Impacts of cryopreservation on phenotype and functionality of mononuclear cells in peripheral blood and ascites.

Background: Mononuclear cells in peripheral blood and ascites are important clinical resources commonly used in translational and basic research. However, the impact of different cryopreservation durations and extra freeze-thaw cycles on the number and function of mononuclear cells is unknown. Methods: Peripheral blood samples (n = 21) and ascites samples (n = 8) were collected from healthy volunteers and ovarian cancer patients. Mononuclear cells were isolated, frozen, and thawed at 6 and 12 months. The impact of cryopreservation on cell viability, the phenotype, and the activation and proliferation of T cells were analyzed by flow cytometry. Single-cell sequencing was applied to investigate the underlying mechanism. Results: The cell number and viability of mononuclear cells in peripheral blood and ascites were significantly decreased after cryopreservation. The T lymphocytes, especially CD4+ T cells, were affected the most significantly. By contrast, monocytes, natural killer (NK) cells, natural killer T (NKT) cells, and B cells were more tolerant. Meanwhile, T cell proliferation and IL-2 secretion are significantly affected after long-term cryopreservation. Mechanistically, the cell death induced by elevated reactive oxygen species (ROS) was involved in the reduction of CD4+ T cells after cryopreservation. Conclusions: Our data indicates that different subtypes of mononuclear cells exhibit different tolerance capacities upon cryopreservation. Thus, our research can provide evidence and support for individuals who are conducting experiments using frozen clinical patient-derived mononuclear cells, for basic research or clinical trials. In addition, extra caution is worthwhile when researchers compare immune cell functionality from peripheral blood or ascites across datasets obtained in different cryopreservation conditions.

Mesenchymal stem cell-regulated miRNA-mRNA landscape in acute-on-chronic liver failure.

BACKGROUND: Acute-on-chronic liver failure (ACLF) is a major challenge in the field of hepatology. While mesenchymal stem cell (MSC) therapy can improve the prognosis of patients with ACLF, the molecular mechanisms through which MSCs attenuate ACLF remain poorly understood. We performed global miRNA and mRNA expression profiling via next-generation sequencing of liver tissues from MSC-treated ACLF mice to identify important signaling pathways and major factors implicated in ACLF alleviation by MSCs. METHODS: Carbon tetrachloride-induced ACLF mice were treated with saline or mouse bone marrow-derived MSCs. Mouse livers were subjected to miRNA and mRNA sequencing. Related signal transduction pathways were obtained through Gene Set Enrichment Analysis. Functional enrichment, protein-protein interaction, and immune infiltration analyses were performed for the differentially expressed miRNA target genes (DETs). Hub miRNA and mRNA associated with liver injury were analyzed using LASSO regression. The expression levels of hub genes were subjected to Pearson's correlation analysis and verified using RT-qPCR. The biological functions of hub genes were verified in vitro. RESULTS: The tricarboxylic acid cycle and peroxisome proliferator-activated receptor pathways were activated in the MSC-treated groups. The proportions of liver-infiltrating NK resting cells, M2 macrophages, follicular helper T cells, and other immune cells were altered after MSC treatment. The expression levels of six miRNAs and 10 transcripts correlated with the degree of liver injury. miR-27a-5p was downregulated in the mouse liver after MSC treatment, while its target gene E2f2 was upregulated. miR-27a-5p inhibited E2F2 expression, suppressed G1/S phase transition and proliferation of hepatocytes, in addition to promoting their apoptosis. CONCLUSIONS: This is the first comprehensive analysis of miRNA and mRNA expression in the liver tissue of ACLF mice after MSC treatment. The results revealed global changes in hepatic pathways and immune subpopulations. The miR-27a-5p/E2F2 axis emerged as a central regulator of the MSC-induced attenuation of ACLF. The current findings improve our understanding of the molecular mechanisms through which MSCs alleviate ACLF.

Advances in regulation and function of stearoyl-CoA desaturase 1 in cancer, from bench to bed.

Stearoyl-CoA desaturase 1 (SCD1) converts saturated fatty acids to monounsaturated fatty acids. The expression of SCD1 is increased in many cancers, and the altered expression contributes to the proliferation, invasion, sternness and chemoresistance of cancer cells. Recently, more evidence has been reported to further support the important role of SCD1 in cancer, and the regulation mechanism of SCD1 has also been focused. Multiple factors are involved in the regulation of SCD1, including metabolism, diet, tumor microenvironment, transcription factors, non-coding RNAs, and epigenetics modification. Moreover, SCD1 is found to be involved in regulating ferroptosis resistance. Based on these findings, SCD1 has been considered as a potential target for cancer treatment. However, the resistance of SCD1 inhibition may occur in certain tumors due to tumor heterogeneity and metabolic plasticity. This review summarizes recent advances in the regulation and function of SCD1 in tumors and discusses the potential clinical application of targeting SCD1 for cancer treatment.

Dual-regulation by Cx32 in hepatocyte to trigger and worsen liver graft injury.

Liver transplantation is the ultimate treatment option for end-stage liver failure. However, liver graft injury remains a challenge. This study aimed to investigate the role of connexin32 (Cx32) in liver graft injury and elucidate its mechanism of action. Through detecting liver graft samples from 6 patients, we observed that changes in the Cx32 level coincided with liver graft injury. Therefore, we established autologous orthotopic liver transplantation (AOLT) models using Cx32-knockout and wild-type mice and hypoxia/reoxygenation (H/R) and lipopolysaccharide (LPS) pretreatment models using alpha mouse liver 12 (AML12) cells, to explore Cx32 mechanisms in liver graft injury. Following in vivo and in vitro Cx32 knockout, oxidative stress and inflammatory response were inhibited through the regulation of PKC-α/NF-κB/NLRP3 and Nrf2/NOX4/ROS signaling pathways, thereby reducing Bak/Bax-related apoptosis and ameliorating liver graft injury. When the Cx32-based gap junction (GJ) was blocked with 2-aminoethoxydiphenyl borate (2-APB), ROS transfer was attenuated between neighboring cells, exacerbated oxidative stress and inflammatory response were prevented, and aggravation of liver graft injury was mitigated. These results highlight the dual regulation mechanism of Cx32 in liver graft injury. Through interaction with PKC-α, Cx32 regulated the NF-κB/NLRP3 and Nrf2/NOX4/ROS signaling pathways, thus directly triggering oxidative stress and inflammatory response. Simultaneously, mass-produced ROS were transferred to neighboring cells through Cx32 channels, for which oxidative stress and the inflammatory response were aggravated indirectly. Finally, Bak/Bax-related apoptosis was activated, thereby worsening liver graft injury. Our findings propose Cx32 as a dual mechanistic factor for oxidative stress and inflammatory signaling pathways in regulating cell apoptosis on liver graft injury, which suggests a promising therapeutic targets for liver graft injury.

Bone marrow mesenchymal stem cell-derived small extracellular vesicles promote liver regeneration via miR-20a-5p/PTEN.

Balancing hepatocyte death and proliferation is key to non-transplantation treatments for acute liver failure (ALF), which has a high short-term mortality rate. Small extracellular vesicles (sEVs) may act as mediators in the repair of damaged liver tissue by mesenchymal stem cells (MSCs). We aimed to investigate the efficacy of human bone marrow MSC-derived sEVs (BMSC-sEVs) in treating mice with ALF and the molecular mechanisms involved in regulating hepatocyte proliferation and apoptosis. Small EVs and sEV-free BMSC concentrated medium were injected into mice with LPS/D-GalN-induced ALF to assess survival, changes in serology, liver pathology, and apoptosis and proliferation in different phases. The results were further verified in vitro in L-02 cells with hydrogen peroxide injury. BMSC-sEV-treated mice with ALF had higher 24 h survival rates and more significant reductions in liver injury than mice treated with sEV-free concentrated medium. BMSC-sEVs reduced hepatocyte apoptosis and promoted cell proliferation by upregulating miR-20a-5p, which targeted the PTEN/AKT signaling pathway. Additionally, BMSC-sEVs upregulated the mir-20a precursor in hepatocytes. The application of BMSC-sEVs showed a positive impact by preventing the development of ALF, and may serve as a promising strategy for promoting ALF liver regeneration. miR-20a-5p plays an important role in liver protection from ALF by BMSC-sEVs.

Transplanting neurofibromatosis-1 gene knockout neural stem cells improve functional recovery in rats with spinal cord injury by enhancing the mTORC2 pathway.

The poor survival and low efficiency of neuronal differentiation limits the therapeutic effects of transplanted neural stem cells in the treatment of spinal cord injury. Neurofibromatosis-1 (NF-1) is a tumor suppressor gene that restricts the rapid and abnormal growth and differentiation of neural cells. In the present study, lentiviral vectors were used to knock out NF-1, Ricotr (the core member of mTORC2) or NF-1+Ricotr in neural stem cells in vitro, and the NF-1, Ricotr or NF-1+Ricotr knockout neural stem cells were transplanted at the lesion site in a rat model of spinal cord injury (SCI). We first demonstrated that targeted knockout of NF-1 had an antiapoptotic effect and improved neuronal differentiation by enhancing the mTORC2/Rictor pathway of neural stem cells in vitro. Subsequently, transplanting NF-1 knockout neural stem cells into the injured site sufficiently promoted the tissue repair and functional recovery of rats with spinal cord injury by enhancing the survival and neuronal differentiation of grafted neural stem cells. Collectively, these findings reveal a prominent role of NF-1 in neural stem cell biology, which is an invaluable step forward in enhancing the benefit of neural stem cell-mediated regenerative cell therapy for spinal cord injury and identifies the transplantation of NF-1 knockout neural stem cells as a promising strategy for spinal cord injury.

Comparative intra- and inter-observer reliability of two methods for evaluating intraoperative ultrasonography-based spinal cord hyperechogenicity intensity in degenerative cervical myelopathy.

OBJECTIVES: During French-door laminoplasty, a linear array transducer of IOUS was used to observe and record the spinal cord decompression. To acquire a higher-reliability method, and compare the in-observer and inter-observer reliability of two methods in evaluating the hyperechoic intensity of spinal cord ultrasound in degenerative cervical myelopathy (DCM). BACKGROUND: The intensity of spinal cord hyperechogenicity is considered as a potential predictor of neurological recovery in DCM after decompression, but the accuracy of gray value ratio (GVR) is affected by many factors. METHODS: Totally 28 patients (20 males and 8 females) who had been followed up for 12 months were included. Their mean age at surgery was 61.2 ± 10.8 years and the average symptom duration was 23.36 ± 22.11 months. The gray values of circles 1, 2 and 3 were recorded as Gcompression, Gnorml and Gsac, respectively. Circle 1 was drawn with the maximum brightness point within the spinal cord as the center, circle 2 with the same area was plotted on the spinal cord with uniform echogenicity, without compression and at least 1 cm away from the circle 1, and circle 3 was drawn on the dorsal dural sac at the same segment as circle 1. GVR was calculated as follows: GVR-A = Gcompression/Gnorml (method A), and GVR-B = Gcompression/Gsac (method B). The in-observer and inter-observer reliabilities of the two methods were compared. It is generally believed a reliability coefficient < 0.40 and > 0.75 indicate poor and good reliability respectively. The images-based GVR-B using this protocol demonstrates higher inter- and intraobserver reliabilities than GVR-A, and can be used as the basis for prognostic prediction and future studies. RESULTS: All examination acquisitions were successfully completed. GVR-A averaged 2.043 (0.318-5.56), and GVR-B averaged 0.578(0.06-1.41). GVR-B has better repeatability of gray value measurement, smaller relative standard deviation (RSD%) (0.298 vs. 0.32) and larger inter-group correlation coefficient compared with GVR-A. The mean value (MD) of the GVR difference calculated by GVR-B between the two clinicians was closer to 0. CONCLUSIONS: For DCM patients routinely using ultrasound for real-time cord visualization during spinal cord decompression by French-door laminoplasty, the images-based GVR-B using this protocol demonstrates better inter- and intraobserver reliabilities compared with GVR-A.

A new side-effect of sufentanil: increased monocyte-endothelial adhesion.

BACKGROUND: Opioids have been identified by the World Health Organization to be 'indispensable for the relief of pain and suffering'. Side-effects, such as nausea, vomiting, postoperative delirium, and effects on breathing, of opioids have been well investigated; however, the influence of opioids on monocyte-endothelial adherence has never been reported. Therefore, we explored the effects of representative opioids, fentanyl, sufentanil, and remifentanil, on monocyte-endothelial adherence and the underlying mechanisms. METHODS: We built a cell adhesion model with U937 monocytes and human umbilical vein endothelial cells (HUVECs). Two kinds of connexin43 (Cx43) channel inhibitors, 18-α-GA and Gap 27, were used to alter Cx43 channel function in U937 monocytes and HUVECs, respectively, to determine the effects of Cx43 channels on U937-HUVEC adhesion. Subsequently, the effects of fentanyl, sufentanil and remifentanil on Cx43 channel function and U937-HUVEC adhesion were explored. RESULTS: When fentanyl, sufentanil and remifentanil acted on monocytes or endothelial cells, their effects on monocyte-endothelial adherence differed. When acting on U937 monocytes, sufentanil significantly increased U937-HUVEC adhesion which was associated with reduced release of ATP from Cx43 channels, while fentanyl and remifentanil did not have these influences. Although sufentanil could also inhibit Cx43 channel function in HUVECs, it had no effect on ATP release from HUVECs or U937-HUVECs adhesion. CONCLUSIONS: We demonstrated that sufentanil application increases monocyte-endothelial adherence which was associated with reduced release of ATP from Cx43 channels in monocytes. This side-effect of sufentanil should be considered seriously by clinicians.

Transcriptomic landscape of circulating mononuclear phagocytes in Langerhans cell histiocytosis at the single-cell level.

Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasm caused by aberrant activation of the mitogen-activated protein kinase (MAPK) pathway. Circulating myeloid cells from patients often carry disease-associated mutations and can be differentiated into langerinhigh LCH-like cells in vitro, but their detailed immune-phenotypic and molecular profiles are lacking and could shed key insights into disease biology. Here we recruited 217 pediatric LCH patients and took blood and tissue samples for BRAFV600E analysis. Immune-phenotyping of the circulating Lin-HLA-DR+ immune population in 49 of these patients revealed that decreased frequency of plasmacytoid dendritic cells was significantly linked to disease severity. By single-cell RNA sequencing of samples from 14 patients, we identified key changes in expression of RAS-MAPK-extracellular signal-regulated kinase (ERK) signaling-related genes and transcription factors in distinct members of the mononuclear phagocyte system in the presence of BRAFV600E. Moreover, treatment of patients with the BRAF inhibitor dabrafenib resulted in MAPK cascade inhibition, inflammation prevention, and regulation of cellular metabolism within mononuclear phagocytes. Finally, we also observed elevated expression of RAS-MAPK-ERK signaling-related genes in a CD207+CD1a+ cell subcluster in skin. Taken together, our data extend the molecular understanding of LCH biology at single-cell resolution, which might contribute to improvement of clinical diagnostics and therapeutics, and aid in the development of personalized medicine approaches.

Publication Trends and Hot Spots in Femoroacetabular Impingement Research: A 20-Year Bibliometric Analysis.

BACKGROUND: Femoroacetabular impingement (FAI) has attracted increasing attention over the past few decades. We aim to evaluate FAI research and predict research hot spots quantitatively and qualitatively. METHODS: The publications in FAI research between 2000 and 2019 were assimilated from the Web of Science Core Collection of Clarivate Analytics. The retrieved data were evaluated by the bibliometric method. Software CiteSpace 5.7.R1, VOSviewer 1.6.15, and the Online Analysis Platform of Literature Metrology (http://bibliometric.com/) were used to analyze and identify the hot spots and trends in this field. RESULTS: A total of 2471 originals articles that fulfilled the study requirements were obtained. The number of manuscripts on FAI has experienced rapid growth, especially after 2009. The United States of America was the leading country for publication and to the collaboration network. FAI, osteoarthritis, hip arthroscopy, labral reconstruction, pathomorphology, outcome, rehabilitation, and joint cartilage are some of the high-frequency keywords in co-occurrence cluster analysis and cocited reference cluster analysis. Burst detection analysis of top keywords revealed that outcomes, instability, labral reconstruction, adolescent, and risk factor were newly emerged research hot spots. CONCLUSION: The understanding of FAI has been improved significantly during the past two decades. Present studies focused on identifying the optimal method to treat labral pathology, outcome assessment of either surgeries or conservative managements, and predicting midterm and long-term outcomes. Together these studies exert critical implications for decision-making and management for FAI.

Hyperglycemia aggravates monocyte-endothelial adhesion in human umbilical vein endothelial cells from women with gestational diabetes mellitus by inducing Cx43 overexpression.

BACKGROUND: Gestational diabetes mellitus (GDM) is among the most common metabolic diseases during pregnancy and inevitably leads to maternal and fetal complications. Hyperglycemia results in injury to vascular endothelial cells, including monocyte-endothelial adhesion, which is considered to be the initiating factor of vascular endothelial cell injury. Connexin 43 (Cx43) plays a key role in this adhesion process. Therefore, this study aimed to explore the effects of Cx43 on monocyte-endothelial adhesion in GDM-induced injury of vascular endothelial cells. METHODS: Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords from pregnant women with and without GDM. THP-1 cells (a human leukemia monocytic cell line) adhering to HUVECs, related molecules [intracellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1)], and the activity of the phosphoinositide 3-kinase/protein kinase B/Nuclear factor- kappa B (PI3K/AKT/NF-κB) signaling pathway were compared between the normal and GDM-HUVECs. Oleamide and specific small interfering ribonucleic acids (siRNAs) were used to inhibit Cx43 expression in GDM-HUVECs to observe the effects of Cx43 on the adhesion of THP-1 cells and HUVECs. RESULTS: A much higher number of THP-1 cells adhered to GDM-HUVECs than to normal HUVECs. This was accompanied by an increased expression of Cx43, ICAM-1, and VCAM-1, as well as activation of the PI3K/AKT/NF-κB signaling pathway. After the inhibition of Cx43 expression in GDM-HUVECs with oleamide and specific siRNA, THP-1-HUVEC adhesion, ICAM-1 and VCAM-1 expression, and activation of PI3K/AKT/NF-κB signaling pathway were all attenuated. Hyperglycemia was able to increase expression of Cx43 in HUVECs. CONCLUSIONS: For the first time, Cx43 expression was found to be substantially higher in GDM-HUVECs than in normal HUVECs. Hyperglycemia caused the overexpression of Cx43 in HUVECs, which resulted in the activation of the PI3K/AKT/NF-κB signaling pathway and the increase of its downstream adhesion molecules, including ICAM-1 and VCAM-1, ultimately leading to increased monocyte-endothelial adhesion.

Deciphering human macrophage development at single-cell resolution.

Macrophages are the first cells of the nascent immune system to emerge during embryonic development. In mice, embryonic macrophages infiltrate developing organs, where they differentiate symbiotically into tissue-resident macrophages (TRMs)1. However, our understanding of the origins and specialization of macrophages in human embryos is limited. Here we isolated CD45+ haematopoietic cells from human embryos at Carnegie stages 11 to 23 and subjected them to transcriptomic profiling by single-cell RNA sequencing, followed by functional characterization of a population of CD45+CD34+CD44+ yolk sac-derived myeloid-biased progenitors (YSMPs) by single-cell culture. We also mapped macrophage heterogeneity across multiple anatomical sites and identified diverse subsets, including various types of embryonic TRM (in the head, liver, lung and skin). We further traced the specification trajectories of TRMs from either yolk sac-derived primitive macrophages or YSMP-derived embryonic liver monocytes using both transcriptomic and developmental staging information, with a focus on microglia. Finally, we evaluated the molecular similarities between embryonic TRMs and their adult counterparts. Our data represent a comprehensive characterization of the spatiotemporal dynamics of early macrophage development during human embryogenesis, providing a reference for future studies of the development and function of human TRMs.

Methylation-mediated miR-214 regulates proliferation and drug sensitivity of renal cell carcinoma cells through targeting LIVIN.

LIVIN, a member of the inhibitor of apoptosis proteins (IAPs), is reported playing important roles in the development and progression of multiple human cancers. However, its underlined mechanisms in human renal cell carcinoma (RCC) are still needed to be clarified. In the present study, we reported that inhibition of miR-214 promoted the expression of LIVIN, then facilitated RCC cells growth and reduced the sensitivity of RCC cells to chemotherapeutic drugs. In constant, overexpression of miR-214 had contradictory effects. Further investigation showed that miR-214 was down-regulated in RCC because of abnormal methylation. In addition, DNA methyltransferase DNMT1, miR-214 and LIVIN are directly correlated in RCC patients. In conclusion, these results suggest that abnormal miR-214 methylation negatively regulates LIVIN, which may promote RCC cells growth and reduced the sensitivity of RCC cells to chemotherapeutic drugs.

Propofol attenuates monocyte-endothelial adhesion via modulating connexin43 expression in monocytes.

AIMS: Monocyte-endothelial adhesion is considered to be the primary initiator of inflammatory vascular diseases, such as atherosclerosis. Connexin 43 (Cx43) has been reported to play an important part in this process, however, the underlying mechanisms are not fully understood. Intravenous anesthetics, propofol is commonly used in the perioperative period and in the intensive care unit, and considered to have good anti-inflammatory and antioxidant effects. Thus, we speculate that propofol could influence monocyte-endothelial adhesion, and explore whether its possible mechanism is relative with Cx43 expression in U937 monocytes influencing cell adhesion of U937 monocytes to human umbilical vein endothelial cells (HUVEC). MAIN METHODS: Cx43-siRNAs or pc-DNA-Cx43 were used to alter Cx43 expression in U937 monocytes. Propofol was given as pretreatments to U937 monocytes. Then, cell adhesion, ZO-1, LFA-1, VLA-4, COX and MCP-1 were determined. PI3K/AKT/NF-κB signaling pathway was explored to clarify the possible mechanism. KEY FINDINGS: Alternation of Cx43 expression affects cell adhesion and adhesion molecules significantly, such as ZO-1, LFA-1, VLA-4, COX-2 and MCP-1, the mechanism of which is relative with Cx43 influencing the activation of PI3K/AKT/NF-κB signaling pathway. Preconditioning with propofol at its clinically relevant anesthesia concentration attenuates cell adhesion. Propofol not only decreases Cx43 expression in U937 monocytes, but also depresses the activation of PI3K/AKT/NF-κB signaling pathway. SIGNIFICANCE: Modulation Cx43 expression in U937 monocytes could affect cell adhesion via regulating the activation of PI3K/AKT/NF-κB signaling pathway. Propofol attenuates cell adhesion via inhibiting Cx43 and its downstream signaling pathway of PI3K/AKT/NF-κB.

Combined Single-Cell Profiling of lncRNAs and Functional Screening Reveals that H19 Is Pivotal for Embryonic Hematopoietic Stem Cell Development.

The generation of hematopoietic stem cells (HSCs) from embryonic endothelial precursors and pre-HSCs is precisely regulated by signaling pathways and transcription factors. Nevertheless, regulatory roles of non-coding RNAs remain unknown. Taking advantage of our ability to capture rare pre-HSCs and HSCs in vivo, we generated a single-cell landscape of long non-coding RNAs (lncRNAs) during HSC development. Combining bioinformatics and functional screening, we identified 6 lncRNAs influencing hematopoiesis in vitro. We further revealed that H19 lncRNA is pivotal for in vivo HSC emergence in aorta-gonads-mesonephros region. Early H19 lncRNA deficiency blocked endothelial-to-hematopoietic transition, which was independent of the H19-derived miR, miR-675. Moreover, H19-deficient pre-HSCs displayed promoter hypermethylation and concomitant downregulation of several master hematopoietic transcription factors, including Runx1 and Spi1. H19 deficiency increased the activity of S-adenosylhomocysteine hydrolase, a regulator of DNA methylation, which partially contributed to the observed hematopoietic defect. Our findings provide a resource for further analysis of lncRNAs in embryonic HSC development.

Comparison of dexmedetomidine vs. remifentanil combined with sevoflurane during radiofrequency ablation of hepatocellular carcinoma: a randomized controlled trial.

BACKGROUND: Remifentanil is widely used for ultrasound-guided percutaneous radiofrequency ablation (RFA) of small hepatocellular carcinoma (HCC). We determined whether dexmedetomidine could be an alternative to remifentanil for RFA of HCC under general anesthesia with sevoflurane. METHODS: We prospectively randomized patients scheduled to undergo RFA for HCC to a dexmedetomidine (DEX) group or remifentanil (REMI) group (47 patients each). In the DEX group, a bolus infusion (0.4 µg kg- 1) was started 15 min before anesthesia induction and continued at 0.2 µg kg- 1 h- 1 until 10 min before the end of surgery. In the REMI group, 3 µg kg- 1 h- 1 of remifentanil was administered from 15 min before anesthesia induction to the end of the surgery. The primary endpoint was postoperative pain intensity. Secondary endpoints included analgesic requirement, postoperative liver function, patient comfort, and hemodynamic changes. Group allocation was concealed from patients and data analysts but not from anesthesiologists. RESULTS: Postoperative pain intensity, analgesic consumption, comfort, liver function, and time to emergence and extubation did not differ between the two groups. Heart rate, but not mean arterial pressure, was significantly lower in the DEX group than in the REMI group, at 1 min after intubation and from 30 min after the start of the surgery until anesthesia recovery. Sevoflurane concentration and dosage were significantly lower in the DEX group than in the REMI group. CONCLUSION: During RFA for HCC, low-dose dexmedetomidine reduced the heart rate and need for inhalational anesthetics, without exacerbating postoperative discomfort or liver dysfunction. Although it did not exhibit outstanding advantages over remifentanil in terms of pain management, dexmedetomidine could be a safe alternative adjuvant for RFA under sevoflurane anesthesia. TRIAL REGISTRATION: Chinese Clinical Trial Registry, ChiCTR-OPC-15006613 . Registered on 16 June 2015.

Single-cell multiomics sequencing and analyses of human colorectal cancer.

Although genomic instability, epigenetic abnormality, and gene expression dysregulation are hallmarks of colorectal cancer, these features have not been simultaneously analyzed at single-cell resolution. Using optimized single-cell multiomics sequencing together with multiregional sampling of the primary tumor and lymphatic and distant metastases, we developed insights beyond intratumoral heterogeneity. Genome-wide DNA methylation levels were relatively consistent within a single genetic sublineage. The genome-wide DNA demethylation patterns of cancer cells were consistent in all 10 patients whose DNA we sequenced. The cancer cells' DNA demethylation degrees clearly correlated with the densities of the heterochromatin-associated histone modification H3K9me3 of normal tissue and those of repetitive element long interspersed nuclear element 1. Our work demonstrates the feasibility of reconstructing genetic lineages and tracing their epigenomic and transcriptomic dynamics with single-cell multiomics sequencing.

Prevalence and risk factors for hypokalemia in patients scheduled for laparoscopic colorectal resection and its association with post-operative recovery.

BACKGROUND: Perioperative serum potassium levels are closely associated with postoperative clinical outcomes after gastrointestinal surgery. The aim of our retrospective study was to identify the prevalence and risk factors for preoperative hypokalemia (before pneumoperitoneum) and to evaluate the influence of preoperative hypokalemia on the recovery of postoperative gastrointestinal function. METHODS: In this retrospective study, patients scheduled for laparoscopic colorectal resection from November 11 2014 to October 20 2016, were considered for inclusion. A blood potassium level between 3.5 and 5.5 mmol/L was defined as normal, with levels between 3.0 to 3.5 mmol/L, 2.5 to 3.0 mmol/L and < 2.5 mmol/L considered as slight, moderate, and severe level of hypokalemia. The factors including age, gender, ASA grade, BMI, hypertension, diabetes, anti-hypertension drugs, lactose oral soluble, oral cathartics, oral cathartics, cathartic enemas, and blood potassium level before gastrointestinal preparation which might be associated with blood potassium level before pneumoperitoneum were analysed. The time to postoperative first flatus (FFL) and first feces (FFE) was compared between patients with and without hypokalemia. RESULTS: The final analysis was based on the data of 108 patients. Hypokalemia was identified in 70.37% patients, with the following distribution of blood potassium levels before pneumoperitoneum: slight, 49 (45.37%) patients; moderate, 23 (21.30%); and severe, 4 (3.70%) patients. Hypokalemia was significantly associated with hypertension and the use of ≥2 types of oral cathartics for preoperative gastrointestinal preparation. With treatment, potassium levels recovered to normal levels in all patients within 48 h postoperatively. Hypokalemia was associated with a longer postoperative time to first feces, compared to patients with a normal potassium level before pneumoperitoneum. CONCLUSIONS: Our findings underlie the importance of early monitoring and management of serum potassium levels in these patients.