The USA300 clone of community‐associated MRSA is reported to be hypervirulent and epidemic in the United States. This clone causes a variety of diseases from lethal pneumonia to mild skin infections. We hypothesized that evolutionary diversity may exist among USA300 clones, which may link virulence traits with host responses and mortality rates.USA300 isolates from severe pneumonia (IP) and skin infection (IS) were characterized by pulsed‐field gel electrophoresis (PFGE) and next‐generation sequencing. Their virulence traits and host responses were compared in a lung infection model.hla (α‐toxin) and lukS‐PV , but not coa, agrA, or rnaIII, was confirmed in vitro. Strain IP also induced strong hemolysis in red blood cells.The two USA300 isolates were found to be identical in genomic analysis. Robust IL‐6 production, aggregation of bacteria, and hemorrhaging were observed in IP‐infected lungs, which were associated with a higher rate of mortality than that observed with strain IS. Few neutrophils were detected in the lungs infected with strain IP, even at high bacterial loads. Massive production of α‐toxin and coagulase were evident during the early phase of IP infection, and robust gene expression of The present data demonstrated latent diversity in the virulence of USA300 clones. Unknown regulatory mechanisms, probably involving a host factor(s) as a trigger, may govern the virulence expression and resultant high mortality in certain sub‐clones of USA300. Staphylococcus aureus (MRSA) was first reported in 1961 Methicillin‐resistant S. aureus strains are classified into 12 types from USA100 to USA1200 according to their banding patterns on pulsed‐field gel electrophoresis (PFGE) by the Centers for Disease Control and Prevention (CDC) USA300 clone has been reported to produce multiple virulence factors that are responsible for life‐threatening outcomes S. aureus is tightly regulated. Global regulatory systems, such as Agr and SaeRS, govern the expression of a range of virulence traits. There is limited information available for the effects and involvement of host‐derived factors and/or cells on virulence factor regulation at the site of infection.Expression of the multiple virulence factors of In this study, we demonstrated the hypervirulence of USA300 clones isolated from a severe pneumonia case (IP) compared with that isolated from a mild skin disease case (IS) using a lung infection mouse model. We hypothesized that evolutionary diversity may exist among USA300 clones, which may link virulence traits with host responses and mortality rates. There are negative perceptions of using mouse models for examining the virulence of CA‐MRSA, in particular, it is known that PVL confers strong cytotoxic activity against neutrophils in humans and rabbits, but not mice Strains IP, IS, and BAA‐1556 showed the same PFGE banding pattern as USA300‐0114, which is a USA300 type strain on PFGE . N315 strain is one of the New York/Japan clone belonging to USA100 clone, so it shows a completely different banding pattern to USA300‐0114. WGS analysis revealed that the predicted ORFs of IP and IS were 2,757 and 2,726 bp in length, respectively. Comparison between the IP and IS genome sequences with that of BAA‐1556 showed that 19 ORFs were present only in the IP genome and nine ORFs were present only in the IS genome, and the role of these strain‐specific ORFs was unknown. The sequences of the major virulence‐associated genes were the same between strains IP and IS, as shown in Sup Table S1. In addition, bacterial growth curves showed similar increases in turbidity in the four strains  .The mice were intra‐tracheally infected with several MRSA strains, including IP, IS, BAA‐1556, and N315, and then survival and body weights were monitored for 2 weeks. Mortality was observed in the mice infected with strains IP and BAA soon after infection. Approximately 80% and 40% mortality rates were recorded in IP‐ and BAA‐1556‐infected mice on Day 3, respectively, with no changes in survival being observed thereafter , macroscopic hemolysis was exhibited in the culture supernatant of strain IP, but not strain IS. To explore the factors contributing to this phenomenon, the transcription levels of several virulence genes were determined for strains IP and IS cultured with murine red blood cells. Among the factors examined, significantly higher hla and lukS‐PV transcription and expression were detected for strain IP than for strain IS, whereas no differences were observed for other factors, such as agrA, rnaIII, clpA (clumping factor), and coa . These data suggested that hla and/or lukS‐PV may be responsible for the strong hemolysis induced by strain IP.agrA, rnaIII, or clpA between strains IP and IS. By contrast, significantly higher expression of the hla (encoding α‐toxin) and lukS‐PV genes was detected, along with lower expression of the coa gene in strain IP  that when triggered lead to the induction of host defense systems. For bacteria, it is plausible to acquire the ability to sense, respond to, and evade host killing mechanisms, and bacteria including There are several limitations in the present study. Although clear differences in virulence were seen between two genetically identical strains, we do not know whether this is a rare occurrence or common phenomenon in nature. We therefore need more investigations using large USA300 strain sets to examine infection site‐specific virulence differences. In addition, we could not identify the molecular mechanisms responsible for the higher virulence of strain IP in the lung infection model. Agr did not appear to be involved in this mechanism, and other regulatory factors therefore need to be investigated. Finally, most of these data were obtained using the mouse model of pneumonia. The host species may therefore be another variable, because PVL was reported to be active against human‐derived cells, but not equally active against other cell types. Future studies using human cells would therefore be of interest.In conclusion, the present data demonstrated latent ongoing molecular diversity and evolution in USA300 clones. Further studies are now needed using novel techniques to differentiate between more and less virulent sub‐clones of USA300.Specific‐pathogen‐free Balb/c mice were purchased from Charles River Laboratories . Mice were maintained under specific‐pathogen‐free conditions within the animal care facility in the Laboratory Animal Research Center of Toho University School of Medicine until 6–8 weeks of age. Animal and pathogen protocols were approved by the Institutional Care and Use Committee .We used two clinical MRSA strains that were isolated from cases of severe pneumonia (IP) and skin infection (IS) http://www.migap.org/). The sequence data for strains IP and IS had been deposited in the DNA Data Bank of Japan (DDBJ) under accession numbers SAMD00106551 and SAMD00106552, respectively.PFGE typing was performed as previously described, using a contour‐clamped homogeneous electric field apparatus CHEF Mapper  S. aureus was cultured overnight in 5 ml of BHI broth  at 35°C with shaking. At the designated time points, the absorbance at 600 nm was measured using the Bio‐Spec mini .For the analysis of growth curves, 10 CFU/ml. Balb/c mice aged 6–8 weeks were anesthetized with a mixture of ketamine (12.5 mg/kg) and xylazine (15 mg/kg). Then, 30 μl  of S. aureus suspension were administered via tracheal instillation. The actual numbers of infected bacteria were confirmed by plate counts. The survival rate and weight changes were monitored for 2 weeks. For the analysis of bacterial numbers and toxin production, mice were euthanized at the designated time points and the lungs were carefully removed for further examination.The overnight culture was washed by centrifugation and re‐suspended in saline to 1.0–3.0 × 10g for 10 min at 4°C, and the supernatant was stored at −20°C. For quantification of IL‐6, IL‐1β, TNF‐α, IFN‐γ, CXCL‐1, and CXCL‐2, commercially available ELISAs were used .The removed lungs were homogenized using a homogenizer  in 1 ml of PBS with protease inhibitor. The homogenates were centrifuged at 5000−1 (Roche) and 150 µg DNase ml−1  for 50 min at 37°C. To the blood samples, 10 μl of heparin was added, and the cells were washed with PBS. Cell suspensions from the lung and blood samples were passed through a strainer and centrifuged at 600g for 5 min. Pellets were washed with PBS, containing 0.5% BSA and 2 mM EDTA, and centrifuged. Then red blood cells were lysed using BD Pharm Lyse™  according to the manufacturer's instructions. Cell suspensions with stain buffer (PBS with 2% BSA) were incubated with an anti‐Fc receptor‐blocking antibody  from BioLegend  for 15 min on ice to reduce nonspecific antibody binding. Cells were then washed with stain buffer and surface stained for 30 min on ice using each experimental design combination of FITC anti‐mouse Ly6G antibody, PerCP/Cy5.5 anti‐mouse CD11b antibody, PE/Cy7 anti‐mouse F4/80 antibody, PE‐CD45 anti‐mouse antibody from BioLegend, and APC‐CD3e anti‐mouse antibody from eBioscience Inc. . Isotype‐matched controls and single‐conjugate controls were always included. Cells were washed with stain buffer and fixed with 4% paraformaldehyde in PBS for 15 min, then washed and stored at 4°C prior to analysis by flow cytometry. Flow cytometry was performed on a FACSCanto II (BD Biosciences) and analyzed using FlowJo software . Controls were performed using cells stained with each labeled antibody individually.At the designed time points, small pieces of PBS‐washed lung tissue were incubated in RPMI 1640 media  containing 2% FBS, 0.5 mg collagenase D mlStaphylococcus alpha‐hemolysin antibody  or anti‐coagulase antibody . The Amersham Imager 600  was used for visualization The extracts from murine lung homogenates at 3, 6, and 12 h after infection were examined by Western blotting. Primary antibodies were anti‐2O2 for blocking peroxidase, and serum‐free Protein Block  for blocking protein artifacts. The primary polyclonal antibodies used were anti‐coagulase antibodies (Denka‐Seiken) at a dilution of 1:100.Lungs were removed 12 h after infection and were fixed in buffered 4% paraformaldehyde for histopathological examination. After embedding in paraffin wax, 6‐µm thick tissue sections were cut perpendicular to the anterior–posterior axis. The sections were placed on each polylysine‐treated slide and stained with hematoxylin and eosin, as reported g, 15 min, 4°C), and the bacterial RNA was protected using an RNAprotect Bacteria Reagent Kit , and isolated using the RNeasy Mini kit (Qiagen) as previously described Each strain was cultured in BHI broth for 12 h after inoculation. The cultures were then pelleted by centrifugation , which were collected from at least three independent experiments, were analyzed for significance using a Log‐rank test, two‐way ANOVA, and one‐way ANOVA, respectively. Statistical analysis was performed using the GraphPad Prism 6 software .All authors: No reported conflicts of interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.Additional supporting information may be found online in the Supporting Information section at the end of the article.Figure S1. (A) PFGE of the SmaI‐digested strains.Click here for additional data file.Figure S2. (A) Macroscopic findings from the broth supernatants after 3 hours growth in BHI broth containing murine blood.Click here for additional data file.Table S1. (A) Major virulence‐associated genes of MRSA. Gene sequences of over 50 toxins and regulators were compared .Click here for additional data file.Table S2. PCR primers used in this studyClick here for additional data file.


Listeria monocytogenes causes the potentially lethal disease listeriosis. Within food-associated environments, L. monocytogenes can persist for long periods and increase the risk of contamination by continued presence in processing facilities or other food-associated environments. Most research on phenotyping of persistent L. monocytogenes’ has explored biofilm formation and sanitizer resistance, with less data examining persistent L. monocytogenes’ phenotypic responses to extrinsic factors, such as variations in osmotic pressure, pH, and energy source availability. It was hypothesized that isolates of persistent strains are able to grow, and grow faster, under a broader range of intrinsic and extrinsic factors compared to closely related isolates of sporadic strains.The foodborne pathogen 600, growth curves. All stress conditions reduced growth rates in isolates compared to control (p < 0.05). In addition, growth varied by the tested energy sources. In chemically defined, minimal media there was a trend toward more isolates showing growth in all replicates using cellobiose (p = 0.052) compared to the control (glucose) and fewer isolates able to grow in glycogen (p = 0.02), lactose (p = 2.2 × 10− 16), and sucrose (p = 2.2 × 10− 16). Still, at least one isolate was able to consistently grow in every replicate for each energy source.To test this hypothesis, 95 isolates (representing 74 isolates of 20 persistent strains and 21 isolates of sporadic strains) from a series of previous studies in retail delis, were grown at 37 °C, in (i) stress conditions: salt , pH , and sanitizer  and (ii) energy sources: 25 mM glucose, cellobiose, glycogen, fructose, lactose, and sucrose; the original goal was to follow up with low temperature experiments for treatments where significant differences were observed. Growth rate and the ability to grow of 95 isolates were determined using high-throughput, ODThe central hypothesis was rejected, as there was not a significant difference in growth rate or ability to grow for retail deli isolates of persistent strains compared to sporadic strains for any treatments at 37 °C. Therefore, these data suggest that persistence is likely not determined by a phenotype unique to persistent strains grown at 37 °C and exposed to extrinsic stresses or variation in energy sources. Listeria monocytogenes is a foodborne pathogen that causes listeriosis and is estimated to cause 1600 illnesses and 260 deaths per year in the US ), some (Variable [VAR]), or no (No Growth [NG]) replicates of each treatment , there were only fives isolates that were in variable growth. Those five isolates with variable growth at pH 5.2 and pH 9.2 were not the same isolates across the two treatments.Isolates’ ability to grow was not significantly different from control BHI media for the 5% NaCl and pH 5.2 & 9.2 conditions. Isolates had significantly reduced ability to grow in 10% NaCl and 2 & 5 μg/mL BAC. BAC 5 μg/mL media was the least likely to support growth, with just above 50% (49/95) of isolates having variable growth and the rest of the panel not growing at all. While pH 5.2 and 9.2 trended to be significantly different . When comparing all extrinsic stress conditions, all treatment means were significantly lower than the control of normal Brain Heart Infusion (BHI) media . The conditions of 5% NaCl, BAC 2 μg/mL, and pH 9.2 were all indistinguishable   persistent and 21 23%] sporadic) that represent 20 putative persistence events and 21 sporadic strains were analyzed. These isolates were included in a previous study that used WGS based phylogenetics to identify persistent strains from retail delis , 32. The% sporadiWhile the referenced study  analyzedIsolates were maintained at − 80 °C in 12.5% v/v glycerol-brain-heart infusion (BHI) media in 96-well microplate format . Before being assayed in the Bioscreen C, isolates were resuscitated from frozen stocks by pre-growth in control media  at 37 °C, for optimal growth, for 18 to 24 h  with shaking at 150 rpm, again in the 96-well microplates.Listeria species [L. monocytogenes strains come from retail delis, three different extrinsic stresses were tested, that are commonly associated with persistence – osmotic pressure, acidic and alkali pH, and a sanitizer. Therefore, the following media were made: BHI with 5% or 10% w/v NaCl; BHI at pH 5.2 or 9.2 BHI (adjusted with 3 M HCl or 3 M NaOH); BHI with 2 or 5 μg/mL benzalkonium chloride (BAC), a QAC and common industry sanitizer [There were multiple treatment media used in this project Table . This pr species , which uanitizer . Specifianitizer , 68, 69.L. monocytogenes’ ability to grow on plant matter (vegetables). Fructose was representative of fruit sugars (fruits). Glycogen was representative of muscle tissue (meats). Lactose was representative of milk sugars (dairy products). Sucrose was representative of refined sugar (sweets).Energy sources were also assayed, such as glucose (control), cellobiose, fructose, glycogen, lactose, and sucrose. DM was used to focus in on the growth rate and ability to grow given different carbohydrate sources. The DM energy sources were substituted at the same initial concentration (25 mM) as directed in previous literature . Each en600 of each of the 100 wells from time zero to 24 h later in 15 min intervals, with shaking at medium-intensity 15 s before each interval reading. This data collection scheme allowed for capture of the starting inoculation levels, final growth level, lag phase, and growth rate, as described below. Cultures were assayed for 3–6 biological replicates of each treatment by individual resuscitations from frozen stock cultures.O/N cultures were transferred from the resuscitated 96-well plate to a 100-well Honeycomb Bioscreen Plate  in the treatment specific media . A 10-fold dilution inoculation was chosen so that the initial inoculum was above the machine’s detection limit, and therefore initial density and lag phase could be obtained at a time point zero. Cultures were then grown for 24 h, at 37 °C, in the Bioscreen C  Automated Growth Curve Analysis System. The Bioscreener software recorded the ODThe specific growth temperature of 37 °C was utilized in the initial screen for relevant phenotypes because this temperature was experimentally convenient compared to refrigerated temperature work. Originally, the design of the experiment was for a high-throughput screen at 37 °C and to perform follow-up experiments at 4 °C, if there were observed significant differences, to gather data even more relevant to environmental survival. As there were no significant differences observed between growth of isolates of persistent and sporadic strains, at 37 °C, the 4 °C work was not attempted in this study.600 data. Curves were only fit to data where growth was observed, which is defined as ΔOD600 ≥ 0.1. Outputs would include the initial and final OD600, lag time, maximum exponential growth rate, doubling time, and ΔOD600 of calculated from each well. The initial analysis included isolates’ lag time, ΔOD, and growth rate. However, only growth rate analyses are presented, as the lag time was inversely related to growth rate and ΔOD was directly proportional to growth rate. Each isolate was grown at a minimum of three times and a maximum of six times. For each treatment, the growth rates were averaged for the control treatments. Initial OD600 readings for all wells of BHINormal and DM Glucose had means of 0.195 and 0.112 with standard deviations of 0.062 and 0.021, respectively, suggesting these isolates were inoculated to similar initial densities within their respective media.Growth data was analyzed using an open-source regression tool specifically adapted to fitting food microbiological growth models to OD data . The too600 ≥ 0.1) every replicate of a treatment, grew in at least one replicate but not all, or did not grow in any of the replicates of a treatment, respectively. Significant differences were tested in the number of isolates for each growth category for each treatment compared to its respective control  using χ2 tests (or Fisher’s Exact tests if any cell had < 5 counts). When persistence was examined for its effect on growth, comparisons to a control were not used. Fisher’s Exact tests were utilized for singular treatments split by persistent and sporadic connotations.Growth parameter data was analyzed to compare both if isolates were able to grow and growth rate, if growth was observed. As for the isolates’ ability to grow, isolates were given the designations of “Growth,” (G) “Variable,” (VAR) or “No Growth” (NG) if they either grew in (ΔOD600 ≥ 0.1). Then, the data was tested for the effects of treatment, persistence, and the interaction of treatment*persistence on growth parameters using Analysis of Variance (ANOVA). Plotting and further statistical analyses were performed in JMP . Phylogenetic clades were also examined for their effect as a main effect into the previously described models for both growth rates and ability to grow.To analyze growth rate data, data was aggregated across replicates by calculating the mean lag time, max growth rate, and ΔOD, for each strain for each treatment where growth was observed , in log scale. The box represents the interquartile range (IQR), the line represents the median of the treatment, whiskers are drawn to the furthest point within 1.5 x IQR from the box, and the points are outliers of the data. Groups were created by use of Tukey’s HSD, where the same letters indicate means that are not different from each other.Additional file 2: Figure S2. Box plots of average growth rate for L. monocytogenes isolates grown in chemically defined media (DM) at 25 mM concentrations of each energy source. Data includes averages of only replicates whose growth was observed (ΔOD600 ≥ 0.1), in log scale. Groups were created by use of Tukey’s HSD, where the same letters indicate means that are not different from each other.Additional file 3: Table S1. Metadata of the 95 L. monocytogenes isolates analyzed.


Alternatively, transplant of autologous adipose tissue mechanically fragmented into submillimeter clusters has recently showed remarkable efficacy in diverse therapeutic indications. To document the biologic basis of the regenerative potential of microfragmented adipose tissue, we first analyzed the distribution of perivascular presumptive MSCs in adipose tissue processed with the Lipogems technology, observing a significant enrichment in pericytes, at the expense of adventitial cells, as compared to isogenic enzymatically processed lipoaspirates. The importance of MSCs as trophic and immunomodulatory cells, due to the secretion of specific factors, has been described. Therefore, we investigated protein secretion by cultured adipose tissue clusters or enzymatically derived SVF using secretome arrays. In culture, microfragmented adipose tissue releases many more growth factors and cytokines involved in tissue repair and regeneration, noticeably via angiogenesis, compared to isogenic SVF. Therefore, we suggest that the efficient tissue repair/regeneration observed after transplantation of microfragmented adipose tissue is due to the secretory ability of the intact perivascular niche. S The fat collected during a liposuction contains tissue‐repairing cells used diversely to erase wrinkles, reconstruct breasts, or treat arthritic joints. These cells, dissociated after enzymatic digestion of the fat, secrete diverse regenerative molecules playing important roles in tissue repair. Results of this study show that keeping the micro‐architecture of the fat intact, by physical fragmentation into sub‐millimetric units in the absence of enzyme treatment, guarantees optimal maintenance of regenerative cells and dramatically improves factor secretion thereby. These results likely explain the observed therapeutic superiority of micro‐fragmented adipose tissue and suggest it should be preferred to routinely used, enzymatically produced single cell suspensions.Pioneered at the end of the nineteenth century for face and breast reconstruction, adipose tissue (AT) grafting later met with scepticism and mostly fell out of favor until new successful attempts reinvigorated this surgical approach in the 1980s Vascularized tissues in general have the capacity to give rise to MSCs in culture, and fittingly peri‐endothelial cells, namely pericytes that enclose capillaries and microvessels It is, however, important to note that although resident pericytes can replenish different cell lineages in vivo, such as adipocytes In summary, AT is used therapeutically for the treatment of different conditions, either as an intact tissue or enzymatically derived SVF, used either immediately or cultured into MSCs. Alternatively, undissociated AT transplants have been supplemented with MSC injections to gain higher therapeutic efficiency  clusters through a mild mechanical size reduction using a sequence of sieves and steel marbles. It is a full immersion closed system that can be used directly in the operating room, reducing contamination risk that results from tissue exposure and/or extended processing methods. The generated AT clusters are a few hundred micrometers in diameter and free from blood and free lipids. Autologous transplantation of such MAT has been used with success in multiple indications, spanning cosmetics, orthopaedics, proctology, and gynaecology To further investigate the regenerative potential of MAT compared to that of enzymatically derived SVF, we characterized the perivascular cell distribution and in vitro protein secretion of Lipogems processed human AT versus isogenic collagenase digested LPA. We show how mechanical fragmentation of LPA modifies the resulting perivascular cell content of the tissue, and additionally that enzymatic dissociation negatively influences growth factor and cytokine secretion in vitro.AT was collected with prior written informed consent from healthy female patients (26–71 years old) undergoing cosmetic liposuction or abdominoplasty. Ethical approval for the use of human tissues in research was obtained from the South East Scotland Research Ethics Committee (reference: 10/S1103/45).Subcutaneous AT from abdominoplasty samples was injected with 50 to 100 ml of 0.9% NaCl solution, warmed at 37°C, using a disposable tissue infiltration cannula (17Gx185 mm‐VG 17/18). LPA were obtained either manually using a 10‐cc luer lock syringe connected to a disposable liposuction cannula (LGI 13Gx185 mm ‐ AR 13/18) or using a standard vacuum pump‐assisted liposuction technique. All instruments used in the manual lipoaspiration procedure were provided in the Lipogems Surgical Kit .A total of 60 ml of manual lipoaspirate was processed each time with the Lipogems 60 device following manufacturer's instructions. Briefly, the system was connected to a 0.9% NaCl solution supply until the cylinder was completely filled and no air was present in the system. First, 30 ml of the manual lipoaspirate were pushed into the cylinder through the blue size reduction filter inlet. The cylinder was shaken for 1 minute to emulsify oil. During the whole process, blood components and emulsified oil residues were removed by the flow of saline. When the solution inside the cylinder appeared clear, floating MAT was expelled from the cylinder through the gray size reduction filter outlet into 10 ml syringes connected to the device. This procedure was repeated, until 60 ml of lipoaspirate were fully processed, yielding from 20 to 30 ml of MAT.4Cl, 170 mM Tris, pH 7.65, all from Sigma‐Aldrich) for 15 minutes at room temperature. Cells were washed again with 2% FCS/PBS and filtered through 40‐μm cell strainers  to obtain single cell suspensions. Viable cells were counted following trypan blue staining  on a haemocytometer.Fresh AT specimens (LPA and MAT) were dissociated enzymatically to obtain SVF. Briefly, samples were digested with type‐II collagenase  for 45 minutes at 37°C in a shaking water bath. Samples were then washed with 2% FCS/PBS  and filtered sequentially through 100‐ and 70‐μm cell strainers . After centrifugation, pellets were resuspended in erythrocyte lysis buffer (155 mM NH+ CD34−) or adventitial cells (CD146− CD34+).The SVF was stained with the following antibodies: CD31‐V450 (1:400) or CD144‐PerCP Cy5.5 (1:100), CD34‐PE (1:100), CD45‐V450 (1:400) or CD45‐APC Cy7 (1:100), and CD146‐BV711 (1:100) . Cells were stained for 30 minutes at 4°C in the dark, followed by washing with 2% FCS/PBS. Analysis was performed on a BD LSR Fortessa 5‐laser flow cytometer (BD Biosciences) using Diva software . Single stained beads were used as compensation controls. Data were analyzed using FlowJo . Forward scatter area (FSC‐A) versus side scatter area (SSC‐A) gate was used to identify cells, followed by FSC‐A versus forward scatter height (FSC‐H) to select single cells. Viable cells were gated as negative for 4′,6‐diamidino‐2‐phenylindole  staining. Hematopoietic and endothelial cells were excluded by gating on CD31 and CD45 negative cells. Perivascular cells were identified as pericytes  and incubated at 4°C overnight. After washing with PBS, sections were incubated for 1 hour at room temperature with species‐specific secondary antibodies diluted 1:300. The following fluorochrome‐conjugated secondary antibodies were used: anti‐mouse‐Alexa 555 IgG, anti‐rabbit‐Alexa 647 IgG, and streptavidin conjugated 488 . Directly biotinylated Ulex europaeus lectin (UEA‐1) was used as an endothelial cell marker for long‐term cultured cells . Nuclei were stained with DAPI (Life Technologies) for 10 minutes at room temperature. Slides were mounted using Fluoramount G  and images were acquired using a fluorescence microscope . Images were processed using Fiji software AT specimens  were fixed in 4% buffered paraformaldehyde (PFA) at 4°C overnight. Samples were immersed for 24 hours in 15% sucrose in PBS (w/w), then embedded in 15% sucrose and 7% gelatin in PBS. After 4 hours at 37°C, samples were transferred to 4°C. After 24 hours, samples were frozen on dry ice. Embedded samples were stored at −80°C and cryosectioned at 8–10 μm thickness. Sections were fixed in 4% PFA prior to staining. Nonspecific antibody binding was blocked with 10% goat serum in PBS (Sigma‐Aldrich) for 1 hour at room temperature. The following uncoupled primary antibodies were used: mouse anti‐human‐NG2 , rabbit anti‐human‐PDGFRβ . All primary antibodies were diluted in antibody diluent  supplemented with 100 μg/ml streptomycin (Sigma‐Aldrich), 100 U/ml penicillin (Sigma‐Aldrich) and 20% heat‐inactivated foetal calf serum (Sigma‐Aldrich). 200 mg (corresponding to 200 μl of MAL) were plated in each well of a six‐well plate and cultured in basal medium. After 8 days in culture under standard conditions  culture media from SVF and MAT were collected and stored at −20°C.SVF cells derived from MAT or LPA were plated at a density of 6,000 cells/cmg for 5 minutes at room temperature to remove debris, filtered through a 70‐μm cell strainer to get rid of adipocytes/small residues of MAT, and incubated with both arrays. The signal was detected using the LiCOR Odyssey Fc apparatus , exposing array membranes for 10 minutes. Positive signals on the membranes were quantified using Image Studio Lite Software (LICOR). The average signal (pixel density) of the duplicate spots corresponding to each protein was normalized on the average signal of paired spots on the negative control. Normalized signals of each protein were then used for comparative analysis.Secretomes were analyzed using the Proteome Profiler Human XL Cytokine Array kit (ARY022b) and Human Angiogenesis Array kit (ARY007), following manufacturer's instructions . Conditioned media collected from cultured SVF and MAT were centrifuged at 500t test using Microsoft Excel or GraphPad Prism5 software. Results are presented as means ± SEM. A p value of less than .05 was considered statistically significant.Statistical analysis was performed by using the Student's Ulex europaeus agglutinin 1 (UEA‐1) receptor on sections of MAT, LPA, and AT illustrated the vascular network present in AT, with microvessels located between adipocytes. Larger vessels were observed principally in the unprocessed AT and LPA, while MAT was mainly characterized by the presence of small, capillary‐like vessels  and 69 × 103 ± 56 × 103 cells per milliliter of LPA (n = 7). Viable cells were selected excluding debris, dead cells, and doublets. Endothelial cells and leukocytes were excluded from the analysis using CD31 and CD45, respectively. Pericytes were identified as CD146+CD34−, and adventitial cells as CD34+CD146− cells p < .05, n = 10; Fig. AT samples (MAT and LPA) were digested using collagenase and analyzed by flow cytometry. The average yield of nucleated cells in the SVF was 27 × 10MSCs are known to secrete growth factors and cytokines, either free or via microvesicle cargoes, involved in tissue repair and regeneration On detailed analysis of secreted angiogenic growth factors, we found higher secretion by MAT of angiogenin, angiostatin/plasminogen, DPPIV, endoglin, hepatocyte growth factor (HGF), leptin, PDGFAB/BB, placental growth factor (PlGF), thrombospondin 2, TIMP4, and uPA Fig. C.Regarding cytokine secretion, adiponectin, CD14, CD31, CD40 ligand (CD154), chitinase 3‐like 1, complement factor D, EMMPRIN , GDF‐15, IGFBP‐2, IL1RA, IP‐10, M‐CSF, MIF, MIG (CXCL9), MIP‐3β (CCL19), PDGFAA, RANTES (CCL5), RBP‐4, relaxin‐2, ST2, TNF‐α, and uPAR (CD87) were significantly more abundant in MAT supernatants compared to SVF ones  has yielded a product of high therapeutic value. In the present study, we used immunohistochemistry to describe MAT clusters and compared those, side by side, with SVF derived from the same donors in terms of perivascular presumptive MSC content and secretory activity in culture. As expected, the microanatomy of MAT is essentially similar to that of intact AT, with capillaries and microvessels organized and distributed between adipocytes, and pericytes wrapped around endothelial cells. However, quantification by flow cytometry of pericytes and adventitial stromal cells revealed that pericytes are encountered at a higher frequency in MAT than in LPA and derived SVF, confirming previous observations Remarkably, higher amounts of diverse mediators of immuno‐inflammation, leukocyte recruitment, and migration were detected in MAT supernatants, as compared to SVF cultures: GDF‐15, a regulator of inflammation and biomarker in diverse pathologies Why does MAT secrete more growth factors and cytokines than SVF cultured under the same conditions? Besides adipocytes, secretory cells within fat tissue include hematopoietic cells, responsible for the production of most factors involved in the regulation of immuno‐inflammatory reactions, endothelial and perivascular cells, including pericytes, and other cell compartments loosely designated as stromal or “mesenchymal.” The demonstration that mesenchymal stem/stromal cells are of perivascular origin has supported the development of a model whereby pericytes and other perivascular cells can, in adverse pathologic conditions, lose contact with blood vessel walls, migrate away from blood vessels and become reprogrammed into regenerative cells B.V.: conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing; I.S.: conception and design, collection and assembly of data; H.L.: collection and assembly of data; L.Y.: provision of study material; N.K.: collection and assembly of data; C.T.: conception and design, provision of study material; B.P.: conception and design, data analysis and interpretation, manuscript writing, final approval of manuscript.L.Y. declared research funding with Small Pump Priming Grant, Royal College of Surgeons Edinburgh. C.T. is the President and founder of Lipogems and declared employment, patent holder and stock ownership. B.P. declared consultancy and researcher funding. All other authors indicated no potential conflicts of interest.


Participants completed emotion recognition and Theory of Mind tasks. Teachers and parents rated children’s emotional and behavioural problems. Children with behavioural problems showed impaired emotion and intention recognition. Emotion recognition and intention recognition were positively related and inversely associated with behavioural problem severity and, independently of one another, predicted behavioural problems. This study is the first to show that children with behavioural problems are impaired in identifying others’ emotions as well as intentions. These social cognitive processes were found to be related and inversely associated with severity of behavioural problems. This has important implications for intervention and prevention programmes for children with behavioural difficulties.Research indicates that the misinterpretation of other’s emotions or intentions may lead to antisocial behaviour. This study investigated emotion and intention recognition in children with behavioural problems and examined their relationship and relations with behaviour problem severity. Participants were 7–11 year old children with behavioural problems (The online version of this article (10.1007/s10802-019-00594-7) contains supplementary material, which is available to authorized users. Antisocial behaviour (ASB) in children is an umbrella term that covers a broad range of behaviours that can be operationalised and studied in different ways, including psychiatric diagnoses of oppositional defiant disorder (ODD), conduct disorder (CD) and disruptive behaviour disorder (DBD). Behavioural problems in childhood are associated with subsequent ASB during adulthood  delivered the Cardiff Emotion Recognition Training  took part from schools across England and Wales. Participants were assigned to one of two groups: the behavioural problems group (BP) or the typically developing group (TD). Participants in the BP group were part of the Early Intervention Hub and were referred into the study by teachers, family support workers or PCSOs. The children in the Hub do not have a formal mental health diagnosis but already show behavioural and/or emotional problems. After referral to the study, the child’s teacher completed the Strengths and Difficulties Questionnaire  aged 7–11 years old  were run to determine any between group differences for emotion recognition and for intentionality and appropriateness scores on the AST. Bonferroni corrections were used to adjust for multiple comparisons.Differences in demographic and behavioural characteristics between groups were analysed using independent samples t-tests for continuous variables and Xp < 0.05). IQ was significantly correlated with performance on the FER but not with performance on the AST. A hierarchical regression analysis showed that FER significantly predicted behavioural problems even when controlling for IQ. Age was significantly correlated with performance on the AST, but not with performance on the FER. A hierarchical regression analysis showed that while performance on the AST significantly predicted behavioural problems, age did not. As FER and AST predicted behavioural problems even when controlling for IQ and age respectively these demographic variables were not included as covariates in the analyses.Due to the verbal nature of the FER and AST tasks and prior research that shows an association between verbal ability and emotional intelligence  = 0.22, p = 0.009, but not with performance on the FER, and IQ was significantly correlated with performance on the FER, r(135) = 0.33, p < 0.001, but not with performance on the AST. See Supplement t(135) = −0.16, p = 0.877, nor was there an effect of gender performance on the AST, t(135) = 0.63, p = 0.530. Participants in the TD group exhibited significantly fewer conduct and peer problems, had a lower total SDQ score and higher prosocial score than the BP group.Participants in the TD group were significantly older, had a higher SES, IQ and verbal IQ than the BP group  = 8.43, p = 0.004, np2 = 0.06, 95% CI , negative, F = 4.66, p = 0.033, np2 = 0.04, 95% CI  and neutral recognition, F = 6.11, p = 0.015, np2 = 0.05, 95% CI . There was no difference between BP and TD participants for happy recognition, F = 3.04, p = 0.084, np2 = 0.02, 95% CI   = 4.72, p = 0.032, np2 = 0.04. There was no difference between BP and TD participants on intentionality for GD clips or appropriateness for either GD or ToM clips  = −0.21, p = 0.014, peer problems, r(130) = −0.28, p = 0.001, and total SDQ score, r(130) = −0.39, p < 0.001, while intentionality scores for ToM clips was inversely associated with conduct problems, r(130) = −0.20, p = 0.021, and total SDQ score only, r(130) = −0.25, p = 0.004. The ability to recognise others’ emotions and others’ intentions were significantly related, r(130) = 0.20, p = 0.019 and there was no relationship between prosocial behaviour and emotion recognition, r(130) = 0.08, p = 0.349, nor intentionality scores for ToM clips, r(130) = 0.10, p = 0.234  = 14.80, p < 0.001, adjusted R2 = 0.17. Both emotion recognition and intention recognition significantly added to the prediction, p < 0.05  predicted behavioural problems . The multiple regression model significantly predicted total SDQ score, The first aim of the current study was to assess and compare emotion recognition and intention recognition and attribution in children with behavioural problems and controls. Our results confirm previous findings of emotion recognition impairments in children with behavioural problems. However, for the first time, our results also indicate that children with behavioural problems are impaired in recognising other people’s intentions. Specifically, children in the BP group were found to be impaired in ascribing intentions and mental states to the triangles for ToM clips, but showed no impairment in their understanding of the clips or in ascribing intentions and mental states to the triangles for GD clips. However, in addition to differing in behavioural problems, the BP and TD groups differed on demographic variables and although unlikely to have influenced the results this needs to be taken into account when considering the findings of the current study. The second aim of the study was to explore relationships between emotion recognition, intention recognition and behavioural characteristics. We found that emotion and intention recognition were positively related and were inversely associated with behavioural problems and these two cognitive skills, independently of each other, predicted behavioural problems.The results of the current study are in contrast to the findings of the only other study to look at intentionality in children with behavioural problems using the AST. Jones et al.  found noMohammadeszadeh et al. 2016) suggest that displays of ASB in individuals with behaviour problems may be due to their misinterpretation of another’s intention which then leads them to act inappropriately. The specific impairment in intentionality displayed by the BP group in the current study provides support for Mohammadzadeh et al.’s  suggest . In antiThe current study found an inverse association between emotion recognition ability and the severity of both conduct and peer problems. Work conducted by Hubble et al.  further Like emotion recognition, intention recognition was inversely associated with conduct problem severity. This is line with previous research showing that lower levels of ToM in childhood are associated with more externalising problems  to show an impairment in ToM. Although in the current study participants were referred due to displays of disruptive behaviour and/or a lack of prosocial behaviour, there was no measure of ASD traits so we cannot rule out a contribution of ASD traits to our findings. Similarly, there was no direct measure of CU traits and previous research has shown deficits in social cognition can be dependent on the presence of CU traits


Alcohol dependence is characterised by persistent drinking despite health, social and economic costs. Behavioural economics has proposed two explanations for the persistence of alcohol use despite costs. Dependent individuals may (a) ascribe excessively high value to alcohol, such that costs associated with alcohol are exceeded, and/or (b) they may discount (neglect) the costs associated with alcohol.To test these predictions, the current study recruited 127 student drinkers who reported varied alcohol use disorder symptom severity in the Alcohol Use Disorders Inventory Test . Participants made concurrent forced choices between alcohol and food points under conditions that manipulated the magnitude of points  and the delay to receive points (0 or 3 s). Alcohol value was indexed by preferential choice of alcohol versus food points, whereas sensitivity to costs was indexed by the decrease in alcohol choice when food points were of greater magnitude (sensitivity to opportunity costs) and when alcohol points were delayed (sensitivity to delay costs).p < .001) and with time delays imposed on reward points (p < .001). AUDIT scores were associated with greater alcohol versus food choice across all conditions (p = .001). As alcohol use disorder symptom severity increased, the sensitivity of alcohol choice to the relative magnitude of points (p = .29) and time delays (p = .62) remained unchanged, suggesting no differential discounting of opportunity or delay costs imposed on alcohol. In contrasts of AUDIT categories, there was comparable sensitivity to costs across groups defined as low-risk (N = 39), hazardous (n = 57), harmful (n = 20) and possible dependent drinkers (n = 11).Percent choice of alcohol over food varied consistently with the relative magnitude of reward points offered (Alcohol use disorder symptom severity is associated with greater relative value ascribed to alcohol, but not with greater discounting of opportunity or delay costs imposed on alcohol. Despite limitations of the current study, it may be concluded that cost discounting plays a lesser role in dependence than previously thought. A key diagnostic feature of alcohol dependence is that dependent individuals will continue to drink even when doing so brings about negative health, social and economic consequences  is considered to be a relatively pure index of the value of alcohol unaffected by sensitivity to costs, whereas peak expenditure (or Omax) and elasticity may reflect both alcohol value and cost sensitivity. Intensity of demand for alcohol correlates with various proxies for dependence, including drinks consumed per week . It is typically found that alcohol use disorder symptoms are associated with a greater preference for the smaller immediate reward  show weaker suppression of drug self-administration by contingent shock punishment, despite comparable baseline self-administration rates to control animals . The claim that percent drug choice indexes the relative value ascribed to the drug versus natural reward is supported by the finding that percent drug choice reliably increases with the severity of dependence to alcohol  and greater discounting of delay costs imposed on alcohol choice . As far as we are aware, only two experiments have utilised such a method (Vuchinich and Tucker M = 21.4). At baseline, participants completed the Alcohol Use Disorders Identification Test (AUDIT) to index alcohol use disorder symptom severity , i.e. the mean was above the hazardous cutoff. Based on the AUDIT categories, there were 39 (31%) low-risk subjects, 57 (45%) hazardous subjects, 20 (16%) harmful subjects and 11 (9%) possible dependent subjects. The TLFB questionnaire indicated that the sample as a whole consumed an average of 14.17 units of alcohol per week  estimated from the 2 weeks prior to testing. This average is right on the limit of 14 units per week proposed by the UK chief medical officers’ guidelines. Of the sample, 81 (64%) subjects drank less than this limit, and 46 (36%) drank more than this limit. There was a significant correlation between AUDIT total scores and average units per week estimated by the TLFB questionnaire, r = .69, p < .001. These findings suggest that the student sample contained a substantial proportion of drinkers above the hazardous cutoff (69%) and that the AUDIT total score was a valid estimate of alcohol use. Ethical approval was obtained from the University of Exeter Research Ethics Committee and subjects provided informed written consent.One hundred and twenty-seven students who reported drinking at least occasionally  were recruited at the University of Exeter. Participants were aged between 18 and 51 . Across these 30 trials, there were five conditions that manipulated the magnitude of the alcohol and chocolate points on offer. Alcohol could be worth two fewer points than chocolate , 1 less point  equal points , 1 more point  or 2 more points . These five conditions were coded as − 2, − 1, 0, + 1 and +2 respectively, reflecting the relative difference in the alcohol versus chocolate points on offer. There were 30 identical trials with the delay imposed on the alcohol choice and another 30 identical trials with the delay imposed on the chocolate choice. The 90 trials were selected at random without replacement. The dependent variable was percent choice of alcohol over chocolate in the five conditions that manipulated the relative magnitude of alcohol points  and three conditions that manipulated delay to reward points .F = 20.79, p < .001, ηp2 = .143, indicating that alcohol choice tracked the relative magnitude of the alcohol points. As can be seen in Fig. M = 18.24, SD = 22.32), − 1 , 0 , + 1  and + 2 . Within-subjects ANOVAs contrasting all possible pairs of the five relative magnitude conditions indicated all contrasts were significant, Fs(1126) > 12.25, ps ≤ .001, ηp2s > .089.Figure F = 11.75, p = .001, ηp2 = .086, indicating that alcohol use disorder symptom severity was associated with an increased preference for alcohol over chocolate, across conditions. The Pearson correlation between AUDIT scores and overall percent alcohol choice was r = .29, p = .001.In the overall GLM, there was also a main effect of AUDIT, F = 1.25, p = .289, ηp2 = .010. This finding indicates that as alcohol use disorder symptom severity increased, there was no difference in the sensitivity of alcohol choice to manipulation of the relative magnitude of alcohol points. Both the decrease in alcohol choice when alcohol was worth relatively less  and the increase in alcohol choice when alcohol was worth relatively more (+ 1 and + 2 conditions), compared to the 0 condition , were comparable as a function of alcohol use disorder symptom severity. These findings suggest that alcohol use disorder symptoms are not associated with greater discounting of opportunity costs imposed on alcohol.Finally and most importantly, in the overall GLM, there was no significant interaction between AUDIT scores and the relative magnitude of alcohol points, F = 24.17, p < .001, ηp2 = .162, indicating that choice was modified by the delays imposed on rewards. As can be seen in Fig. M = 19.97, SD = 22.86), intermediate with no delay  and the greatest when the delay was imposed on chocolate . Within-subjects ANOVAs contrasting all possible pairs of the three delay conditions indicated that every contrast was significant, Fs(1126) > 44.73, ps ≤ .001, ηp2s > .262.Figure F4,50 = 24.1F = 0.48, p = .622, ηp2 = .004. This finding indicated that as alcohol use disorder symptom severity increased, there was no difference in the sensitivity of alcohol choice to the delays imposed on alcohol and chocolate rewards. Both the decrease in alcohol choice when alcohol was delayed (i.e. the impact of delay costs) and the increase in alcohol choice when chocolate was delayed, relative to the no delay condition, were comparable as a function of alcohol use disorder symptom severity. These findings suggest that alcohol use disorder symptoms are not associated with greater discounting of delay costs imposed on alcohol.In the overall GLM, there was also a main effect of AUDIT identical to the GLM that tested the relative magnitude of points, above. Finally, and most importantly, there was no significant interaction between AUDIT scores and delay condition, F = 40.01, p < .001, ηp2 = .245, and a significant main effect of AUDIT category, F = 4.51, p = .005, ηp2 = .099, but no significant interaction between relative magnitude and AUDIT category, F = 1.36, p = .258, ηp2 = .032. These findings confirm the conclusions of the primary analysis  = 41.55, p < .001, ηp2 = .253, and a significant main effect of AUDIT category, F = 3.14, p = .028, ηp2 = .071, but no significant interaction between delay condition and AUDIT category, F = 1.53, p = .211, ηp2 = .036. These findings confirmed the conclusions of the primary analysis  and by imposing delays upon the two rewards, suggesting drug choice is an economic decision based on the weighing of rewards and costs. These findings are consistent with previous concurrent choice studies which have demonstrated that alcohol choice can be lawfully modified by manipulating the magnitude and delay of the alternative money reward  may not have been strong enough to reveal individual differences, such as those found with shock punishment in animals. This could be tested straightforwardly by using shock within the current paradigm. Third, our use of chocolate as the alternative reinforcer may have increased variance in the preferential choice measure due to individual differences in chocolate liking, thereby reducing sensitivity to individual differences in cost discounting. Future studies might negate this risk by utilising an alternative reinforcer for which there is more homogenous liking, such as money. Fourth, participants were deceived that they could earn alcohol and chocolate rewards contingent on their choices in the task. This deception could have been communicated between participants, which would increase variance in the preferential choice measure, thereby reducing sensitivity to individual differences in the cost discounting. Finally, our lab procedure may have failed to detect individual differences in cost discounting because the costs imposed were too specific and were not ecologically valid. For instance, alcohol dependence may be associated with discounting of real delayed costs such as negative educational, career, health or legal consequences, but because the 3 s delay manipulation did not adequately model this cost, we failed to detect differential sensitivity to cost discounting. By contrast, demand tasks measure hypothetical alcohol consumption under costs such as price  decreasing the value of alcohol, (b) increasing the costs of alcohol, (c) increasing the value of competing rewards and (d) decreasing the costs of competing rewards. There are many interventions which address these four decision variables including health education (Kleinot and Rogers


Some patients who achieve a sustained virological response (SVR) to interferon (IFN) treatment for chronic hepatitis C prior to hepatic resection for hepatocellular carcinoma (HCC) experience postoperative recurrence. This study investigated the relationship between obesity and postoperative HCC recurrence in SVR patients.Fifty‐nine patients who had achieved SVR before hepatic resection were evaluated. Patients had a solitary tumor ≤5 cm in diameter or ≤3 lesions each ≤3 cm in size with no macroscopic vascular invasion (Milan criteria). Patient characteristics potentially associated with recurrence risk were investigated.P < .01), hypertension (P = .038), and non‐anatomical resection (P = .022) were significantly associated with a lower recurrence‐free survival rate. In a multivariate analysis, obesity  and non‐anatomical resection  were independently associated with postoperative recurrence. Three‐, 5‐, and 7‐year overall survival rates after surgery were 100%, 80%, and 64% in obese patients and 100%, 92%, and 82% in non‐obese patients, respectively (P = .014). However, other variables showed no significant difference in the overall survival rate.Three‐, 5‐, and 7‐year recurrence‐free survival after surgery were 65%, 44%, and 41%, respectively. Univariate analysis showed that obesity (Obesity and non‐anatomical resection were independent risk factors for HCC recurrence after hepatic resection and successful IFN therapy. Obesity is an important clinical problem to consider to improve postoperative outcomes in such patients. Obesity is closely associated with the development of metabolic syndrome, which includes hypertension, cardiovascular disease, and diabetes mellitus, and is independently associated with the risk of developing several malignancies, including breast, renal, and pancreatic cancer.In the present study, we estimated the impact of obesity on HCC recurrence after hepatic resection following successful IFN therapy for HCV infection.22.1A total of 68 patients who were seronegative for hepatitis B surface antigen and seropositive for anti‐HCV antibodies but had an SVR and a history of curative hepatic resection for HCC between January 1994 and December 2015 at the Department of Hepato‐Biliary‐Pancreatic Surgery, Osaka City University Hospital, were eligible. SVR was defined as a serum HCV‐RNA under the detection sensitivity limit at 6 months after the termination of IFN therapy.Of the 68 eligible patients, we excluded nine with HCC outside the Milan criteria, which specify a solitary tumor ≤5 cm in diameter or ≤3 lesions, each ≤3 cm in diameter, without macroscopic vascular invasion. Tumors exceeding the Milan criteria are considered advanced and to have a potentially increased risk of recurrence. Curative hepatic resection was defined as complete resection of recognizable tumors and the histological absence of tumor cells along the parenchymal transection line.Of the 59 remaining enrolled patients, 27 had received IFN monotherapy . Five patients had received IFN alpha‐2b/ribavirin combination therapy, six had received pegylated (PEG)‐IFN monotherapy , and 21 had received PEG‐IFN/ribavirin combination therapy . HCV strains were identified by genotyping in 36 patients and by serotyping in 21. The HCV strains in two patients were unidentified, as the test was not available at the regional hospital.The study was conducted following the guidelines of the Ethics Committee of our institution and the Declaration of Helsinki and approved by the Ethics Committee of Osaka City University (No. 3815). Informed consent was obtained from each patient.2.22 following the criterion for adult Asian populations.The metabolic syndrome‐related factors assessed were obesity, diabetes mellitus, dyslipidemia, and a history of hypertension. Obesity was defined as a body mass index (BMI) ≥25 kg/m2.3Indication for surgery was based on an algorithm consisting of the presence/absence of ascites, serum total bilirubin level, and results of the indocyanine green (ICG) retention test.2.4Patients were evaluated by serum alpha‐fetoprotein, ultrasonography, computed tomography, or magnetic resonance imaging 1 month after surgery and every 3 months thereafter. If recurrence of HCC was suspected, selective hepatic angiography and an ultrasound‐guided biopsy were used to establish a definitive diagnosis, as required. Sites of recurrence were categorized into near the resection site (NR), which was the residual part of the tumor‐bearing third‐order portal branches and the part adjacent to the liver transection surface, or distant from the resection site (DR).2.5Histological evaluation followed the guidelines of the Liver Cancer Study Group of Japan.2.6U‐test and Fisher's exact probability method. Kaplan‐Meier method was used to calculate recurrence‐free survival rates, and between‐group differences in the survival were evaluated by log‐rank test. Cox's proportional hazard model was used for multivariate analysis.Differences in clinicopathological findings were tested with the Mann‐Whitney 4 or ≤15 × 104/mL); serum alpha‐fetoprotein (AFP) (≤20 or >20 ng/mL); largest diameter of the main tumor (>2.0 or ≤2.0 cm); single or multiple tumors, including intrahepatic metastases; microscopic vascular invasion; degree of differentiation of the main tumor , grading (0‐2 or 3‐4); staging ; steatosis (negative 0 or positive 1‐3); lobular inflammation (negative 0/1 or positive 2/3); ballooning (negative 0 or positive 1/2); and type of hepatic resection .Variables potentially associated with recurrence were selected based on previous study results or on our own clinical experienceP‐values were derived from two‐tailed tests, and the threshold for statistical significance was set at P < .05.Influence of metabolic syndrome‐related variables and clinical and pathological characteristics on recurrence‐free survival was analyzed. 33.1Clinicopathological characteristics of the 59 patients are shown in Table 3.2P < .01), hypertension (P = .038), and non‐anatomical resection (P = .022) were significantly associated with a lower recurrence‐free survival rate (Table P < .01) and non‐anatomic resection  were independent risk factors for postoperative recurrence  experienced recurrence, all of which were in the remnant liver. Univariate analysis showed that obesity (on P = .02 were siP = .014). Other variables showed no significant difference in the overall survival rate . Furthermore, we evaluated the change in BMI after surgery in obese and non‐obese patients. Of 59 patients, 50 patients had body weight measurements taken at 3, 6, or 12 months after surgery without recurrence. In obese patients (n = 17), median pre‐ and postoperative BMI were 27.2  and 26.8 , respectively. (P = .58) In non‐obese patients (n = 33), median pre‐ and postoperative BMI were 22.6  and 23 , respectively. (P = .93) No significant difference in BMI change after surgery was confirmed in obese and non‐obese patients. Among obese patients, improvement of obesity (from BMI ≥25 kg/m2 to BMI < 25 kg/m2) was observed in four patients. Median recurrence‐free and overall survival times were 26.8 months and 85.9 months in patients with improvement of obesity (n = 4), and 19.6 months and 84.7 months in patients without improvement of obesity (n = 13), respectively.Obesity was an independent risk factor for recurrence in this patient series Table . Compari3.4Table Table 4Multivariate analysis confirmed obesity and non‐anatomical resection as independent risk factors for postoperative HCC recurrence after successful IFN therapy, as the risk of recurrence was increased 2.8‐ and 2.7‐fold, respectively, in patients with these factors. To our knowledge, this is the first report of obesity as an independent risk factor for postoperative recurrence of HCC in patients with an SVR. An SVR might be expected to reduce the background hepatocarcinogenic potential in patients with a history of chronic hepatitis C and HCC surgery,Obesity may promote hepatocarcinogenesis.Hepatocellular carcinoma recurrence after curative treatment includes intrahepatic metastasis of original tumors and newly developed tumors (multicentric recurrence).Non‐anatomical resection was also an independent risk factor for HCC recurrence. Anatomical resection along the portal tributaries is considered an effective way to remove occult intrahepatic HCC metastases.In the present study, several common risk factors for HCC recurrence showed no significant difference between obese and non‐obese patients, including liver‐related factorsOn comparing the overall survival, obese patients showed a more unfavorable outcome than non‐obese patients. Obesity is considered a risk factor for the development of hypertension, cardiovascular disease, diabetes mellitus, and several malignancies,Improvement of obesity theoretically can be related to less recurrence or better overall survival. However, in the present study, only four patients showed improvement of obesity. Therefore, further investigation is necessary to clarify the impact of improvement of obesity on recurrence or death.Several limitations associated with the present study warrant mention. First, this study had a retrospective design and enrolled a small number of patients who achieved an SVR. Second, the current results may have been influenced by the long study duration, as medical techniques and treatment approaches may have changed over time. However, the long observational period might have clinical significance in the treatment of SVR patients with HCC. Third, because the treatment of HCV patients with direct‐acting antiviral agents (DAA) is now the standard of care, the findings might not fully apply to future HCV patients. However, the beneficial effects of DAA on HCC development or recurrence have not been determined.In conclusion, obesity and non‐anatomical resection were independent risk factors for HCC recurrence after liver resection following successful IFN therapy for chronic hepatitis C. Obesity is an important clinical problem to consider in order to improve postoperative outcomes in such patients.Conflicts of Interest: Authors declare no conflicts of interest for this article. Click here for additional data file. Click here for additional data file.


Limited studies have preliminarily identified a positive association between nonalcoholic fatty liver disease (NAFLD) and hemoglobin glycation index (HGI). However, this association has not been fully established. We aim to investigate the association between NAFLD and HGI in Chinese nondiabetic individuals and to construct a risk score based on HGI to predict a person's risk of NAFLD.  After strict exclusion criteria, 5,903 individuals were included in this retrospective cross-sectional study. We randomly selected 1,967 subjects in the enrollment to obtain an equation of linear regression, which was used to calculate predicted HbA1c and drive HGI. The other subjects were classified into four categories according to HGI level . All subjects retrospectively reviewed the baseline characteristics, laboratory examinations, and abdominal ultrasonography. P < 0.001). Adjusted to multiple factors, this trend still remained significant ). The combined NAFLD risk score based on HGI resulted in an area under the receiver operator characteristic curve (AUROC) of 0.85 provided sensitivity, specificity, positive predictive value, and a negative predictive value for NAFLD of 84.4%, 71.3%, 65.0%, and 88.0%, respectively.  The prevalence of NAFLD in this population was 20.7%, which increases along with the growth of HGI levels ( NAFLD is independently associated with HGI levels in Chinese nondiabetic individuals. And, NAFLD risk score may be used as one of the risk predictors of NAFLD in nondiabetic population. Nonalcoholic fatty liver disease (NAFLD) includes a wide spectrum of progressive liver disorders ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), which may lead to liver cirrhosis and even hepatocellular carcinoma –3. NAFLDGlycated hemoglobin (HbA1c) represents a 2-3- month average of blood glucose concentration , which in = 4363) and/or hepatitis C (n = 946) virus; (2) fasting plasma glucose (FPG) concentration ≥126 mg/dl or HbA1c ≥6.5% (n = 771); (3) thyrotoxicosis or hypothyroidism with free T4 > 1.2 ng/dl or <0.6 ng/dl (n = 177); (4) abnormal ultrasonographic findings including liver cirrhosis or malignancy (n =60), hepatitis A (n = 1), alcoholic hepatitis (n = 2), chronic kidney disease including polycystic kidney disease (n = 3); (5) age <18 (n = 8); (6) anemia with hemoglobin levels <12 g/dl (n = 142); (7) alcohol intake >20 g per day (n = 1009). After applying the above exclusion criteria, the total number of subjects eligible for the study was 5,903   from July 2014 to August 2017. Initial data were obtained from 13,399 subjects volunteered for a comprehensive health checkup. Finally, 5,903 subjects were excluded from the initial study population according to the following exclusion criteria: (1) positive serologic markers for hepatitis B  .2) was calculated as weight divided by the height. Blood pressure was measured in the right arm at the same level as the left atrium in a seated state with a standard automatic sphygmomanometer . After 12 h of overnight fasting, blood samples were collected from the antecubital vein by experienced nurses. Blood routine including hemoglobin, platelets, white blood cells (WBC), and biochemical markers such as albumin, HbA1c, FPG, total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglyceride (TG), gamma glutamyltransferase (GGT), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were subsequently analyzed by an automated analyzer . All the participants routinely underwent the abdominal ultrasonography scanning  by experienced radiologists, all of whom were blinded to the clinical status of the subjects. And, the fatty liver was diagnosed based on the guidelines for the diagnosis and treatment of nonalcoholic fatty liver disease, including (1) the near field echo of the liver is diffusely enhanced and stronger than the echo of the kidney; (2) liver brightness; and (3) vascular blurring [Patients' heights (measured to the nearest 0.1 cm) and weights (measured to the nearest 0.1 kg) were measured with light clothing by well-trained nurses in the morning. Body mass index +4.663. And, HGI is expressed as measured HbA1c minus predicted HbA1c. In a word, HGI is calculated as the difference between the observed HbA1c and the predicted value of HbA1c based on plasma glucose concentration [We randomly selected 1,967 subjects in the enrollment, and by inserting FPG into a population regression equation expressing the linear association between HbA1c and FPG; we drove the predicted HbA1c, which is defined as follows: HbA1c=0.146ntration , 24. The∗(ALT/AST ratio)+BMI. Additionally, NAFLD was defined as HSI above 36 [The Hepatic Steatosis Index (HSI) is a preliminary screening tool for NAFLD, which uses a formula based on BMI, ALT (IU/L), and AST (IU/L), and the presence or absence of diabetes as follows: HSI=8above 36 .∗age(years)+0.094∗BMI(kg/m2)+1.13∗impaired fasting glucose (IFG)  or  diabetes  +0.99∗AST/ALT  ratio–0.013∗platelet(∗109/L)–0.66∗albumin(g/dL). NFS <−1.455 predicts a low probability of advanced fibrosis [The NAFLD Fibrosis Score (NFS) is an invasive approach to detect liver fibrosis as follows: NFS=−1.675+0.037fibrosis .Data for continuous variables were expressed as mean values with standard deviation and as percentages for categorical variables. One-way analysis of variance with Bonferroni's method, post hoc analysis, and Chi-squared test were used to compare statistical differences in the characteristics of the study participants at baseline among the groups. Univariate logistic regression analysis was acted out to explore the risk factors of NAFLD by ultrasonography. Multiple logistic regression analysis was executed to estimate the OR and 95% CI for the association of categorized HGI levels with the risk of NAFLD. The area under the curve of the receiving operating characteristic (ROC) was used to evaluate the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the proposed risk score for the prediction of NAFLD. Statistical data analysis was performed by using the SPSS version 25.0 for Windows. And, statistical significance was set at <0.05.2. The baseline of anthropometric features and biochemical findings of the study population was stratified according to quartiles of HGI value showed in Study population comprised 3,936 participants, of whom 2,349 (59.7%) were male. Of the subjects at baseline, the participants older than 50 years old and with a BMI ≥25 were 1,437 (36.5%) and 1,278 (32.5%), respectively. Overall, the participants were relatively young and not obese, with the mean age of 47.7 ± 11.0 years old and mean BMI 23.8 ± 3.2 kg/mTo investigate the potential interactions affecting the prevalence of NAFLD, univariate logistic regression analysis was performed as shown in P < 0.05). We put them into the model, which determined the risk of NAFLD (NAFLD risk score=0.363 HGI+0.744 ln TG+1.674 ln FPG+0.213 BMI+1.361 ln ALT − 0.425 HDL − C+0.130 WBC − 14.381). The area below the ROC curve of this model was 0.85, and an optimal cutoff value of 0.174 generated sensitivity, specificity, positive predictive value, and a negative predictive value for NAFLD of 84.4%, 71.3%, 65.0%, and 88.0%, respectively. The AUC of HSI was 0.82, and a cutoff value of 33.1 gave a sensitivity of 82.1% and a specificity of 68.6% for detecting the presence of NAFLD , it has been proved that the higher the HGI, the greater the risk of retinopathy and nephropathy in the patients with type 1 diabetes . SimilarThe first study demonstrated the relationship between NAFLD and HGI in 1,120 white individuals without diabetes was in 2017. When adjusted for age, gender, and BMI, individuals in the highest quartile of HGI exhibited a 1.6-fold increased odd of having hepatic steatosis compared with subjects in the lowest. Another one published recently of 14,465 nondiabetic, Korea subjects showed that a high HGI was associated with a 1.56-fold increase in the risk of hepatic steatosis after adjusting more factors than the previous one. However, they did not further construct a risk score based on HGI to predict a person's risk of NAFLD, which can be useful for the clinician.Our study demonstrated that HGI was one of the independent risk factors for incident NAFLD among nondiabetic Chinese medical examination population. The result that 20.7% of the nondiabetic population who was affected by NAFLD was equivalent to the ultrasound results of one in every four adults with NAFLD in China . In compNowadays, not only the association between HGI and NAFLD has not been fully evaluated but also NAFLD pathogenesis is still unclear, so the pathophysiological mechanisms involved in the associations between each other are still undefined. Through this retrospective cross-sectional study, there are three suggested mechanisms to explain the association between HGI and NAFLD. First, we found a positive association between HGI and inflammatory biomarkers including WBC and platelet count. Obviously, chronic inflammation plays a key role in the progress of NAFLD, and insulin resistance is thought to be a core component of NAFLD. Impaired mitochondria can result in incomplete fat oxidation and generation of toxic lipid intermediates, which generate a large amount of reactive oxygen species (ROS) and reactive nitrogen free radicals (RNS) and lead to inflammation. Then, inflammation may impair insulin signaling and exacerbate liver fatty infiltration, even fuel the transition from NAFLD to NASH, and liver cirrhosis, even hepatocellular carcinoma , 31, 32.Our study had some limitations that should be taken into account. First, our study subjects were derived from a single center; for each population, new regression models should be derived, and multicentered research should be performed to further confirm the association in the next step. Second, our biochemical parameters, such as plasma glucose and HbA1c, were measured once. Even though this approach is commonly used in clinical research, between-individual variability of glucose homeostasis parameters may have led to some imprecisions in the stratification of study population into HGI quartiles. Third, the diagnosis of liver steatosis was performed by ultrasound scanning rather than by invasive methods such as liver biopsy or expensive noninvasive approaches such as proton magnetic resonance spectroscopy or computed tomographic scanning. However, ultrasonography is the most commonly used method to diagnose hepatic steatosis in clinical practice and epidemiological studies. Although liver biopsy is a standard criterion for NAFLD diagnosis, the diagnosis of fatty liver was based on ultrasound imaging with 90% sensitivity and 80% specificity .In conclusion, we further confirmed that NAFLD had an association with HGI level in nondiabetic individuals, and these associations were independent of obesity and other metabolic components. And, NAFLD risk score could be used as one of the risk predictors of NAFLD in nondiabetic population. But the causal relationship between HGI level and NAFLD in nondiabetic individuals is not undefined, while further perspective study should be taken.