Boosting Cancer Immunotherapy via Reversing PD-L1-Mediated Immunosuppression with a Molecularly Imprinted Lysosomal Nanodegrader.

Immune checkpoint blockade therapy has achieved important clinical advances in several types of tumors, particularly via targeting the PD-1/PD-L1 axis. However, existing therapeutic strategies that suppress the PD-1/PD-L1 signal pathway usually experience low treatment efficacy and the risk of causing autoimmune diseases. Herein, we report a cancer cell-targeted molecularly imprinted lysosomal nanodegrader (MILND) for boosting immune checkpoint blockade therapy against tumors. The MILND, imprinted with the N-terminal epitope of PD-L1 as an imprinting template, could specifically target the PD-L1 on tumor cells to promote cellular uptake. This process further induces the transport of PD-L1 into lysosomes for degradation, ultimately resulting in the downregulation of PD-L1 expression levels on tumor cells. As a result, a T cell-mediated immune response in the body was activated via the blockade of the PD-1/PD-L1 signaling pathway, which triggered a durable antitumor efficacy. In vivo experiments demonstrated that the MILND could effectively accumulate in tumor sites and exhibit strong tumor growth suppression efficacy in a xenograft tumor model without obvious side effects. Therefore, the MILND provides not only a promising strategy for boosting cancer immunotherapy but also insights for developing molecular imprinting-empowered nanomedicines.

Catalytic NIR chemiluminescence sensor with enhanced persistence and intensity for in vivo imaging.

Chemiluminescence (CL) is a self-illumination phenomenon that involves the emission of light from chemical reactions, and it provides favorable spatial and temporal information on biological processes. However, it is still a great challenge to construct effective CL sensors that equip strong CL intensity, long emission wavelength, and persistent luminescence for deep tissue imaging. Here, we report a liposome encapsulated polymer dots (Pdots)-based system using catalytic CL substrates (L-012) as energy donor and fluorescent polymers and dyes (NIR 695) as energy acceptors for efficient Near-infrared (NIR) CL in vivo imaging. Thanks to the modulation of paired donor and acceptor distance and the slow diffusion of biomarker by liposome, the Pdots show a NIR emission wavelength (λ em, max = 720 nm), long CL duration (>24 h), and a high chemiluminescence resonance energy transfer efficiency (46.5 %). Furthermore, the liposome encapsulated Pdots possess excellent biocompatibility, sensitive response to H2O2, and persistent whole-body NIR CL imaging in the drug-induced inflammation and the peritoneal metastatic tumor mouse model. In a word, this NIR-II CL nanoplatform with long-lasting emission and high spatial-temporal resolution will be a concise strategy in deep tissue imaging and clinical diagnostics.

Molecularly Imprinted Nanomedicine for Anti-angiogenic Cancer Therapy via Blocking Vascular Endothelial Growth Factor Signaling.

The VEGF-VEGFR2 (VEGF = vascular endothelial growth factor) signaling has been a promising target in cancer therapy. However, because conventional anti-angiogenic therapeutics suffer from drawbacks, particularly severe side effects, novel anti-angiogenic strategies are much needed. Herein, we report the rational engineering of VEGF-targeted molecularly imprinted polymer nanoparticles (nanoMIP) for anti-angiogenic cancer therapy. The anti-VEGF nanomedicine was prepared via a state-of-the-art molecular imprinting approach using the N-terminal epitope of VEGF as the template. The nanoMIP could target the two major pro-angiogenic isoforms (VEGF165 and VEGF121) with high affinity and thereby effectively block the VEGF-VEGFR2 signaling, yielding a potent anti-angiogenic effect of "killing two birds with one stone". In vivo experiments demonstrated that the anti-VEGF nanoMIP effectively suppressed tumor growth via anti-angiogenesis in a xenograft model of human colon carcinoma without apparent side effects. Thus, this study not only proposes an unprecedented anti-angiogenic strategy for cancer therapy but also provides a new paradigm for the rational development of MIPs-based "drug-free" nanomedicines.

ROS-Responsive Fluorescent Sensor Array for Precise Diagnosis of Cancer via pH-Controlled Multicolor Gold Nanoclusters.

Intracellular reactive oxygen species (ROS) are closely associated with cancer cell types. Therefore, ROS-based pattern recognition is a promising strategy for precise diagnosis of cancer, but such a possibility has never been reported yet. Herein, we proposed an ROS-responsive fluorescent sensor array based on pH-controlled histidine-templated gold nanoclusters (AuNCs@His) to distinguish cancer cell types and their proliferation states. In this strategy, three types of AuNCs@His with diverse fluorescence profiles were first synthesized by only adjusting the pH value. Upon the addition of various ROS, fluorescence quenching of three types of AuNCs@His occurred with different degrees, thereby forming unique optical "fingerprints", which were well-clustered into several separated groups without overlap by principal component analysis (PCA). The sensing mechanism was attributable to the oxidation of AuNCs@His by ROS, as revealed by X-ray photoemission spectroscopy, Fourier transform infrared spectroscopy, 1H nuclear magnetic resonance spectroscopy, and electrospray ionization mass spectrometry. Based on the ROS-responsive sensing pattern, cancer cell types were successfully differentiated via PCA with 100% accuracy. Additionally, the proposed sensor array exhibited excellent performance in distinguishing the proliferation states of cancer cells, which was supported by the results of the Ki-67 immunohistochemistry assay. Overall, the ROS-responsive fluorescent sensor array can serve as a promising tool for precise diagnosis of cancer, indicating great potential for clinical application.

Leveraging Macrophage-Mediated Cancer Immunotherapy via a Cascading Effect Induced by a Molecularly Imprinted Nanocoordinator.

Reprogramming tumor-associated macrophages (TAMs) has emerged as a promising strategy in cancer immunotherapy. Targeted therapeutics integrating multiple functions to fully leverage the antitumor immune functions of macrophages without affecting systemic or tissue-resident macrophages are crucial for TAM reprogramming. Herein, by integrating molecular imprinting and nanotechnology, we rationally designed and engineered an unprecedented nanocoordinator for targeted remolding of TAMs to fully leverage the antitumor efficacy of macrophages by inducing a cascade effect. The nanocoordinator features a magnetic iron oxide nanoinner core and sialic acid-imprinted shell. Intravenously administered into systemic circulation, the nanocoordinator can rapidly accumulate at the tumor site in response to an external magnet. Then, by specifically binding to sialic acid overexpressed on tumor cells, the nanocoordinator anchors at the tumor site with prolonged retention time. Via binding with the nanocoordinator, tumor cells are tagged with a foreign substance, which promotes the intrinsic phagocytosis of macrophages. Subsequently, the nanocoordinator taken up by macrophages effectively promotes the polarization of macrophages toward the M1 phenotype, thus activating the immunotherapeutic efficacy of macrophages. Synergized by the cascade effect, this nanocoordinator effectively harnesses TAMs for macrophage-mediated immunotherapy. This study offers new TAM-targeted therapeutics that allows us to fully leverage the antitumor immune functions of macrophages without affecting the normal tissue.

Molecularly Imprinted Nanobeacons Redirect Innate Immune Killing towards Triple Negative Breast Cancer.

Harnessing innate immunity is an appealing strategy for cancer treatment. Herein, we report a new strategy called molecularly imprinted nanobeacons (MINBs) for redirecting innate immune killing towards triple-negative breast cancer (TNBC). The MINBs were molecularly imprinted nanoparticles with the N-epitope of glycoprotein nonmetastatic B (GPNMB) as the template and grafted with plentiful fluorescein moieties as the hapten. The MINBs could tag the TNBC cells via binding with GPNMB and thereby provide navigation for recruiting hapten-specific antibodies. The gathered antibodies could further trigger effective Fc-domain-mediated immune killing towards the tagged cancer cells. In vivo experiments showed that the TNBC growth was significantly inhibited after MINBs treatment by intravenous injection as compared with control groups. This study not only opens a new access for redirecting innate immunity towards TNBC but also paves the way for innate immunity-based therapy of other diseases.

Molecular imprinting and cladding produces antibody mimics with significantly improved affinity and specificity.

Molecularly imprinted polymers (MIPs), as important mimics of antibodies, are chemically synthesized by polymerization in the presence of a target compound. MIPs have found wide applications in important fileds. However, the current molecular imprinting technology suffers from a dilemma; there is often a compromise between the best affinity and the best specificity for MIPs prepared under optimized conditions. Herein, we proposed a new strategy called molecular imprinting and cladding (MIC) to solve this issue. The principle is straightforward; after molecular imprinting, a chemically inert cladding thinlayer is generated to precisely cover non-imprinted area. We further proposed a special MIC approach for controllably engineering protein binders. The prepared cladded MIPs (cMIPs) exhibited significantly improved affinity and specificity. The general applicability of the proposed strategy and method was verified by engineering of cMIPs for the recognition of a variety of different proteins. The feasibility of cMIPs for real applications was demonstrated by fluorescence imaging of cancer cells against normal cells and immunoassay of C-peptide in human urine. This study opened up a new avenue for controllably engineering protein-specific antibody mimics with excellent recognition properties, holding great prospective in important applications such as disease diagnosis and nanomedicine.

Boosting Chemodynamic Therapy by Tumor-Targeting and Cellular Redox Homeostasis-Disrupting Nanoparticles.

Chemodynamic therapy (CDT) that kills tumor cells by converting low-reactivity H2O2 into highly toxic hydroxyl radicals (•OH) is an emerging tumor therapeutic modality, but its therapeutic efficacy is largely limited by both the lack of tumor targeting and redox homeostasis in tumor cells. Herein, we report Cu2+-encapsulated and GalNAc-imprinted biodegradable silica nanoparticles (nanoMIP) for boosting CDT. In this strategy, the Cu2+ was first encapsulated into disulfide-bridged silica nanoparticles with a high loading capacity of ∼18.3%, followed by in situ functionalization via molecular imprinting using GalNAc as a template. Such a nanovector could specifically target tumor cells overexpressing the Tn antigen to promote the cellular uptake. After internalization into tumor cells, the degradation of nanoMIP occurred in response to the tumor microenvironment, spontaneously releasing Cu2+/Cu+ via redox cycles, which in turn promoted highly potent GSH depletion and triggered •OH generation by a Fenton-like reaction. Notably, we found that the catalase activity could be effectively inhibited by the produced Cu+, which indirectly upregulated the endogenous H2O2 level. As a result, the "maladjusted" tumor cells lost the resistance against •OH damage, finally resulting in the apoptosis of tumor cells. In vitro and in vivo experiments demonstrated that our nanoMIP exhibited excellent cytotoxicity against tumor cells and high efficacy of tumor inhibition in the xenograft tumor model with negligible side effects. Taken together, our study provides not only a promising strategy for maximizing the CDT efficacy but also a new insight for developing MIP-based nanomedicine.

Photothermal Therapy of Neuroblastoma via Polysialic Acid-Targeting Nanomissiles.

Exploring new targets and developing novel targeted therapies are urgently needed for neuroblastoma therapy. Polysialic acid (polySia), a linear homopolymer of sialic acid units that correlates well with tumor progression and poor prognosis, has emerged as a potential target for neuroblastoma. However, the lack of polySia-specific binding reagents has severely limited the development of polySia-targeting therapeutics for neuroblastoma. Herein, the construction of polySia-targeting nanomissiles via molecular imprinting for the photothermal therapy of neuroblastoma is reported. Oligosialic acid (oligoSia) containing 3-4 units is considered as a characteristic structure for the recognition of polySia, while oligoSia containing 4-7 units digested from polySia is employed as the template. Via boronate-affinity controllable oriented surface imprinting, oligoSia-imprinted nanoparticles (oSia-MIP) are prepared. The oSia-MIP allows for specifically recognizing polySia and targeting polySia overexpressed neuroblastoma cells in vitro and in vivo. oSia-MIP loaded with indocyanine green is prepared and experimentally demonstrated to be a potent targeted photothermal therapeutic for neuroblastoma. Equipping the core substrate with functional entities, the developed polySia targeting nanoplatform can be accommodated to various therapeutic modalities, holding great promise for neuroblastoma targeted therapy.

Probing Queuosine Modifications of Transfer RNA in Single Living Cells via Plasmonic Affinity Sandwich Assay.

Queuosine (Q) modification on tRNA plays an essential role in protein synthesis, participating in many tRNA functions such as folding, stability, and decoding. Appropriate analytical tools for the measurement of tRNA Q modifications are essential for the exploration of new roles of Q-modified tRNAs and the rationalization of their exact mechanisms. However, conventional methods for Q modification analysis suffer from apparent disadvantages, such as destructive cells, tedious procedure, and low sensitivity, which much hamper in-depth studies of Q modification-related biological questions. In this study, we developed a new approach called plasmonic affinity sandwich assay that allows for facile and sensitive determination of Q-modified tRNAs in single living cells. This method relies on the combination of plasmon-enhanced Raman scattering detection, base-paring affinity in-cell microextraction, and a set of boronate affinity and molecularly imprinted labeling nanotags for selective recognition of individual Q modifications, including queuosine, galactosyl queuosine (Gal-Q), and mannosyl queuosine (Man-Q). The developed method exhibited high affinity extraction and high specificity recognition. It allowed for the measurement of tRNA Q modifications in not only Q-rich cultured tumor cells but also Q-deficient primary tumor cells. Usefulness of this approach for investigation of the change of the Q modification level in single cells under oxidative stress was demonstrated. Because of its significant advantages over conventional methods, this approach provides a promising analytical tool for the exploration of more roles of Q-modified tRNAs and elucidation of their mechanisms.

Controllable Engineering and Functionalizing of Nanoparticles for Targeting Specific Proteins towards Biomedical Applications.

Nanoparticles have been widely used in important biomedical applications such as imaging, drug delivery, and disease therapy, in which targeting toward specific proteins is often essential. However, current targeting strategies mainly rely on surface modification with bioligands, which not only often fail to provide desired properties but also remain challenging. Here an unprecedented approach is reported, called reverse microemulsion-confined epitope-oriented surface imprinting and cladding (ROSIC), for facile, versatile, and controllable engineering coreless and core/shell nanoparticles with tunable monodispersed size as well as specific targeting capability toward proteins and peptides. Via engineering coreless imprinted and cladded silica nanoparticles, the effectiveness and superiority over conventional imprinting of the proposed approach are first verified. The prepared nanoparticles exhibit both high specificity and high affinity. Using quantum dots, superparamagnetic nanoparticles, silver nanoparticles, and upconverting nanoparticles as a representative set of core substrates, a variety of imprinted and cladded single-core/shell nanoparticles are then successfully prepared. Finally, using imprinted and cladded fluorescent nanoparticles as probes, in vitro targeted imaging of triple-negative breast cancer (TNBC) cells and in vivo targeted imaging of TNBC-bearing mice are achieved. This approach opens a new avenue to engineering of nanoparticles for targeting specific proteins, holding great prospects in biomedical applications.

Redox-Responsive Molecularly Imprinted Nanoparticles for Targeted Intracellular Delivery of Protein toward Cancer Therapy.

Although protein therapeutics is of significance in therapeutic intervention of cancers, controlled delivery of therapeutic proteins still faces substantial challenges including susceptibility to degradation and denaturation and poor membrane permeability. Herein, we report a sialic acid (SA)-imprinted biodegradable silica nanoparticles (BS-NPs)-based protein delivery strategy for targeted cancer therapy. Cytotoxic ribonuclease A (RNase A) was effectively caged in the matrix of disulfide-hybridized silica NPs (encapsulation efficiency of ∼64%), which were further functionalized with cancer targeting capability via surface imprinting with SA as imprinting template. Such nanovectors could not only maintain high stability in physiological conditions but also permit redox-triggered biodegradation for both concomitant release of the loaded therapeutic cargo and in vivo clearance. In vitro experiments confirmed that the SA-imprinted RNase A@BS-NPs could selectively target SA-overexpressed tumor cells, promote cells uptake, and subsequently be cleaved by intracellular glutathione (GSH), resulting in rapid release kinetics and enhanced cell cytotoxicity. In vivo experiments further confirmed that the SA-imprinted RNase A@BS-NPs had specific tumor-targeting ability and high therapeutic efficacy of RNase A in xenograft tumor model. Due to the specific targeting and traceless GSH-stimulated intracellular protein release, the SA-imprinted BS-NPs provided a promising platform for the delivery of biomacromolecules in cancer therapy.

P2X4R Contributes to Central Disinhibition Via TNF-α/TNFR1/GABAaR Pathway in Post-stroke Pain Rats.

Central post-stroke pain (CPSP) is a disabling condition in stroke patients. It is a type of neuropathic pain for which the mechanism and relevant drug pathways remain unknown. Inflammatory response and central disinhibition have been suggested recently. Our previous research has shown targeting P2X4 receptors (P2X4R) may be effective in the treatment of CPSP, but the downstream pathway of the P2X4R has not been studied. In this study, we found the increase in tumor necrosis factor alpha (TNF-α) level and endocytosis of surface gamma-aminobutyric acid a receptors (GABAaR) in CPSP, and these effects were inhibited by blocking P2X4R. Furthermore, antagonizing TNF-α can increase surface GABAaR expression and mechanical pain threshold. Meanwhile, knocking down TNFR1 but not TNFR2 reversed the endocytosis of surface GABAaR and alleviated mechanical allodynia. Thus, the neuropathic pain was mediated, in part, through P2X4R/TNF-α/TNFR1/GABAaR signaling, which was induced after stroke. PERSPECTIVE: P2X4R regulates the pathophysiological mechanism of CPSP through central disinhibition mediated by TNF-α/TNFR1. Our results suggest that modulation of P2X4R-TNF-α/TNFR1-GABAaR signaling could provide a new therapeutic strategy to treat CPSP.

Periodontitis in elderly patients with type 2 diabetes mellitus: impact on gut microbiota and systemic inflammation.

Elderly patients with type 2 diabetes mellitus (T2DM) exhibit considerable periodontitis frequency, which causes tooth loss and poor quality of life. To investigate the impact of periodontitis on gut microbiota, we used 16S rRNA amplicon sequencing to characterize the composition and structure of gut microbiota among elderly patients with T2DM and periodontitis (T2DM_P), elderly patients with T2DM alone (T2DM_NP), and healthy volunteers. We identified 34 key gut microbiota markers that distinguished participants with different periodontal conditions and investigated their connections to other gut bacteria, as well as their clinical correlates. The most striking differences in co-occurrence networks between the T2DM_P and T2DM_NP groups comprised interactions involving dominant genera in the oral cavity (i.e., Streptococcus and Veillonella). Of the 34 identified key gut microbiota markers that distinguished participants with different periodontal conditions, 25 taxa were correlated with duration of diabetes, dry mouth or the peripheral levels of pro-inflammatory cytokines (e.g., tumor necrosis factor-α, interferon-γ, prostaglandin E2, interleukin-17, and interleukin-6) and metabolic parameters (e.g., hemoglobin A1c), respectively. Our findings suggest that gut microbial shifts driven by periodontitis may contribute to systemic inflammation and metabolic dysfunction during the progression of T2DM.

Integrated analysis of microbiome and host transcriptome reveals correlations between gut microbiota and clinical outcomes in HBV-related hepatocellular carcinoma.

BACKGROUND: The gut-liver axis plays a pivotal role in the pathogenesis of hepatocellular carcinoma (HCC). However, the correlations between the gut microbiome and the liver tumor transcriptome in patients with HCC and the impact of the gut microbiota on clinical outcome are less well-understood. METHODS: Fecal samples collected from HBV-related HCC patients (n = 113) and healthy volunteers (n = 100) were subjected to 16S rRNA sequencing of the microbiome. After a rigorous selection process, 32 paired tumor and adjacent non-tumor liver tissues from the HCC group were subjected to next-generation sequencing (NGS) RNA-seq. The datasets were analyzed individually and integrated with clinical characteristics for combined analysis using bioinformatics approaches. We further verified the potential of the gut microbiota to predict clinical outcome by a random forest model and a support vector machine model. RESULTS: We found that Bacteroides, Lachnospiracea incertae sedis, and Clostridium XIVa were enriched in HCC patients with a high tumor burden. By integrating the microbiome and transcriptome, we identified 31 robust associations between the above three genera and well-characterized genes, indicating possible mechanistic relationships in tumor immune microenvironment. Clinical characteristics and database analysis suggested that serum bile acids may be important communication mediators between these three genera and the host transcriptome. Finally, among these three genera, six important microbial markers associated with tumor immune microenvironment or bile acid metabolism showed the potential to predict clinical outcome (AUC = 81%). CONCLUSIONS: This study revealed that changes in tumor immune microenvironment caused by the gut microbiota via serum bile acids may be important factors associated with tumor burden and adverse clinical outcome. Gut microbes can be used as biomarkers of clinical features and outcomes, and the microbe-associated transcripts of host tumors can partly explain how gut microbiota promotes HCC pathogenesis.

Alterations in glycolytic/cholesterogenic gene expression in hepatocellular carcinoma.

Metabolic reprogramming is a hallmark of tumors, including hepatocellular carcinoma (HCC). We used data from The Cancer Genome Atlas and the International Cancer Genome Consortium to assess the alterations in glycolytic and cholesterogenic genes in HCC and to determine their association with clinical features in HCC patients. Based on the gene expression profiles from these databases, we established four subtypes of HCC: cholesterogenic, glycolytic, mixed, and quiescent. The prognosis of the cholesterogenic subgroup was poorer than that of the glycolytic group. Tumors in the glycolytic group were more sensitive to chemotherapy. We also explored the relationships between these metabolic subtypes and previously established HCC subgroups. Glycolytic gene expression correlated strongly with poorer prognostic gene expression in the Hoshida classification of HCC. Whole-genome analyses indicated that aberrant amplification of TP53 and MYC in HCC were associated with abnormal anabolic cholesterol metabolism. The mRNA levels of mitochondrial pyruvate carriers 1 and 2 differed among the HCC metabolic subtypes. In a bioinformatics analysis we identified genomic characteristics of tumor metabolism that varied among different cancer types. These findings demonstrate that metabolic subtypes may be valuable prognostic indicators in HCC patients.

Impact Behavior of Hydrophilic Micron Particles on a Planar Gas-Liquid Interface.

The behavior of hydrophilic micron particles impacting on the gas-liquid interface has been further experimentally studied using a high-speed camera at different surface tensions and dynamic viscosities of liquids. The results show that the impact behavior exhibits suspension and submergence modes, whose boundary cannot be clearly identified because the overlap between the impact velocity ranges occurs because of the unstable pinning of the three-phase contact line on the surface of hydrophilic particles. The liquid properties have little effect on the wettability of hydrophilic particles but greatly influence the hydrodynamic and capillary force exerted on the particles, leading to the expansion of the suspension mode range. In addition, the penetration probability changes little with the decrease in surface tension, while it significantly reduces with the increase in dynamic viscosity. A penetration probability model is predicted as an exponential function of the inertial and supporting forces, and the experimental values agree well with the predicted values. The outcomes of this research will be helpful for understanding the mechanism of particle-interface interaction and providing guidance for enhancing the separation of hydrophilic fine ash via a bubble scrubbing system.

Tongue coating microbiome data distinguish patients with pancreatic head cancer from healthy controls.

Background: The microbiota plays a critical role in the process of human carcinogenesis. Pancreatic head carcinoma (PHC)-associated tongue coating microbiome dysbiosis has not yet been clearly defined.Objective: Our aim is to reveal the bacterial composition shifts in the microbiota of the tongue coat of PHC patients.Design: The tongue coating microbiota was analyzed in 30 PHC patients and 25 healthy controls using 16S rRNA gene sequencing technology.Results: The microbiome diversity of the tongue coat in PHC patients was significantly increased, as shown by the Shannon, Simpson, inverse Simpson, Obs and incidence-based coverage estimators. Principal component analysis revealed that PHC patients were colonized by remarkably different tongue coating microbiota than healthy controls and liver cancer patients. Linear discriminant analysis effect size revealed that Leptotrichia, Fusobacterium,Rothia, Actinomyces, Corynebacterium, Atopobium, Peptostreptococcus, Catonella, Oribacterium, Filifactor, Campylobacter, Moraxella and Tannerella were overrepresented in the tongue coating of PHC patients, and Haemophilus, Porphyromonas and Paraprevotella were enriched in the tongue coating microbiota of healthy controls. Strikingly, Haemophilus, Porphyromonas, Leptotrichia and Fusobacterium could distinguish PHC patients from healthy subjects, and Streptococcus and SR1 could distinguish PHC patients from liver cancer patients. Conclusions: These findings identified the microbiota dysbiosis of the tongue coat in PHC patients, and provide insight into the association between the human microbiome and pancreatic cancer.

Gut microbiome analysis as a tool towards targeted non-invasive biomarkers for early hepatocellular carcinoma.

OBJECTIVE: To characterise gut microbiome in patients with hepatocellular carcinoma (HCC) and evaluate the potential of microbiome as non-invasive biomarkers for HCC. DESIGN: We collected 486 faecal samples from East China, Central China and Northwest China prospectively and finally 419 samples completed Miseq sequencing. We characterised gut microbiome, identified microbial markers and constructed HCC classifier in 75 early HCC, 40 cirrhosis and 75 healthy controls. We validated the results in 56 controls, 30 early HCC and 45 advanced HCC. We further verified diagnosis potential in 18 HCC from Xinjiang and 80 HCC from Zhengzhou. RESULTS: Faecal microbial diversity was increased from cirrhosis to early HCC with cirrhosis. Phylum Actinobacteria was increased in early HCC versus cirrhosis. Correspondingly, 13 genera including Gemmiger and Parabacteroides were enriched in early HCC versus cirrhosis. Butyrate-producing genera were decreased, while genera producing-lipopolysaccharide were increased in early HCC versus controls. The optimal 30 microbial markers were identified through a fivefold cross-validation on a random forest model and achieved an area under the curve of 80.64% between 75 early HCC and 105 non-HCC samples. Notably, gut microbial markers validated strong diagnosis potential for early HCC and even advanced HCC. Importantly, microbial markers successfully achieved a cross-region validation of HCC from Northwest China and Central China. CONCLUSIONS: This study is the first to characterise gut microbiome in patients with HCC and to report the successful diagnosis model establishment and cross-region validation of microbial markers for HCC. Gut microbiota-targeted biomarkers represent potential non-invasive tools for early diagnosis of HCC.

Biogas liquid digestate grown Chlorella sp. for biocrude oil production via hydrothermal liquefaction.

Microalgae can not only purify and recover the nutrients from wastewater, but also be harvested as wet biomass for the production of biocrude oil via hydrothermal liquefaction (HTL). Chlorella sp. cultivated in the ultrafiltration (UF) membrane treated anaerobic digestion (AD) liquid digestate of chicken manure was used as the feedstock in this study. The present study characterized the products and investigated the elemental migration during HTL of Chlorella sp. fed with AD effluent wastewater (WW) and BG11 standard medium (ST) in 100mL and 500mL reactors under different operational conditions. Results showed that the highest oil yield of WW (38.1%, daf) was achieved at 320°C, 60min and 15% TS in 500mL reactor, which was 14.1% higher than that of ST (33.4%, daf) at 320°C, 30min and 20% TS in the same reactor. WW had a similar carbon and hydrogen distribution in the four product fractions under HTL conditions compared with ST. 43.4% and 32.4% of carbon in WW11 and ST11 were released into the biocrude and aqueous phase in 500mL reactor, respectively. As much as 64.5% of the hydrogen was transferred to the aqueous phase. GC-MS results showed that the chemical compounds in the biocrude oil from WW consist of a variety of chemical constituents, such as hydrocarbons, acids, alcohols, ketones, phenols and aldehydes. These two biocrude oils contained 17.5% wt. and 8.64% wt. hydrocarbons, and 63.7% wt. and 79.8% wt. oxygen-containing compounds, respectively. TGA results showed that 69.3%-66.7% of the biocrude oil was gasified in 30°C-400°C. This study demonstrates the great potential for biocrude oil production from microalgae grown in biogas effluent via HTL.