GigaAssay - An adaptable high-throughput saturation mutagenesis assay platform.

High-throughput assay systems have had a large impact on understanding the mechanisms of basic cell functions. However, high-throughput assays that directly assess molecular functions are limited. Herein, we describe the "GigaAssay", a modular high-throughput one-pot assay system for measuring molecular functions of thousands of genetic variants at once. In this system, each cell was infected with one virus from a library encoding thousands of Tat mutant proteins, with each viral particle encoding a random unique molecular identifier (UMI). We demonstrate proof of concept by measuring transcription of a GFP reporter in an engineered reporter cell line driven by binding of the HIV Tat transcription factor to the HIV long terminal repeat. Infected cells were flow-sorted into 3 bins based on their GFP fluorescence readout. The transcriptional activity of each Tat mutant was calculated from the ratio of signals from each bin. The use of UMIs in the GigaAssay produced a high average accuracy (95%) and positive predictive value (98%) determined by comparison to literature benchmark data, known C-terminal truncations, and blinded independent mutant tests. Including the substitution tolerance with structure/function analysis shows restricted substitution types spatially concentrated in the Cys-rich region. Tat has abundant intragenic epistasis (10%) when single and double mutants are compared.

Functionalized graphene nanosheets as absorbent for copper (II) removal from water.

Functionalized graphene nanosheets (FGNs) with high surface area and various functional groups were prepared by oxidation method. The characteristics of FGNs were studied by nitrogen adsorption using the Brunauer-Emmett-Teller (BET) method, Fourier transform infrared spectroscopy (FTIR), transmission electron microscope (TEM), scanning electron microscope (SEM), energy dispersion spectrum (EDS), and atomic force microscopy (AFM). The specific surface area of obtained FGNs was measured as 834.06 m2 g-1, which was 20-40% higher than graphene nanosheets (GNs) before oxidation. An abundance of oxygen-containing functional groups, such as carboxyl, hydroxyl and epoxy groups, was grafted on the edge and surface of GNs. Moreover, FGNs demonstrated excellent adsorption and desorption performance when used as absorbent to remove Cu (II) from aqueous solution. The removal percentage could reach 96% within 1 h and remain 72% after 5 adsorption-desorption cycles. Adsorption process and mechanism were elucidated by kinetics models and isotherm models. The results showed FGNs has a great potential to be an adsorbent for removal copper ions from water.

Prognostic roles for IL-2-producing and CD69+ T cell subsets in colorectal cancer patients.

Tumor infiltrating T cells are a predictor of patient outcome in patients with colorectal cancer (CRC). However, many T cell populations have been associated with both poor and positive patient prognoses, indicating a need to further understand the role of different T cell subsets in CRC. In this study, the T cell infiltrate from the tumor and nontumor bowel (NTB) was examined in 95 CRC patients using flow cytometry and associations with cancer stage and disease recurrence made. Our findings showed that IFN-γ-producing T cells were associated with positive patient outcomes, and CD69+ T cells were associated with disease recurrence. Inflammatory (IL-17) and regulatory T cells were not associated with disease recurrence. Surprisingly, in a second cohort of 32 patients with long-term clinical follow up data, tumor infiltrating IL-2-producing T cells correlated negatively with disease free survival (DFS) and a higher frequency of IL-2-producing T cells was found in the NTB of patients with poorly differentiated tumors. These results point toward the possibility of a negative impact of IL-2 in tumor immune responses, which may influence future immunotherapy treatments in CRC patients.

Flexible Thermoelectric Reduced Graphene Oxide/Ag2S/Methyl Cellulose Composite Film Prepared by Screen Printing Process.

As an organic-inorganic thermoelectric composite material, a flexible, reduced graphene oxide (rGO)/silver sulfide (Ag2S)/methyl cellulose (MC) film was fabricated by a two-step method. Firstly, a rGO/Ag2S composite powder was prepared by a chemical synthesis method, and then, the rGO/Ag2S/MC composite film was prepared by a combined screen printing and annealing treatment process. The rGO and rGO/Ag2S composite powders were evenly dispersed in the rGO/Ag2S/MC composite films. A power factor of 115 µW m-1 K-2 at 520 K was acquired for the rGO/Ag2S/MC composite film, which is ~958 times higher than the power factor at 360 K (0.12 µW m-1 K-2), mainly due to the significant increase in the electrical conductivity of the composite film from 0.006 S/cm to 210.18 S/cm as the test temperature raised from 360 K to 520 K. The as-prepared rGO/Ag2S/MC composite film has a good flexibility, which shows a huge potential for the application of flexible, wearable electronics.

Hazard profiling of a combinatorial library of zinc oxide nanoparticles: Ameliorating light and dark toxicity through surface passivation.

Zinc oxide (ZnO) is one of the high-volume production nanoparticles (NPs) currently used in a wide range of consumer and industrial goods. The inevitable seepage into environmental matrices and the photoactive nature of ZnO NPs warrants hazard profiling under environmentally related conditions. In this paper, the influence of simulated solar light (SSL) on dissolution behaviour and phototoxicity of ZnO NPs was studied using a combinatorial library of ZnO NPs with different sizes, surface coatings, dopant chemistry, and aspect ratios in a fish cell line (BF2) and zebrafish embryos. Generally, the cytotoxicity and embryo mortality increased when exposed concomitantly to SSL and ZnO NPs. The increase in toxic potential of ZnO NPs during SSL exposure concurred with release of Zn ions and ROS generation. Surface modification of NPs with poly(methacrylic acid) (PMAA), silica or serum coating decreased toxicity and ZnO with serum coating was the only NP that had no significant effect on any of the cytotoxicity parameters when tested under both dark and SSL conditions. Results from our study show that exposure to light could increase the toxic potential of ZnO NPs to environmental lifeforms and mitigation of ZnO NP toxicity is possible through modifying the surface chemistry.

Urban monitoring of antimicrobial resistance during a COVID-19 surge through wastewater surveillance.

During the early phase of the COVID-19 pandemic, infected patients presented with symptoms similar to bacterial pneumonias and were treated with antibiotics before confirmation of a bacterial or fungal co-infection. We reasoned that wastewater surveillance could reveal potential relationships between reduced antimicrobial stewardship, specifically misprescribing antibiotics to treat viral infections, and the occurrence of antimicrobial resistance (AMR) in an urban community. Here, we analyzed microbial communities and AMR profiles in sewage samples from a wastewater treatment plant (WWTP) and a community shelter in Las Vegas, Nevada during a COVID-19 surge in December 2020. Using a respiratory pathogen and AMR enrichment next-generation sequencing panel, we identified four major phyla in the wastewater, including Actinobacteria, Firmicutes, Bacteroidetes and Proteobacteria. Consistent with antibiotics that were reportedly used to treat COVID-19 infections (e.g., fluoroquinolones and beta-lactams), we also measured a significant spike in corresponding AMR genes in the wastewater samples. AMR genes associated with colistin resistance (mcr genes) were also identified exclusively at the WWTP, suggesting that multidrug resistant bacterial infections were being treated during this time. We next compared the Las Vegas sewage data to local 2018-2019 antibiograms, which are antimicrobial susceptibility profile reports about common clinical pathogens. Similar to the discovery of higher levels of beta-lactamase resistance genes in sewage during 2020, beta-lactam antibiotics accounted for 51 ± 3 % of reported antibiotics used in antimicrobial susceptibility tests of 2018-2019 clinical isolates. Our data highlight how wastewater-based epidemiology (WBE) can be leveraged to complement more traditional surveillance efforts by providing community-level data to help identify current and emerging AMR threats.

Data supporting a saturation mutagenesis assay for Tat-driven transcription with the GigaAssay.

The data in this article are associated with the research paper "GigaAssay - an adaptable high-throughput saturation mutagenesis assay" [1]. The raw data are sequence reads of HIV-1 Tat cDNA amplified from cellular genomic DNA in a new single-pot saturation mutagenesis assay designated the "GigaAssay". A bioinformatic pipeline and parameters used to analyze the data. Raw, processed, analyzed, and filtered data are reported. The data is processed to calculate the Tat-driven transcription activity for cells with each possible single amino acid substitution in Tat. This data can be reused to interpret Tat intermolecular interactions and HIV latency. This is one of the largest and most complete datasets regarding the impact of amino acid substitutions within a single protein on a molecular function.

Use of wastewater surveillance for early detection of Alpha and Epsilon SARS-CoV-2 variants of concern and estimation of overall COVID-19 infection burden.

A decline in diagnostic testing for SARS-CoV-2 is expected to delay the tracking of COVID-19 variants of concern and interest in the United States. We hypothesize that wastewater surveillance programs provide an effective alternative for detecting emerging variants and assessing COVID-19 incidence, particularly when clinical surveillance is limited. Here, we analyzed SARS-CoV-2 RNA in wastewater from eight locations across Southern Nevada between March 2020 and April 2021. Trends in SARS-CoV-2 RNA concentrations (ranging from 4.3 log10 gc/L to 8.7 log10 gc/L) matched trends in confirmed COVID-19 incidence, but wastewater surveillance also highlighted several limitations with the clinical data. Amplicon-based whole genome sequencing (WGS) of 86 wastewater samples identified the B.1.1.7 (Alpha) and B.1.429 (Epsilon) lineages in December 2020, but clinical sequencing failed to identify the variants until January 2021, thereby demonstrating that 'pooled' wastewater samples can sometimes expedite variant detection. Also, by calibrating fecal shedding (11.4 log10 gc/infection) and wastewater surveillance data to reported seroprevalence, we estimate that ~38% of individuals in Southern Nevada had been infected by SARS-CoV-2 as of April 2021, which is significantly higher than the 10% of individuals confirmed through clinical testing. Sewershed-specific ascertainment ratios (i.e., X-fold infection undercounts) ranged from 1.0 to 7.7, potentially due to demographic differences. Our data underscore the growing application of wastewater surveillance in not only the identification and quantification of infectious agents, but also the detection of variants of concern that may be missed when diagnostic testing is limited or unavailable.

How Can We Provide Additively Manufactured Parts with a Fingerprint? A Review of Tagging Strategies in Additive Manufacturing.

Additive manufacturing (AM) is rapidly evolving from "rapid prototyping" to "industrial production". AM enables the fabrication of bespoke components with complicated geometries in the high-performance areas of aerospace, defence and biomedicine. Providing AM parts with a tagging feature that allows them to be identified like a fingerprint can be crucial for logistics, certification and anti-counterfeiting purposes. Whereas the implementation of an overarching strategy for the complete traceability of AM components downstream from designer to end user is, by nature, a cross-disciplinary task that involves legal, digital and technological issues, materials engineers are on the front line of research to understand what kind of tag is preferred for each kind of object and how existing materials and 3D printing hardware should be synergistically modified to create such tag. This review provides a critical analysis of the main requirements and properties of tagging features for authentication and identification of AM parts, of the strategies that have been put in place so far, and of the future challenges that are emerging to make these systems efficient and suitable for digitalisation. It is envisaged that this literature survey will help scientists and developers answer the challenging question: "How can we embed a tagging feature in an AM part?".

Effects of Preparation Methods on the Thermoelectric Performance of SWCNT/Bi2Te3 Bulk Composites.

Single-walled carbon nanotube (SWCNT)/Bi2Te3 composite powders were fabricated via a one-step in situ reductive method, and their corresponding bulk composites were prepared by a cold-pressing combing pressureless sintering process or a hot-pressing process. The influences of the preparation methods on the thermoelectric properties of the SWCNT/Bi2Te3 bulk composites were investigated. All the bulk composites showed negative Seebeck coefficients, indicating n-type conduction. A maximum power factor of 891.6 µWm-1K-2 at 340 K was achieved for the SWCNT/Bi2Te3 bulk composites with 0.5 wt % SWCNTs prepared by a hot-pressing process, which was ~5 times higher than that of the bulk composites (167.7 µWm-1K-2 at 300 K) prepared by a cold-pressing combing pressureless sintering process, and ~23 times higher than that of the bulk composites (38.6 µWm-1K-2 at 300 K) prepared by a cold-pressing process, mainly due to the enhanced density of the hot-pressed bulk composites.

Exposure, assessment and health hazards of particulate matter in metal additive manufacturing: A review.

Metal additive manufacturing (AM), also known as metal three-dimensional (3D) printing, is a new technology offering design freedom to create complex structures that has found increasing applications in industrial processes. However, due to the fine metal powders and high temperatures involved, the printing process is likely to generate particulate matter (PM) that has a detrimental impact on the environment and human health. Therefore, comprehensive assessement of the exposure and health hazards of PM pollution related to this technique is urgently required. This review provides general knowledge of metal AM and its possible particle release. The health issues of metal PM are described considering the exposure routes, adverse human health outcomes and influencing factors. Methods of evaluating PM exposure and risk assessment techniques are also summarized. Lastly, future research needs are suggested. The information and knowledge presented in this review will contribute to the understanding, assessment, and control of possible risks in metal AM and benefit the wider metal 3D printing community, which includes machine operators, consumers, R&D scientists, and policymakers.

Effect of Macro-, Micro- and Nano-Calcium Carbonate on Properties of Cementitious Composites-A Review.

Calcium carbonate is wildly used in cementitious composites at different scales and can affect the properties of cementitious composites through physical effects (such as the filler effect, dilution effect and nucleation effect) and chemical effects. The effects of macro (>1 mm)-, micro (1 µm⁻1 mm)- and nano (<1 µm)-sizes of calcium carbonate on the hydration process, workability, mechanical properties and durability are reviewed. Macro-calcium carbonate mainly acts as an inert filler and can be involved in building the skeletons of hardened cementitious composites to provide part of the strength. Micro-calcium carbonate not only fills the voids between cement grains, but also accelerates the hydration process and affects the workability, mechanical properties and durability through the dilution, nucleation and even chemical effects. Nano-calcium carbonate also has both physical and chemical effects on the properties of cementitious composites, and these effects behave even more effectively than those of micro-calcium carbonate. However, agglomeration of nano-calcium carbonate reduces its enhancement effects remarkably.

How rheological behaviors of concentrated starch affect graft copolymerization of acrylamide and resultant hydrogel.

Corn starches with different amylose/amylopectin ratios were used to explore the effect of rheological behaviors of concentrated system on the graft copolymerization of acrylamide and resultant hydrogels, which sheds a light on their reactive extrusion process. The viscoelastic moduli of starch melts increased with increasing amylose content (AC), leading to a decreased extent of micro-mixing detected by a reduced rheokinetic rate. With increasing AC, the graft efficiency was decreased but with almost similar monomer conversion (about 87.5%) and nearly equivalent graft content. XRD and SAXS spectra revealed that the extent of retrogradation of the starches were increased and two-phase separation was enhanced for hydrogels with increasing AC. Interestingly, microscopic analysis showed the superabsorbent hydrogel from the starch with AC of 50% exhibited a gridding membrane porous structure, resulting in a higher water absorbent capacity of 550 g/g. This was attributed to the moderate crosslinking and the slightly greater graft content.

Morphologies Tuning of Polypyrrole and Thermoelectric Properties of Polypyrrole Nanowire/Graphene Composites.

Polypyrrole (PPy) with different morphologies (e.g., particles, nanotubes, and nanowires) were successfully prepared by adding or without adding different kinds of surfactants through a chemical oxidative polymerization method, respectively. The results show that the morphologies of PPy can be effectively controlled and have a significantly effects on their thermoelectric properties. The PPy nanowires exhibit the highest electrical conductivity and Seebeck coefficient among the various PPy morphologies, such as particles, nanotubes, and nanowires, so PPy nanowires were chosen to prepare PPy nanowire/graphene thermoelectric composites via a soft template polymerization method using cetyltrimethyl ammonium bromide as the template. Both electrical conductivity and Seebeck coefficient of the PPy nanowire/graphene composites increased as the content of graphene increases from 0 to 20 wt %, and as the measured temperature increases from 300 K to 380 K, which leds to the same trend for the power factor. A highest power factor of 1.01 µWm-1K-2 at ~380 K was obtained for the PPy nanowire/graphene composites with 20 wt % PPy nanowire, which is about 3.3 times higher than that of the pure PPy nanowire.

Rheokinetics of graft copolymerization of acrylamide in concentrated starch and rheological behaviors and microstructures of reaction products.

A widely recognized challenge in starch chemistry is to manipulate the graft copolymerization onto starch melt by reactive extrusion (REX). To understand the complex in-situ graft copolymerization in highly concentrated systems, we firstly used a mixer to achieve a homogeneous viscous starch melt, and then undertook dynamic rheological measurements to study the rheokinetics of the reaction. The in-situ synthesis also facilitated the characterization of the microstructures of reaction products. The melt mixture could be regarded to be completely micromixed since the rheokinetics was predominated by the reaction kinetics. The rheological characterization revealed that G'of hydrogels followed a linear progression with the crosslinker concentration. Nevertheless, the reaction temperature and initiator content had little influence on the final microstructure of hydrogels, most likely due to the strong chain transfer reaction in the melt. Additionally, high-amylose starches tended to form grafted hydrogels with a high physical crosslinking density.

Thermoelectric Properties of Flexible PEDOT:PSS/Polypyrrole/Paper Nanocomposite Films.

Flexible poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate)/polypyrrole/paper (PEDOT:PSS/PPy/paper) thermoelectric (TE) nanocomposite films were prepared by a two-step method: first, PPy/paper nanocomposite films were prepared by an in situ chemical polymerization process, and second, PEDOT:PSS/PPy/paper TE composite films were fabricated by coating the as-prepared PPy/paper nanocomposite films using a dimethyl sulfoxide-doped PEDOT:PSS solution. Both the electrical conductivity and the Seebeck coefficient of the PEDOT:PSS/PPy/paper TE nanocomposite films were greatly enhanced from 0.06 S/cm to ~0.365 S/cm, and from 5.44 µV/K to ~16.0 µV/K at ~300 K, respectively, when compared to the PPy/paper TE nanocomposite films. The thermal conductivity of the PEDOT:PSS/PPy/paper composite film (0.1522 Wm-1K-1 at ~300 K) was, however, only slightly higher than that of the PPy/paper composite film (0.1142 Wm-1K-1 at ~300 K). As a result, the ZT value of the PEDOT:PSS/PPy/paper composite film (~1.85 × 10-5 at ~300 K) was significantly enhanced when compared to that of the PPy/paper composite film (~4.73 × 10-7 at ~300 K). The as-prepared nanocomposite films have great potential for application in flexible TE devices.

Research and industrialization progress of recovering alumina from fly ash: A concise review.

Fly ash, a by-product of high temperature combustion of coal in coal-fired power plants, is one of the most complex and largest amount of industrial solid wastes generated in China. Its improper disposal has become an environmental problem. Now it is widely realized that fly ash should be considered as a useful and potential mineral resource. Fly ash is rich in alumina, making it a potential substitute for bauxite. With the diminishing reserves of bauxite resources, as well as the increasing demand for alumina, recovery of alumina from fly ash has attracted extensive attention world-wide. The present review describes, firstly, the generation and physicochemical properties of high alumina fly ash found in northern China and then focuses on the various alumina recovery technologies, the advantages and disadvantages of these processes, and in particular, the latest industrial developments. Finally, the directions for future research are also considered.

Design and construction of polymerized-chitosan coated Fe3O4 magnetic nanoparticles and its application for hydrophobic drug delivery.

In this study, a novel hydrogel, chitosan (CS) crosslinked carboxymethyl-ß-cyclodextrin (CM-ß-CD) polymer modified Fe3O4 magnetic nanoparticles was synthesized for delivering hydrophobic anticancer drug 5-fluorouracil (CS-CDpoly-MNPs). Carboxymethyl-ß-cyclodextrin being grafted on the Fe3O4 nanoparticles (CDpoly-MNPs) contributed to an enhancement of adsorption capacities because of the inclusion abilities of its hydrophobic cavity with insoluble anticancer drugs through host-guest interactions. Experimental results indicated that the amounts of crosslinking agent and bonding times played a crucial role in determining morphology features of the hybrid nanocarriers. The nanocarriers exhibited a high loading efficiency (44.7±1.8%) with a high saturation magnetization of 43.8emu/g. UV-Vis spectroscopy results showed that anticancer drug 5-fluorouracil (5-Fu) could be successfully included into the cavities of the covalently linked CDpoly-MNPs. Moreover, the free carboxymethyl groups could enhance the bonding interactions between the covalently linked CDpoly-MNPs and anticancer drugs. In vitro release studies revealed that the release behaviors of CS-CDpoly-MNPs carriers were pH dependent and demonstrated a swelling and diffusion controlled release. A lower pH value led to swelling effect and electrostatic repulsion contributing to the protonation amine impact of NH3(+), and thus resulted in a higher release rate of 5-Fu. The mechanism of 5-Fu encapsulated into the magnetic chitosan nanoparticles was tentatively proposed.

Thermoelectric fabrics: toward power generating clothing.

Herein, we demonstrate that a flexible, air-permeable, thermoelectric (TE) power generator can be prepared by applying a TE polymer (e.g. poly(3,4-ethylenedioxythiophene):poly(4-styrenesulfonate)) coated commercial fabric and subsequently by linking the coated strips with a conductive connection (e.g. using fine metal wires). The poly(3,4-ethylenedioxythiophene):poly(4-styrenesulfonate) coated fabric shows very stable TE properties from 300 K to 390 K. The fabric device can generate a TE voltage output (V) of 4.3 mV at a temperature difference (ΔT) of 75.2 K. The potential for using fabric TE devices to harvest body temperature energy has been discussed. Fabric-based TE devices may be useful for the development of new power generating clothing and self-powered wearable electronics.