Intravascular Casting Radiopaque Hydrogel Systems for Transarterial Chemo/Cascade Catalytic/Embolization Therapy of Hepatocellular Carcinoma.

This paper introduces catheter-directed intravascular casting hydrogels for transarterial chemo/starvation/chemodynamic embolization (TACSCE) therapy of hepatocellular carcinoma (HCC). Comprising Mn ion-crosslinked hyaluronic acid-dopamine (HD) with glucose oxidase (for glucose decomposition to H2O2 in starvation therapy), doxorubicin (for chemotherapy), and iopamidol (for X-ray imaging), these hydrogels are fabricated for transarterial embolization therapy guided by X-ray fluoroscopy. Mn4+ (from MnO2) demonstrates strong coordination with the catechol group of HD, providing hypoxia relief through O2 generation and cellular glutathione (GSH) consumption, compared to the OH radical generation potential of Mn2+. The gelation time-controlled, catheter-injectable, and rheologically tuned multitherapeutic/embolic gel system effectively reaches distal arterioles, ensuring complete intravascular casting with fewer complications related to organic solvents. Glucose deprivation, cascade reactive oxygen species (ROS) generation, GSH depletion, and sustained release profiles of multiple drug cargos from the hydrogel system are also achieved. The combined chemo/starvation/chemodynamic efficacies of these designed hydrogel systems are confirmed in HCC cell cultures and HCC-bearing animal models. The developed radiopaque/injectable/embolic/sol-to-gel transformable systems for TACSCE therapy may offer enhanced therapeutic efficacies compared to typical transarterial embolization and transarterial chemoembolization procedures for HCC.

Cuproptosis-Inducible Chemotherapeutic/Cascade Catalytic Reactor System for Combating with Breast Cancer.

Cascade hydroxyl radical generating hydrogel reactor structures including a chemotherapeutic agent are invented for multiple treatment of breast cancer. Glucose oxidase (GOx) and cupric sulfate (Cu) are introduced for transforming accumulated glucose (in cancer cells) to hydroxyl radicals for starvation/chemodynamic therapy. Cu may also suppress cancer cell growth via cuproptosis-mediated cell death. Berberine hydrochloride (BER) is engaged as a chemotherapeutic agent in the hydrogel reactor for combining with starvation/chemodynamic/cuproptosis therapeutic modalities. Moreover, Cu is participated as a gel crosslinker by coordinating with catechol groups in hyaluronic acid-dopamine (HD) polymer. Controlling viscoelasticity of hydrogel reactor can extend the retention time following local injection and provide sustained drug release patterns. Low biodegradation rate of designed HD/BER/GOx/Cu hydrogel can reduce dosing frequency in local cancer therapy and avoid invasiveness-related inconveniences. Especially, it is anticipated that HD/BER/GOx/Cu hydrogel system can be applied for reducing size of breast cancer prior to surgery as well as tumor growth suppression in clinical application.

Opportunistic detection of Fusobacterium nucleatum as a marker for the early gut microbial dysbiosis.

BACKGROUND: The essential roles of gut microbiome have been emphasized in modulating human health and disease. Fusobacterium nucleatum (F. nucleatum), an obligate Gram-negative microorganism residing in oral cavity, gastrointestinal tract and elsewhere, has been recently considered as a potential oncobacterium associated with human cancers. However, the consequence of its enrichment was not extensively explored in terms of microbial homeostasis and stability at the early stage of disease development. RESULT: Our analysis on longitudinal metagenomic data generated by the Integrative Human Microbiome Project (iHMP) showed that F. nucleatum was frequently found in inflammatory bowel diseases (IBD) subjects with reduced microbial diversity. Using non-parametric logarithmic linear discriminant analysis (LDA) effect size (LEfSe) algorithm, 12 IBD- and 14 non-IBD-specific bacterial species were identified in the fecal metagenome and the IBD-specific ones were over-represented in the F. nucleatum-experienced subjects during long-term surveillance. In addition, F. nucleatum experience severely abrogated intra-personal stability of microbiome in IBD patients and induced highly variable gut microbiome between subjects. From the longitudinal comparison between microbial distributions prior and posterior to F. nucleatum detection, 41 species could be proposed as indicative "classifiers" for dysbiotic gut state. By multiple logistic regression models established on these classifiers, the high probability of experiencing F. nucleatum was significantly correlated with decreased alpha-diversity and increased number of biomarker species for IBD and colorectal cancer (CRC). Finally, microbial clustering confirmed that biomarker species for IBD and non-IBD conditions as well as CRC signature markers were well distinguishable and could be utilized for explaining gut symbiosis and dysbiosis. CONCLUSION: F. nucleatum opportunistically appeared under early dysbiotic condition in gut, and discriminative classifier species associated with F. nucleatum were successfully applied to predict microbial alterations in both IBD and non-IBD conditions. Our prediction model and microbial classifier biomarkers for estimating gut dysbiosis should provide a novel aspect of microbial homeostasis/dynamics and useful information on non-invasive biomarker screening.

UNC93B1 is essential for the plasma membrane localization and signaling of Toll-like receptor 5.

The proper trafficking and localization of Toll-like receptors (TLRs) are important for specific ligand recognition and efficient signal transduction. The TLRs sensing bacterial membrane components are expressed on the cell surface and recruit signaling adaptors to the plasma membrane upon stimulation. On the contrary, the nucleotide-sensing TLRs are mostly found inside cells and signal from the endolysosomes in an acidic pH-dependent manner. Trafficking of the nucleotide-sensing TLRs from the endoplasmic reticulum to the endolysosomes strictly depends on UNC93B1, and their signaling is completely abolished in the 3d mutant mice bearing the H412R mutation of UNC93B1. In contrast, UNC93B1 was considered to have no role for the cell surface-localized TLRs and signaling via TLR1, TLR2, TLR4, and TLR6 is normal in the 3d mice. Unexpectedly, we discovered that TLR5, a cell surface receptor for bacterial protein flagellin, also requires UNC93B1 for plasma membrane localization and signaling. TLR5 physically interacts with UNC93B1, and the cells from the 3d or UNC93B1-deficient mice not only lack TLR5 at the plasma membrane but also fail to secret cytokines and to up-regulate costimulatory molecules upon flagellin stimulation, demonstrating the essential role of UNC93B1 in TLR5 signaling. Our study reveals that the role of UNC93B1 is not limited to the TLRs signaling from the endolysosomes and compels the further probing of the mechanisms underlying the UNC93B1-assisted differential targeting of TLRs.

Hydrogel design to overcome thermal resistance and ROS detoxification in photothermal and photodynamic therapy of cancer.

Responsive heat resistance (by heat shock protein upregulation) and spontaneous reactive oxygen species (ROS) detoxification have been regarded as the major obstacles for photothermal/photodynamic therapy of cancer. To overcome the thermal resistance and improve ROS susceptibility in breast cancer therapy, Au ion-crosslinked hydrogels including indocyanine green (ICG) and polyphenol are devised. Au ion has been introduced for gel crosslinking (by catechol-Au3+ coordination), cellular glutathione depletion, and O2 production from cellular H2O2. ICG can generate singlet oxygen from O2 (for photodynamic therapy) and induce hyperthermia (for photothermal therapy) under the near-infrared laser exposure. (-)-Epigallocatechin gallate downregulates heat shock protein to overcome heat resistance during hyperthermia and exerts multiple anticancer functions in spite of its ironical antioxidant features. Those molecules are concinnously engaged in the hydrogel structure to offer fast gel transformation, syringe injection, self-restoration, and rheological tuning for augmented photo/chemotherapy of cancer. Intratumoral injection of multifunctional hydrogel efficiently suppressed the growth of primary breast cancer and completely eliminated the residual tumor mass. Proposed hydrogel system can be applied to tumor size reduction prior to surgery of breast cancer and the complete remission after its surgery.

Enterotypical Prevotella and three novel bacterial biomarkers in preoperative stool predict the clinical outcome of colorectal cancer.

BACKGROUND: A significant proportion of colorectal cancer (CRC) patients suffer from early recurrence and progression after surgical treatment. Although the gut microbiota is considered as a key player in the initiation and progression of CRC, most prospective studies have been focused on a particular pathobionts such as Fusobacterium nucleatum. Here, we aimed to identify novel prognostic bacteria for CRC by examining the preoperative gut microbiota through 16S ribosomal RNA gene sequencing. RESULTS: We collected stool samples from 333 patients with primary CRC within 2 weeks before surgery and followed up the patients for a median of 27.6 months for progression and 43.6 months for survival. The sequence and prognosis data were assessed using the log-rank test and multivariate Cox proportional hazard analysis. The gut microbiota was associated with the clinical outcomes of CRC patients (Pprogress = 0.011, Pdecease = 0.007). In particular, the high abundance of Prevotella, a representative genus of human enterotypes, indicated lower risks of CRC progression (P = 0.026) and decease (P = 0.0056), while the occurrence of Alistipes assigned to Bacteroides sp., Pyramidobacter piscolens, Dialister invisus, and Fusobacterium nucleatum indicated a high risk of progression. A microbiota-derived hazard score considering the five prognostic bacteria accurately predicted CRC progression in 1000 random subsamples; it outperformed widely accepted clinical biomarkers such as carcinoembryonic antigen and lymphatic invasion, after adjustment for the clinicopathological stage (adjusted HR 2.07 [95% CI, 1.61-2.64], P = 7.8e-9, C-index = 0.78). PICRUSt2 suggested that microbial pathways pertaining to thiamine salvage and L-histidine degradation underlie the different prognoses. CONCLUSIONS: The enterotypical genus Prevotella was demonstrated to be useful in improving CRC prognosis, and combined with the four pathobionts, our hazard score based on the gut microbiota should provide an important asset in predicting medical outcomes for CRC patients. Video Abstract.

N-glycosylation of UNC93B1 at a Specific Asparagine Residue Is Required for TLR9 Signaling.

Toll-like receptors (TLRs) play critical roles in the first line of host defense against pathogens through recognition of pathogen-associated molecular patterns and initiation of the innate immune responses. The proper localization of TLRs in specific subcellular compartments is crucial for their ligand recognition and downstream signaling to ensure appropriate responses against pathogens while avoiding erroneous or excessive activation. Several TLRs, including TLR7 and TLR9 but not TLR4, depend on UNC93B1 for their proper intracellular localization and signaling. Accumulating evidence suggest that UNC93B1 differentially regulates its various client TLRs, but the specific mechanisms by which UNC93B1 controls individual TLRs are not well understood. Protein N-glycosylation is one of the most frequent and important post-translational modification that occurs in membrane-localized or secreted proteins. UNC93B1 was previously shown to be glycosylated at Asn251 and Asn272 residues. In this study, we investigated whether N-glycosylation of UNC93B1 affects its function by comparing wild type and glycosylation-defective mutant UNC93B1 proteins. It was found that glycosylation of Asn251 and Asn272 residues can occur independently of each other and mutation of neither N251Q or N272Q in UNC93B1 altered expression and localization of UNC93B1 and TLR9. In contrast, CpG DNA-stimulated TLR9 signaling was severely inhibited in cells expressing UNC93B1(N272Q), but not in cells with UNC93B1(N251Q). Further, it was found that glycosylation at Asn272 of UNC93B1 is essential for the recruitment of MyD88 to TLR9 and the subsequent downstream signaling. On the other hand, the defective glycosylation at Asn272 did not affect TLR7 signaling. Collectively, these data demonstrate that the glycosylation at a specific asparagine residue of UNC93B1 is required for TLR9 signaling and the glycosylation status of UNC93B1 differently affects activation of TLR7 and TLR9.

Gut microbiota of the young ameliorates physical fitness of the aged in mice.

BACKGROUND: Aging is a natural process that an organism gradually loses its physical fitness and functionality. Great efforts have been made to understand and intervene in this deteriorating process. The gut microbiota affects host physiology, and dysbiosis of the microbial community often underlies the pathogenesis of host disorders. The commensal microbiota also changes with aging; however, the interplay between the microbiota and host aging remains largely unexplored. Here, we systematically examined the ameliorating effects of the gut microbiota derived from the young on the physiology and phenotypes of the aged. RESULTS: As the fecal microbiota was transplanted from young mice at 5 weeks after birth into 12-month-old ones, the thickness of the muscle fiber and grip strength were increased, and the water retention ability of the skin was enhanced with thickened stratum corneum. Muscle thickness was also marginally increased in 25-month-old mice after transferring the gut microbiota from the young. Bacteria enriched in 12-month-old mice that received the young-derived microbiota significantly correlated with the improved host fitness and altered gene expression. In the dermis of these mice, transcription of Dbn1 was most upregulated and DBN1-expressing cells increased twice. Dbn1-heterozygous mice exhibited impaired skin barrier function and hydration. CONCLUSIONS: We revealed that the young-derived gut microbiota rejuvenates the physical fitness of the aged by altering the microbial composition of the gut and gene expression in muscle and skin. Dbn1, for the first time, was found to be induced by the young microbiota and to modulate skin hydration. Our results provide solid evidence that the gut microbiota from the young improves the vitality of the aged. Video Abstract.

Flagellin-Stimulated Production of Interferon-ß Promotes Anti-Flagellin IgG2c and IgA Responses.

Flagellin, a major structural protein of the flagellum found in all motile bacteria, activates the TLR5- or NLRC4 inflammasomedependent signaling pathway to induce innate immune responses. Flagellin can also serve as a specific antigen for the adaptive immune system and stimulate anti-flagellin antibody responses. Failure to recognize commensal-derived flagellin in TLR5-deficient mice leads to the reduction in antiflagellin IgA antibodies at steady state and causes microbial dysbiosis and mucosal barrier breach by flagellated bacteria to promote chronic intestinal inflammation. Despite the important role of anti-flagellin antibodies in maintaining the intestinal homeostasis, regulatory mechanisms underlying the flagellin-specific antibody responses are not well understood. In this study, we show that flagellin induces interferon-ß (IFN-ß) production and subsequently activates type I IFN receptor signaling in a TLR5- and MyD88-dependent manner in vitro and in vivo . Internalization of TLR5 from the plasma membrane to the acidic environment of endolysosomes was required for the production of IFN-ß, but not for other proinflammatory cytokines. In addition, we found that antiflagellin IgG2c and IgA responses were severely impaired in interferon-alpha receptor 1 (IFNAR1)-deficient mice, suggesting that IFN-ß produced by the flagellin stimulation regulates anti-flagellin antibody class switching. Our findings shed a new light on the regulation of flagellin-mediated immune activation and may help find new strategies to promote the intestinal health and develop mucosal vaccines.

Disrupted-in-schizophrenia 1 (DISC1) and Syntaphilin collaborate to modulate axonal mitochondrial anchoring.

In neuronal axons, the ratio of motile-to-stationary mitochondria is tightly regulated by neuronal activation, thereby meeting the need for local calcium buffering and maintaining the ATP supply. However, the molecular players and detailed regulatory mechanisms behind neuronal mitochondrial movement are not completely understood. Here, we found that neuronal activation-induced mitochondrial anchoring is regulated by Disrupted-in-schizophrenia 1 (DISC1), which is accomplished by functional association with Syntaphilin (SNPH). DISC1 deficiency resulted in reduced axonal mitochondrial movement, which was partially reversed by concomitant SNPH depletion. In addition, a SNPH deletion mutant lacking the sequence for interaction with DISC1 exhibited an enhanced mitochondrial anchoring effect than wild-type SNPH. Moreover, upon neuronal activation, mitochondrial movement was preserved by DISC1 overexpression, not showing immobilized response of mitochondria. Taken together, we propose that DISC1 in association with SNPH is a component of a modulatory complex that determines mitochondrial anchoring in response to neuronal activation.