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88; 95% CI 0.35-1.92; P = .7692) had no relationship with OS. LGR5 expression is associated with tumor necrosis of TNBC and suggested higher malignant potential. Enzyme design and engineering strategies rely almost exclusively on nature's alphabet of twenty canonical amino acids. Recent years have seen the emergence of powerful genetic code expansion methods that allow hundreds of structurally diverse amino acids to be installed into proteins in a site-selective manner. Here, we will highlight how the availability of an expanded alphabet of amino acids has opened new avenues in enzyme engineering research. Genetically encoded noncanonical amino acids have provided new tools to probe complex enzyme mechanisms, improve biocatalyst activity and stability, and most ambitiously to design enzymes with new catalytic mechanisms that would be difficult to access within the constraints of the genetic code. We anticipate that the studies highlighted in this article, coupled with the continuing advancements in genetic code expansion technology, will promote the widespread use of noncanonical amino acids in biocatalysis research in the coming years. BACKGROUND Acyclovir (ACV) is the most commonly used drug for herpes simplex virus (HSV) infection therapy. Prolonged antiviral therapy or prophylaxis in immunocompromised patients may promote the development of drug-resistant strains. Due to the high polymorphism in genes involved in drug resistance, phenotypic methods, although work-intensive, are still required to test drug susceptibility. Real-time cell analysis (RTCA) based methods could offer a rapid and less labor-intensive alternative for phenotypic testing of ACV resistance. OBJECTIVE To investigate the utility of a new RTCA based assay (RTCAA) to test acyclovir susceptibility of HSV clinical isolates. STUDY DESIGN Four reference strains and 93 clinical isolates (60 HSV-1 and 33 HSV-2) were tested by RTCAA. In the presence of ACV concentrations from 2.2 to 140.8 μM, Vero cells were infected with different virus dilutions. IC50 values were calculated by dose-response curve (DRC) with area-under-curve (AUC) method. The reference strains and 22 clinical isolates were additionally tested by dye-uptake assay, and IC50 values of both methods were compared. RESULTS IC50 values from RTCAA and dye-uptake assays were positively correlated (Spearman's rho = 0.897, p  less then  0.001) and quantitatively agreed (Bland-Altman plot). Based on a cut-off of 4 μM for HSV-1 and 13 μM for HSV-2, 87 isolates were classified as ACV-sensitive and 6 isolates as ACV-resistant. The reference strains showed the expected results of ACV susceptibility. CONCLUSION RTCAA agrees well with the dye-uptake assay. Compared with other phenotypic methods, RTCAA requires less manipulation, reduces the workload and the turnaround time, and appears to be an objective and reliable method to test ACV susceptibility. The present study evaluates the physicochemical properties of maple leaf-derived biochars (M-BCs) produced at different pyrolytic temperatures (i.e., 350, 550, and 750 °C) and their adsorptive properties for tetracycline onto M-BCs. The increase in pyrolysis temperature to produce M-BCs led to a significant increase in the biochar's hydrophobicity, surface area, and calcite (CaCO3) crystallization. The M-BC750 produced without functionalization or activation possessed a high calcite composition and a hydrophobic nature with lower O/C and H/C, hydroxyl groups (-OH) on the surface, and functional groups (i.e., O-containing) as H-bond acceptors. Among M-BCs, the M-BC750 present a highest TC adsorption capacity owing to possible mechanisms such as metal complexation, H-bonding, and hydrophobic interactions. The isotherm and kinetic models for TC adsorption followed the Freundlich models and pseudo-second-order models, respectively. M-BCs produced from the waste fallen maple leaves could be applied as low-cost environmental adsorbents for TC removal. The present investigation was design to evaluate the impact of different dosages of biochar on heavy metal resistant bacterial (HMRB) dynamic in biosolid (BS) compost. The bacterial abundance map was reveals that all the samples have 35 prominent genera and showed significant alteration of HMRB among the all biochar applied treatment. The main phyla identify in each treatments were Proteobacteria, Firmicutes and Chloroflexi, however, Pseudomonas, T78, Acinetobacter and Urebacillus were most abundant genera in all the treatment. The visualization of HMRB bio-diversity by bioinformatic tools and krona were clearly indicated a considerable difference in classification tree complexes and network analysis among the all biochar added treatments. Thus, in present study was found that HMRB like Paracoccus, Planomicrobium, Devosia and Agrobacterium viable hyper-thermo tolerant in BS compost. In addition, heat map analysis also confirmed that Proteobacteria, Firmicutes and Chloroflexi have significant correlation with physicochemical parameters. Aiming to improve the economy and sustainability of biodiesel production, the scale-up of lipid production by heterotrophic Thraustochytrium sp. BM2 utilizing crude glycerol as a low cost carbon source was optimized in stirred tank fermenter. The issues of impurities such as excess ions, methanol, soap and other organic impurities as well as different pretreatment techniques were explored and tackled for industrial application of crude glycerol as carbon source. For process engineering strategies to enhance lipid production, semi-batch operation outperformed fed-batch cultivation and achieved higher lipid yield and overall lipid productivity primarily due to shorter fermentation time. The two-step esterification/transesterification method achieved high fatty acid methyl ester (FAME) conversion rate up to 91.8%, which was two to three folds higher compared with the one-step process. Humic acid (HA), considered the main component of organic matter in the ash of waste wheat straw (WWS), has the potential to improve autohydrolysis through its function as a surfactant. In this work, a pre-washed WWS (PWWS) was subjected to autohydrolysis with addition of HA to explore whether its surfactant properties can provide benefit to biorefinery operations. Acquired results showed that HA acted as delignification agent likely due to its surfactant properties. Delignification was more than doubled at the maximum HA dosage (30 g/L) relative to the control, which allowed for enzymatic hydrolysis efficiency to also increase from 64.9% to 81.8%. The pretreated materials were further subjected to analysis structure characterization. The results showed that HA effectively reduced the surface lignin area of PWWS, lowering non-specific adsorption of lignin to enzymes. The autohydrolysis with HA was an effective technique to improve the subsequent cellulose enzymatic digestion by enhancing the delignification. Pulsed electric field (PEF) was conducted for the extraction of proteins/C-Phycocyanins from Arthrospira platensis. The cyanobacterial suspension was treated with 1 µs long pulses at an electric field strength of 40 kV·cm-1 and a treatment energy of 114 kJ·kgsus-1 and 56 kJ·kgsus-1. For benchmarking, additional biomass was processed by high pressure homogenization. Homogeneity of the suspension prior to PEF-treatment influenced the protein/C-phycocyanin extraction efficiency. Stability of C-Phycocyanin during post-PEF incubation time was affected by incubation temperature and pH of the external medium. Biomass concentration severely affect proteins/C-Phycocyanins extraction yield via PEF-treatment. The optimum conditions for extraction of proteins/C-Phycocyanin was obtained at 23 °C while incubating in pH 8-buffer. The energy demand for PEF-treatment amounts to 0.56 MJ·kgdw-1 when processing biomass at 100 gdw·kgsus-1. PEF treatment enhances the protein/C-Phycocyanin extraction yield, thus, it can be suggested as preferential downstream processing method for the production of C-Phycocyanin from A. platensis biomass. This study aimed to evaluate the potential of live and lyophilized fungi-algae pellets as biosorbents for gold recovery and their regeneration ability. The optimum conditions determined by Taguchi method were 1 g/L co-pellets, 9-10 mm size at 250 rpm of agitation speed and pH 3.5 and 2.0 for live and lyophilized co-pellets, respectively. The porous characteristics of fungi-algae pellets played an important role on gold adsorption. Lyophilized co-pellets achieved adsorption capacity of 112.36 mg/g which were comparable with some synthesized granular adsorbents and performed better than the live co-pellets due to more cell-wall polysaccharides involved in gold interaction. 97.77% of gold was selectively absorbed by the lyophilized co-pellets from multi-metal wastewater in column reactor. This study may provide new insights into the application of fungi-algae pelletized reactor in bioremediation of contaminated wastewater by precious metals and their recovery and the in-situ regeneration of biosorbents. Two full-scale high-rate bioreactors, i.e. external circulation sludge bed (ECSB) and expanded granular sludge bed (EGSB), were monitored for three years. Their performances for treating wastewater in a whiskey distillery were compared in terms of COD, pH, alkalinity and VFA. Even though feed flowrate highly fluctuated, COD removals of ECSB and EGSB were both excellent (95.7 ± 1.3% and 94.8 ± 3.0%, respectively). The influent and effluent characteristics of ECSB reactor were profiled and urea and urethane were also detected. High-strength properties of raw spent wash were exhibited in TOC, soluble COD and BOD5,20°C of 13500, 37750, and 1950 mg·L-1, respectively and characterized by GC-MS. Anaerobic granular sludge sampled from different heights of ECSB reactor were fractionated for demonstrating vertical size distributions. Moreover, major species found by next-generation sequencing technique were archaea, i.e. Methanosaeta and Methanolinea, while major bacteria were Bacteroidetes with minor Nitrospiraceae. This metagenomic analysis provided an insight of anaerobic microbial consortium. INTRODUCTION/OBJECTIVES Balloon instability is commonly encountered during balloon pulmonary valvuloplasty (BPV) and may result in an unsuccessful procedure. The NuCLEUS-X™ catheter is a recently developed BPV catheter with a unique barbell shape and an ordered pattern of inflation that stabilizes the balloon to span the valve annulus before expansion of the balloon center. ANIMALS Ten client-owned dogs with severe valvular pulmonic stenosis (PS). MATERIALS AND METHODS Prospective observational study. The BPV procedure was performed by standard technique with use of NuCLEUS-X™ catheters targeting a balloon-to-annulus ratio between 1.2 and 1.5. Balloon stability, safety, and procedural success were assessed. Procedural success was defined as either a reduction in the Doppler transpulmonic PG by at least 50% of the pre-procedural PG or less then 80 mmHg one month post procedure. RESULTS Balloon stability centered at the pulmonic valve on the first inflation was achieved in 10/10 cases. Heptadecanoic acid activator The mean PG before BPV was 141 mmHg ±41 mmHg, and the PG after BPV at one month was 83 mmHg ±41 mmHg.