ExtraCorporeal Membrane Oxygenation in the therapy for REfractory Septic shock with Cardiac function Under Estimated (ECMO-RESCUE): study protocol for a prospective, multicentre, non-randomised cohort study.

INTRODUCTION: Severe septic cardiomyopathy (SCM) is one of the main causes of refractory septic shock (RSS), with a high mortality. The application of venoarterial extracorporeal membrane oxygenation (ECMO) to support the impaired cardiac function in patients with septic shock remains controversial. Moreover, no prospective studies have been taken to address whether venoarterial ECMO treatment could improve the outcome of patients with sepsis-induced cardiogenic shock. The objective of this study is to assess whether venoarterial ECMO treatment can improve the 30-day survival rate of patients with sepsis-induced refractory cardiogenic shock. METHODS AND ANALYSIS: ExtraCorporeal Membrane Oxygenation in the therapy for REfractory Septic shock with Cardiac function Under Estimated is a prospective, multicentre, non-randomised, cohort study on the application of ECMO in SCM. At least 64 patients with SCM and RSS will be enrolled in an estimated ratio of 1:1.5. Participants taking venoarterial ECMO during the period of study are referred to as cohort 1, and patients receiving only conventional therapy without ECMO belong to cohort 2. The primary outcome is survival in a 30-day follow-up period. Other end points include survival to intensive care unit (ICU) discharge, hospital survival, 6-month survival, quality of life for long-term survival (EQ-5D score), successful rate of ECMO weaning, long-term survivors' cardiac function, the number of days alive without continuous renal replacement therapy, mechanical ventilation and vasopressor, ICU and hospital length of stay, the rate of complications potentially related to ECMO treatment. ETHICS AND DISSEMINATION: The trial has been approved by the Clinical Research and Application Institutional Review Board of the Second Affiliated Hospital of Guangzhou Medical University (2020-hs-51). Participants will be screened and enrolled from ICU patients with septic shock by clinicians, with no public advertisement for recruitment. Results will be disseminated in research journals and through conference presentations. TRIAL REGISTRATION NUMBER: NCT05184296.

Afterload-related cardiac performance identifies cardiac impairment and associates with outcome in patients with septic shock: a retrospective cohort study.

BACKGROUND: Septic patients with cardiac impairment are with high mortality. Afterload-related cardiac performance (ACP), as a new tool for diagnosing septic cardiomyopathy (SCM), still needs to be evaluated for its impact on the prognosis for patients with septic shock. METHODS: In this retrospective study, 100 patients with septic shock undertaken PiCCO monitoring were included. The ability of ACP, cardiac index (CI), and cardiac power index (CPI) to discriminate between survivors and non-survivors was tested by comparing the area under the receiver operating characteristic curve (AUROC) analysis. Cox proportional hazards regression analyses were performed to assess the associations of ACP with day-28 mortality. Curve estimation was used to describe the relationship between the hazard ratio (HR) of death and ACP. RESULTS: ACP had a strong linear correlation with CI and CPI (P < 0.001). ACP demonstrated significantly greater discrimination for day-28 mortality than CI before adjusted [AUROC 0.723 (95% CI 0.625 to 0.822) vs. 0.580 (95% CI 0.468 to 0.692), P = 0.007] and CPI after adjusted [AUROC 0.693 (95% CI 0.590 to 0.797) vs. 0.448 (0.332 to 0.565), P < 0.001]. Compared with ACP > 68.78%, HR for ACP ≤ 68.78% was 3.55 (1.93 to 6.54) (P < 0.001). When adjusted with age, APACHE-II score, Vasoactive Inotropic Score, Lactate, CRRT, day-1 volume, fibrinogen and total bilirubin as possible confounders, and decrease ACP are still associated with increasing day-28 mortality (P < 0.05). An exponential relationship was observed between ACP12h and HR of day-28 death. CONCLUSIONS: Our results suggested thatACP could improve mortality predictions when compared to CI and CPI. Decreased ACP was still an independent risk factor for increased day-28 mortality.

The timing of continuous renal replacement therapy initiation in sepsis-associated acute kidney injury in the intensive care unit: the CRTSAKI Study (Continuous RRT Timing in Sepsis-associated AKI in ICU): study protocol for a multicentre, randomised controlled trial.

INTRODUCTION: Acute kidney injury (AKI) is one of the most common organ dysfunction in sepsis, and increases the risk of unfavourable outcomes. Renal replacement therapy (RRT) is the predominant treatment for sepsis-associated AKI (SAKI). However, to date, no prospective randomised study has adequately addressed whether initiating RRT earlier will attenuate renal injury and improve the outcome of sepsis. The objective of the trial is to compare the early strategy with delayed strategy on the outcomes in patients with SAKI in the intensive care unit (ICU). METHODS AND ANALYSIS: This is a large-scale, multicentre, randomised controlled trial about SAKI. In total, 460 patients with sepsis and evidence of AKI stage 2 of Kidney Disease Improving Global Outcomes (KDIGO) will be recruited and equally randomised into the early group and the delay group in a ratio of 1:1. In the early group, continuous RRT (CRRT) will be started immediately after randomisation. In the delay group, CRRT will initiated if at least one of the following criteria was met: stage 3 of KDIGO, severe hyperkalaemia, pulmonary oedema, blood urea nitrogen level higher than 112 mg/dL after randomisation. The primary outcome is overall survival in a 90-day follow-up period (90-day all-cause mortality). Other end points include 28-day, 60-day and 1-year mortality, recovery rate of renal function by day 28 and day 90, ICU and hospital length of stay, the numbers of CRRT-free days, mechanical ventilation-free days and vasopressor-free days, the rate of complications potentially related to CRRT, CRRT-related cost, and concentrations of inflammatory mediators in serum. ETHICS AND DISSEMINATION: The trial has been approved by the Clinical Research and Application Institutional Review Board of the Second Affiliated Hospital of Guangzhou Medical University (2017-31-ks-01). Participants will be screened and enrolled from patients in the ICU with SAKI by clinicians, with no public advertisement for recruitment. Results will be disseminated in research journals and through conference presentations. TRIAL REGISTRATION: NCT03175328.

Puerarin-7-O-glucuronide, a water-soluble puerarin metabolite, prevents angiotensin II-induced cardiomyocyte hypertrophy by reducing oxidative stress.

This study aimed to investigate the anti-oxidant and anti-hypertrophic effects of puerarin-7-O-glucuronide, a water-soluble puerarin metabolite. The anti-oxidant effects of puerarin-7-O-glucuronide were assessed by measurement of intracellular superoxide levels, total superoxide dismutase (SOD) activity, total anti-oxidant capacity, and glutathione (GSH)/glutathione disulfide (GSSG) ratio in cultured neonatal rat cardiomyocytes (NRCMs) stimulated with the xanthine oxidase (XO)/xanthine (X) system or angiotensin II. The activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and expression of NADPH oxidase subunits p22phox and p47phox were determined. The anti-hypertrophic effects of puerarin-7-O-glucuronide in angiotensin II-challenged NRCMs were characterized by changes in cell morphology and expression of hypertrophic genes. In the pharmacokinetic study, the plasma concentration of puerarin-7-O-glucuronide was determined by rapid resolution-liquid chromatography-tandem mass spectrometry (RR-LC-MS/MS). Puerarin-7-O-glucuronide prevented XO/X-induced increase in intracellular superoxide production and decreases in total SOD activity, GSH/GSSG ratio, and total anti-oxidant capacity. Puerarin-7-O-glucuronide also reversed angiotensin II-induced increases in intracellular superoxide production and NADPH oxidase activity and decreases in total SOD activity. These anti-oxidant effects of puerarin-7-O-glucuronide were accompanied by downregulation of p22phox and p47phox. Furthermore, puerarin-7-O-glucuronide prevented angiotensin II-induced increases in cell surface area and perimeter, as well as changes in Nppa, Myh7, and Myh6. In the pharmacokinetic study, puerarin-7-O-glucuronide was cleared with a half-life of 0.94 h after intravenous administration. Puerarin could be detected in rat plasma, albeit in low concentration, as early as 5 min after intravenous administration of puerarin-7-O-glucuronide. These anti-oxidant and anti-hypertrophic properties of puerarin-7-O-glucuronide were similar to those of its parent compound puerarin. These results support the development of puerarin-7-O-glucuronide as a novel pharmaceutical agent for therapeutic application.

Dysregulated miR-133a Mediates Loss of Type II Collagen by Directly Targeting Matrix Metalloproteinase 9 (MMP9) in Human Intervertebral Disc Degeneration.

STUDY DESIGN: A microRNA (miRNA) study using Solexa sequencing. OBJECTIVE: The purpose of this study was to identify intervertebral disc degeneration (IDD)-specific miRNA expression profile, and to validate its biological function. SUMMARY OF BACKGROUND DATA: Accumulating evidence indicates that miRNAs play a critical role in IDD, but the role of specific miRNAs involved in this entity remains unclear. METHODS: MiRNA expression profile was determined in nucleus pulposus (NP) tissues from patients with IDD and controls, employing Solexa sequencing and quantitative real-time PCR (qRT-PCR). Biological functions of differential expression miRNAs were further investigated. Luciferase reporter assays and western blotting were performed to determine miRNA targets. RESULTS: We identified 31 miRNAs that were differentially expressed (22 upregulated and nine downregulated) in patients compared with controls. After qRT-PCR confirmation, miR-133a was significantly down-regulated in degenerative NP tissues. Moreover, its level was inversely correlated with grade of disc degeneration. Through gain- and loss-of-function studies, miR-133a was demonstrated to significantly promote type II collagen expression in NP cells. MMP9 was identified as a target of miR-133a. Knockdown of MMP9 induced effects on NP cells similar to those induced by miR-133a. Expression of MMP9 was inversely correlated with miR-133a expression in degenerative NP tissues. CONCLUSION: These results suggest that the downregulation of miR-133a induces type II collagen loss by directly targeting MMP9. Our findings also highlight miR-133a as a novel hopeful therapeutic target for IDD. LEVEL OF EVIDENCE: 3.

MicroRNA-221 Regulates Hypertrophy of Ligamentum Flavum in Lumbar Spinal Stenosis by Targeting TIMP-2.

STUDY DESIGN: A study of lumbar ligamentum flavum (LF). OBJECTIVE: The aim of this study was to identify LF hypertrophy related microRNAs (miRNAs) expression profile and to investigate the role of miRNAs in the development of LF hypertrophy in lumbar spinal stenosis (LSS). SUMMARY OF BACKGROUND DATA: Although histologic and biologic literature on LF hypertrophy is available, the pathomechanism is still unknown. Accumulating evidence suggests that microRNAs (miRNAs) participate in many physiologic processes, including cell proliferation, differentiation, and fibrosis, but the role of specific miRNAs involved in LF hypertrophy remains elusive. METHODS: An initial screening of LF tissues miRNA expression by miRNA microarray was performed using samples from 10 patients and 10 controls, respectively. Subsequently, differential expression was validated using qRT-PCR. Then, functional analysis of the miRNAs in regulating collagens I and III expression was carried out. Western blotting and luciferase reporter assay were also used to detect the target gene. In addition, the thickness of the LF at the level of the facet joint was measured on axial T1-weighted magnetic resonance images. RESULTS: We identified 18 miRNAs that were differentially expressed in patients compared with controls. Following qRT-PCR confirmation, miR-221 was significantly lower in LF tissues of patients than controls. The LF was significantly thicker in patients than that in controls. Bioinformatics target prediction identified tissue inhibitors of matrix metalloproteinase (TIMP)-2 as a putative target of miR-221. Furthermore, luciferase reporter assays demonstrated that miR-221 directly targets TIMP-2 and affects the protein expression of TIMP-2 in fibroblasts isolated from LF. Of note, miR-221 mimic reduced mRNA and protein expression of collagens I and collagen III in fibroblasts isolated from LF. CONCLUSION: The downregulation of miR-221 might contribute to LF hypertrophy by promoting collagens I and III expression via the induction of TIMP-2. Our study also underscores the potential of miR-221 as a novel therapeutic target in LSS. LEVEL OF EVIDENCE: 3.

[Role of microRNA-141 in the regulation of synthesis of high mobility group protein B1 in THP-1 cells].

OBJECTIVE: To investigate the regulatory effect of microRNA-141 (miR-141) on expression of high mobility group protein B1(HMGB1) in human monocytes THP-1 cell line. METHODS: THP-1 cells were transfected with miR-141 mimic or inhibitor (100 nmol/L) for 48 hours with lipofectamine RNAi MAX. The levels of miR-141 and HMGB1 mRNA in the THP-1 cells were detected by real-time fluorescence quantitation reverse transcription-polymerase chain reaction (RT-PCR), and HMGB1 protein was determined with Western blotting. RESULTS: The levels of miR-141 could be up regulated (35.33±7.24 vs. 1.21±0.20, t=-8.408, P=0.010) or down regulated (0.55±0.12 vs 1.09±0.05, t=7.473, P=0.002) after being transfected with 100 nmol/L miR-141 mimic or inhibitor for 48 hours by lipofectamine RNAi MAX in THP-1, and the level of HMGB1 mRNA and protein decreased (mRNA: 0.43±0.06 vs. 0.97±0.08, t=9.760, P=0.001; protein: 0.63±0.12 vs. 1.00±0.11, t=2.991, P=0.040) or increased (mRNA: 2.13±0.11 vs. 1.16±0.13, t=-9.977, P=0.001; protein: 1.78±0.04 vs. 0.96±0.09, t=-13.778, P=0.000) simultaneously compared with the control group. CONCLUSIONS: miR-141 is involved in regulation of inflammation through HMGB1 gene and protein pathway, suggesting that miR-141 plays an important role in regulating immune cells during the inflammatory response.

miR-10a restores human mesenchymal stem cell differentiation by repressing KLF4.

miRNAs have recently been shown to play a significant role in human aging. However, data demonstrating the effects of aging-related miRNAs in human mesenchymal stem cells (hMSCs) are limited. We observed that hMSC differentiation decreased with aging. We also identified that miR-10a expression was significantly decreased with age by comparing the miRNA expression of hMSCs derived from young and aged individuals. Therefore, we hypothesized that the downregulation of miR-10a may be associated with the decreased differentiation capability of hMSCs from aged individuals. Lentiviral constructs were used to up- or downregulate miR-10a in young and old hMSCs. Upregulation of miR-10a resulted in increased differentiation to adipogenic, osteogenic, and chondrogenic lineages and in reduced cell senescence. Conversely, downregulation of miR-10a resulted in decreased cell differentiation and increased cell senescence. A chimeric luciferase reporter system was generated, tagged with the full-length 3'-UTR region of KLF4 harboring the seed-matched sequence with or without four nucleotide mutations. These constructs were cotransfected with the miR-10a mimic into cells. The luciferase activity was significantly repressed by the miR-10a mimic, proving the direct binding of miR-10a to the 3'-UTR of KLF4. Direct suppression of KLF4 in aged hMSCs increased cell differentiation and decreased cell senescence. In conclusion, miR-10a restores the differentiation capability of aged hMSCs through repression of KLF4. Aging-related miRNAs may have broad applications in the restoration of cell dysfunction caused by aging.

MicroRNA-26 was decreased in rat cardiac hypertrophy model and may be a promising therapeutic target.

MicroRNA (miR)-26 was found to be downregulated in cardiac diseases. In this study, the critical role of miR-26 in myocardial hypertrophy in both in vivo and in vitro was investigated. Sixteen male Wistar rats that underwent sham or transverse abdominal aortic constriction (TAAC) surgery were divided into control or TAAC group. Cardiomyocytes were isolated from neonatal Sprague-Dawley rats. Our study demonstrated that miR-26a/b was downregulated in both TAAC rat model and cardiomyocytes. The results of luciferase assays also suggested that glycogen synthase kinase 3ß (GSK3ß) may be a direct target of miR-26. The overexpression of miR-26 attenuated GSK3ß expression and inhibited myocardial hypertrophy. The downregulation of miR-26 reversed these effects. Furthermore, silence of GSK3ß gene phenocopied the anti-hypertrophy effects of miR-26, whereas overexpression of this protein attenuated the effects of miR-26. Taken together, these data suggest that miR-26 regulates pathological structural changes in the rat heart, which may be associated with suppression of the GSK3ß signaling pathway, and implicate the potential application of miR-26 in diagnosis and therapy of cardiac hypertrophy.

[Screening and identification of polypeptides specifically binding to human B type natriuretic peptide from 12TM phage display peptide library].

OBJECTIVE: To identify and characterize the polypeptides specifically binding to human B type natriuretic peptide (BNP) screened from 12TM phage display peptide library. METHODS: The BNP-binding peptides were screened from 12TM phage display peptide library and identified by ELISA. RESULTS: After 4 rounds of screening, 10 of the 16 phage clones were identified as the positive clones which could bind to BNP. Five amino acid sequences were obtained in the 10 positive clones. Dose-dependent ELISA results demonstrated that the screened polypeptides could specifically bind to BNP. CONCLUSION: These screened polypeptides can bind specifically to BNP, which provides a basis for further research on expression and purification of anti-BNP polypeptides and the development of the detection kit of BNP.

[Role of macrophage migration inhibitory factor in mice with sepsis and after effect of its intervention].

OBJECTIVE: To investigate the expression profile of macrophage migration inhibitory factor (MIF) in serum and lung tissues of mice with sepsis, and to explore the effect of MIF antagonist ISO-1 on sepsis in a murine sepsis model. METHODS: Sepsis was reproduced in 40 mice by cecal ligation and puncture (CLP). Heart blood was obtained from 8 mice each at 12, 24, 36, 48 hours after CLP. The content of MIF in serum was determined by enzyme linked immunosorbent assay (ELISA). MIF mRNA and protein expressions in lung tissues of septic mice were assessed by reverse transcription-polymerase chain reaction (RT-PCR) or Western blotting. Another group of 40 mice were selected to investigate the role and the impact of MIF antagonist ISO-1 in septic mice. RESULTS: The content of MIF in serum was higher in septic mice than that in sham operation group, and it peaked at 36 hours, and decreased at 48 hours, but still higher than that in sham operation group (all P<0.01). The MIF mRNA and protein expression in lung tissues of septic mice were higher than those in sham operation group, beginning at 12 hours, and peaked at 48 hours (P<0.05 or P<0.01). ISO-1, which was the antagonist of MIF, could elevate the surviving rate of animals with sepsis [60% (12/20) vs. 25% (5/20), P<0.05]. CONCLUSION: MIF plays a role as a late mediator in sepsis, with a high expression of MIF in serum and lung tissue. ISO-1 can elevate the surviving rate in murine model of sepsis. It is concluded that MIF could be taken as a potential target of treatment of sepsis.

A novel method for apoptosis protein subcellular localization prediction combining encoding based on grouped weight and support vector machine.

Apoptosis proteins have a central role in the development and homeostasis of an organism. These proteins are very important for understanding the mechanism of programmed cell death. Based on the idea of coarse-grained description and grouping in physics, a new feature extraction method with grouped weight for protein sequence is presented, and applied to apoptosis protein subcellular localization prediction associated with support vector machine. For the same training dataset and the same predictive algorithm, the overall prediction accuracy of our method in Jackknife test is 13.2% and 15.3% higher than the accuracy based on the amino acid composition and instability index. Especially for the else class apoptosis proteins, the increment of prediction accuracy is 41.7 and 33.3 percentile, respectively. The experiment results show that the new feature extraction method is efficient to extract the structure information implicated in protein sequence and the method has reached a satisfied performance despite its simplicity. The overall prediction accuracy of EBGW_SVM model on dataset ZD98 reach 92.9% in Jackknife test, which is 8.2-20.4 percentile higher than other existing models. For a new dataset ZW225, the overall prediction accuracy of EBGW_SVM achieves 83.1%. Those implied that EBGW_SVM model is a simple but efficient prediction model for apoptosis protein subcellular location prediction.

[Assessment on heavy metal pollution in the sediment of Poyang Lake].

In order to know the heavy metals content and the degree of the potential ecological risk in the sediment of Poyang Lake in high-water period and low-water period, based on the detailed survey and analysis of the current state of pollution in Poyang Lake, using the Index of Geoaccumulation and the Potential Ecological Risk Index evaluate the heavy metals pollution of Poyang Lake. The results indicate that Poyang Lake has been polluted by heavy metals in various degrees. According to the Index of Geoaccumulation, the order of the analyzed heavy metals, arranged from highest to lowest pollution degree, is as follows: Cu > Pb > Zn > Cd; The ecological risk to Poyang Lake is: Cd>Pb> Zn>Cu;And the pollution is different from space and time, the pollution degree of each sampling site is arranged in the following order: Rao River> Longkou > Nanji shan > Dongku shan > Yumin village, and the degrees in low-water season are bigger than the degrees that in the high-water period commonly.