Building RNA-Mediated Artificial Signaling Pathways between Endogenous Genes.

ConspectusSophisticated genetic networks play a pivotal role in orchestrating cellular responses through intricate signaling pathways across diverse environmental conditions. Beyond the inherent complexity of natural cellular signaling networks, the construction of artificial signaling pathways (ASPs) introduces a vast array of possibilities for reshaping cellular responses, enabling programmable control of living organisms. ASPs can be integrated with existing cellular networks and redirect output responses as desired, allowing seamless communication and coordination with other cellular processes, thereby achieving designable transduction within cells. Among diversified ASPs, establishing connections between originally independent endogenous genes is of particular significance in modifying the genetic networks, so that cells can be endowed with new capabilities to sense and deal with abnormal factors related to differentiated gene expression (i.e., solve the issues of the aberrant gene expression induced by either external or internal stimuli). In a typical scenario, the two genes X and Y in the cell are originally expressed independently. After the introduction of an ASP, changes in the expression of gene X may exert a designed impact on gene Y, subsequently inducing the cellular response related to gene Y. If X represents a disease signal and Y serves as a therapeutic module, the introduction of the ASP empowers cells with a new spontaneous defense system to handle potential risks, which holds great potential for both fundamental and translational studies.In this Account, we primarily review our endeavors in the construction of RNA-mediated ASPs between endogenous genes that can respond to differentiated RNA expression. In contrast to other molecules that may be restricted to specific pathways, synthetic RNA circuits can be easily utilized and expanded as a general platform for constructing ASPs with a high degree of programmability and tunability for diversified functionalities through predictable Watson-Crick base pairing. We first provide an overview of recent advancements in RNA-based genetic circuits, encompassing but not limited to utilization of RNA toehold switches, siRNA and CRISPR systems. Despite notable progress, most reported RNA circuits have to contain at least one exogenous RNA X as input or one engineered RNA Y as a target, which is not suitable for establishing endogenous gene connections. While exogenous RNAs can be engineered and controlled as desired, constructing a general and efficient platform for manipulation of naturally occurring RNAs poses a formidable challenge, especially for the mammalian system. With a focus on this goal, we are devoted to developing efficient strategies to manipulate cell responses by establishing RNA-mediated ASPs between endogenous genes, particularly in mammalian cells. Our step-by-step progress in engineering customized cell signaling circuits, from bacterial cells to mammalian cells, from gene expression regulation to phenotype control, and from small RNA to long mRNA of low abundance and more complex secondary structures, is systematically described. Finally, future perspectives and potential applications of these RNA-mediated ASPs between endogenous genes are also discussed.

Binary conformers of a flexible, long-chain fluoroalcohol: dispersion controlled selectivity and relative abundances in a jet.

The complex conformational panorama of binary 4,4,4-trifluoro-1-butanol (TFB) aggregates was investigated using chirped-pulse Fourier transform microwave spectroscopy, aided by conformational searches using CREST (Conformer-Rotamer Ensemble Sampling Tool) and quantum chemistry calculations. From nearly 1500 initial dimer geometries, 16 most stable binary candidates were obtained within a relative energy window of ∼4 kJ mol-1. Rotational spectra of five binary conformers were experimentally observed in supersonic expansion and assigned. Interestingly, three out of the five observed binary conformers are composed solely of monomer conformers, which were not observed in their isolated gas phase forms in jet expansion. In addition, an observed dimer that is made exclusively of the most stable TFB monomer subunits does not correspond to the global minimum. The intricate kinetically and thermodynamically controlled dimer formation mechanisms are discussed, and a modified kinetic-thermodynamic model was developed, providing conformational abundances that are in good agreement with the experiment. Subsequent non-covalent interaction analyses reveal that the observed conformers are held together by one primary O-H⋯O hydrogen bond and secondary intermolecular C-H⋯O, C-H⋯F, and/or O-H⋯F interactions, as well as C-H⋯H-C London dispersion interactions between the methylene groups. Further symmetry-adapted perturbation theory analyses of the TFB dimer conformers and related alcohol dimers reveal a considerable rise in dispersion contributions with increasing n-alkyl carbon chain length and highlight the role of dispersion interactions in preferentially stabilizing the global minimum of the TFB dimer.

Plant-derived flavonoids are a potential source of drugs for the treatment of liver fibrosis.

Liver fibrosis is a dynamic pathological process that can be triggered by any chronic liver injury. If left unaddressed, it will inevitably progress to the severe outcomes of liver cirrhosis or even hepatocellular carcinoma. In the past few years, the prevalence and fatality of hepatic fibrosis have been steadily rising on a global scale. As a result of its intricate pathogenesis, the quest for pharmacological interventions targeting liver fibrosis has remained a formidable challenge. Currently, no pharmaceuticals are exhibiting substantial clinical efficacy in the management of hepatic fibrosis. Hence, it is of utmost importance to expedite the development of novel therapeutics for the treatment of this condition. Various research studies have revealed the ability of different natural flavonoid compounds to alleviate or reverse hepatic fibrosis through a range of mechanisms, which are related to the regulation of liver inflammation, oxidative stress, synthesis and secretion of fibrosis-related factors, hepatic stellate cells activation, and proliferation, and extracellular matrix synthesis and degradation by these compounds. This review summarizes the progress of research on different sources of natural flavonoids with inhibitory effects on liver fibrosis over the last decades. The anti-fibrotic effects of natural flavonoids have been increasingly studied, making them a potential source of drugs for the treatment of liver fibrosis due to their good efficacy and biosafety.

Structural and Electronic Evolution of Ethanolamine upon Microhydration: Insights from Hyperfine Resolved Rotational Spectroscopy.

Ethanolamine hydrates containing from one to seven water molecules were identified via rotational spectroscopy with the aid of accurate quantum chemical methods considering anharmonic vibrational corrections. Ethanolamine undergoes significant conformational changes upon hydration to form energetically favorable hydrogen bond networks. The final structures strongly resemble the pure (H2O)3-9 complexes reported before when replacing two water molecules by ethanolamine. The 14N nuclear quadrupole coupling constants of all the ethanolamine hydrates have been determined and show a remarkable correlation with the strength of hydrogen bonds involving the amino group. After addition of the seventh water molecule, both hydrogen atoms of the amino group actively contribute to hydrogen bond formation, reinforcing the network and introducing approximately 21-27% ionicity towards the formation of protonated amine. The findings highlight the critical role of microhydration in altering the electronic environment of ethanolamine, enhancing our understanding of amine hydration dynamics.

Rotational spectroscopy of hydrogen-bonded binary trifluoro-propanol conformers: conformational diversity, preference and abundances in a jet expansion.

The rich conformational landscape including the associated conformational conversion paths of the hydrogen-bonded binary 3,3,3-trifluoropropanol (TFP) aggregate was explored using chirped pulse Fourier transform microwave spectroscopy and computational chemistry. To appropriately identify the binary TFP conformers responsible for the five sets of candidate rotational transitions assigned, we established a set of important conformational assignment criteria. These include an extensive conformational search, good agreement between the experimental and theoretical rotational constants, relative magnitude of the three dipole moment components, and quartic centrifugal distortion constants, and observation and non-observation of the predicted conformers. Extensive conformational searches were carried out using CREST, a conformational search tool, producing hundreds of structural candidates. The CREST candidates were screened using a multitier approach and subsequently the low energy conformers (<25 kJ mol-1) were optimized at the B3LYP-D3BJ/def2-TZVP level, leading to 62 minima within an energy window of 10 kJ mol-1. Good agreement with the predicted spectroscopic properties mentioned above allowed us to clearly identify five binary TFP conformers as the molecular carriers. Particularly, a combined kinetic and thermodynamic model was developed, which provides a satisfactory explanation for the observation and non-observation of the low energy conformers predicted. The role of the intra- and intermolecular hydrogen bonding interactions in the stability ordering of the binary conformers is discussed.

Effects of tamarind seed polysaccharides on physicochemical characteristics of frozen dough: structure-function relationship.

BACKGROUND: Recently, frozen dough has become more popular because of its ability to be quickly transformed into freshly baked foods. During the storage and transport process, frozen dough can suffer some degree of damage caused by ice crystallization and recrystallization. Adding polysaccharides to frozen dough is a good way to solve this problem. Tamarind seed polysaccharide (TSP) has excellent ice crystal steady ability and has also been widely used in frozen foods. However, there is no study on the use of TSP in frozen dough. RESULTS: TSP can stabilize the bound water content, inhibit the freezable water content, and increase elasticity. However, the dough with different structures of TSP added was less firm after 30 days of freezing compared to the dough without TSP, and the porosity and stomatal density of the prepared steamed bread gradually decreased. The addition of TSP reduced gluten deterioration during the freezing process, thus decreasing the collapse and uneven porosity of the steamed bread. CONCLUSIONS: The results could provide new insights into the structure of TSP and its effect on the quality characteristics of frozen dough. © 2023 Society of Chemical Industry.

CO2 Aggregation on Monoethanolamine: Observations from Rotational Spectroscopy.

The initial stages of the gas-phase nucleation between CO2 and monoethanolamine were investigated via broadband rotational spectroscopy with the aid of extensive theoretical structure sampling. Sub-nanometer-scale aggregation patterns of monoethanolamine-(CO2 )n , n=1-4, were identified. An interesting competition between the monoethanolamine intramolecular hydrogen bond and the intermolecular interactions between monoethanolamine and CO2 upon cluster growth was discovered, revealing an intriguing CO2 binding priority to the hydroxyl group over the amine group. These findings are in sharp contrast to the general results for aqueous solutions. In the quinary complex, a cap-like CO2 tetramer was observed cooperatively surrounding the monoethanolamine. As the cluster approaches the critical size of new particle formation, the contribution of CO2 self-assembly to the overall stability increases.

Hydrogen-Atom Tunneling in a Homochiral Environment.

The role-exchanging concerted torsional motion of two hydrogen atoms in the homochiral dimer of trans-1,2-cyclohexanediol was characterized through a combination of broadband rotational spectroscopy and theoretical modeling. The results reveal that the concerted tunneling motion of the hydrogen atoms leads to the inversion of the sign of the dipole moment components along the a and b principal axes, due to the interchange motion that cooperatively breaks and reforms one intermolecular hydrogen bond. This motion is also coupled with two acceptor switching motions. The energy difference between the two ground vibrational states arising from this tunneling motion was determined to be 29.003(2) MHz. The corresponding wavefunctions suggest that the two hydrogen atoms are evenly delocalized on two equivalent potential wells, which differs from the heterochiral case where the hydrogen atoms are confined in separate wells, as the permutation-inversion symmetry breaks down. This intriguing contrast in hydrogen-atom behavior between homochiral and heterochiral environments could further illuminate our understanding of the role of chirality in intermolecular interactions and dynamics.

Programming Non-Nucleic Acid Molecules into Computational Nucleic Acid Systems.

Nucleic acid (NA) computation has been widely developed in the past years to solve kinds of logic and mathematic issues in both information technologies and biomedical analysis. However, the difficulty to integrate non-NA molecules limits its power as a universal platform for molecular computation. Here, we report a versatile prototype of hybridized computation integrated with both nucleic acids and non-NA molecules. Employing the conformationally controlled ligand converters, we demonstrate that non-NA molecules, including both small molecules and proteins, can be computed as nucleic acid strands to construct the circuitry with increased complexity and scalability, and can be even programmed to solve arithmetical calculations within the computational nucleic acid system. This study opens a new door for molecular computation in which all-NA circuits can be expanded with integration of various ligands, and meanwhile, ligands can be precisely programmed by the nuclei acid computation.

Evolution, gene expression, and protein‒protein interaction analyses identify candidate CBL-CIPK signalling networks implicated in stress responses to cold and bacterial infection in citrus.

BACKGROUND: Cold is a major abiotic stress and Huanglongbing and citrus canker disease are two devastating bacterial diseases for citrus. The Ca2+-CBL-CIPK network is known to regulate different types of stress signalling in plants. How do CBL-CIPK signalling networks function in response to cold and infection by CLas or Xcc in citrus? RESULTS: Eight calcineurin B-like proteins (CBLs) and seventeen CBL-interacting protein kinases (CIPKs) were identified from the cold-tolerant satsuma mandarin 'Guijing2501' (Citrus. unshiu) and CLas/Xcc-sensitive sweet orange (C. sinensis). Phylogenetic analysis revealed that both CBL and CIPK family members in citrus were classified into an ancient and a recent clade according to their conserved domain characteristics and/or intron/exon structures. Genome duplication analysis suggested that both tandem and segmental duplications contributed to the amplification of the CBL and CIPK gene families in citrus under intense purifying selection, and the duplication events only existed in the recent clades. Expression comparison of the duplicated gene pairs indicated that the duplicated CBL and CIPK genes underwent functional differentiation. Further expression analysis identified that CBL1, 5, 6, and 8 and CIPK2, 8, 12, 15, 16, and 17 were significantly regulated by multiple stresses, including cold, Xcc infection and/or CLas infection, in citrus, whereas CBL2/7 and CIPK1/4/5/11/13/14 were independently highly regulated by cold and CIPK3 was uniquely responsive to Xcc infection. The combination analyses of targeted Y2H assay and expression analysis revealed that CBL6-CIPK8 was the common signalling network in response to cold and Xcc infection, while CBL6/CBL8-CIPK14 was uniquely responsive to cold in citrus. Further stable transformation and cold tolerance assay indicated that overexpression of CuCIPK16 enhanced the cold tolerance of transgenic Arabidopsis with higher POD activity and lower MDA content. CONCLUSIONS: In this study, evolution, gene expression and protein‒protein interaction analyses of citrus CBLs and CIPKs were comprehensively conducted over a genome-wide range. The results will facilitate future functional characterization of individual citrus CBLs and CIPKs under specific stresses and provide clues for the clarification of cold tolerance and disease susceptibility mechanisms in corresponding citrus cultivars.

Rotational Spectroscopy of 2-Furoic Acid and Its Dimer: Conformational Distribution and Double Proton Tunneling.

Structural and tunneling properties of the 2-furoic acid (FA) monomer and dimer were investigated using rotational spectroscopy and DFT calculations. CREST, a conformational ensemble space exploration tool, was used to identify all possible low-energy conformations of the FA monomer and dimer, followed by the DFT geometry optimization and harmonic frequency calculations. Broadband rotational spectra in the 2-6 and 8-12 GHz regions were recorded in a supersonic jet expansion. The monomeric FA was found to exist dominantly as three different conformers: I, II, and III in a jet, with I and II taking on the cis-COOH configuration while III having the trans-COOH configuration. For the FA dimer, only the I-II conformer was observed experimentally, whereas the symmetric I-I and II-II conformers were not observed because of their zero dipole moments. The analysis of the splittings in the rotational transitions of I-II allowed one to extract the tunneling splitting to be 1056.0(12) MHz. The barrier height was determined to be ∼442 cm-1 using the scaled potential energy scans at several different levels of theory.

Development of pyrazolo[3,4-d]pyrimidin-4-one scaffold as novel CDK2 inhibitors: Design, synthesis, and biological evaluation.

A series of pyrazolo[3,4-d]pyrimidin-4-one scaffold were designed and synthesized as novel CDK2 inhibitors. By analyzing the common motifs of various known inhibitors, the designed compounds 1 were virtually screen for their inhibitory activity by docking into the active pocket of CDK2. The influence of different substitutes on the docking results was investigated. A total of 15 pyrazolo[3,4-d]pyrimidin-4-ones 1 were synthesized by Paal-Knorr reaction, pyrimidine ring closure, bromination, Suzuki coupling reaction, amide formation and Knoevenagel condensation. The Cell Counting Kit-8 (CCK-8) was used to evaluate the inhibitory activity of pyrazolo[3,4-d]pyrimidin-4-ones 1 in the breast cancer cell line MCF-7 in vitro using Etoposide as a reference control substance. The screening results demonstrated that the designed compounds have significant antiproliferative activity, and compounds 1e and 1j were the most active compounds with IC50 values of 10.79 µM and 10.88 µM, respectively, being better than that of Etoposide (IC50 = 18.75 µM). The enzyme inhibition assay was carried out against CDK2, the results indicated that the compounds 1e and 1j significantly inhibited CDK2 with IC50 values of 1.71 µM and 1.60 µM.

Fractional laser-assisted administration of human umbilical cord mesenchymal stem cells to reduce hypertrophic scars in rabbit ears.

BACKGROUND: The formation of hypertrophic scar is due to the abnormal accumulation and remodeling of the extracellular matrix, especially collagen tissue. Our research was designed to investigate the treatment effect of different administrations of human umbilical cord-derived stem cells and to hypertrophic scars on rabbit ears. METHODS: Thirty New Zealand female white rabbits were treated as hypertrophic scar models. PBS was injected into the scars on the right ear of each group as control, while human umbilical cord-derived stem cells or condition medium of human umbilical cord-derived stem cells were administrated into the left ear through subcutaneous injection or fractional laser-assisted administration. Gross examination, scar elevation index (SEI) calculation and sampling were executed 5 weeks after administration. Then H&E and Masson staining analysis and the expression levels detections of α-SMA, Collagen I, TGF-ß1, IL-1ß, and IL-6 were performed. RESULTS: Our results demonstrated that the severity of hyperplasia was lower than the model group after stem cells and conditioned medium treatment. H&E and Masson staining results showed that the inflammation in scars was greatly alleviated and the degree of fibrosis was reduced after treatment. There was no significant difference in the therapeutic effect between subcutaneous injection or fractional laser-assisted administration. Both stem cells and conditioned medium can down-regulate SEI and factors expression levels in all groups. However, compared with the stem cells, the therapeutic effects of the conditioned medium were lower. CONCLUSIONS: The results confirmed that stem cells had an available treatment effect on hypertrophic scars of rabbit ears. In addition to the paracrine pathway, stem cells may have other ways to treat hypertrophic scars. Fractional laser-assisted administration may become a potential administration of stem cell clinical application in the future.

The review of alpha-linolenic acid: Sources, metabolism, and pharmacology.

α-linolenic acid (ALA, 18:3n-3) is a carboxylic acid composed of 18 carbon atoms and three cis double bonds, and is an essential fatty acid indispensable to the human body. This study aims to systematically review related studies on the dietary sources, metabolism, and pharmacological effects of ALA. Information on ALA was collected from the internet database PubMed, Elsevier, ResearchGate, Web of Science, Wiley Online Library, and Europe PMC using a combination of keywords including "pharmacology," "metabolism," "sources." The following findings are mainly contained. (a) ALA can only be ingested from food and then converted into eicosapentaenoic acid and docosahexaenoic acid in the body. (b) This conversion process is relatively limited and affected by many factors such as dose, gender, and disease. (c) Pharmacological research shows that ALA has the anti-metabolic syndrome, anticancer, antiinflammatory, anti-oxidant, anti-obesity, neuroprotection, and regulation of the intestinal flora properties. (d) There are the most studies that prove ALA has anti-metabolic syndrome effects, including experimental studies and clinical trials. (e) The therapeutic effect of ALA will be affected by the dosage. In short, ALA is expected to treat many diseases, but further high quality studies are needed to firmly establish the clinical efficacy of ALA.

Conformational Landscape, Chirality Recognition and Chiral Analyses: Rotational Spectroscopy of Tetrahydro-2-Furoic Acid⋠⋠⋠Propylene Oxide Conformers.

A chiral adduct formed between a chiral carboxylic acid, tetrahydro-2-furoic acid (THFA), and a chiral ester, propylene oxide (PO), was investigated using rotational spectroscopy and DFT calculations. Isolated THFA exists dominantly as three different conformers: I, II, and III in a jet, with I and II taking on the trans-COOH configuration and III having the cis-COOH configuration. We utilized CREST, a conformational ensemble space exploration tool, to identify the possible conformations of the binary adduct, THFA⋅⋅⋅PO. Subsequent DFT geometry optimizations predicted about two hundred homochiral and heterochiral binary structures with 28 low energy structures within an energy window of 15 kJ mol-1 . A rich broadband rotational spectrum was obtained with a mixture of trace amounts of THFA+PO in neon in a supersonic jet expansion. Six THFA⋅⋅⋅PO conformers were identified experimentally. Kinetically favored binary products which contain trans-COOH I dominate among the observed conformers, while thermodynamically more stable adducts were also detected. Detailed analyses of the structures of the observed conformers show interesting chirality-controlled structural preferences. Such non-covalently bound chiral contact pairs are the foundation of chiral-tag rotational spectroscopy, an exciting new analytical application of rotational spectroscopy for determination of enantiomeric excess. Enantiomeric excess analyses were performed and the results are discussed.

Modifying conformational distribution of chiral tetrahydro-2-furoic acid through its interaction with water: a rotational spectroscopic and theoretical investigation.

Rotational spectrum of a binary complex formed between tetrahydro-2-furoic acid (THFA) and water was measured using a chirped pulse Fourier transform microwave spectrometer. A comprehensive theoretical conformational search procedure was carried out using CREST, a conformational searching tool, and DFT calculations to aid the spectral assignment and interpretation. The final conformer ensemble is classified into two structural groups: Type 1 conformers showing a classic carboxylic acid monohydrate structure with two strong hydrogen-bonds formed between the COOH group of cis-THFA and water, and the much less stable Type 2 conformers with trans-THFA and weaker intermolecular interactions with water. The 'cis-' and 'trans-' labels refer to the configurations where the carboxylic C[double bond, length as m-dash]O and OH functional groups are on the same or opposite side, respectively. Only the two most stable Type 2 conformers containing trans-THFA I and II were observed experimentally in a neon jet expansion with an abundance ratio of 1 : 1. This relative abundance observation differs greatly from that of the THFA monomer, i.e. with trans-THFA I : trans-THFA II : cis-THFA III of 10 : 1 : 1 in a neon jet expansion, reported previously. The observation indicates a kinetically controlled formation process of different types of the monohydrates in a jet expansion, whereas a thermodynamically controlled process dominates within each type of structures. The relative stability of the THFA ring conformations is altered by interaction with water, showing a noticeable water induced conformational preference.

Research on the circadian clock gene HNF4a in different malignant tumors.

Background: The circadian rhythm is produced by multiple feedback loops formed by the core clock genes after transcription and translation, thus regulating various metabolic and physiological functions of the human body. We have shown previously that the abnormal expression of 14 clock genes is related closely to the occurrence and development of different malignant tumors, and these genes may play an anti-cancer or pro-cancer role in different tumors. HNF4a has many typical properties of clock proteins involved in the clock gene negative feedback loop regulation process. We need to explore the function of HNF4a as a circadian clock gene in malignant tumors further. Methods: We used The Cancer Genome Atlas (TCGA) database to download the clinicopathological information of twenty malignant tumors and the corresponding RNA-seq data. The HNF4a RNA-seq data standardized by R language and clinical information were integrated to reveal the relationship between HNF4a and prognosis of patients. Results: Analysis of TCGA data showed that the prognosis of HNF4a was significantly different in BLCA, KIRC, LUSC, and READ. High HNF4a expression is correlated with good prognosis in BLCA, KIRC, and READ but poor prognosis in LUSC. However, HNF4a was associated with the stages, T stages, and lymph node status only in BLCA. Conclusions: HNF4a plays different roles in different malignancies, and the abnormal expression of HNF4a has a great correlation with the biological characteristics of BLCA. The low expression of HNF4a could be a reference index for the metastasis, recurrence, and prognosis of BLCA.

TEMPO-Oxidized Cellulose Beads as Potential pH-Responsive Carriers for Site-Specific Drug Delivery in the Gastrointestinal Tract.

The development of controlled drug delivery systems based on bio-renewable materials is an emerging strategy. In this work, a controlled drug delivery system based on mesoporous oxidized cellulose beads (OCBs) was successfully developed by a facile and green method. The introduction of the carboxyl groups mediated by the TEMPO(2,2,6,6-tetramethylpiperidine-1-oxyradical)/NaClO/NaClO2 system presents the pH-responsive ability to cellulose beads, which can retain the drug in beads at pH = 1.2 and release at pH = 7.0. The release rate can be controlled by simply adjusting the degree of oxidation to achieve drug release at different locations and periods. A higher degree of oxidation corresponds to a faster release rate, which is attributed to a higher degree of re-swelling and higher hydrophilicity of OCBs. The zero-order release kinetics of the model drugs from the OCBs suggested a constant drug release rate, which is conducive to maintaining blood drug concentration, reducing side effects and administration frequency. At the same time, the effects of different model drugs and different drug-loading solvents on the release behavior and the physical state of the drugs loaded in the beads were studied. In summary, the pH-responsive oxidized cellulose beads with good biocompatibility, low cost, and adjustable release rate have shown great potential in the field of controlled drug release.

Inhibitory effect of lignin from Canna edulis Ker residues on trypsin: kinetics and molecular docking studies.

BACKGROUND: Lignin extracted from Canna edulis Ker residues shows a strong inhibitory effect on α-glucosidase and a promoting effect on α-amylase. Protease activity inhibition may play a key role in disease processes, such as metastasis, tumor invasion and bacterial colonization. Hence, in the present study, the inhibitory mechanism of lignin on trypsin was examined, including the interaction type, thermodynamic parameters, structure, reaction site and molecular docking. RESULTS: The isolated lignin presented an inhibitory effect on trypsin activity with an IC50 value of 1.35 µmol L-1 . This inhibition was a mixed linear type with a constant Ki of 3.92 µmol L-1 . The lignin could bind with the key amino acid residue Ser195 on the active site of the trypsin molecule to inhibit its activity, and the phenolic hydroxyl group and -OH on the ß-O-4 structure of the lignin molecule were the major groups bound with trypsin. CONCLUSION: These results illustrate the inhibitory effects of Canna edulis residue lignin on protease, which helps with respect to understanding the possible application of lignin in the food industry in functional foods. © 2020 Society of Chemical Industry.

Heterogeneous immunogenomic features and distinct escape mechanisms in multifocal hepatocellular carcinoma.

BACKGROUND & AIMS: The presence of multifocal tumors, developed either from intrahepatic metastasis (IM) or multicentric occurrence (MO), is a distinct feature of hepatocellular carcinoma (HCC). Immunogenomic characterization of multifocal HCC is important for understanding immune escape in different lesions and developing immunotherapy. METHODS: We combined whole-exome/transcriptome sequencing, multiplex immunostaining, immunopeptidomes, T cell receptor (TCR) sequencing and bioinformatic analyses of 47 tumors from 15 patients with HCC and multifocal lesions. RESULTS: IM and MO demonstrated distinct clonal architecture, mutational spectrum and genetic susceptibility. The immune microenvironment also displayed spatiotemporal heterogeneity, such as less T cell and more M2 macrophage infiltration in IM and higher expression of inhibitory immune checkpoints in MO. Similar to mutational profiles, shared neoantigens and TCR repertoires among tumors from the same patients were abundant in IM but scarce in MO. Combining neoantigen prediction and immunopeptidomes identified T cell-specific neoepitopes and achieved a high verification rate in vitro. Immunoediting mainly occurred in MO but not IM, due to the relatively low immune infiltration. Loss of heterozygosity of human leukocyte antigen (HLA) alleles, identified in 17% of multifocal HCC, hampered the ability of major histocompatibility complex to present neoantigens, especially in IM. An integrated analysis of Immunoscore, immunoediting, TCR clonality and HLA loss of heterozygosity in each tumor could stratify patients into 2 groups based on whether they have a high or low risk of recurrence (p = 0.038). CONCLUSION: Our study comprehensively characterized the genetic structure, neoepitope landscape, T cell profile and immunoediting status that collectively shape tumor evolution and could be used to optimize personalized immunotherapies for multifocal HCC. LAY SUMMARY: Immunogenomic features of multifocal hepatocellular carcinoma (HCC) are important for understanding immune-escape mechanisms and developing more effective immunotherapy. Herein, comprehensive immunogenomic characterization showed that diverse genomic structures within multifocal HCC would leave footprints on the immune landscape. Only a few tumors were under the control of immunosurveillance, while others evaded the immune system through multiple mechanisms that led to poor prognosis. Our study revealed heterogeneous immunogenomic landscapes and immune-constrained tumor evolution, the understanding of which could be used to optimize personalized immunotherapies for multifocal HCC.