temperatures (27-37 °C) and pH (5-8). Strains PyB-6 and PyB-10 almost completely degraded 50 mg L-1 PYR within 15 days, and 75.5% and 98.9% of 100 mg L-1 PYR in 27 days, respectively. The kinetics of PYR biodegradation was well represented by the Gompertz model. Ten and twelve PYR metabolites were identified in PYR degradation process by strains PyB-6 and PyB-10, respectively. Chemical analyses demonstrated that the degradation mechanisms of PYR were the same for strains PyB-6 and PyB-10 with initial dioxygenation mainly on C-4,5 positions of PYR. The degradation of 4,5-phenanthrenedicarboxylic acid was branched to 4-phenanthrenecarboxylic acid pathway and 5-hydroxy-4-phenanthrenecarboxylic acid pathway, both of which played important roles in PYR degradation by strains PyB-6 and PyB-10. To our knowledge, Shigella sp. and Agromyces sp. were found for the first time to possess the capability for PAHs degradation. These findings contributed to upgrading the bank of microbial resource and knowledge on PAH biodegradation.

Large-Area Electronics-Enabled High-Resolution Digital Microfluidics for Parallel Single-Cell Manipulation.

Large-area electronics as switching elements are an ideal option for electrode-array-based digital microfluidics. With support of highly scalable thin-film semiconductor technology, high-resolution digital droplets (diameter around 100 µm) containing single-cell samples can be manipulated freely on a two-dimensional plane with programmable addressing logic. In addition, single-cell generation and manipulation as foundations for single-cell research demand ease of operation, multifunctionality, and accurate tools. In this work, we reported an active-matrix digital microfluidic platform for single-cell generation and manipulation. The active device contained 26,368 electrodes that could be independently addressed to perform parallel and simultaneous droplet generation and achieved single-cell manipulation. We demonstrate a high-resolution digital droplet generation with a droplet volume limit of 500 pL and show the continuous and stable movement of droplet-contained cells for over 1 h. Furthermore, the success rate of single droplet formation was higher than 98%, generating tens of single cells within 10 s. In addition, a pristine single-cell generation rate of 29% was achieved without further selection procedures, and the droplets containing single cells could then be tested for on-chip cell culturing. After 20 h of culturing, about 12.5% of the single cells showed cell proliferation.

Structure of the platelet glycoprotein Ib receptor in complex with a novel antithrombotic agent.

Agkisacucetin, a snake C-type lectin-like protein isolated from the venom of Deinagkistrodon acutus (formerly Agkistrodon acutus), is a novel antithrombotic drug candidate in phase 2 clinical trials. Agkisacucetin specifically recognizes the platelet surface receptor glycoprotein Ib α chain (GPIbα) to block GPIb and von Willebrand factor (VWF). In this study, we solved the crystal structure of the GPIbα N-terminal domain (residues 1-305) in complex with agkisacucetin to understand their molecular recognition mechanism. The crystal structure showed that agkisacucetin primarily contacts GPIbα at the C-terminal part of the conserved leucine-rich repeat (LRR) domain (LRR-6 to LRR-8) and the previously described "ß-switch" region through the ß chain. In addition, we found that agkisacucetin α chain contacts part of the GPIbα C-terminal peptide after the LRR domain through complementary charge interactions. This C-terminal peptide plays a key role in GPIbα and thrombin recognition. Therefore, our structure revealed that agkisacucetin can sterically block the interaction between the GPIb receptor and VWF and thrombin proteins to inhibit platelet function. Our structural work provides key molecular insights into how an antithrombotic drug candidate recognizes the GPIb receptor to modulate platelet function to inhibit thrombosis.

Synthesis and Spectroscopic Properties of Selenophene[3, 2-b]-Fused BODIPYs.

Fusion selenophene endows the chromophore with more intrinsic and special functions. Herein, nonsymmetric selenophene-fused BODIPYs were designed and synthesized starting from the selenophene unit. The fused ring of selenophene not only maintains the rigid structure of BODIPY but also further modulates its spectral properties. The newly prepared dyes possessed many promising properties including large molar extinction coefficients, low fluorescence quantum yields, and moderate singlet oxygen generation. Quantum calculations affirmed that the smaller singlet-triplet energy gap and larger spin-orbit coupling cause efficient intersystem crossing, thus enhancing the singlet oxygen generation yield. Furthermore, selenophene-fused BODIPY exhibited significant phototoxicity with negligible dark cytotoxicity, based on the fluorescence imaging of the reactive oxygen species detection experiment.

Enhanced absorbance detection system for online bacterial monitoring in digital microfluidics.

We report a fully integrated digital microfluidic absorbance detection system with an enhanced sensitivity for online bacterial monitoring. Through a 100 µm gap in the chip, our optical detection system has a detection sensitivity for a BCA protein concentration of 0.1 mg mL-1. The absorbance detection limit of our system is 1.4 × 10-3 OD units, which is one order of magnitude better than that of the existing studies. The system's linear region is 0.1-7 mg mL-1, and the dynamic range is 0-25 mg mL-1. We measured the growth curves of wild-type and E. coli transformed with resistance plasmids and mixed at different ratios on chip. We sorted out the bacterial species including highly viable single cells based on the difference in absorbance data of growth curves. We explored the changes in the growth curves of E. coli under different concentrations of resistant media. In addition, we successfully screened for the optimal growth environment of the bacteria, in which the growth rate of PET30a-DH5α (in a medium with 33 µg mL-1 kanamycin resistance) was significantly higher than that of a 1 mg mL-1 resistance medium. In conclusion, the enhanced digital microfluidic absorbance detection system exhibits exceptional sensitivity, enabling precise bacterial monitoring and growth curve analysis, while also laying the foundation for DMF-based automated bioresearch platforms, thus advancing research in the life sciences.

Oxidative Dissolution of Sulfide Minerals Tends to Accumulate More Dissolved Heavy Metals in Deep Seawater Environments than in Shallow Seawater Environments.

Deep-sea mining magnifies the release of heavy metals into seawater through oxidative dissolution of seafloor massive sulfide (SMS). At present, there is little information about how the metals released into seawater might be affected by the mineral assemblages, seawater conditions, and solid percentages. Here, leaching experiments were carried out to examine the behavior of three sulfides from the Southwest Indian Ridge, under conditions that replicated deep and shallow seawater environments at three solid-liquid ratios. The results demonstrated that sphalerite dissolved rapidly, and the metals released in both experimental conditions were comparable, potentially reflecting galvanic interactions between the sulfide minerals. Large quantities of the released metals were removed from the solutions when hydrous ferric oxides formed, especially for shallow seawater conditions. A comparison of metal concentrations in the leachates with the baseline metal concentrations in natural seawater indicated that most of the released metals, when diluted with seawater, would not have widespread impacts on ecosystems. Based on the obtained unique oxidative dissolution properties of each SMS at variable solid-liquid ratios, targeted wastewater discharge treatments are proposed to minimize impacts from the dissolved metals. This study will support the development of robust guidelines for deep-sea mining activities.

Dihydrochalcones in Sweet Tea: Biosynthesis, Distribution and Neuroprotection Function.

Sweet tea is a popular herbal drink in southwest China, and it is usually made from the shoots and tender leaves of Lithocarpus litseifolius. The sweet taste is mainly attributed to its high concentration of dihydrochalcones. The distribution and biosynthesis of dihydrochaldones in sweet tea, as well as neuroprotective effects in vitro and in vivo tests, are reviewed in this paper. Dihydrochalones are mainly composed of phloretin and its glycosides, namely, trilobatin and phloridzin, and enriched in tender leaves with significant geographical specificity. Biosynthesis of the dihydrochalones follows part of the phenylpropanoid and a branch of flavonoid metabolic pathways and is regulated by expression of the genes, including phenylalanine ammonia-lyase, 4-coumarate: coenzyme A ligase, trans-cinnamic acid-4-hydroxylase and hydroxycinnamoyl-CoA double bond reductase. The dihydrochalones have been proven to exert a significant neuroprotective effect through their regulation against Aß deposition, tau protein hyperphosphorylation, oxidative stress, inflammation and apoptosis.

Epigenetic upregulation of galanin-like peptide mediates deoxynivalenol induced-growth inhibition in pituitary cells.

Deoxynivalenol (DON) is an unavoidable contaminant in human food, animal feeds, and agricultural products. Growth retardation in children caused by extensive DON pollution has become a global problem that cannot be ignored. Previous studies have shown that DON causes stunting in children through intestinal dysfunction, insulin-like growth factor-1 (IGF-1) axis disorder and peptide YY (PYY). Galanin-like peptide (GALP) is an important growth regulator, but its role in DON-induced growth retardation is unclear. In this study, we report the important role of GALP during DON-induced growth inhibition in the rat pituitary tumour cell line GH3. DON was found to increase the expression of GALP through hypomethylationin the promoter region of the GALP gene and upregulate the expression of proinflammatory factors, while downregulate the expression of growth hormone (GH). Furthermore, GALP overexpression promoted proinflammatory cytokines, including TNF-α, IL-1ß, IL-11 and IL-6, and further reduced cell viability and cell proliferation, while the inhibitory effect of GALP was the opposite. The expression of GALP and insulin like growth factor binding protein acid labile subunit (IGFALS) showed the opposite trend, which was the potential reason for the regulation of cell proliferation by GALP. In addition, GALP has anti-apoptotic effects, which could not eliminate the inflammatory damage of cells, thus aggravating cell growth inhibition. The present findings provide new mechanistic insights into the toxicity of DON-induced growth retardation and suggest a therapeutic potential of GALP in DON-related diseases.

Phage display screening of TIGIT-specific antibody for antitumor immunotherapy.

The fully synthetic humanized phage antibody library has the advantages including the minimized immunogenicity, which frequently happened in hybridoma cell-based antibody production. In this paper, using the constructed diverse complementarity determining region gene library and the germline gene as the backbone, we constructed eight single-chain antibody libraries and a combinatorial antibody library with a big capacity of 1.41 × 1010. M13EEA helper phage that was engineered from M13KO7 was applied to prepare phage antibody library. The eukaryotic expression of T-cell immune receptor with Ig and ITIM domain (TIGIT) antigen was used as a target antigen for screening. The screening of antigen-specific single-chain Fc-fused protein was performed through evaluation of binding affinity based on ELISA analysis. The IgG antibody was prepared with the screened single-chain protein. Finally, the CB3 antibody was screened out which exhibits the highest binding affinity with TIGIT with the Kd value of 8.155 × 10-10 M.

OCT imaging detection of brain blood vessels in mouse, based on semiconducting polymer nanoparticles.

Optical Coherence Tomography (OCT) is a valuable technology that has been used to obtain microstructure images of tissue, and has several advantages, though its applications are limited in high-scattering tissues. Therefore, semiconducting polymer nanoparticles (SPNs) that possess strong absorption characteristics are applied to decrease light scattering in tissues and used as exogenous contrast agents for enhancing the contrast of OCT imaging detection. In this paper, we prepared two kinds of SPNs, termed PIDT-TBZ SPNs and PBDT-TBZ SPNs, as the contrast agents for OCT detection to enhance the signal. Firstly, we proved that they were good contrast agents for OCT imaging in agar-TiO2. After that, the contrast effects of these two SPNs were quantitatively analyzed, and then cerebral blood vessels were monitored by a home-made SD-OCT system. Finally, we created OCT images in vitro and in vivo with these two probes and performed quantitative analysis using the images. The results indicated that these SPNs created a clear contrast enhancement of small vessels in the OCT imaging process, which provides a basis for the application of SPNs as contrast agents for bioimaging studies.

Four-photon-excited fluorescence resonance energy transfer in an aqueous system from ZnSe:Mn/ZnS quantum dots to hypocrellin A.

In this work, we established a fluorescence resonance energy transfer (FRET) system between ZnSe:Mn/ZnS quantum dots and Hypocrellin A (HA, a photosensitizer used for photodynamic therapy of cancer) in aqueous solution, excited by four-photon. Here, the QDs are the donors and the HA are the acceptors. The four-photon-excited fluorescence resonance energy transfer spectrum was obtained under 1300nm femtosecond laser pluses. The experimental results indicated that the highest efficiency of FRET can reach up to 61.3%. Furthermore, the viability test in cancer cells was further demonstrated for biological applications of FRET system. When FRET occurs the cell killing rate of the cancer cells will reach to 84.8% with the 1mM concentration of HA. Our work demonstrates that while the four-photon excited FRET system is promising in both optics and biological applications, is also needs further investigation.

Optimization of the aqueous synthesis of Cu2S quantum dots with different surface ligands.

Surface functionalization of quantum dots (QDs) is one of the most important aspects of designing and preparing the desired QDs for intended optical and biomedical applications. In this paper, we synthesized aqueous-phase Cu2S quantum dots coating by three different stabilizers, i.e. mercaptoacetic acid, mercaptopropionic acid and glutathione (GSH). Different stabilizers can influence the coordination modes between Cu(+) on the surface of Cu2S and S(2-) of the ligand. The Cu2S QDs were characterized by UV-vis spectroscopy, energy dispersive spectrometry, transmission electron microscopy, Fourier transform infrared spectroscopy, x-ray diffraction and Raman spectra. Then, we performed a systematic study to evaluate the colloidal stability and in vitro toxicity of the formulations of Cu2S QDs with different stabilizers. Our results show that Cu2S QDs modified with different stabilizers have distinct functional groups on their surface and these groups make Cu2S produce different vibrations according to Raman spectra. The Cu2S-GSH exhibit the best colloidal stability in all pH buffer solutions and the lowest toxicity compare to the other two stabilizers. These properties make the Cu2S-GSH quantum dots a candidate for bioapplications in the future.

Efficient fluorescence energy transfer system between fluorescein isothiocyanate and CdTe quantum dots for the detection of silver ions.

We report a fluorescence resonance energy transfer (FRET) system in which the fluorescent donor is fluorescein isothiocyanate (FITC) dye and the fluorescent acceptor is CdTe quantum dot (QDs). Based on FRET quenching theory, we designed a method to detect the concentration of silver ions (Ag(+)). The results revealed a good linear trend over Ag(+) concentrations in the range 0.01-8.96 nmol/L, a range that was larger than with other methods; the quenching coefficient is 0.442. The FRET mechanism and physical mechanisms responsible for dynamic quenching are also discussed.

Development and Characterization of a Humanized Anti-HER2 Antibody HuA21 with Potent Anti-Tumor Properties in Breast Cancer Cells.

Human epidermal growth factor receptor 2 (HER2) is one of the most studied tumor-associated antigens for cancer immunotherapy. An engineered anti-HER-2 chimeric A21 antibody (chA21) is a chimeric antibody targeted to subdomain I of the HER2 extracellular domain. Here, we report the anti-tumor activity of the novel engineered monoclonal antibody humanized chA21 (HuA21) that targets HER2 on the basis of chA21, and we describe the underlying mechanisms. Our results reveal that HuA21 markedly inhibits the proliferation and migration of HER2-overexpressing breast cancer cells and causes enhanced antibody-dependent cell-mediated cytotoxicity potency against HER2-overexpressing tumor cells. In particular, HuA21, but not trastuzumab (Tra), markedly suppresses growth and enhances the internalization of the antibody in Tra-resistant BT-474 breast cancer cells. These characteristics are highly associated with the intrinsic ability of HuA21 to down-regulate HER2 activation and inhibit the extracellular signal-regulated kinase 1/2 (ERK1/2) and protein kinase B (Akt) signaling pathways. Furthermore, the combination of HuA21 with Tra synergistically enhances the anti-tumor effects in vitro and in vivo and inhibits HER2 activation and the ERK1/2 and Akt signaling pathways. Altogether, our results suggest that HuA21 may represent a unique anti-HER2 antibody with potential as a therapeutic candidate alone or in combination with other anti-HER2 reagents in cancer therapy.

The composition effect on the optical properties of aqueous synthesized Cu-In-S and Zn-Cu-In-S quantum dot nanocrystals.

Multiternary quantum dots (QDs), because of the large degree of freedom in their structure and composition, have a wide tunability in their bandgap but also exhibit an increased uncertainty and complexity in their optical properties. In this work, we synthesized the ternary Cu-In-S (CIS) and quaternary Zn-Cu-In-S (ZCIS) QDs with different composition ratios via a facile aqueous route. The CIS QDs show multi-peak photoluminescence with their peak intensity dependent on the Cu : In ratio, which was illustrated using a donor-acceptor pair recombination process. Upon incorporation of Zn into the CIS QDs under similar conditions, the acquired ZCIS QDs exhibit blue-shifted photoluminescence (PL) spectra with an enhanced emission intensity and a narrowed spectral width (∼100 nm). A comparative study reveals that, reducing the Cu : In ratio in the CIS QDs and increasing the Zn content in the alloyed ZCIS QDs are both feasible strategies for bandgap engineering, although the influences on optical properties of the QDs were different. The XRD and EDX spectra revealed that the widening of the bandgap of the ZCIS QDs was correlated with the alloyed nanostructures and the preferential substitution of Cu by Zn. Compared to the Cu : In ratio variation, incorporation of Zn into CIS QDs is an effective strategy to achieve a more homogeneous absorption band and a wide range of emission wavelength tunability. After ZnS shell coating, the ZCIS/ZnS QDs show a further enhanced PL intensity with a prolonged fluorescence lifetime. Unlike CIS QDs, the blue shift in PL upon the shell growth was not pronounced for ZCIS QDs, for which a surface reconstruction mechanism was proposed and discussed. Finally, the as-prepared ZCIS/ZnS QDs were employed for in vitro cell imaging and exhibited good biocompatibility to macrophage cells.

Quantitative evaluation main of the components in Paeoniae Radix Alba-Atractylodis Macrocephalae Rhizoma herbal pair by high-performance liquid chromatography.

The aim of this study was to investigate the influence of compatibility on the contents of main compounds in Paeoniae Radix Alba and Atractylodis Macrocephalae Rhizoma. Ten compounds were separated on an Inertsil ODS-SP Extend C18 column (250 mm × 4.6 mm, 5 µm) and detected by a diode array detector with the mobile phase consisting of aqueous phosphoric acid (0.1%, v/v; A) and acetonitrile (B) by linear gradient elution. All analytes showed good linearity over a wide concentration range (r(2) ≥ 0.9989). The limits of detection and quantification were <8.10 and 10.80 µg/mL, respectively. The intra- and interday variations were <4.36%. The average recoveries were observed from 94.90 to 103.38%, with relative standard deviation ranging from 1.23 to 3.15% for the analytes. The established method was reliable enough for global quality evaluation of Paeoniae Radix Alba, Atractylodis Macrocephalae Rhizoma, and their co-decoctions.

Preparation of biofunctionalized quantum dots using microfluidic chips for bioimaging.

Biofunctionalized quantum dots (QDs), especially protein-coated QDs, are known to be useful targeted fluorescent labels for cellular and deep-tissue imaging. These nanoparticles can also serve as efficient energy donors in fluorescence resonance energy transfer (FRET) binding assays for the multiplexed sensing of tumor markers. However, current preparation processes for protein-functionalized QDs are laborious and require multiple synthesis steps (e.g. preparing them in high temperature, making them dispersible in water, and functionalizing them with surface ligands) to obtain a high quality and quantity of QD formulations, significantly impeding the progress of employing QDs for clinical diagnostics use such as a QD-based immunohistofluorescence assay. Herein, we demonstrate a one-step synthesis approach for preparing protein-functionalized QDs using a microfluidic (MF) chip setup. Using bovine serum albumin (BSA) molecules as the surface ligand model, we first studied and optimized the MF reaction synthesis parameters (e.g. reaction temperature, and channel width and length) for making protein-functionalized QDs using COMSOL simulation modeling, followed by experimental verification. Moreover, in comparison with the BSA-functionalized QDs synthesized using the conventional bench-top method, BSA-QDs prepared using the MF approach exhibit a significantly higher protein-functionalization efficiency, photostability and colloidal stability. The proposed one-step MF synthesis approach provides a rapid, cost effective, and a small-scale production of nanocrystals platform for developing new QD formulations in applications ranging from cell labeling to biomolecular sensing. Most importantly, this approach will considerably reduce the amount of chemical waste generated during the trial-and-error stage of developing and perfecting the desired physical and optical properties of new QD materials.

Boosted photo-immunotherapy via near-infrared light excited phototherapy in tumor sites and photo-activation in sentinel lymph nodes.

Phototherapy is a promising modality that could eradicate tumor and trigger immune responses via immunogenic cell death (ICD) to enhance anti-tumor immunity. However, due to the lack of deep-tissue-excitable phototherapeutic agents and appropriate excitation strategies, the utility of phototherapy for efficient activation of the immune system is challenging. Herein, we report functionalized ICG nanoparticles (NPs) with the capture capability of tumor-associated antigens (TAAs). Under near-infrared (NIR) light excitation, the ICG NPs exhibited high-performance phototherapy, i.e., synergistic photothermal therapy and photodynamic therapy, thereby efficiently eradicating primary solid tumor and inducing ICD and subsequently releasing TAAs. The ICG NPs also captured TAAs and delivered them to sentinel lymph nodes, and then the sentinel lymph nodes were activated with NIR light to trigger efficient T-cell immune responses through activation of dendritic cells with the assistance of ICG NP generated reactive oxygen species, inhibiting residual primary tumor recurrence and controlling distant tumor growth. The strategy of NIR light excited phototherapy in tumor sites and photo-activation in sentinel lymph nodes provides a powerful platform for active immune systems for anti-tumor photo-immunotherapy.

Open Thermal Control System for Stable Polymerase Chain Reaction on a Digital Microfluidic Chip.

In this paper, a digital microfluidic thermal control system was introduced for the stable polymerase chain reaction (PCR). The system consists of a thermoelectric cooler unit, a thermal control board, and graphical-user-interface software capable of simultaneously achieving temperature control and on-chip droplet observation. A fuzzy proportional-integral-derivative (PID) method was developed for this system. The simulation analysis was performed to evaluate the temperature of different reagents within the chip. Based on the results, applying fuzzy PID control for PCR will enhance the thermal stability by 67.8% and save the time by 1195 s, demonstrating excellent dynamic response capability and thermal robustness. The experimental results are consistent with the simulation results on the planar temperature distribution, with a data consistency rate of over 99%. The PCR validation was carried out on this system, successfully amplifying the rat GAPDH gene at a concentration of 193 copies/µL. This work has the potential to be useful in numerous existing lab-on-a-chip applications.

AIEgens-enhanced rapid sensitive immunofluorescent assay for SARS-CoV-2 with digital microfluidics.

BACKGROUND: Sensitive and rapid antigen detection is critical for the diagnosis and treatment of infectious diseases, but conventional ELISAs including chemiluminescence-based assays are limited in sensitivity and require many operation steps. Fluorescence immunoassays are fast and convenient but often show limited sensitivity and dynamic range. RESULTS: To address the need, an aggregation-induced emission fluorgens (AIEgens) enhanced immunofluorescent assay with beads-based quantification on the digital microfluidic (DMF) platform was developed. Portable DMF devices and chips with small electrodes were fabricated, capable of manipulating droplets within 100 nL and boosting the reaction efficiency. AIEgen nanoparticles (NPs) with high fluorescence and photostability were synthesized to enhance the test sensitivity and detection range. The integration of AIEgen probes, transparent DMF chip design, and the large magnetic beads (10 µm) as capture agents enabled rapid and direct image-taking and signal calculation of the test result. The performance of this platform was demonstrated by point-of-care quantification of SARS-CoV-2 nucleocapsid (N) protein. Within 25 min, a limit of detection of 5.08 pg mL-1 and a limit of quantification of 8.91 pg mL-1 can be achieved using <1 µL sample. The system showed high reproducibility across the wide dynamic range (10-105 pg mL-1), with the coefficient of variance ranging from 2.6% to 9.8%. SIGNIFICANCE: This rapid, sensitive AIEgens-enhanced immunofluorescent assay on the DMF platform showed simplified reaction steps and improved performance, providing insight into the small-volume point-of-care testing of different biomarkers in research and clinical applications.