Dry Ball-Milled Quinoa Starch as a Pickering Emulsifier: Preparation, Microstructures, Hydrophobic Properties and Emulsifying Properties.

This research supplied a "cleaner-production" way to produce "clean-label" quinoa starch-based Pickering emulsifier with excellent emulsifying properties. The effects of dry ball-milling time and speed on the multi-scale structures and emulsifying properties of quinoa starch were studied. With increasing ball-milling time and speed, particle size first decreased and then increased, the crystallinity, lamellar structure and short-range ordered structure gradually decreased, and contact angle gradually increased. The increased contact angle might be related to the increased oil absorption properties and the decreased water content. The emulsification properties of ball-milled quinoa starch (BMQS)-based Pickering emulsions increased with the increase in ball-milling time and speed, and the emulsions of BMQS-4 h, 6 h, 8 h, and 600 r reached the full emulsification state. After 120 days' storage, the oil droplets of BMQS-2 h (BMQS-400 r) deformed, the oil droplets increased, and the emulsification index decreased. The emulsification index and the oil droplets of BMQS-4 h, 6 h, 8 h and 600 r-based emulsions did not show obvious changes after storage, indicating the good emulsifying stability of these BMQS-based emulsions, which might be because that the relatively larger amount of starch particles that dispersed in the voids among the oil droplets could act as stronger network skeletons for the emulsion gel. This Pickering emulsifier was easily and highly efficiently produced and low-cost, having great potential to be used in the food, cosmetic and pharmaceutical industries.

Microascaceae from the Marine Environment, with Descriptions of Six New Species.

Most reported members of Microascaceae that have been reported originate from the terrestrial environment, where they act as saprobes or plant pathogens. However, our understanding of their species diversity and distribution in the marine environment remains vastly limited, with only 22 species in nine genera having been reported so far. A survey of the fungal diversity in intertidal areas of China's mainland has revealed the discovery of several Microascaceae strains from 14 marine algae and 15 sediment samples. Based on morphological characteristics and LSU-ITS-tef1-tub2 multilocus phylogeny using Bayesian inference and maximum likelihood methods, 48 strains were identified as 18 species belonging to six genera. Among these, six new species were discovered: Gamsia sedimenticola, Microascus algicola, M. gennadii, Scedosporium ellipsosporium, S. shenzhenensis, and S. sphaerospermum. Additionally, the worldwide distribution of the species within this family across various marine habitats was briefly reviewed and discussed. Our study expands the knowledge of species diversity and distribution of Microascaceae in the marine environment.

2D Ag Ion-Loaded Anionic Nanosheets for Polymer-Based Film with Durable Antibacterial Activities.

Silver (Ag) has been demonstrated to have excellent performance to kill bacteria, fungi, and some viruses because it can release positively charged Ag ions with highly antibacterial and antifungal activities. However, effectively controlling the slow release of Ag ions is the key to preparing high-performance Ag-based antibacterial agents, which remains a challenge. In this work, we have developed a new Ag-based antibacterial agent composed of Ag ions loaded on 2D anionic 2D Sb3P2O14 3- nanosheets (denoted as Ag-Sb3P2O14). 2D anionic nanosheets not only adsorb a large amount of Ag ions but also control their slow release through electrostatic interaction between nanosheets and Ag ions. 2D Ag-Sb3P2O14 nanofillers enable excellent high antibacterial activities for the poly(vinylidene fluoride) (PVDF) film composites against microorganisms including Escherichia coli and Staphylococcus aureus. Moreover, the PVDF membrane with 5 wt % 2D Ag-Sb3P2O14 nanofillers can kill almost all bacterial after 50 times washing, demonstrating its excellent durable antibacterial activities. This work opens up a new and promising route to durable Ag-based antibacterial agents for polymer-based composites.

The therapeutic effect of balloon dilatation with different duration for biliary duct calculi: A network meta-analysis.

Objective: To systematically evaluate the application effect of endoscopic papillary balloon dilatation (EPBD) with different balloon dilatation duration for biliary duct calculi, and find the most appropriate dilatation duration for EPBD using a network meta-analysis. Materials and Methods: PubMed, Embase and Cochrane Library databases were searched for relevant randomised controlled trials (RCTs) published up to August 2020. Node split, consistency and inconsistency models analysis were all conducted in network meta-analysis. Results: Eighteen RCTs with 2256 participants were finally analysed. EPBD was divided into four categories based on balloon dilatation duration, including EPBD (P0.5), EPBD (>0.5, ≤1), EPBD (1, ≤2) and EPBD (>2, ≤5). Compared with EPBD (>0.5, ≤1), EPBD (>2, ≤5) had a lower risk of early complications (odds ratio [OR] = 0.23, 95% credible interval [CI] = 0.05-0.96) and post-endoscopic procedure pancreatitis (PEP) (OR = 0.17, 95% CI = 0.03-0.72). Endoscopic sphincterotomy (EST) tended to have less need for mechanical lithotripsy (OR = 0.37, 95% CI = 0.16-0.88) and PEP (OR = 0.26, 95% CI = 0.08-0.71) than EPBD (>0.5, ≤1). EPBD (>2, ≤5) was the safest endoscopic procedure with respect to early complications (surface area under cumulative ranking curves [SUCRA] = 79.0) and PEP (SUCRA = 85.3). In addition, EPBD (>2, ≤5) and EST had the highest probability of being the best (SUCRA = 82.6) and the worst (SUCRA = 10.8), respectively, regarding late complications. Conclusion: EPBD and EST are two methods used to treat uncomplicated choledocholithiasis (stone diameter <10 mm and stone number <3). The extension of balloon dilatation duration has no significant influence on successful stone removal in the first endoscopic session or preventing the need for mechanical lithotripsy. However, it can reduce the risk of early complications, especially PEP. What's more, EPBD seems to have less late complications compared with EST, and the effect of prolonged balloon dilatation duration on late complications still needs to be further explored. Therefore, 2-5 min is the recommended dilatation duration range for EPBD using balloon with ≤10 mm diameter. Further research based on a specific population and with a longer follow-up time are needed.

Roles of calpain in the apoptosis of Eimeria tenella host cells at the middle and late developmental stages.

This study investigated the role of calpain in Eimeria tenella-induced host cell apoptosis. Chick embryo cecal epithelial cell culture technology, flow cytometry, enzyme-linked immunosorbent assays, and fluorescence quantitative PCR were used to detect the E. tenella host cell apoptotic rate, Bax and Bid expression levels, and calpain activity. The results demonstrated that Bax, Bid, and calpain levels were upregulated and apoptosis was increased following E. tenella infection at 24-120 h. Calpain levels were reduced by pharmacological inhibition of calpain using SJA6017 or by blocking Ca2+ entry into the cell using BAPTA/AM at 24-120 h. The mRNA and protein levels of Bax and Bid, the E. tenella infection rate, and the early apoptotic and late apoptotic (necrosis) rates were decreased by using SJA6017 at 24-120 h. These results indicated that E. tenella-promoted host cell apoptosis is regulated by calpain via Bid and Bax at 24-120 h. Thus, manipulation of calpain levels could be used to manage E. tenella infection in chickens in the middle and late developmental stages.

Ambroxol reverses tau and α-synuclein accumulation in a cholinergic N370S GBA1 mutation model.

Cognitive impairment is a common non-motor complication of Parkinson's disease (PD). Glucocerebrosidase gene (GBA1) variants are found in 10-15% of PD cases and are numerically the most important risk factor for PD and dementia with Lewy bodies. Accumulation of α-synuclein and tau pathology is thought to underlie cognitive impairment in PD and likely involves cholinergic as well as dopaminergic neurons. Neural crest stem cells were isolated from both PD patients with the common heterozygous N370S GBA1 mutation and normal subjects without GBA1 mutations. The stem cells were used to generate a cholinergic neuronal cell model. The effects of the GBA1 variant on glucocerebrosidase (GCase) protein and activity, and cathepsin D, tau and α-synuclein protein levels in cholinergic neurons were examined. Ambroxol, a GCase chaperone, was used to investigate whether GCase enhancement was able to reverse the effects of the GBA1 variant on cholinergic neurons. Significant reductions in GCase protein and activity, as well as in cathepsin D levels, were found in GBA1 mutant (N370S/WT) cholinergic neurons. Both tau and α-synuclein levels were significantly increased in GBA1 mutant (N370S/WT) cholinergic neurons. Ambroxol significantly enhanced GCase activity and decreased both tau and α-synuclein levels in cholinergic neurons. GBA1 mutations interfere with the metabolism of α-synuclein and tau proteins and induce higher levels of α-synuclein and tau proteins in cholinergic neurons. The GCase pathway provides a potential therapeutic target for neurodegenerative disorders related to pathological α-synuclein or tau accumulation.

Increased expression of IGF-1Ec with increasing colonic polyp dysplasia and colorectal cancer.

PURPOSE: IGF-1Ec is an isoform of Insulin-like growth factor 1 (IGF-1) and has recently been identified to be overexpressed in cancers including prostate and neuroendocrine tumours. The aim of this paper is to investigate the expression of IGF-1Ec in colorectal cancer and polyps compared to normal colon tissues and its association with recurrent disease using semi-quantitative immunohistochemistry. METHODS: Immunohistochemistry for IGF-1Ec expression was performed for colorectal cancer, colorectal polyps and normal colonic tissues. The quantification of IGF-1Ec expression was performed with the use of Image J software and the IHC profiler plugin. Following ethics approval from the National Research Ethics Service (Reference 11/LO/1521), clinical information including recurrent disease on follow-up was collected for patients with colorectal cancer. RESULTS: Immunohistochemistry was performed in 16 patients with colorectal cancer and 11 patients with colonic polyps and compared to normal colon tissues and prostate adenocarcinoma (positive control) tissues. Significantly increased expression of IGF-1Ec was demonstrated in colorectal cancer (p < 0.001) and colorectal polyps (p < 0.05) compared to normal colonic tissues. Colonic adenomas with high-grade dysplasia had significantly higher expression of IGF-1Ec compared to low-grade dysplastic adenomas (p < 0.001). Colorectal cancers without lymph node metastases at the time of presentation had significantly higher IGF-1Ec expression compared to lymph node-positive disease (p < 0.05). No correlation with recurrent disease was identified with IGF-1Ec expression. CONCLUSION: IGF-1Ec is significantly overexpressed in colorectal cancer and polyps compared to normal colon tissues offering a potential target to improve colonoscopic identification of colorectal polyps and cancer and intraoperative identification of colorectal tumours.

Glucocerebrosidase activity, cathepsin D and monomeric α-synuclein interactions in a stem cell derived neuronal model of a PD associated GBA1 mutation.

The presence of GBA1 gene mutations increases risk for Parkinson's disease (PD), but the pathogenic mechanisms of GBA1 associated PD remain unknown. Given that impaired α-synuclein turnover is a hallmark of PD pathogenesis and cathepsin D is a key enzyme involved in α-synuclein degradation in neuronal cells, we have examined the relationship of glucocerebrosidase (GCase), cathepsin D and monomeric α-synuclein in human neural crest stem cell derived dopaminergic neurons. We found that normal activity of GCase is necessary for cathepsin D to perform its function of monomeric α-synuclein removal from neurons. GBA1 mutations lead to a lower level of cathepsin D protein and activity, and higher level of monomeric α-synuclein in neurons. When GBA1 mutant neurons were treated with GCase replacement or chaperone therapy; cathepsin D protein levels and activity were restored, and monomeric α-synuclein decreased. When cathepsin D was inhibited, GCase replacement failed to reduce monomeric α-synuclein levels in GBA1 mutant neurons. These data indicate that GBA1 gene mutations increase monomeric α-synuclein levels via an effect on lysosomal cathepsin D in neurons.

Assessment of ctDNA in CSF may be a more rapid means of assessing surgical outcomes than plasma ctDNA in glioblastoma.

We aimed to develop a high-throughput deep DNA sequencing assay of cerebrospinal fluid (CSF) to identify clinically relevant oncogenic mutations that contribute to the development of glioblastoma (GBM) and serve as biomarkers to predict patients' responses to surgery. For this purpose, we recruited five patients diagnosed with highly suspicious GBM according to preoperative magnet resonance imaging. Subsequently, patients were histologically diagnosed with GBM. CSF was obtained through routine lumbar puncture, and plasma from peripheral blood was collected before surgery and 7 days after. Fresh tumor samples were collected using routine surgical procedures. Targeted deep sequencing was used to characterize the genomic landscape and identify mutational profile that differed between pre-surgical and post-surgical samples. Sequence analysis was designed to detect protein-coding exons, exon-intron boundaries, and the untranslated regions of 50 genes associated with cancers of the central nervous system. Circulating tumor DNAs (ctDNAs) were prepared from the CSF and plasma from peripheral blood. For comparison, DNA was isolated from fresh tumor tissues. Non-silent coding variants were detected in CSF and plasma ctDNAs, and the overall minor allele frequency (MAF) of the former corresponded to an earlier disease stage compared with that of plasma when the tumor burden was released (surgical removal). Gene mutation loads of GBMs significantly correlated with overall survival (OS, days) (Pearson correlation = -0.95, P = 0.01). We conclude that CSF ctDNAs better reflected the sequential mutational changes of driver genes compared with those of plasma ctDNAs. Deep sequencing of the CSF of patients with GBM may therefore serve as an alternative clinical assay to improve patients' outcomes.

Mechano-growth Factor Expression in Colorectal Cancer Investigated With Fluorescent Gold Nanoparticles.

BACKGROUND/AIM: Fluorescent gold nanoparticles demonstrate strong photoluminescence, photostability, and low cellular toxicity, making them attractive agents for biomedical applications. Mechano-growth factor (MGF) is an isoform of IGF1 and its expression has been demonstrated in malignancies including prostate cancer. MATERIALS AND METHODS: Near-infrared-emitting gold nanoparticles (AuNPs) were synthesized and conjugated to MGF. Following characterization and confirmation of conjugation, these AuNPs were used to investigate the expression of MGF in colon cancer cell lines (HT29 and SW620) and tissues comparing normal and colon cancer. The prostate cancer cell line PC3 and adenocarcinoma tissues were used as positive controls. RESULTS: Colon cancer cell lines, adenocarcinoma tissues and polyp tissues demonstrated evidence of MGF peptide expression, which was not found in normal colon tissues and human umbilical vein endothelial cells. CONCLUSION: MGF appears to be overexpressed in colon cancer tissues, offering a potential unique target for imaging and drug delivery in colon cancer.

A Human Neural Crest Stem Cell-Derived Dopaminergic Neuronal Model Recapitulates Biochemical Abnormalities in GBA1 Mutation Carriers.

Numerically the most important risk factor for the development of Parkinson's disease (PD) is the presence of mutations in the glucocerebrosidase GBA1 gene. In vitro and in vivo studies show that GBA1 mutations reduce glucocerebrosidase (GCase) activity and are associated with increased α-synuclein levels, reflecting similar changes seen in idiopathic PD brain. We have developed a neural crest stem cell-derived dopaminergic neuronal model that recapitulates biochemical abnormalities in GBA1 mutation-associated PD. Cells showed reduced GCase protein and activity, impaired macroautophagy, and increased α-synuclein levels. Advantages of this approach include easy access to stem cells, no requirement to reprogram, and retention of the intact host genome. Treatment with a GCase chaperone increased GCase protein levels and activity, rescued the autophagic defects, and decreased α-synuclein levels. These results provide the basis for further investigation of GCase chaperones or similar drugs to slow the progression of PD.

Novel POSS-PCU Nanocomposite Material as a Biocompatible Coating for Quantum Dots.

Quantum dots (QDs) are fluorescent nanoparticles with unique photophysical properties that enable them to potentially replace traditional organic dyes and fluorescent proteins in various bioimaging applications. However, the inherent toxicity of their cores based on cadmium salts limits their widespread biomedical use. We have developed a novel nanocomposite polymer emulsion based on polyhedral oligomeric silsesquioxane poly(carbonate-urea) urethane (POSS-PCU) that can be used to coat quantum dots to nullify their toxicity and enhance photostability. Here we report the synthesis and characterization of a novel POSS-PCU nanocomposite polymer emulsion and describe its application for coating QDs for biological application. The polymer was synthesized by a process of emulsion polymerization and formed stable micelles of ∼33 nm in diameter. CdTe/CdS/ZnS QDs were efficiently stabilized by the polymer emulsion through encapsulation within the polymer micelles. Characterization studies showed no significant change in the unique photophysical properties of QDs after coating. The polymer was biocompatible to HepG2, HUVECs, and mouse skeletal muscle cells at 2.5% after 24 h exposure on in vitro testing. Polymer encapsulated QDs showed enhanced photostability on exposure to high degrees of UV irradiation and air as well as significantly reduced cytotoxicity on exposure to HepG2 cells at 30 µg/mL for 24 h. We have therefore concluded that the POSS-PCU polymer emulsion has the potential to make a biocompatible and photostable coating for QDs enabling a host of biomedical applications to take this technology to the next level.

Near-infrared quantum dots for HER2 localization and imaging of cancer cells.

BACKGROUND: Quantum dots are fluorescent nanoparticles with unique photophysical properties that allow them to be used as diagnostic, therapeutic, and theranostic agents, particularly in medical and surgical oncology. Near-infrared-emitting quantum dots can be visualized in deep tissues because the biological window is transparent to these wavelengths. Their small sizes and free surface reactive groups that can be conjugated to biomolecules make them ideal probes for in vivo cancer localization, targeted chemotherapy, and image-guided cancer surgery. The human epidermal growth factor receptor 2 gene (HER2/neu) is overexpressed in 25%-30% of breast cancers. The current methods of detection for HER2 status, including immunohistochemistry and fluorescence in situ hybridization, are used ex vivo and cannot be used in vivo. In this paper, we demonstrate the application of near-infrared-emitting quantum dots for HER2 localization in fixed and live cancer cells as a first step prior to their in vivo application. METHODS: Near-infrared-emitting quantum dots were characterized and their in vitro toxicity was established using three cancer cell lines, ie, HepG2, SK-BR-3 (HER2-overexpressing), and MCF7 (HER2-underexpressing). Mouse antihuman anti-HER2 monoclonal antibody was conjugated to the near-infrared-emitting quantum dots. RESULTS: In vitro toxicity studies showed biocompatibility of SK-BR-3 and MCF7 cell lines with near-infrared-emitting quantum dots at a concentration of 60 µg/mL after one hour and 24 hours of exposure. Near-infrared-emitting quantum dot antiHER2-antibody bioconjugates successfully localized HER2 receptors on SK-BR-3 cells. CONCLUSION: Near-infrared-emitting quantum dot bioconjugates can be used for rapid localization of HER2 receptors and can potentially be used for targeted therapy as well as image-guided surgery.

The nitric oxide pathway--evidence and mechanisms for protection against liver ischaemia reperfusion injury.

Ischaemia reperfusion (IR) injury is a clinical entity with a major contribution to the morbidity and mortality of liver surgery and transplantation. A central pathway of protection against IR injury utilizes nitric oxide (NO). Nitric oxide synthase (NOS) enzymes manufacture NO from L-arginine. NO generated by the endothelial NOS (eNOS) isoform protects against liver IR injury, whereas inducible NOS (iNOS)-derived NO may have either a protective or a deleterious effect during the early phase of IR injury, depending on the length of ischaemia, length of reperfusion and experimental model. In late phase hepatic IR injury, iNOS-derived NO plays a protective role. In addition to NOS consumption of L-arginine during NO synthesis, this amino acid may also be metabolized by arginase, an enzyme whose release is increased during prolonged ischaemia, and therefore diverts L-arginine away from NOS metabolism leading to a drop in the rate of NO synthesis. NO most commonly acts through the soluble guanylyl cyclase-cyclic GMP- protein kinase G pathway to ameliorate hepatic IR injury. Both endogenously generated and exogenously administered NO donors protect against liver IR injury. The beneficial effects of NO on liver IR are not, however, universal, and certain conditions, such as steatosis, may influence the protective effects of NO. In this review, the evidence for, and mechanisms of these protective actions of NO are discussed, and areas in need of further research are highlighted.

Peptide Vaccine Therapy in Colorectal Cancer.

Colorectal cancer is the third most common cause of cancer-related deaths and the second most prevalent (after breast cancer) in the western world. High metastatic relapse rates and severe side effects associated with the adjuvant treatment have urged oncologists and clinicians to find a novel, less toxic therapeutic strategy. Considering the limited success of the past clinical trials involving peptide vaccine therapy to treat colorectal cancer, it is necessary to revise our knowledge of the immune system and its potential use in tackling cancer. This review presents the efforts of the scientific community in the development of peptide vaccine therapy for colorectal cancer. We review recent clinical trials and the strategies for immunologic monitoring of responses to peptide vaccine therapy. We also discuss the mechanisms underlying the therapy and potential molecular targets in colon cancer.

Nerve regeneration with aid of nanotechnology and cellular engineering.

Repairing nerve defects with large gaps remains one of the most operative challenges for surgeons. Incomplete recovery from peripheral nerve injuries can produce a diversity of negative outcomes, including numbness, impairment of sensory or motor function, possibility of developing chronic pain, and devastating permanent disability. In the last few years, numerous microsurgical techniques, such as coaptation, nerve autograft, and different biological or polymeric nerve conduits, have been developed to reconstruct a long segment of damaged peripheral nerve. A few of these techniques are promising and have become popular among surgeons. Advancements in the field of tissue engineering have led to development of synthetic nerve conduits as an alternative for the nerve autograft technique, which is the current practice to bridge nerve defects with gaps larger than 30 mm. However, to date, despite significant progress in this field, no material has been found to be an ideal alternative to the nerve autograft. This article briefly reviews major up-to-date published studies using different materials as an alternative to the nerve autograft to bridge peripheral nerve gaps in an attempt to assess their ability to support and enhance nerve regeneration and their prospective drawbacks, and also highlights the promising hope for nerve regeneration with the next generation of nerve conduits, which has been significantly enhanced with the tissue engineering approach, especially with the aid of nanotechnology in development of the three-dimensional scaffold. The goal is to determine potential alternatives for nerve regeneration and repair that are simply and directly applicable in clinical conditions.

Recent advances in artificial nerve conduit design: strategies for the delivery of luminal fillers.

Artificial nerve conduits offer an attractive alternative to nerve autografts for the repair of peripheral nerve injuries and several commercially-available conduits are currently on the market. However, at present, utilization of these conduits is limited to the repair of nerve gaps less than 3 cm in length. Thus, current research is focused on how best to design artificial conduits with improved nerve regeneration potential over longer distances. Successful nerve regeneration necessitates that the cells, extracellular matrix components, and growth factors involved interact in a highly specific manner that is tightly coordinated. Combinatorial approaches that take into account these interactions and conduits that utilize supportive factors, such as neurotrophins and stem cells, may be key components of the next generation of artificial conduits. Additionally, design strategies that combine physical cues for contact guidance and biochemical signals to enhance cellular function have shown promise. This review highlights recent advances in artificial nerve conduit design, focusing on the use of luminal fillers, with special focus on the various techniques for accessory cell and/or growth factor delivery into artificial nerve conduits.

Role of endothelial nitric oxide synthase in remote ischemic preconditioning of the mouse liver.

Hindlimb remote ischemic preconditioning (RIPC) reduces liver ischemia/reperfusion (IR) injury in wild-type mice. The underlying mechanisms of RIPC are currently unknown. In this study, we investigated the role of endothelial nitric oxide synthase (eNOS) in mediating the protective effects of RIPC. Endothelial nitric oxide synthase knockout (eNOS(-/-) ) mice were divided into 4 groups: (1) a sham surgery group, (2) an RIPC group (6 cycles of 4 minutes of hindlimb ischemia and 4 minutes of hindlimb reperfusion), (3) an IR group [40 minutes of lobar (70%) hepatic ischemia and 2 hours of reperfusion], and (4) an RIPC+IR group (RIPC followed by the IR group procedures). Plasma liver aminotransferases, hepatic histopathological injury scores, transmission electron microscopy studies, and hepatic microcirculatory blood flow (MBF) were assessed. eNOS protein expression was analyzed in the livers and hindlimb muscles of wild-type mice. Hindlimb RIPC did not protect against subsequent liver IR injury in eNOS(-/-) mice; this was demonstrated by the lack of reduction in the plasma aminotransferase levels, histopathological scores, or ultrastructural features of IR injury in the RIPC+IR group versus the IR group. Hepatic MBF did not recover during liver reperfusion in the RIPC+IR group versus the IR group. eNOS protein expression was similar among all wild-type groups. In conclusion, eNOS is essential for the protective effects of hindlimb RIPC on liver IR injury. eNOS exerts its protective effects through the preservation of hepatic MBF. At 2 hours of reperfusion, eNOS protection is likely due to the increased activation of eNOS rather than increased expression.

Nitric oxide is an essential mediator of the protective effects of remote ischaemic preconditioning in a mouse model of liver ischaemia/reperfusion injury.

NO (nitric oxide) may protect the liver from IR (ischaemia/reperfusion) injury. RIPC (remote ischaemic preconditioning) also protects against liver IR injury; however, the molecular mediator(s) of RIPC are currently unknown. The aim of the present study was to assess the role of NO in hindlimb RIPC-induced protection against liver IR injury. Mice were allocated to the following groups: sham group; RIPC group (six cycles of 4×4 min IR of hindlimb); IR group [40 min lobar (70%) hepatic ischaemia and 2-h reperfusion]; RIPC+IR group (RIPC followed by IR group procedures); and C-PTIO [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt]+RIPC+IR group [C-PTIO (a direct NO scavenger) was administered, followed by the RIPC+IR group procedure]. Hepatic MBF (microcirculatory blood flow) was measured throughout the experiment. Circulating NOx (nitrite and nitrate) levels, plasma liver transaminases, hepatic histopathological and TEM (transmission electron microscopy) studies were performed at the end of the experiment. NOx concentrations were significantly elevated (P<0.05) in the RIPC and RIPC+IR groups. Compared with liver IR alone, RIPC+IR preserved hepatic MBF during liver reperfusion (P<0.05). In contrast, C-PTIO+RIPC+IR reduced MBF compared with RIPC+IR (P<0.05). RIPC+IR reduced plasma transaminases (P<0.05), and histopathological and ultrastructural features of injury compared with IR alone. The protective effects of RIPC+IR in reducing liver IR injury were abrogated in the group that received antecedent C-PTIO (C-PTIO+RIPC+IR). In conclusion, NO is an essential mediator of the protection afforded by hindlimb RIPC against liver IR injury. The mechanisms underlying this protection involve preservation of the sinusoidal structure and maintenance of blood flow through the hepatic microcirculation.