Activation of STING by the novel liposomal TLC388 enhances the therapeutic response to anti-PD-1 antibodies in combination with radiotherapy.

Current immune checkpoint inhibiters (ICIs) have contrasting clinical results in poorly immunogenic cancers such as microsatellite-stable colorectal cancer (MSS-CRC). Therefore, understanding and developing the combinational therapeutics for ICI-unresponsive cancers is critical. Here, we demonstrated that the novel topoisomerase I inhibitor TLC388 can reshape the tumor immune landscape, corroborating their antitumor effects combined with radiotherapy as well as immunotherapy. We found that TLC388 significantly triggered cytosolic single-stranded DNA (ssDNA) accumulation for STING activation, leading to type I interferons (IFN-Is) production for increased cancer immunogenicity to enhance antitumor immunity. TLC388-treated tumors were infiltrated by a vast number of dendritic cells, immune cells, and costimulatory molecules, contributing to the favorable antitumor immune response within the tumor microenvironment. The infiltration of cytotoxic T and NK cells were more profoundly existed within tumors in combination with radiotherapy and ICIs, leading to superior therapeutic efficacy in poorly immunogenic MSS-CRC. Taken together, these results showed that the novel topoisomerase I inhibitor TLC388 increased cancer immunogenicity by ssDNA/STING-mediated IFN-I production, enhancing antitumor immunity for better therapeutic efficacy in combination with radiotherapy and ICIs for poorly immunogenic cancer.

The InterPro protein families and domains database: 20 years on.

The InterPro database (https://www.ebi.ac.uk/interpro/) provides an integrative classification of protein sequences into families, and identifies functionally important domains and conserved sites. InterProScan is the underlying software that allows protein and nucleic acid sequences to be searched against InterPro's signatures. Signatures are predictive models which describe protein families, domains or sites, and are provided by multiple databases. InterPro combines signatures representing equivalent families, domains or sites, and provides additional information such as descriptions, literature references and Gene Ontology (GO) terms, to produce a comprehensive resource for protein classification. Founded in 1999, InterPro has become one of the most widely used resources for protein family annotation. Here, we report the status of InterPro (version 81.0) in its 20th year of operation, and its associated software, including updates to database content, the release of a new website and REST API, and performance improvements in InterProScan.

Interrelationships among growth hormone, thyroid function, and endocrine-disrupting chemicals on the susceptibility to attention-deficit/hyperactivity disorder.

Abnormal growth hormones and thyroid function may be linked to pathophysiology of attention-deficit/hyperactivity disorder (ADHD). Phthalates and bisphenol-A (BPA), two endocrine-disrupting chemicals (EDCs), may affect the human endocrine system. In this study, we aimed to perform a comprehensive investigation of whether growth hormone, thyroid function, and EDCs exhibited differential levels between ADHD patients and healthy controls. In total, 144 children with ADHD and 70 healthy control subjects were enrolled. Their endocrine systems were evaluated using the serum levels of insulin-like growth factor-1 (IGF-1), IGF-binding protein-3 (IGFBP-3), thyroid-stimulating hormone (TSH), triiodothyronine (T3), thyroxine (T4), and Free T4. The urinary levels of EDCs, including monoethyl phthalate (MEP), mono-methyl phthalate (MMP), monoethylhexyl phthalate (MEHP), mono-n-butyl phthalate (MnBP), monobenzyl phthalate (MBzP), and BPA, were also examined. Patients with ADHD had lower IGF-1 levels than healthy controls (p = 0.003), but we observed no significant difference in IGFBP-3, TSH, T3, T4, or Free T4. Compared to the control group, patients with ADHD demonstrated higher MEHP levels (p = 0.043), MnBP (p = 0.033), and MBzP (p = 0.040). Furthermore, MEHP levels (p < 0.001) and BPA levels (p = 0.041) were negatively correlated with IGF-1 levels, while IGF-1 levels were negatively correlated with principal components consisting of ADHD clinical symptoms and neuropsychological performance variables. We suggest that MEHP exposure may be associated with decreased serum levels of IGF-1 and increased risk of ADHD. The mechanism underlying this association may be important for protecting children from environmental chemicals that adversely affect neurodevelopment.

Atypical vesicles and membranes with monolayer and multilayer structures formed by graft copolymers with diblock side-chains: nonlamellar structures and curvature-enhanced permeability.

Graft copolymers with diblock side-chains Am(-graft-B3Ay)n in a selective solvent have been reported to self-assemble into vesicles, but the structure is expected to differ distinctly from those of lipid bilayers. Surprisingly, the number of alternating hydrophobic A-block and hydrophilic B-block layers in the vesicle can vary from a monolayer to multilayers such as the hepta-layer, subject to the same copolymer concentration. The area density of the copolymer layer is not uniform across the membrane. This structural difference among different layers is attributed to the neighboring environment and the curvature of the layer. Because of the unusual polymer conformations, nonlamellar structures of polymersomes are formed, and they are much more intricate than those of liposomes. In fact, a copolymer can contribute to a single or two hydrophilic layers, and it can provide up to three hydrophobic layers. The influence of the backbone length (m) and side-chain length (y) and the permeation dynamics are also studied. The thickness of hydrophobic layers is found to increase with increasing side-chain length but is not sensitive to the backbone length. Although the permeation time increases with the layer number for planar membranes, the opposite behavior is observed for spherical vesicles owing to the curvature-enhanced permeability associated with Laplace pressure.

ATAD3A stabilizes GRP78 to suppress ER stress for acquired chemoresistance in colorectal cancer.

AAA domain containing 3A (ATAD3A) is a nucleus-encoded mitochondrial protein with vital function in communication between endoplasmic reticulum (ER) and mitochondria which is participated in cancer metastasis. Here we show that elevated ATAD3A expression is clinically associated with poor 5-year disease-free survival in patients with colorectal cancer (CRC), especially high-risk CRC patients who received adjuvant chemotherapy. Our results indicated ATAD3A is significantly upregulated to reduce chemotherapy-induced cancer cell death. We found that knockdown of ATAD3A leads to dysregulation in protein processing for inducing ER stress by RNA sequencing (RNA-seq). In response to chemotherapy-induced ER stress, ATAD3A interacts with elevated GRP78 protein to assist protein folding and alleviate ER stress for cancer cell survival. This reduction of ER stress leads to reduce the surface exposure of calreticulin, which is the initiator of immunogenic cell death and antitumor immunity. However, silencing of ATAD3A enhances cell death, triggers the feasibility of chemotherapy-induced ER stress for antitumor immunity, increases infiltration of T lymphocytes and delays tumor regrowth in vitro and in vivo. Clinically, CRC patients with less ATAD3A have high density of CD45+ intratumoral infiltrating lymphocytes (TILs) and memory CD45RO+ TILs. Taken together, our results suggest that pharmacologic targeting to ATAD3A might be a potential therapeutic strategy to enhance antitumor immunity for CRC patients who received adjuvant chemotherapy.

Floating and Diving Loops of ABA Triblock Copolymers in Lipid Bilayers and Stability Enhancement for Asymmetric Membranes.

Hybrid membranes of lipids and AxByAz triblock copolymers can possess better biocompatibility and mechanical stability. In this work, triblock copolymer conformations and stability of asymmetric membranes are explored by dissipative particle dynamics. The triblock copolymers in the membranes exhibit either the bridge or loop conformation. As hydrophobic B-blocks interact attractively with lipid heads, bridge-shaped copolymers are significantly inhibited and loop-shaped copolymers prefer to stay at the interface between hydrophilic and hydrophobic layers. This floating loop has a flattened conformation, consistent with the experimental findings. In contrast, for repulsive interactions between B-blocks and lipid heads, bridge-shaped copolymers are abundant and loop-shaped copolymers tend to plunge into the hydrophobic layer. This diving loop displays a random coil conformation. The asymmetric membrane in which the fractions of loop-shaped copolymers in the upper and lower leaflets are different is thermodynamically unstable. Two approaches are proposed to acquire kinetically stable asymmetric membranes. First, membrane symmetrization is arrested by eliminating bridge-shaped copolymers, which is achieved by B-block/lipid head attraction and B-block/lipid tail repulsion. Second, asymmetric triblock copolymers (x ≠ z) are used to prevent the passage of the long A-block through the hydrophobic layer.

Strengthening mechanism of the mechanical properties of graft copolymers with incompatible pendant groups: nano-clusters and weak cross-linking.

It is known that the adhesive property and mechanical strength of an apolar polymer can be improved by grafting with polar side chains, whereas the underlying mechanism is still elusive. In this work, the equilibrium structure and mechanical moduli of the melt of graft copolymers have been explored by dissipative particle dynamics. Due to the strong immiscibility of the non-polar backbone and polar side chains, nano-clusters of side chains formed and acted as physical crosslinkers. Moreover, non-affinity adsorption of polar side chains in the melt to the wall was observed, revealing an improvement in the adhesion property. Subjecting graft copolymers to cyclic deformation, the storage and loss moduli were acquired, and they grew with increasing grafting density. The melt strength in terms of the crossover frequency ascended with more side chains on the backbone. Our findings reveal that the strengthening of the mechanical properties of graft copolymers can be attributed to the formation of weakly cross-linked structures, thus offering an insight into the molecular design to aid the development of stronger graft copolymers.

InterPro in 2019: improving coverage, classification and access to protein sequence annotations.

The InterPro database (http://www.ebi.ac.uk/interpro/) classifies protein sequences into families and predicts the presence of functionally important domains and sites. Here, we report recent developments with InterPro (version 70.0) and its associated software, including an 18% growth in the size of the database in terms on new InterPro entries, updates to content, the inclusion of an additional entry type, refined modelling of discontinuous domains, and the development of a new programmatic interface and website. These developments extend and enrich the information provided by InterPro, and provide greater flexibility in terms of data access. We also show that InterPro's sequence coverage has kept pace with the growth of UniProtKB, and discuss how our evaluation of residue coverage may help guide future curation activities.

Analysis of fibroblast migration dynamics in idiopathic pulmonary fibrosis using image-based scaffolds of the lung extracellular matrix.

Idiopathic pulmonary fibrosis (IPF) is characterized by a profound remodeling of the collagen in the extracellular matrix (ECM), where the fibers become both denser and more highly aligned. However, it is unknown how this reconfiguration of the collagen matrix affects disease progression. Here, we investigate the role of specific alterations in collagen fiber organization on cell migration dynamics by using biomimetic image-based collagen scaffolds representing normal and fibrotic lung, where the designs are derived directly from high-resolution second harmonic generation microscopy images. The scaffolds are fabricated by multiphoton-excited (MPE) polymerization, where the process is akin to three-dimensional printing, except that it is performed at much greater resolution (∼0.5 microns) and with collagen and collagen analogs. These scaffolds were seeded with early passaged primary human normal and IPF fibroblasts to enable the decoupling of the effect of cell-intrinsic characteristics (normal vs. IPF) versus ECM structure (normal vs. IPF) on migration dynamics. We found that the highly aligned IPF collagen structure promoted enhanced cell elongation and F-actin alignment along with increased cell migration speed and straightness relative to the normal tissues. Collectively, the data are consistent with an enhanced contact guidance mechanism on the aligned IPF matrix. Although cell intrinsic effects were observed, the aligned collagen matrix morphology had a larger effect on these metrics. Importantly, these biomimetic models of the lung cannot be synthesized by conventional fabrication methods. We suggest that the MPE image-based fabrication method will enable additional hypothesis-based testing studies of cell-matrix interactions in the context of tissue fibrosis.

Tylophorine-based compounds are therapeutic in rheumatoid arthritis by targeting the caprin-1 ribonucleoprotein complex and inhibiting expression of associated c-Myc and HIF-1α.

Interruption of the Warburg effect - the observation that un-stimulated macrophages reprogram their core metabolism from oxidative phosphorylation toward aerobic glycolysis to become pro-inflammatory M1 macrophages upon stimulation - is an emerging strategy for the treatment of cancer and anti-inflammatory diseases such as rheumatoid arthritis. We studied this process with view to the discovery of novel therapeutics, and found that tylophorine-based compounds targeted a ribonucleoprotein complex containing caprin-1 and mRNAs of c-Myc and HIF-1α in LPS/IFN-γ stimulated Raw264.7 cells, diminished the protein levels of c-Myc and HIF-1α, and consequently downregulated their targeted genes that are associated with the Warburg effect, as well as the pro-inflammatory iNOS and COX2. The tylophorine-based compound DBQ 33b significantly meliorated the severity and incidence of type II collagen-monoclonal antibody-induced rheumatoid arthritis and diminished gene expressions of c-Myc, HIF-1α, iNOS, COX2, TNFα, and IL-17A in vivo. Moreover, pharmacological inhibition of either c-Myc or HIF-1α exhibited similar effects as the tylophorine-based compound DBQ 33b, even though inhibition of c-Myc reversed the induction of iNOS and COX2 in LPS/IFN-γ stimulated Raw264.7 cells to a lesser degree. Therefore, simultaneous inhibition of both c-Myc and HIF-1α is efficacious for anti-inflammation in vitro and in vivo and merits further study.

InterPro in 2017-beyond protein family and domain annotations.

InterPro (http://www.ebi.ac.uk/interpro/) is a freely available database used to classify protein sequences into families and to predict the presence of important domains and sites. InterProScan is the underlying software that allows both protein and nucleic acid sequences to be searched against InterPro's predictive models, which are provided by its member databases. Here, we report recent developments with InterPro and its associated software, including the addition of two new databases (SFLD and CDD), and the functionality to include residue-level annotation and prediction of intrinsic disorder. These developments enrich the annotations provided by InterPro, increase the overall number of residues annotated and allow more specific functional inferences.

The cardenolide ouabain suppresses coronaviral replication via augmenting a Na+/K+-ATPase-dependent PI3K_PDK1 axis signaling.

Cardenolides are plant-derived toxic substances. Their cytotoxicity and the underlying mechanistic signaling axes have been extensively documented, but only a few anti-viral activities of cardenolides and the associated signaling pathways have been reported. Previously, we reported that a variety of cardenolides impart anti-transmissible gastroenteritis coronavirus (TGEV) activity in swine testicular (ST) cells, through targeting of the cell membrane sodium/potassium pump, Na+/K+-ATPase. Herein, we further explore the potential signaling cascades associated with this anti-TGEV activity in ST cells. Ouabain, a representative cardenolide, was found to potently diminish TGEV titers and inhibit the TGEV-induced production of IL-6 in a dose dependent manner, with 50% inhibitory concentrations of 37 nM and 23 nM respectively. By pharmacological inhibition and gene silencing, we demonstrated that PI3K_PDK1_RSK2 signaling was induced in TGEV-infected ST cells, and ouabain imparted a degree of anti-TGEV activity via further augmentation of this existing PI3K_PDK1 axis signaling, in a manner dependent upon its association with the Na+/K+-ATPase. Finally, inhibition of PI3K by LY294002 or PDK1 by BX795 antagonized the anti-viral activity of ouabain and restored the TGEV virus titer and yields. This finding is the first report of a PI3K_PDK1 signaling axis further induced by ouabain and implicated in the suppression of TGEV activity and replication; greatly illuminates the underlying mechanism of cardenolide toxicity; and is expected to result in one or more anti-viral applications for the cardenolides in the future.

Identification of anti-viral activity of the cardenolides, Na+/K+-ATPase inhibitors, against porcine transmissible gastroenteritis virus.

A series of naturally occurring cardenolides that exhibit potent anti-transmissible gastroenteritis virus (TGEV) activity in swine testicular (ST) cells has been identified. In an immunofluorescence assay, these cardenolides were found to diminish the expressions of TGEV nucleocapsid and spike protein, which was used as an indication for viral replication; block TGEV infection induced apoptosis and cytopathic effects; and impart the same trend of inhibitory activity against Na+/K+-ATPase as for anti-TGEV activity. The viral titer inhibition was found to take place in a dose-dependent manner. Knocking down expression of Na+/K+-ATPase, the cellular receptor of cardenolides, in ST cells was found to significantly impair the susceptibility of ST cells to TGEV infectivity. Thus, we have identified Na+/K+-ATPase as an anti-viral drug target and its antagonists, cardenolides, a novel class of anti- TGEV agents.

Three-dimensional patterned graphene oxide-quantum dot microstructures via two-photon crosslinking.

The two-photon crosslinking of graphene oxide-quantum dots (GOQDs) adopts rose Bengal as the photoactivator to induce the GOQD assembly process. Based on the Förster resonance energy transfer mechanism with oxygen as the crosslinking medium, three-dimensional patterned GOQD microstructures with near diffraction-limit spatial resolution have been fabricated and analyzed by a multiphoton excited fabrication instrument/microscope.

The InterPro protein families database: the classification resource after 15 years.

The InterPro database (http://www.ebi.ac.uk/interpro/) is a freely available resource that can be used to classify sequences into protein families and to predict the presence of important domains and sites. Central to the InterPro database are predictive models, known as signatures, from a range of different protein family databases that have different biological focuses and use different methodological approaches to classify protein families and domains. InterPro integrates these signatures, capitalizing on the respective strengths of the individual databases, to produce a powerful protein classification resource. Here, we report on the status of InterPro as it enters its 15th year of operation, and give an overview of new developments with the database and its associated Web interfaces and software. In particular, the new domain architecture search tool is described and the process of mapping of Gene Ontology terms to InterPro is outlined. We also discuss the challenges faced by the resource given the explosive growth in sequence data in recent years. InterPro (version 48.0) contains 36,766 member database signatures integrated into 26,238 InterPro entries, an increase of over 3993 entries (5081 signatures), since 2012.

InterProScan 5: genome-scale protein function classification.

MOTIVATION: Robust large-scale sequence analysis is a major challenge in modern genomic science, where biologists are frequently trying to characterize many millions of sequences. Here, we describe a new Java-based architecture for the widely used protein function prediction software package InterProScan. Developments include improvements and additions to the outputs of the software and the complete reimplementation of the software framework, resulting in a flexible and stable system that is able to use both multiprocessor machines and/or conventional clusters to achieve scalable distributed data analysis. InterProScan is freely available for download from the EMBl-EBI FTP site and the open source code is hosted at Google Code.

Graphene oxide-based micropatterns via high-throughput multiphoton-induced reduction and ablation.

In this study, a developed temporal focusing-based femtosecond laser system provides high-throughput multiphoton-induced reduction and ablation of graphene oxide (GO) films. Integrated with a digital micromirror device to locally control the laser pulse numbers, GO-based micropatterns can be quickly achieved instantly. Furthermore, the degree of reduction and ablation can be precisely adjusted via controlling the laser wavelength, power, and pulse number. Compared to point-by-point scanning laser direct writing, this approach offers a high-throughput and multiple-function approach to accomplish a large area of micro-scale patterns on GO films. The high-throughput micropatterning of GO via the temporal focusing-based femtosecond laser system fulfills the requirement of mass production for GO-based applications in microelectronic devices.

Phyllanthus emblica L. polysaccharides ameliorate colitis via microbiota modulation and dual inhibition of the RAGE/NF-κB and MAPKs signaling pathways in rats.

Pharmacological treatments for colitis have limited efficacy and side effects. Plant polysaccharides improve colitis by modulating the gut microbiota. However, the specific benefits of Phyllanthus emblica L. polysaccharides (PEPs) in colitis remain unclear. Therefore, this study aimed to assess the physical characteristics and health advantages of PEP in rats subjected to 2,4,6-trinitrobenzene sulfonic acid (TNBS) treatment. The results showed that PEP (1.226 × 103 kDa) was an α-acidic pyran heteropolysaccharide rich in galactose and galacturonic acid. Prefeeding rats with PEP significantly decreased the levels of NO, MDA, proinflammatory cytokines (IL-6, IL-1ß, TNF-α), apoptosis, and the activities of mucinase and ß-glucuronidase. These changes were accompanied by increases in the levels of anti-inflammatory cytokines (IL-4, IL-10) and antioxidant enzymes (SOD, catalase, GPx) in colitis rats. Mechanistically, PEP suppressed the abundance of inflammatory-related bacteria (Bacteroides, Intestinimonas, and Parabacteroides) while promoting the growth of short-chain fatty acid (SCFA)-producing bacteria (Romboutsia, Clostridium_sensu_stricto_1, and Lactobacillus), along with an increase in SCFA secretion. SCFAs may engage with the GPR43 receptor and inhibit downstream HDAC3, consequently downregulating the activation of the RAGE/NF-κB and MAPK pathways. In conclusion, PEP demonstrated preventive effects through its antioxidant, anti-inflammatory, and microbiota modulation properties, thereby ameliorating TNBS-induced colitis in rats.

Phyllanthus emblica Fruit Improves Obesity by Reducing Appetite and Enhancing Mucosal Homeostasis via the Gut Microbiota-Brain-Liver Axis in HFD-Induced Leptin-Resistant Rats.

The impact of leptin resistance on intestinal mucosal barrier integrity, appetite regulation, and hepatic lipid metabolism through the microbiota-gut-brain-liver axis has yet to be determined. Water extract of Phyllanthus emblica L. fruit (WEPE) and its bioactive compound gallic acid (GA) effectively alleviated methylglyoxal (MG)-triggered leptin resistance in vitro. Therefore, this study investigated how WEPE and GA intervention relieve leptin resistance-associated dysfunction in the intestinal mucosa, appetite, and lipid accumulation through the microbiota-gut-brain-liver axis in high-fat diet (HFD)-fed rats. The results showed that WEPE and GA significantly reduced tissues (jejunum, brain, and liver) MG-evoked leptin resistance, malondialdehyde (MDA), proinflammatory cytokines, SOCS3, orexigenic neuropeptides, and lipid accumulation through increasing leptin receptor, tight junction proteins, antimicrobial peptides, anorexigenic neuropeptides, excretion of fecal triglyceride (TG), and short-chain fatty acids (SCFAs) via a positive correlation with the Allobaculum and Bifidobacterium microbiota. These novel findings suggest that WEPE holds the potential as a functional food ingredient for alleviating obesity and its complications.

Inhibition of DNMTs increases neoantigen-reactive T-cell toxicity against microsatellite-stable colorectal cancer in combination with radiotherapy.

The therapeutic efficacy of immunotherapy is limited in the majority of colorectal cancer patients due to the low mutational and neoantigen burdens in this immunogenically "cold" microsatellite stability-colorectal cancer (MSS-CRC) cohort. Here, we showed that DNA methyltransferase (DNMT) inhibition upregulated neoantigen-bearing gene expression in MSS-CRC, resulting in increased neoantigen presentation by MHC class I in tumor cells and leading to increased neoantigen-specific T-cell activation in combination with radiotherapy. The cytotoxicity of neoantigen-reactive T cells (NRTs) to DNMTi-treated cancer cells was highly cytotoxic, and these cells secreted high IFNγ levels targeting MSS-CRC cells after ex vivo expansion of NRTs with DNMTi-treated tumor antigens. Moreover, the therapeutic efficacy of NRTs further increased when NRTs were combined with radiotherapy in vivo. Administration of DNMTi-augmented NRTs and radiotherapy achieved an ∼50 % complete response and extended survival time in an immunocompetent MSS-CRC animal model. Moreover, remarkably, splenocytes from these mice exhibited neoantigen-specific T-cell responses, indicating that radiotherapy in combination with DNMTi-augmented NRTs prolonged and increased neoantigen-specific T-cell toxicity in MSS-CRC patients. In addition, these DNMTi-augmented NRTs markedly increase the therapeutic efficacy of cancer vaccines and immune checkpoint inhibitors (ICIs). These data suggest that a combination of radiotherapy and epi-immunotherapeutic agents improves the function of ex vivo-expanded neoantigen-reactive T cells and increases the tumor-specific cytotoxic effector population to enhance therapeutic efficacy in MSS-CRC.