Rationale: Fc engineering has become the focus of antibody drug development. The current mutagenesis and in silico protein design methods are confined by the limited throughput and high cost, while the high-throughput phage display and yeast display technologies are not suitable for screening glycosylated Fc variants. Here we developed a mammalian cell display-based Fc engineering platform. Methods: By using mammalian cell display and next generation sequencing, we screened millions of Fc variants for optimized affinity and specificity for FcγRIIIa or FcγRIIb. The identified Fc variants with improved binding to FcγRIIIa were substituted into trastuzumab and rituximab and the effector function of antibodies were examined in the PBMC-based assay. On the other hand, the identified Fc variants with selectively enhanced FcγRIIb binding were applied to CD40 agonist antibody and the activities of the antibodies were measured on different cell assays. The immunostimulatory activity of CD40 antibodies was also evaluated by OVA-specific CD8+ T cell response model in FcγR/CD40-humanized mice. Results: Using this approach, we screened millions of Fc variant and successfully identified several novel Fc variants with enhanced FcγRIIIa or FcγRIIb binding. These identified Fc variants displayed a dramatic increase in antibody-dependent cellular cytotoxicity in PBMC-based assay. Novel variants with selectively enhanced FcγRIIb binding were also identified. CD40 agonist antibodies substituted with these Fc variants displayed activity more potent than the parental antibody in the in vitro and in vivo models.Conclusions: This approach increased the throughput of Fc variant screening from thousands to millions magnitude, enabled screening variants containing multiple mutations and could be integrated with glycoengineering technology, represents an ideal platform for Fc engineering. The initial efforts demonstrated the capability of the platform and the novel Fc variants could be substituted into nearly any antibody for the next generation of antibody therapeutics.

The acetohydroxyacid synthase (AHAS) is an essential enzyme involved in branched amino acids. Several herbicides wither weeds via inhibiting AHAS activity, and the AHAS mutants show tolerance to these herbicides. However, most AHAS mutations are residue substitutions but not residue deletion. Here, residue deletion was used to engineering the AHAS gene and herbicide-tolerant rice. Molecular docking analysis predicted that the W548 of the AHAS was a residue deletion to generate herbicide tolerance. The AHAS-ΔW548 protein was generated in vitro to remove the W548 residue. Interestingly, the deletion led to the tetramer dissociation of the AHAS, while this dissociation did not reduce the activity of the AHAS. Moreover, the W548 deletion contributed to multi-family herbicides tolerance. Specially, it conferred more tolerance to sulfometuron-methyl and bispyribac-sodium than the W548L substitution. Further analysis revealed that AHAS-ΔW548 had the best performance on the sulfometuron-methyl tolerance compared to the wild-type control. Over-expression of the AHAS-ΔW548 gene into rice led to the tolerance of multiple herbicides in the transgenic line. The T-DNA insertion and the herbicide treatment did not affect the agronomic traits and yields, while more branched-chain amino acids were detected in transgenic rice seeds. Residue deletion of W548 in the AHAS could be a useful strategy for engineering herbicide tolerant rice. The increase of branched-chain amino acids might improve the umami tastes of the rice.

The healthy human heart has special directional arrangement of cardiomyocytes and a unique electrical conduction system, which is critical for the maintenance of effective contractions. The precise arrangement of cardiomyocytes (CMs) along with conduction consistency between CMs is essential for enhancing the physiological accuracy of in vitro cardiac model systems. Here, we prepared aligned electrospun rGO/PLCL membranes using electrospinning technology to mimic the natural heart structure. The physical, chemical and biocompatible properties of the membranes were rigorously tested. We next assembled human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) on electrospun rGO/PLCL membranes in order to construct a myocardial muscle patch. The conduction consistency of cardiomyocytes on the patches were carefully recorded. We found that cells cultivated on the electrospun rGO/PLCL fibers presented with an ordered and arranged structure, excellent mechanical properties, oxidation resistance and effective guidance. The addition of rGO was found to be beneficial for the maturation and synchronous electrical conductivity of hiPSC-CMs within the cardiac patch. This study verified the possibility of using conduction-consistent cardiac patches to enhance drug screening and disease modeling applications. Implementation of such a system could one day lead to in vivo cardiac repair applications.

The filamentous fungus Trichoderma reesei is a major workhorse employed to produce cellulase, which hydrolyzes lignocellulosic biomass for the production of cellulosic ethanol and bio-based products. However, the economic efficiency of biorefineries is still low.

Genetically engineered mesenchymal stem cells (MSCs), as non-viral gene delivery platforms, are rapidly evolving in tumor therapy due to their low immunogenicity and natural tumor-homing capacity. Methods: In this paper, we selected reconstituted high-density lipoprotein (rHDL), a lipoprotein-bioinspired nanovector with specific binding ability to scavenger receptor B type I (SR-BI) expressed on MSCs, as a transfection agent to genetically modify MSCs. pDNA encoding tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) was used as a functional gene to be transfected into the nucleus of MSCs for TRAIL expression. Lauric acid-coupled polyethyleneimine (PEI-LA) as an amphiphilic cationic polymer was synthesized to electrostatically bind to pDNA, and then incorporated into rHDL to form rHDL/PEI-LA/pDNA nanoparticles. Results: The nanoparticles exhibited homogenous particle size and excellent serum stability in vitro. Meanwhile, this SR-BI-targeted rHDL performed efficient intracellular gene delivery, specific lysosome-independent mechanism of cellular uptake and high transfection of pDNA towards MSCs. Moreover, high TRAIL expression in MSCs was detected after rHDL-mediated transfection. In vitro and in vivo results indicated that genetically engineered MSCs could accurately target to B16F10 cells, thereby producing significant apoptosis-inducing effect on aggressive melanoma. Conclusion: TRAIL-expressing MSCs engineered by rHDL nanovector was an efficient and hypotoxic method for stem cells-based pulmonary melanoma metastasis-targeting therapy.

The chemical compositions in atmospheric aerosols, which often evolve with environmental factors, have significant impact on climate and human health, while our fundamental understanding of chemical process is limited owing to their sensitive to atmospheric conditions. pH and RH are critical chemical factors of aerosols, impacting reaction pathways and kinetics that ultimately govern final components in particles. Herein, we monitored the chemical composition in internally mixed malonic acid/calcium nitrate with the mole ratio of 1:1 as a function of pH and relative humidity (RH). At 30% RH, lower than efflorescence relative humidity (ERH) of pure malonic acid aerosols, malonic acid still exhibits solution feature reflected by IR spectra, which was observed to transform to malonate, along with water loss and nitrate depletion. At another RH of 54% and 80%, the similar chemical process happened with less reaction rate. The response of chemical reaction between malonic acid and calcium nitrate to pH was studied by manipulating the starting pH of the bulk solution through dropping aqueous sodium hydroxide. Due to lower H+ concentration at higher pH, the formation and liberation of HNO3 slow down, as well as water loss. After a down-up RH cycle, the water loss was obvious and grew with the decrease in pH. These measurements are improving our understanding of chemical composition evolution dependent upon pH and RH from a fundamental physical chemistry perspective and are critical for connecting chemistry and climate.

Paper mulberry (Broussonetia papyrifera, PM) is high protein but unutilized as a feed source. The study explores the different parts (leaf, stem, and whole plant) of PM chemical composition, silage fermentation, and in vitro and in situ digestibility, aiming to give some guidelines to PM usage as feed. The result showed that the leaf had a higher fresh weight than the stem (p < 0.05). The dry matter contents of the three groups had no differences. The highest crude protein, ether extract, water-soluble carbohydrate, ash, calcium, phosphorus, amino acid contents, and butter capacity were observed in the leaf (p < 0.05). The stem had the highest (p < 0.05) neutral detergent fiber, acid detergent fiber, and lignin contents. After ensiling, the stem silage had the lowest pH value, ammonia nitrate (NH3-N), lactate, acetate, and propionate (p < 0.05). The leaf silage had the highest pH value (p < 0.05). The lactate, acetate, and propionate in the leaf and whole plant silage had no difference. The butyrate was not detected in all silage. The in vitro and in situ digestibility experiments showed the leaf had the highest digestibility (p < 0.05), which could produce more volatile fatty acids and have a higher effective digestibility. These results allow a greater understanding of PM to be used as a feedstuff.

Aurantii fructus (AF) is a traditional Chinese medicine that has been used to improve gastrointestinal motility disorders for over a thousand years, but there is no exhaustive identification of the basic chemical components and comprehensive quality control of this herb. In this study, high-performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (HPLC-Q-TOF-MS) and gas chromatography coupled mass spectrometry (GC-MS) were employed to identify the basic chemical compounds, and high-performance liquid chromatography (HPLC) was developed to determine the major biochemical markers from AF extract. There were 104 compounds belonging to eight structure types, including 13 amino acids or peptides, seven alkaloids, 18 flavanones, 14 flavones, 15 polymethoxyflavonoids, six triterpenoids, nine coumarins, and 18 volatile oils, as well as four other compounds that were systematically identified as the basic components from AF, and among them, 41 compounds were reported for the first time. Twelve bioactive ingredients were chosen as the benchmark markers to evaluate the quality of AF. The analysis was completed with a gradient elution at a flow rate of 0.7 mL/min within 55 min. This efficient method was validated showing good linearity, precision, stability, repeatability and recovery. Furthermore, the method was successfully applied to the simultaneous determination of 12 chemical markers in different samples of AF. This study could be applied to the identification of multiple bioactive substances and improve the quality control of AF.

Compared with steviol glycosides, the taste of glucosylated steviol glycosides is better and more similar to that of sucrose. At present, cyclodextrin glucanotransferase (CGTase) is primarily used to catalyze the conversion of steviol glycosides to glucosylated steviol glycosides, with soluble starch serving as a glycosyl donor. The main disadvantages of enzymatic transglycosylation are the limited number of enzymes available, the low conversion rates that result in low yields, and the lack of selectivity in the degree of glycosylation of the products. In order to fill these gaps, the proteome of Alkalihalobacillus oshimensis (also named Bacillus oshimensis) was used for mining novel CGTases.

Yarrowia lipolytica, known to accumulate lipids intracellularly, lacks the cellulolytic enzymes needed to break down solid biomass directly. This study aimed to evaluate the potential metabolic burden of expressing core cellulolytic enzymes in an engineered high lipid-accumulating strain of Y. lipolytica. Three fungal cellulases, Talaromyces emersonii-Trichoderma reesei chimeric cellobiohydrolase I (chimeric-CBH I), T. reesei cellobiohydrolase II (CBH II), and T. reesei endoglucanase II (EG II) were expressed using three constitutive strong promoters as a single integrative expression block in a recently engineered lipid hyper-accumulating strain of Y. lipolytica (HA1). In yeast extract-peptone-dextrose (YPD) medium, the resulting cellulase co-expressing transformant YL165-1 had the chimeric-CBH I, CBH II, and EG II secretion titers being 26, 17, and 132 mg L-1, respectively. Cellulase co-expression in YL165-1 in culture media with a moderate C/N ratio of ∼4.5 unexpectedly resulted in a nearly two-fold reduction in cellular lipid accumulation compared to the parental control strain, a sign of cellular metabolic drain. Such metabolic drain was ameliorated when grown in media with a high C/N ratio of 59 having a higher glucose utilization rate that led to approximately twofold more cell mass and threefold more lipid production per liter culture compared to parental control strain, suggesting cross-talk between cellulase and lipid production, both of which involve the endoplasmic reticulum (ER). Most importantly, we found that the chemical chaperone, trimethylamine N-oxide dihydride increased glucose utilization, cell mass and total lipid titer in the transformants, suggesting further amelioration of the metabolic drain. This is the first study examining lipid production in cellulase-expressing Y. lipolytica strains under various C/N ratio media and with a chemical chaperone highlighting the metabolic complexity for developing robust, cellulolytic and lipogenic yeast strains.

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading threat to public health as it is resistant to most currently available antibiotics. Prodigiosin is a secondary metabolite of microorganisms with broad-spectrum antibacterial activity. This study identified a significant antibacterial effect of prodigiosin against MRSA with a minimum inhibitory concentration as low as 2.5 mg/L. The results of scanning electron microscopy, crystal violet staining, and confocal laser scanning microscopy indicated that prodigiosin inhibited biofilm formation in S. aureus USA300, while also destroying the structure of the cell wall and cell membrane, which was confirmed by transmission electron microscopy. At a prodigiosin concentration of 1.25 mg/L, biofilm formation was inhibited by 76.24%, while 2.5 mg/L prodigiosin significantly reduced the vitality of MRSA cells in the biofilm. Furthermore, the transcriptomic results obtained at 1/8 MIC of prodigiosin indicated that 235and 387 genes of S. aureus USA300 were significantly up- and downregulated, respectively. The downregulated genes were related to two-component systems, including the transcriptional regulator LytS, quorum sensing histidine kinases SrrB, NreA and NreB, peptidoglycan biosynthesis enzymes (MurQ and GlmU), iron-sulfur cluster repair protein ScdA, microbial surface components recognizing adaptive matrix molecules, as well as the key arginine synthesis enzymes ArcC and ArgF. The upregulated genes were mainly related to cell wall biosynthesis, as well as two-component systems including vancomycin resistance-associated regulator, lipoteichoic acid biosynthesis related proteins DltD and DltB, as well as the 9 capsular polysaccharide biosynthesis proteins. This study elucidated the molecular mechanisms through which prodigiosin affects the cell envelope of MRSA from the perspectives of cell wall synthesis, cell membrane and biofilm formation, providing new potential targets for the development of antimicrobials for the treatment of MRSA.

Introducing functionalities into the interior of metal-organic cage complexes can confer properties and utilities (e.g. catalysis, separation, drug delivery, and guest recognition) that are distinct from those of unfunctionalized cages. Endohedral functionalization of such cage molecules, for decades, has largely relied on modifying their organic linkers to covalently append targeted functional groups to the interior surface. We herein introduce an effective coordination method to bring in functionalities at the metal sites instead, for a set of polyhedral cages where the nodes are in situ formed polyoxovanadate clusters, [VIV 6O6(OCH3)9(μ6-SO4)(COO)3]2-. Replacing the central sulfates of these hexavanadate clusters with more strongly coordinating phosphonate groups allows the installation of functionalities within the cage cavities. Organophosphonates with phenyl, biphenyl, and terphenyl tails were examined for internalization. Depending on the size/shape of the cavities, small phosphonates can fit into the molecular containers whereas larger ones inhibit or transform the framework architecture, whereby the first non-cage complex was isolated from a reaction that otherwise would lead to entropically favored regular polyhedra cages. The results highlight the complex and dynamic nature of the self-assembly process involving polyoxometalates and the scope of molecular variety accessible by the introduction of endo functional groups.

Branched-chain amino acid (BCAA) metabolism fulfills numerous physiological roles and can be harnessed to produce valuable chemicals. However, the lack of eukaryotic biosensors specific for BCAA-derived products has limited the ability to develop high-throughput screens for strain engineering and metabolic studies. Here, we harness the transcriptional regulator Leu3p from Saccharomyces cerevisiae to develop a genetically encoded biosensor for BCAA metabolism. In one configuration, we use the biosensor to monitor yeast production of isobutanol, an alcohol derived from valine degradation. Small modifications allow us to redeploy Leu3p in another biosensor configuration that monitors production of the leucine-derived alcohol, isopentanol. These biosensor configurations are effective at isolating high-producing strains and identifying enzymes with enhanced activity from screens for branched-chain higher alcohol (BCHA) biosynthesis in mitochondria as well as cytosol. Furthermore, this biosensor has the potential to assist in metabolic studies involving BCAA pathways, and offers a blueprint to develop biosensors for other products derived from BCAA metabolism.

A new low-cost composite of ZnCo x Fe2-x O4 loaded on rice hull carbon (ZnCo x Fe2-x O4-RHC) was synthesized via waste ferrous sulfate (the industrial waste produced in the process of producing titanium dioxide) and rice hull as raw materials, which was applied for the degradation of bisphenol A (BPA) by heterogeneous activated peroxodisulfate (PS). A series of characterizations including XRD, SEM, FTIR, and BET analysis were carried out to analyze the structure and morphology of the materials. It is confirmed that the ZnCo x Fe2-x O4-RHC composites show better catalytic activity and performance than other control samples, which can be attributed to the synergistic effect of Fe and Co, ZnCo x Fe2-x O4 and RHC based on these analyses. The degradation rate of BPA by ZnCo1.3Fe0.7O4-50%RHC reached 100% within 15 min, and it can still maintain good catalytic efficiency after 5 cycles. ESR test and XPS results showed that free radical and non-free radical processes were involved in BPA degradation. These findings offer a novel, low cost and simple strategy for rational design and modulation of catalysts for the industrial degradation of organic pollutants, and provide a new idea for the utilization of waste ferrous sulphate in titanium dioxide industry.

Glycine cleavage system (GCS) plays a central role in one-carbon (C1) metabolism and receives increasing interest as a core part of the recently proposed reductive glycine pathway (rGlyP) for assimilation of CO2 and formate. Despite decades of research, GCS has not yet been well understood and kinetic data are barely available. This is to a large degree because of the complexity of GCS, which is composed of four proteins (H, T, P, and L) and catalyzes reactions involving different substrates and cofactors. In vitro kinetics of reconstructed microbial multi-enzyme glycine cleavage/synthase system is desired to better implement rGlyP in microorganisms like Escherichia coli for the use of C1 resources. Here, we examined in vitro several factors that may affect the rate of glycine synthesis via the reverse GCS reaction. We found that the ratio of GCS component proteins has a direct influence on the rate of glycine synthesis, namely higher ratios of P protein and especially H protein to T and L proteins are favorable, and the carboxylation reaction catalyzed by P protein is a key step determining the glycine synthesis rate, whereas increasing the ratio of L protein to other GCS proteins does not have significant effect and the ratio of T protein to other GCS proteins should be kept low. The effect of substrate concentrations on glycine synthesis is quite complex, showing interdependence with the ratios of GCS component proteins. Furthermore, adding the reducing agent dithiothreitol to the reaction mixture not only results in great tolerance to high concentration of formaldehyde, but also increases the rate of glycine synthesis, probably due to its functions in activating P protein and taking up the role of L protein in the non-enzymatic reduction of Hox to Hred. Moreover, the presence of some monovalent and divalent metal ions can have either positive or negative effect on the rate of glycine synthesis, depending on their type and their concentration.

Histones and DNA associate to form the nucleosomes of eukaryotic chromatin. Chromatin assembly factor 1 (CAF-1) complex and histone regulatory protein A (HIRA) complex mediate replication-couple (RC) and replication-independent (RI) nucleosome assembly, respectively. CHAF1B and HIRA share a similar domain but play different roles in nucleosome assembly by binding to the different interactors. At present, there is limited understanding for the similarities and differences in their respective functions. Tetrahymena thermophila contains transcriptionally active polyploid macronuclei (MAC) and transcriptionally silent diploid micronuclei (MIC). Here, the distribution patterns of Caf1b and Hir1 exhibited both similarities and distinctions. Both proteins localized to the MAC and MIC during growth, and to the MIC during conjugation. However, Hir1 exhibited additional signaling on parental MAC and new MAC during sexual reproduction and displayed a punctate signal on developing anlagen. Caf1b and Hir1 only co-localized in the MIC with Pcna1 during conjugation. Knockdown of CAF1B impeded cellular growth and arrested sexual reproductive development. Loss of HIR1 led to MIC chromosome defects and aborted sexual development. Co-interference of CAF1B and HIR1 led to a more severe phenotype. Moreover, CAF1B knockdown led to the up-regulation of HIR1 expression, while knockdown of HIR1 also led to an increase in CAF1B expression. Furthermore, Caf1b and Hir1 interacted with different interactors. These results showed that CAF-1 and Hir1 have independent and complementary functions for chromatin assembly in T. thermophila.

Komagataeibacter xylinus ATCC 23770 was statically cultivated in eight culture media based on different carbon sources, viz. seven biomass-derived sugars and one sugar mixture. The productivity and quality of the bacterial nanocellulose (BNC) produced in the different media were compared. Highest volumetric productivity, yield on consumed sugar, viscometric degree of polymerization (DPv , 4350-4400) and thermal stability were achieved using media based on glucose or maltose. Growth in media based on xylose, mannose or galactose resulted in lower volumetric productivity and DPv , but in larger fibril diameter and higher crystallinity (76-78%). Growth in medium based on a synthetic sugar mixture resembling the composition of a lignocellulosic hydrolysate promoted BNC productivity and yield, but decreased fibril diameter, DPv , crystallinity and thermal stability. This work shows that volumetric productivity, yield and properties of BNC are highly affected by the carbon source, and indicates how industrially relevant sugar mixtures would affect these characteristics.

Nuclear autophagy is an important selective autophagy process. The selective autophagy of sexual development micronuclei (MICs) and the programmed nuclear degradation of parental macronucleus (paMAC) occur during sexual reproduction in Tetrahymena thermophila. The molecular regulatory mechanism of nuclear selective autophagy is unclear. In this study, the autophagy-related protein Atg5 was identified from T. thermophila. Atg5 was localized in the cytoplasm in the early sexual-development stage and was localized in the paMAC in the late sexual-development stage. During this stage, the degradation of meiotic products of MIC was delayed in atg5i mutants. Furthermore, paMAC was abnormally enlarged and delayed or failed to degrade. The expression level and lipidation of Atg8.2 significantly decreased in the mutants. All these results indicated that Atg5 was involved in the regulation of the selective autophagy of paMAC by regulating Atg8.2 in Tetrahymena.

Integrative analyses of transcriptomic and neuroimaging data have generated a wealth of information about biological pathways underlying regional variability in imaging-derived brain phenotypes in humans, but rarely in nonhuman primates due to the lack of a comprehensive anatomically-defined atlas of brain transcriptomics. Here we generate complementary bulk RNA-sequencing dataset of 819 samples from 110 brain regions and single-nucleus RNA-sequencing dataset, and neuroimaging data from 162 cynomolgus macaques, to examine the link between brain-wide gene expression and regional variation in morphometry. We not only observe global/regional expression profiles of macaque brain comparable to human but unravel a dorsolateral-ventromedial gradient of gene assemblies within the primate frontal lobe. Furthermore, we identify a set of 971 protein-coding and 34 non-coding genes consistently associated with cortical thickness, specially enriched for neurons and oligodendrocytes. These data provide a unique resource to investigate nonhuman primate models of human diseases and probe cross-species evolutionary mechanisms.

Histone modification and nucleosome assembly are mainly regulated by various histone-modifying enzymes and chaperones. The roles of histone-modification enzymes have been well analyzed, but the molecular mechanism of histone chaperones in histone modification and nucleosome assembly is incompletely understood. We previously found that the histone chaperone Nrp1 is localized in the micronucleus (MIC) and the macronucleus (MAC) and involved in the chromatin stability and nuclear division of Tetrahymena thermophila. In the present work, we found that truncated C-terminal mutant HA-Nrp1TrC abnormally localizes in the cytoplasm. The truncated-signal-peptide mutants HA-Nrp1TrNLS1 and HA-Nrp1TrNLS2 are localized in the MIC and MAC. Overexpression of Nrp1TrNLS1 inhibited cellular proliferation and disrupted micronuclear mitosis during the vegetative growth stage. During sexual development, Nrp1TrNLS1 overexpression led to abnormal bouquet structures and meiosis arrest. Furthermore, Histone H3 was not transported into the nucleus; instead, it formed an abnormal speckled cytoplastic distribution in the Nrp1TrNLS1 mutants. The acetylation level of H3K56 in the mutants also decreased, leading to significant changes in the transcription of the genome of the Nrp1TrNLS1 mutants. The histone chaperone Nrp1 regulates the H3 nuclear import and acetylation modification of H3K56 and affects chromatin stability and genome transcription in Tetrahymena.