In patients at high risk of heart failure following myocardial infarction (MI) as a result of residual left ventricular systolic dysfunction (LVSD), the angiotensin receptor neprilysin inhibitor sacubitril/valsartan may result in a greater attenuation of adverse left ventricular (LV) remodelling than renin angiotensin aldosterone system inhibition alone, due to increased levels of substrates for neprilysin with vasodilatory, anti‐hypertrophic, anti‐fibrotic, and sympatholytic effects.We designed a randomized, double‐blinded, active‐comparator trial to examine the effect of sacubitril/valsartan to the current standard of care in reducing adverse LV remodelling in patients with asymptomatic LVSD following MI. Eligible patients were ≥3 months following MI, had an LV ejection fraction ≤40% as measured by echocardiography, were New York Heart Association functional classification I, tolerant of an angiotensin‐converting enzyme inhibitor or angiotensin receptor blocker at equivalent dose of ramipril 2.5 mg twice daily or greater, and taking a beta‐blocker unless contraindicated or intolerant. Patients were randomized to sacubitril/valsartan (target dose 97/103 mg twice daily) or valsartan (target dose 160 mg twice daily). The primary endpoint will be change in LV end‐systolic volume indexed for body surface area measured using cardiac magnetic resonance imaging over 52 weeks from randomization. Secondary endpoints include other magnetic resonance imaging‐based metrics of LV remodelling, biomarkers associated with LV remodelling and neurohumoral activation, and change in patient well‐being assessed using a patient global assessment questionnaire.This trial will investigate the effect of neprilysin inhibition on LV remodelling and the neurohumoral actions of sacubitril/valsartan in patients with asymptomatic LVSD following MI. Routine use of coronary reperfusion therapy  has reduced the degree of ventricular damage sustained at the time of acute myocardial infarction (MI) and improved survival.Not all neurohumoral systems activated in patients after MI (or in HF) are harmful, and some endogenous neurohumoral systems may be protective. A‐type natriuretic peptide (ANP) and B‐type natriuretic peptide (BNP) are secreted by the heart in response to increased wall stress, and these peptides promote vasodilation , stimulate renal sodium and water excretion , and inhibit pathological growth, that is, hypertrophy and fibrosis (key components of the adverse LV remodelling that occurs after MI and in HFrEF).To further explore this hypothesis, we have designed a randomized‐controlled trial comparing the angiotensin receptor neprilysin inhibitor, sacubitril/valsartan, with the ARB, valsartan, to provide information on the effect of neprilysin inhibition on LV remodelling in patients at high risk after MI as a result of residual LVSD.The trial was conceived and designed by the Trial Steering Committee. The trial was co‐sponsored by the University of Glasgow and the National Health Service Greater Glasgow and Clyde health board. The trial protocol and any substantial amendments to the protocol were approved by the East of Scotland Research Ethics Committee.This trial was funded by the British Heart Foundation (PG/17/23/32850), and trial medication along with funding for trial drug packaging, labelling, distribution, storage and destruction was supplied by Novartis Pharmaceuticals UK limited who had no input to the design. J. J. V. M. and M. C. P. are supported by a British Heart Foundation Centre of Research Excellence Grant (RE/18/6/34217).ClinicalTrials.gov Identifier: NCT03552575.The trial is a multi‐centre, prospective, randomized, double‐blind, active‐comparator trial designed to evaluate the effect of sacubitril/valsartan at a target dose of 97/103 mg twice daily, compared with valsartan at a target dose of 160 mg twice daily, on LV remodelling in patients with asymptomatic LVSD following MI, considered to be at high risk of developing HFrEF. The trial is registered as 2, and/or a serum potassium level of >5.2 mmol/L. Full inclusion and exclusion criteria are detailed in TableConsenting patients at least 3 months after acute MI  was performed prior to randomization and at 12 months following randomization with a single 3 Tesla Siemens MAGNETOM Prisma scanner at the Queen Elizabeth University Hospital Glasgow Imaging Centre of Excellence. Images were obtained with a phased‐array chest coil, during breath‐hold, and gated to the electrocardiogram. The MRI protocol is outlined in FigureVenous blood and spot urine samples were collected at baseline and at 6 and 12 months following randomization Figure1. SampPatients completed a patient global assessment of change questionnaire at the 12 months of visit. Details of the questionnaire and available responses are in the 2/>45 mL/m2) and by use of diuretics. The randomization schedule was generated by a computer using permuted blocks, with block lengths of 4 and 6. All participants and trial staff were blind to treatment allocation.Following baseline measurements, participants were randomly assigned to sacubitril/valsartan or valsartan in a 1:1 ratio. Patients were provided with two packs of tablets—valsartan or matching placebo and sacubitril/valsartan or matching placebo—and instructed to take one pill from each pack (i.e. one active treatment and one placebo pill) twice daily. Randomization was stratified by baseline left ventricular end‐systolic volume index (LVESVI) measured using cardiac MRI  because of safety or tolerability concerns related to (i) symptomatic hypotension, (ii) a clinically significant decline in renal function, or (iii) hyperkalaemia , with a two‐step titration to target dose over Visits 3 and 4, if systolic blood pressure at Visit 1 was 100 to 110 mmHg or if estimated glomerular filtration rate was 30–60 mL/min/1.73 mia Figure3.Patients continued standard background therapy.Patients developing HF during follow‐up were offered open‐label sacubitril/valsartan. Patients starting open‐label sacubitril/valsartan or withdrawing from study medication (or study follow‐up) ≥6 months after randomization were asked to undergo an ‘end‐of‐study’ cardiac MRI examination (patients withdrawing before 6 months were not asked to have a second cardiac MRI as an effect of LV remodelling was unlikely to be detected before that time point).The primary and secondary endpoints will be measured as change from baseline to 12 months of follow‐up. The between‐treatment differences in these changes will be analysed.The primary endpoint is change in LV end‐systolic volume, measured using cardiac MRI, and indexed for body surface area (LVESVI).change in N‐terminal pro‐B‐type natriuretic peptide;change in high‐sensitivity troponin I;change in other cardiac MRI‐based metrics of LV remodelling:LV end‐diastolic volume indexed for body surface arealeft atrial volume indexed for body surface areaLVEFLV mass index; andchange in patient well‐being, assessed using a patient global assessment questionnaire.The pre‐specified secondary endpoints are as follows: change in biomarkers of LV remodelling: soluble suppression of tumourigenicity‐2 (ST2), galectin‐3, tissue inhibitor matrix metalloproteinase‐1 (TIMP‐1), matrix metallopeptidase‐9 (MMP‐9), Type III procollagen peptide, growth differentiation factor‐15 (GDF‐15), and other relevant biomarkers of interest;change in neurohormonal levels: BNP, mid‐regional pro‐ANP, C‐terminal ANP, C‐type natriuretic peptide, mid‐regional pro‐adrenomedullin, guanosine 3',5' cyclic monophosphate (cGMP), endothelin‐1, neprilysin antigen, renin and aldosterone, and other relevant biomarkers of interest; andchange in extracellular volume fraction, LV global function index, T1 relaxation time, and LV strain as measured using cardiac MRI.The exploratory endpoints are as follows: α level = 0.05) to detect a difference of 6 mL/m2 in LVESVI (standard deviation = 7.8 mL/m2),2 difference in LVESVI was selected as it is believed to represent a minimally important difference.The study size was 100 patients, based on the calculation that 45 patients in each treatment group provided >90% power  and use of diuretic at baseline. MRI outcomes will also include adjustment for the time from baseline to follow‐up MRI. Efficacy outcome measures will be summarized at baseline, 12 months, and for the change from baseline to 12 months, presented overall and by treatment group. The effect of treatment will be presented with 95% confidence intervals and a two‐sided Results from this trial will provide detailed insight into the effects of neprilysin inhibition, added to standard care, in patients at high risk of developing HF as a result of residual LVSD following MI. It will be the first adequately powered, randomized, and long‐term examination of the effect of sacubitril/valsartan on LV remodelling and the only comparison with valsartan, that is, the only study to ensure identical background renin angiotensin blocking therapy in both randomized treatment groups. The use of multi‐parametric cardiac MRI will provide high‐fidelity information about the effects of neprilysin inhibition on cardiac structure and function and, along with comprehensive biomarker profiling of patients, will provide further understanding of the mechanisms of action underlying the clinical benefits observed with sacubitril/valsartan in patients with HFrEF.The presence of LVSD and LV dilatation are powerful predictors of the risk of developing HF following MI. Prior to reperfusion therapy, the major therapeutic breakthrough in MI was the demonstration that ACE inhibitors, given to prevent adverse LV remodelling in high‐risk patients, reduced the likelihood of developing HF and the risk of death.What evidence do we have for a reversal of LV remodelling with neprilysin inhibition? A series of experimental models of MI and HFrEF have reported positive effects of neprilysin inhibition on metrics of LV remodelling.There are also few data demonstrating the neurohumoral effects of neprilysin inhibition potentially underlying the clinical benefits of sacubitril/valsartan in patients with HFrEF. The substrates for neprilysin are ubiquitous and include the natriuretic peptides, adrenomedullin, endothelin, angiotensin II, substance P, bradykinin, vasoactive intestinal peptide, calcitonin gene‐related peptide, and glucagon‐like peptide‐1, among others. Following on from PARADIGM‐HF, subsequent analyses have reported only modest increases in BNP in contrast to ANP, a finding that is perhaps not unsurprising given the greater affinity neprilysin has for ANP relative to BNP.per se without the uncertainty about comparing renin angiotensin system blockade with an ACE inhibitor, compared with an ARB. Our choice of primary endpoint, LV end‐systolic volume, has been shown to be a major determinant of survival after MI, and improvements in LV end‐systolic volume are associated with improved outcomes in high‐risk patients following MI.Several other features of the trial design merit further discussion. We recruited patients with asymptomatic LVSD identified at least 3 months after MI to ensure resolution of ‘stunning’ (reversible LVSD). This will distinguish our study from the Prospective ARNI vs. ACE Inhibitor Trial to DetermIne Superiority in Reducing Heart Failure Events After MI (PARADISE‐MI), which is enrolling patients up to 7 days after acute infarction and does not require all patients to have LVSD.Despite advances in the management of acute MI, patients remain at substantial risk of developing HF as a result of residual LVSD. Inhibition of neprilysin activity in combination with RAAS blockade, using sacubitril/valsartan, has been demonstrated to improve outcomes in patients with established symptomatic HFrEF. However, the mechanisms of action underlying these clinical benefits remain unclear. They may, in part, be due to a reverse‐remodelling effect of neprilysin inhibition and increased levels of the vasoactive substrates of the enzyme, including the natriuretic peptides, among others. Results from this trial will provide comprehensive information regarding the effects of neprilysin inhibition on LV remodelling and the neurohumoral actions of sacubitril/valsartan in patients at high risk of HF following MI.K.F.D. reports personal fees from Eli Lilly, outside the submitted work. P.F. reports personal fees from Novartis for lectures and scientific advice, outside the submitted work. A.M. and B.S. report grants from the British Heart Foundation during the conduct of the study. P.W. reports receiving grants from Roche Diagnostics, Astrazeneca, and Boehringer Ingelheim outside the submitted work. P.S.J. reports personal fees from Novartis and payments made to his employer (the University of Glasgow) for work on the PARADIGM‐HF and PARAGON‐HF trials during the conduct of the study; personal fees from Astrazeneca; and grants from Boehringer Ingelheim, outside the submitted work. M.C.P. reports receiving grants and personal fees from Novartis lecture fees during the conduct of the study and personal fees from Novo Nordisk, AstraZeneca, Eli Lilly NAPP Pharmaceuticals, Takeda Pharmaceutical, Alnylam, Bayer, Resverlogix, and Cardiorentis and grants and personal fees from Boehringer Ingelheim outside the submitted work. J.J.V.M.'s employer employer, Glasgow University, has been paid by Novartis  for his time spent as committee member for the trials listed , meetings related to these trials, and other activities related to sacubitril/valsartan, for example, lectures, advisory boards, and other meetings. Novartis has also paid his travel and accommodation for some of these meetings. These payments were made through a Consultancy with Glasgow University, and he has not received personal payments in relation to these trials/this drug. The trials include PARADIGM‐HF: co‐PI; PARAGON‐HF: co‐PI; and PERSPECTIVE, PARADISE‐MI, and UK HARP III Trial: executive/steering committees. No other conflicts of interest were declared.This trial was funded by the British Heart Foundation (PG/17/23/32850), and trial medication along with funding for trial drug packaging, labelling, distribution, storage and destruction was supplied by Novartis Pharmaceuticals UK Limited who had no input to the design. J.J.V.M. and M.C.P. are supported by a British Heart Foundation Centre of Research Excellence Grant (RE/18/6/34217).Data S1. Cardiac MRI protocol.Table S1. Total daily doses of commonly used ACE inhibitors or ARBs corresponding to ramipril 2.5mg twice daily (dose level 2 of study drug).Table S2. Schedule of Assessments.Click here for additional data file.


SC5D gene and by plasma sterol analysis to confirm elevated lathosterol levels. In this report, we describe a girl with transaminitis in association with developmental delay/intellectual disability, facial dysmorphism, limb anomalies, and bilateral cataracts. Fibroscan showed severe liver fibrosis. Plasma sterol analysis and exome sequencing confirmed the diagnosis of lathosterolosis. Simvastatin treatment resulted in lowering of plasma lathosterol levels, improvement in transaminitis, and liver fibrosis grade, suggesting that children with this condition should be actively treated in order to prevent progression of liver disease.Lathosterolosis is a rare autosomal recessive disorder of cholesterol biosynthesis. It is caused by defects in the SC5D (sterol C5‐desaturase) gene which encodes for the 3‐beta‐hydroxysteroid‐delta‐5‐desaturase . Only six cases have been described in the literature, but it is possible that a number of patients with milder forms of the condition might have been missed. Lathosterolosis manifests as microcephaly, bilateral cataracts, dysmorphism, limb anomalies, and developmental delay/intellectual disability. Liver involvement is variable and can range from normal liver function tests to portal fibrosis and cirrhosis. Diagnosis is made by demonstration of specific mutations in the  If the presence of distinctive facial features and limb anomalies raises the suspicion of cholesterol synthesis defect, testing of full sterol profile is of utmost importance since an early intervention improving the metabolic imbalance may have an impact on the patient's prognosis. Simvastatin, an inhibitor of the proximal cholesterol biosynthesis, may be useful in decreasing the level of sterol precursors in disorders of cholesterol biosynthesis.1SC5D (sterol C5‐desaturase) gene which encodes for the enzyme 3‐beta‐hydroxysteroid‐delta‐5‐desaturase . The enzyme 3‐beta‐hydroxysteroid‐delta‐5‐desaturase catalyses the conversion of lathosterol to 7‐dehydrocholesterol, the penultimate enzymatic reaction in the cholesterol synthesis pathway. Lathosterolosis manifests as microcephaly, bilateral cataracts, dysmorphism, limb anomalies, developmental delay/intellectual disability, and liver involvement. Only six cases have been described in the literature thus far.Cholesterol is an essential metabolite of cell membrane structure and myelin and is the precursor of steroid hormones, bile acids and other oxysterols which play a crucial role in biological functions throughout the body. Defects in several stages of cholesterol biosynthesis have been identified and their phenotypes described.2inv (9)(q13q21.12), that was assessed as likely benign. She had a birth weight of 2680 g (10th centile), and perinatal history was unremarkable. She demonstrated early motor and language skills delay. She sat unassisted at 9 months of age, crawled at 10 months, and walked at age 22 months. She spoke her first words at 18 months of age and two word utterances at 2.5 years of age. By 8 years of age, she was behind her peers in all academic areas and attending mainstream school with an integration aid. A formal assessment using Wechsler Intelligence Scale for Children—Fifth Edition (WISC‐V) showed that her general intellectual functioning was in the extremely low range (first percentile). At 5 years of age, she was noted to have bilateral cataracts and subsequently underwent capsulotomies. Examination findings at age 7 years included height and weight on the 10th centile and head circumference 1.8 cm below the second centile. She had subtle facial dysmorphism including mild hypotelorism, epicanthic folds and retrognathia  and detection of horseshoe kidney at 20 week ultrasound. Amniocentesis was performed and karyotype showed a paternally inherited paracentric inversion, inv (9)q3q21.12, Liver function was first assessed at age 7 years and showed alanine aminotransferase (ALT) 321 IU/L (5‐30), gamma‐glutamyl transferase (GGT) 317 U/L (5‐20), and alkaline phosphatase (ALP) 436 U/L (120‐350). Serum bilirubin, albumin, urea, creatinine and coagulation profile were within normal range. A full blood count was essentially normal except for large platelets as evidenced by increased mean platelet volume (MPV) values ranging from 10.9 to 11.3 fL . Chromosome microarray, fragile X test, urine amino and organic acids and galactoscreen were normal. Abdominal ultrasound showed a liver and spleen of normal size and echotexture, and a horseshoe kidney. Investigations for infection‐related hepatitis, autoimmune liver disease, alpha‐1‐antitrypsin deficiency, Wilson disease and coeliac disease were negative. Fasting total bile acid levels were performed using a recombinant 3‐alpha hydroxysteroid dehydrogenase enzymatic kit that measures 3‐alpha hydroxyl steroids only and results showed slightly elevated level at 10 μmol/L . Plasma very long chain fatty acid (VLCFA) and phytanic acid levels were normal.She underwent liver biopsy at 8 years of age which demonstrated a mild portal and lobular hepatitis with preserved lobular architecture. Portal tracts showed mild focal fibrosis with occasional bridging fibrosis. There were no features of autoimmune hepatitis or metabolic liver disease. Electron microscopy revealed prominent and dilated endoplasmic reticulum containing finely flocculent content. The ultrastructure of mitochondria, lysosomes, peroxisomes and bile canaliculi appeared normal.SC5D gene. Based on ACMG variant classification guidelines both variants were classified as likely pathogenic. The presence of these two variants in trans confirmed a compound heterozygous mode of inheritance, consistent with lathosterolosis.Exome sequencing of the patient was performed. A missense variant, NM_006918.4 (SC5D):c.212A>T;p.(Asn71Ile) and a nonsense variant, NM_006918.4(SC5D):c.214C>T; p.(Gln72*) were identified in exon 3 of the Subsequent analysis of plasma sterol levels revealed abnormally high lathosterol level , normal 7‐dehydrocholesterol, total cholesterol, and elevated 8(9)‐cholestenol levels, confirming the diagnosis of lathosterolosis.Her fibroscan results suggested severe fibrosis . 25‐OH vitamin D levels were 65 to 80 nmol/L (RR > 50). Bone mineral density was low when compared to age matched controls, but it was in the lower 25% of normal with height adjustment.The patient was treated with the cholesterol‐lowering agent simvastatin, 5 mg (0.2 mg/kg/day) initially, and the dose was increased to 10 mg daily (0.4 mg/kg/day) 8 weeks later. Serum creatine kinase levels were regularly monitored and ranged from 82 to 139 IU/L (RR: 30‐150 U/L). Serial plasma sterol assays showed near normalization of lathosterol levels  and 8‐dehydrocholesterol (8DHC). It is therefore important to be aware of the limitations of the sterol analysis offered in each laboratory so that a complete sterol analysis can be pursued especially when there is a clinical suspicion of a defect in cholesterol biosynthesis and a normal 7DHC.Simvastatin is an inhibitor of HMG‐CoA reductase which converts HMG‐CoA to mevalonate, an early but rate limiting step in cholesterol synthesis. It has an advantage over other statins because of its lipophilic properties allowing it to cross the blood brain barrier and potentially inhibits cerebral cholesterol precursor accumulation.Simvastatin was given to three lathosterolosis patients to reduce lathosterol levels using the same rationale as its use in SLOS patients.Liver transplantation (LT) was performed in one patient who had intrahepatic cholestasis and fibrosis progressing to liver failure with portal hypertension and this led to complete biochemical recovery and halted progression of neurological damage.4This report describes a rare cause of abnormal liver function tests in association with dysmorphic features, cataracts, limb anomalies, and developmental delay/intellectual disability. Simvastatin treatment resulted in lowering of plasma lathosterol levels, improvement in liver function tests and liver fibrosis grade, suggesting that children with this condition should be actively treated in order to prevent progression of liver disease. Furthermore, our case illustrates the importance of including additional sterols such as lathosterol in sterol analysis particularly if the clinical features strongly suggest a disorder in cholesterol biosynthetic pathway.Joy Yaplito‐Lee, Gautham Pai, Winita Hardikar, Kai M. Hong, James Pitt, Justine Marum, and David J. Amor declare that they have no conflict of interest.Joy Yaplito‐Lee, Gautham Pai, David J. Amor, and Winita Hardikar were involved in clinical management of the patient, collecting the data, and drafting the manuscript. David J. Amor, Kai M. Hong, James Pitt, and Justine Marum were involved with the laboratory and genetic studies, collecting the data, and provided intellectual inputs in drafting the manuscript. All authors have inspected and approved the final draft of the manuscript. Joy Yaplito‐Lee will serve as the guarantor.All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation  and with the Helsinki Declaration of 1975, as revised in 2000 (5). Informed consent was obtained from the patient for being included in the case report.Additional informed consent was obtained from the patient for which identifying information was included in this article.This article does not contain any studies with human or animal subjects performed by the any of the authors.Joy Yaplito‐Lee.Not required.


Mirogabalin, which is approved for the treatment of peripheral neuropathic pain in Japan, is a ligand for the α2δ subunit of voltage-gated calcium channels. Both pregabalin and mirogabalin act as nonselective ligands at the α2δ-1 and α2δ-2 subunits. Mirogabalin has a unique binding profile and long duration of action. Pregabalin has been reported to produce intolerable adverse effects in some patients. This study investigated outcomes associated with mirogabalin administration in patients with peripheral neuropathic pain who ceased treatment with pregabalin.We retrospectively assessed peripheral neuropathic pain using the neuropathic pain screening questionnaire (NeP score) in 187 patients  who were treated with mirogabalin. All patients had switched from pregabalin to mirogabalin due to lack of efficacy or adverse events. Differences in the treatment course  scores) were compared using one-way analysis of variance with Bonferroni post hoc tests.The mean age of the patients was 72.3 years , and the mean duration of disease was 37 months . After treatment with mirogabalin for 1 week, NRS scores significantly decreased compared with baseline and continued to decrease over time. After 8 weeks, NRS scores improved by ≥ 30% from baseline in 113 patients (69.3%). Twenty-four patients (12.8%) stopped mirogabalin treatment due to adverse events. Somnolence (26.7%), dizziness (12.3%), edema (5.9%), and weight gain (0.5%) were noted as adverse events of mirogabalin.The results of this investigation indicate that mirogabalin is safe and effective for reducing peripheral neuropathic pain. The International Association for the Study of Pain defined neuropathic pain as “pain caused by a lesion or disease of the somatosensory nervous system” . NeuropaPregabalin is a ligand for the α2δ subunit of voltage-sensitive calcium channels and is recommended as the first-line drug for neuropathic pain in guidelines around the world . It decrMirogabalin, which is a potent and specific ligand for the α2δ subunit of voltage-gated calcium channels, is an orally administered gabapentinoid developed for the treatment of peripheral neuropathic pain in Japan , 16. ThiAlthough there are many positive reports associated with pregabalin, some patients have moderate adverse events . TetsunaThis retrospective study included outpatients with peripheral neuropathic pain who consulted our hospital between April 2019 and October 2019. Data were collected from medical records. The diagnosis of peripheral neuropathic pain was based on a history of neuropathic pain and confirmatory findings on examination. The inclusion criteria were a diagnosis of peripheral neuropathic pain based on the flow chart of the grading system for neuropathic pain , lack ofAt least 1 month after cessation from pregabalin, treatment with mirogabalin  was prescribed. The patients received mirogabalin 10 mg/day orally for the first week. In patients with decreased renal function, the dose of mirogabalin was decreased to 5 mg/day. Depending on patient age and symptoms, the dose of mirogabalin was decreased or increased as required to between 2.5 mg and 15 mg/dose twice daily. All patients visited the hospital at 1, 2, 4, and 8 weeks to ensure compliance with the study regimen. The patients with an adequate effect received the same dose of mirogabalin. In patients with an inadequate effect, the dose of mirogabalin was increased up to 30 mg/day. If adverse events were observed, the dose was decreased. In this study, no other conservative treatments  or surgeries were performed. During the study, only the dispensing pharmacist had knowledge of the patient codes. The manufacturer and provider of mirogabalin  was not involved in the protocol development, data collection and management, statistical analysis, or manuscript preparation.The neuropathic pain screening questionnaire (NeP score), developed by Ogawa et al., was used for the peripheral neuropathic pain survey Table  19–21].–21.19–21The primary endpoint of this study was to analyze the treatment course of peripheral neuropathic pain using NRS scores in patients receiving mirogabalin.The secondary endpoint of the current study was to investigate the adverse events of mirogabalin.p < 0.05 were considered significant. Statistical analyses were conducted using SPSS software version 25.0 for Windows .Factors associated with the cessation of mirogabalin treatment due to adverse events were identified using univariate analyses between the patients who continued treatment and those who ceased from treatment. Differences in the treatment course  were compared using one-way analysis of variance with Bonferroni post hoc tests. Moderate improvements in pain are considered to be 30%h Bonferroni postnivariate 50% pain relief is considered a good outcome , 24. TheOf the 337 outpatients with peripheral neuropathic pain who had been treated with pregabalin, 187 (55.5%) met the inclusion criteria and were included in this study Table . Adversep = 0.0017) and significantly higher incidences of dizziness (p = 0.015) and edema (p = 0.012) with pregabalin than those who continued mirogabalin. The patients who discontinued mirogabalin also had significantly higher incidences of dizziness (p = 0.0069) and edema (p = 0.016) than the patients who continued mirogabalin, and these effects contributed to the cessation of treatment.Fifty-nine patients received mirogabalin 10 mg/day orally for the first week, and 128 patients received mirogabalin 5 mg/day due to decreased renal function. Thirty-three patients were treated with 5 mg/day for 8 weeks of treatment. Eighty-one patients increased the dose of mirogabalin to 10 mg/day due to lack of efficacy. Thirty patients increased the dose of mirogabalin to 20 mg/day. Nineteen patients increased the dose of mirogabalin to 30 mg/day; none of these patients experienced severe adverse events with the increased dose. Twenty-four patients (12.8%) withdrew from mirogabalin treatment because of adverse events. Adverse events associated with mirogabalin included somnolence , dizziness , edema , epigastric pain , weight gain , and fatigue . We investigated factors associated with the cessation of mirogabalin treatment due to adverse events. There were no significant differences in age, sex, body mass index (BMI), or diagnosis between the patients who discontinued mirogabalin and those who continued its use . We examined whether there were differences in the adverse events and magnitude of NRS improvement between the patients with initial doses of 5 mg/day (n = 128) and 10 mg/day (n = 59). The incidence of adverse events in patients with an initial dose of 5 mg/day  was not significantly different from that in patients with 10 mg/day . There were no significant differences in the magnitude of NRS improvement between the groups with initial doses of 5 mg/day (46.5% ± 24.8%) and 10 mg/day . Similarly, there were no significant differences in the magnitude of NRS improvement in patients taking maximum doses of 5 mg/day (n = 47), 10 mg/day (n = 89), 20 mg/day (n = 32), and 30 mg/day (n = 19, p = 0.79; Fig. We also investigated changes in NRS scores over the course of treatment with mirogabalin for 8 weeks. After treatment with mirogabalin for 1 week, NRS scores significantly decreased compared with baseline (day n = 18 and 10 In this study, we used mirogabalin to treat patients with peripheral neuropathic pain who switched from pregabalin treatment to mirogabalin treatment due to lack of efficacy or adverse events. Mirogabalin is a new drug for the treatment of peripheral neuropathic pain, and to our knowledge, there have been no reports of its clinical outcomes for patients who were previously treated with pregabalin. Mirogabalin exerted a significant analgesic effect within 1 week and was associated with mild CNS adverse effects.Guidelines for the pharmacologic management of neuropathic pain recommend drugs acting at α2δ subunits of voltage-gated calcium channels, serotonin–norepinephrine reuptake inhibitors, and tricyclic antidepressants as first-line agents, neurotropin and tramadol as second-line agents, and opioids as third-line agents . SeveralIn vitro studies of its pharmacologic action have demonstrated that mirogabalin had a higher binding affinity for the human and rat α2δ subunit than pregabalin . In a diThe α2δ-1 subunit plays an important role in the onset and pathological persistence of neuropathic pain. Α2δ-1 expression levels correlated with tactile allodynia development were significantly increased in rats with spinal cord injury . KnockdoAlthough the results of the present study suggest that mirogabalin might be an alternative treatment option for the treatment of peripheral neuropathic pain, the present study has some limitations. First, both pregabalin and mirogabalin are ligands for the α2δ subunit of voltage-sensitive calcium channels. However, this study had no pretrial protocol and is a case series without a comparison or placebo group. We did not compare these two drugs in this study. Thus, the results cannot be clearly attributed to mirogabalin administration. Second, this study had a short observation period. Although most patients treated with mirogabalin for 8 weeks maintained their weight within ± 5% of their baseline weight, weight gain can be an issue with mirogabalin when used for a longer period. A third limitation was a lack of determination of the best screening questionnaire for neuropathic pain. Neuropathic pain screening questionnaires include painDETECT , the spiThis investigation indicated that mirogabalin is safe and effective for reducing peripheral neuropathic pain in patients who ceased treatment with pregabalin due to adverse events or lack of efficacy.


The effect of erector spinae plane block has been evaluated by clinical trials leading to a diversity of results. The main objective of the current investigation is to compare the analgesic efficacy of erector spinae plane block to no block intervention in patients undergoing surgical procedures.We performed a quantitative systematic review of randomized controlled trials in PubMed, Embase, Cochrane Library, and Google Scholar electronic databases from their inception through July 2019. Included trials reported either on opioid consumption or pain scores as postoperative pain outcomes. Methodological quality of included studies was evaluated using Cochrane Collaboration’s tool.P < 0.001) IV mg morphine equivalents. The effect of erector spinae plane block on post surgical pain at 6 h compared to control revealed a significant effect weighted mean difference of − 1.31 (95% CI: − 2.40 to − 0.23), P < 0.02. At 12 h, the weighted mean difference was of − 0.46 (95% CI: − 1.01 to 0.09), P = 0.10. No block related complications were reported.Thirteen randomized controlled trials evaluating 679 patients across different surgical procedures were included. The aggregated effect of erector spinae plane block on postoperative opioid consumption revealed a significant effect, weighted mean difference of − 8.84 (95% CI: − 12.54 to − 5.14), (Our results provide moderate quality evidence that erector spinae plane block is an effective strategy to improve postsurgical analgesia. The misuse of prescribed opioids leading to the current opioid epidemic crisis has put greater emphasis on the development of non-opioid analgesic techniques to manage postoperative pain –3. A larThe erector spinae plane block has been used clinically by anesthesiologists as a potential non-opioid analgesic strategy across multiple surgical procedures –14. The The objective of our study was to examine the analgesic efficacy of erector spinae plane block for postoperative analgesia outcomes in patients undergoing surgical procedures. In addition, we also investigated the potential side effects related to the use of the erector spinae plane block.We performed a quantitative systematic review according to the PRISMA guidelines . The stuA comprehensive search of randomized trials investigating the effects of erector spinae plane block to control (i.e. no block or sham block) on postoperative surgical analgesia was performed using web-based electronic databases PubMed, Google Scholar, the Cochrane Database of Systematic Reviews, and Embase from inception up to July 2019. The search words ‘erector spine block’, ‘erector spinae plane block’, or “ESPB” were used in various combinations using Boolean operators. Search strategy is shown in Additional file Studies were excluded if a direct comparison of erector spinae plane block and no block could not be determined. Non-randomized controlled trials, anatomical or cadaver studies, case reports, or editorials were not considered for inclusion.Two investigators (MCK and LA) independently reviewed the abstracts obtained from the initial search using the predetermined inclusion and exclusion criteria. The trials that were not relevant based on the inclusion criteria were omitted. Any discrepancies encountered during the selection process were resolved by discussion among the evaluators (MCK and LA). If there was a disagreement then the final decision was determined by the senior investigator (GDO). Data extraction was carried out by using a pre-designed data collection form. The primary source of data extraction was from either the text or tables. If the data could not be found in either location, we extracted the data manually from available figures or plots. The extracted data obtained from studies included the sample size, number of study participants in treatment/control groups, surgery description, type of local anesthetic dose, single-shot or bilateral block placement, use of ultrasonography for block placement, postoperative opioid consumption, postoperative pain scores, postoperative nausea and vomiting, and adverse events. Postoperative opioid consumption was converted to intravenous morphine milligram equivalents assuming no cross-tolerance (morEq) . ContinuThe validity of the included studies was evaluated in accordance with Cochrane risk-of-bias tool (RoB-2) . This rePostoperative opioid consumption (IV morEq) reported at 24 h following surgery.Postoperative pain scores  at rest and with activity at 6 h, at 12 h, and at 24 h after surgery, block complications, and postoperative nausea and vomiting displayed as (n).P < 0.05 was considered as an indication of an asymmetric funnel plot. In the presence of an asymmetric funnel plot, a file drawer analysis was performed, which estimates the lowest number of additional studies that if they would become available, it would reduce the combined effect to non-significance assuming the average z-value of the combined P values of these missing studies would be 0. Heterogeneity was considered to be high if the I2 statistic was greater than 50%. If heterogeneity was high, we performed a sensitivity analysis by removing individual studies and investigated surgical procedures by examining its effect on the overall heterogeneity. A P value < 0.05 was required to reject the null hypothesis. Analyses was performed using Stata version 15  and Comprehensive Meta-analysis software version 3 .The weighted mean differences (WMD) with 95% confidence interval (CI) were calculated and reported for continuous data for total opioid consumption up to 24 h and pain scores (NRS) at 6 h, 12 h and at 24 h. Statistical significance required that the 95% CI for continuous data did not include zero and for dichotomous data, the 95% confidence interval did not include 1.0. Due to the variety of surgical procedures, we chose to use the random-effects model in an attempt to generalize our findings to studies not included in our meta-analysis . A one sA flow diagram of the literature search and reasons for exclusion are shown in Fig. The initial query identified 903 articles and 884 articles were excluded after review of the study abstracts. A total of 19 articles were evaluated and after full-text review 6 articles were omitted. Thirteen studies involving 679 subjects fulfilled the inclusion criteria and were included in the final analysis –38. The All included trials reported inclusion and exclusion criteria and described baseline characteristics. The risk of bias assessment according to the Cochrane Handbook using the Cochrane risk-of-bias assessment tool RoB-2) is presented in Table  is preseP < 0.001) mg IV morEq  and could be partially explained by whether the block was placed bilaterally or as a single-shot procedure (I2 = 91%). The type of surgery did not substantially reduce the heterogeneity any further (I2 = 86%). Potential sources of heterogeneity were further tested by a sensitivity analysis by removing individual studies which did not significantly reduce the heterogeneity among the studies.The pooled effect of twelve studies , 32–38 eP < 0.001 and in patients who underwent spine or orthopedic procedures WMD of − 4.13 (95% CI: − 5.78 to − 2.48), P < 0.001. Patients who had abdominal surgery did not experience statistical significance, WMD of − 12.05 (95% CI: − 25.88 to 1.79), P = 0.09. Visual examination of the funnel plot and Egger’s regression test (P = 0.06) revealed no apparent publication bias.A subgroup analysis of surgery type revealed the reduction of opioid consumption compared to control was statistically significant in patients who underwent chest surgical procedures WMD of − 9.04 (95% CI: − 11.37 to − 6.70), P < 0.02  and could be partially explained by the type of block placement in which the heterogeneity decreased to I2 = 89% for studies utilizing single-shot blocks. When investigating the type of surgical procedure the heterogeneity decreased to 10% for studies of spine/orthopedic procedures.The combined effect of nine studies –35, 37 eP = 0.003, or spine/orthopedic procedures WMD of − 0.95 (95% CI: − 1.60 to − 0.31), P = 0.004. However, postsurgical pain compared to control was not different in patients who had chest surgical procedures WMD of − 1.34 (95% CI: − 3.56 to 0.88), P = 0.24). A sensitivity analysis was performed by omitting individual studies which did not considerably reduce heterogeneity. An examination of the funnel plot to test publication bias did reveal asymmetry. The Egger’s regression test result was P = < 0.001.A subgroup analysis looking at type of surgery indicated that the reduction in postsurgical pain compared to control was statistically different in patients who underwent abdominal surgical procedures WMD of − 1.35 (95% CI: − 2.25 to − 0.45), P = 0.01 [One study reported the effect of erector spinae plane block on postsurgical pain during movement compared to control at 6 h after surgery and demonstrated a mean difference of − 0.55 (95% CI) -1.21 to 0.11, P = 0.01 .P = 0.10,  and was slightly decreased to I2 = 62% for studies using single-shot block placement. The heterogeneity decreased to I2 = 0% for studies involving only abdominal surgical procedures. A sensitivity analysis was performed by omitting individual studies which did not significantly reduce heterogeneity.The effect of ten studies , 37, 38 P = 0.003. However, postsurgical pain at rest compared to control was not different in patients 12 h after chest surgical procedures WMD of − 0.70 (95% CI: − 1.51 to 0.12), P = 0.09 or spine/orthopedic procedures WMD of − 0.11 (95% CI: − 1.22 to 0.99), P = 0.84. An examination of the funnel plot to test publication bias did not reveal asymmetry. The Egger’s test result was P = 0.47.A subgroup analysis involving the type of surgery revealed that the reduction in postsurgical pain compared to control was statistically different in patients who underwent abdominal surgical procedures WMD of − 0.57 (95% CI: − 0.95 to − 0.19), P = 0.02. Yayik et al. [P < 0.01.There were two studies that reported on postoperative surgical pain at activity 12 h after surgery. Tulgar et al.  reportedk et al.  reportedP = 0.23, . The heterogeneity decreased to I2 = 30% for studies using bilateral block placement. A sensitivity analysis by deleting individual studies did not substantially reduce heterogeneity.The pooled effect of ten studies , 37, 38 P = 0.35, spine/orthopedic surgical procedures WMD of − 0.17 (95% CI: − 0.85 to 0.51), P = 0.63, or after chest procedures WMD of − 0.70 (95% CI: − 1.43 to 0.03), P < 0.06. An examination of the funnel plot did not reveal asymmetry; Egger’s regression test result was P = 0.40.A subgroup analysis involving the type of surgery demonstrated that reduction in postsurgical pain compared to control was not statistically different in patients who underwent abdominal surgical procedures WMD of 0.11 (95% CI: − 0.13 to 0.35), P = 0.09. Heterogeneity was I2 = 89% [The pooled effect of three studies evaluating the effect of erector spinae plane block on postoperative surgical pain during activity compared to control did not show a significant effect, weighted mean difference of − 0.65 (95% CI: − 1.40 to 0.11), I2 = 89% , 37, 38.P = 0.001), , Fig. . HeterogP = 0.03. In contrast, the postoperative nausea and vomiting compared to no block or sham block was not different in patients who had chest surgery, WMD of 0.22 (95% CI: 0.05 to 1.04), P = 0.06 or abdominal surgery WMD of 0.39 (95% CI: 0.10 to 0.1.47), P = 0.16.A subgroup analysis involving the type of surgery revealed that postoperative nausea and vomiting compared to control was reduced in spine/orthopedic surgical procedures WMD of 0.29 (95% CI: 0.09 to 0.91), All thirteen studies reported either no adverse events  or did not report any adverse events. One study  reportedThe most important finding of the current investigation was the reduction of postoperative pain in patients who received an erector spinae plane block compared to a control group across multiple surgical procedures. Patients in the erector spinae plane group reported substantially less pain in the immediate postoperative phase . Our results suggest that the erector spinae plane block is an effective strategy to reduce postsurgical pain.Our results are important as pain continues to be poorly controlled after surgery. A recent study by Herbst et al. showed that 23.3% of postsurgical readmissions were related to poor postoperative pain control . ApproprAnother important finding of our current investigation was the effect of the erector spinae plane block on the reduction of postoperative nausea and vomiting. This is interesting as not all analgesic interventions have been shown to reduce opioid-related side effects –44. In aOne of the main advantages of the erector spinae plane block is that the block is considered easy to be performed, especially when compared to paravertebral blocks or thoracic epidurals. This is important because it not only maximizes efficacy of the block, but also allows its implementation across multiple surgical procedures. The injection is performed deep in the erector spinae muscle and superficial to the tips of the thoracic transverse processes. The block has an excellent safety profile since the local anesthetic injection is distant from the pleura, major blood vessels, and spinal cord.The anatomical localization of the spinal nerves and the different anatomy of the vertebral column may be a major factor for the various postoperative outcomes following the placement of an erector spinae plane block. Recent literature has reported that different volumes of local anesthetic injectate and its corresponding spread are influenced by the site of injection. For example, a 5 mL of injectate was needed to cover one vertebral level in the lumbar region, whereas only 3.3  to 3.5 (cadaveric dissections studies) mL are needed in thoracic region . In our The findings of our study should only be interpreted within the context of its limitations. First, in order to minimize heterogeneity, we compared erector spinae plane block to an “inactive” control group. More recently, studies have compared the erector spinae plane blocks to other commonly performed blocks  –48. NoneIn summary, our results provide moderate-quality evidence the erector spinae plane block may be an effective analgesic strategy to minimize postoperative pain and reduce postoperative opioid consumption across several types of surgeries. In addition, a high quality of evidence demonstrated that erector spinae plane block also reduced postoperative nausea and vomiting. More studies are necessary to confirm our findings of a possible short-term analgesic benefit of the erector spinae plane block.Additional file 1. Search strategy.


Silicosis is a pneumoconiosis caused by inhaled crystalline silica microparticles, which trigger inflammatory responses and granuloma formation in pulmonary parenchyma, thus affecting lung function. Although systemic administration of mesenchymal stromal cells (MSCs) ameliorates lung inflammation and attenuates fibrosis in experimental silicosis, it does not reverse collagen deposition and granuloma formation. In an attempt to improve the beneficial effects of MSCs, magnetic targeting (MT) has arisen as a potential means of prolonging MSC retention in the lungs. In this study, MSCs were incubated with magnetic nanoparticles and magnets were used for in vitro guidance of these magnetized MSCs and to enhance their retention in the lungs in vivo. In vitro assays indicated that MT improved MSC transmigration and expression of chemokine receptors. In vivo, animals implanted with magnets for 48 hours had significantly more magnetized MSCs in the lungs, suggesting improved MSC retention. Seven days after magnet removal, silicotic animals treated with magnetized MSCs and magnets showed significant reductions in static lung elastance, resistive pressure, and granuloma area. In conclusion, MT is a viable technique to prolong MSC retention in the lungs, enhancing their beneficial effects on experimentally induced silicosis. MT may be a promising strategy for enhancing MSC therapies for chronic lung diseases. Magnetic targeting technique has emerged as a new strategy to aid delivery, increase retention, and enhance the effects of mesenchymal stromal cells (MSCs) but, so far, has not been performed in lung diseases. With the aid of magnets, magnetized MSCs remained longer in the lungs, and this was associated with increased beneficial effects for the treatment of silicosis in mice. This study established an MSC magnetization protocol using citrate functionalized magnetic nanoparticles that kept cells viable and made them magnetically responsive. With the aid of magnets, more magnetized MSCs remained in the lungs, and this was associated with enhanced beneficial effects for the treatment of silicosis in mice. Magnetic targeting may be a promising strategy for enhancing the beneficial effects of MSC‐based cell therapies for silicosis and other chronic lung diseases.2Silicosis is an occupational lung disease caused by inhaled crystalline silica microparticles, which trigger an inflammatory response and granuloma formation in the pulmonary parenchyma, thus impairing lung function.Bone marrow‐derived MSCs have demonstrated beneficial effects in preclinical studies,In murine models of experimental silicosis, systemic MSC administration ameliorates inflammation and fibrosis, but it does not reverse all lung histological changes, such as collagen deposition and granuloma formation.2O3) nanoparticles functionalized with dimercaptosuccinic acid (DMSA) have been tested as potential agents for MT of MSCs to injured lungs.Iron oxide . All animals received humane care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the 3.12O3‐Cit) were used to magnetize MSCs. The nanoparticles were prepared following protocols published by van Ewijk et al.Superparamagnetic maghemite nanoparticles functionalized with citrate . γFe2O3‐Cit crystallite size and morphology was assessed by transmission electron microscopy (TEM) . Hydrodynamic diameter, polydispersity index, and zeta potential measurements were obtained using a Malvern Zetasizer Nano‐ZS . Finally, magnetization of γFe2O3‐Cit was measured in a commercial Physical Properties Measurement System  at 37°C, in the magnetic field range of −80 to 80 kOe.Total iron content in the suspensions was determined by atomic absorption spectrophotometry in a commercial Perkin‐Elmer 5000 system . The content of Fe3.22, humidified chamber). Cells from passage 3 to 6 at less than 80% confluence were used in this study.MSCs used in the present study were obtained from male C57BL/6 mouse femurs and tibias and characterized as MSCs as described elsewhere.3.32O3‐Cit for 24 hours. The nanoparticles were initially diluted in DMEM‐LG supplemented with 1% L‐glutamine, 1% antibiotic, and 10% fetal bovine serum, at a concentration of 100 μg iron/mL. Nonmagnetized MSCs were cultured in the same medium, but without γFe2O3‐Cit nanoparticles. Quantitative and qualitative evaluation of γFe2O3‐Cit uptake by MSCs was performed using Prussian blue staining and TEM, as described in previous reports.Magnetization was performed by co‐culturing MSCs with γFe3.4Nonmagnetized and magnetized MSCs were then harvested by trypsinization with 1X TryPLe Express Enzyme (Gibco), and their viability was determined by flow cytometry using a live/dead viability kit , in accordance with manufacturer instructions. Stained cells were then analyzed by flow cytometry ; each reading acquired 20 000 events. Data were analyzed using Flowing software . Tests were performed in triplicate.3.54 cells/well). A pair of permanent rare‐earth (NdFeB) magnets  was then attached to the outside bottom of the plate, exposing cells to their static magnetic fields (SMFs).Nonmagnetized and magnetized MSCs were seeded at 12‐well plates  isolated from the bronchoalveolar lavage fluid of C57BL/6 mice were used. AMs were expanded in culture with Roswell Park Memorial Institute (RPMI) medium (Gibco), supplemented with 1% antibiotic and 10% fetal bovine serum. The AMs were then seeded onto 12‐well plates (3 × 105 cells/well) and cultured in the presence of silica particles   for 24 hours.4 cells/insert, 8 μm pore size) and co‐cultured with the silica‐activated AMs. In addition to chemotactic stimuli from AMs, in the lower chamber, some wells were also exposed to SMFs provided by pairs of magnets. As a negative control, MSCs were cultured without any stimuli.Magnetic responsivity of γFeMSC migration was evaluated after 15 hours. Lower surface of membrane was then fixed in 4% paraformaldehyde in PBS for 15 minutes and stained in 0.5% crystal violet for 10 minutes. The number of migrating cells was determined by counting 40 random fields per well under the microscope at ×400 magnification. Three independent experiments in duplicate were performed.3.66 cells/50 μL saline) or of saline (50 μL) in the internal jugular vein. MT was performed attaching a pair of circular magnets to the chest of each mouse, immediately after injection of magnetized MSCs, with the aid of a surgical tape jacket.Female C57BL/6 mice aged 8 to 10 weeks were anesthetized with sevoflurane, and a 1‐cm‐long midline incision was made to expose the trachea. Silica  was then instilled intratracheally using a 31‐gauge needle. The neck incision was closed with 5‐0 silk sutures and mice returned to their cages.3.75 cells in 50 μL saline); and animals treated with magnetized MSCs plus exposure to magnets for 48 hours. MSC retention after 48 hours was evaluated by ex vivo fluorescent imaging of the lungs and by counting iron‐loaded cells in histology, as described below.Mice were initially divided into two groups: control (n = 6) and silicosis (n = 18). After 15 days, silicotic mice were further randomized into three groups (n = 6/group): animals treated with intravenous administration of saline (50 μL); animals treated with magnetized MSCs , a lipophilic near‐infrared fluorescent dye (absorption/emission: 748/780 nm). DiR was initially dissolved in ethanol, and this solution then mixed with PBS. MSCs were incubated with 32 μg/mL DiR solution in a 37°C incubator for 30 minutes. After incubation, the cells were centrifuged and washed with PBS to remove free dye, in accordance with the manufacturer's instructions.In vivo tracking of MSCs was then performed using an IVIS Lumina XR system . The filters were configured at 710 nm for excitation and 760 nm for emission. Fluorescence images were acquired immediately, 24 hours, and 48 hours after cell transplantation. At 48 hours, mice were euthanized by cervical dislocation under anesthesia and ex vivo imaging of their lungs was performed. Fluorescent signal intensities  were measured and analyzed in Living Image 4.3.1 software. Two independent experiments were performed in triplicate.3.7.2Lungs were fixed with 4% paraformaldehyde in PBS and paraffin‐embedded. Sections (4 μm thick) were obtained and stained by the Prussian blue technique (1867).3.85 cells, suspended in 50 μL of saline, intravenously. Animals from the Sil‐MT group had magnets placed on their chests for 48 hours  and silicosis . After 15 days, animals from the Sil group were further randomized to receive a single dose of saline , nonmagnetized MSCs , magnetized MSCs , or magnetized MSCs plus magnets for the MT technique . Each animal received 3 × 10s Figure .Two and seven days after saline/MSC injection, noninvasive analysis of lung mechanics was performed by whole‐body plethysmography. Nine days after saline/MSC administration, lung mechanics were analyzed invasively, mice were euthanized, and the lungs harvested for histological and molecular biology analysis Figure .3.8.1whole‐body plethysmography, mice were placed conscious and unrestrained in single cylindrical Plexiglas chambers that were connected to a barometric whole‐body plethysmography system  for measurement of enhanced pause responses. Readings were recorded for 10 minutes and data expressed as Penh, an indirect measurement that correlates with airway resistance, impedance, and intrapleural pressure.For 3.8.2Left lungs of mice were fixed with 4% paraformaldehyde, and paraffin‐embedded. Slices (4 μm thick) slices were cut and stained with hematoxylin‐eosin. Then, the fractional area of the lung occupied by collapsed or normal alveoli, as well as percentage of neutrophils, mononuclear cells, and total cells in the alveolar septa, were determined by the point‐counting technique, as described elsewhere.Collagen fiber content was quantified in lung tissue using Masson's trichrome staining.3.8.3Levels of interleukin (IL)‐1β, IL‐10 , and transforming growth factor (TGF)‐β  were quantified by enzyme‐linked immunosorbent assay (ELISA) in lung tissue homogenate as instructed in the manufacturer's protocol, and normalized to the total protein content quantified by Bradford's reagent (Sigma‐Aldrich). Lung tissue was homogenized in lysis buffer  using a bead mill  with a 3‐mm stainless steel bead .3.936B4) and given as the fold change relative to samples from the control group.Reverse transcription followed by quantitative polymerase chain reaction (RT‐qPCR) was performed to measure mRNA expression in MSCs and in lung tissue homogenates. For this purpose, cells/tissues were lysed for RNA extraction through the ReliaPrep RNA Miniprep System  in accordance with manufacturer recommendations. The total RNA concentration and purity was measured by spectrophotometry in a Nanodrop ND‐1000 system . A260/A230 and A260/A280 ratios approximately equal to 2 were considered ideal for RNA purity. First‐strand cDNA was synthesized from 1 μg RNA using a high‐capacity cDNA reverse transcription kit (Thermo Fisher Scientific). Relative mRNA levels were measured by SYBR Green detection (Promega) in a PCR Mastercycler ep Realplex system . All samples were measured in triplicate. The relative level of each gene was calculated as the ratio of the study gene to the control gene , C‐X‐C chemokine receptor type 4 (CXCR‐4), Integrin α4, Monocyte chemoattractant protein‐1 (MCP‐1), Procollagen type I, Procollagen type III, and stromal cell‐derived factor 1 (SDF‐1). Primers 5′‐3′ sequences are presented in Table 3.10t test, one‐way analysis of variance (ANOVA) (followed by Tukey's test), or two‐way ANOVA (followed by Bonferroni's test) were used. If the assumption of normality was rejected, the Mann‐Whitney test or Kruskal‐Wallis test (followed by Dunn's test) was used. Differences were considered statistically significant at P < .05.Statistical analyses were performed in GraphPad Prism version 6.0 . First, the normality of the data was tested using the Shapiro‐Wilk test; then, the ROUT test was performed to identify outliers. If data were normally distributed, Student's 44.12+/Fe3+ molar ratio of approximately 0.05, characterizes the magnetic nanoparticles as maghemite (γ‐Fe2O3). TEM photomicrographs  or free in the cytoplasm , corresponding to the region rich in iron oxide . Resistive pressure was reduced after treatments with nonmagnetized or magnetized MSC; however, Silicosis‐MT animals were the only group in which the difference was significant when compared to Silicosis‐Saline animals  exhibited significantly less alveolar collapse when compared to untreated silicotic animals Figure .MT was the only treatment that significantly reduced granuloma area compared to saline control Figures  and 5.To corroborate these functional and morphological results, we measured the levels of mediators associated with inflammation and fibrosis in lung tissues Figure . MT sign5The results obtained in this study suggest that MT of MSCs improves their therapeutic effects in experimental silicosis. After placing a pair of magnets on the chest of silicotic mice for 48 hours, there was a significant increase in the number of magnetized MSCs retained in their lungs. This higher retention was associated with more pronounced reductions in static lung elastance, resistive pressure, and granuloma.In the context of MSC‐based therapies, MT has been used to promote more effective or focused cell delivery to areas MSCs usually do not reach after systemic administration.2O3‐cit) nanoparticles, which can be synthesized on a large scale, are more colloidal and chemically stable, are less expensive than nanoparticles functionalized with DMSA, and are biocompatible with several cell lines, including MSCs.In a previous study by our group, we observed a significant increase in iron concentration in lung homogenates from silicotic mice 48 hours after administration of magnetized MSCs and application of MT. Although this suggested higher MSC retention, its impact in therapeutic outcomes was not directly assessed.CCR2 and CXCR4 are involved in MSC chemotaxis in response to MCP‐1 and SDF‐1, respectively,In the present study, magnetized MSCs co‐cultured with silica‐activated macrophages transmigrated more when exposed to the SMFs from the magnets. However, the presence of magnetic fields itself is not sufficient to explain significant transmigration Figure . We thenProlonged retention of MSCs in injured lungs may increase secretion of reparative factors at the site of injury. In fact, we observed increased IL‐10 levels in the lung tissue of Silicosis‐MT group animals, while there was a significant reduction in IL‐1β and TGF‐β Figure . Since tSuch attenuation of inflammation and reduction of granuloma area could be the reasons why we observed significant improvement in lung mechanics. During noninvasive analyses of pulmonary mechanics Figure , Penh inDuring invasive analyses of pulmonary mechanics, only animals from the Silicosis‐MT group showed a significant reduction in static lung elastance and resistive pressure compared to silicotic animals treated with saline Figure . InteresNevertheless, such MT effects were not statistically different compared to those observed in Silicosis‐Nonmagnetized MSC Figures  and 6. W2O3 from nanoparticles could be released to the pulmonary microenvironment through EVs,2O3 release is not enough to be detrimental to animals, but is enough to limit MSC therapeutic efficacy. Importantly, we previously described that magnetized MSCs exposed to magnetic fields in vitro maintained associated iron in cytoplasm, unlike unexposed MSCs.2O3 release must be further investigated before implementing MT in clinical practice.Last, treatment with magnetized MSCs without magnet exposure did not lead to significantly reduced collagen deposition in granuloma; values in this group were different than those of animals treated with natural MSCs or with MT Figure . We hypo6In this study, we established an MSC magnetization protocol using citrate functionalized magnetic nanoparticles that kept cells viable and made them magnetically responsive. With the aid of magnets, these magnetized MSCs remained longer in the lungs, and this was associated with reduction of granuloma area and improved lung mechanics—beneficial effects for the treatment of experimentally induced silicosis in mice. Therefore, this MT technique may be a promising strategy to potentialize MSC‐based cell therapies for silicosis.D.J.W. declared research funding from United Therapeutics, Inc. The other authors declared no potential conflicts of interest.L.H.A.S.: conducted the experiments and study, contributed to data collection and analysis, interpreted the data, wrote the first draft; M.C.S., J.B.V., E.C.D.L., R.C.S.: conducted the experiments, contributed to data collection and analysis; D.J.W., M.M.M.: interpreted the data, wrote and edited the manuscript; F.F.C., P.R.M.R.: contributed to idea, conception, and design of study, interpreted the data, edited and reviewed the manuscript. All authors approved the final version of the manuscript.7Supplemental Figure 1 Study design and timeline. At day zero, 45 C57BL/6 mice received intratracheal saline (negative control group ‐ Ctrl) or silica (Sil). After 15 days, once changes in lung histology resembling human silicosis were established, the animals were treated with saline, non‐magnetized MSCs, or magnetized MSCs. Magnetic targeting (Sil‐MT group) was performed by holding a pair of magnets for 48 hours on the anterior chest wall of animals inoculated with magnetized MSCs. Functional evaluations were performed 2, 7, and 9 days after treatments. Lungs were harvested for further analysis at day 24. i.t., intratracheal; i.v.; intravenous.Click here for additional data file.Supplemental Figure 2 Characterization of γ‐Fe2O3‐Citrate nanoparticles (γ‐Fe2O3‐Cit). (A) x‐ray diffraction pattern of samples. (B and C) Representative photomicrographs of transmission electron microscopy. Bars are 50 nm and 5 nm, respectively. (D) Dynamic light scattering analysis. The graph shows size distribution of γ‐Fe2O3‐Cit by intensity, when dispersed in water (red) or in cell culture medium (green), at 100 μg/mL. Three independent samples were read in triplicate (n = 9). (E) Magnetic behavior of γ‐Fe2O3‐Cit. The graph shows the magnetic moment as a function of the magnetic field, at 300 K/27°C. The saturation magnetic moment was 40.1 emu (emu)/g.Click here for additional data file.Supplemental Figure 3 Representative photographs of MSC tracking by in vivo fluorescence imaging. Silicotic mice injected with saline solution are presented on the left of each image, while animals inoculated with magnetized MSCs (labeled with Xenolight DiR) are presented on the right. In the Sil‐MT group, a pair of magnets was secured onto the dorsal thorax of the mice immediately after intravenous injection of magnetized MSCs (0 hour time point) and kept in place for 48 hours.Click here for additional data file.Supplemental Figure 4 Effects of magnetic targeting on lung mechanics. Non‐invasive whole‐body plethysmography. Boxes show the interquartile range, while whiskers encompass the range (minimum‐maximum) of Penh index values (n = 8). (**) Different from Control of respective time point; (†) Different from Silicosis‐Saline of respective time point; (‡) Different from Silicosis‐Non‐magnetized MSC of respective time point; (#) Different from Silicosis‐Magnetized MSC of respective time point.Click here for additional data file.Supplemental Figure 5 Molecular biology analysis. MCP‐1 and SDF‐1 mRNA expression (measured by RT‐qPCR), normalized to control (n = 6). Boxes show the interquartile range (25th‐75th percentile), while whiskers encompass the range (minimum‐maximum).Click here for additional data file.Supplemental table 1 Forward and reverse oligonucleotide sequences of target gene primersClick here for additional data file.Supplemental table 2 Cell viability test by annexin/propidium iodide staining. Data refer to the mean and SD (n = 3) of the percentage of viable, early apoptotic, late apoptotic, and necrotic MSCs.Click here for additional data file.Video S1Click here for additional data file.


Chip global knockout (KO) mice, nuclear factor‐κB signaling is activated, osteoclast formation is increased, osteoblast differentiation is inhibited, and bone mass is decreased in postnatal Chip KO mice. To determine the role of Chip in different cell types at different developmental stages, we created Chipflox/flox mice. We then generated Chip conditional KO mice ChipCMV and ChipOsxER and demonstrated defects in skeletal development and postnatal bone growth in Chip conditional KO mice. Our findings indicate that Chip conditional KO mice could serve as a critical reagent for further investigations of functions of CHIP in bone cells and in other cell types.C terminus of Hsc70‐interacting protein (CHIP) is a chaperone‐dependent and U‐box containing E3 ubiquitin ligase. In previous studies, we found that CHIP regulates the stability of multiple tumor necrosis factor receptor‐associated factor proteins in bone cells. In  In this study, we observed defects in skeletal development and postnatal bone growth in C terminus of Hsc70‐interacting protein (CHIP) conditional knockout mice. All of these steps are regulated precisely by multiple growth factors and signaling molecules. Nuclear factor‐κB (NF‐κB) signaling controls osteoclast formation  is a chaperone‐dependent and U‐box containing E3 ubiquitin ligase. It targets the degradation of proteins critical for multiple cellular functions and signaling pathways. In previous studies, we found that CHIP controls the degradation of multiple tumor necrosis factor receptor‐associated factor proteins and regulates NF‐κB signaling in both osteoclasts and osteoblasts and NF‐κB signaling is activated in Chip in bone and cartilage and other organs in the body, we have generated Chipflox/flox mice. To demonstrate that Chipflox/flox mice have been properly developed, we then created ChipCMV and ChipOsxER conditional KO mice. We analyzed bone phenotype of Chip conditional KO mice and demonstrated that Chipflox/flox mice could be used as an important tool to investigate functions of Chip in multiple tissues at different developmental stages.To study the role of CHIP in bone remodeling and in aging in adult mice and to determine the tissue‐specific effects of 22.1Chipflox/flox mice were generated in Nanjing Biomedical Research Institute . In these mice the Chip gene was floxed at the flanking sites of exon 1 and exon 3. The primer sequences for genotyping Chipflox/flox mice were: P1 (forward primer): 5′‐CATATCTCACCAGGCTCAT‐3′, P2 (reverse primer): 5′‐ACACACAATGACCCACAT‐3′ and P3 (reverse primer): 5′‐GGCCTACCCGCTTCCATTGCTC‐3′. The genotyping result was presented in Figure CMV‐Cre and Osx‐CreER transgenic mice were obtained from the Jackson Laboratory .2.2Embryos at E14.5 and E18.5 were collected and the skin, viscera, and adipose tissues were carefully removed. Whole skeletons were fixed in 95% ethanol for 2 days followed by fixation in acetone for an additional day, and stained with 0.015% alcian blue and 0.005% alizarin red for 3 days. Images of the skeletons were taken when most of the soft tissue was digested in 1% potassium chloride.2.3We used a Scanco microcomputed tomography 35 scanner  with 55 kVp source and 145 μA current for formalin‐fixed mouse legs with a resolution of 10 μm. The scanned images from each group were evaluated at the same thresholds to allow three‐dimensional structural rendering of each sample.2.4Tibiae were harvested and fixed in 10% neutral‐buffered formalin for 3 days and decalcified for 14 days in 14% EDTA and then paraffin‐embedded. Three‐micrometer sections were cut and alcian blue/H&E orange G staining and tartrate‐resistant acid phosphatase (TRAP) staining were performed . The results of a significant decrease in body weight and slightly reduced body size were found in OxsERChip KO mice  is a stress‐responsive protein deacetylase and mono‐ADP ribosyltransferase enzyme encoded by the on Frye, . SirT6 pon Frye, . CHIP prChipflox/flox mice to other fields will make studies of CHIP functions in other organ systems easier.The roles of CHIP in physiological functions and disease initiation and progression have been extensively investigated in recent years. For example, CHIP plays a critical role in neurodegenerative diseases, inflammation, and cardiovascular diseases. Generation and making the availability of The authors declare that there are no conflict of interests.W. W., J. L., F. C. K., X. Z., Y. Q., and R. S. L. carried out experiments. T. W. and D. C. prepared the manuscript, contributed to the experimental design, data interpretation, and finalized the manuscript. D. R. S. revised the manuscript.The animal protocol of this study has been approved by the IACUC of the Rush University Medical Center and all experimental methods and procedures were carried out in accordance with the approved guidelines.


In a patient with very long-chain acyl-Coenzyme A dehydrogenase (VLCAD) deficiency, metabolism of fatty acids is impaired and a supply of alternative energy is limited when glucose level is insufficient on starvation.A 37-year-old woman with VLCAD deficiency was diagnosed with an ovarian cyst and was scheduled for laparoscopic ovarian cystectomy. Glucose was administered intravenously with the start of fasting. Anesthesia was induced with remifentanil, midazolam, and thiamylal, maintained with desflurane and remifentanil. Body temperature was maintained at 36.2–36.7 °C. During anesthesia, hypoglycemia did not occur, creatine kinase levels were in the normal range, and myoglobinuria was not detected. No shivering was observed after extubation.Glucose was administered to avoid perioperative hypoglycemia. Body temperature was controlled to avoid shivering, which would otherwise increase skeletal muscle energy needs. Blood creatine kinase did not increase, and myoglobinuria was not detected; thus, rhabdomyolysis was unlikely to develop. Very long-chain acyl-Coenzyme A dehydrogenase (VLCAD) deficiency is one of the disorders of mitochondrial fatty acid metabolism . β-OxidaA 37-year-old woman  with VLCAD deficiency was diagnosed with an ovarian cyst and was scheduled for laparoscopic ovarian cystectomy. Rhabdomyolysis due to VLCAD deficiency first appeared at age 6 with skeletal muscle pain and myoglobinuria after swimming. Since then, she has exhibited similar symptoms several times a year during fasting, exercise, and mental stress. At the age of 31, VLCAD deficiency was diagnosed by acylcarnitine profile analysis. At the same time, genetic mutations c.1349G>A (p.R450H) and c.1639G>A (p.V547M) were identified in her ACADVL gene.−1 kg−1 h−1 intravenously to prevent hypoglycemia with the start of fasting at 9 p.m. on the night before the surgery. On the day of surgery, when the patient entered the operating room, her blood glucose level was 108 mg dL−1  (Table −1 kg−1 h−1. Anesthesia was induced with 0.5 μg−1 kg−1 min−1 of remifentanil, 3 mg of midazolam, and 200 mg of thiamylal, and muscle relaxation was achieved by 30 mg of rocuronium. After intubation with a cuffed tracheal tube, mechanical ventilation was started, and anesthesia was maintained with 40% oxygen and 5% desflurane in combination with the continuous infusion of 0.25–0.3 μg−1 kg−1 min−1 of remifentanil. Routine monitoring of vital signs and invasive radial artery pressure was performed. Blood glucose, creatine kinase, myoglobinuria, and lactate were monitored during anesthesia (Table −1 kg−1 h−1 until oral intake was started. The blood glucose level of the patient was 142 mg dL−1  about 1 h after the surgery. The patient was discharged 2 days after the surgery.Glucose was administered at 2 mgVLCAD deficiency is one of disorders of fatty acid oxidation in the mitochondria and was first reported in 1993 . VLCAD iSince the present case developed skeletal muscle symptoms at the age of 6 years, it was considered that the residual activity of VLCAD remains moderate. In addition, a R450H ACADVL gene mutation found in this case is often found in patients with skeletal muscle symptoms .−1 and did not result in hypoglycemia (Table The ultimate goal of anesthetic management in a patient with VLCAD deficiency is to prevent rhabdomyolysis by supplying sufficient glucose and suppressing the increase in energy demand. In order to accomplish this purpose, adequate glucose supply and avoidance of increased energy demand by postoperative shivering are necessary. More stringent management of glucose supply and energy demand is required when residual activity of VLCAD is reduced. In this case, supplementation of intravenous glucose infusion was performed continuously from the start of fasting 6 h before the surgery to the start of oral intake. Blood glucose levels ranged from 108 to 190 mg dLShivering generates heat by involuntary movements of skeletal muscles. If the energy demand of skeletal muscles markedly increases, then rhabdomyolysis may occur in the patients. Therefore, the body surface was actively warmed during anesthesia, and the body temperature was maintained around 36.5 °C Table . FortunaAs for anesthetics, benzodiazepines, barbiturates, non-depolarizing muscle relaxants, and opioids have been previously used without problems , 11–13. Iwata et al.  reportedIn conclusion, we experienced anesthesia management in a patient with VLCAD deficiency. Continuous glucose administration was used to avoid perioperative hypoglycemia. Body temperature was controlled to avoid shivering, which would otherwise increase skeletal muscle energy needs. Creatine kinase level did not increase, myoglobinuria was not detected, and thus, rhabdomyolysis was unlikely to develop.


The aim of this study was to evaluate the efficacy of postoperative radiotherapy (PORT) in stage pIIIA‐N2 non‐small cell lung cancer (NSCLC) patients with positive surgical margins.P < 0.05.Between January 2003 and December 2015, patients who had undergone lobectomy or pneumonectomy plus mediastinal lymph node dissection or systematic sampling in our single institution were retrospectively reviewed. Those with pIIIA‐N2 NSCLC and positive surgical margins were enrolled into the study. The Kaplan‐Meier method was used for survival analysis, and the log‐rank test was used to analyze differences between the groups. Univariate and multivariate analyses using Cox proportional hazards regression models were performed to evaluate potential prognostic factors for OS. Statistically significant difference was set as P = 0.568). Subset analysis showed that for patients with R1 resection, the median OS was 52.4 months in the PORT group which was nonsignificantly longer than that of 22.6 months in the non‐PORT group (P = 0.127), whereas PORT combined with chemotherapy could significantly improve OS, with a median OS of 52.4 months versus 17.2 months (P = 0.027). For patients with R2 resection, PORT made no significant difference in OS .Of all the 1547 patients with pIIIA‐N2 NSCLC reviewed, 113 patients had positive surgical margins, including 76 patients with R1 resection and 37 with R2 resection. The median overall survival (OS) was 28.3 months in the PORT group and 22.6 months in the non‐PORT group  has been recommended to treat patients with positive surgical margins. However, the existing evidence is controversial and high‐level evidence is lacking.What this study adds: The PORT group had markedly, but not statistically significant, longer median OS compared with the non‐PORT group in patients with R1 resection. OS was significantly longer in the patients with R1 resection receiving adjuvant CRT than the surgery alone group. Postoperative radiotherapy (PORT) has been recommended to treat patients with positive surgical margins. However, the existing evidence is controversial and high‐level evidence is lacking. In our study, the PORT group had markedly, but not statistically significant, longer median OS compared with the non‐PORT group in R1 resection patients. OS was significantly longer in the R1 resection patients receiving adjuvant CRT than the surgery alone group. Surgery is the mainstay of curative treatment for operable non‐small cell lung cancer (NSCLC). Although intraoperative frozen section evaluation has been routinely performed, some patients are left with microscopic (R1 resection) or macroscopic (R2 resection) residual tumor at the surgical margins, especially among those with pN2,In this retrospective case control study, inclusion criteria were as follows: (i) Patients undergoing surgery in our center between January 2003 and December 2015; (ii) with postoperative microscopic or macroscopic residual disease identified; (iii) pathologically diagnosed as stage IIIA‐N2 NSCLC, according to the seventh edition of the American Joint Committee on Cancer Staging System; and (iv) complete medical records available. Study variables included gender, age, smoking history, tumor location, surgery type, status of surgical margins, histological subtype, tumor size, number of positive lymph nodes and postoperative treatment.Radiotherapy techniques included intensity modulated radiotherapy (IMRT) and three‐dimensional conformal radiotherapy (3DCRT) using 6 MV X‐rays. All patients underwent computed tomography (CT) simulation for PORT. The gross tumor volume (GTV) was defined as the area of residual tumor (those that were well located). The clinical target volume (CTV) enclosed the GTV with a 6 or 8 mm margin, bronchial stump, subcarinal nodes, ipsilateral mediastinum and ipsilateral hilum. The planning target volume (PTV) comprised the CTV with a 5 mm margin. The main dose‐volume constraints (DVCs) for lung were set as follows: V20 <20% for lobectomy or <10% for pneumonectomy. The prescription dose of PTV was 60–66 Gy, with a conventional‐fraction schedule.Platinum‐based double‐agent regimens of 4–6 cycles were recommended for patients, and some were given concurrently or sequentially with PORT.We used the National Cancer Institute Common Toxicity Criteria for Adverse Events version 4.0 (CTCAE 4.0) to assess side effects.P‐value of less than 0.05 was set as the threshold for significance.OS was calculated from surgical resection until death or last follow‐up. Progression‐free survival (PFS) was measured from surgical resection to failure at any site, death or last follow‐up. Local progression‐free survival (LPFS) was calculated from surgery to recurrence at the surgical resection margins, death or last follow‐up. Regional recurrence‐free survival (RRFS) was defined as the duration from surgery to mediastinal relapse , death or last follow‐up. Distant metastasis‐free survival (DMFS) was defined as the time between surgery and recurrence in any other organ, contralateral lobe or malignant pleural/pericardial effusion, death or last follow‐up. SPSS version 25.0 (IBM) was used to perform the statistical analysis. Kaplan‐Meier method was used for survival analysis, and log‐rank test was adopted to analyze differences. Cox proportional hazards regression models were applied in univariate and multivariate analyses to evaluate potential prognostic factors for OS. A Of all the 1547 patients with pIIIA‐N2 NSCLC receiving lobectomy or pneumonectomy plus mediastinal lymph node dissection or systematic sampling, 113 (7.3%) were diagnosed with positive surgical margins, including 76 (67.3%) with R1 resection and 37 (32.7%) with R2 resection. As for the sites of microscopic residual tumors for R1 resection, 70 cases (92.1%) were at the bronchial stumps, three at lymph nodes and three at the vessel walls. For R2 resection, the sites of macroscopic residual tumors were as follows: 22 cases (59.5%) at the bulky metastatic lymph nodes, 11 (29.7%) at the bronchial stumps, four (10.8%) at the vessel walls and one at the pericardium. The median age was 58 (range: 38–83) years. Adenocarcinoma was identified in 57 patients (50.4%), followed by squamous cell carcinoma in 42 (37.2%), adenosquamous carcinoma in seven, large cell carcinoma in four and others in three. Of the 113 patients in this study, 36 (31.9%) received PORT. The demographic and clinicopathological details between the PORT and non‐PORT groups are presented in Table Of all patients included in this study, 89 (78.8%) patients underwent lobectomy and 24 (21.2%) underwent pneumonectomy. For 36 patients receiving PORT, the median total dose of PORT was 60 (range: 45–70) Gy including two patients who failed to complete the prescription dose due to distant metastasis . A total of 46 (40.7%) patients received chemotherapy with a median course of four (range: 1–6). In this study, 24 (21.2%) patients received chemoradiotherapy (CRT), including seven with concurrent CRT, 15 with chemotherapy followed by radiotherapy and two with radiotherapy followed by chemotherapy.P = 0.568, Fig P = 0.466), RRFS  or DMFS  were demonstrated between the PORT and non‐PORT groups.The median follow‐up was 19.8 (range: 0.5–165.8) months. A total of 70 (61.9%) patients died during follow‐up. The median OS was 23.9 months and the median PFS was 17.1 months of all patients. Survival analysis showed that the median OS in PORT group (28.3 months) was a little longer than non‐PORT group (22.6 months) with no significant difference (P = 0.018) and receiving chemotherapy  were significantly associated with better OS. In the multivariate analysis, only age was the independent prognostic factors for OS   Table .P = 0.127, Fig P = 0.027, Fig P = 0.692). In patients receiving CRT, no significant difference was detected in OS between concurrent and sequential CRT .When OS was assessed by surgical margin status, the PORT group had markedly, but not statistically significant, longer median OS of 52.4 months compared with the non‐PORT group of 22.6 months (n = 21) and 36.4% (n = 28), respectively. Six (16.7%) and nine (11.7%) patients in the PORT and non‐PORT groups were diagnosed with local progression. Eleven (30.6%) and 18 (23.4%) patients in the two groups suffered from regional recurrence. Compared with the non‐PORT group, PORT did not change the failure pattern in patients with positive surgical margins  cases of grade 1/2 esophagitis were reported in the PORT group, but for patients receiving concurrent CRT, the rate was relatively higher. Five of seven patients were diagnosed with esophagitis. There was no radiation pneumonitis observed.In this study, although PORT did not improve the overall survival of pIIIA‐N2 NSCLC patients with positive surgical margins, the results still deserve further consideration and also provide reference for clinical practice.et al.et al.According to the newly published NCCN guidelines,P = 0.016), while not in S + R (PORT).P = 0.027). The result is in accordance with our previous study17 which emphasized that only when combined with chemotherapy was PORT able to effectively improve survival of patients who had undergone R1 resection.For patients with R1 resection, the tumor burden is relatively low and the prognosis is favorable compared with R2 resection; therefore, local treatment should be administered more actively to enhance tumor control, coupled with systematic treatment to prolong survival. Our previously published study which included stage I–III NSCLC patients with R1 resection showed that S + R + C (PORT plus postoperative chemotherapy) significantly improved OS, compared with S (surgery) alone  cases were due to bulky metastatic lymph nodes which could not be completely resected. Additionally, postoperative immunosuppression may further contribute to the progression of residual disease.With the intraoperative frozen section evaluationP = 0.037), but not in the multivariate analysis. This in combination with the recurrence pattern mainly seen in patients with distant metastasis indicates that adjuvant chemotherapy should be strengthened or more precise treatment such as adjuvant TKIs for those with EGFR mutations is needed.Our analysis showed that distant metastasis was the most predominant type of relapse, accounting for 58.3% in the PORT group and 36.4% in the non‐PORT group. This reflects the nature of stage III disease, which is more aggressive and prone to distant metastasis, and thus, adjuvant chemotherapy could, in part, compromise the risk of failure. Previous retrospective studies also reported that for patients with positive surgical margins, adjuvant chemotherapy could significantly improve OS.As for the safety profile of PORT, a total of 27.8% patients with grade 1/2 radiation esophagitis were reported and no radiation pneumonitis was observed in our study. The low incidence of PORT‐related adverse events could be due to the strict dose‐volume constraints with IMRT or 3DCRT techniques and one third of patients received radiotherapy without chemotherapy. In patients receiving concurrent CRT, there was a relatively high incidence rate of esophagitis (71.4%), but all of the cases were grade 1/2.As a retrospective analysis, this study had some limitations. First, all patients came from a single institution and the sample size was limited, although to our knowledge, this is the only study aimed at patients with stage pIIIA‐N2 NSCLC with positive margins. Second, the ratio of patients receiving concurrent CRT was low, which was different from the recommendation of the guidelines, by reason of the low patient acceptance and tolerance in Chinese clinical practice. In addition, with the development of immunotherapy and targeted therapy in cancer treatment, the role of local radiotherapy needs to be re‐evaluated.In conclusion, for pIIIA‐N2 NSCLC patients with positive surgical margins in this study, PORT did not improve OS, although was found to improve OS of those with R1 resection when combined with chemotherapy. The result is insufficient to support the recommendation of routine use of PORT. Further prospective studies are warranted to identify the value of PORT for NSCLC patients with positive surgical margins.The authors declare that they have no known competing financial interests or personal relationships.