Identifying Novel Proteins for Chronic Pain: Integration of Human Brain Proteomes and Genome-wide Association Data.

Numerous genome-wide association studies have identified risk genes for chronic pain, yet the mechanisms by which genetic variants modify susceptibility have remained elusive. We sought to identify key genes modulating chronic pain risk by regulating brain protein expression. We integrated brain proteomic data with the largest genome-wide dataset for multisite chronic pain (N = 387,649) in a proteome-wide association study (PWAS) using discovery and confirmatory proteomic datasets (N = 376 and 152) from the dorsolateral prefrontal cortex. Leveraging summary data-based Mendelian randomization and Bayesian colocalization analysis, we pinpointed potential causal genes, while a transcriptome-wide association study integrating 452 human brain transcriptomes investigated whether cis-effects on protein abundance extended to the transcriptome. Single-cell RNA-sequencing data and single-nucleus transcriptomic data revealed cell-type-specific expression patterns for identified causal genes in the dorsolateral prefrontal cortex and dorsal root ganglia (DRG), complemented by RNA microarray analysis of expression profiles in other pain-related brain regions. Of the 22 genes cis-regulating protein abundance identified by the discovery PWAS, 18 (82%) were deemed causal by summary data-based Mendelian randomization or Bayesian colocalization analysis analyses, with 7 of these 18 genes (39%) replicating in the confirmatory PWAS, including guanosine diphosphate-mannose pyrophosphorylase B, which also associated at the transcriptome level. Several causal genes exhibited selective expression in excitatory and inhibitory neurons, oligodendrocytes, and astrocytes, while most identified genes were expressed across additional pain-related brain regions. This integrative proteogenomic approach identified 18 high-confidence causal genes for chronic pain, regulated by cis-effects on brain protein levels, suggesting promising avenues for treatment research and indicating a contributory role for the DRG. PERSPECTIVE: The current post genome-wide association study analyses identified 18 high-confidence causal genes regulating chronic pain risk via cis-modulation of brain protein abundance, suggesting promising avenues for future chronic pain therapies. Additionally, the significant expression of these genes in the DRG indicated a potential contributory role, warranting further investigation.

Sevoflurane suppresses colorectal cancer malignancy by modulating ß-catenin ubiquitination degradation via circSKA3.

BACKGROUND: Sevoflurane (SEV), a commonly used inhalational anesthetic, reportedly inhibits colorectal cancer (CRC) malignancy, but whether SEV can inhibit the malignancy of CRC by regulating circular RNAs (circRNAs) remains unclear. Therefore, we aimed to identify specific circRNAs that may be affected by SEV and to investigate their functional roles in CRC. METHODS: RT-qPCR was employed to detect the expression of circRNAs and mRNAs in CRC cells and tissues. Fluorescence in situ hybridization (FISH) was used to determine the location of circSKA3. Protein expression was assessed by western blot analysis. Function-based in vitro and in vivo experiments, including CCK-8, colony formation, transwell, and apoptosis assays and mouse xenograft tumor models, were conducted using circSKA3-knockdown and circSKA3-overexpression cell lines. RNA immunoprecipitation, RNA pull-down and mass spectrometry analyses were performed to explore the related mechanism. RESULTS: Our findings revealed that SEV could inhibit CRC cell activity, proliferation and migration and promote apoptosis in CRC cells. We found that circSKA3 was upregulated in CRC and associated with poorer survival and that its expression could be reduced by SEV. The overexpression of circSKA3 reversed the effects of SEV on inhibiting cell activity, proliferation and migration and promoting apoptosis. The mechanistic analysis revealed that circSKA3 could bind to the ARM structural domain of ß-catenin and thereby disrupt its interaction with the CK1/GSK3ß/ß-TrCP1 destruction complex, resulting in the ubiquitinated degradation of ß-catenin and the activation of Wnt/ß-catenin signaling. In addition, SEV downregulated circSKA3 in vivo to inhibit tumor growth. CONCLUSIONS: All the results showed that SEV could inhibit CRC progression via circSKA3 by increasing ß-catenin ubiquitination degradation.

THE EFFECTS OF EARLY-PHASE FUROSEMIDE USE ON THE PROGRESSION OF OLIGURIC ACUTE KIDNEY INJURY ACROSS DIFFERENT CENTRAL VENOUS PRESSURE: A RETROSPECTIVE ANALYSIS.

ABSTRACT: Background: Furosemide is a commonly used loop diuretic in critical care. However, its effect on the progression of oliguric acute kidney injury across different central venous pressure (CVP) remains unknown. This study therefore aims to investigate the association between furosemide 6-12h (defined as the use of furosemide within 6 h after the diagnosis of AKI according to the urine output [UO] criteria set by the Kidney Disease: Improving Global Outcomes [KDIGO] guidelines) and the progression of AKI across different CVP 6-12h (defined as CVP within 6 h after the diagnosis of AKI by the KDIGO UO criteria) levels. Methods: Patients involved in this study were identified from the Medical Information Mart for Intensive Care IV database with the following criteria: (i) adults with UO <0.5 mL/kg per hour for the first 6 h upon admission to the intensive care unit (ICU) (meeting stage 1 AKI by UO) and (ii) CVP 6-12h ranging from 0 to 30 mm Hg. From there on, the target primary outcome would be progression to stage 3 AKI by UO among these chosen patients. The secondary outcome was 28-d mortality since ICU admission. The risks of severe-stage AKI progression and 28-d mortality were respectively examined against furosemide 6-12h (vs. without furosemide 6-12h ) within the full cohort and across different subgroups of CVP 6-12h , using multivariate adjusted logistic regression and inverse probability treatment weighting (IPTW). Sensitivity analyses were performed to assess the robustness of our findings. Results: One thousand one hundred eighty patients were ultimately selected for this study, of whom 643 (54.5%) progressed to stage 3 AKI from stage 1 based on the UO criteria by KDIGO. Multivariate analysis showed that furosemide 6-12h is significantly associated with this severe-stage progression within the full cohort (odds ratio [OR] was 0.62 at 95% confidence interval [CI] of 0.43-0.90, P = 0.011). After dividing the patients into CVP 6-12h subgroups according to their CVP during the early phases, lower risk of AKI progression was observed only in furosemide 6-12h application at CVP 6-12h of ≥12 mm Hg (adjusted OR was 0.40 at 95% CI of 0.25-0.65, P < 0.001), as confirmed by the IPTW analysis (OR was 0.47 at 95% CI of 0.29-0.76, P = 0.002). The robustness of these findings was confirmed by sensitivity analyses. In addition, for patients with CVP 6-12h ≥12 mm Hg, furosemide 6-12h is also significantly associated with lower risk of 28-d mortality (adjusted OR was 0.47 at 95% CI of 0.25-0.92, P = 0.026) in the multivariate logistic regression analysis, and there was a similar trend in the IPTW analysis (adjusted OR was 0.55 at 95% CI of 0.28-1.10, P = 0.092). Conclusions: Among the identified early-stage AKI patients in critical care, the use of furosemide was associated only with lower risk of oliguric AKI progression and 28-d mortality within the high CVP group. These findings suggest the potential of CVP as a guidance or reference point in the usage of furosemide among early-stage oliguric AKI patients in the ICU.

Lymphatic metastasis-associated circRNA‒miRNA‒mRNA network for exploring the pathogenesis and therapeutic target of triple negative breast cancer based on whole-transcriptome sequencing analysis: an experimental verification study.

BACKGROUND: The metastatic mechanisms of axillary lymph nodes (ALNs) in triple-negative breast cancer (TNBC) remain unclear. We aimed to identify the potential circRNA regulatory network in ALN metastasis. METHODS: We performed whole transcriptome sequencing (WTS) to determine the expression profiles of RNAs and screen out differentially expressed messenger RNAs (DEMs), microRNAs (DEMis), and circRNAs (DECs) between ALN-positive and ALN-negative TNBC patients. Functional enrichment analysis and Kaplan-Meier survival analysis were utilized to unearth the potential regulatory mechanisms of the DEMs. A competing endogenous RNA (ceRNA) network was constructed using computational biology. The expression levels of DECs in cell lines were confirmed by real-time polymerase chain reaction (RT‒PCR). RESULTS: Following WTS and differential expression analysis, 739 DEMs, 110 DEMis, and 206 DECs were identified between ALN-positive and ALN-negative TNBC patients. Functional analysis indicated that the DEMs mainly functioned in carcinogenesis and tumor progression-related pathways. ceRNA networks containing eight circRNAs, six miRNAs, and eighteen mRNAs were developed. In the ceRNA network, two mRNAs (RAB3D and EDARADD) that were significantly associated with better overall survival and one mRNA (GSR) that predicted favorable recurrence-free survival in TNBC patients were chosen for further analysis. Then, a survival-related ceRNA network containing two DECs (hsa_circ_0061260 and hsa_circ_0060876), two DEMis (hsa-miR-5000-3p and hsa-miR-4792), and three mRNAs (GSR, RAB3D, and EDARADD) was identified. Then, two candidate DECs were validated by real-time PCR. CONCLUSION: Our research constructed a ceRNA network that provides novel insights into the molecular mechanism of ALN metastasis and potential therapeutic targets in TNBC.

Infusion management associated with prolonged length of stay following free flap reconstruction of head and neck defects: A propensity score matching study.

BACKGROUND: Surgical resection of the primary lesion and reconstruction of the defects with free flaps are common treatments for head and neck cancer (HNC). However, various variables can lead to prolonged length of stay (LOS). The aim of this study is to investigate risk factors correlated with prolonged LOS following free flap reconstruction of head and neck defects. METHODS: A retrospective study of patients with all types of free flaps reconstruction of HNC between January 2011 and January 2019 at Sun Yat-sen Memorial Hospital was performed. We recorded predictive variables and divided them into: personal and clinical, hemodynamic, anesthetic and surgical. The primary endpoint was prolonged length of stay. Univariate and multivariate analyses were applied to identify risk factors that associated with prolonged LOS. Propensity score matching was performed with the identified risk variables and other perioperative factors that may impact transfusion decision to explore the independent influence of intraoperative blood transfusion on prolonged LOS. RESULTS: A total of 1047 patients were included in this study. The median LOS was 13.00 (11.00, 16.00) days. Multivariate analysis suggested that blood transfusion, duration of surgery, postoperative complications and unplanned reoperation were associated with prolonged LOS. After propensity score matching, unnecessary blood transfusion and inadequate fluid rate over 24 h, postoperative complications and unplanned reoperation were identified risk factors that led to prolonged LOS. CONCLUSION: Unnecessary blood transfusion and inadequate fluid infusion rate over 24 h were independent risk factors associated with prolonged LOS in HNC patients who underwent free flap reconstruction. Our results indicated consideration of restrictive blood transfusion and adequate fluid infusion over postoperative 24 h in these patients.

Dexmedetomidine Ameliorates Postoperative Cognitive Dysfunction in Aged Mice.

Neuroinflammation and oxidative stress coexist and interact in the progression of postoperative cognitive dysfunction (POCD) and other neurodegenerative disease. Mounting studies reveal that Dexmedetomidine (Dex) possesses anti-inflammatory and antioxidant properties. Nevertheless, whether Dex exerts neuroprotective effect on the cognitive sequelae of oxidative stress and inflammatory process remains unclear. A mouse model of abdominal exploratory laparotomy-induced cognitive dysfunction was employed to explore the underlying mechanism of neuroprotective effects exerted by Dex in POCD. Aged mice were treated with Dex (20 µg/kg) 20 min prior to surgery. Open field test (OFT) and Morris water maze (MWM) were employed to examine the cognitive function on postoperative day 3 (POD 3) or POD 7. In the present study, mice underwent surgery exhibited cognitive impairment without altering spontaneous locomotor activity, while the surgery-induced cognitive impairment could be alleviated by Dex pretreatment. Dex inhibited surgery-induced pro-inflammatory cytokines accumulation and microglial activation in the hippocampi of mice. Furthermore, Dex decreased MDA levels, enhanced SOD activity, modulated CDK5 activity and increased BDNF expression in the hippocampus. In addition, Dex remarkably reduced the surgery-induced increased ratio of Bax/Bcl-2 and apoptotic neurons in the hippocampi of aged mice. Collectively, our study provides evidence that Dex may exert neuroprotective effects against surgery-induced cognitive impairment through mechanisms involving its anti-inflammatory and antioxidant properties, as well as the suppression on the mitochondrial permeability transition pore and apoptosis-related pathway.

Galectin-1 ameliorates perioperative neurocognitive disorders in aged mice.

INTRODUCTION: The incidence of perioperative neurocognitive disorders (PND) is higher in the elderly patients undergoing surgery. Microglia activation-mediated neuroinflammation is one of the hallmarks of PND. Galectin-1 has been identified as a pivotal modulator in the central nervous system (CNS), while the role of galectin-1 in PND induced by microglia-mediated neuroinflammation is still undetermined. METHODS: An exploratory laparotomy model anesthetized with isoflurane was employed to investigate the role of galectin-1 on PND in aged mice. Open field test and Morris water maze were used to test the cognitive function 3- or 7-days post-surgery. The activation of microglia in the hippocampus of aged mice was tested by immunohistochemistry. Western blot, enzyme-linked immunosorbent assay (ELISA), and quantitative real-time polymerase chain reaction (qRT-PCR) were employed to elucidate the underlying mechanisms. RESULTS: Galectin-1 attenuated the cognitive dysfunction induced by surgery in aged mice and inhibited microglial activity. Moreover, galectin-1 decreased the expression level of inflammatory proteins (interleukin-1ß, interleukin-6, and tumor necrosis factor-α), and prevented neuronal loss in the hippocampus. Galectin-1 inhibited the inflammation of BV2 microglial cells induced by lipopolysaccharide via decreasing the translocation of NF-κB p65 and c-Jun, while this kind of inhibition was rescued when overexpressing IRAK1. CONCLUSION: Our findings provide evidence that galectin-1 may inhibit IRAK1 expression, thus suppressing inflammatory response, inhibiting neuroinflammation, and improving ensuing cognitive dysfunction. Collectively, these findings unveil that galectin-1 may elicit protective effects on surgery-induced neuroinflammation and neurocognitive disorders.

Intravenous versus Volatile Anesthetic Effects on Postoperative Cognition in Elderly Patients Undergoing Laparoscopic Abdominal Surgery.

BACKGROUND: Delayed neurocognitive recovery after surgery is associated with poor outcome. Most surgeries require general anesthesia, of which sevoflurane and propofol are the most commonly used inhalational and intravenous anesthetics. The authors tested the primary hypothesis that patients with laparoscopic abdominal surgery under propofol-based anesthesia have a lower incidence of delayed neurocognitive recovery than patients under sevoflurane-based anesthesia. A second hypothesis is that there were blood biomarkers for predicting delayed neurocognitive recovery to occur. METHODS: A randomized, double-blind, parallel, controlled study was performed at four hospitals in China. Elderly patients (60 yr and older) undergoing laparoscopic abdominal surgery that was likely longer than 2 h were randomized to a propofol- or sevoflurane-based regimen to maintain general anesthesia. A minimum of 221 patients was planned for each group to detect a one-third decrease in delayed neurocognitive recovery incidence in propofol group compared with sevoflurane group. The primary outcome was delayed neurocognitive recovery incidence 5 to 7 days after surgery. RESULTS: A total of 544 patients were enrolled, with 272 patients in each group. Of these patients, 226 in the propofol group and 221 in the sevoflurane group completed the needed neuropsychological tests for diagnosing delayed neurocognitive recovery, and 46 (20.8%) in the sevoflurane group and 38 (16.8%) in the propofol group met the criteria for delayed neurocognitive recovery (odds ratio, 0.77; 95% CI, 0.48 to 1.24; P = 0.279). A high blood interleukin-6 concentration at 1 h after skin incision was associated with an increased likelihood of delayed neurocognitive recovery (odds ratio, 1.04; 95% CI, 1.01 to 1.07; P = 0.007). Adverse event incidences were similar in both groups. CONCLUSIONS: Anesthetic choice between propofol and sevoflurane did not appear to affect the incidence of delayed neurocognitive recovery 5 to 7 days after laparoscopic abdominal surgery. A high blood interleukin-6 concentration after surgical incision may be an independent risk factor for delayed neurocognitive recovery.

HIF-1α Promotes the Metastasis of Esophageal Squamous Cell Carcinoma by Targeting SP1.

Background: In microenvironment of malignant tumors, Hypoxia-Inducible Factors (HIF), most importantly HIF-1α, play an important role in regulation of adaptive biological response to hypoxia, promoting angiogenesis and metastasis. However, the underlying mechanism that HIF-1α regulates metastasis needs to be further clarified. Methods: The expressions of HIF-1α and SP1 were detected in 182 samples of esophageal squamous cell carcinoma (ESCC) and adjacent normal tissues by immunohistochemistry (IHC), and the correlation between the expression levels of HIF-1α and SP1 was analyzed. The expression of HIF-1α in ESCC cell lines TE1 and KYSE30 was then detected using qRT-PCR and western blot. The potential binding sites of HIF-1α on the SP1 promoter were analyzed using UCSC and JASPAR databases, verified by chromosomal immunoprecipitation (ChIP) assay and qRT-PCR. The effects of HIF-1α and SP1 on ESCC cell migration and invasion were then tested with Transwell and Matrigel experiments. Results: The expression of HIF-1α in cancer tissues is higher than adjacent normal tissues, and is correlated with metastasis, recurrence and poor prognosis. Upon silencing HIF-1α by siRNA, the invasion and migration ability of ESCC cells were significantly inhibited, which could be restored by the overexpression of SP1. Hypoxic conditions significantly increased the expression of HIF-1α and SP1 at both protein and mRNA levels in ESCC cells. HIF-1α enhanced SP1 transcription through binding to the promoter region. The expression of protein and mRNA levels of SP1 was decreased by silencing HIF-1α in cells. In contrast, overexpression of HIF-1α significantly increased the mRNA and protein levels of SP1. The expression of SP1 in ESCC was positively correlated with the protein expression of HIF-1α and poor prognosis. Conclusion: The results of our study indicate that HIF-1α promotes metastasis of ESCC by targeting SP1 in a hypoxic microenvironment. Further study on this mechanism may elucidate the possibility of HIF-1α and SP1 as new targets for the treatment of ESCC.

Development and validation of a nomogram incorporating selected systemic inflammation-based prognostic marker for complication prediction after vascularized fibula flap reconstruction.

OBJECTIVE: To develop and validate a nomogram incorporating systemic inflammatory markers (the Albumin/NLR Score [ANS]) to predict postoperative complications after vascularized fibula flap reconstruction. PATIENTS AND METHODS: A total of 238 patients who underwent vascularized fibula flap reconstruction between March 2012 and December 2016 were collected as the primary cohort. Univariable and multivariable analysis were performed to identify independent risk factors for postoperative complications. Backward stepwise logistic regression analysis was then applied with and without the ANS; and nomograms were established based on these criteria. Independent validation of these nomograms was carried out in an independent validation cohort including 106 consecutive patients from December 2016 and January 2018. RESULTS: Radiotherapy history (odds ratio [OR] = 0.336; 95% CI, 0.157-0.717; P = 0.005), the ANS (OR = 0.248; 95% CI, 0.093-0.661; P = 0.005) and fluid infusion rate over 24 h (OR = 0.671; 95% CI, 0.479-0.94; P = 0.02) were identified as independent risk factors for postoperative complications. A higher C-index was found in both the primary (0.759; 95% CI, 0.719-0.739) and validation cohort (0.704; 95% CI, 0.613-0.659) for the nomogram incorporating the ANS, and NRI was 0.496 (95% CI, 0.072-0.920; P = 0.022) comparing of these nomograms. Furthermore, a wider threshold probability (0.2-0.9) and superior clinical value were observed in the nomogram incorporating the ANS on the decision curve. CONCLUSION: The ANS was an independent risk factor for postoperative complications associated with vascularized fibula flap reconstruction. The nomogram incorporating the ANS was established with better accuracy and showed more potential clinical benefit for the estimation of postoperative complications.

CCL18-induced HOTAIR upregulation promotes malignant progression in esophageal squamous cell carcinoma through the miR-130a-5p-ZEB1 axis.

Accumulating evidence indicates that CCL18 and the long non-coding RNA, HOTAIR, have critical roles in cancer progression and metastasis, but the correlation between CCL18 and HOTAIR in esophageal squamous cell carcinoma (ESCC) and their downstream molecular mechanisms remain unclear. Overexpression of CCL18 in ESCC tissues was associated with a worse survival in patients with ESCC. CCL18 enhanced the invasiveness of ESCC cells in a dose-dependent manner, whereas CCL18 knockdown inhibited their invasiveness. In particular, CCL18 expression was positively associated with HOTAIR expression in ESCC tissues. Furthermore, CCL18 upregulated the expression of HOTAIR, and knockdown of HOTAIR alleviated the CCL18-induced invasiveness of ESCC cells. HOTAIR may act as a competing endogenous RNA and could effectively becoming a sponge for miR-130a-5p, thereby modulating the derepression of ZEB1 and promoting epithelial-mesenchymal transition in ESCC. Our study suggests that CCL18 contributes to the malignant progression of esophageal cancer by upregulating HOTAIR expression. HOTAIR overexpression may promote tumor invasiveness and progression in ESCC, given that HOTAIR functions as a miR-130a-5p sponge, positively regulating ZEB1. This provides new therapeutic targets for early diagnosis and treatment of ESCC.

LncRNA THOR promotes tongue squamous cell carcinomas by stabilizing IGF2BP1 downstream targets.

THOR, a highly conserved lncRNA, is potentially involved in various cancer development. However, its involvement in tongue squamous cell carcinoma (TSCC) remains unclear. The present study aims to explore the biological function and molecular mechanism of THOR in TSCC progression. The expressions of THOR and IGF2BP1 in TSCC tissues and adjacent non-cancerous tongue tissues (ANT) were examined through qRT-PCR. THOR levels were manipulated in TSCC cells to explore its function in cancer progression in vitro and in vivo, which were subsequently evaluated by CCK8, colony formation assay, flow cytometry, xenograft tumor assays. In situ hybridization, RIP and Western blot assay were performed to explore the underlying molecular mechanisms. We discovered that THOR and IGF2BP1 were dramatically upregulated in TSCC tissues. The expression of THOR is positively correlated with IGF2BP1 mRNA level. THOR mediated IGF2 expression via interacting with IGF2BP1, and affected the downstream MEK-ERK signaling pathway to regulate TSCC cells proliferation. THOR/IGF2BP1/IGF2-MEK-ERK axis regulated the proliferation of TSCC cells, implying that THOR would be a promising therapeutic target for TSCC patients.

Ligustrazine Enhances the Hypnotic and Analgesic Effect of Ketamine in Mice.

The purpose of this study was to determine the effects of different concentrations of ligustrazine, an extract from Chinese herb, on ketamine requirement for hypnosis and analgesia in mice. In the hypnotic response study, mice were randomly allocated to receive saline or ligustrazine at 10, 20, 40, 80 or 160 mg·kg-1 by intraperitoneal injection. Ketamine was administrated 15 min after ligustrazine injection. The hypnotic response was determined by assessing loss of the righting reflex (LORR) after ketamine injection. The dose of ketamine was determined by modified Dixon's up-and-down method in each group. In the analgesia study, different doses of ligustrazine were administrated 15 min before 50 mg·kg-1 ketamine injection. The analgesia effects (pain threshold) were determined by heat radiation-induced tail-flick latency and evaluated before ligustrazine administration or 5, 15, 30 and 60 min after ketamine administration. The ED50 [95% confidence interval (CI)] for hypnosis induced by ketamine was 54.1 (44.8, 65.3) mg·kg-1. Ligustrazine dose-dependently decreased the ED50 for ketamine to induce hypnosis, which was [31.6 (26.2, 38.1)] mg·kg-1 with the addition of 80 mg·kg-1 ligustrazine and [27.7 (22.6, 33.7)] mg·kg-1 with the addition of 160 mg·kg-1 ligustrazine, respectively (p<0.05). Ligustrazine at 160 mg·kg-1 also increased pain threshold in the presence of ketamine. Ligustrazine enhanced the hypnotic effect of ketamine in a dose-dependent manner. Ligustrazine at a large dose also increased the analgesic effect of ketamine.