Microfluidic chain reaction of structurally programmed capillary flow events.

Chain reactions, characterized by initiation, propagation and termination, are stochastic at microscopic scales and underlie vital chemical (for example, combustion engines), nuclear and biotechnological (for example, polymerase chain reaction) applications1-5. At macroscopic scales, chain reactions are deterministic and limited to applications for entertainment and art such as falling dominoes and Rube Goldberg machines. On the other hand, the microfluidic lab-on-a-chip (also called a micro-total analysis system)6,7 was visualized as an integrated chip, akin to microelectronic integrated circuits, yet in practice remains dependent on cumbersome peripherals, connections and a computer for automation8-11. Capillary microfluidics integrate energy supply and flow control onto a single chip by using capillary phenomena, but programmability remains rudimentary with at most a handful (eight) operations possible12-19. Here we introduce the microfluidic chain reaction (MCR) as the conditional, structurally programmed propagation of capillary flow events. Monolithic chips integrating a MCR are three-dimensionally printed, and powered by the free energy of a paper pump, autonomously execute liquid handling algorithms step-by-step. With MCR, we automated (1) the sequential release of 300 aliquots across chained, interconnected chips, (2) a protocol for severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) antibodies detection in saliva and (3) a thrombin generation assay by continuous subsampling and analysis of coagulation-activated plasma with parallel operations including timers, iterative cycles of synchronous flow and stop-flow operations. MCRs are untethered from and unencumbered by peripherals, encode programs structurally in situ and can form a frugal, versatile, bona fide lab-on-a-chip with wide-ranging applications in liquid handling and point-of-care diagnostics.

Astragalin ameliorates renal injury in diabetic mice by modulating mitochondrial quality control via AMPK-dependent PGC1α pathway.

Diabetic kidney disease (DKD) is a common microvascular complication of diabetes mellitus, and oxidative stress and mitochondrial dysfunction play an important role in this process. It has been shown that aldose reductase (ALR2) catalyzes NADPH-dependent reduction of glucose to sorbitol, resulting in oxidative stress and mitochondrial dysfunction in diabetic patients. Astragalin (AG), a flavonoid extracted from Thesium chinense Turcz., shows an inhibitory activity on ALR2. In this study, we investigated the therapeutic effects of AG against renal injury in streptozocin (STZ)-induced diabetic mouse model. Diabetic mice were orally administered AG (5, 10 mg·kg-1·d-1) for 4 weeks. We showed that AG treatment greatly improved the proteinuria and ameliorated renal pathological damage without affecting the elevated blood glucose in diabetic mice. Furthermore, AG treatment significantly suppressed highly activated ALR2, and reduced oxidative stress in the kidney of diabetic mice and in high glucose and lipids-stimulated HK2 cells in vitro. We demonstrated that AG treatment modulated mitochondrial quality control and ameliorated apoptosis, boosting mitochondrial biogenesis, maintaining mitochondrial dynamic homeostasis, and improving energy metabolism disorder in vivo and in vitro. In high glucose and lipids-stimulated HK2 cells, we found that AG (20 µM) restored the phosphorylation level of AMPK, and upregulated the expression and transcriptional activity of PGC1α, whereas treatment with H2O2, blockade of AMPK with Compound C or knockdown of AMPKα with siRNA abolished the protective effect of AG on mitochondrial function, suggesting that antioxidant effects and activation of AMPK-dependent PGC1α pathway might be the molecular mechanisms underlying the protective effects of AG on mitochondrial quality control. We conclude that AG could be a promising drug candidate for the treatment of diabetic renal injury through activating AMPK.

Dapagliflozin ameliorates hepatic steatosis via suppressing LXRα-mediated synthesis of lipids and bile acids.

Nonalcoholic fatty liver disease (NAFLD) prevalence is rising globally with no pharmacotherapies approved. Hepatic steatosis is closely associated with progression and prognosis of NAFLD. Dapagliflozin, kind of sodium-glucose cotransporter 2 (SGLT2) inhibitor, was found to improve NAFLD in clinical trials, while the underlying mechanism remains poorly elucidated. Here, we reported that dapagliflozin effectively mitigated liver injury and relieved lipid metabolism disorders in vivo. Further investigation showed that dapagliflozin markedly suppressed Liver X Receptor α (LXRα)-mediated synthesis of de novo lipids and bile acids (BAs). In AML12 cells, our results proved dapagliflozin decreased lipid contents via inhibiting the expression of LXRα and downstream liposynthesis genes. Proteosome inhibitor MG132 eliminated the effect of dapagliflozin on LXRα-mediated signaling pathway, which suggested that dapagliflozin downregulated LXRα expression through increasing LXRα degradation. Knockdown of LXRα with siRNA abolished the reduction of lipogenesis from dapagliflozin treatment, indicating that LXRα might be the pivotal target for dapagliflozin to exhibit the aforementioned benefits. Furthermore, the data showed that dapagliflozin reversed gut dysbiosis induced by BAs disruption and altered gut microbiota profile to reduce intestinal lipids absorption. Together, our study deciphered a novel mechanism by which dapagliflozin relieved hepatic steatosis and highlighted the potential benefit of dapagliflozin in treating NAFLD.

Dapagliflozin ameliorates diabetic renal injury through suppressing the self-perpetuating cycle of inflammation mediated by HMGB1 feedback signaling in the kidney.

Dapagliflozin, the Sodium-glucose cotransporter 2 (SGLT2) inhibitor class of glucose-lowering agents, has shown the significantly nephroprotective effects to reduce the risk of kidney failure in diabetes. However, the underlying mechanisms are incompletely understood to explain the beneficial effects of dapagliflozin on kidney function. Here, we demonstrated that the administered of dapagliflozin for 12 weeks improved the proteinuria, histomorphology damage, oxidative stress, and macrophage infiltrations in the kidney of streptozotocin (STZ)-induced diabetic mice. Meanwhile, dapagliflozin attenuated the renal inflammation and fibrosis by reducing the pro-inflammatory factors interleukin-6 (IL-6), IL-1ß, and tumor necrosis factor α (TNF-α) and anti-fiber factor fibronectin (FN) and elevating the anti-inflammatory factor IL-10. Our data revealed that dapagliflozin exerted anti-inflammatory effects by inhibiting the activation of high mobility group box 1 (HMGB1)/TLR2/4/NF-κB signaling pathway. Consistently, we found that dapagliflozin suppressed the expression of HMGB1 and downstream TLR2/4/NF-κB signaling proteins in the human proximal tubular (HK-2) stimulated by high glucose and lipids or HMGB1 and RAW264.7 cells stimulated by IL-1ß, respectively. Further experiments were performed in the indirect co-culture model of RAW264.7 and HK-2 cells induced by high glucose and lipids. The results again confirmed the effects of dapagliflozin on alleviating inflammatory response and regulating the proportions of M1/M2 macrophage. It is indicated that the feedback signaling of HMGB1 between the tubules and macrophage involves in the persistence of the inflammation. These data demonstrate that dapagliflozin suppress the self-perpetuating inflammation by blocking the feedback loop of HMGB1 in the kidney, which contribute to ameliorate the renal injury in diabetes.

Effects of sodium-glucose co-transporter 2 inhibitors on liver fibrosis in non-alcoholic fatty liver disease patients with type 2 diabetes mellitus: An updated meta-analysis of randomized controlled trials.

BACKGROUND AND AIM: Sodium-glucose co-transporter 2 inhibitors (SGLT2i) has been verified to improve Non-alcoholic fatty liver disease (NAFLD) in previous clinical practice. We mainly aim to investigate the effects of SGLT2i on liver fibrosis in NAFLD patients with type 2 diabetes mellitus (T2DM). METHODS: We conducted a comprehensive literature search utilizing the databases PubMed, Embase, Web of Science, and Cochrane Library, and extracted continuous data in the form of mean and standard deviation of the difference before and after treatment. RevMan 5.3 software was used to chart the pooled forest plot and perform heterogeneity, sensitivity and subgroup analysis. This study is conducted under the protocol registered with the Platform of Registered Systematic Review and Meta-analysis Protocols (INPLASY protocol 4946, INPLASY202360058). RESULTS: A total of 16 articles involving 699 patients were included. Indicators of liver fibrosis, containing Liver Stiffness Measurement (LSM), Controlled Attenuation Parameter (CAP), Serum ferritin, Serum type 4 collagen 7s, and FIB-4 index, were found to be considerably reduced by SGLT2i medication and subgroup analysis manifested pronounced dose-dependence. Additionally, SGLT2i therapy decreased BMI, lipid buildup and insulin resistance. CONCLUSIONS: SGLT2 inhibitors significantly ameliorated liver fibrosis and liver fat content, improved body conditions and insulin resistance, demonstrating that SGLT2i might reduce the risk of the progression of liver fibrosis and have a positive effect on NAFLD patients with T2DM.

FTY-720 alleviates diabetes-induced liver injury by inhibiting oxidative stress and inflammation.

We aimed to investigate the protective effect of FTY-720 on liver injury and explore its potential mechanism in diabetic mice. The diabetic mouse model was induced with streptozotocin and FTY-720 was administered for 12 weeks. We assayed biocharacters and liver function and used histopathology staining to evaluate the protective effects of FTY-720 against diabetic liver injury. Levels of oxidative stress and inflammation in the liver were observed. mRNA and protein levels of essential enzymes for glucose metabolism were quantified in the liver and the protein expression of TLR4, HIF1α and NF-κB was determined. In vivo results revealed that FTY-720 significantly lowered blood glucose and lipids and improved liver function and alleviated liver fibrosis in diabetic mice. FTY-720 reduced oxidative stress and inflammation, with the increased catalase activity and reduced levels of malondialdehyde, myeloperoxidase, IL-1ß, IL-6, TNF-α, TGF-ß, and MCP1. Furthermore, FTY-720 modulated glucose metabolism in liver and elevated the ATP production, showing the promotion of glycogenesis and glycolysis and inhibition of gluconeogenesis. Moreover, FTY-720 inhibited the expression of TLR4 and HIF1α, contributing to restoration of liver function. In conclusion, FTY-720 ameliorates diabetes-induced liver injury and improves glucose homeostasis by inhibiting oxidative stress and inflammation and may be a promise drug for treatment of liver disease.

TAGET: a toolkit for analyzing full-length transcripts from long-read sequencing.

Single-molecule Real-time Isoform Sequencing (Iso-seq) of transcriptomes by PacBio can generate very long and accurate reads, thus providing an ideal platform for full-length transcriptome analysis. We present an integrated computational toolkit named TAGET for Iso-seq full-length transcript data analyses, including transcript alignment, annotation, gene fusion detection, and quantification analyses such as differential expression gene analysis and differential isoform usage analysis. We evaluate the performance of TAGET using a public Iso-seq dataset and newly sequenced Iso-seq datasets from tumor patients. TAGET gives significantly more precise novel splice site prediction and enables more accurate novel isoform and gene fusion discoveries, as validated by experimental validations and comparisons with RNA-seq data. We identify and experimentally validate a differential isoform usage gene ECM1, and further show that its isoform ECM1b may be a tumor-suppressor in laryngocarcinoma. Our results demonstrate that TAGET provides a valuable computational toolkit and can be applied to many full-length transcriptome studies.

The Mini Colon Model: a benchtop multi-bioreactor system to investigate the gut microbiome.

In vitro fermentation systems allow for the investigation of gut microbial communities with precise control of various physiological parameters while decoupling confounding factors from the human host. Current systems, such as the SHIME and Robogut, are large in footprint, lack multiplexing, and have low experimental throughput. Alternatives which address these shortcomings, such as the Mini Bioreactor Array system, are often reliant on expensive specialized equipment, which hinders wide replication across labs. Here, we present the Mini Colon Model (MiCoMo), a low-cost, benchtop multi-bioreactor system that simulates the human colon environment with physiologically relevant conditions. The device consists of triplicate bioreactors working independently of an anaerobic chamber and equipped with automated pH, temperature, and fluidic control. We conducted 14-d experiments and found that MiCoMo was able to support a stable complex microbiota community with a Shannon Index of 3.17 ± 0.65, from individual fecal samples after only 3-5 d of inoculation. MiCoMo also retained inter-sample microbial differences by developing closely related communities unique to each donor, while maintaining both minimal variations between replicate reactors (average Bray-Curtis similarity 0.72 ± 0.13) andday-to-day variations (average Bray-Curtis similarity 0.81±0.10) after this short stabilization period. Together, these results establish MiCoMo as an accessible system for studying gut microbial communities with high throughput and multiplexing capabilities.

Single-cell gene fusion detection by scFusion.

Gene fusions can play important roles in tumor initiation and progression. While fusion detection so far has been from bulk samples, full-length single-cell RNA sequencing (scRNA-seq) offers the possibility of detecting gene fusions at the single-cell level. However, scRNA-seq data have a high noise level and contain various technical artifacts that can lead to spurious fusion discoveries. Here, we present a computational tool, scFusion, for gene fusion detection based on scRNA-seq. We evaluate the performance of scFusion using simulated and five real scRNA-seq datasets and find that scFusion can efficiently and sensitively detect fusions with a low false discovery rate. In a T cell dataset, scFusion detects the invariant TCR gene recombinations in mucosal-associated invariant T cells that many methods developed for bulk data fail to detect; in a multiple myeloma dataset, scFusion detects the known recurrent fusion IgH-WHSC1, which is associated with overexpression of the WHSC1 oncogene. Our results demonstrate that scFusion can be used to investigate cellular heterogeneity of gene fusions and their transcriptional impact at the single-cell level.

Global immune characterization of HBV/HCV-related hepatocellular carcinoma identifies macrophage and T-cell subsets associated with disease progression.

Diverse immune cells in the tumor microenvironment form a complex ecosystem, but our knowledge of their heterogeneity and dynamics within hepatocellular carcinoma (HCC) still remains limited. To assess the plasticity and phenotypes of immune cells within HBV/HCV-related HCC microenvironment at single-cell level, we performed single-cell RNA sequencing on 41,698 immune cells from seven pairs of HBV/HCV-related HCC tumors and non-tumor liver tissues. We combined bio-informatic analyses, flow cytometry, and multiplex immunohistochemistry to assess the heterogeneity of different immune cell subsets in functional characteristics, transcriptional regulation, phenotypic switching, and interactions. We identified 29 immune cell subsets of myeloid cells, NK cells, and lymphocytes with unique transcriptomic profiles in HCC. A highly complex immunological network was shaped by diverse immune cell subsets that can transit among different states and mutually interact. Notably, we identified a subset of M2 macrophage with high expression of CCL18 and transcription factor CREM that was enriched in advanced HCC patients, and potentially participated in tumor progression. We also detected a new subset of activated CD8+ T cells highly expressing XCL1 that correlated with better patient survival rates. Meanwhile, distinct transcriptomic signatures, cytotoxic phenotypes, and evolution trajectory of effector CD8+ T cells from early-stage to advanced HCC were also identified. Our study provides insight into the immune microenvironment in HBV/HCV-related HCC and highlights novel macrophage and T-cell subsets that could be further exploited in future immunotherapy.