Metastable hexagonal close-packed palladium hydride in liquid cell TEM.

Metastable phases-kinetically favoured structures-are ubiquitous in nature1,2. Rather than forming thermodynamically stable ground-state structures, crystals grown from high-energy precursors often initially adopt metastable structures depending on the initial conditions, such as temperature, pressure or crystal size1,3,4. As the crystals grow further, they typically undergo a series of transformations from metastable phases to lower-energy and ultimately energetically stable phases1,3,4. Metastable phases sometimes exhibit superior physicochemical properties and, hence, the discovery and synthesis of new metastable phases are promising avenues for innovations in materials science1,5. However, the search for metastable materials has mainly been heuristic, performed on the basis of experiences, intuition or even speculative predictions, namely 'rules of thumb'. This limitation necessitates the advent of a new paradigm to discover new metastable phases based on rational design. Such a design rule is embodied in the discovery of a metastable hexagonal close-packed (hcp) palladium hydride (PdHx) synthesized in a liquid cell transmission electron microscope. The metastable hcp structure is stabilized through a unique interplay between the precursor concentrations in the solution: a sufficient supply of hydrogen (H) favours the hcp structure on the subnanometre scale, and an insufficient supply of Pd inhibits further growth and subsequent transition towards the thermodynamically stable face-centred cubic structure. These findings provide thermodynamic insights into metastability engineering strategies that can be deployed to discover new metastable phases.

Localized Inhibition of Protein Phosphatase 1 by NUAK1 Promotes Spliceosome Activity and Reveals a MYC-Sensitive Feedback Control of Transcription.

Deregulated expression of MYC induces a dependence on the NUAK1 kinase, but the molecular mechanisms underlying this dependence have not been fully clarified. Here, we show that NUAK1 is a predominantly nuclear protein that associates with a network of nuclear protein phosphatase 1 (PP1) interactors and that PNUTS, a nuclear regulatory subunit of PP1, is phosphorylated by NUAK1. Both NUAK1 and PNUTS associate with the splicing machinery. Inhibition of NUAK1 abolishes chromatin association of PNUTS, reduces spliceosome activity, and suppresses nascent RNA synthesis. Activation of MYC does not bypass the requirement for NUAK1 for spliceosome activity but significantly attenuates transcription inhibition. Consequently, NUAK1 inhibition in MYC-transformed cells induces global accumulation of RNAPII both at the pause site and at the first exon-intron boundary but does not increase mRNA synthesis. We suggest that NUAK1 inhibition in the presence of deregulated MYC traps non-productive RNAPII because of the absence of correctly assembled spliceosomes.

Staphylococcus aureus induces drug resistance in cancer T cells in Sézary syndrome.

ABSTRACT: Patients with Sézary syndrome (SS), a leukemic variant of cutaneous T-cell lymphoma (CTCL), are prone to Staphylococcus aureus infections and have a poor prognosis due to treatment resistance. Here, we report that S aureus and staphylococcal enterotoxins (SE) induce drug resistance in malignant T cells against therapeutics commonly used in CTCL. Supernatant from patient-derived, SE-producing S aureus and recombinant SE significantly inhibit cell death induced by histone deacetylase (HDAC) inhibitor romidepsin in primary malignant T cells from patients with SS. Bacterial killing by engineered, bacteriophage-derived, S aureus-specific endolysin (XZ.700) abrogates the effect of S aureus supernatant. Similarly, mutations in major histocompatibility complex (MHC) class II binding sites of SE type A (SEA) and anti-SEA antibody block induction of resistance. Importantly, SE also triggers resistance to other HDAC inhibitors (vorinostat and resminostat) and chemotherapeutic drugs (doxorubicin and etoposide). Multimodal single-cell sequencing indicates T-cell receptor (TCR), NF-κB, and JAK/STAT signaling pathways (previously associated with drug resistance) as putative mediators of SE-induced drug resistance. In support, inhibition of TCR-signaling and Protein kinase C (upstream of NF-κB) counteracts SE-induced rescue from drug-induced cell death. Inversely, SE cannot rescue from cell death induced by the proteasome/NF-κB inhibitor bortezomib. Inhibition of JAK/STAT only blocks rescue in patients whose malignant T-cell survival is dependent on SE-induced cytokines, suggesting 2 distinct ways SE can induce drug resistance. In conclusion, we show that S aureus enterotoxins induce drug resistance in primary malignant T cells. These findings suggest that S aureus enterotoxins cause clinical treatment resistance in patients with SS, and antibacterial measures may improve the outcome of cancer-directed therapy in patients harboring S aureus.

In Vivo Base Editing of Scn5a Rescues Type 3 Long QT Syndrome in Mice.

BACKGROUND: Pathogenic variants in SCN5A can result in long QT syndrome type 3, a life-threatening genetic disease. Adenine base editors can convert targeted A T base pairs to G C base pairs, offering a promising tool to correct pathogenic variants. METHODS: We generated a long QT syndrome type 3 mouse model by introducing the T1307M pathogenic variant into the Scn5a gene. The adenine base editor was split into 2 smaller parts and delivered into the heart by adeno-associated virus serotype 9 (AAV9-ABEmax) to correct the T1307M pathogenic variant. RESULTS: Both homozygous and heterozygous T1307M mice showed significant QT prolongation. Carbachol administration induced Torsades de Pointes or ventricular tachycardia for homozygous T1307M mice (20%) but not for heterozygous or wild-type mice. A single intraperitoneal injection of AAV9-ABEmax at postnatal day 14 resulted in up to 99.20% Scn5a transcripts corrected in T1307M mice. Scn5a mRNA correction rate >60% eliminated QT prolongation; Scn5a mRNA correction rate <60% alleviated QT prolongation. Partial Scn5a correction resulted in cardiomyocytes heterogeneity, which did not induce severe arrhythmias. We did not detect off-target DNA or RNA editing events in ABEmax-treated mouse hearts. CONCLUSIONS: These findings show that in vivo AAV9-ABEmax editing can correct the variant Scn5a allele, effectively ameliorating arrhythmia phenotypes. Our results offer a proof of concept for the treatment of hereditary arrhythmias.

Mass spectrometry-based draft of the mouse proteome.

The laboratory mouse ranks among the most important experimental systems for biomedical research and molecular reference maps of such models are essential informational tools. Here, we present a quantitative draft of the mouse proteome and phosphoproteome constructed from 41 healthy tissues and several lines of analyses exemplify which insights can be gleaned from the data. For instance, tissue- and cell-type resolved profiles provide protein evidence for the expression of 17,000 genes, thousands of isoforms and 50,000 phosphorylation sites in vivo. Proteogenomic comparison of mouse, human and Arabidopsis reveal common and distinct mechanisms of gene expression regulation and, despite many similarities, numerous differentially abundant orthologs that likely serve species-specific functions. We leverage the mouse proteome by integrating phenotypic drug (n > 400) and radiation response data with the proteomes of 66 pancreatic ductal adenocarcinoma (PDAC) cell lines to reveal molecular markers for sensitivity and resistance. This unique atlas complements other molecular resources for the mouse and can be explored online via ProteomicsDB and PACiFIC.

An intra-tumoral niche maintains and differentiates stem-like CD8 T cells.

Tumour-infiltrating lymphocytes are associated with a survival benefit in several tumour types and with the response to immunotherapy1-8. However, the reason some tumours have high CD8 T cell infiltration while others do not remains unclear. Here we investigate the requirements for maintaining a CD8 T cell response against human cancer. We find that CD8 T cells within tumours consist of distinct populations of terminally differentiated and stem-like cells. On proliferation, stem-like CD8 T cells give rise to more terminally differentiated, effector-molecule-expressing daughter cells. For many T cells to infiltrate the tumour, it is critical that this effector differentiation process occur. In addition, we show that these stem-like T cells reside in dense antigen-presenting-cell niches within the tumour, and that tumours that fail to form these structures are not extensively infiltrated by T cells. Patients with progressive disease lack these immune niches, suggesting that niche breakdown may be a key mechanism of immune escape.

The role of PCSK9 in glomerular lipid accumulation and renal injury in diabetic kidney disease.

AIMS/HYPOTHESIS: Glomerular lipid accumulation is a defining feature of diabetic kidney disease (DKD); however, the precise underlying mechanism requires further elucidation. Recent evidence suggests a role for proprotein convertase subtilisin/kexin type 9 (PCSK9) in intracellular lipid homeostasis. Although PCSK9 is present in kidneys, its role within kidney cells and relevance to renal diseases remain largely unexplored. Therefore, we investigated the role of intracellular PCSK9 in regulating lipid accumulation and homeostasis in the glomeruli and podocytes under diabetic conditions. Furthermore, we aimed to identify the pathophysiological mechanisms responsible for the podocyte injury that is associated with intracellular PCSK9-induced lipid accumulation in DKD. METHODS: In this study, glomeruli were isolated from human kidney biopsy tissues, and glomerular gene-expression analysis was performed. Also, db/db and db/m mice were used to perform glomerular gene-expression profiling. We generated DKD models using a high-fat diet and low-dose intraperitoneal streptozocin injection in C57BL/6 and Pcsk9 knockout (KO) mice. We analysed cholesterol and triacylglycerol levels within the kidney cortex. Lipid droplets were evaluated using BODIPY staining. We induced upregulation and downregulation of PCSK9 expression in conditionally immortalised mouse podocytes using lentivirus and siRNA transfection techniques, respectively, under diabetic conditions. RESULTS: A significant reduction in transcription level of PCSK9 was observed in glomeruli of individuals with DKD. PCSK9 expression was also reduced in podocytes of animals under diabetic conditions. We observed significantly higher lipid accumulation in kidney tissues of Pcsk9 KO DKD mice compared with wild-type (WT) DKD mice. Additionally, Pcsk9 KO mouse models of DKD exhibited a significant reduction in mitochondria number vs WT models, coupled with a significant increase in mitochondrial size. Moreover, albuminuria and podocyte foot process effacement were observed in WT and Pcsk9 KO DKD mice, with KO DKD mice displaying more pronounced manifestations. Immortalised mouse podocytes exposed to diabetic stimuli exhibited heightened intracellular lipid accumulation, mitochondrial injury and apoptosis, which were ameliorated by Pcsk9 overexpression and aggravated by Pcsk9 knockdown in mouse podocytes. CONCLUSIONS/INTERPRETATION: The downregulation of PCSK9 in podocytes is associated with lipid accumulation, which leads to mitochondrial dysfunction, cell apoptosis and renal injury. This study sheds new light on the potential involvement of PCSK9 in the pathophysiology of glomerular lipid accumulation and podocyte injury in DKD.

UHMK1 is a novel splicing regulatory kinase.

The U2AF Homology Motif Kinase 1 (UHMK1) is the only kinase that contains the U2AF homology motif, a common protein interaction domain among splicing factors. Through this motif, UHMK1 interacts with the splicing factors SF1 and SF3B1, known to participate in the 3' splice site recognition during the early steps of spliceosome assembly. Although UHMK1 phosphorylates these splicing factors in vitro, the involvement of UHMK1 in RNA processing has not previously been demonstrated. Here, we identify novel putative substrates of this kinase and evaluate UHMK1 contribution to overall gene expression and splicing, by integrating global phosphoproteomics, RNA-seq, and bioinformatics approaches. Upon UHMK1 modulation, 163 unique phosphosites were differentially phosphorylated in 117 proteins, of which 106 are novel potential substrates of this kinase. Gene Ontology analysis showed enrichment of terms previously associated with UHMK1 function, such as mRNA splicing, cell cycle, cell division, and microtubule organization. The majority of the annotated RNA-related proteins are components of the spliceosome but are also involved in several steps of gene expression. Comprehensive analysis of splicing showed that UHMK1 affected over 270 alternative splicing events. Moreover, splicing reporter assay further supported UHMK1 function on splicing. Overall, RNA-seq data demonstrated that UHMK1 knockdown had a minor impact on transcript expression and pointed to UHMK1 function in epithelial-mesenchymal transition. Functional assays demonstrated that UHMK1 modulation affects proliferation, colony formation, and migration. Taken together, our data implicate UHMK1 as a splicing regulatory kinase, connecting protein regulation through phosphorylation and gene expression in key cellular processes.

KDM5 family as therapeutic targets in breast cancer: Pathogenesis and therapeutic opportunities and challenges.

Breast cancer (BC) is the most frequent malignant cancer diagnosis and is a primary factor for cancer deaths in women. The clinical subtypes of BC include estrogen receptor (ER) positive, progesterone receptor (PR) positive, human epidermal growth factor receptor 2 (HER2) positive, and triple-negative BC (TNBC). Based on the stages and subtypes of BC, various treatment methods are available with variations in the rates of progression-free disease and overall survival of patients. However, the treatment of BC still faces challenges, particularly in terms of drug resistance and recurrence. The study of epigenetics has provided new ideas for treating BC. Targeting aberrant epigenetic factors with inhibitors represents a promising anticancer strategy. The KDM5 family includes four members, KDM5A, KDM5B, KDM5C, and KDMD, all of which are Jumonji C domain-containing histone H3K4me2/3 demethylases. KDM5 proteins have been extensively studied in BC, where they are involved in suppressing or promoting BC depending on their specific upstream and downstream pathways. Several KDM5 inhibitors have shown potent BC inhibitory activity in vitro and in vivo, but challenges still exist in developing KDM5 inhibitors. In this review, we introduce the subtypes of BC and their current therapeutic options, summarize KDM5 family context-specific functions in the pathobiology of BC, and discuss the outlook and pitfalls of KDM5 inhibitors in this disease.

Novel Use of the Falciform Ligament for MHV Reconstruction During Laparoscopic Hepatectomy of Colorectal Liver Metastasis.

BACKGROUND: Laparoscopic hepatectomy (LH) with oncological R0 resection combined with systemic therapy offers the best chance of cure for colorectal liver metastasis. However, tumors in vicinity of major hepatic veins require complex technique. Parenchyma-sparing resection with involved vein resection and peritoneal patch reconstruction could be an efficacious alternative to preserve liver volume for adjuvant chemotherapy and avoid venous congestion of the remnant liver.1,2 METHODS: A 64-year-old female, with history of colon cancer, had new diagnosis of liver metastatic tumor of S8 (2.8 cm), which was considering encroached on middle hepatic vein (MHV) with distal part patent. Thus margin-negative, parenchyma-sparing liver resection with involved vein resection and proximal MHV reconstruction was indicated for oncological radicality. RESULTS: With the patient in modified French position, we dissected falciform ligament and right coronary ligament to expose the crypt between right hepatic vein (RHV) and MHV. Intraoperative ultrasound localized the tumor and resection margin. Parenchymal dissection was performed caudally to cranially, left to right, to ligate dorsal branch of G8 (G8d) and V8 and expose main trunk of MHV. The involved side-wall of MHV was incised after the proximal and distal parts clamped. Peritoneal patch was harvested from falciform ligament to repair MHV side-wall before clamps released. The patient had an uneventful recovery and remained disease-free at 1 year postoperatively with patency of distal MHV by image. CONCLUSIONS: LH with MHV reconstruction by falciform ligament for metastatic lesion is technically demanding but feasible with oncological radicality and volume preservation for adjuvant chemotherapy.

The changing morphology of the ventricular walls of mouse and human with increasing gestation.

That the highly trabeculated ventricular walls of the developing embryos transform to the arrangement during the fetal stages, when the mural architecture is dominated by the thickness of the compact myocardium, has been explained by the coalescence of trabeculations, often erroneously described as 'compaction'. Recent data, however, support differential rates of growth of the trabecular and compact layers as the major driver of change. Here, these processes were assessed quantitatively and visualized in standardized views. We used a larger dataset than has previously been available of mouse hearts, covering the period from embryonic day 10.5 to postnatal day 3, supported by images from human hearts. The volume of the trabecular layer increased throughout development, in contrast to what would be expected had there been 'compaction'. During the transition from embryonic to fetal life, the rapid growth of the compact layer diminished the proportion of trabeculations. Similarly, great expansion of the central cavity reduced the proportion of the total cavity made up of intertrabecular recesses. Illustrations of the hearts with the median value of left ventricular trabeculation confirm a pronounced growth of the compact wall, with prominence of the central cavity. This corresponds, in morphological terms, to a reduction in the extent of the trabecular layer. Similar observations were made in the human hearts. We conclude that it is a period of comparatively slow growth of the trabecular layer, rather than so-called compaction, that is the major determinant of the changing morphology of the ventricular walls of both mouse and human hearts.

Wavy water-to-air optical camera communication system using rolling shutter image sensor and long short term memory neural network.

We propose and experimentally demonstrate for the first time up to the authors' knowledge a wide field-of-view (FOV) water-to-air optical transmission using rolling-shutter (RS) based optical camera communication (OCC). Here, we evaluate the proposed OCC system without water ripple and with different percentage increases of water ripple. Long short term memory neural network (LSTM-NN) is utilized to mitigate the wavy water turbulence induced link outage and to decode 4-level pulse-amplitude-modulation (PAM4) RS pattern by meeting the pre-forward error correction bit-error-rate (pre-FEC BER = 3.8 × 10-3). We also evaluate the FOVs of the proposed water-to-air RS-based OCC system. This can be implemented by using different angular rotations of the camera. Experimental results show that the proposed OCC system can support ±70°, ± 30°, and ±30° rotations around the z-, y- and x-directions, respectively when operated at 6 kbit/s and decoded using LSTM-NN.

Screening and genetic engineering of marine-derived Aspergillus terreus for high-efficient production of lovastatin.

BACKGROUND: Lovastatin has widespread applications thanks to its multiple pharmacological effects. Fermentation by filamentous fungi represents the major way of lovastatin production. However, the current lovastatin productivity by fungal fermentation is limited and needs to be improved. RESULTS: In this study, the lovastatin-producing strains of Aspergillus terreus from marine environment were screened, and their lovastatin productions were further improved by genetic engineering. Five strains of A. terreus were isolated from various marine environments. Their secondary metabolites were profiled by metabolomics analysis using Ultra Performance Liquid Chromatography-Mass spectrometry (UPLC-MS) with Global Natural Products Social Molecular Networking (GNPS), revealing that the production of secondary metabolites was variable among different strains. Remarkably, the strain of A. terreus MJ106 could principally biosynthesize the target drug lovastatin, which was confirmed by High Performance Liquid Chromatography (HPLC) and gene expression analysis. By one-factor experiment, lactose was found to be the best carbon source for A. terreus MJ106 to produce lovastatin. To improve the lovastatin titer in A. terreus MJ106, genetic engineering was applied to this strain. Firstly, a series of strong promoters was identified by transcriptomic and green fluorescent protein reporter analysis. Then, three selected strong promoters were used to overexpress the transcription factor gene lovE encoding the major transactivator for lov gene cluster expression. The results revealed that compared to A. terreus MJ106, all lovE over-expression mutants exhibited significantly more production of lovastatin and higher gene expression. One of them, LovE-b19, showed the highest lovastatin productivity at a titer of 1512 mg/L, which represents the highest production level reported in A. terreus. CONCLUSION: Our data suggested that combination of strain screen and genetic engineering represents a powerful tool for improving the productivity of fungal secondary metabolites, which could be adopted for large-scale production of lovastatin in marine-derived A. terreus.

Cutting Edge: Neutrophils License the Maturation of Monocytes into Effective Antifungal Effectors.

Neutrophils are critical for the direct eradication of Aspergillus fumigatus conidia, but whether they mediate antifungal defense beyond their role as effectors is unclear. In this study, we demonstrate that neutrophil depletion impairs the activation of protective antifungal CCR2+ inflammatory monocytes. In the absence of neutrophils, monocytes displayed limited differentiation into monocyte-derived dendritic cells, reduced formation of reactive oxygen species, and diminished conidiacidal activity. Upstream regulator analysis of the transcriptional response in monocytes predicted a loss of STAT1-dependent signals as the potential basis for the dysfunction seen in neutrophil-depleted mice. We find that conditional removal of STAT1 on CCR2+ cells results in diminished antifungal monocyte responses, whereas exogenous administration of IFN-γ to neutrophil-depleted mice restores monocyte-derived dendritic cell maturation and reactive oxygen species production. Altogether, our findings support a critical role for neutrophils in antifungal immunity not only as effectors but also as important contributors to antifungal monocyte activation, in part by regulating STAT1-dependent functions.

The emerging role of deubiquitylating enzyme USP21 as a potential therapeutic target in cancer.

Although certain members of the Ubiquitin-specific peptidases (USPs) have been recognized as promising therapeutic targets for various diseases, research progress regarding USP21 has been relatively sluggish in its early stages. USP21 is a crucial member of the USPs subfamily, involved in diverse cellular processes such as apoptosis, DNA repair, and signal transduction. Research findings from the past decade demonstrate that USP21 mediates the deubiquitination of multiple well-known target proteins associated with critical cellular processes relevant to both disease and homeostasis, particularly in various cancers.This reviewcomprehensively summarizes the structure and biological functions of USP21 with an emphasis on its role in tumorigenesis, and elucidates the advances on the discovery of tens of small-molecule inhibitors targeting USP21, which suggests that targeting USP21 may represent a potential strategy for cancer therapy.

Question prompt list intervention for patients with advanced cancer: a systematic review and meta-analysis.

BACKGROUND: Enhanced communication in end-of-life care (EOL) improves preparation and treatment decisions for patients with advanced cancer, affecting their quality of life at the end of life. Question prompt list (QPL) has been shown to enhance physician-patient communication in patients with cancer, but there is a lack of systematic review and meta-analysis for those with advanced cancer. Enhanced communication in end-of-life care improves preparation and treatment decisions for patients with advanced cancer, affecting their quality of life at the end of life. OBJECTIVE: To review the effectiveness of QPL intervention on physician-patient communication and health outcomes during consultation in patients with advanced cancer. METHODS: CINAHL, Embase, Scopus, and PsycINFO databases were undertaken using inclusion criteria for relevant articles up to August 2021. Pooled standardized mean difference (SMD) and 95% confidence intervals (CIs) were calculated using random-effects models. We used the Cochrane risk-of-bias assessment tool and modified Jadad scale to assess the quality of the studies. RESULTS: Seven RCTs with 1059 participants were included, of which six studies were eligible for the meta-analysis. The pooled meta-analysis results indicated that QPL in patients with advanced cancer had a significant positive effect on the total number of questions asked (SMD, 0.73; 95% CI, 0.28 to 1.18; I2 = 83%) and on the patients' expectations for the future (SMD, 0.67; 95% CI, 0.08 to 1.25; I2 = 88%). There were no significant improvements in health-related outcomes such as end of life, anxiety, and quality of life. CONCLUSIONS: Using QPL in advanced cancer consultations boosts patient questions which helps communication but not health-related indicators. Optimal results depend on full reading, but timing varies. Future research should examine the relationship between communication and health outcomes, including patient/physician behavior and social context.

Risk of Type 1 Diabetes Mellitus in Patients with Atopic Dermatitis: A Nationwide Population-Based Cohort Study.

INTRODUCTION: Atopic dermatitis (AD) is a disease frequently occurring in children. The immune response is characterized by T-helper (Th)-2-dependent inflammation. Type 1 diabetes mellitus (T1DM) is an autoimmune disease that destroys pancreatic islet beta cells. In contrast, it is mainly mediated by a Th-1-dependent response. An inverted association has been hypothesized between T1DM and AD since Th1 and Th2 responses are mutually inhibitory. METHODS: Data was retrieved from a nationwide healthcare database in Taiwan. A logistic regression model was used to evaluate the association of T1DM in patients with AD within a year. A Cox proportional hazards analysis was used to evaluate the subsequent risk of developing T1DM 1 year after AD diagnosis. RESULTS: We identified 396,461 patients with AD and 1,585,844 age- and sex-matched controls. During the first year of follow-up, after adjusting variates, the association between T1DM and AD showed no statistical differences (odds ratio: 1.40; 95% confidence interval [CI]: 0.83-2.38, p = 0.207). After excluding those T1DM cases within 1 year of AD diagnosis and those with a follow-up duration of less than 1 year, AD did not significantly increase the risk of T1DM (hazard ratio [HR]: 1.02; 95% CI, 0.83-1.25, p = 0.843). CONCLUSIONS: Our study revealed that there was no significant association between AD and T1DM in the first year after AD diagnosis, and there was no increased risk of T1DM in AD patients in the average 5-year follow-up in our study.

Novel anti-inflammatory diketopiperazine alkaloids from the marine-derived fungus Penicillium brasilianum.

Diketopiperazine alkaloids have proven the most abundant heterocyclic alkaloids up to now, which usually process diverse scaffolds and rich biological activities. In our search for bioactive diketopiperazine alkaloids from marine-derived fungi, two novel diketopiperazine alkaloids, penipiperazine A (1) and its biogenetically related new metabolite (2), together with a known analogue neofipiperzine C (3), were obtained from the strain Penicillium brasilianum. Their planar structures and absolute configurations were elucidated by extensive spectroscopic analyses, 13C NMR calculation, Marfey's, ECD, and ORD methods. Compound 1 featured a unique 6/5/6/6/5 indole-pyrazino-pyrazino-pyrrolo system, and its plausible biogenetic pathway was also proposed. Additionally, compounds 1-3 have been tested for their inflammatory activities. 1 and 2 significantly inhibited the release of NO and the expression of related pro-inflammatory cytokines on LPS-stimulated RAW264.7 cells, suggesting they could be attracting candidate for further development as anti-inflammatory agent. KEY POINTS: • A novel diketopiperazine alkaloid featuring a unique 6/5/6/6/5 indole-pyrazino-pyrazino-pyrrolo system was isolated from the marine fungus Penicillium brasilianum. • The structure of 1 was elucidated by detailed analysis of 2D NMR data, 13C NMR calculation, Marfey's, ECD, and ORD methods. • Compounds 1 and 2 significantly inhibited the release of NO and the expression of related pro-inflammatory cytokines on LPS-stimulated RAW264.7 cells.

An unexpected role of EasDaf: catalyzing the conversion of chanoclavine aldehyde to chanoclavine acid.

Ergot alkaloids (EAs) are a diverse group of indole alkaloids known for their complex structures, significant pharmacological effects, and toxicity to plants. The biosynthesis of these compounds begins with chanoclavine-I aldehyde (CC aldehyde, 2), an important intermediate produced by the enzyme EasDaf or its counterpart FgaDH from chanoclavine-I (CC, 1). However, how CC aldehyde 2 is converted to chanoclavine-I acid (CC acid, 3), first isolated from Ipomoea violacea several decades ago, is still unclear. In this study, we provide in vitro biochemical evidence showing that EasDaf not only converts CC 1 to CC aldehyde 2 but also directly transforms CC 1 into CC acid 3 through two sequential oxidations. Molecular docking and site-directed mutagenesis experiments confirmed the crucial role of two amino acids, Y166 and S153, within the active site, which suggests that Y166 acts as a general base for hydride transfer, while S153 facilitates proton transfer, thereby increasing the acidity of the reaction. KEY POINTS: • EAs possess complicated skeletons and are widely used in several clinical diseases • EasDaf belongs to the short-chain dehydrogenases/reductases (SDRs) and converted CC or CC aldehyde to CC acid • The catalytic mechanism of EasDaf for dehydrogenation was analyzed by molecular docking and site mutations.