Changes in Metabolite Profiles of Chinese Soy Sauce at Different Time Durations of Fermentation Studied by 1H-NMR-Based Metabolomics.

Fermentation parameters, especially the duration, are important in imparting a peculiar taste and flavor to soy sauce. The main purpose of this research was to monitor metabolic changes occurring during the various time intervals of the fermentation process. NMR-based metabolomics was used to monitor the compositional changes in soy sauce during fermentation. The 1H-NMR spectra of the soy sauce samples taken from the fermentation tanks at 0 to 8 months were analyzed using 1H-NMR spectroscopy, and the obtained spectra were analyzed by multivariate statistical analysis. The Principal Component Analysis (PCA) and Partial Least Square Discriminate analysis (PLSDA) revealed the separation of samples fermented for various time durations under identical conditions. Key metabolites shown by corresponding loading plots exhibited variations in amino acids (lysine, threonine, isoleucine, etc.), acetate, glucose, fructose, sucrose, ethanol, glycerol, and others. The levels of ethanol in soy sauce increased with longer fermentation durations, which can be influenced by both natural fermentation and the intentional addition of ethanol as a preservative. The study shows that the variation in metabolite can be very efficiently monitored using 1H-NMR-based metabolomics, thus suggestion to optimize the time duration to get the soy sauce product with the desired taste and flavor.

Cryopreserved leukapheresis material can be transferred from controlled rate freezers to ultracold storage at warmer temperatures without affecting downstream CAR-T cell culture performance and in-vitro functionality.

Chimeric antigen receptor (CAR) T-cell therapies are increasingly adopted as a commercially available treatment for hematologic and solid tumor cancers. As CAR-T therapies reach more patients globally, the cryopreservation and banking of patients' leukapheresis materials is becoming imperative to accommodate intra/inter-national shipping logistical delays and provide greater manufacturing flexibility. This study aims to determine the optimal temperature range for transferring cryopreserved leukapheresis materials from two distinct types of controlled rate freezing systems, Liquid Nitrogen (LN2)-based and LN2-free Conduction Cooling-based, to the ultracold LN2 storage freezer (≤-135 °C), and its impact on CAR T-cell production and functionality. Presented findings demonstrate that there is no significant influence on CAR T-cell expansion, differentiation, or downstream in-vitro function when employing a transfer temperature range spanning from -30 °C to -80 °C for the LN2-based controlled rate freezers as well as for conduction cooling controlled rate freezers. Notably, CAR T-cells generated from cryopreserved leukapheresis materials using the conduction cooling controlled rate freezer exhibited suboptimal performance in certain donors at transfer temperatures lower than -60 °C, possibly due to the reduced cooling rate of lower than 1 °C/min and extended dwelling time needed to reach the final temperatures within these systems. This cohort of data suggests that there is a low risk to transfer cryopreserved leukapheresis materials at higher temperatures (between -30 °C and -60 °C) with good functional recovery using either controlled cooling system, and the cryopreserved materials are suitable to use as the starting material for autologous CAR T-cell therapies.

Introgression of an all-stage and broad-spectrum powdery mildew resistance gene Pm3VS from Dasypyrum villosum chromosome 3V into wheat.

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a serious disease tothat threatens wheat production globally. It is imperative to explore novel resistance genes in order to control this disease throughby developing and planting resistant varieties. Here, we identified a wheat-Dasypyrum villosum 3V (3D) disomic substitution line, NAU3815 (2n=42), with a high level of powdery mildew resistance at both the seedling and adult-plant stages. Subsequently, NAU3815 was used to generate recombination between chromosomes 3V and 3D. Through genomic in situ hybridization (GISH), fluorescence in situ hybridization (FISH)GISH/FISH and 3VS, 3VL-specific markers analysis, four introgression lines were developed from the selfing progenies of 3V and 3D double monosomic line NAU3816, which was derived from the F1 hybrids of NAU3815/NAU0686. There were t3VS (3D) ditelosomic substitution line NAU3817, t3VL (3D) ditelosomic substitution line NAU3818, homozygous T3DL·3VS translocation line NAU3819, and homozygous T3DS·3VL translocation line NAU3820. Powdery mildew tests of these lines confirmed the presence of an all-stage and broad-spectrum powdery mildew resistance gene, Pm3VS, located on chromosome arm 3VS. When compared with the recurrent parent NAU0686 plants, the T3DL·3VS translocation line NAU3819 showed no obvious negative effect on yield-related traits. However, the introduction of the T3DL·3VS translocated chromosome had a strong effect on reducing the flag-leaf length. Consequently, the T3DL·3VS translocation line NAU3819 provides a new germplasm in breeding for both resistance and plant architecture.

Wheat Pm55 alleles exhibit distinct interactions with an inhibitor to cause different powdery mildew resistance.

Powdery mildew poses a significant threat to wheat crops worldwide, emphasizing the need for durable disease control strategies. The wheat-Dasypyrum villosum T5AL·5 V#4 S and T5DL·5 V#4 S translocation lines carrying powdery mildew resistant gene Pm55 shows developmental-stage and tissue-specific resistance, whereas T5DL·5 V#5 S line carrying Pm5V confers resistance at all stages. Here, we clone Pm55 and Pm5V, and reveal that they are allelic and renamed as Pm55a and Pm55b, respectively. The two Pm55 alleles encode coiled-coil, nucleotide-binding site-leucine-rich repeat (CNL) proteins, conferring broad-spectrum resistance to powdery mildew. However, they interact differently with a linked inhibitor gene, SuPm55 to cause different resistance to wheat powdery mildew. Notably, Pm55 and SuPm55 encode unrelated CNL proteins, and the inactivation of SuPm55 significantly reduces plant fitness. Combining SuPm55/Pm55a and Pm55b in wheat does not result in allele suppression or yield penalty. Our results provide not only insights into the suppression of resistance in wheat, but also a strategy for breeding durable resistance.

LILRB3 Supports Immunosuppressive Activity of Myeloid Cells and Tumor Development.

The existing T cell-centered immune checkpoint blockade therapies have been successful in treating some but not all patients with cancer. Immunosuppressive myeloid cells, including myeloid-derived suppressor cells (MDSC), that inhibit antitumor immunity and support multiple steps of tumor development are recognized as one of the major obstacles in cancer treatment. Leukocyte Ig-like receptor subfamily B3 (LILRB3), an immune inhibitory receptor containing tyrosine-based inhibitory motifs (ITIM), is expressed solely on myeloid cells. However, it is unknown whether LILRB3 is a critical checkpoint receptor in regulating the activity of immunosuppressive myeloid cells, and whether LILRB3 signaling can be blocked to activate the immune system to treat solid tumors. Here, we report that galectin-4 and galectin-7 induce activation of LILRB3 and that LILRB3 is functionally expressed on immunosuppressive myeloid cells. In some samples from patients with solid cancers, blockade of LILRB3 signaling by an antagonistic antibody inhibited the activity of immunosuppressive myeloid cells. Anti-LILRB3 also impeded tumor development in myeloid-specific LILRB3 transgenic mice through a T cell-dependent manner. LILRB3 blockade may prove to be a novel approach for immunotherapy of solid cancers.

P450-catalyzed atom transfer radical cyclization.

Cytochromes P450 have been extensively studied for both fundamental enzymology and biotechnological applications. Over the past decade, by taking inspiration from synthetic organic chemistry, new classes of P450-catalyzed reactions that were not previously encountered in the biological world have been developed to address challenging problems in organic chemistry and asymmetric catalysis. In particular, by repurposing and evolving P450 enzymes, stereoselective biocatalytic atom transfer radical cyclization (ATRC) was developed as a new means to impose stereocontrol over transient free radical intermediates. In this chapter, we describe the detailed experimental protocol for the directed evolution of P450 atom transfer radical cyclases. We also delineate protocols for analytical and preparative scale biocatalytic atom transfer radical cyclization processes. These methods will find application in the development of new P450-catalyzed radical reactions, as well as other synthetically useful processes.

Aldehyde modified cellulose-based dual stimuli responsive multiple cross-linked network ionic hydrogel toward ionic skin and aquatic environment communication sensors.

Recently, materials with complicated environmentally-sensitive abilities, high stretchability and excellent conductive sensitivity are interesting actuators in future applications. Herein, we fabricated a versatile and facile polyvinyl alcohol/polyacrylic acid/dialdehyde cellulose nanofibrils-Fe3+ hydrogel integrated with programmable dual-shape memory properties, high mechanical strength, good recoverability, and heat-induced self-healing capability. Benefiting from the synergistic effect of hydrogen bonds and dual metal coordination bonds of cellulose-based dialdehyde and carboxyl with Fe3+and then heating-freeze-thawing cycle treatment, the obtained hydrogel exhibited dual shape memory abilities, high tensile strain (up to 600 %), good self-recovery, and anti-fatigue properties. Moreover, the resultant hydrogel sensors showed revealed high strain sensitivity (gauge factor = 2.95) and satisfactory electrochemical performance; and such hydrogel-based sensor could be used as ionic skin to detect various human motions in real-time and barrier-free communication in the aquatic environment. The composite hydrogel with superior and versatile performances reported in this study could offer a great promise to be applied under extreme conditions as multifunctional sensors.

Low-temperature torrefaction assisted with solid-state KOH/urea pretreatment for accelerated methane production in wheat straw anaerobic digestion.

Low-temperature torrefaction assisted with solid-state KOH/urea applied onto wheat straw was proposed to break down the lignocellulosic material to enhance biomethane production in anaerobic digestion (AD). The optimization of key parameters applying the Box-Behnken design and response surface methodology showed that an addition of 0.1 g/gstraw KOH/urea at 180 °C while torrefying for 30 min was the optimal condition for producing biomethane. Results indicate that co-applying KOH and urea in torrefaction synergistically enhanced the biodegradability of straw by effectively removing lignin and largely retaining cellulose, giving rise to a 41 % increase in the cumulative methane production compared to untreated straw (213 mL/g-volatile solids (VSraw)) from batch AD. Additionally, the nitrogen- and potassium-rich digestates helped to improve soil fertility, thus achieving a zero-waste discharge. This study demonstrated the feasibility of using solid-state KOH/urea assisted low-temperature torrefaction as an effective pretreatment method to promote methane production during AD.

Blocking LAIR1 signaling in immune cells inhibits tumor development.

The current immune checkpoint blockade therapy has been successful in treating some cancers but not others. New molecular targets and therapeutic approaches of cancer immunology need to be identified. Leukocyte associated immunoglobulin like receptor 1 (LAIR1) is an immune inhibitory receptor expressing on most immune cell types. However, it remains a question whether we can specifically and actively block LAIR1 signaling to activate immune responses for cancer treatment. Here we report the development of specific antagonistic anti-LAIR1 monoclonal antibodies and studied the effects of LAIR1 blockade on the anti-tumor immune functions. The anti-LAIR1 antagonistic antibody stimulated the activities of T cells, natural killer cells, macrophages, and dendritic cells in vitro. The single-cell RNA sequencing analysis of intratumoral immune cells in syngeneic human LAIR1 transgenic mice treated with control or anti-LAIR1 antagonist antibodies indicates that LAIR1 signaling blockade increased the numbers of CD4 memory T cells and inflammatory macrophages, but decreased those of pro-tumor macrophages, regulatory T cells, and plasmacytoid dendritic cells. Importantly, the LAIR1 blockade by the antagonistic antibody inhibited the activity of immunosuppressive myeloid cells and reactivated T cells from cancer patients in vitro and impeded tumor metastasis in a humanized mouse model. Blocking LAIR1 signaling in immune cells represents a promising strategy for development of anti-cancer immunotherapy.

Combined effects of liquid digestate recirculation and biochar on methane yield, enzyme activity, and microbial community during semi-continuous anaerobic digestion.

The combined effects of liquid digestate recirculation (LDR) and biochar on methanogenesis and microbial communities were studied in semi-continuous anaerobic reactors fed with wheat straw and swine manure. The tolerated organic loading rate (OLR) was expanded from 5 g- volatile solids (VS)∙L-1∙d-1 in the control to higher than 6 g-VS∙L-1∙d-1 in the LDR. At the OLR of 5.0 g-VS∙L-1∙d-1, average special methane yield in LDR with biochar was 0.234 L∙g-VS-1, which was 5.4 % higher than that of the LDR alone. Moreover, enzyme activity and microbial community analysis indicated that LDR with biochar enhanced the processes of hydrolysis and methanogenesis, and balanced the pathway between hydrogenotrophic and acetoclastic methanogenesis. The co-application of LDR and biochar synergistically enhanced the degradation pathways of substrates and the loading shock resistance of anaerobic digestion system. This study could offer strategies for developing sustainable applications of full and continuous LDR in industrial biogas projects.

Engineered P450 Atom-Transfer Radical Cyclases are Bifunctional Biocatalysts: Reaction Mechanism and Origin of Enantioselectivity.

New-to-nature radical biocatalysis has recently emerged as a powerful strategy to tame fleeting open-shell intermediates for stereoselective transformations. In 2021, we introduced a novel metalloredox biocatalysis strategy that leverages the innate redox properties of the heme cofactor of P450 enzymes, furnishing new-to-nature atom-transfer radical cyclases (ATRCases) with excellent activity and stereoselectivity. Herein, we report a combined computational and experimental study to shed light on the mechanism and origins of enantioselectivity for this system. Molecular dynamics and quantum mechanics/molecular mechanics (QM/MM) calculations revealed an unexpected role of the key beneficial mutation I263Q. The glutamine residue serves as an essential hydrogen bond donor that engages with the carbonyl moiety of the substrate to promote bromine atom abstraction and enhance the enantioselectivity of radical cyclization. Therefore, the evolved ATRCase is a bifunctional biocatalyst, wherein the heme cofactor enables atom-transfer radical biocatalysis, while the hydrogen bond donor residue further enhances the activity and enantioselectivity. Unlike many enzymatic stereocontrol rationales based on a rigid substrate binding model, our computations demonstrate a high degree of rotational flexibility of the allyl moiety in an enzyme-substrate complex and succeeding intermediates. Therefore, the enantioselectivity is controlled by the radical cyclization transition states rather than the substrate orientation in ground-state complexes in the preceding steps. During radical cyclization, anchoring effects of the Q263 residue and steric interactions with the heme cofactor concurrently control the π-facial selectivity, allowing for highly enantioselective C-C bond formation. Our computational findings are corroborated by experiments with ATRCase mutants generated from site-directed mutagenesis.

Tandem Imine Formation and Alkyne Metathesis Enabled by Catalyst Choice.

Three-rung molecular ladder 8 was prepared in one pot via tandem imine condensation and alkyne metathesis. Catalyst VI is demonstrated to successfully engender the metathesis of imine-bearing substrate 7, while catalyst III does not. The susceptibility of catalyst VI to deactivation by hydrolysis and ligand exchange is demonstrated. Assembly and disassembly of ladder 8 in one pot were demonstrated in the presence and absence of a Lewis acid catalyst.

Photoenzymes for Radical C-C Coupling.

General catalytic methods for free radical-mediated asymmetric transformations have long eluded synthetic organic chemists. Now, NAD(P)H-dependent ketoreductases (KREDs) are repurposed and engineered as highly efficient photoenzymes to catalyse asymmetric radical C-C couplings.

Leukocyte immunoglobulin-like receptor subfamily B: therapeutic targets in cancer.

Inhibitory leukocyte immunoglobulin-like receptors (LILRBs 1-5) transduce signals via intracellular immunoreceptor tyrosine-based inhibitory motifs that recruit phosphatases to negatively regulate immune activation. The activation of LILRB signaling in immune cells may contribute to immune evasion. In addition, the expression and signaling of LILRBs in cancer cells especially in certain hematologic malignant cells directly support cancer development. Certain LILRBs thus have dual roles in cancer biology-as immune checkpoint molecules and tumor-supporting factors. Here, we review the expression, ligands, signaling, and functions of LILRBs, as well as therapeutic development targeting them. LILRBs may represent attractive targets for cancer treatment, and antagonizing LILRB signaling may prove to be effective anti-cancer strategies.

LILRB3 supports acute myeloid leukemia development and regulates T-cell antitumor immune responses through the TRAF2-cFLIP-NF-κB signaling axis.

Leukocyte immunoglobulin-like receptor B (LILRB), a family of immune checkpoint receptors, contributes to acute myeloid leukemia (AML) development, but the specific mechanisms triggered by activation or inhibition of these immune checkpoints in cancer is largely unknown. Here we demonstrate that the intracellular domain of LILRB3 is constitutively associated with the adaptor protein TRAF2. Activated LILRB3 in AML cells leads to recruitment of cFLIP and subsequent NF-κB upregulation, resulting in enhanced leukemic cell survival and inhibition of T-cell-mediated anti-tumor activity. Hyperactivation of NF-κB induces a negative regulatory feedback loop mediated by A20, which disrupts the interaction of LILRB3 and TRAF2; consequently the SHP-1/2-mediated inhibitory activity of LILRB3 becomes dominant. Finally, we show that blockade of LILRB3 signaling with antagonizing antibodies hampers AML progression. LILRB3 thus exerts context-dependent activating and inhibitory functions, and targeting LILRB3 may become a potential therapeutic strategy for AML treatment.

Sustainable Shape-Memory Polyurethane from Abietic Acid: Superior Mechanical Properties and Shape Recovery with Tunable Transition Temperatures.

A new chemical architecture from abietic acid, consisting of a cycloaliphatic unsaturated terminal diisocyanate (AADI) structure, is synthesized and fully characterized. The AADI is then used to construct an amorphous and biocompatible shape-memory polyurethane (SMPU) network system with adjustable cross-linking densities over a wide range. The SMPU network exhibits good shape-memory properties with a shape fixing rate of greater than 98 % and a shape recovery rate of 95 %. In vitro hydrolytic biodegradation weight loss ratio of SMPUs reaches 71 % within eight weeks. The physical properties of these SMPUs are comparable to those reported for SMPUs obtained from commercially available petroleum-derived diisocyanates. This is the first time that multiple SMPU networks based on abietic acid have been reported. These environmentally-friendly SMPUs display a wide range of physicomechanical properties with promising hydrolytic degradability, showing good potential for practical application.

Antagonistic anti-LILRB1 monoclonal antibody regulates antitumor functions of natural killer cells.

BACKGROUND: Current immune checkpoint blockade strategies have been successful in treating certain types of solid cancer. However, checkpoint blockade monotherapies have not been successful against most hematological malignancies including multiple myeloma and leukemia. There is an urgent need to identify new targets for development of cancer immunotherapy. LILRB1, an immunoreceptor tyrosine-based inhibitory motif-containing receptor, is widely expressed on human immune cells, including B cells, monocytes and macrophages, dendritic cells and subsets of natural killer (NK) cells and T cells. The ligands of LILRB1, such as major histocompatibility complex (MHC) class I molecules, activate LILRB1 and transduce a suppressive signal, which inhibits the immune responses. However, it is not clear whether LILRB1 blockade can be effectively used for cancer treatment. METHODS: First, we measured the LILRB1 expression on NK cells from cancer patients to determine whether LILRB1 upregulated on NK cells from patients with cancer, compared with NK cells from healthy donors. Then, we developed specific antagonistic anti-LILRB1 monoclonal antibodies and studied the effects of LILRB1 blockade on the antitumor immune function of NK cells, especially in multiple myeloma models, in vitro and in vivo xenograft model using non-obese diabetic (NOD)-SCID interleukin-2Rγ-null mice. RESULTS: We demonstrate that percentage of LILRB1+ NK cells is significantly higher in patients with persistent multiple myeloma after treatment than that in healthy donors. Further, the percentage of LILRB1+ NK cells is also significantly higher in patients with late-stage prostate cancer than that in healthy donors. Significantly, we showed that LILRB1 blockade by our antagonistic LILRB1 antibody increased the tumoricidal activity of NK cells against several types of cancer cells, including multiple myeloma, leukemia, lymphoma and solid tumors, in vitro and in vivo. CONCLUSIONS: Our results indicate that blocking LILRB1 signaling on immune effector cells such as NK cells may represent a novel strategy for the development of anticancer immunotherapy.

Correction to: LILRB4 ITIMs mediate the T cell suppression and infiltration of acute myeloid leukemia cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Thermally stable, enhanced water barrier, high strength starch bio-composite reinforced with lignin containing cellulose nanofibrils.

Lignin containing cellulose nanofibrils (LCNF) were obtained by mechanically fibrillating unbleached tree bark after alkaline extraction and used as a reinforcement in thermoplastic starch (TPS) to develop novel biodegradable composite films. With the addition of 15 wt % LCNF, the tensile strength and modulus of the composites increased by 319 % and 800 % compared to neat TPS films, respectively. The crystalline property of cellulose and the high interaction between TPS and LCNF improved the mechanical property of the composite films. The composite film Tonset and Tmax were 263.1 °C and 316.5 °C, respectively, compared to 250.5 °C and 297.3 °C for neat TPS. The composite films also showed higher water barrier property. Experimental results showed that LCNF features a high lignin content. Lignin, a natural polymer, contains hydrophobic and aromatic groups and, thus, can increase the water barrier property and thermal stability of TPS/LCNF composite films.

LILRB4 ITIMs mediate the T cell suppression and infiltration of acute myeloid leukemia cells.

We recently demonstrated that leukocyte Ig-like receptor 4 (LILRB4) expressed by monocytic acute myeloid leukemia (AML) cells mediates T-cell inhibition and leukemia cell infiltration via its intracellular domain. The cytoplasmic domain of LILRB4 contains three immunoreceptor tyrosine-based inhibitory motifs (ITIMs); the tyrosines at positions 360, 412, and 442 are phosphorylation sites. Here, we analyzed how the ITIMs of LILRB4 in AML cells mediate its function. Our in vitro and in vivo data show that Y412 and Y442, but not Y360, of LILRB4 are required for T-cell inhibition, and all three ITIMs are needed for leukemia cell infiltration. We constructed chimeric proteins containing the extracellular domain of LILRB4 and the intracellular domain of LILRB1 and vice versa. The intracellular domain of LILRB4, but not that of LILRB1, mediates T-cell suppression and AML cell migration. Our studies thus defined the unique signaling roles of LILRB4 ITIMs in AML cells.