Early detection and stratification of colorectal cancer using plasma cell-free DNA fragmentomic profiling.

Timely accurate and cost-efficient detection of colorectal cancer (CRC) is of great clinical importance. This study aims to establish prediction models for detecting CRC using plasma cell-free DNA (cfDNA) fragmentomic features. Whole-genome sequencing (WGS) was performed on cfDNA from 620 participants, including healthy individuals, patients with benign colorectal diseases and CRC patients. Using WGS data, three machine learning methods were compared to build prediction models for the stratification of CRC patients. The optimal model to discriminate CRC patients of all stages from healthy individuals achieved a sensitivity of 92.31% and a specificity of 91.14%, while the model to separate early-stage CRC patients (stage 0-II) from healthy individuals achieved a sensitivity of 88.8% and a specificity of 96.2%. Additionally, the cfDNA fragmentation profiles reflected disease-specific genomic alterations in CRC. Overall, this study suggests that cfDNA fragmentation profiles may potentially become a noninvasive approach for the detection and stratification of CRC.

Identification and validation of cuproptosis and disulfidptosis related genes in colorectal cancer.

Colorectal cancer, the third most prevalent malignant cancer, is associated with poor prognosis. Recent studies have investigated the mechanisms underlying cuproptosis and disulfidptosis in colorectal cancer. However, whether genes linked to these processes impact the prognosis of colorectal cancer patients through analogous mechanisms remains unclear. In this study, we developed a model of cuproptosis and disulfidptosis in colorectal cancer and concurrently explored the role of the pivotal model gene HSPA8 in colorectal cancer cell lines. Our results revealed a positive correlation between cuproptosis and disulfidptosis, both of which are emerging as protective factors for the prognosis of CRC patients. Consequently, a prognostic model encompassing HSPA8, PDCL3, CBX3, ATP6V1G1, TAF1D, RPL4, and RPL14 was constructed. Notably, the key gene in our model, HSPA8, exhibited heightened expression and was validated as a protective prognostic factor in colorectal cancer, exerting inhibitory effects on colorectal cancer cell proliferation. This study offers novel insights into the interplay between cuproptosis and disulfidptosis. The application of the prognostic model holds promise for more effectively predicting the overall survival of colorectal cancer patients.

The apple autophagy-related gene MdATG10 improves drought tolerance and water use efficiency in transgenic apple plants.

The Loess Plateau is the main apple production area in China; low precipitation is one of the most important factors limiting apple production here. Autophagy is a conserved process in eukaryotes that recycles cell contents or damaged macromolecules. Previously, we identified an autophagy-related gene MdATG10 from apple plants, which was involved in the responses to stressed conditions. In this study, we found that MdATG10 improved the drought tolerance and water use efficiency (WUE) of transgenic apple plants. MdATG10-overexpressing (OE) apple plants were more tolerant of short-term drought stress, as evidenced by their fewer drought-related injuries, compared with wild-type (WT) apple plants. In addition, the WUE of OE plants was higher than that of WT plants under long-term moderate water deficit conditions. The growth rate, biomass accumulation, photosynthetic efficiency, and stomatal aperture were higher in OE plants than in WT plants under long-term moderate drought conditions. During the process of adapting to drought, the expressions of genes involved in the abscisic acid (ABA) pathway were reduced in OE plants to decrease the synthesis of ABA, which helped maintain the stomatal opening for gas exchange. Furthermore, autophagic activity was higher in OE plants than in WT plants, as evidenced by the higher expressions of ATG genes and the greater number of autophagy bodies. In sum, our results suggested that overexpression of MdATG10 improved drought tolerance and WUE in apple plants, possibly by regulating stomatal movement and enhancing autophagic activity, which then enhanced the photosynthetic efficiency and reduced damage, as well as the reactive oxygen species (ROS) accumulation in apple plants.

MdHB7-like positively modulates apple salt tolerance by promoting autophagic activity and Na+ efflux.

Salt stress adversely affects the yield and quality of crops and limits their geographical distribution. Studying the functions and regulatory mechanisms of key genes in the salt stress response is important for breeding crops with enhanced stress resistance. Autophagy plays an important role in modulating the tolerance of plants to various types of abiotic stressors. However, the mechanisms underlying salt-induced autophagy are largely unknown. Cation/Ca2+ exchanger proteins enhance apple salt tolerance by inhibiting Na+ accumulation but the mechanism underlying the response to salt stress remains unclear. Here, we show that the autophagy-related gene MdATG18a modulated apple salt tolerance. Under salt stress, the autophagic activity, proline content, and antioxidant enzyme activities were higher and Na+ accumulation was lower in MdATG18a-overexpressing transgenic plants than in control plants. The use of an autophagy inhibitor during the salt treatment demonstrated that the regulatory function of MdATG18a depended on autophagy. The yeast-one-hybrid assay revealed that the homeodomain-leucine zipper (HD-Zip) transcription factor MdHB7-like directly bound to the MdATG18a promoter. Transcriptional regulation and genetic analyses showed that MdHB7-like enhanced salt-induced autophagic activity by promoting MdATG18a expression. The analysis of Na+ efflux rate in transgenic yeast indicated that MdCCX1 expression significantly promoted Na+ efflux. Promoter binding, transcriptional regulation, and genetic analyses showed that MdHB7-like promoted Na+ efflux and apple salt tolerance by directly promoting MdCCX1 expression, which was independent of the autophagy pathway. Overall, our findings provide insight into the mechanism underlying MdHB7-like-mediated salt tolerance in apple through the MdHB7-like-MdATG18a and MdHB7-like-MdCCX1 modules. These results will aid future studies on the mechanisms underlying stress-induced autophagy and the regulation of stress tolerance in plants.

MdNAC104 positively regulates apple cold tolerance via CBF-dependent and CBF-independent pathways.

Low temperature is the main environmental factor affecting the yield, quality and geographical distribution of crops, which significantly restricts development of the fruit industry. The NAC (NAM, ATAF1/2 and CUC2) transcription factor (TF) family is involved in regulating plant cold tolerance, but the mechanisms underlying these regulatory processes remain unclear. Here, the NAC TF MdNAC104 played a positive role in modulating apple cold tolerance. Under cold stress, MdNAC104-overexpressing transgenic plants exhibited less ion leakage and lower ROS (reactive oxygen species) accumulation, but higher contents of osmoregulatory substances and activities of antioxidant enzymes. Transcriptional regulation analysis showed that MdNAC104 directly bound to the MdCBF1 and MdCBF3 promoters to promote expression. In addition, based on combined transcriptomic and metabolomic analyses, as well as promoter binding and transcriptional regulation analyses, we found that MdNAC104 stimulated the accumulation of anthocyanin under cold conditions by upregulating the expression of anthocyanin synthesis-related genes, including MdCHS-b, MdCHI-a, MdF3H-a and MdANS-b, and increased the activities of the antioxidant enzymes by promoting the expression of the antioxidant enzyme-encoding genes MdFSD2 and MdPRXR1.1. In conclusion, this study revealed the MdNAC104 regulatory mechanism of cold tolerance in apple via CBF-dependent and CBF-independent pathways.

Novel Compounds with Promising IDO1 Inhibitory Activity As New Cancer Drug Candidates: Derivatives Of N, N'-diphenylurea Linked With 1,2,3-triazole.

To explore potential indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors, we designed a series of compounds incorporating urea and 1,2,3-triazole structures. IDO1 enzymatic activity experiments with the synthesized compounds were used to verify their molecular-level activity; for instance, the half maximal inhibitory concentration value of compound 3c was 0.07 µM. Our research has yielded a series of novel IDO1 inhibitors which may be beneficial in the development of drugs targeting IDO1 for cancer treatment.

The ubiquitin-binding protein MdRAD23D1 mediates drought response by regulating degradation of the proline-rich protein MdPRP6 in apple (Malus domestica).

RAD23 (RADIATION SENSITIVE23) proteins are a group of UBL-UBA (ubiquitin-like-ubiquitin-associated) proteins that shuttle ubiquitylated proteins to the 26S proteasome for breakdown. Drought stress is a major environmental constraint that limits plant growth and production, but whether RAD23 proteins are involved in this process is unclear. Here, we demonstrated that a shuttle protein, MdRAD23D1, mediated drought response in apple plants (Malus domestica). MdRAD23D1 levels increased under drought stress, and its suppression resulted in decreased stress tolerance in apple plants. Through in vitro and in vivo assays, we demonstrated that MdRAD23D1 interacted with a proline-rich protein MdPRP6, resulting in the degradation of MdPRP6 by the 26S proteasome. And MdRAD23D1 accelerated the degradation of MdPRP6 under drought stress. Suppression of MdPRP6 resulted in enhanced drought tolerance in apple plants, mainly because the free proline accumulation is changed. And the free proline is also involved in MdRAD23D1-mediated drought response. Taken together, these findings demonstrated that MdRAD23D1 and MdPRP6 oppositely regulated drought response. MdRAD23D1 levels increased under drought, accelerating the degradation of MdPRP6. MdPRP6 negatively regulated drought response, probably by regulating proline accumulation. Thus, "MdRAD23D1-MdPRP6" conferred drought stress tolerance in apple plants.

Fruit crops combating drought: Physiological responses and regulatory pathways.

Drought is a common stress in agricultural production. Thus, it is imperative to understand how fruit crops respond to drought and to develop drought-tolerant varieties. This paper provides an overview of the effects of drought on the vegetative and reproductive growth of fruits. We summarize the empirical studies that have assessed the physiological and molecular mechanisms of the drought response in fruit crops. This review focuses on the roles of calcium (Ca2+) signaling, abscisic acid (ABA), reactive oxygen species signaling, and protein phosphorylation underlying the early drought response in plants. We review the resulting downstream ABA-dependent and ABA-independent transcriptional regulation in fruit crops under drought stress. Moreover, we highlight the positive and negative regulatory mechanisms of microRNAs in the drought response of fruit crops. Lastly, strategies (including breeding and agricultural practices) to improve the drought resistance of fruit crops are outlined.

mTORC2-AKT-LAT1 signalling participates in methionine-induced ß-CASEIN expression in mammary epithelial cells of dairy cows.

This study investigated the role of the mammalian target of rapamycin complex 2 (mTORC2)-protein kinase B (AKT) signalling in methionine (Met)-induced L-type amino acid transporter 1 (LAT1) expression and milk protein production. Primary mammary epithelial cells (MECs) from mammary parenchymal tissues of three lactating cows and MAC-T bovine MECs were cultured with or without 0.6 mM Met. Rapamycin-insensitive companion of mTOR (RICTOR) siRNA, the mTORC1 inhibitor rapamycin and the AKT activator SC79 were used to evaluate the effects of mTORC2-AKT signalling on Met-induced LAT1 expression and function. Each experiment was performed three times. Data were analysed with a two-sided unpaired t test or ANOVA with the Bonferroni multiple-comparison test. Western blotting showed that Met stimulation increased RICTOR expression (~244.67%; p < 0.05; control, 0.15 ± 0.026; Met, 0.517 ± 0.109) and AKT-S473 levels (~281.42%; p < 0.01; control, 0.253 ± 0.067; Met, 0.965 ± 0.019) in both primary MECs and MAC-T cells. Rapamycin-induced mTORC1 signalling inhibition decreased only Met-induced ß-CASEIN expression by ~21.24% (p < 0.01; Met, 0.777 ± 0.01; Met and rapamycin, 0.612 ± 0.04) and did not affect Met-stimulated AKT-S473 levels, suggesting that mTORC2-AKT activation upon Met stimulation also contributes to milk protein synthesis. LAT1 participates in Met-induced ß-CASEIN expression. In dairy cow MECs, mTORC2 inhibition by RICTOR siRNA decreased LAT1 levels on the plasma membrane by ~45.13% (p < 0.01; control, 0.359 ± 0.006; siRICTOR, 0.197 ± 0.004). However, SC79-induced AKT activation had the opposite effect (p < 0.01). In primary MECs and MAC-T cells, Met stimulation increased cytosolic and plasma membrane LAT1 expression respectively (MECs, 113.98% and 58.43%; MAC-T, 165.85% and 396.39%; p < 0.05). However, RICTOR siRNA significantly reduced Met-induced plasma membrane LAT1 expression (~76.48%; Met, 0.539 ± 0.05; Met and siRICTOR, 0.127 ± 0.012; p < 0.05). Thus, Met increased LAT1 expression and function via mTORC2-AKT signalling, upregulating milk protein synthesis in dairy cow MECs.

Identification of CBPA as a New Inhibitor of PD-1/PD-L1 Interaction.

Targeting of the PD-1/PD-L1 immunologic checkpoint is believed to have provided a real breakthrough in the field of cancer therapy in recent years. Due to the intrinsic limitations of antibodies, the discovery of small-molecule inhibitors blocking PD-1/PD-L1 interaction has gradually opened valuable new avenues in the past decades. In an effort to discover new PD-L1 small molecular inhibitors, we carried out a structure-based virtual screening strategy to rapidly identify the candidate compounds. Ultimately, CBPA was identified as a PD-L1 inhibitor with a KD value at the micromolar level. It exhibited effective PD-1/PD-L1 blocking activity and T-cell-reinvigoration potency in cell-based assays. CBPA could dose-dependently elevate secretion levels of IFN-γ and TNF-α in primary CD4+ T cells in vitro. Notably, CBPA exhibited significant in vivo antitumor efficacy in two different mouse tumor models (a MC38 colon adenocarcinoma model and a melanoma B16F10 tumor model) without the induction of observable liver or renal toxicity. Moreover, analyses of the CBPA-treated mice further showed remarkably increased levels of tumor-infiltrating CD4+ and CD8+ T cells and cytokine secretion in the tumor microenvironment. A molecular docking study suggested that CBPA embedded relatively well into the hydrophobic cleft formed by dimeric PD-L1, occluding the PD-1 interaction surface of PD-L1. This study suggests that CBPA could work as a hit compound for the further design of potent inhibitors targeting the PD-1/PD-L1 pathway in cancer immunotherapy.

The Valsa Mali effector Vm1G-1794 protects the aggregated MdEF-Tu from autophagic degradation to promote infection in apple.

Macroautophagy/autophagy is a conserved degradation pathway in eukaryotes that is required for recycling unwanted intracellular components, maintaining homeostasis, and coping with biotic and abiotic stresses. Pathogens have evolved to subvert autophagic machinery by secreting host cell-entering effector proteins. Here, we provided evidence that an apple autophagy-related gene MdATG8i, activated autophagy and contributed to resistance against Valsa canker caused by Valsa Mali (Vm) when being overexpressed in apple. MdATG8i interacted with a plastid elongation factor Tu (MdEF-Tu) which became insoluble and aggregated during Vm infection and was degraded through the autophagy pathway. Intriguingly, we identified a highly-induced effector secreted from Vm, Vm1G-1794, which competitively interacted with MdATG8i, suppressed autophagy, and depleted MdEF-Tu out of MdATG8i complexes. The formation of stable MdEF-Tu aggregates caused by Vm1G-1794 promoted the susceptibility of apple to Vm. Overall, our study demonstrated that MdATG8i contributed to Vm resistance by targeting and degrading MdEF-Tu, and Vm1G-1794 competed with MdEF-Tu to target MdATG8i and prevent MdEF-Tu degradation, thus favoring infection.Abbreviations: 35S: cauliflower mosaic virus 35S promoter; AIM: ATG8-interacting motif; ATG8-PE: ATG8 conjugated with phosphatidylethanolamine; BiFC: biomolecular fluorescence complementation; Con A: concanamycin A; Co-IP: co-immunoprecipitation; DEPs: differentially expressed proteins; DMSO: dimethyl sulfoxide; GFP: green fluorescent protein; hpt: hours post-treatment; LCI: luciferase complementation imaging; MdATG8i: autophagy-related protein 8i in Malus domestica; MDC: monodansylcadaverine; MdEF-Tu: elongation factor Tu in Malus domestica; MdNBR1: neighbor of BRCA1 in Malus domestica; N. benthamiana: Nicotiana benthamiana; OE: overexpression; PAMP: pathogen-associated molecular pattern; PTI: pattern-triggered immunity; qRT-PCR: quantitative reverse transcription PCR; RFP: red fluorescent protein; RNAi: RNA interference; ROS: reactive oxygen species; Ub: ubiquitin; V. Mali: Valsa Mali; WT: wild-type plant; YFP: yellow fluorescent protein.

Discovery of Novel 1,2,3-triazole Derivatives as IDO1 Inhibitors.

Indoleamine 2,3-dioxygenase 1 (IDO1) has received much attention as an immunomodulatory enzyme in the field of cancer immunotherapy. While several IDO1 inhibitors have entered clinical trials, there are currently no IDO1 inhibitor drugs on the market. To explore potential IDO1 inhibitors, we designed a series of compounds with urea and 1,2,3-triazole structures. Organic synthesis and IDO1 enzymatic activity experiments verified the molecular-level activities of the designed compounds, and the IC50 value of compound 3a was 0.75 µM. Molecular docking and quantum mechanical studies further explained the binding mode and reaction potential of compound 3a with IDO1. Our research has resulted in a series of novel IDO1 inhibitors, which is beneficial to the development of drugs targeting IDO1 in numerous cancer diseases.

PPARγ-AGPAT6 signaling mediates acetate-induced mTORC1 activation and milk fat synthesis in mammary epithelial cells of dairy cows.

This research communication investigated the role and the underlying mechanism of sn-1-acylglycerol-3-phosphate O-acyltransferase 6 (AGPAT6) in acetate-induced mTORC1 signaling activation and milk fat synthesis in dairy cow mammary epithelial cells. The data showed AGPAT6 knockdown significantly decreased acetate-induced phosphorylation of mTORC1 signaling molecules and intracellular triacylglycerol (TAG) content, whereas this inhibition effect was reversed after the addition of 16:0,18:1 phosphatidic acid (PA), suggesting that AGPAT6 could generate PA in response to acetate simulation, that in turn activates mTORC1 signaling. PPARγ is the upstream regulator of AGPAT6 upon acetate stimulation. Luciferase assay with clones containing various deletions and mutation in AGPAT6 promoter showed that there is a RXRα binding sequence located at -96 bp of AGPAT6 promoter. Acetate stimulation significantly increased the interaction between PPARγ and AGPAT6 via this RXRα binding site. Taken together, our data indicated that AGPAT6 could activate mTORC1 signaling by producing PA during acetate-induced milk fat synthesis, and PPARγ acts as a transcription factor to mediate the effect of acetate on AGPAT6 via RXRα.

A C1q domain-containing protein in Pinctada fucata contributes to the innate immune response and elimination of the pathogen.

The C1q domain-containing proteins (C1qDCs) in bivalve mollusks primarily exist as the globular head C1q proteins (ghC1qs), for the N-terminal collagen domains were very rare in bivalves, although widespread in C1qDCs of vertebrates. In this work, the C1qDC protein with only a ghC1q domain (named as Pf-ghC1q) was identified from Pinctada fucata, and molecular characterization, gene expression, and functional studies were also conducted. The full-length cDNA sequence of Pf-ghC1q was 738 bp long, containing a signal peptide of 23 residues encoded. Pf-ghC1q was clustered with some C1qDCs from other invertebrates in the phylogenetic tree analysis, rather than vertebrates. Pf-ghC1q was detected in all tested tissues, including the mantle, hemocyte, digestive gland, gill, and adductor muscle. Moreover, the expression levels of Pf-ghC1q were up-regulated in all tested tissues after the challenge with Vibrio alginolyticus 4 h later. The expression level of Pf-ghC1q was inhibited by specific si-276, and the low level of Pf-ghC1q affected the phagocytosis efficiency of V. alginolyticus by hemocytes. These results indicated that Pf-ghC1q may participate in the target recognition of V. alginolyticus and the phagocytosis process in the immune response of P. fucata.

Target of rapamycin (TOR) regulates the response to low nitrogen stress via autophagy and hormone pathways in Malus hupehensis.

Target of rapamycin (TOR) is a highly conserved master regulator in eukaryotes; it regulates cell proliferation and growth by integrating different signals. However, little is known about the function of TOR in perennial woody plants. Different concentrations of AZD8055 (an inhibitor of TOR) were used in this study to investigate the role of TOR in the response to low nitrogen (N) stress in the wild apple species Malus hupehensis. Low N stress inhibited the growth of M. hupehensis plants, and 1 µM AZD alleviated this effect. Plants supplied with 1 µM AZD had higher photosynthetic capacity, which promoted the accumulation of biomass, as well as higher contents of N and anthocyanins and lower content of starch. Exogenous application of 1 µM AZD also promoted the development of the root system. Plants supplied with at least 5 µM AZD displayed early leaf senescence. RNA-seq analysis indicated that TOR altered the expression of genes related to the low N stress response, such as genes involved in photosystem, starch metabolism, autophagy, and hormone metabolism. Further analysis revealed altered autophagy in plants supplied with AZD under low N stress; the metabolism of plant hormones also changed following AZD supplementation. In sum, our findings revealed that appropriate inhibition of TOR activated autophagy and jasmonic acid signaling in M. hupehensis, which allowed plants to cope with low N stress. Severe TOR inhibition resulted in the excessive accumulation of salicylic acid, which probably led to programmed cell death in M. hupehensis.

Design, synthesis and biological evaluation of erlotinib-based IDO1 inhibitors.

Erlotinib is a highly specific and reversible epidermal growth factor receptor tyrosine kinase inhibitor for the targeted therapy of non-small-cell lung cancer (NSCLC) However, the efficacy of erlotinib is limited because the development of drug resistance during chemotherapy. Indoleamine 2,3-dioxygenase-1 (IDO1) is a rate-limiting tryptophan catabolic enzyme that is activated in many human cancers. In this study, we designed a series of erlotinib-based 1,2,3-triazole compounds by combining erlotinib with phenyl or benzyl azide. Attentive FP prediction model was used to predict the bioactivity of those compounds. We discovered that most of the erlotinib-based 1,2,3-triazole compounds are capable of suppressing IDO1 activities in vitro experiments. Among them, compound 14b (IC50 = 0.59 ± 0.05 µM) had the strongest inhibitory effect on IDO1. In addition, compound 14b significantly inhibited tumor growth comparable to the antitumor activity of erlotinib and the IDO1 inhibitor epacadostat in murine tumor models.

Novel 1,2,3-Triazole Erlotinib Derivatives as Potent IDO1 Inhibitors: Design, Drug-Target Interactions Prediction, Synthesis, Biological Evaluation, Molecular Docking and ADME Properties Studies.

Indoleamine 2,3-dioxygenase 1 (IDO1) plays a predominant role in cancer immunotherapy which catalyzes the initial and rate limiting steps of the kynurenine pathway as a key enzyme. To explore novel IDO1 inhibitors, five derivatives of erlotinib-linked 1,2,3-triazole compounds were designed by using a structure-based drug design strategy. Drug-target interactions (DTI) were predicted by DeePurpose, an easy-to-use deep learning library that contains more than 50 algorithms. The DTI prediction results suggested that the designed molecules have potential inhibitory activities for IDO1. Chemical syntheses and bioassays showed that the compounds exhibited remarkable inhibitory activities against IDO1, among them, compound e was the most potent with an IC50 value of 0.32 ± 0.07 µM in the Hela cell assay. The docking model and ADME analysis exhibited that the effective interactions of these compounds with heme iron and better drug-likeness ensured the IDO1 inhibitory activities. The studies suggested that compound e was a novel and interesting IDO1 inhibitor for further development.

Genome-Wide Analysis of the WRKY Gene Family in Malus domestica and the Role of MdWRKY70L in Response to Drought and Salt Stresses.

The WRKY transcription factors are unique regulatory proteins in plants, which are important in the stress responses of plants. In this study, 113 WRKY genes were identified from the apple genome GDDH13 and a comprehensive analysis was performed, including chromosome mapping, and phylogenetic, motif and collinearity analysis. MdWRKYs are expressed in different tissues, such as seeds, flowers, stems and leaves. We analyzed seven WRKY proteins in different groups and found that all of them were localized in the nucleus. Among the 113 MdWRKYs, MdWRKY70L was induced by both drought and salt stresses. Overexpression of it in transgenic tobacco plants conferred enhanced stress tolerance to drought and salt. The malondialdehyde content and relative electrolyte leakage values were lower, while the chlorophyll content was higher in transgenic plants than in the wild-type under stressed conditions. In conclusion, this study identified the WRKY members in the apple genome GDDH13, and revealed the function of MdWRKY70L in the response to drought and salt stresses.

Thermodynamic integration combined with molecular dynamic simulations to explore the cross-resistance mechanism of isoniazid and ethionamide.

Tuberculosis is an ancient disease of mankind, and its causative bacterium is Mycobacterium tuberculosis. Isoniazid is one of the most effective first-line antituberculosis drugs. As prodrugs, it and its derivative ethionamide act on enoyl-acyl carrier protein reductase (InhA) after being oxidized in bacteria, and kill the bacteria by inhibiting the formation of M. tuberculosis cell walls. However, the S94A mutation of InhA causes M. tuberculosis to develop cross-resistance to isoniazid and ethionamide. This work is dedicated to studying the cross-resistance mechanism of isoniazid and ethionamide through theoretical calculations. First, thermodynamic integral simulations are used to accurately calculate the relative binding energy of two drugs in the mutant and wild-type system. Furthermore, through classic molecular dynamic simulations and molecular mechanics generalized-Born surface area calculation, some key residues are identified and the binding affinity of isoniazid and ethionamide reduced by 9-13 kcal/mol due to S94A mutation. The hydrogen bond between Ala94 and isoniazid (ethionamide) disappeared and the energy contribution of Ala94 decreased after the mutation. In addition, the dynamic network analysis indicated that the mutation of Ser94 also indirectly affected the conformation of key residues such as Met147, Thr196, and Leu97, resulting in a reduction in the energy contribution of these residues. Finally, the binding conformation of isoniazid and ethionamide has also undergone major changes. The obtained results could provide valuable information for the future molecular design to overcome the drug resistance.

Overexpression of MdASMT9, an N-acetylserotonin methyltransferase gene, increases melatonin biosynthesis and improves water-use efficiency in transgenic apple.

Improving apple water-use efficiency (WUE) is increasingly desirable in the face of global climate change. Melatonin is a pleiotropic molecule that functions in plant development and stress tolerance. In apple, exogenous application of melatonin has been largely investigated, but melatonin biosynthesis and its physiological roles remain elusive. In the plant biosynthetic pathway of melatonin, the last and key step is that N-acetylserotonin methyltransferase (ASMT) converts N-acetylserotonin into melatonin. Here, we identified an apple ASMT gene, MdASMT9, using homology-based cloning and in vitro enzyme assays. Overexpression of MdASMT9 significantly increased melatonin accumulation in transgenic apple lines. Moreover, an enhanced WUE was observed in the MdASMT9-overexpressing apple lines. Under well-watered conditions, this increase in WUE was attributed to an enhancement of photosynthetic rate and stomatal aperture via a reduction in abscisic acid biosynthesis. By contrast, under long-term moderate water deficit conditions, regulations in photoprotective mechanisms, stomatal behavior, osmotic adjustment and antioxidant activity enhanced the WUE in transgenic apple lines. Taken together, our findings shed light on the positive effect of MdASMT9 on improving WUE of apple by modulating melatonin biosynthesis.