Activation of ERß hijacks the splicing machinery to trigger R-loop formation in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with aggressive behavior and poor prognosis. Current therapeutic options available for TNBC patients are primarily chemotherapy. With our evolving understanding of this disease, novel targeted therapies, including poly ADP-ribose polymerase (PARP) inhibitors, antibody-drug conjugates, and immune-checkpoint inhibitors, have been developed for clinical use. Previous reports have demonstrated the essential role of estrogen receptor ß (ERß) in TNBC, but the detailed molecular mechanisms downstream ERß activation in TNBC are still far from elucidated. In this study, we demonstrated that a specific ERß agonist, LY500307, potently induces R-loop formation and DNA damage in TNBC cells. Subsequent interactome experiments indicated that the residues 151 to 165 of U2 small nuclear RNA auxiliary factor 1 (U2AF1) and the Trp439 and Lys443 of ERß were critical for the binding between U2AF1 and ERß. Combined RNA sequencing and ribosome sequencing analysis demonstrated that U2AF1-regulated downstream RNA splicing of 5-oxoprolinase (OPLAH) could affect its enzymatic activity and is essential for ERß-induced R-loop formation and DNA damage. In clinical samples including 115 patients from The Cancer Genome Atlas (TCGA) and 32 patients from an in-house cohort, we found a close correlation in the expression of ESR2 and U2AF1 in TNBC patients. Collectively, our study has unraveled the molecular mechanisms that explain the therapeutic effects of ERß activation in TNBC, which provides rationale for ERß activation-based single or combined therapy for patients with TNBC.

[Tracking Research Progress in the Modulatory Role of Gut Microbiome in Immune Checkpoint Inhibitors Applied in Cancer Treatment].

In recent years, immunotherapy, as an emerging anti-tumor therapy, has shown great potential in the treatment of both solid and hematologic tumors. There is increasing preclinical and clinical evidence linking the composition of gut microbiome with the efficacy as well as adverse effects of immune checkpoint inhibitor anti-tumor therapy. We summarized in this review the modulatory role of the gut microbiome in antitumor therapy with different immune checkpoint inhibitors. We also discussed the limitations of existing research and prospective development of the further clinical strategies.

Proteomic Identification of Immune-Related Silkworm Proteins Involved in the Response to Bacterial Infection.

Bombyx mori (Lepidoptera: Bombycidae) is an important economic insect and a classic Lepidopteran model system. Although immune-related genes have been identified at a genome-wide scale in the silkworm, proteins involved in immune defense of the silkworm have not been comprehensively characterized. In this study, two types of bacteria were injected into the silkworm larvae, Gram-negative Escherichia coli (Enterobacteriales: Enterobacteriaceae), or Gram-positive Staphylococcus aureus (Bacillales: Staphylococcaceae). After injection, proteomic analyses of hemolymph were performed by liquid chromatography-tandem mass spectrometry. In total, 514 proteins were identified in the uninduced control group, 540 were identified in the E. coli-induced group, and 537 were identified in the S. aureus-induced group. Based on Uniprot annotations, 32 immunological recognition proteins, 28 immunological signaling proteins, and 21 immunological effector proteins were identified. We found that 127 proteins showed significant upregulation, including 10 immunological recognition proteins, 4 immunological signaling proteins, 11 immunological effector proteins, and 102 other proteins. Using real-time quantitative polymerase chain reaction in the fat body, we verified that immunological recognition proteins, signaling proteins, and effector proteins also showed significant increases at the transcriptional level after infection with E. coli and S. aureus. Five newly identified proteins showed upregulation at both protein and transcription levels after infection, including 30K protein, yellow-d protein, chemosensory protein, and two uncharacterized proteins. This study identified many new immune-related proteins, deepening our understanding of the immune defense system in B. mori. The data have been deposited to the iProX with identifier IPX0001337000.

Improved strength of silk fibers in Bombyx mori trimolters induced by an anti-juvenile hormone compound.

BACKGROUND: Bombyx mori silk fibers with thin diameters have advantages of lightness and crease-resistance. Many studies have used anti-juvenile hormones to induce trimolters in order to generate thin silk; however, there has been comparatively little analysis of the morphology, structure and mechanical properties of trimolter silk. METHODS: This study induced two kinds of trimolters by appling topically anti-juvenile hormones and obtained thin diameter silk. Scanning electron microscope (SEM), FTIR analysis, tensile mechanical testing, chitin staining were used to reveal that the morphology, conformation and mechanical property of the trimolter silk. RESULTS: Cocoon of trimolters were highly densely packed by thinner fibers and thus had small apertures. We found that the conformation of trimolter silk fibroin changed and formed more ß-sheet structures. In addition, analysis of mechanical parameters yielded a higher Young's modulus and strength in trimolter silk than in the control. By chitin staining of silk gland, we postulated that the mechanical properties of trimolters' silk was enhanced greatly during to the structural changes of silk gland. CONCLUSION: We induced trimolters by anti-juvenile hormones and the resulting cocoons were more closely packed and had smaller silk fiber diameters. We found that the conformation of trimolters silk fibroin had a higher content of ß-sheet structures and better mechanical properties. GENERAL SIGNIFICANCE: Our study revealed the structures and mechanical properties of trimolter silk, and provided a valuable reference to improve silk quality by influencing molting in silkworms.

The genomic and immunogenomic landscape of mechanics pathway informs clinical prognosis and response to mechanotherapy.

Mechanics shape cell and tissue plasticity and maintain their homeostasis. In cancers, mechanical signals regulate cancer hallmarks via mechanotransduction pathways, such as proliferation, metastasis and metabolic reprogramming. However, comprehensive characterization of mechanotransduction pathway genes and their clinical relevance across different cancer types remains untouched. Herein, we systematically portrayed the alterations of mechanotransduction pathway genes across 31 cancer types using The Cancer Genome Atlas (TCGA) databases. All the cancer types could be categorized into 6 subtypes based upon the transcriptional pattern of mechanics pathway genes. Each subtype has its own unique molecular expression pattern, mutation landscapes, immune infiltrates, and patient clinical outcome. We further found that the responses of two subtypes of cancers, one with the optimal outcome and the other with the worst prognosis, to a classical mechanotherapeutic agent (Fasudil, RhoA/ROCK inhibitor) were totally different, indicating that our cancer stratification system based upon mechanotransduction pathway genes could inform clinical responses of patients to mechanotherapeutic agents. Collectively, our study provides a novel pan-cancer landscape of the mechanotransduction pathways and underscores its potential clinical significance in the prediction of clinical prognosis and therapeutic responses to mechanotherapy among cancer patients.

SPINK7 Recognizes Fungi and Initiates Hemocyte-Mediated Immune Defense Against Fungal Infections.

Serine protease inhibitors of Kazal-type (SPINKs) were widely identified in vertebrates and invertebrates, and played regulatory roles in digestion, coagulation, and fibrinolysis. In this study, we reported the important role of SPINK7 in regulating immune defense of silkworm, Bombyx mori. SPINK7 contains three Kazal domains and has 6 conserved cysteine residues in each domain. Quantitative real-time PCR analyses revealed that SPINK7 was exclusively expressed in hemocytes and was upregulated after infection with two fungi, Saccharomyces cerevisiae and Candida albicans. Enzyme activity inhibition test showed that SPINK7 significantly inhibited the activity of proteinase K from C. albicans. Additionally, SPINK7 inhibited the growth of three fungal spores, including S. cerevisiae, C. albicans, and Beauveria bassiana. The pathogen-associated molecular patterns (PAMP) binding assays suggested that SPINK7 could bind to ß-D-glucan and agglutinate B. bassiana and C. albicans. In vitro assays were performed using SPINK7-coated agarose beads, and indicated that SPINK7 promoted encapsulation and melanization of agarose beads by B. mori hemocytes. Furthermore, co-localization studies using immunofluorescence revealed that SPINK7 induced hemocytes to aggregate and entrap the fungi spores of B. bassiana and C. albicans. Our study revealed that SPINK7 could recognize fungal PAMP and induce the aggregation, melanization, and encapsulation of hemocytes, and provided valuable clues for understanding the innate immunity and cellular immunity in insects.

Synthesis, secretion, and antifungal mechanism of a phosphatidylethanolamine-binding protein from the silk gland of the silkworm Bombyx mori.

A silkworm cocoon contains several antimicrobial proteins such as protease inhibitors and seroins to provide protection for the enclosed pupa. In this study, we identified a new Bombyx mori phosphatidylethanolamine-binding protein (BmPEBP) with antimicrobial activity in the cocoon silk using semi-quantitative and quantitative RT-PCR, western blotting, and immunofluorescence. The results indicated that BmPEBP was synthesized in the middle silk gland and secreted into the sericin layer of the cocoon silk. Functional analysis showed that BmPEBP could inhibit the spore growth of four types of fungi, Candida albicans, Saccharomyces cerevisiae, Beauveriabassiana, and Aspergillus fumigates, by binding to the fungal cell membrane. Investigation of the interaction of BmPEBP with membrane phospholipids revealed that the protein showed a strong binding affinity to phosphatidylethanolamine, weak affinity to phosphatidylinositol, and no affinity to phosphatidylserine or phosphatidylcholine. Circular dichroism spectroscopy showed that binding to phosphatidylethanolamine caused conformational changes in the BmPEBP molecule by reducing ß-sheet formation and inducing the appearance of an α-helix motif. We speculate that BmPEBP performs antifungal function in the cocoon silk through interaction with phosphatidylethanolamine in the fungal membrane.

Functional analysis and characterization of antimicrobial phosphatidylethanolamine-binding protein BmPEBP in the silkworm Bombyx mori.

Phosphatidylethanolamine-binding proteins (PEBPs) are a class of highly conserved, biologically diverse proteins, which are widely distributed in plants, insects, and mammals. In this study, a Bombyx mori PEBP (BmPEBP) gene was reported, which encodes a protein composed of 209 amino acid residues. BmPEBP includes a predicted signal peptide, indicating that it is an extracellular protein, which differs from the cytoplasmic PEBPs of plants and mammals. Recombinant soluble BmPEBP was successfully synthesized using a prokaryotic expression system and was then purified effectively by Ni2+-NTA affinity chromatography and gel filtration. Far-ultraviolet circular dichroism spectra indicated that BmPEBP had a well-defined ß-sheet structure, with the ß-sheet content accounting for about 41% of the protein. BmPEBP had a relatively stable structure at temperatures ranging from 15 °C to 57.5 °C. The Tm, ΔH, and ΔS of BmPEBP were 62.27 °C ±â€¯0.14 °C, 570.10 ±â€¯0.17 kJ/mol, and 1.70 ±â€¯0.03 KJ/(mol·K), respectively. Homology modeling analysis suggested that the active sites of BmPEBP were conserved, comprising Pro96, His111, and His143. Quantitative real-time PCR showed that BmPEBP was highly expressed in the silk gland and had very low expression in other tissues. However, BmPEBP expression was significantly upregulated in the larval fat body after infection with two kinds of fungi, Beauveria bassiana and Candida albicans. Moreover, in vitro fungal inhibition tests showed that BmPEBP could significantly inhibit the sporular growth of Saccharomyces cerevisiae, C. albicans, B. bassiana, and Aspergillus fumigatus. To our knowledge, this is the first report to reveal the antifungal role of a PEBP in insects.

Identification of Bombyx mori sericin 4 protein as a new biological adhesive.

Sericins are large proteins with molecular weights >70 kDa. Three sericin genes were reported in the silkworm, including sericin 1, sericin 2 and sericin 3. In this study, we have identified a new sericin gene and designated it as sericin 4. The sequence, exon-intron structure, alternative splicing, and translation products of this gene have been described in this study. Quantitative RT-PCR analysis indicates that sericin 4 is expressed in the middle silk gland. Immunofluorescence results show co-localization of sericin 1 and sericin 4 in the MSG. Western blot analysis revealed that sericin 4 was found in the larval silk produced from the second instar to the fourth instar. Two protein bands at approximately 280 kDa and 260 kDa, were detected by western blot for sericin 4. Two repetitive motifs that are rich in charged amino acids and glutamine have been identified, and they are likely to be responsible for the adhesiveness of sericin 4. Overall, this study identifies a novel biological adhesive protein and provides new information for understanding how sericins contribute to the adhesive properties of larval silks.