Social interaction in an alternative context can be beneficial against drugs of abuse. Stress is known to be a risk factor that can exacerbate the effects of addictive drugs. In this study, we investigated whether the positive effects of social interaction are mediated through a decrease in stress levels. For that purpose, rats were trained to express cocaine or social interaction conditioned place preference (CPP). Behavioural, hormonal, and molecular stress markers were evaluated. We found that social CPP decreased the percentage of incorrect transitions of grooming and corticosterone to the level of naïve untreated rats. In addition, corticotropin‐releasing factor (CRF) was increased in the bed nucleus of stria terminalis after cocaine CPP. In order to study the modulation of social CPP by the CRF system, rats received intracerebroventricular CRF or alpha‐helical CRF, a nonselective antagonist of CRF receptors. The subsequent effects on CPP to cocaine or social interaction were observed. CRF injections increased cocaine CPP, whereas alpha‐helical CRF injections decreased cocaine CPP. However, alpha‐helical CRF injections potentiated social CPP. When social interaction was made available in an alternative context, CRF‐induced increase of cocaine preference was reversed completely to the level of rats receiving cocaine paired with alpha‐helical CRF. This reversal of cocaine preference was also paralleled by a reversal in CRF‐induced increase of p38 MAPK expression in the nucleus accumbens shell. These findings suggest that social interaction could contribute as a valuable component in treatment of substance use disorders by reducing stress levels. SI has anti‐stress effects? Rats that express SI CPP, show decreased markers of stress to the level of naive untreated rats. SI preference is potentiated by alpha‐helical CRF (a non‐selective CRF receptor antagonist) but is not affected by CRF icv injections. Cocaine preference is potentiated by icv CRF injections. However, alpha‐helical CRF icv injections or the availability of positive social interaction as an alternative to cocaine, abolish cocaine preference and decrease markers of stress. Thus, SI reward in an alternative context in rats is beneficial against cocaine by reducing stress levels. SI = social interaction; CPP = conditioned place preference; icv = intracerebroventricular; CRF = corticotropin‐releasing factor. Indeed, p38 MAPK was reported to be activated after stressAn important question that needs to be addressed is whether the beneficial effects of social interaction reward are mediated through a reverse of stress levels. For that, we analysed stress markers specifically altered behavioural transitions of grooming, urine corticosterone levels, and CRF expression, after cocaine or social CPP expression in rats. With the purpose of examining whether social interaction CPP could also be modulated by the CRF system, we investigated the modulation of CPP to cocaine and social interaction by intracerebroventricular (icv) injections of CRF or alpha‐helical CRF, a nonselective antagonist of CRF receptors. In order to check for potential beneficial protective effects of social interaction against drug abuse and stress, we investigated the availability of social interaction as mutual choice in an alternative context on CRF‐induced effects on cocaine preference and on the subsequent p38 MAPK activation in the nucleus accumbens shell.22.1Male Sprague Dawley rats  were housed at controlled environmental conditions (12‐hour light/dark schedule) with food and water supplied ad libitum. Experiments were performed during the light phase of the circadian cycle. All animals were group‐housed until 10 days before the start of the behavioural experiments, from which time on they were single housed.The Austrian National Animal Experiment Ethics Committee approved all experiments in the present study (BMWF‐66.011/0112‐WF/V/3b/2015).2.2The CPP apparatus (64 cm wide × 32 cm deep × 31 cm high) is made of unplasticized polyvinyl chloride and consists of a three‐compartment chamber with a middle  compartment (10 × 30 × 30 cm) connected to the two outer/conditioning compartments (25 × 30 × 30 cm each). The compartments differ in floor (white floor in the middle compartment/stainless steel floor with either holes or stripes in the two conditioning compartments) and in wall .The acquisition protocolIn the concurrent CPP paradigm, the protocol started with a pretest session on day 1 followed by eight consecutive training days in an alternate‐day‐design, one 15‐minute training session per day, a total of four training sessions each for social interaction in one compartment or cocaine in th7e other compartment of the CPP, then a CPP test on day 10.Time that animals spent in each compartment in the pretest and test was recorded and evaluated using Any‐Maze Software.Preference for a stimulus was defined by the time spent in the stimulus‐associated compartment during the test minus the time spent in the stimulus‐associated compartment during the pretest (preference score). For the saline control group, the preference score was calculated as the time spent in the less preferred compartment during the test minus the time spent in the less preferred compartment during the pretest.2.3Grooming is an important element of rodent behaviour with a general pattern of cephalocaudal progression . Incorrect transitions of cephalocaudal progression between different grooming patterns can be used as a stress marker in rats.2.4Twenty‐four hours after the CPP test,2.5Twenty‐four hours after the CPP test, rats were sacrificed by an overdose of isoflurane. This time point was chosen according to our previous findingsBrains were removed and immediately frozen in −40°C isopentane. The amygdala, BNST, the hypothalamic paraventricular nucleus, and the nucleus accumbens shell were punched out using a sample corer  while viewing sections using magnifying goggles from thaw‐mounted coronal 200‐μm sections at −15°C in a cryostat.Total RNA was isolated from the dissected brain regions using Trizol (Invitrogen) according to the manufacturer's recommendations. To avoid contamination with genomic DNA, total RNA was treated with DNase (2 U/μL) using the TURBO DNA‐free Kit (Ambion). RNA was reverse transcribed in the presence of random hexamer primers and MultiScribe Reverse Transcriptase (50 U/μL) in a total volume of 20 μL employing the high‐capacity cDNA reverse transcription kit with RNase Inhibitor (Applied Biosystems).After dilution with 80 μL of water, 3 μL of the diluted cDNA was used as a template for amplification (duplicates) with AB Fast SYBR Green mastermix (Applied Biosystems). Real‐time polymerase chain reaction (RT‐PCR) quantification was performed on a 7500 Fast Real‐Time PCR system (Applied Biosystems) using the following cycle settings: 20 seconds 95°C, 40 cycles of 95°C for 3 seconds, and 60°C for 30 seconds. All PCR primers were designed using PrimerSelect 5.05 software (DNASTAR). Ct and ΔCt values were calculated by using the 7500 software v2.0.1 and GAPDH as a reference gene.The primers used in this study are detailed in Table 2.6Rats were anesthetized with isoflurane. Then, each rat was implanted with a 22‐gauge guide cannula above one of the lateral ventricles. The stereotaxic coordinates used were −0.9 mm from bregma, 1.4 mm lateral from the midline, and −3.0 mm from the skull surface. The incisor bar was set at −3.5 mm. The cannulae were secured by dental cement and anchored to stainless steel screws fixed to the skull. Postoperative analgesia was provided to the animals during the recovery period. The animals were allowed to recover for 6 days from surgery.Human/rat CRF, alpha‐helical CRF (9‐41), and SB203580 were purchased from Tocris.CRF (1 μg/rat) was dissolved in physiological saline; alpha‐helical CRF (10 μg/rat) was dissolved in distilled watericv injections of CRF and alpha‐helical CRF were administered 1 hour prior to conditioning. icv injections of SB203580 were administered 30 minutes prior to conditioning.For cocaine CPP and social CPP, injections were given before cocaine or social interaction conditioning. In control rats, injections were given before conditionings in the less preferred compartment. In concurrent CPP, injections were given prior to both cocaine and social interaction conditionings.A general time line for the experiments performed in the study is represented in Figure 2.7t test using Graphpad Prism and Statview programs were used. Results showing significant overall changes were subjected to Tukey's multiple comparison post hoc test. Effect sizes were calculated as Cohen's d. The level of statistical significance was predefined at a P < .05.All data were expressed as mean ± SEM (standard error of the mean). According to the number of effects implicated, one‐ or two‐way analysis of variance (ANOVA) or two‐tailed unpaired 33.1F = 8.504; P = .0016; control [n = 8] vs cocaine CPP [n = 11], P = .0021, Cohen's d = 4.86; control vs social CPP [n = 8], P = .008, Cohen's d = 4.84). Cocaine and social interaction had the same reward value as the preference score after cocaine or social interaction conditioning was not different   = 14.73; P < .0001; naïve [n = 10] vs control [n = 7], P = .0002, Cohen's d = 3.08; naïve vs cocaine CPP [n = 11], P < .0001, Cohen's d = 2.38). However, the percentage of incorrect transitions in the social interaction CPP group was significantly decreased to the level of naïve rats   = 11.91; P < .0001; naïve [n = 7] vs control [n = 6], P = .0003, Cohen's d = 2.07; naïve vs cocaine CPP [n = 9], P = .0037, Cohen's d = 2.95). However, corticosterone levels in urine after social CPP were not different from naïve rats   = 10.12, P < .0001; marker effect, F = 3.735, P = .0313; treatment × marker interaction, F = 2.996, P = .0151; naïve [n = 4] vs cocaine CPP [n = 5], P < .0001, Cohen's d = 1.84; control [n = 4] vs cocaine CPP, P < .0001, Cohen's d = 1.81; cocaine CPP vs social CPP [n = 5], P < .0001, Cohen's d = 1.65). Expression of CRFR1 and CRFR2 in the BNST region did not differ among the groups  = 42.92, P < .0001; icv treatment effect, F = 4.474, P = .0176; CPP treatment × icv treatment interaction, F = 24.54, P < .0001)  Figure A‐C.P < .0001, Cohen's d = 3.08; CRF vs alpha‐helical CRF [n = 5], P < .0001, Cohen's d = 5.00). Alpha‐helical CRF injections did not affect the CPP in the saline control group of rats  , whereas icv alpha‐helical CRF injections decreased cocaine CPP significantly  , but icv alpha‐helical CRF injections increased social CPP   = 16.04, P < .0001; icv treatment effect, F = 15.75, P < .0001; CPP treatment × icv treatment interaction, F = 3.970, P = .0083) . However, icv alpha‐helical CRF injections decreased significantly the percentage of incorrect transitions   and decreased by icv alpha‐helical CRF injections    = 8.551, P = .0007; CRF vs CRF + [social] [n = 8], P = .0005, Cohen's d = 3.83; alpha‐helical CRF vs CRF+ [social], P = .8829, n.s.)  = 22.66, P < .0001; CRF vs CRF + [social] [n = 8], P < .0001, Cohen's d = 3.83; alpha‐helical CRF vs CRF + [social], P = .9950, n.s.)  = 39.35, P < .0001; vehicle [n = 4] vs CRF [n = 5], P = .0004, Cohen's d = 3.33; vehicle vs alpha‐helical CRF, P = .0101, Cohen's d = 3.13; CRF vs alpha‐helical CRF [n = 5], P < .0001, Cohen's d = 4.89; CRF vs CRF + [social] [n = 5], P < .0001, Cohen's d = 5.09; alpha‐helical CRF vs CRF + [social], P = .9418, n.s.)   in a concurrent CPP paradigm, in which rats received cocaine conditionings in one compartment and social conditionings in the other compartment of the CPP, were performed. SB203580 did not induce a shift in preference to any of the compartment associated with either cocaine or social interaction (two‐tailed unpaired ) Figure .4The main finding of the present study is that social interaction reward decreased stress markers at the behavioural, hormonal, and molecular levels. In addition, social interaction CPP was promoted by CRFR antagonist and alpha‐helical CRF but not affected by CRF. Furthermore, social interaction, when available as an alternative choice to cocaine and CRF, was able to reverse not only cocaine preference but also CRF‐induced increased levels of stress.The percentage of incorrect transitions can be used as a behavioural stress marker in rats behaviour.Several studies have shown that social stress strongly increases basal corticosterone levelsOne of the most important neurotransmitters involved in behavioural components of the stress response is the CRF.CRF, CRFR1, and CRFR2 expression in the amygdala and the hypothalamus was not different among the investigated groups. This is in line with other studies showing changes in CRF mRNA only in BNST but not in the amygdala. For example, environmental switch from enriched to standard environment, which produces a negative emotional state, is associated with increased levels in mRNA levels of CRF only in the BNST but not in the amygdala.In order to investigate the effects of enhancing stress levels on CPP, we investigated the effect of CRF icv injections on saline control, cocaine CPP, and social CPP rats. We found that saline control rats injected with vehicle did not express any preference for any of the compartments of the CPP. However, saline control rats that received icv CRF expressed conditioned place aversion to the compartment associated with CRF injections as shown by Cador et al.The CRF system has been shown to have major roles in drug‐seeking behaviour.On the other hand, pharmacological activation of the CRF system has been shown to decrease social interaction.When drug and social interaction rewards were presented as a series of mutually exclusive choices, it was reported that operant social reward eliminated drug self‐administration, even in rats that met criteria for addiction.Previous studies have shown that p38 MAPK antagonists impair the amphetamine‐induced CPPStress is a key risk factor in the development of addiction and in addiction relapse, and hence treatment failure.CL and AS performed the experiments with the help of IMA and VF. RER and CL designed the experiments and interpreted the data. RER analysed the data and drafted the manuscript. NS, GD, and AH provided critical revision of the manuscript for important intellectual content. All authors critically reviewed content and approved final version for publication.The authors declare that they have no conflicts of interest.Figure S1: Effects of icv injections of SB203580 on concurrent CPP (n=7‐11). Rats received icv injection of either VEH or SB before each cocaine and social interaction conditioning. Preference score is the time that the rat spent in the cocaine‐associated compartment during the test – pretest. VEH (n=7); SB (n=11). Statistical test two‐tailed unpaired t‐test. VEH= Vehicle; SB= SB203580Click here for additional data file.Figure S2: Time in the three compartments of the CPP during pretest. Overall, there was no difference in the time spent between the vertical and the horizontal compartments of the CPP in any of the groups used in the study. Statistical test: one‐way ANOVA, effect: time spent in compartments, followed by Tukey's post hoc test. Pretest future Sal CTR, Vertical vs horizontal, p=0.8274; Pretest future cocaine, Vertical vs horizontal, p=0.9996; Pretest future SI, Vertical vs horizontal, p=0.9986; Pretest future Sal CTR – with surgery, Vertical vs horizontal, p=0.4983; Pretest future cocaine – with surgery, Vertical vs horizontal, p=0.9871; Pretest future SI‐ with surgery, Vertical vs horizontal, p=0.8359. Control=Sal CTR; SI= Social CPP.Click here for additional data file.Table S1: CRF, CRFR1 and CRFR2 expression in the hypothalamus and the amygdala 24 hours after the CPP test (n=4). Naïve rats are untreated rats. Control=Sal CTR; COC= cocaine CPP; SI= Social CPPClick here for additional data file.Table S2: List of Primers used in the q‐PCR experiment.Click here for additional data file.


Skeletal muscle is a plastic tissue that can adapt to different stimuli. It is well established that Mammalian Target of Rapamycin Complex 1 (mTORC1) signalling is a key modulator in mediating increases in skeletal muscle mass and function. However, the role of mTORC1 signalling in adult skeletal muscle homeostasis is still not well defined.Inducible, muscle‐specific Raptor and mTOR k.o. mice were generated. Muscles at 1 and 7 months after deletion were analysed to assess muscle histology and muscle force.P < 0.01), muscle fibrillation, and neuromuscular junction (NMJ) fragmentation in 24% of examined fibres (P < 0.001). Mechanistically, reactivation of autophagy with the small peptide Tat‐beclin1 is sufficient to prevent mitochondrial dysfunction and the appearance of NCAM‐positive fibres in Raptor k.o. muscles.We found no change in muscle size or contractile properties 1 month after deletion. Prolonging deletion of Raptor to 7 months, however, leads to a very marked phenotype characterized by weakness, muscle regeneration, mitochondrial dysfunction, and autophagy impairment. Unexpectedly, reduced mTOR signalling in muscle fibres is accompanied by the appearance of markers of fibre denervation, like the increased expression of the neural cell adhesion molecule (NCAM). Both muscle‐specific deletion of mTOR or Raptor, or the use of rapamycin, was sufficient to induce 3–8% of NCAM‐positive fibres (Our study shows that mTOR signalling in skeletal muscle fibres is critical for maintaining proper fibre innervation, preserving the NMJ structure in both the muscle fibre and the motor neuron. In addition, considering the beneficial effects of exercise in most pathologies affecting the NMJ, our findings suggest that part of these beneficial effects of exercise are through the well‐established activation of mTORC1 in skeletal muscle during and after exercise. In most cell types, the kinase mTOR is a major regulator of cellular metabolism, protein synthesis, and turnover.While these findings clearly show an important role of mTOR signalling during adult muscle hypertrophy, results obtained from transgenic mice show a less straight‐forward role of mTOR signalling in muscle growth. Deletion of Rictor specifically from skeletal muscle does not compromise muscle growth or function, while muscle‐specific ablation of Raptor from birth leads to a myopathy around 8 weeks of age and premature death.With regard to pathology, reduction of mTORC1 signalling by rapamycin is beneficial for numerous diseases, like certain cancers, aging, or neurological diseases like Huntington's.In order to identify the functional role of mTORC1 signalling during adult muscle homeostasis, we generated inducible muscle specific knockout mice in which Raptor or mTOR are acutely and specifically deleted in adult animals. Interestingly, we observed that loss of mTORC1 signalling for 1 month is not sufficient to induce a muscle phenotype. However, adding pharmacological mTORC1 inhibition to Raptor k.o. and mTOR k.o. mice was sufficient to induce a very rapid myopathy, while not affecting wild‐type mice, suggesting the presence of residual mTORC1 signalling in the Raptor k.o. and mTOR k.o. 1 month after deletion, which is sufficient for maintaining muscle homeostasis.Deletion of Raptor for longer periods, however, leads to a significant myopathy, characterized by central nuclei, mitochondrial dysfunction, and reduced muscle function, but without premature death after 7 months of deletion. Surprisingly, while rapamycin treatment leads to the expected increase in autophagy, long‐term Raptor k.o. mice showed an impairment of both bulk autophagic flux and mitophagy flux. Surprisingly, this block of autophagy and consequently increased mitochondrial dysfunction significantly contributes to an instability of the neuromuscular junction. Indeed, both loss of mTOR and Raptor, or the use of rapamycin, are sufficient to induce the appearance of NCAM‐positive fibres, a marker of fibre denervation, and neuromuscular junction degeneration. Importantly, prolonged loss of mTORC1 leads to muscle fibrillation and a significant NMJ fragmentation. Reactivation of autophagy using the Tat‐beclin1 peptide is able to prevent mitochondrial dysfunction and the appearance of NCAM‐positive fibres. Taken together, these results show that basal mTORC1 signalling in adult muscle fibres is necessary for the maintenance of the NMJ.fl/fl or mTORfl/fl, Jackson Labs) with mice carrying Cre recombinase fused to a mutated estrogen receptor domain under the control of human skeletal actin promoter.Inducible, muscle‐specific Raptor knockout mice and mTOR knockout mice were generated crossing mice expressing Raptor gene or mTOR gene between two LoxP sites ; 10 μm cryosections of TA or gastrocnemius muscles were stained for H&E, PAS, and for succinate dehydrogenase. Dystrophin was used to determine fibre CSA measurement, by using ImageJ software .Twenty micrometres cryosections of frozen TA or gastrocnemius muscles were lysed in a buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 10 mM MgClWe used the following antibodies for western blotting: P‐AKT , P‐S6 , S6 (ref. 2217), AKT (ref. 9272), GSK‐3β (ref. 9315), P‐GSK‐3β , P‐4E‐BP1 , P‐4E‐BP1 , 4E‐BP1 (ref. 9644), RAPTOR (ref. 2280), RICTOR (ref. 2114), mTOR (ref. 2983), P‐AMPK , P‐ULK1 , SDH (ref. 11998) from Cell Signalling, LC3 (ref. L7543) and p62 (ref. P0067) from Sigma, GAPDH (ref. 8245), Mitoprofile (ref. 110413) and TOM20 (ref. 56783) from Abcam, actin (ref. 56459), and GRP‐75 (ref. 13967) and Porin1 (ref. 390996) from Santa Cruz. For immunofluorescence, LAMP1 (ref. 1D4B), embryonic MyHC (ref. BF‐G6), and types I, IIa, and IIb MyHC  were from Developmental Studies Hybridoma Bank, mTOR was from Cell Signalling, distrophin (ref. 15277) from Abcam, NCAM (ref. 5032) was from Millipore, and α‐bungarotoxin Alexa Fluor 555‐conjugate (ref. B35451) was from Invitrogen.Sample preparation and in‐solution digest, frozen tissue was cryogenically ground using a mortar and pestle and resuspended in ice‐cold lysis buffer  with protease inhibitor cocktail. After homogenization on a rotating wheel at 4°C for 30 min and sonication the proteins where precipitated with ice‐cold acetone at −20°C over night. The next day, 50 μg of protein dissolved in 8 M urea were reduced with 5 mM DDT for 60 min, followed by carbamidomethylation with 40 mM iodoacetamide for 45 min in the dark at room temperature. A predigest with the protease LysC  was performed for 3 h followed by a digest with trypsin (Promega) for 16 h. The resulting peptides were separated from salts and detergents using the C18‐based Stop and Go extraction tips.6 with a maximum injection time of 20 ms. The dynamic exclusion was set to 20 s. The MS2 measurements were acquired using a resolution of 17 500 at 200 m/z with a top 10 data dependent mode. Here, the automated gain control target was set to 5 × 105 with an injection time of 60 ms. The normalized collision energy in the HCD cell was 25.An Easy nLC 1000 ultra‐high performance liquid chromatography coupled to a QExactive Plus Hybrid Quadrupole‐Orbitrap mass spectrometer (Thermo Fisher Scientific) was used for the proteomic analysis with the following settings. Peptides were fractionated using in‐house made 50 cm columns packed with 1.7 μm C18 beads using a binary buffer system, consisting of Buffer A (0.1% FA) and Buffer B (80% ACN in 0.1% FA). All samples were analysed over a 240 min gradient, raising the content of Buffer B from 5% to 34% over 215 min and then from 34% to 55% over 5 min, followed by washing with 90% Buffer B. Spectra for full MS were acquired at a resolution of 70 000 at 200 m/z and the automated gain control target was set to 3 × 10t‐tests, and data visualization were performed using the Perseus software and the Instant Clue software.The raw data were analysed using the Andromeda search engine implemented in the MaxQuant software 1.5.3.8.in vivo using a 305B muscle lever system (Aurora Scientific Inc.). Force was normalized to the muscle mass as an estimate of specific force. Animals were then sacrificed by cervical dislocation, and muscles were dissected, weighted, and frozen.Gastrocnemius muscle force was measured in living mice as previously described.Resting EMGraphic activity was recorded by inserting one needle electrode in the middle region of TA muscle and the second electrode close to the tendon region. Ground electrode was placed around the tail. Recordings were made at room temperature (20–22°C). Calibration bars in Raptor k.o. mice is the same as in wild‐type mice.Mitochondrial membrane potential was measured in isolated fibres from the Flexor Digitorum Brevis (FDB) muscles as previously described.To analyse mitophagy flux, we used mitoKEIMA. Electroporation experiments were performed on FDB muscles from wild‐type and knockout animals. Muscles were analysed 12 days later. FDB muscles were collected in 1% P/S Dulbecco's modified Eagle's medium, dissociated and plated on glass coverslips coated with 10% Matrigel in Tyrode's salt solution (pH 7.4). Mitochondria‐targeted mitoKeima plasmid (mito‐Keima)  was used to monitor mitophagy in transfected FDB single fibres. mito‐Keima is a coral‐derived protein that exhibits both pH‐dependent excitation and resistance to lysosomal proteases. These properties allow rapid determinations to whether the protein is in mitochondria or the in lysosome.Muscle mitochondria from the indicated genotype were isolated from quadriceps muscles as described.Total RNA was extracted from muscles using TRIzol (Invitrogen). Complementary DNA was generated from 0.4 μg of RNA reverse‐transcribed with SuperScript III Reverse Transcriptase (Invitrogen). cDNA samples were then amplified on the 7900HT Fast Real‐Time PCR System (Applied Biosystems) using the Power SYBR Green RT‐PCR kit (Applied Biosystems). All data were normalized to HPRT and to GAPDH expression.4 and 0.8% K3Fe(CN)6 for 1–2 h, rinsed with 0.1 M sodium cacodylate buffer with 75 mM CaCl2, en‐block stained with saturated uranyl acetate, and embedded for EM in epoxy resin (Epon 812). Ultrathin sections (~40 nm) were cut in a Leica Ultracut R microtome  using a Diatome diamond knife  and examined at 60 kV after double staining with uranyl acetate and lead citrate, with a FP 505 Morgagni Series 268D electron microscope , equipped with Megaview III digital camera  and Soft Imaging System (Germany).For EM, EDL and Soleus muscles were dissected from sacrificed animals, pinned on a Sylgard dish, fixed at room temperature with 3.5% glutaraldehyde in 0.1 M NaCaCO buffer (pH 7.4), and stored in the fixative at 4°C. Fixed muscles were then post‐fixed in a mixture of 2% OsORaptor knockout and wild‐type mice performed 1 day of concentric exercise on a treadmill , at increasing velocity, according to the protocol of acute exercise previously described.t‐test and one‐way or two‐way analysis of variance (ANOVA), using Tukey's multiple comparison test. Significance was defined as a P‐value of less than 0.05.All data are expressed as means ± SEMs. Differences between groups were assessed using Student's FigureFigureFigureFigureFigureFigurein vivo produced by the plantar flexors and muscle histology by H&E staining are the same for both groups.In order to address the role of Raptor, and therefore mTORC1, in adult skeletal muscle, we generated a tamoxifen inducible, muscle‐specific Raptor k.o. mouse. We crossed a line expressing a tamoxifen‐inducible Cre placed under a human skeletal actin promoter, with a line containing two LoxP sites on exon 6 of the Raptor gene  rapamycin has off target effects, which are accentuated in Raptor k.o. mice; (ii) there are Raptor‐independent, mTOR‐dependent effects of rapamycin; and (iii) there is a residual amount of mTORC1 activity, which is sufficient to maintain most of its basic tasks. Because rapamycin is very specific in its effects on mTOR, we wondered if there could be a Raptor‐independent, mTOR‐dependent effect. To address this issue, we generated mice in which we crossed the inducible, muscle‐specific Cre‐line to a line expressing two LoxP sites flanking exon 1‐5 of the mTOR gene. As can be seen in Supporting Information, FigureFigureFigureFigureFigureIt has been reported that loss of Raptor from skeletal muscle leads to a myopathic phenotype, which starts manifesting itself after the postnatal growth period, culminating in premature death between 5–7 months of age.n vivo approach. As can be seen in FigureFigureFigureFigureFigureFigureFigureFigureThe previously reported conditional muscle‐specific Raptor k.o. mice showed a lethal myopathy, despite the fact that grip strength and specific muscle force of either EDL or soleus muscles was unaffected.FigureFigureFigureFigureFigureFigureFigureFigureMitochondrial dysfunction can occur when organelle turnover is reduced. In addition to regulating protein synthesis, mTORC1 activation is also a well‐established inhibitor of autophagy, which is critical for mitochondrial turnover. In order to assess how long‐term loss of Raptor affects muscle autophagy, we evaluated the inhibitory phosphorylation of mTORC1 on ULK1, an important step in autophagy induction. As can be seen in FigureFigureFigureFigureFigureFigureFigureOne of the most striking features of long‐term Raptor deletion is the presence of numerous small, angulated fibres FigureA. IntereFigureFigureFigureFigureNext, we analysed the histology of the neuromuscular junction in the most affected group, that is, after 7 months of Raptor deletion. In the images shown in FigureFigureFigureBecause mitochondrial dysfunction and a block in autophagy are linked to NMJ dysfunction,One of the major players in the regulation of adult muscle mass and function is mTORC1.A surprising finding in this study was the fact that deletion of either Raptor or mTOR from skeletal muscle for 1 month does not lead to a muscle myopathy or muscle wasting. It was reported that electroporation of a siRNA leads to a reduction in fibre size 4 to 6 weeks after electroporation.While we did not observe any basal atrophy, we do observe an important number of regenerating fibres and a significant drop in muscle force when administering rapamycin for only 2 weeks in Raptor k.o. mice. While these observations can have multiple interpretations, the most likely explanation is that low levels of residual mTORC1 can maintain a lot of the basic cell functions. Indeed, the muscle‐specific deletion of mTOR or Raptor from birth does not impair postnatal growth. Considering that this early growth period is characterized by an important activation and fusion of satellite cells, it is possible that these newly added myonuclei, in which mTOR and Raptor are not deleted, maintain mTORC1 levels sufficiently high to sustain muscle growth. Indeed, the combination of muscle‐specific deletion of mTOR, with the concomitant expression of a kinase inactive form of mTOR, is sufficient to significantly reduce body growth and lead to premature death around 8 weeks of age.Taken together, our results suggest that reduced mTORC1 activation levels are able to maintain skeletal muscle homeostasis for short periods; however, complete inhibition leads to a very rapid loss in muscle histology and function.FigureOne interesting finding from conditional muscle‐specific Raptor and mTOR k.o. mice is that the loss of muscle mTORC1 signalling reduces longevity. Indeed, no conditional Raptor k.o. mice survive for more than 5–6 months. This reduced survival, however, is difficult to explain by muscle dysfunction, as normalized force and resistance to fatigue are not reduced, and muscle atrophy in the diaphragm is less pronounced than in other myopathies, which are compatible with life.The role of mTORC1 signalling in the regulation of autophagy in skeletal muscle has been debated. More than 10 years ago, it was shown that FoxO transcription factors are able to induce autophagy, independently of mTORC1 signalling.Taken together, these results suggest that mTORC1 inhibition is important for the induction of autophagy; however, long‐term mTORC1 inhibition leads to a block in autophagic flux.One of the most surprising findings in this study was the observation that loss of muscle mTORC1 signalling during homeostasis was sufficient to induce fibre denervation. This study adds mechanistic insights to some recently published papers in which genetic alterations in muscle fibres is sufficient to impact the NMJ, without directly affecting the motor neuron.In addition to reducing autophagy, also the inhibition of protein synthesis in muscle fibres is sufficient to affect NMJ stability and the presynaptic component.Here, we use various genetic and pharmacological loss‐of‐function approaches to show that mTORC1 signalling in adult skeletal muscle plays a critical role in muscle autophagy, force production, and maintenance of the neuromuscular junction. Its role in the NMJ is particularly intriguing, as numerous pathologies affecting the NMJ stability are significantly improved by exercise,B. B. conceived the project and wrote the manuscript. M. B., A. G., M. P., L. N., C. T., H. N., V. R., A. M., and S. B. performed experiments and analysed data. M. S. and M. K. contributed intellectually and provided technical advice. All authors discussed the results and commented on the manuscript.The authors declare that they have no conflict of interest.Figure S1.A) Western blot for 4E‐BP1 and relative quantification. Phosphorylation of 4E‐BP1 on Ser65 and Thr37/46 is reduced upon Raptor deletion B) Western blot for IRS‐1 and relative quantification. Increased IRS‐1 protein levels in Raptor k.o. mice 1 month after Raptor deletion C) No colocalization between mTOR and Lamp2 (n = 4‐6 muscles/group). Data are shown as mean ± SEM. Statistical analysis was performed using two‐tailed Student t‐test. Statistical significance: *P < 0.05, **P < 0.01Figure S2. Efficient reduction of mTOR transcript (A) and protein level in the inducible mTOR k.o. mice (n = 4‐5 muscles/group). No effect on muscle weight two months after mTOR deletion. Data are shown as mean ± SEM. Statistical analysis was performed using two‐tailed Student t‐test. Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001Figure S3. No change in epididymal fat or heart weight between wt and Raptor k.o. longterm mice Figure S4. A) Volcano plot of the differences in the proteome 1 month after Raptor deletion. Mitochondrial proteins are indicated in red B) No reduction in mitochondrial number as evidenced by mitoprofile and blots for TOM20 and SDH (n = 4‐5 muscles/group) C) Treadmill performance of Raptor k.o. mice is significantly impaired (n = 4 mice/group). Data are shown as mean ± SEM. Statistical analysis was performed using two‐tailed Student t‐test. Statistical significance: *P < 0.05Figure S5. Expression levels of genes involved in the autophagy‐lysosome system and the ubiquitin‐proteasome system in wildtype and long‐term Raptor k.o. muscles (n = 6/group). Data are shown as mean ± SEM. Statistical analysis was performed using two‐tailed Student t‐test. Statistical significance: *P < 0.05, **P < 0.01Figure S6. Representative image of NCAM‐positive fibers (red) in Raptor k.o. longterm miceClick here for additional data file.


Sander vitreus). Lake Winnipeg walleye represent the largest component of the second‐largest freshwater fishery in Canada. In the present study, large female walleye were sampled via nonlethal gill biopsy over two years at three spawning sites representing a latitudinal gradient in the lake. Genetic variation from sequenced mRNA was analyzed for neutral and adaptive markers to investigate population structure and possible adaptive variation. We find low population divergence (FST = 0.0095), possible northward gene flow, and outlier loci that vary latitudinally in transcripts associated with cell membrane proteins and cytoskeletal function. These results indicate that Lake Winnipeg walleye may be effectively managed as a single demographically connected metapopulation with contributing subpopulations and suggest genomic differences possibly underlying observed phenotypic differences. Despite its high cost relative to other genotyping methods, RNA sequencing data can yield physiological in addition to genetic information discussed here. We therefore argue that it is useful for addressing diverse molecular questions in the conservation of freshwater species.RNA sequencing is an effective approach for studying aquatic species yielding both physiological and genomic data. However, its population genetic applications are not well‐characterized. We investigate this possible role for RNA sequencing for population genomics in Lake Winnipeg, Manitoba, Canada, walleye  called from RNA seq data, 386 showed signatures of selection across a latitudinal gradient in Lake Winnipeg, Manitoba, Canada. Lake Winnipeg is characterized by a north and a south basin connected by a narrow channel . With n = 16 individuals per site over both years, sample sizes can predict mean population allele frequency by site, because sample sizes as low as n = 12 have been shown to be sufficient with RNA sequencing data  in accordance with approved animal use protocols of Fisheries and Oceans Canada , the University of Manitoba (F2018‐019), and the University of Nebraska‐Lincoln (Project ID: 1208). Fish were sampled nonlethally for gill tissue, where 2–3 mm of the terminal ends of 3–4 filaments from the left side each fish were collected and placed in RNAlater (Thermo Fisher Scientific) that was kept at 4°C for 24 hr prior to storage at −80°C. Total RNA extractions were performed on gill tissue using RNeasy Plus Mini Prep Kits (QIAGEN) following manufacturer's protocols, with minor modifications (provided in the Gill tissue was collected from large ≥1.2 kg) predominately female  Table  walleye .2 kg prehttp://gqinnovationcenter.com) for cDNA library preparation and sequencing. A mRNA isolation approach was performed on 250 nanograms of total RNA using the NEBNext Poly(A) Magnetic Isolation Module (New England BioLabs). From mRNA, stranded cDNA libraries were created using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs). Each library was individually barcoded with NEBNext dual adaptors (New England Biolabs) prior to sequencing 100 base pair paired‐end reads. All 48 fish were sequenced on a single lane of a NovaSeq 6,000 (Illumina). 2.17 billion reads total were sequenced, with an average of 45,225,548 reads per sample collected   and electrophoresis on a 1% agarose gel, respectively. Total RNA was normalized to 80 ng/µl and sent to the McGill University and Génome Québec Innovation Centre sequencing facility (D) Table .2.2https://www.westgrid.ca/). Read files were checked for quality using FastQC version 0.11.8  using information from Corset and the original transcriptome, where 263,272 unique gene features were identified using Trinotate   Table .p‐value < .005 . Ten replicates of K = 2–5 were tested.To investigate population structure using the 52,372 putatively neutral SNPs, we used a combination of exploratory analyses, either with no prior information or with sampling location provided as priors, and population reassignment and differentiation tests to find genetic clusters despite possible signals of admixture or gene flow. Structure version 2.3.4  were identified as having a large effect between years and were thus removed, leaving 38,732 SNPs. Analyses for population structure were then rerun with this smaller set of SNPs. FST was calculated both between sites and between two reassigned clusters described by Adegenet . The Colony command‐line input file was then generated to run the program with updated allele frequencies, dioecy, inbreeding possible, polygamy allowed, no clones, full sibship scaling, no sibship prior, unknown population allele frequencies, ten runs of medium length, full likelihood inference, and high precision. This Colony input file was generated using a script originally written by M. Ackerman  to that transcript's putative function and gene ID from the annotated reference transcriptome, a database of transcripts which diverged by sampling location or year was created for the Lake Winnipeg walleye in the present study. From this database, a list of transcripts relevant to either sampling location or year was used for gene set enrichment analysis using EnrichR , while the Dauphin River fish were the most genetically distinct group sampled   and twelve in the other , ten fish were in another group (Cluster 2), and 19 fish in a final group (Cluster 3). Clusters 1 and 3 were characterized by a year effect, where every individual in Cluster 1 was captured in 2018 and every individual in Cluster 3 was captured in 2017. Both Clusters 1 and 3 had 16 out of 19 fish coming from the Red River or Matheson Island sites. Meanwhile, all ten fish in Cluster 2 were from the Dauphin River with five fish each collected in 2017 and 2018 Table .3.3FST estimates overlapped over both sampling years, indicating consistent patterns of between‐site divergence in 2017 and 2018. However, values between the Dauphin River and Matheson Island varied the most, with an estimate of 0.0044 (0.0035–0.0052) in 2017, and 0.0060 (0.0051–0.0070) in 2018 , where a kinship coefficient of approximately 0.5 would indicate full‐siblings.We found no evidence of kinship using either Colony or the method of moments. Over ten replicate runs in Colony, individuals belonged to separate families with inclusive and exclusive probabilities of 1.0000 each. Using the method of moments implemented in SNPRelate  in the pcadapt analysis Figure . In totar Figure . For ther Figure , 120 uni05 Table . By filtp‐value < .05. For transcripts with ≥2 PC2 outlier SNPs, 17 uniquely annotated genes were identified of which six were either transposons, transposable elements, or fragments of transposons , which varied by year Figure , 130 unins Table . Two genns Table .4FST of 0.0095, but with gene flow between them primarily at the channel connecting the two basins. Consistent with results in the present study, a study using microsatellites found a similar weak, but significant, differentiation between Lake Winnipeg walleye from some sites in the north and south basins , the greatest pairwise FST confidence interval difference between years was between walleye collected at Matheson Island and the Dauphin River, where confidence intervals for FST estimates overlapped by only 0.0001 between 2017 and 2018. Following Amrhein, Greenland, and McShane . Nevertheless, confidence intervals for FST overlapped between years between the Matheson Island and Dauphin River sites; therefore, patterns of environment‐dependent spawning site fidelity must be corroborated with other data, such as by telemetry.While pairwise site  McShane , we inteNotably, gene flow appears to be one way from the southern Red River, northward. Going by capture location, no fish caught in the Red River showed a genetic background consistent with the Dauphin River fish, while with the Adegenet‐reassigned clusters, no fish assigned to the mostly Dauphin River group was found in the Red River. On the other hand, fish which showed a genetic background consistent with the Red River group were found in the Dauphin River, both based on capture location and population reassignment.4.2Osmerus mordax), after which walleye body condition has decreased since 2010  or Atlantic salmon  ; Data curation ; Formal analysis (lead); Methodology ; Software (lead); Validation ; Visualization ; Writing‐original draft (lead); Writing‐review & editing . Jennifer D. Jeffrey: Investigation (supporting); Validation ; Writing‐review & editing (supporting). Jason R. Treberg: Funding acquisition ; Investigation (supporting); Methodology (supporting); Project administration ; Supervision ; Writing‐review & editing (supporting). Douglas A. Watkinson: Investigation ; Methodology ; Resources ; Supervision ; Writing‐review & editing (supporting). Eva C. Enders: Conceptualization ; Funding acquisition (lead); Investigation ; Project administration (lead); Resources (lead); Supervision ; Writing‐review & editing (supporting). Ken M. Jeffries: Conceptualization ; Data curation ; Formal analysis (supporting); Funding acquisition ; Investigation (lead); Methodology ; Project administration ; Resources ; Software ; Supervision (lead); Validation (supporting); Visualization ; Writing‐original draft (supporting); Writing‐review & editing (lead).https://www.ncbi.nlm.nih.gov/sra/PRJNA596986; https://github.com/BioMatt/Walleye_RNAseq.This article has been awarded Open Materials, Open Data Badges. All materials and data are publicly accessible via the Open Science Framework at Supplementary MaterialsClick here for additional data file.


Racial/ethnic differences in mortality have not been well studied for either non‐cardia gastric cancer (NCGC) or cardia gastric cancer (CGC). The aim of this study was to examine the US mortality rates for these cancer subtypes, as well as esophageal adenocarcinoma (EAC) as a comparator.We identified 14 164 individuals who died from NCGC, 5235 from CGC, and 13 982 from EAC in the Surveillance, Epidemiology, and End Results database between 2004 and 2016. Age‐adjusted incidence‐based mortality rates and corresponding annual percent changes (APCs) were calculated. Analyses were stratified by race/ethnicity, age, and stage of disease at diagnosis.P < .01). Mortality in CGC was higher in non‐Hispanic whites than blacks and Asians/PI, particularly in individuals in the 50‐64 year age group and those with stage IV disease. Mortality in EAC was two‐ to sixfold higher in non‐Hispanic whites than all other groups across all age groups and stages of disease. From 2004 to 2016, mortality rates were stable across all racial/ethnic groups in NCGC and CGC, and in minority groups with EAC, but have been rising in non‐Hispanic whites with EAC .The mortality rate in NCGC was two‐ to threefold higher in blacks, Hispanics, and Asians/Pacific Islanders (PI) than non‐Hispanic whites, and was significant across all age groups and stages of disease (This is the largest study of incidence‐based mortality in CGC and NCGC and demonstrates racial/ethnic differences in mortality between these subtypes. Mortality rates for NCGC are highest in minority groups, and have been stable in recent years despite declining incidence. Mortality rates for CGC are marginally higher in middle‐aged non‐Hispanic whites with advanced disease, though have remained stable. In contrast, mortality in EAC has been rising for non‐Hispanic whites, in parallel to incidence. Further studies are needed to refine prevention strategies for high‐risk individuals dying from these specific cancer subtypes. This study examined the US incidence‐based mortality rates for cardia and non‐cardia gastric cancer, as well as esophageal adenocarcinoma as a comparator, and found distinct trends. Mortality was higher in blacks, Hispanics, and Asians/Pacific Islanders compared to non‐Hispanic whites in non‐cardia gastric cancer, but higher in non‐Hispanic whites in cardia gastric cancer and esophageal adenocarcinoma. Further studies are needed to refine prevention strategies for high‐risk individuals dying from these specific cancer subtypes. Several studies assessed survival and death certificate mortality in gastric cancer overall, yielding mixed results.A major limitation in these studies was that they did not stratify outcomes by anatomic subtype for different racial/ethnic groups. Given that CGC and NCGC appear to be distinct disease processes, it is important to understand which subpopulations are at highest risk of dying from each so that interventions can be targeted appropriately. The only study that did investigate this breakdown in both NCGC and CGC utilized the California Cancer Registry and showed no significant differences in survival with respect to race/ethnicity in CGC, but better survival in Asians/Pacific Islanders than non‐Hispanic whites in NCGC.Incidence‐based mortality is a mortality assessment that allows for the partitioning of data by variables associated with cancer onset, such as age and stage, which is not possible with death certificate mortality alone.22.1www.seer.cancer.gov) SEER*Stat Database (2004‐2016), National Cancer Institute, DCCPS, and Surveillance Research Program, released April 2019, based on the November 2018 submission. Underlying mortality data were provided by NCHS (www.cdc.gov/nchs).24 Given that all SEER data are de‐identified, Institutional Review Board (IRB) approval was not required for this study.This study utilized the database of the Surveillance, Epidemiology, and End Results (SEER18) Program of the National Cancer Institute, which includes 18 cancer registry areas , Greater Bay Area (2 registry areas) and Greater California Cancer Registries, Hawaii, Iowa, Kentucky, Los Angeles, Louisiana, New Mexico, Seattle–Puget Sound, Utah, Detroit, and New Jersey) and covers 28% of the US population.2.2Data for patients aged 20 years or older who were diagnosed with NCGC, CGC, or EAC from 2004 to 2016 were extracted from the SEER18 database using SEER*Stat (version 8.3.5).CGC patients were defined as those with a primary site of C16.0 (cardia), and NCGC patients were defined as those with a primary site of C16.1‐C16.6 . EAC patients were defined using International Classification of Diseases for Oncology (ICD‐O‐3) histology codes 8050, 8140‐8147, 8160‐8162, 8180‐8221, 8250‐8507, 8514, 8520‐8551, 8560, 8570‐8574, 8576, and 8940‐8941. Patients with cancers with overlapping tumor locations or cancers for which the tumor location was not specified were excluded.2.3P‐value was calculated for each APC using t tests to determine whether the trend was significantly different from zero. Mortality analyses were stratified by race/ethnicity, age, and stage of disease at diagnosis (I‐IV). Rates of black, Hispanic, and Asian patients were each compared to non‐Hispanic whites within each age and stage category using the rate ratio method. All statistical analyses were performed using SEER*Stat 8.3.5 software.P < .05.The primary outcome in this study was incidence‐based mortality due to NCGC, CGC, or EAC, which was characterized by age‐adjusted incidence‐based mortality rates. Unlike death certificate mortality, incidence‐based mortality is an assessment that involves tracking cases from diagnosis to death so each mortality event can be linked to characteristics at the time of diagnosis, such as age and cancer stage.3A total of 14 164 deaths from NCGC, 5235 from CGC, and 13 982 from EAC were identified in the SEER18 database. Patients with cancers with overlapping tumor locations (n = 3648) or cancers for which tumor location was not specified (n = 6989) were excluded. Patients with unknown disease stage at the time of diagnosis  were excluded. Among individuals with known stage at diagnosis, non‐Hispanic Native Americans/Alaska Natives, and those with unknown race/ethnicity were excluded due to small sample size .Table P = .26) and NCGC , but were rising for EAC . This is illustrated in Figure The age‐adjusted incidence‐based mortality rate (per 100 000 population) for NCGC was 0.675, for CGC was 1.859, and for EAC was 2.281. Overall mortality rates between 2004 and 2016 were stable for CGC , Hispanics (3.47), and Asians/Pacific Islanders (3.77) than in non‐Hispanic whites (1.13). These differences were statistically significant, with P‐values < .01, see Figure Mortality in CGC was slightly lower in blacks (0.61 per 100 000 population) and Asians/PI (0.64 per 100 000 population) than non‐Hispanic whites (0.69 per 100 000 population), and these differences were statistically significant (P < .01 for each group, see Figure P‐values < .01, see Table On the other hand, mortality was 2.3‐ to 6.4‐fold higher in EAC in non‐Hispanic whites than blacks, Hispanics, and Asians/Pacific Islanders (4In this study, we found that overall incidence‐based mortality rates have been stable for CGC and NCGC in recent years, but have been rising for EAC. Mortality was significantly higher in minority groups with NCGC than non‐Hispanic whites. Mortality rates for CGC are marginally higher in non‐Hispanic whites than blacks or Asian/PIs, particularly in middle‐aged individuals with advanced disease. In contrast, mortality in EAC is higher in non‐Hispanic whites than minority groups. Therefore, this study found distinct racial/ethnic mortality patterns among these three disease processes.This is the first study to demonstrate a difference in mortality between different races/ethnicities in CGC, though this difference is marginal and mainly seen in middle‐aged individuals with advanced disease. One prior study found no significant differences in survival; however, that study utilized the California Cancer Registry and it is possible that survival and mortality trends may differ by region in the US.While one prior study demonstrated a rising incidence‐based mortality in EAC, ours is the first study to assess this outcome by race/ethnicity.In NCGC, we found that incidence‐based mortality was significantly higher in blacks, Hispanics, and Asians/Pacific Islanders than in non‐Hispanic whites, across all age groups and stages of disease. This is in line with the findings of Schlansky et al, who showed higher death certificate mortality in non‐whites than whites in NCGC.We demonstrate that mortality in NCGC has been stable in recent years, despite the fact that studies show that incidence in this cancer subtype has been declining.H pylori infection was highest in the US among Asians but low in non‐Asian minorities, including Hispanics and blacks, despite higher rates of NCGC in these groups.The reasons behind these unfavorable mortality trends in gastric cancer in recent years are not clear. Some studies have shown that screening for Another possibility is that disparities may exist in management practices. Studies have shown that race and geographic region of diagnosis impact treatment recommendations and gastric adenocarcinoma‐specific survival among individuals with resectable tumors.Finally, it is possible that there are underlying biological and pathophysiological factors that predispose certain racial and ethnic groups to have more aggressive disease and worse mortality rates, such as minorities in NCGC and non‐Hispanic whites in CGC and EAC. Early findings suggest that differences in molecular subtypes of advanced gastric cancer impact efficacy of various treatment modalities.Our study has many strengths. To our knowledge, this is the largest study to compare incidence‐based mortality in gastric cancer stratified by anatomic subtype. Our findings further confirm and extend the concept that these subtypes are distinct disease entities with different patient characteristics and should be considered separately in further studies. Finally, by using incidence‐based mortality rather than death certificate mortality, we are able to adjust our analysis by age and stage of diagnosis, which helped identify distinctions in subgroups among patients with CGC.H pylori infection, smoking, and obesity. While the SEER registry includes data from 18 sites across the US, it is possible that sampling may not be completely representative, particularly given evidence of in‐state variability in data for gastric cancer among racial/ethnic groups in other studies.Our study also has several limitations. First, we were unable to adjust for certain factors associated with the risk of gastric cancer, such as In conclusion, our study found distinct trends in incidence‐based mortality in CGC and NCGC, as well as EAC. Mortality was higher in blacks, Hispanics, and Asians/Pacific Islanders compared to non‐Hispanic whites in NCGC, but higher in non‐Hispanic whites in CGC and EAC. Further research is needed to better understand the underlying mechanisms driving these differences, so that cancer prevention strategies can be targeted to groups who are at highest risk of dying from these specific cancer subtypes.Monika Laszkowska: Nothing to disclose. Angela C. Tramontano: Nothing to disclose. Judith Kim: Nothing to disclose. M. Constanza Camargo: Nothing to disclose. Alfred I. Neugut: Consulted for Eisai, Otsuka, United Biosource Corp, Hospira, and EHE Intl.Julian A. Abrams: Nothing to disclose. Chin Hur: Nothing to disclose.Conceptualization: ML, ACT, JK, MCC, AIN, JA, CH. Methodology: ML, ACT, JK, MCC, AIN, JA, CH. Formal Analysis: ML, ACT, JK, MCC, AIN, JA, CH. Investigation: ML, ACT, CH. Data Curation: ML, ACT, CH. Writing—Original Draft: ML, ACT, CH. Writing—Review and Editing: ML, ACT, JK, MCC, AIN, JA, CH. Visualization: ML, ACT, CH. Project Administration: CH. Supervision: CH.


Evidence on the associations of carotid intima‐media thickness and carotid plaque characteristics with stroke subtypes and coronary artery disease risks in Asians is limited. This study investigated these associations in the Japanese general population.Maximum intima‐media thicknesses of both the common carotid artery and internal carotid artery and carotid plaque characteristics were evaluated in 2943 Japanese subjects aged 40 to 75 years without history of cardiovascular disease. Subjects were followed up for a median of 15.1 years. Using a multivariate Cox proportional hazard model, we found that hazard ratios (HRs) and 95% CIs for the highest (≥1.07 mm) versus lowest (≤0.77 mm) quartiles of maximum intima‐media thicknesses of the common carotid artery were 1.97 (1.26–3.06) for total stroke, 1.52 (0.67–3.41) for hemorrhagic stroke, 2.45 (1.41–4.27) for ischemic stroke, 3.60 (1.64–7.91) for lacunar infarction, 1.53 (0.69–3.41) for nonlacunar cerebral infarction, 2.68 (1.24–5.76) for coronary artery disease, and 2.11 (1.44–3.12) for cardiovascular disease . HRs(95% CIs) for heterogeneous plaque versus no plaque were 1.58 (1.09–2.30) for total stroke, 1.25 (0.58–2.70) for hemorrhagic stroke, 1.74 (1.13–2.67) for ischemic stroke, 1.84 (1.03–3.19) for lacunar infarction, 1.58 (0.80–3.11) for nonlacunar cerebral infarction, 2.11 (1.20–3.70) for coronary artery disease, and 1.71 (1.25–2.35) for cardiovascular disease.Maximum intima‐media thicknesses of the common carotid artery, maximum intima‐media thicknesses of the internal carotid artery, and heterogeneous plaque were associated with the risks of stroke, lacunar infarction, coronary artery disease, and cardiovascular disease in Asians. CCAcommon carotid arteryCIMTcarotid intima‐media thicknessCIRCSCirculatory Risk in Communities StudyICAinternal carotid arterymax CCA‐IMTmaximum intima‐media thickness of common carotid arterymax ICA‐IMTmaximum intima‐media thickness of internal carotid arteryMaximum intima‐media thicknesses of both the common carotid artery and internal carotid artery were positively associated with the risk of lacunar infarction in the Asian general population.Maximum intima‐media thickness of the common carotid artery was positively associated with the risk of coronary artery disease in the Asian general population.Carotid plaque heterogeneity was positively associated with the risks of total stroke, ischemic stroke, lacunar infarction, coronary artery disease, and cardiovascular disease.Carotid ultrasound imaging is useful for identifying patients at high risk for stroke and coronary artery disease in Asians.Carotid intima‐media thickness (CIMT) and carotid plaque are considered as markers of atherosclerosis, and they are known to be associated with the risk of stroke.In Western countries, CIMT and carotid plaque have been found associated with increased risk of coronary artery disease (CAD)The data supporting the findings of this study are available from the corresponding author upon reasonable request.We conducted a cohort study using data from the CIRCS (Circulatory Risk in Communities Study). The CIRCS is an ongoing dynamic community cohort study in Japan from 1963. Details of the CIRCS design have been described elsewhere.A total of 3073 subjects (2338 men and 735 women) who participated in the cardiovascular risk factor surveys underwent ultrasound examination, including 903 from Ikawa town, 886 from Yao City, 631 from Noichi town, and 653 from Kyowa town. Subjects with a history of stroke (n=75) or CAD (n=40) were excluded from the analysis. Further, 15 subjects without data for cardiovascular risk factors (n=6) or for CIMT (n=9) were excluded. Additionally, 1 subject without data for presence of carotid plaque was excluded from the analysis of carotid plaque characteristics. Finally, 2943 subjects (2226 men and 717 women) were included in the analyses for CIMT and 2942 subjects in the analyses for carotid plaque characteristics.Informed consent was implied by participation in cardiovascular risk factor surveys and was obtained from representatives in communities according to the guidelines of the Council for International Organizations of Medical Science,A single epidemiologist (A.K.) trained in carotid ultrasonography and certified from the Cardiovascular Health Study conducted ultrasonography using a high‐resolution B‐mode ultrasound imaging unit  with a 7.5 MHz annular array probe (Toshiba SMA‐736s). The protocol was based on that of the Cardiovascular Health Study,2). Blood pressure was measured by trained observers using standard mercury sphygmomanometers in a quietly seated position after a ≥5‐minute rest. We identified current smokers as those who smoked ≥1 cigarette per day, ex‐smokers as those who had quit smoking for ≥3 months, current drinkers as those who drank ≥1 times per week, and ex‐drinkers as those who had not drunk for ≥3 months according to the subjects' reports. We defined diabetes mellitus as fasting glucose ≥7.0 mmol/L, nonfasting glucose ≥11.1 mmol/L, or current use of antidiabetic drugs. Non–high‐density lipoprotein cholesterol was calculated by subtracting the high‐density lipoprotein cholesterol (mmol/L) value from the total cholesterol (mmol/L) value. Creatinine was assayed using the Jaffe method before 2001 and with an enzymatic method after 2001. Creatinine values obtained by Jaffe method were converted into those obtained by an enzymatic method and the estimated glomerular filtration rate (eGFR) was calculated using the following equation proposed by the working group of the Japanese Chronic Kidney Disease initiative2)=194×(creatinine [an enzymatic method])−1.094×(age)−0.287×(0.739 for women). A 12‐lead ECG was obtained, and atrial fibrillation was determined according to Minnesota codes 8‐3‐1.The protocols for the cardiovascular risk factor surveys have been described in detail in our previous report.Subjects were followed until the end of 2017 for Ikawa, 2016 for Yao, 2009 for Noichi, and 2014 for Kyowa. Subjects were censored when they withdrew from the cohort because of deaths without prior CVD incidence (n=753) or moved out of the communities (n=103).We defined CVD as CAD and stroke. Stroke subtypes were identified as intraparenchymal hemorrhage, subarachnoid hemorrhage, lacunar infarction, large‐artery embolism, large‐artery thrombosis, unclassified large‐artery infarction, or unclassified stroke. CAD was defined as definite or possible myocardial infarction, angina pectoris, and sudden cardiac death. The details of the criteria for diagnosis of stroke and CAD are shown in Data P values for statistical tests were 2‐tailed, and those <0.05 were considered statistically significant.Baseline demographic characteristics of the subjects were summarized according to the quartiles of max CCA‐IMT or max ICA‐IMT. We reported the percentage of men and the mean value with standard deviation for age. For other values, except for age and sex, we reported age‐, sex‐, and community‐adjusted means with standard errors for continuous variables or percentages for categorical variables. We performed linear tests for trend using the median values of max CCA‐IMT or max ICA‐IMT within each quartile as ordinal variables. Cox proportional hazards regression models were used to estimate the hazard ratios (HRs) and 95% CIs for incident total stroke, hemorrhagic stroke, ischemic stroke, lacunar infarction, nonlacunar cerebral infarction, CAD, and CVD for each quartile of maximum CIMT relative to the lowest quartile of maximum CIMT. We selected adjustment variables based on previous studies.Table During the median follow‐up of 15.1 years (maximum 21.6 years), stroke occurred in 186 subjects, and the incidence rate per 1000 person‐years was 4.3. Among subjects with total stroke, 137 subjects had ischemic stroke. Lacunar infarction occurred in 77 subjects, which accounted for 41.4% of total stroke cases and 56.2% of ischemic stroke cases. CAD occurred in 88 subjects, and the incidence rate per 1000 person‐years was 1.9.Table The results according to max ICA‐IMT quartiles Table  showed sTable The corresponding HRs (95% CIs) according to plaque surface characteristics are shown in Table The present study showed that max CCA‐IMT was positively associated with the risks of total stroke, CAD, and CVD in the Japanese population. When we examined the associations by stroke subtypes, we observed positive associations for ischemic stroke and lacunar infarction, but not for hemorrhagic stroke and nonlacunar cerebral infarction although caution should be taken to interpret the results for hemorrhagic and nonlacunar cerebral infarction because the statistical power may be limited. We observed similar associations for max ICA‐IMT.To the best of our knowledge, our study is the first to prospectively report on the association between CIMT and the risk of lacunar infarction in the Asian general population. Subjects in the highest quartile of max CCA‐IMT or the highest quartile of max ICA‐IMT had ≈3‐fold higher risks for lacunar infarction compared with those in the lowest quartiles. The Atherosclerosis Risk in Communities study in the United States was the only population‐based cohort study that examined the association between CIMT and incident lacunar infarction, in which the significant association was found for Black individuals, but not for White individuals.The associations of the risk of CAD with CIMT and carotid plaque in the Asian population are unclear. Several previous Western studies showed positive associations between CIMT and the risk of CAD. The Rotterdam Study, a case‐cohort study of 2073 subjects aged ≥55 years with a mean follow‐up of 4.6 years, showed that the relative risks (95% CIs) of myocardial infarction for the highest versus the lowest quartiles of maximum CIMT were 2.43 (1.38–4.27) for CCA, 4.81 (1.51–14.35) for ICA, and 3.91 (1.87–8.18) for bifurcation.We found that heterogeneous plaque was associated with the risks of total stroke, ischemic stroke, lacunar infarction, CAD, and CVD, similar to what we found for max CCA‐IMT and max ICA‐IMT. To the best of our knowledge, no previous study has examined the association between carotid plaque characteristics and the risk of lacunar infarction. Furthermore, our study is the first to report the association between carotid plaque heterogeneity and the risk of CAD.There are different pathophysiologic pathways of lacunar infarction.Heterogeneous plaque was reported to be correlated with intraplaque hemorrhage, ulceration, deposits of lipids, and cholesterol or proteinaceous material accumulation,A common cause of CAD is coronary plaque rupture, which accounts for as many as 73% of coronary thrombi.This study has some limitations. First, we did not consider the long‐term changes in cardiovascular risk factors, CIMT, and plaque characteristics. However, it is a highly challenging problem to examine these variables repeatedly with the same accuracy, and no previous study has examined these changes. Second, we could not test interobserver and intraobserver reproducibility for ultrasound imaging. However, the reproducibility of ultrasonography findings in the present study may be comparable with those from the Cardiovascular Health Study,In conclusion, we showed that max CCA‐IMT and heterogeneous plaque were associated with increased risks of total stroke, ischemic stroke, lacunar infarction, CAD, and CVD but not with hemorrhagic stroke and nonlacunar cerebral infarction in the Japanese general population. Max ICA‐IMT had similar associations, although it did not have a significant association with the risk of CAD. In addition, we found strong associations of markedly irregular or ulcerated plaque with CAD and CVD.This study was supported by grants‐in‐aid research B (11877069 from 1998 to 1999 and 12470092 from 2000 to 2001) from the Japan Society for the Promotion of Science, and the Japan Arteriosclerosis Prevention Fund for the Japan Arteriosclerosis Longitudinal Study (2001–2012).None.Data S1Table S1ReferencesClick here for additional data file.


To evaluate the efficacy and feasibility of minimally invasive oblique lumbar interbody debridement and fusion for the treatment of conservatively ineffective lumbar spondylodiscitis.This is a retrospective study. Between December 2016 and November 2017, a total of 14 consecutive patients  with single‐level lumbar spondylodiscitis were included in the study. The inclusion criteria include single‐level spondylodiscitis without spinal deformity or epidural abscess, ineffective conservative treatment , minimally invasive oblique lumbar interbody fusion surgery (Mis‐OLIF) and iliac graft for the treatment of lumbar spondylodiscitis, and postoperative follow‐up >12 months. Each patient was treated Mis‐OLIF. Clinical outcomes including demographic characteristics, erythrocyte sedimentation rate (ESR), C‐reactive protein (CRP), visual analog scale (VAS), the Oswestry Disability Index (ODI), American Spinal Injury Association neurological classification, and lordotic angle were analyzed.Staphylococcus aureus (5), brucellosis (6), and enterobacterium (2). The pathogen was undefined in one patient. The mean duration of the surgery, mean blood loss, and mean follow‐up were 89.3 ± 17.5 min, 155.0 ± 49.4 mL, and 16.8 ± 4.2 months, respectively. The ESR and CRP decreased after Mis‐OLIF and antibiotic administration. The average preoperative VAS score was 6.9 ± 0.9, then decreased to 3.0 ± 1.0  and 0.6 ± 0.7  before discharge and at final follow‐up, respectively. The average preoperative ODI score was 58.4 ± 13.0, then decreased to 28.3 ± 6.1  and 8.0 ± 4.6  before discharge and at final follow‐up, respectively. None of the patients developed postoperative ileus, vascular injury, nerve injury, and ureteral injury. One patient suffered incision‐related complication that healed by debridement and dressing change. One patient developed subsidence of autologous iliac bone before discharge and achieved complete bony fusion after staying in bed and fixing it with a brace at 3 months follow‐up. All patients achieved bony fusion at final follow‐up.The infectious levels included L1/2 (one patient), L2/3 (two patients), L3/4 (eight patients), and L4/5 (three patients). The pathogens found in these patients included Mis‐OLIF without anterior or posterior instrumentation and iliac graft is an effective and viable approach for the treatment of conservatively ineffective lumbar spondylodiscitis without spinal deformity or epidural abscess. Spondylodiscitis is defined as an infection of the intervertebral disc and vertebral body. The incidence of spinal spondylodiscitis has increased over the period 1995–2008, and the elderly have the highest risk of suffering from spondylodiscitisNo treatment consensus has yet been reached due to the heterogeneity of spondylodiscitis. Furthermore, diagnose with delay and inaccuracy and inadequate treatment may result in devastating complications of spondylodiscitisPercutaneous endoscopic discectomy and drainage or CT‐guided percutaneous puncture and drainage and antibiotic are safe procedures for early‐stage spinal spondylodiscitisOblique lumbar interbody fusion (OLIF) has been widely used in lumbar disease and OLIF surgical approach has the potential advantage over conventional anterior approachTherefore, in this study, patients with single‐level lumbar spondylodiscitis that were treated with Mis‐OLIF without anterior or posterior instrumentation were retrospectively reviewed. The purpose of this study was to: (i) describe Mis‐OLIF strategy without anterior or posterior instrumentation for the treatment of single‐level lumbar spondylodiscitis; (ii) evaluate the efficacy and feasibility of Mis‐OLIF without anterior or posterior instrumentation in treating lumbar spondylodiscitis; and (iii) analyse clinical fusion effect of iliac graft for the treatment of lumbar spondylodiscitis. We found that Mis‐OLIF without anterior or posterior instrumentation and iliac graft is an effective and viable approach for the treatment of conservatively ineffective lumbar spondylodiscitis without spinal deformity or epidural abscess.The inclusion criteria included: (i) diagnosis of single‐level spondylodiscitis without spinal deformity or epidural abscess; (ii) ineffective conservative treatment ; (iii) Mis‐OLIF and iliac graft for the treatment of lumbar spondylodiscitis; (iv) postoperative follow‐up >12 months; and (v) retrospective study. Exclusion criteria were as follows: (i) lumbar infections after other lumbar surgeries; (ii) treatment with other surgical approaches; and (iii) lumbar trauma.A retrospective study was performed between December 2016 and November 2017 for 14 consecutive patients with single‐level lumbar spondylodiscitis treated with Mis‐OLIF without anterior or posterior instrumentation. The characteristics of patients and individual data, including age, gender, surgical segments, clinical symptoms, radiographic data, surgical approach, operating duration, blood loss, follow‐up, pathogens, and the American Spinal Injury Association (ASIA) neurological classification, for the 14 patients are shown in Table For an accurate diagnosis, the X‐ray, CT, and MRI were performed in all patients. The preoperative laboratory examinations, including blood routine examination, ESR, CRP, procalcitonin, bacterial culture, hepatorenal function, and other routine laboratory examinations provided effective diagnostic references. For four of the patients using needle biopsies of pathological vertebrae, a conclusive diagnosis was made, but the specific pathogen in one of the patients was not confirmed. Antibiotic treatment was principally based on the results of bacterial culture, and secondly on the clinical experience. Patients received irregular anti‐infection therapy prior to admission and preoperative antibiotic therapy for approximately 2 weeks in our hospital. In this study, the indications of Mis‐OLIF surgery were ineffective conservative antibiotic treatment (approximately >6 weeks), the existence of paravertebral abscess or sequestrum, and persistent and unmitigated low back pain with or without neurological symptoms.The patients were placed in the right lateral decubitus position after general anesthesia was administered. A 4‐cm long incision parallel to the musculus obliquus externus abdominis was made in the left lateral abdominal region, and a blunt dissection of the musculus obliquus externus and internus abdominis and musculus transversus abdominis was performed to approach the retroperitoneal intermuscular space. After clearing the retroperitoneal space and palpating the psoas, initial dilator was inserted to the intervertebral space and positioned by fluoroscopy. Then the sequential dilators were introduced and the retractor of OLIF was placed and fixed. The infective focus, including infective abscesses, necrotic vertebras, and intervertebral disc, were completely removed. After the debridement, autologous iliac bone of appropriate size was implanted into the intervertebral space. Autologous iliac bone graft was taken from the left anterosuperior iliac spine, and the size of iliac bone graft was based on the size of bone defect. These procedures were presented in Fig. Thoracolumbosacral bracing was necessary to maintain the location of autologous iliac bone and help to achieve bony fusion. Patients received postoperative antibiotic therapy for approximately 2–4 weeks before discharge. Antibiotic therapy continued for approximately 4 weeks after discharge, and the antibiotic medication was adjusted according to the blood routine examination, ESR, and CRP during the 1‐month follow‐up. The fusion rates were evaluated by the X‐ray and CT scans at final follow‐up The Visual Analogue Scale (VAS) scoring system was used to evaluate the pain level of patients. The VAS scoring system is self‐completed by the patient. Patients mark the location on the 10‐cm line corresponding to the amount of pain they experienced. 0 is no pain and 10 is severest pain.Oswestry Disability Index (ODI) is a principal condition‐specific outcome measure used in the management of spinal disorders, and to assess patient progress in routine clinical practice. The ODI score system includes 10 sections: pain intensity, personal care, lifting, walking, sitting, standing, sleeping, sex life, social life, and traveling. For each section of six statements, the total score is 5. Intervening statements are scored according to rank. If more than one box is marked in each section, take the highest score. If all 10 sections are completed the score is calculated as follows: total score out of total possible score × 100. If one section is missed (or not applicable), the score is calculated: ) × 100%. Scores are as follows: 0%–20% is considered mild dysfunction; 21%–40% is moderate dysfunction; 41%–60% is severe dysfunction; and 61%–80% is considered as disability. For cases with score of 81%–100%, the patient is either long‐term bedridden, or exaggerating the impact of pain on their life.The American Spinal Injury Association Score (ASIA) is commonly used to evaluate neurological deficits. It is based on motor and sensory scores, neurological levels, a completeness criterion, zones of partial preservation, and an impairment scale. The impairment scale is as follows: (A) no sensory or motor function; (B) incomplete sensory but no motor function; (C) incomplete motor function is preserved below the neurological level and more than half of the key muscles below the neurological level have a muscle grade less than 3; (D) incomplete motor function is preserved below the neurological level, and more than half of the key muscles below the neurological level have a muscle grade greater than or equal to 3; and (E) sensory and motor function are normal.Postoperative complications, including subsidence, ileus, vascular injury, nerve injury, ureteral injury, incision‐related complication, and fusion‐related complication were recorded and analyzed.P values <0.05 was considered statistically significant.The preoperative, postoperative, and final follow‐up ESR, CRP, VAS scores and ODI were analyzed using SPSS 25.0 software . The paired t‐test was used to assess the difference between the preoperative, postoperative, and final follow‐up ESR, CRP, VAS scores and ODI. From December 2016 to November 2017, 14 consecutive patients  with lumbar spondylodiscitis were included. The mean age of the patients at the time of treatment was 49.1 ± 8.0 years . The mean follow‐up was 16.8 ± 4.2 months .All patients underwent Mis‐OLIF without any instrumentation. The mean duration of surgery was 89.3 ± 17.5 min . The mean blood loss was 155.0 ± 49.4 mL . The results and statistics are shown in Table The infectious levels included L1/2 (1), L2/3 (2), L3/4 (8), and L4/5 (3). Intraoperative pus cultures were obtained for all 14 patients. The pathogens found in these patients included Staphylococcus aureus (five patients), brucellosis (six patients), and enterobacterium (two patients). In one of the patients, the pathogen was undefined, even though the clinical symptoms, radiological findings, laboratory, and pathological examinations were all combined to achieve a diagnosis.Clinical symptoms including low back pain, fever, and weakness or pain of lower extremity improved after surgery and significantly improved at the final follow‐up. Infective indicators (ESR and CPR) decreased to normal levels at the final follow‐up.In Table P > 0.05). Compared with preoperative ESR, it significantly decreased at final follow‐up  . Compared with preoperative CRP, it significantly decreased at the final follow‐up   and 0.6 ± 0.7  before discharge and at final follow‐up, respectively.According to Table According to Table P > 0.05).As indicated in Fig. The location of autologous iliac bone had no obvious change at the final follow‐up, except for one patient who had subsidence of autologous iliac bone before discharge. All 14 patients achieved bony fusion at the final follow‐up.Three typical cases with lumbar spondylodiscitis treated with Mis‐OLIF were selected. The preoperative radiological images, intraoperative situation of one of the cases, and bony fusion achieved at the final follow‐up according to plain radiographs and CT scans are shown in Figs , 3, 4.P < 0.001) and 8.0 ± 4.6%  before discharge and at final follow‐up, respectively.Functional disability was improved according to Table Neurological evaluation was assessed in Table None of the patients developed postoperative ileus, vascular injury, nerve injury, and ureteral injury.However, one patient suffered incision‐related complication after surgery that healed by debridement and dressing change. One patient developed subsidence of autologous iliac bone before discharge and achieved complete bony fusion after staying in bed and fixing it with a brace at 3 months follow‐up.Staphylococcus aureusStaphylococcus aureus and brucellosis are the main pathogens in lumbar spondylodiscitis. Moreover, lumbar spondylodiscitis mostly affects the L3/4 level.Spondylodiscitis mostly affects the lumbar spine, followed by the thoracic and cervical spineThe purpose of treatment in spondylodiscitis is to cure the infection, release the symptoms, and restore spinal stability. Treatment methods for spinal spondylodiscitis remain controversial. It has been reported that a 6‐week antibiotic treatment is sufficient for most patients with nonspecific spondylodiscitisSurgery is performed due to spinal instability, local abscesses, compression of the nerve or spinal cord, and spinal deformityRegarding clinical outcomes, the clinical symptoms evidently improved in our study, according to the VAS and ODI scores. Even though the ESR and CRP before discharge showed no significant improvement compared with preoperative values, there was a declining trend, and the ESR and CRP returned to normal at follow‐up. The possible reasons could be stress reaction to surgery and the normal course of the disease. In addition, the patients in our study were diagnosed as lumbar spondylodiscitis without spinal deformity or epidural abscess and the lumbar vertebral bodies were mildly destroyed. Therefore, this may explain why there are no significant differences between the preoperative Lordotic angle and postoperative ones. The main purposes of surgery for lumbar spondylodiscitis include curing the infection and releasing the symptomsFor patients with lumbar spondylodiscitis, anterior or posterior fixation may be necessary due to the spine being unstableThe common complications that occurred in OLIF surgery include subsidence, postoperative ileus, vascular injury, nerve injury, ureteral injury, and blood transfusionOur study has some limitations such as it being a small‐size retrospective study with a lack of control groups. The effectiveness of OLIF in multi‐level lumbar spondylodiscitis is unclear. Moreover, OLIF is usually performed from the left side, and further study is warranted to confirm the effectiveness of Mis‐OLIF to manage psoas abscess on the right side. We believe that a multi‐center, longer follow‐up randomized control study is needed to ameliorate the limitations of this study. Nonetheless, this study shows that Mis‐OLIF is a viable treatment option for lumbar spondylodiscitis.Mis‐OLIF without anterior or posterior instrumentation and the iliac graft is an effective and viable approach in the treatment of conservatively ineffective lumbar spondylodiscitis without spinal deformity or epidural abscess. It has achieved effective clinical and radiological outcomes. Mis‐OLIF reduces clinical manifestations, improves the diagnostic efficiency and treatment, and achieves good bony fusion. Additionally, it may reduce operation time, blood loss, and tissue trauma, thus avoiding disturbance of the spinal canal.