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Patulin is a fatal mycotoxin that is widely detected in drinking water and fruit-derived products contaminated by diverse filamentous fungi. CgSDR from Candida guilliermondii represents the first NADPH-dependent short-chain dehydrogenase/reductase that catalyzes the reduction of patulin to the nontoxic E-ascladiol. To elucidate the catalytic mechanism of CgSDR, we solved its crystal structure in complex with cofactor and substrate. Structural analyses indicate that patulin is situated in a hydrophobic pocket adjacent to the cofactor, with the hemiacetal ring orienting toward the nicotinamide moiety of NADPH. In addition, we conducted structure-guided engineering to modify substrate-binding residue V187 and obtained variant V187F, V187K and V187W, whose catalytic activity was elevated by 3.9-, 2.2- and 1.7-fold, respectively. The crystal structures of CgSDR variants suggest that introducing additional aromatic stacking or hydrogen-bonding interactions to bind the lactone ring of patulin might account for the observed enhanced activity. These results illustrate the catalytic mechanism of SDR-mediated patulin detoxification for the first time and provide the upgraded variants that exhibit tremendous potentials in industrial applications.

Isoprenoids represent the largest group of natural products, whose basal skeletons are synthesized by various isoprenyl diphosphate synthases (IDSs). As majority of IDSs catalyze head-to-tail reaction to produce linear form isoprenoids, some catalyze head-to-middle reaction to produce branched form products. In a previous study, an IDS termed MA1831 from Methanosarcina acetivorans was found to be capable of catalyzing both types of reaction. In addition to the canonical linear product of C

The biosynthesis of brasilane-type sesquiterpenoids (BTSs) attracts much attention owing to their unique skeleton of 5/6 bicyclic structure that contains five Me groups. Here, the crystal structures of a BTS cyclase TaTC6 from Trichoderma atroviride FKI-3849 and its complexes with farnesyl pyrophosphate (FPP) and analogue were reported. These structural information reveal that TaTC6 exploits a hydrophobic pocket to constrain the hydrocarbon region of FPP in a "U-shape" to facilitate the initial C1-C11 bond formation after pyrophosphate ionization. Following, four carbocations of reaction intermediates were molecularly docked into the hydrophobic pocket to reveal critical residues involved in the cyclization cascade. Finally, an S239-stabilized water molecule that is 3.9 Å away from the C8 of the last allyl cation may conduct hydration to quench the reaction cascade. Mutating S239 to alanine led to ca. 40% reduction in activity compared with the wild-type enzyme. The conservation of the residues that constitute the hydrophobic pocket is also discussed. Overall, this study will give an insight into the mechanism of how the active site of STCs constrain the conformation of the flexible FPP and series allylic carbocations for the complicated-ring formation and unusual carbon rearrangement in the biosynthesis of BTSs.

Using enzymes to hydrolyze and recycle poly(ethylene terephthalate) (PET) is an attractive eco-friendly approach to manage the ever-increasing PET wastes, while one major challenge to realize the commercial application of enzyme-based PET degradation is to establish large-scale production methods to produce PET hydrolytic enzyme. To achieve this goal, we exploited the industrial strain Pichia pastoris to express a PET hydrolytic enzyme from Caldimonas taiwanensis termed CtPL-DM. In contrast to the protein expressed in Escherichia coli, CtPL-DM expressed in P. pastoris is inactive in PET degradation. Structural analysis indicates that a putative N-glycosylation site N181 could restrain the conformational change of a substrate-binding Trp and hamper the enzyme action. We thus constructed N181A to remove the N-glycosylation and found that the PET hydrolytic activity of this variant was restored. The performance of N181A was further enhanced via molecular engineering. These results are of valuable in terms of PET hydrolytic enzyme production in industrial strains in the future. Graphical Abstract

Background As the RNA secondary structure is highly related to its stability and functions, the structure prediction is of great value to biological research. The traditional computational prediction for RNA secondary prediction is mainly based on the thermodynamic model with dynamic programming to find the optimal structure. However, the prediction performance based on the traditional approach is unsatisfactory for further research. Besides, the computational complexity of the structure prediction using dynamic programming is $$O(N^3)$$ O ( N 3 ) ; it becomes $$O(N^6)$$ O ( N 6 ) for RNA structure with pseudoknots, which is computationally impractical for large-scale analysis. Results In this paper, we propose REDfold, a novel deep learning-based method for RNA secondary prediction. REDfold utilizes an encoder-decoder network based on CNN to learn the short and long range dependencies among the RNA sequence, and the network is further integrated with symmetric skip connections to efficiently propagate activation information across layers. Moreover, the network output is post-processed with constrained optimization to yield favorable predictions even for RNAs with pseudoknots. Experimental results based on the ncRNA database demonstrate that REDfold achieves better performance in terms of efficiency and accuracy, outperforming the contemporary state-of-the-art methods.

Poly(butylene adipate-co-terephthalate) (PBAT), a polyester made of terephthalic acid (TPA), 1,4-butanediol, and adipic acid, is extensively utilized in plastic production and has accumulated globally as environmental waste. Biodegradation is an attractive strategy to manage PBAT, but an effective PBAT-degrading enzyme is required. Here, we demonstrate that cutinases are highly potent enzymes that can completely decompose PBAT films in 48 h. We further show that the engineered cutinases, by applying a double mutation strategy to render a more flexible substrate-binding pocket exhibit higher decomposition rates. Notably, these variants produce TPA as a major end-product, which is beneficial feature for the future recycling economy. The crystal structures of wild type and double mutation of a cutinase from Thermobifida fusca in complex with a substrate analogue are also solved, elucidating their substrate-binding modes. These structural and biochemical analyses enable us to propose the mechanism of cutinase-mediated PBAT degradation.

1,2-β-Mannobiose phosphorylases (1,2-β-MBPs) from glycoside hydrolase 130 (GH130) family are important bio-catalysts in glycochemistry applications owing to their ability in synthesizing oligomannans. Here, we report the crystal structure of a thermostable 1,2-β-MBP from Thermoanaerobacter sp. X-514 termed Teth514_1789 to reveal the molecular basis of its higher thermostability and mechanism of action. We also solved the enzyme complexes of mannose, mannose-1-phosphate (M1P) and 1,4-β-mannobiose to manifest the enzyme-substrate interaction networks of three main subsites. Notably, a Zn ion that should be derived from crystallization buffer was found in the active site and coordinates the phosphate moiety of M1P. Nonetheless, this Zn-coordination should reflect an inhibitory status as supplementing Zn severely impairs the enzyme activity. These results indicate that the effects of metal ions should be taken into consideration when applying Teth514_1789 and other related enzymes. Based on the structure, a reliable model of Teth514_1788 that shares 61.7% sequence identity to Teth514_1789 but displays a different substrate preference was built. Analyzing the structural features of these two closely related enzymes, we hypothesized that the length of a loop fragment that covers the entrance of the catalytic center might regulate the substrate selectivity. In conclusion, these information provide in-depth understanding of GH130 1,2-β-MBPs and should serve as an important guidance for enzyme engineering for further applications.

Incorporation of branched structures is a major pathway to build macromolecules with desired three-dimensional (3D) structures, which are of high importance in the rational design of functional polymeric scaffolds. Dendrimers and hyperbranched polymers have been extensively studied for this purpose, but proper gain-of-function for these structures usually requires large enough molecular weights and a highly branched interior so that a spherical 3D core-shell architecture can be obtained, yet it is generally challenging to achieve precise control over the structure, high molecular weight, and high degree of branching (DoB) simultaneously. In this article, we present a set of snowflake-shaped star polymers with functional cores and dendronized arms, which ensure a high DoB and an overall globular conformation, thus facilitating the introduction of functional moieties onto the easily achieved scaffold without the need for high-generation dendrons. Using a polyglycerol dendron (PGD) as a proof of concept, we propose that this dendronized arm snowflake polymer (DASP) structure can serve as a better performing alternative to high-generation PGDs. DASPs with molecular weights of 750, 1220, 2120, and 3740 kDa were prepared with >85% yields in all cases, and we show that these DASPs have high encapsulating efficiency of Nile Red due to their high DoB and high biocompatibility due to their hydroxyl-rich nature after ketal removal, as well as high cell permeability that is molecular-weight-dependent. Introduced fluorophores such as fluorescein and difluoroboron 1,3-diphenylaminophenyl β-diketonate with suitable excitation wavelengths may turn the DASPs into stable, endosome-staining fluorophores with ultra-large Stokes shifts, narrowed emission bands, and suitability for long-term cellular tracing. Moreover, the scaffold can encapsulate antibiotic molecules and deliver them into phagolysosomes for efficient elimination of intracellular Staphylococcus aureus, which is insensitive toward many antibiotics but is a key target for the clinical success of methicillin-resistant Staphylococcus aureus infection treatment. Elimination of Staphylococcus aureus could be improved to >99.9% for chloramphenicol at 32 μg/mL with 450 μg/mL DASP.

The effects of transglutaminase (TGase, 1.0 unit/mL) with heat (95 °C, 5 min), 2-mercaptoethanol (2-ME, 0.83 %), and l-cysteine (l-Cys, 50 mM) pretreatment on the cross-linking of ovalbumin (OVA) and ovotransferrin (OVT) were investigated. SDS-PAGE revealed that although the polymerization of OVA and OVT did not occur after 3 h of incubation at 40 °C with TGase, OVA polymerized into high molecular weight polymers following TGase with 2-ME and heat pretreatment after 3 h of incubation. The surface hydrophobicity and reactive sulfhydryl (SH) groups of OVA samples significantly increased from 4065.7 ± 136.7 and 89.3 ± 1.2 SH groups (μmol/g) to 31483.6 ± 342.7 and 119.5 ± 3.7 SH groups (μmol/g), respectively. Similar results were obtained for OVT with TGase and l-Cys pretreatment and a 3-h incubation at 40 °C. The use of TGase, a reducing agent, and/or heat pretreatment can be used for the polymerization of OVA and OVT.

Poly(ethylene terephthalate) (PET) is the most abundant polyester plastic and a major contributor to plastic pollution. IsPETase, from the PET-assimilating bacterium Ideonella sakaiensis, is a unique PET-hydrolytic enzyme that shares high sequence identity to canonical cutinases, but shows substrate preference towards PET and exhibits higher PET-hydrolytic activity at ambient temperature. Structural analyses suggest that IsPETase harbours a substrate-binding residue, W185, with a wobbling conformation and a highly flexible W185-locating β6-β7 loop. Here, we show that these features result from the presence of S214 and I218 in IsPETase, whose equivalents are strictly His and Phe, respectively, in all other homologous enzymes. We found that mutating His/Phe residues to Ser/Ile could enhance the PET-hydrolytic activity of several IsPETase-like enzymes. In conclusion, the Ser/Ile mutations should provide an important strategy to improve the activity of potential PET-hydrolytic enzymes with properties that may be useful for various applications. In the face of global plastic pollution, enzymatic degradation of poly(ethylene terephthalate) (PET) has attracted much attention. Now, structural and biochemical studies reveal a minimal mutational strategy to increase the activity of PET-degrading enzymes, with potential evolutionary implications.

Background. The use of electronic health (e-health) resources is emerging as an alternative method to improve the secondary prevention of coronary artery disease (CAD). The aim of this study was to describe the influence of an e-health application in holistic healthcare for patients with CAD. Methods. A quasiexperiment with nonequivalent groups design recruited outpatients with a high risk of CAD admitted for cardiac catheterization. They were divided into two groups. Before the procedure, the control group received traditional patient education, and the intervention group watched videos on Internet-based social media. EQ-5D and FACIT–Sp-12 questionnaires were used as outcome measures of interest, and they were administered before and after the procedure and at the first return visit to the outpatient clinic after discharge. The effect of each intervention was tested using a linear mixed effects model. In addition, the 90-day readmission rate was also studied. Results. A total of 300 patients were divided into intervention and control groups (150 patients in each group). The interaction effect of EQ-5D was not statistically significant; however, improvements in FACIT–Sp-12 were greater in the intervention group from baseline to before discharge (regression coefficient (B) = 1.70, p < 0.001 ) and from baseline to postdischarge first outpatient visit (B = 1.81, p < 0.001 ). Moreover, the 90-day readmission rate was significantly lower in the intervention group (14% vs. 18.7%; p = 0.016 , log-rank test). Conclusions. e-health intervention with easily accessible Internet-based social media is a promising model to meet the holistic needs of patients with CAD in the modern era.

Cytochrome P450s are heme-thiolate enzymes that participate in carbon source assimilation, natural compound biosynthesis and xenobiotic metabolism in all kingdoms of life. P450s can catalyze various reactions by using a wide range of organic compounds, thus exhibiting great potential in biotechnological applications. The catalytic reactions of P450s are driven by electron equivalents that are sourced from pyridine nucleotides and delivered by cognate or matching redox partners (RPs). The electron transfer (ET) route from RPs to P450s involves one or more redox center-containing domains. As the rate of ET is one of the main determinants of P450 efficacy, an in-depth understanding of the P450 ET pathway should increase our knowledge of these important enzymes and benefit their further applications. Here, the various P450 RP systems along with current understanding of their ET routes will be reviewed. Notably, state-of-the-art structural studies of the two main types of self-sufficient P450 will also be summarized.

Terpenes and isoprenoids are the most diverse small organic molecules on Earth. Here, we obtained the structures of sesquisabinene and santalene synthases from Santalum album L. in the presence or ...

There is interest in the development of drugs to treat fungal infections due to the increasing threat of drug resistance, and here, we report the first crystallographic structure of the catalytic domain of a fungal squalene synthase (SQS)

In 1993, Taiwan has transformed into an aged society. Compared with general patients, older patients differ considerably in body functionality, mentally, and mobility-wise. The primary customers of medical institutions are adult patients in Taiwan, and the needs of older patients are often ignored; however, older patients visiting the case hospital in this study accounted for nearly 30% of the total number of outpatients. This indicates that the needs of older patients are a matter requiring the serious attention of medical personnel. This is a common issue for all countries that were entered an aging structure of the population. This study investigated the attention paid by supervisors and medical personnel involved with medical decisions to the quality of medical services received by older outpatients. An analytic hierarchy process (AHP) was employed to interview 10 experts in older medical care providing age-friendly medical care at the case hospital, medical staff including senior physicians, nursing department supervisors, administration department supervisors, and certified managers. The results indicate that the supervisors and medical personnel considered the “communication and services” provided to older patients during their medical treatments to be the most crucial factor in their treatment process, rating it twice as important as the next most important criteria, the care process and physical environment. Medical personnel paid great attention to communicate with and servicing older patients; listening to their needs and concerns was deemed the most critical, followed by being able to empathize with them to solve problems. Asking patients about their problems when they visited the hospital was considered the most crucial aspect of the care process; in the physical environment, it was spatial planning and traffic flow design. The results of this study enable other countries' medical institution managers and relevant competent authorities to gain insight into the attention paid by front-line employees in promoting age-friendly medical care. If supervisors and medical personnel involved with medical decisions share a similar attitude toward promoting medical service quality, government authorities and medical institutions can implement and enhance an age-friendly medical treatment environment for older patients, as well as increase quality of patient-centered medical services.

African Swine Fever Virus (ASFV) is an enveloped double-stranded DNA icosahedral virus that causes the devastating hemorrhagic fever of pigs. ASFV infections severely impact swine production and cause an enormous economic loss, but no effective vaccine and therapeutic regimen is available. pA151R is a non-structural protein of ASFV, which is expressed at both early and late stages of viral infection. Significantly, pA151R may play a key role in ASFV replication and virus assembly as suppressing pA151R expression can reduce virus replication. However, little is known about the functional and structural mechanisms of pA151R because it shares a very low sequence identity to known structures. It was proposed that pA151R might participate in the redox pathway owing to the presence of a thioredoxin active site feature, the WCTKC motif. In this study, we determined the crystal structure of pA151R. Based on the crystal structure, we found that pA151R comprises of a central five-stranded β-sheet packing against two helices on one side and an incompact C-terminal region containing the WCTKC motif on the other side. Notably, two cysteines in the WCTKC motif, an additional cysteine C116 from the β7-β8 loop together with ND1 of H109 coordinate a Zn2+ ion to form a Zn-binding motif. These findings suggest that the structure of pA151R is significantly different from that of typical thioredoxins. Our structure should provide molecular insights into the understanding of functional and structural mechanisms of pA151R from ASFV and shall benefit the development of prophylactic and therapeutic anti-ASFV agents.

This brief presents an all-digital CMOS pulse-shrinking time-to-digital converter (TDC) with temperature-measuring capability for higher circuit value and cost saving. A pulse generator is proposed to generate either a time-added pulse or a temperature-sensing pulse. A cyclic delay line (DL) composed of a temperature-sensing DL and a pulse-shrinking DL becomes area efficient and achieves sufficiently wide dynamic range. A time subtractor that eliminates the effect of the offset error enhances accuracy. The proposed TDC is fabricated with TSMC 0.35- $\mu \text{m}$ CMOS process and has a small area of approximately 0.019 mm2. The effective time resolution is approximately 45 ps, with an integral nonlinearity of −0.5 to 0.85 least significant bit (LSB). The achieved temperature resolution is nearly 0.1 °C/LSB, with an inaccuracy of −1.35 °C to 0.7 °C for a range of 0 °C–90 °C. Experimental results prove that we have proposed the first TDC that successfully measures time and temperature.

The ubiquitous cyclic di-GMP (c-di-GMP) network is highly redundant with numerous GGDEF domain proteins as diguanylate cyclases and EAL domain proteins as c-di-GMP specific phosphodiesterases comprising those domains as two of the most abundant bacterial domain superfamilies. One hallmark of the c-di-GMP network is its exalted plasticity as c-di-GMP turnover proteins can rapidly vanish from species within a genus and possess an above average transmissibility. To address the evolutionary forces of c-di-GMP turnover protein maintenance, conservation, and diversity, we investigated a Gram-positive and a Gram-negative species, which preserved only one single clearly identifiable GGDEF domain protein. Species of the family Morganellaceae of the order Enterobacterales exceptionally show disappearance of the c-di-GMP signaling network, but Proteus spp. still retained one diguanylate cyclase. As another example, in species of the bovis, pyogenes, and salivarius subgroups as well as Streptococcus suis and Streptococcus henryi of the genus Streptococcus, one candidate diguanylate cyclase was frequently identified. We demonstrate that both proteins encompass PAS (Per-ARNT-Sim)-GGDEF domains, possess diguanylate cyclase catalytic activity, and are suggested to signal via a PilZ receptor domain at the C-terminus of type 2 glycosyltransferase constituting BcsA cellulose synthases and a cellulose synthase-like protein CelA, respectively. Preservation of the ancient link between production of cellulose(-like) exopolysaccharides and c-di-GMP signaling indicates that this functionality is even of high ecological importance upon maintenance of the last remnants of a c-di-GMP signaling network in some of today's free-living bacteria.

Thebaine synthase 2 (THS2) that can transform (7S)-salutaridinol 7-O-acetate to thebaine catalyzes the final step of thebaine biosynthesis in Papaver somniferum. Here, the crystal structures of THS2 and its complex with thebaine are reported, revealing the interaction network in the substrate-binding pocket. Subsequent docking and QM/MM studies was performed to further explore the catalytic mechanism of THS2. Our results suggest that T105 may abstract the proton of C4–OH from the substrate under the assistance of H89. The resulting C4–O- phenolate anion then attacks the nearby C5, and triggers intramolecular SN2′ syn displacement with the elimination of O-acetyl group. Moreover, the latter SN2′ reaction is the rate-determining step of the whole enzymatic reaction with an overall energy barrier of 18.8 kcal/mol. These findings are of pivotal importance to understand the mechanism of action of thebaine biosynthesis, and would guide enzyme engineering to enhance the production of opiate alkaloids via metabolic engineering.

This brief presents a cost-efficient all-digital CMOS digital-to-time converter (DTC) that innovatively applies binary-weighted pulse expansion. The DTC consists of a pulse generator (PG) for pulse generation ( $t_{p}$ ), a binary-weighted pulse-expanding circuit (BWPEC) as a main circuit for digital-to-time conversion, and a time subtractor (TS) for $t_{p}$ removal. Compared with the original pulse-expanding unit (PEU), a novel PEU with reduced complexity and a linear pulse expansion was developed for binary-weighted operation. The use of the BWPEC with the binary-weighted PEUs exhibits lower circuit complexity and considerably reduces the circuit cost compared to the original unary-weighted pulse-expanding circuit (PEC). A prototype of the 4-bit all-digital DTC was fabricated using a Taiwan Semiconductor Manufacturing Company (TSMC) 0.35- $\mu \text{m}$ CMOS process. The fabricated DTC exhibited an area of 0.02 mm2 achieving twofold improvement considerably. The measured resolution was approximately 5 ps, with the integral nonlinearity being −1.2–1 least significant bit (LSB). Without requiring an advanced CMOS process, the pulse variation technique with a simple structure can easily achieve fine resolution. The DTC features cost-efficient pulse expansion with binary weights and low circuit complexity.

Isoprenyl diphosphate synthases (IDSs) catalyze condensation reactions of isoprene units to produce isoprenoids or terpenoids, the largest class of natural products on earth. IDSs are divided into ...

Plant biomass is an abundant renewable resource on Earth. Microorganisms harvest energy from plant material by means of complex enzymatic systems that efficiently degrade natural polymers. Intriguingly, microorganisms have evolved to exploit these ancient mechanisms to also decompose synthetic plastic polymers. In this Review, we summarize the mechanisms by which they decompose non-starch plant biomass and the six major types of synthetic plastics. We focus on the structural features of the enzymes that contribute to substrate recognition and then describe the catalytic mechanisms of polymer metabolism. An understanding of these natural biocatalysts is valuable if we are to exploit their potential for the degradation of synthetic polymers. Microorganisms enzymatically degrade natural polymers and synthetic plastics to enable a sustainable environment. This Review describes common features of these enzymes and how they can be harnessed to convert waste into useful precursors.

PURPOSE Coronary computed tomography angiography (CCTA) has its limitations in evaluating arteries with stents or heavy calcification. This study compares the diagnostic performance of subtracted coronary computed tomography angiography (SCCTA) and nonsubtracted coronary computed tomography angiography (NSCCTA) in evaluating coronary artery disease (CAD) and in-stent restenosis (ISR). MATERIALS AND METHODS Twelve patients with stents and 20 patients with heavy coronary calcifications (total Agatston's score >400) underwent both SCCTA and invasive coronary angiography (ICA) with an interval of

The epimerase MoeE5 from Streptomyces viridosporus converts UDP-glucuronic acid (UDP-GlcA) to UDP-galacturonic acid (UDP-GalA) to provide the first sugar in synthesizing moenomycin, a potent inhibitor against bacterial peptidoglycan glycosyltransferases. The enzyme belongs to the UDP-hexose 4-epimerase family, and uses NAD+ as its cofactor. Here we present the complex crystal structures of MoeE5/NAD+/UDP-GlcA and MoeE5/NAD+/UDP-glucose, determined at 1.48 A and 1.66 A resolution. The cofactor NAD+ is bound to the N-terminal Rossmann-fold domain and the substrate is bound to the smaller C-terminal domain. In both crystals the C4 atom of the sugar moiety of the substrate is in close proximity to the C4 atom of the nicotinamide of NAD+, and the O4 atom of the sugar is also hydrogen bonded to the side chain of Tyr154, suggesting a productive binding mode. As the first complex structure of this protein family with a bound UDP-GlcA in the active site, it shows an extensive hydrogen-bond network between the enzyme and the substrate. We further built a model with the product UDP-GalA, and found that the unique Arg192 of MoeE5 might play an important role in the catalytic pathway. Consequently, MoeE5 is likely a specific epimerase for UDP-GlcA to UDP-GalA conversion, rather than a promiscuous enzyme as some other family members.

Tumor cells and pathogen-infected cells are presented to human γδ T cells based on “inside-out” signaling in which metabolites called phosphoantigens (pAgs) inside target cells are recognized by the intracellular domain of a butyrophilin protein (BTN3A1), leading to an extracellular conformational change. Here, we report that pAgs function as molecular “glues” that initiate a heteromeric association between the intracellular domains of BTN3A1 and the structurally similar BTN2A1. Working with both exogenous and endogenous pAgs, we used x-ray crystallography, mutational studies, cellular assays, synthetic probe as well as molecular dynamics investigations to determine how pAgs glue intracellular BTN3A1 and BTN2A1 together for the “inside-out” signaling that triggers γδ T cell activation. This γδ T cell-specific mode of antigen sensing creates opportunities for the development of alternative immunotherapies against cancer and infectious diseases that do not involve αβ T cells.One Sentence SummaryThe responses of gamma-delta T cells to cancer cells or pathogens are initiated via the intracellular association of heteromeric butyrophilins that are glued together by isoprenoid metabolites.

Soybeans are a good source of protein and functional nutrition for humans and are used in the production of many foods, such as soymilk and tofu, which are popular in Asia. Soymilk is a beverage with a milky appearance and is produced by grinding soybeans in water; this raw soymilk is then filtered and heated. Soymilk can be used to produce tofu, a gelatinous food, in a process that generally involves coagulating, forming, and pressing soymilk. Tofu is also a source of functional ingredients, such as isoflavones, that have received much attention for their antioxidant properties. These isoflavones bind to soy protein to form soy protein–isoflavone complexes in tofu. In this review, we examined the processing, gelation and functional components of tofu. First, we introduced novel processing technologies (such as thermal and nonthermal processing) and hydrocolloids that affect the rheological property and texture of tofu. Then, because the coagulation and gelation of soymilk are important steps in the tofu manufacturing process, we also described detailed coagulation and gelation mechanisms of soybean proteins in tofu. Finally, we described the functional compounds and ingredients in tofu. This review provides basic knowledge for the further application of novel processing technology in tofu processing.

KRAS mutation occurs in nearly 30% of human cancers, yet the most prevalent and oncogenic KRAS mutation (G12D) still lacks inhibitors. Herein, we explored the formation of a salt-bridge between KRAS’s Asp12 residue and a series of potent inhibitors. Our ITC results show that these inhibitors bind to and inhibit both GDP-bound and GTP-bound KRAS G12D, and our crystallographic studies revealed the structural basis of inhibitor binding in the switch-II pocket, experimentally confirming the formation of a salt-bridge between the piperazine moiety of the inhibitors and the 12D residue of the mutant protein. Among KRAS family proteins and mutants, both ITC and enzymatic assays support the selectivity of the inhibitors for KRAS G12D, and the inhibitors disrupt the KRAS-CRAF interaction. We also observed inhibition of cancer cell proliferation and inhibition of MAPK signaling by a representative inhibitor (TH-Z835); however, since this was not fully dependent on KRAS mutation status, it is possible that our inhibitors may have off-target effects via non-KRAS small GTPases. Experiments with a mouse model of pancreatic cancer showed that TH-Z835 significantly reduced tumor volume and synergized with an anti-PD-1 antibody. Collectively, our study demonstrates proof-of-concept for a salt-bridge, induced-fit pocket strategy for KRAS G12D, which warrants future medicinal chemistry efforts for optimal efficacy and minimized off-target effects.

Deoxynivalenol (DON) and its acetylated derivatives such as 3-acetyldeoxynivalenol (3A-DON) and 15-acetyldeoxynivalenol (15A-DON) are notorious mycotoxins in Fusarium contaminated cereals, which pose a great threat to human and livestock health. The specialized glyoxalase I from Gossypium hirsutum (SPG) can lower the toxicity of 3A-DON by conducting isomerization to transfer C8 carbonyl to C7 and double bond from C9-C10 to C8-C9. Here we report that the substrate-flexible SPG can also recognize 15A-DON and DON, probably following the same isomerization mechanism as that for 3A-DON. The crystallographic, mutagenesis, and biochemical analyses revealed that SPG provides a hydrophobic pocket to accommodate the substrate and residue E167 might serve as the catalytic base. A variant SPG