Generating the extended endoplasmic reticulum (ER) network depends on microtubules, which act as tracks for motor-driven ER tubule movement, generate the force to extend ER tubules by means of ...attachment to growing microtubule plus-ends and provide static attachment points. We have analysed ER dynamics in living VERO cells and find that most ER tubule extension is driven by microtubule motors. Surprisingly, we observe that ~50% of rapid ER tubule movements occur in the direction of the centre of the cell, driven by cytoplasmic dynein. Inhibition of this movement leads to an accumulation of lamellar ER in the cell periphery. By expressing dominant-negative kinesin-1 constructs, we show that kinesin-1 drives ER tubule extension towards the cell periphery and that this motility is dependent on the KLC1B kinesin light chain splice form but not on KLC1D. Inhibition of kinesin-1 promotes a shift from tubular to lamellar morphology and slows down the recovery of the ER network after microtubule depolymerisation and regrowth. These observations reconcile previous conflicting studies of kinesin-1 function in ER motility in vivo. Furthermore, our data reveal that cytoplasmic dynein plays a role in ER motility in a mammalian cultured cell, demonstrating that ER motility is more complex than previously thought.
Kinesin‐1 drives the movement of diverse cargoes, and it has been proposed that specific kinesin light chain (KLC) isoforms target kinesin‐1 to these different structures. Here, we test this ...hypothesis using two in vitro motility assays, which reconstitute the movement of rough endoplasmic reticulum (RER) and vesicles present in a Golgi membrane fraction. We generated GST‐tagged fusion proteins of KLC1B and KLC1D that included the tetratricopeptide repeat domain and the variable C‐terminus. We find that preincubation of RER with KLC1B inhibits RER motility, whereas KLC1D does not. In contrast, Golgi fraction vesicle movement is inhibited by KLC1D but not KLC1B reagents. Both RER and vesicle movement is inhibited by preincubation with the GST‐tagged C‐terminal domain of ubiquitous kinesin heavy chain (uKHC), which binds to the N‐terminal domain of uKHC and alters its interaction with microtubules. We propose that although the TRR domains are required for cargo binding, it is the variable C‐terminal region of KLCs that are vital for targeting kinesin‐1 to different cellular structures.
Abstract
We hypothesised that measuring changes in urinary levels of EV and miR will predict the onset of acute kidney injury in cardiac surgery patients. The study was performed in the cohort of the ...REVAKI-2 trial. Urine samples were collected before and 24 h after the procedure from 94 cardiac surgery patients. Urinary particle concentrations and size distribution were assessed using NanoSight. EV derivation and levels were measured using flow cytometry. Samples from 10 selected patients were sequenced, and verification was performed with advanced TaqMan assays in samples from all patients. Urinary particle concentrations significantly increased in patients with AKI after surgery, with the percentage of EV positive for CD105 and β1-integrin also increasing. Pre-surgery podocalyxin-positive EV were significantly lower in patients with AKI. Their levels correlated with the severity of the injury. Pre-operative miR-125a-5p was expressed at lower levels in urine from patients with AKI when adjusted for urinary creatinine. Levels of miR-10a-5p were lower after surgery in AKI patients and its levels correlated with the severity of the injury. Pre-operative levels of podocalyxin EVs, urinary particle concentrations and miR-125a-5p had moderate AKI predictive value and, in a logistic model together with ICU lactate levels, offered good (AUC = 82%) AKI prediction.
We hypothesized that body mass index (BMI) dependent changes in myocardial gene expression and energy-related metabolites underlie the biphasic association between BMI and mortality (the obesity ...paradox) in cardiac surgery. We performed transcriptome profiling and measured a panel of 144 metabolites in 53 and 55, respectively, myocardial biopsies from a cohort of sixty-six adult patients undergoing coronary artery bypass grafting (registration: NCT02908009). The initial analysis identified 239 transcripts with biphasic BMI dependence. 120 displayed u-shape and 119 n-shape expression patterns. The identified local minima or maxima peaked at BMI 28-29. Based on these results and to best fit the WHO classification, we grouped the patients into three groups: BMI < 25, 25 ≤ BMI ≤ 32, and BMI > 32. The analysis indicated that protein translation-related pathways were downregulated in 25 ≤ BMI ≤ 32 compared with BMI < 25 patients. Muscle contraction transcripts were upregulated in 25 ≤ BMI ≤ 32 patients, and cholesterol synthesis and innate immunity transcripts were upregulated in the BMI > 32 group. Transcripts involved in translation, muscle contraction and lipid metabolism also formed distinct correlation networks with biphasic dependence on BMI. Metabolite analysis identified acylcarnitines and ribose-5-phosphate increasing in the BMI > 32 group and α-ketoglutarate increasing in the BMI < 25 group. Molecular differences in the myocardium mirror the biphasic relationship between BMI and mortality.
BACKGROUND:In an apparent paradox, morbidity and mortality are lower in obese patients undergoing cardiac surgery, although the nature of this association is unclear. We sought to determine whether ...the obesity paradox observed in cardiac surgery is attributable to reverse epidemiology, bias, or confounding.
METHODS:Data from the National Adult Cardiac Surgery registry for all cardiac surgical procedures performed between April 2002 and March 2013 were extracted. A parallel systematic review and meta-analysis (MEDLINE, Embase, SCOPUS, Cochrane Library) through June 2015 were also accomplished. Exposure of interest was body mass index categorized into 6 groups according to the World Health Organization classification.
RESULTS:A total of 401 227 adult patients in the cohort study and 557 720 patients in the systematic review were included. A U-shaped association between mortality and body mass index classes was observed in both studies, with lower mortality in overweight (adjusted odds ratio, 0.79; 95% confidence interval, 0.76–0.83) and obese class I and II (odds ratio, 0.81; 95% confidence interval, 0.76–0.86; and odds ratio, 0.83; 95% confidence interval, 0.74–0.94) patients relative to normal-weight patients and increased mortality in underweight individuals (odds ratio, 1.51; 95% confidence interval, 1.41–1.62). In the cohort study, a U-shaped relationship was observed for stroke and low cardiac output syndrome but not for renal replacement therapy or deep sternal wound infection. Counter to the reverse epidemiology hypotheses, the protective effects of obesity were less in patients with severe chronic renal, lung, or cardiac disease and greater in older patients and in those with complications of obesity, including the metabolic syndrome and atherosclerosis. Adjustments for important confounders did not alter our results.
CONCLUSIONS:Obesity is associated with lower risks after cardiac surgery, with consistent effects noted in multiple analyses attempting to address residual confounding and reverse causation.
Aortic stenosis (AS) is the commonest valve lesion requiring surgery in the Western world. The presence of myocardial fibrosis is associated with mortality even after valve replacement. MicroRNAs ...could serve as biomarkers of fibrosis and risk stratify patients for earlier intervention. This study aimed to systematically review reports of micro‐RNA (miR) associated with fibrosis in AS and identify potential biomarkers. We searched EMBASE, Medline, and Web of Science up to May 2020. Studies that reported on the role of miRs in AS and cardiac fibrosis were included. Study quality was assessed using the Newcastle‐Ottawa scale. Of 4230 reports screened, 25 were included. All studies were of low to moderate quality. MiRs were analyzed in myocardial tissue (n = 10), aortic valve tissue (n = 5), plasma (n = 5), and serum (n = 5). A total of 365 miRs were reported, of which only a few were reported in more than one paper (3 in the myocardium, 5 in the aortic valve, and 1 in plasma). miR‐21 was upregulated in plasma and myocardial tissue. MiR‐19b was downregulated in the myocardium. Papers reporting myocardial miR‐1 contradicted each other, and miR‐133a was associated with increased left ventricular mass regression post‐surgery. In the aortic valve, miRs‐665, 602 and 939 were downregulated, and miRs‐193b and 214 were upregulated. The data on miR in fibrosis in AS is scarce and of low to moderate quality. Further studies are needed to identify novel miRs as biomarkers, especially at an earlier asymptomatic phase of the disease.
BACKGROUND:We evaluated the effects of two interventions that modify the red cell storage lesion on kidney and lung injury in experimental models of transfusion.
METHODS:White–landrace pigs (n = 32) ...were allocated to receive sham transfusion (crystalloid), 14-day stored allogeneic red cells, 14-day red cells washed using the red cells washing/salvage system (CATS; Fresenius, Germany), or 14-day red cells rejuvenated using the inosine solution (Rejuvesol solution; Zimmer Biomet, USA) and washed using the CATS device. Functional, biochemical, and histologic markers of organ injury were assessed for up to 24 h posttransfusion.
RESULTS:Transfusion of 14 day red cells resulted in lung injury (lung injury score vs. sham, mean difference −0.3 (95% CI, −0.6 to −0.1; P = 0.02), pulmonary endothelial dysfunction, and tissue leukocyte sequestration. Mechanical washing reduced red cell–derived microvesicles but increased cell-free hemoglobin in 14-day red cell units. Transfusion of washed red cells reduced leukocyte sequestration but did not reduce the lung injury score (mean difference −0.2; 95% CI, −0.5 to 0.1; P = 0.19) relative to 14-day cells. Transfusion of washed red cells also increased endothelial activation and kidney injury. Rejuvenation restored adenosine triphosphate to that of fresh red cells and reduced microvesicle concentrations without increasing cell-free hemoglobin release. Transfusion of rejuvenated red cells reduced plasma cell-free hemoglobin, leukocyte sequestration, and endothelial dysfunction in recipients and reduced lung and kidney injury relative to 14-day or washed 14-day cells.
CONCLUSIONS:Reversal of the red cell storage lesion by rejuvenation reduces transfusion-associated organ injury in swine.
BACKGROUND: HNA‐3 is a diallellic system located on choline transporter‐like protein 2 (CTL2), defined by a polymorphism at Amino Acid 154. HNA‐3a antibodies are of clinical importance in ...transfusion‐related acute lung injury but antibody detection requires labor‐intensive granulocyte isolation from HNA‐typed donors and the use of techniques such as the granulocyte agglutination test or granulocyte immunofluorescence test. Also, there is no commercial test for detection of HNA‐3 antibodies.
STUDY DESIGN AND METHODS: HEK293 cells were transfected to generate stable cell lines expressing CTL2 fragments (Amino Acids 55‐230) and full‐length membrane bound CTL2 with HNA‐3a and ‐3b epitopes. Soluble fragments were used in enzyme‐linked immunosorbent assays to detect HNA‐3 antibodies. The cell lines expressing full‐length proteins were trypsin treated to remove HLA antigens and frozen at −80°C. Thawed cells were then used to detect HNA‐3 antibodies by flow cytometry.
RESULTS: Glycosylated and soluble CTL2 fragments were correctly recognized by 15 of 31 anti‐HNA‐3a sera and by both available anti‐HNA‐3b sera. Twenty‐one anti‐HLA sera reacted variably with untreated cell lines expressing full‐length CTL2. After trypsin treatment of the cell lines, reactivity with HLA antisera was abrogated and all 31 anti‐HNA‐3a and two anti‐HNA‐3b sera bound to the corresponding cell line.
CONCLUSION: Whereas soluble, glycosylated CTL2 fragments cannot be used for the detection of HNA‐3 antibodies, the HEK293 cells expressing full‐length CTL2 proteins were useful in the detection of HNA‐3 antibodies even in the presence of HLA antibodies. Moreover, the cell lines can be stored for at least 6 months before use.
BACKGROUND
Most recently described human platelet antigens (HPAs) have been low‐frequency polymorphisms identified in cases of fetomaternal alloimmune thrombocytopenia (FMAIT). There is limited ...opportunity to study the clinical significance or different antenatal management strategies in cases involving low‐frequency HPA antibodies because many are single pregnancies. We have previously described a low‐frequency platelet (PLT) antigen, HPA‐28bw, implicated in FMAIT in two of the three infants in the same family. This report describes the outcome of an additional two pregnancies in this family.
STUDY DESIGN AND METHODS
The fourth and fifth pregnancies in a HPA‐28bw–alloimmunized mother with a heterozygous partner were investigated to determine the risk of FMAIT. The presence of anti‐HPA‐28bw was assessed by paternal crossmatch studies. Prenatal HPA genotyping of amniocytes was performed to inform antenatal management.
RESULTS
GPIIb/IIIa antibodies reactive only with paternal PLTs were detected. These antibodies had been previously identified as HPA‐28bw specific using recombinant GPIIb glycoprotein mutated to contain the HPA‐28bw (V740L) mutation. The fetus in the fourth pregnancy did not inherit the HPA‐28bw mutation, no antenatal management was required, and the baby had a normal PLT count. The fetus in the fifth pregnancy did inherit the HPA‐28bw mutation. The mother received IVIG (2 g/kg/week) and prednisolone during pregnancy, and the baby was born with a normal PLT count.
CONCLUSION
Study of this family has provided a unique opportunity to assess the clinical significance of antibodies against the low‐frequency PLT antigen (HPA‐28bw) during five pregnancies and to compare the outcomes of different antenatal treatments.
•A systematic review identified 29 studies that evaluated the predictive accuracy of 6 point-of-care devices for coagulopathic bleeding in cardiac surgery cohorts.•Concerns about the applicability of ...the definition of coagulopathic bleeding used in individual studies to consensus definitions, along with concerns about the implementation, timing, or interpretation of the tests in almost all the studies, limited interpretation of the results.•The study did not demonstrate predictive accuracy for commonly used point-of-care devices for coagulopathic bleeding in cardiac surgery, however, the certainty of the evidence was low.•The review identified an important knowledge gap with respect to the role of point-of-care tests in actively bleeding patients; no study was performed in this setting.•The review identifies improved standardisation of reference outcomes, test thresholds, and the definition of the coagulopathic bleeding, as important considerations in future research.
Treatment guidelines recommend the routine use of point-of-care diagnostic tests for coagulopathy in the management of cardiac surgery patients at risk of severe bleeding despite uncertainty as to their diagnostic accuracy. We performed a systematic review and meta-analysis of studies that evaluated the diagnostic accuracy of viscoelastometry, platelet function tests, and modified thromboelastography (TEG) tests, for coagulopathy in cardiac surgery patients. The reference standard included resternotomy for bleeding, transfusion of non-red cell components, or massive transfusion. We searched MEDLINE, EMBASE, CINAHL, and Clinical Trials.gov, from inception to June 2019. Study quality was assessed using QUADAS-2. Bivariate models were used to estimate summary sensitivity and specificity with (95% confidence intervals). All 29 studies (7440 participants) included in the data synthesis evaluated the tests as predictors of bleeding. No study evaluated their role in the management of bleeding. None was at low risk of bias. Four were judged as low concern regarding applicability. Pooled estimates of diagnostic accuracy were; Viscoelastic tests, 12 studies, sensitivity 0.61 (0.44, 0.76), specificity 0.83 (0.70, 0.91) with significant heterogeneity. Platelet function tests, 12 studies, sensitivity 0.63 (0.53, 0.72), specificity 0.75 (0.64, 0.84) with significant heterogeneity. TEG modification tests, 3 studies, sensitivity 0.80 (0.67, 0.89), specificity 0.76 (0.69, 0.82) with no evidence of heterogeneity. Studies reporting the highest values for sensitivity and specificity had important methodological limitations. In conclusion, we did not demonstrate predictive accuracy for commonly used point-of-care devices for coagulopathic bleeding in cardiac surgery. However, the certainty of the evidence was low.
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