ARMH3-mediated recruitment of PI4KB directs Golgi-to-endosome trafficking and activation of the antiviral effector STING.

The cGAS-STING pathway mediates cytoplasmic DNA-triggered innate immunity. STING activation is initiated by cyclic-GMP-AMP (cGAMP)-induced translocation from the endoplasmic reticulum and sulfated glycosaminoglycans-induced polymerization at the Golgi. Here, we examine the mechanisms underlying STING transport and activation beyond the Golgi. A genome-wide CRISPR-Cas9 screen identified Armadillo-like helical domain-containing protein 3 (ARMH3) as critical for STING activation. Upon cGAMP-triggered translocation, ARMH3 interacted with STING at the Golgi and recruited phosphatidylinositol 4-kinase beta (PI4KB) to synthesize PI4P, which directed STING Golgi-to-endosome trafficking via PI4P-binding proteins AP-1 and GGA2. Disrupting PI4P-dependent lipid transport through RNAi of other PI4P-binding proteins impaired STING activation. Consistently, disturbed lipid composition inhibited STING activation, whereas aberrantly elevated cellular PI4P led to cGAS-independent STING activation. Armh3fl/fllLyzCre/Cre mice were susceptible to DNA virus challenge in vivo. Thus, ARMH3 bridges STING and PIK4B to generate PI4P for STING transportation and activation, an interaction conserved in all eukaryotes.

Designing antimicrobial peptides using deep learning and molecular dynamic simulations.

With the emergence of multidrug-resistant bacteria, antimicrobial peptides (AMPs) offer promising options for replacing traditional antibiotics to treat bacterial infections, but discovering and designing AMPs using traditional methods is a time-consuming and costly process. Deep learning has been applied to the de novo design of AMPs and address AMP classification with high efficiency. In this study, several natural language processing models were combined to design and identify AMPs, i.e. sequence generative adversarial nets, bidirectional encoder representations from transformers and multilayer perceptron. Then, six candidate AMPs were screened by AlphaFold2 structure prediction and molecular dynamic simulations. These peptides show low homology with known AMPs and belong to a novel class of AMPs. After initial bioactivity testing, one of the peptides, A-222, showed inhibition against gram-positive and gram-negative bacteria. The structural analysis of this novel peptide A-222 obtained by nuclear magnetic resonance confirmed the presence of an alpha-helix, which was consistent with the results predicted by AlphaFold2. We then performed a structure-activity relationship study to design a new series of peptide analogs and found that the activities of these analogs could be increased by 4-8-fold against Stenotrophomonas maltophilia WH 006 and Pseudomonas aeruginosa PAO1. Overall, deep learning shows great potential in accelerating the discovery of novel AMPs and holds promise as an important tool for developing novel AMPs.

Associations of combined phenotypic ageing and genetic risk with incidence of chronic respiratory diseases in the UK Biobank: a prospective cohort study.

BACKGROUND: Accelerated biological ageing has been associated with an increased risk of several chronic respiratory diseases. However, the associations between phenotypic age, a new biological age indicator based on clinical chemistry biomarkers, and common chronic respiratory diseases have not been evaluated. METHODS: We analysed data from 308 592 participants at baseline in the UK Biobank. The phenotypic age was calculated from chronological age and nine clinical chemistry biomarkers, including albumin, alkaline phosphatase, creatinine, glucose, C-reactive protein, lymphocyte percent, mean cell volume, red cell distribution width and white blood cell count. Furthermore, phenotypic age acceleration (PhenoAgeAccel) was calculated by regressing phenotypic age on chronological age. The associations of PhenoAgeAccel with incident common chronic respiratory diseases and cross-sectional lung function were investigated. Moreover, we constructed polygenic risk scores and evaluated whether PhenoAgeAccel modified the effect of genetic susceptibility on chronic respiratory diseases and lung function. RESULTS: The results showed significant associations of PhenoAgeAccel with increased risk of idiopathic pulmonary fibrosis (IPF) (hazard ratio (HR) 1.52, 95% CI 1.45-1.59), COPD (HR 1.54, 95% CI 1.51-1.57) and asthma (HR 1.18, 95% CI 1.15-1.20) per 5-year increase and decreased lung function. There was an additive interaction between PhenoAgeAccel and the genetic risk for IPF and COPD. Participants with high genetic risk and who were biologically older had the highest risk of incident IPF (HR 5.24, 95% CI 3.91-7.02), COPD (HR 2.99, 95% CI 2.66-3.36) and asthma (HR 2.07, 95% CI 1.86-2.31). Mediation analysis indicated that PhenoAgeAccel could mediate 10∼20% of the associations between smoking and chronic respiratory diseases, while ∼10% of the associations between particulate matter with aerodynamic diameter <2.5 µm and the disorders were mediated by PhenoAgeAccel. CONCLUSION: PhenoAgeAccel was significantly associated with incident risk of common chronic respiratory diseases and decreased lung function and could serve as a novel clinical biomarker.

o-Vanillin binds covalently to MAL/TIRAP Lys-210 but independently inhibits TLR2.

Toll-like receptor (TLR) innate immunity signalling protects against pathogens, but excessive or prolonged signalling contributes to a range of inflammatory conditions. Structural information on the TLR cytoplasmic TIR (Toll/interleukin-1 receptor) domains and the downstream adaptor proteins can help us develop inhibitors targeting this pathway. The small molecule o-vanillin has previously been reported as an inhibitor of TLR2 signalling. To study its mechanism of action, we tested its binding to the TIR domain of the TLR adaptor MAL/TIRAP (MALTIR). We show that o-vanillin binds to MALTIR and inhibits its higher-order assembly in vitro. Using NMR approaches, we show that o-vanillin forms a covalent bond with lysine 210 of MAL. We confirm in mouse and human cells that o-vanillin inhibits TLR2 but not TLR4 signalling, independently of MAL, suggesting it may covalently modify TLR2 signalling complexes directly. Reactive aldehyde-containing small molecules such as o-vanillin may target multiple proteins in the cell.

Structural Basis of the Bivalency of the TRPV1 Agonist DkTx.

Bivalency is a prevalent natural mechanism to enhance receptor avidity. Various two-domain disulfide-rich peptides exhibiting bivalent action have been identified from animal venoms. A unique characteristic of these peptides is that they induce a pharmacological response different from that provoked by any of the constituent domains. The enhanced potency and avidity of such peptides is therefore a consequence of their domain fusion by a peptide linker. The role of the linker itself, beyond conjugation, remains unclear. Here, we investigate how the linker affects the bivalency of the capsaicin receptor (TRPV1) agonist DkTx. We recombinantly produced isotope labelled DkTx using a protein splicing approach, to solve the high-resolution solution structure of DkTx, revealing residual linker order stabilised by linker-domain interactions leading to biased domain orientations. The significance of this was studied using a combination of mutagenesis, spin relaxation studies and electrophysiology measurements. Our results reveal that disrupting the pre-organisation of the domains of DkTx is accompanied by reductions in potency and onset of avidity. Our findings support a model of pre-configured two-domain binding, in favour of the previously suggested sequential binding model. This highlights the significance of ordered elements in linker design and the natural evolution of these in bivalent toxins.

[Influencing Factors and Prediction Model of Performance of Needle Visualization in Fine Needle Aspiration of Thyroid Nodules].

Objective To investigate the influencing factors and establish a model predicting the performance of needle visualization in fine-needle aspiration (FNA) of thyroid nodules. Methods This study prospectively included 175 patients who underwent FNA of thyroid nodules in the Department of Ultrasound in China-Japan Friendship Hospital and compared the display of the needle tips in the examination of 199 thyroid nodules before and after the application of needle visualization.We recorded the location,the positional relationship with thyroid capsule,ultrasonic characteristics,and the distribution of the soft tissue strip structure at the puncture site of the nodules with unclear needle tips display before using needle visualization.Furthermore,according to the thyroid imaging reporting and data system proposed by the American College of Radiology,we graded the risk of the nodules.Lasso-Logistic regression was employed to screen out the factors influencing the performance of needle visualization and establish a nomogram for prediction. Results The needle tips were not clearly displayed in the examination of 135 (67.8%) and 53 (26.6%) nodules before and after the application of needle visualization,respectively,which showed a significant difference (P<0.001).Based on the positional relationship between the nodule and capsule,anteroposterior/transverse diameter (A/T) ratio,blood supply,and the distribution of subcutaneous strip structure at the puncture site,a nomogram was established to predict the probability of unclear display of the needle tips after application of needle visualization.The C-index of the prediction model was 0.75 (95%CI=0.67-0.84) and the area under the receiver operating characteristic curve was 0.72.The calibration curve confirmed the appreciable reliability of the prediction model,with the C-index of 0.70 in internal validation. Conclusions Needle visualization can improve the display of the needle tip in ultrasound-guided FNA of thyroid nodules.The nomogram established based on ultrasound features such as the positional relationship between the nodule and capsule,A/T ratio,blood supply,and the distribution of subcutaneous strip structure at the puncture site can predict whether needle visualization is suitable for the examination of nodules.

A new vector coupling ligation-independent cloning with sortase a fusion for efficient cloning and one-step purification of tag-free recombinant proteins.

We have developed a new ligation independent cloning (LIC) vector - pSrtA9, which can be utilized for one-step purification of recombinant proteins. The new LIC site in the pSrtA9 vector, hosts a DNA sequence centered on a SfoI restriction site and integrates a coding sequence for sortase A (SrtA) recognition. Preceding the LIC site, pSrtA9 incorporates an N-terminal 6xHis-tag and the catalytic core of SrtA from Staphylococcus aureus (SrtAΔ59). Thus, after cloning and protein expression in Escherichia coli, the resultant fusion protein comprises an N-terminal 6xHis-tag, SrtAΔ59, an L-P-E-T-G linker and the protein of interest at the C-terminus. The fusion protein can be captured onto immobilized Ni-NTA resin and any unwanted proteolysis activity of SrtA is suppressed during the purification by optimisation of solution conditions. Upon addition of Ca2+ and triglycine (Gly3), the immobilized fusion protein undergoes on-column SrtA-mediated cleavage at the T-G bond of LPETG linker to selectively release 90% of the protein of interest within 3 h when incubated at room temperature. This new pSrtA9 vector, thus, offers an efficient method for LIC of genes and a one-step purification procedure to obtain a tag-free recombinant protein, and is therefore suitable for the high-throughput proteins production.

Integrated Transcriptomic-Proteomic Analysis Using a Proteogenomic Workflow Refines Rat Genome Annotation.

Proteogenomic re-annotation and mRNA splicing information can lead to the discovery of various protein forms for eukaryotic model organisms like rat. However, detection of novel proteoforms using mass spectrometry proteomics data remains a formidable challenge. We developed EuGenoSuite, an open source multiple algorithmic proteomic search tool and utilized it in our in-house integrated transcriptomic-proteomic pipeline to facilitate automated proteogenomic analysis. Using four proteogenomic pipelines (integrated transcriptomic-proteomic, Peppy, Enosi, and ProteoAnnotator) on publicly available RNA-sequence and MS proteomics data, we discovered 363 novel peptides in rat brain microglia representing novel proteoforms for 249 gene loci in the rat genome. These novel peptides aided in the discovery of novel exons, translation of annotated untranslated regions, pseudogenes, and splice variants for various loci; many of which have known disease associations, including neurological disorders like schizophrenia, amyotrophic lateral sclerosis, etc. Novel isoforms were also discovered for genes implicated in cardiovascular diseases and breast cancer for which rats are considered model organisms. Our integrative multi-omics data analysis not only enables the discovery of new proteoforms but also generates an improved reference for human disease studies in the rat model.

Exome sequencing reveals mutation in GJA1 as a cause of keratoderma-hypotrichosis-leukonychia totalis syndrome.

Keratoderma-hypotrichosis-leukonychia totalis syndrome (KHLS) is an extremely rare, autosomal-dominant disorder characterized by severe skin hyperkeratosis, congenital alopecia and leukonychia totalis. The genetic defect underlying KHLS remained undetermined. By performing whole-exome sequencing in a family with KHLS, we identified a heterozygous mutation (c.23G>T [p.Gly8Val]) in GJA1, which cosegregated with the phenotype in the family. In an additional affected individual, we also found the identical de novo mutation which was absent in his unaffected family members. GJA1 encodes a gap junction protein connexin 43 (Cx43) which is ubiquitously expressed in various organs, including the epidermis and hair follicles. In vitro studies on HEK293 cells expressing Cx43(Gly8Val) found that the protein formed gap junction plaques between adjacent transfected cells, as observed in the wild-type. Dye-transfer experiments by microinjection of Lucifer yellow displayed functional gap junction of the Cx43(Gly8Val) mutant. Using patch clamp and Ca(2+) imaging methods, we observed that the Cx43(Gly8Val) hemichannel had significantly more openings than Cx43(WT), facilitating Ca(2+) influx at resting potential. Such gain-of-function effect might result in cytoplasmic Ca(2+) overload, accelerated apoptosis of keratinocytes and subsequent skin hyperkeratosis. Taken together, our results demonstrated that, with probably enhanced hemichannel activities, a mutation in GJA1 is linked to KHLS without extracutaneous involvement.

A non-uniform sampling approach enables studies of dilute and unstable proteins.

NMR spectroscopy is a powerful method in structural and functional analysis of macromolecules and has become particularly prevalent in studies of protein structure, function and dynamics. Unique to NMR spectroscopy is the relatively low constraints on sample preparation and the high level of control of sample conditions. Proteins can be studied in a wide range of buffer conditions, e.g. different pHs and variable temperatures, allowing studies of proteins under conditions that are closer to their native environment compared to other structural methods such as X-ray crystallography and electron microscopy. The key disadvantage of NMR is the relatively low sensitivity of the method, requiring either concentrated samples or very lengthy data-acquisition times. Thus, proteins that are unstable or can only be studied in dilute solutions are often considered practically unfeasible for NMR studies. Here, we describe a general method, where non-uniform sampling (NUS) allows for signal averaging to be monitored in an iterative manner, enabling efficient use of spectrometer time, ultimately leading to savings in costs associated with instrument and isotope-labelled protein use. The method requires preparation of multiple aliquots of the protein sample that are flash-frozen and thawed just before acquisition of a short NMR experiments carried out while the protein is stable (12 h in the presented case). Non-uniform sampling enables sufficient resolution to be acquired for each short experiment. Identical NMR datasets are acquired and sensitivity is monitored after each co-added spectrum is reconstructed. The procedure is repeated until sufficient signal-to-noise is obtained. We discuss how maximum entropy reconstruction is used to process the data, and propose a variation on the previously described method of automated parameter selection. We conclude that combining NUS with iterative co-addition is a general approach, and particularly powerful when applied to unstable proteins.

A comparative proteomic analysis of bile for biomarkers of cholangiocarcinoma.

Cholangiocarcinoma is a primary malignant tumor of the bile duct epithelium. Cholangiocarcinoma is usually detected at an advanced stage when successful treatment is no longer possible. As the tumor originates from the bile duct epithelium, bile is an ideal source of tumor biomarkers for cholangiocarcinoma. In this study, we used a quantitative proteomics approach to identify potential tumor-associated proteins in the bile fluid of six cholangiocarcinoma patients. Three different gross-appearance tumor types were used in the analysis: mass-forming type ( n = 2), periductal infiltrating type ( n = 2), and intraductal growth type ( n = 2). Two bile samples from non-cancerous patients were used as controls. Isobaric labeling, coupled with Tandem mass spectrometry, was used to quantify protein levels in the bile of cholangiocarcinoma and control patients. In all, 63 proteins were significantly increased in cholangiocarcinoma bile compared to normal bile. Alpha-1-antitrypsin was one of the overexpressed proteins that increased in cholangiocarcinoma bile samples. Immunohistochemical analysis revealed that alpha-1-antitrypsin was detected in 177 (50%) of 354 cholangiocarcinoma tissues from our Tissue Bank. Immunoblotting of 54 cholangiocarcinoma bile samples showed that alpha-1-antitrypsin was positive in 38 (70%) samples. Fecal enzyme-linked immunosorbent assay showed that alpha-1-antitrypsin level was able to distinguish cholangiocarcinoma patients from normal individuals. In conclusion, alpha-1-antitrypsin is a potential marker for early diagnosis of cholangiocarcinoma.

An enzymatic [4+2] cyclization cascade creates the pentacyclic core of pyrroindomycins.

The [4+2] cycloaddition remains one of the most intriguing transformations in synthetic and natural products chemistry. In nature, however, there are remarkably few enzymes known to have this activity. We herein report an unprecedented enzymatic [4+2] cyclization cascade that has a central role in the biosynthesis of pyrroindomycins, which are pentacyclic spirotetramate natural products. Beginning with a linear intermediate that contains two pairs of 1,3-diene and alkene groups, the dedicated cyclases PyrE3 and PyrI4 act in tandem to catalyze the formation of two cyclohexene rings in the dialkyldecalin system and the tetramate spiro-conjugate of the molecules. The two cyclizations are completely enzyme dependent and proceed in a regio- and stereoselective manner to establish the enantiomerically pure pentacyclic core. Analysis of a related spirotetronate pathway confirms that homologs are functionally exchangeable, establishing the generality of these findings and explaining how nature creates diverse active molecules with similar rigid scaffolds.

Memory T cell RNA rearrangement programmed by heterogeneous nuclear ribonucleoprotein hnRNPLL.

Differentiation of memory cells involves DNA-sequence changes in B lymphocytes but is less clearly defined in T cells. RNA rearrangement is identified here as a key event in memory T cell differentiation by analysis of a mouse mutation that altered the proportions of naive and memory T cells and crippled the process of Ptprc exon silencing needed to generate CD45RO in memory T cells. A single substitution in a memory-induced RNA-binding protein, hnRNPLL, destabilized an RNA-recognition domain that bound with micromolar affinity to RNA containing the Ptprc exon-silencing sequence. Hnrpll mutation selectively diminished T cell accumulation in peripheral lymphoid tissues but not proliferation. Exon-array analysis of Hnrpll mutant naive and memory T cells revealed an extensive program of alternative mRNA splicing in memory T cells, coordinated by hnRNPLL. A remarkable overlap with alternative splicing in neural tissues may reflect a co-opted strategy for diversifying memory T cells.

Solution structure, membrane interactions, and protein binding partners of the tetraspanin Sm-TSP-2, a vaccine antigen from the human blood fluke Schistosoma mansoni.

The tetraspanins (TSPs) are a family of integral membrane proteins that are ubiquitously expressed at the surface of eukaryotic cells. TSPs mediate a range of processes at the surface of the plasma membrane by providing a scaffold for the assembly of protein complexes known as tetraspanin-enriched microdomains (TEMs). We report here the structure of the surface-exposed EC2 domain from Sm-TSP-2, a TSP from Schistosoma mansoni and one of the better prospects for the development of a vaccine against schistosomiasis. This is the first solution structure of this domain, and our investigations of its interactions with lipid micelles provide a general model for interactions between TSPs, membranes, and other proteins. Using chemical cross-linking, eight potential protein constituents of Sm-TSP-2-mediated TEMs were also identified. These include proteins important for membrane maintenance and repair, providing further evidence for the functional role of Sm-TSP-2- and Sm-TSP-2-mediated TEMs. The identification of calpain, Sm29, and fructose-bisphosphate aldolase, themselves potential vaccine antigens, suggests that the Sm-TSP-2-mediated TEMs could be disrupted via multiple targets. The identification of further Sm-TSP-2-mediated TEM proteins increases the available candidates for multiplex vaccines and/or novel drugs targeting TEMs in the schistosome tegument.

Semienzymatic cyclization of disulfide-rich peptides using Sortase A.

Disulfide-rich cyclic peptides have generated great interest in the development of peptide-based therapeutics due to their exceptional stability toward chemical, enzymatic, or thermal attack. In particular, they have been used as scaffolds onto which bioactive epitopes can be grafted to take advantage of the favorable biophysical properties of disulfide-rich cyclic peptides. To date, the most commonly used method for the head-to-tail cyclization of peptides has been native chemical ligation. In recent years, however, enzyme-mediated cyclization has become a promising new technology due to its efficiency, safety, and cost-effectiveness. Sortase A (SrtA) is a bacterial enzyme with transpeptidase activity. It recognizes a C-terminal penta-amino acid motif, LPXTG, and cleaves the amide bond between Thr and Gly to form a thioacyl-linked intermediate. This intermediate undergoes nucleophilic attack by an N-terminal poly-Gly sequence to form an amide bond between the Thr and N-terminal Gly. Here, we demonstrate that sortase A can successfully be used to cyclize a variety of small disulfide-rich peptides, including the cyclotide kalata B1, α-conotoxin Vc1.1, and sunflower trypsin inhibitor 1. These peptides range in size from 14 to 29 amino acids and contain three, two, or one disulfide bond, respectively, within their head-to-tail cyclic backbones. Our findings provide proof of concept for the potential broad applicability of enzymatic cyclization of disulfide-rich peptides with therapeutic potential.

TEIF associated centrosome activity is regulated by EGF/PI3K/Akt signaling.

Centrosome amplification, which is a characteristic of cancer cells, has been understood as a driving force of genetic instability in the development of cancer. In previous work, we demonstrated that TEIF (transcriptional element-interacting factor) distributes in the centrosomes and regulates centrosome status under both physiologic and pathologic conditions. Here we identify TEIF as a downstream effector in EGF/PI3K/Akt signaling. The addition of EGF or transfection of active Akt stimulates centrosome TEIF distribution, resulting in an increase of centrosome splitting and amplification, while inhibitors of either PI3K or Akt attenuate these changes in TEIF and the associated centrosome status. A consensus motif for Akt phosphorylation (RHRVLT) proved to be involved in centrosomal TEIF localization, and the 469-threonine of this motif may be phosphorylated by Akt both in vitro and in vivo. Elimination of this phosphorylated site on TEIF caused reduced centrosome distribution and centrosome splitting or amplification. Moreover, TEIF closely co-localized with C-NAP1 at the proximal ends of centrioles, and centriolar loading of TEIF stimulated by EGF/Akt could displace C-NAP1, resulting in centrosome splitting. These findings reveal linkage of the EGF/PI3K/Akt signaling pathway to regulation of centrosome status which may act as an oncogenic pathway and induce genetic instability in carcinogenesis.

Transcriptome and venom proteome of the box jellyfish Chironex fleckeri.

BACKGROUND: The box jellyfish, Chironex fleckeri, is the largest and most dangerous cubozoan jellyfish to humans. It produces potent and rapid-acting venom and its sting causes severe localized and systemic effects that are potentially life-threatening. In this study, a combined transcriptomic and proteomic approach was used to identify C. fleckeri proteins that elicit toxic effects in envenoming. RESULTS: More than 40,000,000 Illumina reads were used to de novo assemble ∼ 34,000 contiguous cDNA sequences and ∼ 20,000 proteins were predicted based on homology searches, protein motifs, gene ontology and biological pathway mapping. More than 170 potential toxin proteins were identified from the transcriptome on the basis of homology to known toxins in publicly available sequence databases. MS/MS analysis of C. fleckeri venom identified over 250 proteins, including a subset of the toxins predicted from analysis of the transcriptome. Potential toxins identified using MS/MS included metalloproteinases, an alpha-macroglobulin domain containing protein, two CRISP proteins and a turripeptide-like protease inhibitor. Nine novel examples of a taxonomically restricted family of potent cnidarian pore-forming toxins were also identified. Members of this toxin family are potently haemolytic and cause pain, inflammation, dermonecrosis, cardiovascular collapse and death in experimental animals, suggesting that these toxins are responsible for many of the symptoms of C. fleckeri envenomation. CONCLUSIONS: This study provides the first overview of a box jellyfish transcriptome which, coupled with venom proteomics data, enhances our current understanding of box jellyfish venom composition and the molecular structure and function of cnidarian toxins. The generated data represent a useful resource to guide future comparative studies, novel protein/peptide discovery and the development of more effective treatments for jellyfish stings in humans. (Length: 300).

Bioclojure: a functional library for the manipulation of biological sequences.

MOTIVATION: BioClojure is an open-source library for the manipulation of biological sequence data written in the language Clojure. BioClojure aims to provide a functional framework for the processing of biological sequence data that provides simple mechanisms for concurrency and lazy evaluation of large datasets. RESULTS: BioClojure provides parsers and accessors for a range of biological sequence formats, including UniProtXML, Genbank XML, FASTA and FASTQ. In addition, it provides wrappers for key analysis programs, including BLAST, SignalP, TMHMM and InterProScan, and parsers for analyzing their output. All interfaces leverage Clojure's functional style and emphasize laziness and composability, so that BioClojure, and user-defined, functions can be chained into simple pipelines that are thread-safe and seamlessly integrate lazy evaluation. AVAILABILITY AND IMPLEMENTATION: BioClojure is distributed under the Lesser GPL, and the source code is freely available from GitHub (https://github.com/s312569/clj-biosequence).

A microRNA profile associated with Opisthorchis viverrini-induced cholangiocarcinoma in tissue and plasma.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) is a highly aggressive tumor of the bile duct, and a significant public health problem in East Asia, where it is associated with infection by the parasite Opisthorchis viverrini. ICC is often detected at an advanced stage and with a poor prognosis, making a biomarker for early detection a priority. METHODS: We have comprehensively profiled miRNA expression levels in ICC tumor tissue using small RNA-Seq and validated these profiles using quantitative PCR on matched plasma samples. RESULTS: Distinct miRNA profiles were associated with increasing histological differentiation of ICC tumor tissue. We also observed that histologically normal tissue adjacent to ICC tumor displayed miRNA expression profiles more similar to tumor than liver tissue from healthy donors. In plasma samples, an eight-miRNA signature associated with ICC, regardless of the degree of histological differentiation of its matched tissue, forming the basis of a circulating miRNA-based biomarker for ICC. CONCLUSIONS: The association of unique miRNA profiles with different ICC subtypes suggests the involvement of specific miRNAs during ICC tumor progression. In plasma, an eight-miRNA signature associated with ICC could form the foundation of an accessible (plasma-based) miRNA-based biomarker for the early detection of ICC.

Analyzing and engineering of the biosynthetic pathway of mollemycin A for enhancing its production.

Mollemycin A (MOMA) is a unique glyco-hexadepsipeptide-polyketide that was isolated from a Streptomyces sp. derived from the Australian marine environment. MOMA exhibits remarkable inhibitory activity against both drug-sensitive and multidrug-resistant malaria parasites. Optimizing MOMA through structural modifications or product enhancements is necessary for the development of effective analogues. However, modifying MOMA using chemical approaches is challenging, and the production titer of MOMA in the wild-type strain is low. This study identified and characterized the biosynthetic gene cluster of MOMA for the first time, proposed its complex biosynthetic pathway, and achieved an effective two-pronged enhancement of MOMA production. The fermentation medium was optimized to increase the yield of MOMA from 0.9 mg L-1 to 1.3 mg L-1, a 44% boost. Additionally, a synergistic mutant strain was developed by deleting the momB3 gene and overexpressing momB2, resulting in a 2.6-fold increase from 1.3 mg L-1 to 3.4 mg L-1. These findings pave the way for investigating the biosynthetic mechanism of MOMA, creating opportunities to produce a wide range of MOMA analogues, and developing an efficient strain for the sustainable and economical production of MOMA and its analogues.