Acetyl-CoA synthetase 2 induces pyroptosis and inflammation of renal epithelial tubular cells in sepsis-induced acute kidney injury by upregulating the KLF5/NF-κB pathway.

BACKGROUND: Pyroptosis of the renal tubular epithelial cells (RTECs) and interstitial inflammation are central pathological characteristics of acute kidney injury (AKI). Pyroptosis acts as a pro-inflammatory form of programmed cell death and is mainly dependent on activation of the NLRP3 inflammasome. Previous studies revealed that acetyl-CoA synthetase 2 (ACSS2) promotes inflammation during metabolic stress suggesting that ACSS2 might regulate pyroptosis and inflammatory responses of RTECs in AKI. METHODS AND RESULTS: The expression of ACSS2 was found to be significantly increased in the renal epithelial cells of mice with lipopolysaccharide (LPS)-induced AKI. Pharmacological and genetic strategies demonstrated that ACSS2 regulated NLRP3-mediated caspase-1 activation and pyroptosis through the stimulation of the KLF5/NF-κB pathway in RTECs. The deletion of ACSS2 attenuated renal tubular pathological injury and inflammatory cell infiltration in an LPS-induced mouse model, and ACSS2-deficient mice displayed impaired NLRP3 activation-mediated pyroptosis and decreased IL-1ß production in response to the LPS challenge. In HK-2 cells, ACSS2 deficiency suppressed NLRP3-mediated caspase-1 activation and pyroptosis through the downregulation of the KLF5/NF-κB pathway. The KLF5 inhibitor ML264 suppressed NF-κB activity and NLRP3-mediated caspase-1 activation, thus protecting HK-2 cells from LPS-induced pyroptosis. CONCLUSION: Our results suggested that ACSS2 regulates activation of the NLRP3 inflammasome and pyroptosis by inducing the KLF5/NF-κB pathway in RTECs. These results identified ACSS2 as a potential therapeutic target in AKI.

Comparison of the Watson formula and bioimpedance spectroscopy for measuring body volume and calculating kt/V in patients with peritoneal dialysis.

CONCLUSION: There were significant differences between Vwat and Vbis and between Kt/Vwat and Kt/Vbis. Kt/Vwat may underestimate small-solute dialysis adequacy in most cases. Kt/Vbis instead of Kt/Vwat could be accounted for in creating individualized dialysis prescriptions if the patient has no obvious clinical symptoms.

Identification of RPGR ORF15 mutation for X-linked retinitis pigmentosa in a large Chinese family and in vitro correction with prime editor.

X-linked retinitis pigmentosa (XLRP) is the most severe form of Retinitis Pigmentosa (RP) and one of the leading causes of blindness in the world. Currently, there is no effective treatment for RP. In the present study, we recruited a XLRP family and identified a 4 bp deletion mutation (c. 2234_2237del) in RPGR ORF15 with Sanger sequencing, which was located in the exact same region as the missing XES (X chromosome exome sequencing) coverage. Then, we generated cell lines harboring the identified mutation and corrected it via enhanced prime editing system (ePE). Collectively, Sanger sequencing identified a pathogenic mutation in RPGR ORF15 for XLRP which was corrected with ePE. This study provides a valuable insight for genetic counseling of the afflicted family members and prenatal diagnosis, also paves a way for applying prime editing based gene therapy in those patients.

ABPP-CoDEL: Activity-Based Proteome Profiling-Guided Discovery of Tyrosine-Targeting Covalent Inhibitors from DNA-Encoded Libraries.

DNA-encoded chemical library (DEL) has been extensively used for lead compound discovery for decades in academia and industry. Incorporating an electrophile warhead into DNA-encoded compounds recently permitted the discovery of covalent ligands that selectively react with a particular cysteine residue. However, noncysteine residues remain underexplored as modification sites of covalent DELs. Herein, we report the design and utility of tyrosine-targeting DELs of 67 million compounds. Proteome-wide reactivity analysis of tyrosine-reactive sulfonyl fluoride (SF) covalent probes suggested three enzymes (phosphoglycerate mutase 1, glutathione s-transferase 1, and dipeptidyl peptidase 3) as models of tyrosine-targetable proteins. Enrichment with SF-functionalized DELs led to the identification of a series of tyrosine-targeting covalent inhibitors of the model enzymes. In-depth mechanistic investigation revealed their novel modes of action and reactive ligand-accessible hotspots of the enzymes. Our strategy of combining activity-based proteome profiling and covalent DEL enrichment (ABPP-CoDEL), which generated selective covalent binders against a variety of target proteins, illustrates the potential use of this methodology in further covalent drug discovery.

Modular Click Assembly DNA-Encoded Glycoconjugate Libraries with on-DNA Functional Group Transformations.

Carbohydrates are an important class of naturally active products and play vital roles in regulating various physiological activities. To meet the demand for carbohydrate-based libraries used for the identification of potential drug candidates for pharmaceutical-related targets, we developed a set of on-DNA protocols to construct the DNA-encoded glycoconjugates, including Seyferth-Gilbert homologation, anomeric azidation, and CuAAC cyclization. These on-DNA chemistries enable the generation and modification of DNA-linked glycosyl compounds with good conversions and broad substrate scope. Finally, three DNA-linked glycoconjugate libraries were successfully generated to demonstrate their applicability and feasibility in library preparation.

Renal-on-Chip Microfluidic Platform with a Force-Sensitive Resistor (ROC-FS) for Molecular Pathogenesis Analysis of Hydronephrosis.

Hydronephrosis is one of the most common diseases in urology. However, due to the difficulties in clinical trials and the lack of reliable in vitro platforms, the surgical indicators are not clear. Herein, the renal-on-chip with a force-sensitive resistor microfluidic platform was established to simulate the state of hydronephrosis. Cell counting kit-8 (CCK-8) and tight junction protein claudin-2 were detected on a renal-on-chip microfluidic platform with a force-sensitive resistor (ROC-FS). The results indicated that the ROC-FS had normal physiological functions and the cell viability on ROC-FS declined to around 40% after 48 h of hydronephrosis-simulated treatment. In addition, proteomics analysis of 15 clinical ureteropelvic junction obstruction (UPJO) samples showed that compared with normal children, a total of 50 common proteins were differentially expressed in UPJO children (P < 0.05, |log2fold change| ≥ 1). Metabolomic analysis of 39 clinical UPJO samples showed that a total of 241 metabolisms were dysregulated. Subsequent immunofluorescence and enzyme-linked immunosorbent assay (ELISA) analysis using ROC-FS were performed to identify the clinical multi-omics results for screening. All results pointed out that the TGF-ß-related signaling pathways and arginine-related metabolism signaling pathways were dysregulated and α-SMA, AGT, and AGA might be the potential biomarkers of hydronephrosis. In addition, correlation analysis of AGT and KLK1 with differential renal function (DRF) from clinical samples indicated good correlation coefficients (R2 0.923, 0.8742, 0.6412, and 0.8347). This demonstrates the state of hydronephrosis could be significantly correlated with the biomarkers. These findings could provide a reliable reference for determining surgical biomarkers clinically, and ROC could be further used in the analysis of other kidney diseases.

Investigation of PM2.5-induced carcinogenic effects through mediation of ErbB family based on DNA methylation and transcriptomics analysis by a lung-mimicking microfluidic platform.

Fine particle (PM2.5, less than 2.5 micrometers in diameter) is regarded as a harmful carcinogen. However, the molecular mechanisms of the carcinogenic effects of ambient fine particles have not been fully elucidated, and therapeutic options to address this major public health challenge are lacking. Here, we present global gene-specific DNA methylation and transcriptomic (RNA-Seq) analyses after HBE cells were exposed to fine particles on a portable, small, and all-in-one organ-level lung-mimicking air-liquid interface exposure (MALIE) microfluidic platform. A series of cancer-related signal transduction pathways were activated. ErbB1, ErbB2, and ErbB3 gene expression altered by fine particle exposure was the result of changes in the cellular DNA methylome. The protein expression of ErbB family was inhibited by drugs and could regulate downstream Grb2/Raf pathway and Akt/MDM2 pathway. All of the above results indicated that ErbB family may be promising drug targets for air pollution-related diseases and that inhibitor drugs can be used as therapeutic options to treat these diseases.

Association of PM2.5 with Insulin Resistance Signaling Pathways on a Microfluidic Liver-Kidney Microphysiological System (LK-MPS) Device.

Insulin resistance (IR) is a typical sign of metabolic dysregulation caused by fine particulate matter (PM2.5), but the underlying signaling has not been clearly determined. Herein, a microfluidic liver-kidney microphysiological system (LK-MPS) is presented to assess the signaling pathways of IR generated by PM2.5 at 200 µg/mL for 24 h. The LK-MPS device consisted of a biomimetic liver-kidney architecture and reconstructed two circulation paths: the liver metabolism-kidney excretion (LM-KE) and kidney excretion-liver metabolism (KE-LM), by which PM2.5 is feasibly distributed in the two organs. Transmission electron microscopy (TEM) analysis revealed that PM2.5 can embed in the cytoplasm and nuclei, undergo transport by vesicles, and lead to the destruction of mitochondria. Further comprehensive immunofluorescence, enzyme-linked immunosorbent assays (ELISAs) and untargeted metabolomic analyses confirmed that PM2.5 disturbed the classic IRS-1/AKT signaling pathway (INSR, IRS-1, PI3K, AKT, GLUT2, GLUT4, and FOXO1 downregulated) and IR-related metabolic pathways: UDP-hexosamine (UDP-GlcNAc), gluconeogenesis (ß-d-glucose 6-phosphate), and lipid biosynthesis (ceramide (Cer) and triacylglycerol (TG)) pathways, leading to the disorder of glucose levels. Collectively, these disorders aggravate hepatic and renal IR. Pearson's correlation coefficient test showed that elemental carbon (EC), polycyclic aromatic hydrocarbons (PAHs), and metals (Ca, Co, and V) were negatively correlated to the dysregulated proteins (INSR, IRS-1, AKT, FOXO1, GLUT2, and GLUT4). These findings may partially explain IR-related signaling pathways triggered by PM2.5.

A General Set of DNA-Compatible Reactions for Preparing DNA-Tagged Multisubstituted Pyrroles.

DNA-encoded library (DEL) technology provided a powerful screening platform for identifying potential bioactive small molecules with high affinity to biologically interesting targets. Essential to a successful DEL campaign are the drug-like small molecular moieties of DNA-encoded libraries with expanded chemical space. Our laboratory has been working on developing and producing novel DNA-encoded libraries that complement current reported DELs. Herein, we demonstrated a general set of DNA-compatible reactions that enable the preparation of pyrrole-based DNA-encoded libraries in which the DNA tags are linked to the N position of the pyrrole central core. Further diversification could be rapidly incorporated into the pyrrole scaffold by robust iodination and Suzuki coupling reactions.

The Effect of Peroral Endoscopic Myotomy in Achalasia Patients with Prior Endoscopic Intervention: A Systematic Review and Meta-Analysis.

BACKGROUND: Peroral endoscopic myotomy (POEM) has been reported to be effective in achalasia patients with prior failed endoscopic intervention (PFI). We performed this meta-analysis to compare and summarize the clinical outcome of POEM in patients with or without prior endoscopic intervention. METHOD: We searched relevant studies published up to March 2020. Meta-analysis for technical success, clinical success, Eckardt score, lower esophageal sphincter (LES) pressure, clinical reflux, and adverse event were conducted based on a random-effects model. RESULTS: Eight studies enrolling 1,797 patients who underwent POEM were enrolled, including 1,128 naïve achalasia patients and 669 patients with PFI. In the PFI group, the pooled estimated rate of technical success was 97.7% (95% confidence interval [CI], 95.8-98.8%), the pooled clinical success rate was 91.0% (95% CI, 88.0-93.4%), and the pooled adverse events rate was 23.5% (95% CI, 10.6-44.1%). The Eckardt score significantly decreased by 5.95 points (95% CI, 5.50-6.40, p < 0.00001) and the LES pressure significantly reduced by 19.74 mm Hg (95% CI, 14.10-25.39, p < 0.00001) in the PFI group. There were no difference in the technical success, clinical success, and adverse events rate between the treatment-naïve group and PFI group, with a risk ratio of 1.0 (95% CI, 0.99-1.01, p = 0.89), 1.02 (95% CI, 0.98-1.06, p = 0.36), and 0.88 (95% CI, 0.67-1.16, p = 0.38), respectively. CONCLUSIONS: POEM is an effective and safe treatment for achalasia patients with prior endoscopic intervention. Randomized clinical trials are needed to further verify the efficiency and safety of the POEM in those patients.

Palladium-mediated Suzuki-Miyaura Cross-Coupling Reaction of Potassium Boc-protected aminomethyltrifluoroborate with DNA-Conjugated aryl bromides for DNA-Encoded chemical library synthesis.

A mild reaction for DNA-compatible, palladium promoted Suzuki-Miyaura cross-coupling reaction of potassium Boc-protected aminomethyltrifluoroborate with DNA-conjugated aryl bromides has been developed efficiently. This novel DNA encoded chemistry reaction proceeded well with a wide range of functional group tolerance, including aryl bromides and heteroaryl bromides. Further, the utility our DNA conjugated aminomethylated arene products is demonstrated by reaction with various types of reagents (including amide formation with carboxylic acids, alkylation with aldehydes, and carbamoylation with amines) as would be desired for the production of a DNA encoded library.

Effect of probiotics on the intestinal microbiota of hemodialysis patients: a randomized trial.

BACKGROUND: Intestinal dysbiosis contributes to the progression of renal failure and cardiovascular diseases in patients with chronic kidney disease. Probiotics is a promising intervention to improving intestinal dysbiosis. A double-blind clinical trial to investigate the ability of probiotics to modulate gut microbiota compositions in patients receiving hemodialysis (HD) was undertaken. METHODS: Fifty HD patients were enrolled and randomized, receiving either probiotics or placebo for 6 months. The responses to the interventions on gut microbiome, serum and fecal metabolome, serum albumin and endotoxin, endothelial activation markers and inflammatory markers were assessed. RESULTS: Totally, 22 in the probiotics group (11 males; 14 non-diabetic) and 23 in the placebo group (13 males; 17 non-diabetic) completed the study. Compared to that in the placebo group, probiotics did not significantly alter species diversity of the fecal microbiome. Probiotics did, however, restore the community composition, with particular significance in non-diabetic HD patients (P = 0.007 by Adonis analysis). Specifically, according to the results of linear discriminate analysis effect size, probiotics raised the proportions of family Bacteroidaceae and Enterococcaceae, and reduced Ruminococcaceae, Halomonadaceae, Peptostreptococcaceae, Clostridiales Family XIII. Incertae Sedis and Erysipelotrichaceae in non-diabetic HD patients. Additionally, probiotics reduced the abundances of several uremic retention solutes in serum or feces, including indole-3-acetic acid-O-glucuronide, 3-guanidinopropionic acid, and 1-methylinosine (P < 0.05). In the probiotic arm, no significant changes were observed in other secondary outcomes. CONCLUSIONS: Taken together, outcomes from this study suggest that probiotics do have benefits on improving intestinal imbalances and lowering exposure to several uremic toxins in HD patients.

Homocysteine Aggravates Intestinal Epithelial Barrier Dysfunction in Rats with Experimental Uremia.

BACKGROUND/AIMS: Previous studies have shown that homocysteine (Hcy) is an important intestinal-derived uremic toxin. However, whether Hcy is involved in the epithelial barrier dysfunction observed in uremia remains unclear. This study aimed to investigate the effect of Hcy on intestinal permeability and intestinal barrier structure and function in adenine-induced uremic rats. METHODS: Sprague-Dawley rats were divided into five groups: normal control (group NC), Hcy (group H), uremia (group U), uremia + Hcy (group UH), and uremia + Hcy + VSL#3 (group UHV). Experimental uremia was induced by intragastric adenine administration, and Hcy was injected subcutaneously. The animal models were assessed for renal function and pathological tissue staining. The pathological changes of intestinal tissue were observed by hematoxylin and eosin staining and electron microscopy. The serum and intestinal tissue levels of Hcy, interleukin (IL)-6, tumor necrosis factor (TNF)-α, superoxide dismutase (SOD), and malondialdehyde (MDA) as well as serum endotoxin and intestinal permeability were assessed. The levels of the tight junction proteins claudin-1, occludin, and zonula occludens-1 (ZO-1) were assessed by western blotting. RESULTS: Blood analyses and renal pathology indicated that experimental uremia was induced successfully. Pathological damage to intestinal structure was most obvious in group UH. Serum and tissue Hcy, serum endotoxin, and intestinal permeability were significantly elevated in group UH. The protein levels of claudin-1, occludin, and ZO-1 were decreased to various degrees in group UH compared with groups NC, H, and U. The serum and tissue levels of IL-6, TNF-α, and MDA were significantly increased, while SOD activity was markedly decreased. Supplementation with the probiotic VSL#3 improved these parameters to various degrees and up-regulated the abundance of tight junction proteins, which indicated a role for Hcy in the increase of intestinal permeability and destruction of the epithelial barrier in uremia. CONCLUSION: Hcy aggravates the increase of intestinal permeability and destruction of the epithelial barrier by stimulating inflammatory and oxidative damage. Probiotic administration can ameliorate this damage by reducing the levels of Hcy-induced inflammation and oxidation.

Metabolite Profiling of Feces and Serum in Hemodialysis Patients and the Effect of Medicinal Charcoal Tablets.

BACKGROUND/AIMS: Recently, the colon has been recognized as an important source of various uremic toxins in patients with end stage renal disease. Medicinal charcoal tablets are an oral adsorbent that are widely used in patients with chronic kidney disease in China to remove creatinine and urea from the colon. A parallel fecal and serum metabolomics study was performed to determine comprehensive metabolic profiles of patients receiving hemodialysis (HD). The effects of medicinal charcoal tablets on the fecal and serum metabolomes of HD patients were also investigated. METHODS: Ultra-performance liquid chromatography/mass spectrometry was used to investigate the fecal and serum metabolic profiles of 20 healthy controls and 31 HD patients before and after taking medicinal charcoal tablets for 3 months. RESULTS: There were distinct metabolic variations between the HD patients and healthy controls both in the feces and serum according to multivariate data analysis. Metabolic disturbances of alanine, aspartate and glutamate metabolism, arginine and proline metabolism figured prominently in the serum. However, in the feces, alterations of tryptophan metabolism, lysine degradation and beta-alanine metabolism were pronounced, and the levels of several amino acids (leucine, phenylalanine, lysine, histidine, methionine, tyrosine, and tryptophan) were increased dramatically. Nineteen fecal metabolites and 21 serum metabolites were also identified as biomarkers that contributed to the metabolic differences. Additionally, medicinal charcoal treatment generally enabled the serum and fecal metabolomes of the HD patients to draw close to those of the control subjects, especially the serum metabolic profile. CONCLUSION: Parallel fecal and serum metabolomics uncovered the systematic metabolic variations of HD patients, especially disturbances in amino acid metabolism in the colon. Medicinal charcoal tablets had an impact on the serum and fecal metabolomes of HD patients, but their exact effects still need to be studied further.

Signal Transductions of BEAS-2B Cells in Response to Carcinogenic PM2.5 Exposure Based on a Microfluidic System.

PM2.5 (particulate matter less than 2.5 µm in diameter) is considered as a harmful carcinogen. Determining the precise relationship between the chemical constituents of PM2.5 in the air and cancer progression could aid the treatment of environment related disease and establishing risk reduction strategies. Herein, we used transcriptomics (RNA-seq) and an integrated microfluidic system to identify the global gene expression and differential target proteins expression induced by ambient fine particles collected from the heavy haze in China. The results clearly indicated that cancer related pathways exhibited the strongest dysregulation. The ambient fine particles could be uptaken into the cells by pinocytosis, mainly promoting the PI3K-Akt pathway, FGF/FGFR/MAPK/VEGF signaling, and the JAK-STAT pathway, leading to evading apoptosis, sustained angiogenesis, and cell proliferation, which are the most important hallmarks of cancer. And fine particles also have been demonstrated to create intracellular reactive oxygen species (ROS) and mitochondrial ROS, change intracellular free Ca2+, and induce apoptosis, which are all key players in mediating cancer progression. It was observed by transmission electron microscopy (TEM) that the particles from the haze could enter the mitochondria, resulting in disturbance of the mitochondrial membrane and disruption of the mitochondria, and these particles can even enter inside the nucleus. It was also found in our study of organics (OC, PAHs) and metals (Zn, As, V) that compounds of fine particles were more closely associated with the exacerbation of cancer and secondary aerosols generated by traffic had the largest impact on cancer related signal transductions.

First airborne pathogen direct analysis system.

We report a portable "sample to answer" system for the rapid detection of airborne pathogens for the first time. The system contains a key microfluidic chip which fulfills both pathogen enrichment and biological identification functions. The system realizes simple operation and less human intervention as well as minimum reagent contamination. The operation is user-friendly and suitable for field and point-of-care applications. The system is capable of handling detection of different pathogens by changing the primers.

Microfluidic platform for direct capture and analysis of airborne Mycobacterium tuberculosis.

Airborne Mycobacterium tuberculosis is the main source of tuberculosis infection, which is known as one of the worldwide infectious diseases. Direct capture and analysis of airborne Mycobacterium tuberculosis is essential for disease prevention and control. At present, low concentration of pathogens directly collected from the air is the major drawback for rapid analysis. Herein an integrated microfluidic system capable of airborne Mycobacterium tuberculosis capture, enrichment, and rapid bacteriological immunoassay was developed. The whole detection time was decreased to less than 50 min including 20 min of enrichment and 30 min of immunoreaction analysis. It had the advantages of low detection limit, fast detection speed, and low reagent consumption compared with conventional techniques, showing the potential to become a new airborne pathogen analysis platform.

Characteristics of a microcystin-LR biodegrading bacterial isolate: Ochrobactrum sp. FDT5.

Mass growth of blue-green algae in eutrophic water bodies leads to a large amount of toxins, e.g. microcystins (MCs). How to remove MCs from water bodies is an environmental problem. In this study, an algicidal bacterium Ochrobactrum sp. FDT5 was isolated and found to have microcystin-LR (MC-LR) degradation capacity, which could be enhanced by a domestication process. The FDT5 cell density, MC-LR initial concentration, temperature, and pH on the degradation of MC-LR were investigated. The results indicated that the initial cell density of FDT5 and the initial concentration of MC-LR could influence MC-LR degradation. The optimum conditions were under the temperature of 35°C with pH of 7.0. After FDT5 was exposed to MC-LR for 2 days, FDT5 cells produced active cellular components that degraded MC-LR. These cellular components were heat-inactivated and removed when FDT5 cells were removed by filtration.

FGF1ΔHBS ameliorates retinal inflammation via suppressing TSPO signal in a type 2 diabetes mouse model.

Translocator protein (18 kDa) (TSPO) plays an important role in retinal neuroinflammation in the early stage of diabetic retinopathy (DR). Studies have found that a FGF1 variant (FGF1ΔHBS) with reduced proliferative potency exerts excellent anti-inflammatory effects and potential therapeutic value for diabetic complications. In this study, intravitreal injection of FGF1ΔHBS was administrated every week for one month in db/db mice, which are genetically predisposed to develop type 2 diabetes mellitus and early retinopathy. Changes in retinal function and structure in the animal models were detected by electrophysiology (ERG) and optical tomography coherence (OCT). TSPO expression and retinal inflammation were analyzed by immunofluorescence, Western blot and real-time qPCR. In the retina of T2D (db/db) mice, FGF1 was significantly down-regulated while FGFR1 was up-regulated (both p < 0.05). TSPO and retinal inflammatory factors were all up-regulated. TSPO and FGFR1 were mainly co-stained in the inner retina. After FGF1ΔHBS treatment, ERG showed that the total amplitude of dark-adapted b-wave and oscillating potentials (Ops) was significantly improved, and OCT showed that the thickness of the retina around the optical nerve head was significantly preserved in T2D mice (all p < 0.05). The TSPO signal was significantly suppressed by FGF1ΔHBS. The activation of NF-κB p65 and the expression of inflammatory factors such as TNF-α, IL-1ß, IL-6, COX-2, MIP-1α, and iNOS were all significantly down-regulated (all p < 0.05). Collectively, our current data demonstrated that intravitreal FGF1ΔHBS treatment can effectively inhibit retinal inflammation via suppressing TSPO signal and to preserve retinal function and structure in a T2D mouse model.