Brunonianines A-C, C20-diterpenoid alkaloids with cyano group from Delphinium brunonianum Royle.

Cyano tends to have better biological activity, but it is rarely reported in natural products, especially in the C20-diterpene alkaloids. Herein, three unprecedented C20-diterpenoid alkaloids, brunonianines A-C (1-3), possessing rare cyano functional group as well as an atisine backbone constructed from a phenethyl substituent and a tetrahydropyran ring, along with four C19-alkaloids (4-7) and one amide alkaloids (8), were isolated from the whole plant of Delphinium brunonianum Royle. Compounds 1-3 are also the first atisine type diterpenoid alkaloids with cyano group obtained from nature. The structures of the previously undescribed compounds were elucidated by HR-ESI-MS, 1D/2D NMR spectroscopic data and electronic circular dichroism calculations and single-crystal X-ray diffraction. Reasonable speculations have also been made regarding the biogenic synthetic pathways of compounds 1-3. In addition, the inhibitory activity of all compounds was also tested against four tumor lines: A549, Caco-2, H460 and Skov-3, where compound 2 (IC50 2.20 ± 0.21 µM) showed better inhibitory activity against Skov-3 cells than the hydroxycamptothecin. Using flow cytometry, cell staining, migration and invasion analysis, and Western blot, compound 2 was found to arrest cells in the G2/M phase and was able to effectively inhibit cell motility to achieve potent anti-tumor effects. In addition, compound 2 can effectively induce apoptosis by activating the Bax/Bcl-2/Caspase-3 signaling pathway.

Fangchinoline derivatives inhibits PI3K signaling in vitro and in vivo in non-small cell lung cancer.

Fangchinoline (Fan) are extracted from the traditional Chinese medicine Stephania tetrandra S., which is a bis-benzyl isoquinoline alkaloids with anti-tumor activity. Therefore, 25 novel Fan derivatives have been synthesized and evaluated for their anti-cancer activity. In CCK-8 assay, these fangchinoline derivatives displayed higher proliferation inhibitory activity on six tumor cell lines than the parental compound. Compared to the parent Fan, compound 2h presented the anticancer activity against most cancer cells, especially A549 cells, with an IC50 value of 0.26 µM, which was 36.38-fold, and 10.61-fold more active than Fan and HCPT, respectively. Encouragingly, compound 2h showed low biotoxicity to the human normal epithelial cell BEAS-2b with an IC50 value of 27.05 µM. The results indicated compound 2h remarkably inhibited the cell migration by decreasing MMP-2 and MMP-9 expression and inhibited the proliferation of A549 cells by arresting the G2/M cell cycle. Meanwhile, compound 2h could also induce A549 cell apoptosis by promoting endogenous pathways of mitochondrial regulation. In nude mice presented that the growth of tumor tissues was markedly inhibited by the consumption of compound 2h in a dose-dependent manner, and it was found that compound 2h could inhibit the mTOR/PI3K/AKT pathway in vivo. In docking analysis, high affinity interaction between 2h and PI3K was responsible for drastic kinase inhibition by the compound. To conclude, this derivative compound may be useful as a potent anti-cancer agent for treatment of NSCLC.

Improved colonic inflammation by nervonic acid via inhibition of NF-κB signaling pathway of DSS-induced colitis mice.

BACKGROUND: Nervonic acid (C24:1∆15, 24:1 ω-9, cis-tetracos-15-enoic acid; NA), a long-chain monounsaturated fatty acid, plays an essential role in prevention of metabolic diseases, and immune regulation, and has anti-inflammatory properties. As a chronic, immune-mediated inflammatory disease, ulcerative colitis (UC) can affect the large intestine. The influences of NA on UC are largely unknown. PURPOSE: The present study aimed to decipher the anti-UC effect of NA in the mouse colitis model. Specifically, we wanted to explore whether NA can regulate the levels of inflammatory factors in RAW264.7 cells and mouse colitis model. METHODS: To address the above issues, the RAW264.7 cell inflammation model was established by lipopolysaccharide (LPS), then the inflammatory factors tumor necrosis factor-α (TNF-α), Interleukin-6 (IL-6), Interleukin-1ß (IL-1ß), and Interleukin-10 (IL-10) were detected by Enzyme-linked immunosorbent assay (ELISA). The therapeutic effects of NA for UC were evaluated using C57BL/6 mice gavaged dextran sodium sulfate (DSS). Hematoxylin and eosin (H&E) staining, Myeloperoxidase (MPO) kit assay, ELISA, immunofluorescence assay, and LC-MS/MS were used to assess histological changes, MPO levels, inflammatory factors release, expression and distribution of intestinal tight junction (TJ) protein ZO-1, and metabolic pathways, respectively. The levels of proteins involved in the nuclear factor kappa-B (NF-κB) pathway in the UC were investigated by western blotting and RT-qPCR. RESULTS: In vitro experiments verified that NA could reduce inflammatory response and inhibit the activation of key signal pathways associated with inflammation in LPS-induced RAW264.7 cells. Further, results from the mouse colitis model suggested that NA could restore intestinal barrier function and suppress NF-κB signal pathways to ameliorate DSS-induced colitis. In addition, untargeted metabolomics analysis of NA protection against UC found that NA protected mice from colitis by regulating citrate cycle, amino acid metabolism, pyrimidine and purine metabolism. CONCLUSION: These results suggested that NA could ameliorate the secretion of inflammatory factors, suppress the NF-κB signaling pathway, and protect the integrity of colon tissue, thereby having a novel role in prevention or treatment therapy for UC. This work for the first time indicated that NA might be a potential functional food ingredient for preventing and treating inflammatory bowel disease (IBD).

Activating Innate Immunity by a STING Signal Amplifier for Local and Systemic Immunotherapy.

The number of patients who benefit from acquired immunotherapy is limited. Stimulator of interferon genes (STING) signal activation is a significant component to enhance innate immunity, which has been used to realize broad-spectrum immunotherapy. Here, M@P@HA nanoparticles, as a STING signal amplifier, are constructed to enhance innate immunotherapy. Briefly, when M@P@HA was targeted into tumor cells, the nanoparticles decomposed with Mn2+ and activated the release of protoporphyrin (PpIX). Under light irradiation, the generated reactive oxygen species disrupt the cellular redox homeostasis to lead cytoplasm leakage of damaged mitochondrial double-stranded (ds) DNA, which is the initiator of the STING signal. Simultaneously, Mn2+ as the immunoregulator could significantly increase the activity of related protein of a STING signal, such as cyclic GMP-AMP synthase (cGAS) and STING, to further amplify the STING signal of tumor cells. Subsequently, the STING signal of tumor-associated macrophages (TAM) is also activated by capturing dsDNA and Mn2+ that escaped from tumor cells, so as to enhance innate immunity. It is found that, by amplifying the STING signal of tumor tissue, M@P@HA could not only activate innate immunity but also cascade to activate CD8+ T cell infiltration even in a tumor with low immunogenicity.

Synthesis and bioactivity evaluation of novel nuciferine derivatives with antihyperuricemia and nephroprotective effects.

Hyperuricemia is a common metabolic disease with a series of complications. Nuciferine, a typical aporphine alkaloid natural compound extracted from the leaves of Nelumbo nucifera Gaertn., was confirmed to have an antihyperuricemia effect. In the present study, 30 novel nuciferine derivatives were designed and synthesized. The effects of all derivatives on the regulation of URAT1 were studied in a uric acid-induced HK-2 cell model with benzbromarone as a positive control. The results indicated that Compound 1j showed the optimal URAT1 inhibitory activity through repressing PI3K/Akt pathway in HK-2 cells and the inhibitory effect was similar to that of benzbromarone. In addition, in vivo experiments demonstrated that Compound 1j could reduce uric acid levels and ameliorate kidney damage in hyperuricemic mice. On the one hand, Compound 1j could inhibit the expression of URAT1 and GLUT9 to increase the uric acid excretion index. On the other hand, Compound 1j could regulate the TLR4/IκBα/NF-κB signaling pathway to reduce the levels of inflammatory cytokines, thereby alleviating kidney damage. Meanwhile, a molecular docking assay revealed the potential molecular binding power (-9.79 kcal/mol) between Compound 1j and URAT1, which was more tightly bound than the lead compound nuciferine (-7.44 kcal/mol). Based on these results, Compound 1j may be a future drug for the development of new potential antihyperuricemia and nephroprotective drug candidates.

Pore-Structure Control in Metal-Organic Frameworks (MOFs) for Capture of the Greenhouse Gas SF6 with Record Separation.

In the electronics industry, the efficient recovery and capture of sulfur hexafluoride (SF6 ) from SF6 /N2 mixtures is of great importance. Herein, three metal-organic frameworks with fine-tuning pore structures, Cu(peba)2 , Ni(pba)2 , and Ni(ina)2 , were designed for SF6 capture. Among them, Ni(ina)2 has perfect pore sizes (6 Å) that are comparable to the kinetic diameter of sulfur hexafluoride (5.2 Å), affording the benchmark binding affinity for SF6 gas. Ni(ina)2 exhibits the highest SF6 /N2 selectivity (375.1 at 298 K and 1 bar) and ultra-high SF6 uptake capacity (53.5 cm3 g-1 at 298 K and 0.1 bar) at ambient conditions. The remarkable separation performance of Ni(ina)2 was verified by dynamic breakthrough experiments. Theoretical calculations and the SF6 -loaded single-crystal structure provided critical insight into the adsorption/separation mechanism. This porous coordination network has the potential to be used in industrial applications.

Two new alkaloids from Tibetan medicine of Hypecoum leptocarpum.

Two new alkaloids, leptocarpinine B (1) and corydamine acid (2), with thirteen known alkaloid compounds (3-15), were isolated from Hypecoum leptocarpum. The structures of the isolated compounds were determined based on spectroscopic data analyses, including IR, ESI-MS, 1 D, and 2 D NMR. In addition, all the isolates were evaluated for cytotoxic activities. Compound 6 showed moderate cytotoxicity against human ovarian cancer cell lines (A2780), human cervical cancer cell lines (HeLa), and human hepatocellular carcinomas cell lines (HepG2).[Formula: see text].

Discovery of Chromane-6-Sulfonamide Derivative as a Potent, Selective, and Orally Available Novel Retinoic Acid Receptor-Related Orphan Receptor γt Inverse Agonist.

Interleukin-17 (IL-17) is a proinflammatory cytokine that plays a dominant role in inflammation, autoimmunity, and host defense. RORγt is a key transcription factor mediating T helper 17 (Th17) cell differentiation and IL-17 production, which is able to activate CD8+ T cells and elicit antitumor efficacy. A series of sulfonamide derivatives as novel RORγt inverse agonists were designed and synthesized. Using GSK2981278 (phase II) as a starting point, we engineered structural modifications that significantly improved the activity and pharmacokinetic profile. In animal studies, oral administration of compound d3 showed a robust and dose-dependent inhibition of the IL-17A cytokine expression in a mouse imiquimod-induced skin inflammation model. Docking analysis of the binding mode revealed that the compound d3 occupied the active pocket suitably. Thus, compound d3 was selected as a clinical compound for the treatment of Th17-driven autoimmune diseases.

Design, synthesis and in vitro anticancer research of novel tetrandrine and fangchinoline derivatives.

Cancer treatment is one of the major public health issues in the world. Tetrandrine (Tet) and fangchinoline (d-Tet) are two bis-benzyl isoquinoline alkaloids extracted from Stephania tetrandra S. Moore, and their antitumor activities have been confirmed. However, the effective dose of Tet and d-Tet were much higher than that of the positive control and failed to meet clinical standards. Therefore, in this study, as a continuation of our previous work to study and develop high-efficiency and low-toxic anti-tumor lead compounds, twenty new Tet and d-Tet derivatives were designed, synthesized and evaluated as antitumor agents against six cancer cell lines (H460, H520, HeLa, HepG-2, MCF-7, SW480 cell lines) and BEAS-2B normal cells by CCK-8 analysis. Ten derivatives showed better cytotoxic effects than the parent fangchinoline, of which 4g showed the strongest cell growth inhibitory activity with an IC50 value of 0.59 µM against A549 cells. Subsequently, the antitumor mechanism of 4g was studied by flow cytometry, Hoechst 33258, JC-1 staining, cell scratch, transwell migration, and Western blotting assays. These results showed that compound 4g could inhibit A549 cell proliferation by arresting the G2/M cell cycle and inhibiting cell migration and invasion by reducing MMP-2 and MMP-9 expression. Meanwhile, 4g could induce apoptosis of A549 cells through the intrinsic pathway regulated by mitochondria. In addition, compound 4g inhibited the phosphorylation of PI3K, Akt and mTOR, suggesting a correlation between blocking the PI3K/Akt/mTOR pathway and the above antitumor activities. These results suggest that compound 4g may be a future drug for the development of new potential drug candidates against lung cancer.

Dynamically tunable light responsive silk-elastin-like proteins.

Dynamically tunable biomaterials are of particular interest in the field of biomedical engineering because of the potential utility for shape-change materials, drug and cell delivery and tissue regeneration. Stimuli-responsive proteins formed into hydrogels are potential candidates for such systems, due to the genetic tailorability and control over structure-function relationships. Here we report the synthesis of genetically engineered Silk-Elastin-Like Protein (SELP) photoresponsive hydrogels. Polymerization of the SELPs and monomeric adenosylcobalamin (AdoB12)-dependent photoreceptor C-terminal adenosylcobalamin binding domain (CarHC) was achieved using genetically encoded SpyTag-SpyCatcher peptide-protein pairs under mild physiological conditions. The hydrogels exhibited a partial collapse of the crosslinked molecular network with both decreased loss and storage moduli upon exposure to visible light. The materials were also evaluated for cytotoxicity and the encapsulation and release of L929 murine fibroblasts from 3D cultures. The design of these photo-responsible proteins provides new stimuli-responsive SELP-CarHC hydrogels for dynamically tunable protein-based materials.

Two new diterpenoids with their antiproliferative activities from the supercritical fluid extraction of Croton crassifolius root.

Supercritical fluid extraction was applied to obtain the lower polarity extracts from Croton crassifolius roots, and chemical investigation of which led to the isolation and identification of two new diterpenoids, named crassifolius P (1) and crassifolius Q (2). In vitro anti-proliferative activities of compounds 1 and 2 on A549, Hep-G2 and Hela tumor cell lines were evaluated. The two new compounds exhibited obvious selectivity to tumor cells with IC50 values ranging from 20.43 ± 1.18 µM to 25.72 ± 1.32 µM.

PDB-1 from Potentilla discolor Bunge induces apoptosis and autophagy by downregulating the PI3K/Akt/mTOR signaling pathway in A549 cells.

PDB-1 is a new C-27-carboxylated-lupane-triterpenoid derivative isolated from Potentilla discolor Bunge. In our previous research, PDB-1 was suggested to have an obvious selectivity for tumor cells. This study focused on clarifying PDB-1's anticancer mechanism in the inhibition of proliferation and in the induction of apoptosis and autophagy in A549 cells. In general, A549 cells were treated with PDB-1 for different times, and cell survival was assessed by a CCK8 assay. The assessment of intracellular reactive oxygen species, a mitochondrial membrane potential assay, a cell cycle assay, an annexin V-FITC/PI assay, and MDC staining were performed in A549 cells treated with PDB-1. Moreover, the mRNA and protein expression of cell cycle-, apoptosis- and autophagy-related factors were detected by RT-qPCR and western blotting. The results showed that PDB-1 inhibited A549 cell proliferation and colony formation in a dose- and time-dependent manner. The decrease in the viability of A549 cells was due to a G2/M cell cycle arrest. Moreover, PDB-1 induced cell apoptosis, accompanied by an increase in the Bax/Bcl-2 ratio and an increase in the expression levels of cleaved caspase-3/caspase-9. We also found that PDB-1 induced autophagy by increasing the conversion of LC3-I to LC3-II and elevating Beclin-1. In addition, further studies indicated that pretreatment with a specific PI3K inhibitor (LY294002) enhanced the effects of PDB-1 on the expression of proteins associated with apoptosis and autophagy, demonstrating that the PI3K/Akt/mTOR pathway was related to PDB-1-induced apoptosis and autophagy. These results indicated that PDB-1 may be considered a potential candidate for the future treatment of lung adenocarcinoma. These findings should benefit the development of the C14-COOH type of pentacyclic triterpenoids.

Gut dysbiosis induces the development of pre-eclampsia through bacterial translocation.

OBJECTIVE: Pre-eclampsia (PE) is one of the malignant metabolic diseases that complicate pregnancy. Gut dysbiosis has been identified for causing metabolic diseases, but the role of gut microbiome in the pathogenesis of PE remains unknown. DESIGN: We performed a case-control study to compare the faecal microbiome of PE and normotensive pregnant women by 16S ribosomal RNA (rRNA) sequencing. To address the causative relationship between gut dysbiosis and PE, we used faecal microbiota transplantation (FMT) in an antibiotic-treated mouse model. Finally, we determined the microbiome translocation and immune responses in human and mouse placental samples by 16S rRNA sequencing, quantitative PCR and in situ hybridisation. RESULTS: Patients with PE showed reduced bacterial diversity with obvious dysbiosis. Opportunistic pathogens, particularly Fusobacterium and Veillonella, were enriched, whereas beneficial bacteria, including Faecalibacterium and Akkermansia, were markedly depleted in the PE group. The abundances of these discriminative bacteria were correlated with blood pressure (BP), proteinuria, aminotransferase and creatinine levels. On successful colonisation, the gut microbiome from patients with PE triggered a dramatic, increased pregestational BP of recipient mice, which further increased after gestation. In addition, the PE-transplanted group showed increased proteinuria, embryonic resorption and lower fetal and placental weights. Their T regulatory/helper-17 balance in the small intestine and spleen was disturbed with more severe intestinal leakage. In the placenta of both patients with PE and PE-FMT mice, the total bacteria, Fusobacterium, and inflammatory cytokine levels were significantly increased. CONCLUSIONS: This study suggests that the gut microbiome of patients with PE is dysbiotic and contributes to disease pathogenesis.

Design, synthesis and in vitro evaluation of fangchinoline derivatives as potential anticancer agents.

The isolation and modification of natural products play an important role in the synthesis of anti-tumor drugs for the treatment of cancer. The present study was designed to evaluate the effects of fangchinoline derivatives against cancer cells. In vitro cytotoxicity of all derivatives against five cancer cell lines (A549, Hela, HepG-2, MCF-7 and MDA-MB-231 cell lines) and HL-7702 normal cells was assessed using the CCK-8 assay, and the results showed that most of the synthesized compounds displayed better cytotoxic effects on all the tested cells compared to that of the parent fangchinoline. In particular, compound 3i had the strongest inhibitory effect on cell proliferation, with an IC50 value of 0.61 µM against A549 cells. Compared with fangchinoline and HCPT (hydroxycamptothecine), the anti-proliferative activity of compound 3i was significantly increased. More interestingly, compound 3i had slight toxic side effects on normal cells, with an IC50 value of 27.53 µM. Moreover, the cell viability and cell cycle assays revealed that compound 3i inhibited A549 cell proliferation and arrested A549 cells at the G2/M-phase. The apoptosis-inducing effects of compound 3i and the associated molecular mechanisms were assessed using flow cytometry, cell staining, reactive oxygen species assays, RT-qPCR and Western blot analysis. These results suggested that compound 3i induces apoptosis through a mitochondria-mediated intrinsic pathway. This study revealed that compound 3i is a promising candidate for future development as an anti-tumor drug.

Reliable and reusable whole polypropylene plastic microfluidic devices for a rapid, low-cost antimicrobial susceptibility test.

Using an antimicrobial susceptibility test (AST) as an example, this work demonstrates a practical method to fabricate microfluidic chips entirely from polypropylene (PP) and the benefits for potential commercial use. Primarily caused by the misuse and abuse of antibiotics, antimicrobial resistance (AMR) is a major threat to modern medicine. The AST is a promising technique to help with the optimal use of antibiotics for reducing AMR. However, current phenotypic ASTs suffer from long turnaround time, while genotypic ASTs suffer from low reliability, and both are unaffordable for routine use. New microfluidics based AST methods are rapid but still unreliable as well as costly due to the PDMS chip material. Herein, we demonstrate a convenient method to fabricate whole PP microfluidic chips with high resolution and fidelity. Unlike PDMS chips, the whole PP chips showed better reliability due to their inertness; they are solvent-compatible and can be conveniently reused and recycled, which largely decreases the cost, and are environmentally friendly. We specially designed 3D chambers that allow for quick cell loading without valving/liquid exchange; this new hydrodynamic design satisfies the shear stress requirement for on-chip bacterial culture, which, compared to reported designs for similar purposes, allows for a simpler, more rapid, and high-throughput operation. Our system allows for reliable tracking of individual cells and acquisition of AST results within 1-3 hours, which is among the group of fastest phenotypic methods. The PP chips are more reliable and affordable than PDMS chips, providing a practical solution to improve current culture-based AST and benefiting the fight against AMR through helping doctors prescribe effective, narrow-spectrum antibiotics; they will also be broadly useful for other applications wherein a reliable, solvent-resistant, anti-fouling, and affordable microfluidic chip is needed.

Regional variation limits applications of healthy gut microbiome reference ranges and disease models.

Dysbiosis, departure of the gut microbiome from a healthy state, has been suggested to be a powerful biomarker of disease incidence and progression1-3. Diagnostic applications have been proposed for inflammatory bowel disease diagnosis and prognosis4, colorectal cancer prescreening5 and therapeutic choices in melanoma6. Noninvasive sampling could facilitate large-scale public health applications, including early diagnosis and risk assessment in metabolic7 and cardiovascular diseases8. To understand the generalizability of microbiota-based diagnostic models of metabolic disease, we characterized the gut microbiota of 7,009 individuals from 14 districts within 1 province in China. Among phenotypes, host location showed the strongest associations with microbiota variations. Microbiota-based metabolic disease models developed in one location failed when used elsewhere, suggesting that such models cannot be extrapolated. Interpolated models performed much better, especially in diseases with obvious microbiota-related characteristics. Interpolation efficiency decreased as geographic scale increased, indicating a need to build localized baseline and disease models to predict metabolic risks.

A General Strategy for Extrusion Bioprinting of Bio-Macromolecular Bioinks through Alginate-Templated Dual-Stage Crosslinking.

The recently developed 3D bioprinting technology has greatly improved the ability to generate biomimetic tissues that are structurally and functionally relevant to their human counterparts. The selection of proper biomaterials as the bioinks is a key step toward successful bioprinting. For example, viscosity of a bioink is an important rheological parameter to determine the flexibility in deposition of free-standing structures and the maintenance of architectural integrity following bioprinting. This requirement, however, has greatly limited the selection of bioinks, especially for those naturally derived due to their commonly low mechanical properties. Here the generalization of a mechanism for extrusion bioprinting of bio-macromolecular components, mainly focusing on collagen and its derivatives including gelatin and gelatin methacryloyl, is reported. Specifically, a templating strategy is adopted using a composite bioink containing both the desired bio-macromolecular component and a polysaccharide alginate. The physically crosslinkable alginate component serves as the temporal structural support to stabilize the shape of the construct during bioprinting; upon subsequent chemical or physical crosslinking of the bio-macromolecular component, alginate can be selectively removed to leave only the desired bio-macromolecule. It is anticipated that this strategy is general, and can be readily expanded for use of a wide variety of other bio-macromolecular bioinks.

Multi-channel silk sponge mimicking bone marrow vascular niche for platelet production.

In the bone marrow, the interaction of progenitor cells with the vasculature is fundamental for the release of blood cells into circulation. Silk fibroin, derived from Bombyx mori silkworm cocoons, is a promising protein biomaterial for bone marrow tissue engineering, because of its tunable architecture and mechanical properties, the capacity to incorporate labile compounds without loss of bioactivity and the demonstrated ability to support blood cell formation without premature activation. In this study, we fabricated a custom perfusion chamber to contain a multi-channel lyophilized silk sponge mimicking the vascular network in the bone marrow niche. The perfusion system consisted in an inlet and an outlet and 2 splitters that allowed funneling flow in each single channel of the silk sponge. Computational Fluid Dynamic analysis demonstrated that this design permitted confined flow inside the vascular channels. The silk channeled sponge supported efficient platelet release from megakaryocytes (Mks). After seeding, the Mks localized along SDF-1α functionalized vascular channels in the sponge. Perfusion of the channels allowed the recovery of functional platelets as demonstrated by increased PAC-1 binding upon thrombin stimulation. Further, increasing the number of channels in the silk sponge resulted in a proportional increase in the numbers of platelets recovered, suggesting applicability to scale-up for platelet production. In conclusion, we have developed a scalable system consisting of a multi-channeled silk sponge incorporated in a perfusion chamber that can provide useful technology for functional platelet production ex vivo.

Impaired renal function and dysbiosis of gut microbiota contribute to increased trimethylamine-N-oxide in chronic kidney disease patients.

Chronic kidney disease (CKD) patients have an increased risk of cardiovascular diseases (CVDs). The present study aimed to investigate the gut microbiota and blood trimethylamine-N-oxide concentration (TMAO) in Chinese CKD patients and explore the underlying explanations through the animal experiment. The median plasma TMAO level was 30.33 µmol/L in the CKD patients, which was significantly higher than the 2.08 µmol/L concentration measured in the healthy controls. Next-generation sequence revealed obvious dysbiosis of the gut microbiome in CKD patients, with reduced bacterial diversity and biased community constitutions. CKD patients had higher percentages of opportunistic pathogens from gamma-Proteobacteria and reduced percentages of beneficial microbes, such as Roseburia, Coprococcus, and Ruminococcaceae. The PICRUSt analysis demonstrated that eight genes involved in choline, betaine, L-carnitine and trimethylamine (TMA) metabolism were changed in the CKD patients. Moreover, we transferred faecal samples from CKD patients and healthy controls into antibiotic-treated C57BL/6 mice and found that the mice that received gut microbes from the CKD patients had significantly higher plasma TMAO levels and different composition of gut microbiota than did the comparative mouse group. Our present study demonstrated that CKD patients had increased plasma TMAO levels due to contributions from both impaired renal functions and dysbiosis of the gut microbiota.

Label-Free Isolation and mRNA Detection of Circulating Tumor Cells from Patients with Metastatic Lung Cancer for Disease Diagnosis and Monitoring Therapeutic Efficacy.

We develop an inertial-based microfluidic cell sorter combined with an integrated membrane filter, allowing for size-based, label-free, and high-efficiency separation and enrichment of circulating tumor cells (CTCs) in whole blood. The cell sorter is composed of a double spiral microchannel that hydrodynamically focuses and separates large CTCs from small blood cells. The focused CTCs with the equilibrium position around the midline of microchannel are further captured and enriched by a membrane filter (pore size of 8 µm) attached at the middle outlet. This integrated microfluidic device can process 1 mL of whole blood containing spiked tumor cells (A549, human lung adenocarcinoma epithelial cell line) within 15 min, with the capture efficiency of 74.4% at the concentration as low as tens of A549 cells per mL of whole blood. This microfluidic cell sorter is further adopted for isolation of CTCs from peripheral blood samples of patients with metastatic lung cancer. The immunostaining and CK-19 mRNA detection are applied for identification of captured CTCs, showing that our method can detect 90% of metastatic lung cancer patients before therapy, whereas the commercially used system can only detect 40% of the same patients. We also use the expression of CK-19 mRNA from captured CTCs as an indicator for monitoring the therapeutic efficiency, which correlates well with X-ray computed tomography (CT) assessment of the disease.