Type 2 diabetes (T2D) is associated with increased risk of atherosclerotic vascular disease due to excessive vascular smooth muscle cell (VSMC) proliferation. Here, we investigated the role of mitochondrial dysfunction and Ca2+ levels in VSMC proliferation in T2D. VSMCs were isolated from normoglycemic and T2D-like mice induced by diet. The effects of mitochondrial Ca2+ uptake were studied using mice with selectively inhibited mitochondrial Ca2+/calmodulin-dependent kinase II (mtCaMKII) in VSMCs. Mitochondrial transition pore (mPTP) was blocked using ER-000444793. VSMCs from T2D compared to normoglycemic mice exhibited increased proliferation and baseline cytosolic Ca2+ levels ([Ca2+]cyto). T2D cells displayed lower endoplasmic reticulum Ca2+ levels, reduced mitochondrial Ca2+ entry, and increased Ca2+ leakage through the mPTP. Mitochondrial and cytosolic Ca2+ transients were diminished in T2D cells upon platelet-derived growth factor (PDGF) administration. Inhibiting mitochondrial Ca2+ uptake or the mPTP reduced VSMC proliferation in T2D, but had contrasting effects on [Ca2+]cyto. In T2D VSMCs, enhanced activation of Erk1/2 and its upstream regulators was observed, driven by elevated [Ca2+]cyto. Inhibiting mtCaMKII worsened the Ca2+ imbalance by blocking mitochondrial Ca2+ entry, leading to further increases in [Ca2+]cyto and Erk1/2 hyperactivation. Under these conditions, PDGF had no effect on VSMC proliferation. Inhibiting Ca2+-dependent signaling in the cytosol reduced excessive Erk1/2 activation and VSMC proliferation. Our findings suggest that altered Ca2+ handling drives enhanced VSMC proliferation in T2D, with mitochondrial dysfunction contributing to this process. Approximately 30% of individuals with T2D are affected by atherosclerosis and coronary artery disease . AlteredVSMCs derived from patients with T2D or treated with insulin and glucose exhibit excessive proliferation ,8. VarioThe role of cytosolic Ca2+ in regulating VSMC proliferation has received less attention ,12. IndeIn VSMCs, [Ca2+]cyto is regulated by plasma membrane export and uptake into the ER and mitochondria ,21,22. PHowever, T2D alters multiple aspects of mitochondrial function, including density, fission/fusion, respiration, and reactive oxygen species production . It remaThis study aims to investigate whether VSMC proliferation in a diet-plus-low-dose streptozotocin-based mouse model of T2D is driven by increased [Ca2+]cyto, and the extent to which mitochondrial dysfunction contributes to this process. Specifically, we explore whether manipulating mitochondrial Ca2+ entry or extrusion alters VSMC proliferation in T2D. To block Ca2+ entry via the mitochondrial Ca2+ uniporter (MCU), we expressed the inhibitor peptide mtCaMKIIN, which inhibits Ca2+/calmodulin-dependent kinase II (CaMKII) selectively in mitochondria ,28,29. ILastly, we connect our findings to the activation of cytosolic growth pathways. Our data emphasize the significance of mitochondrial Ca2+ handling in cell proliferation in T2D and propose it as a potential strategy to combat neointimal hyperplasia in T2D patients.To determine the dependence of VSMC proliferation on [Ca2+]cyto, we performed proliferation assays in aortic VSMCs from T2D and normoglycemic mice in the presence of the Ca2+ chelator BAPTA (1 µM). The addition of BAPTA eliminated the differences in cell proliferation between PDGF-treated VSMCs from T2D and normoglycemic mice A.Next, we investigated the impact of inhibiting mitochondrial Ca2+ entry on VSMC proliferation. We cultured aortic VSMCs derived from T2D and normoglycemic mice and observed that VSMCs from T2D mice exhibited significantly increased proliferation, particularly in the presence of PDGF B.We then examined the effect of inhibiting mitochondrial Ca2+ entry on proliferation. In these experiments, we used VSMCs from WT and transgenic mice expressing mtCaMKIIN in smooth muscle. Interestingly, the expression of mtCaMKIIN led to a significant reduction in VSMC proliferation in T2D mice, but not in normoglycemic mice B. FurthePreincubating VSMCs overexpressing mtCaMKIIN with the pharmacological MCU inhibitor Ru265 had no effect on cell proliferation. In WT VSMCs, it had a similar effect to mtCaMKIIN expression. These results support the notion that inhibiting mtCaMKII in mitochondria hampers proliferation by blocking the mitochondrial calcium uniporter (MCU) and, consequently, mitochondrial Ca2+ entry D,E.Based on these findings, we investigated the impact of T2D on [Ca2+]cyto levels and cytosolic Ca2+ transients in response to PDGF stimulation. We observed that VSMCs from WT T2D mice displayed higher baseline [Ca2+]cyto compared to normoglycemic mice of the same background A,B. In pWe also recorded cytosolic Ca2+ transients triggered by PDGF in the presence of thapsigargin and observed attenuated responses in VSMCs from T2D mice C–E. NotaFurthermore, we examined the release of endoplasmic reticulum calcium ([Ca2+]ER) by thapsigargin and found that it was lower in T2D conditions compared to normoglycemic WT conditions A–C. TherNext, we measured the baseline [Ca2+]mito, which was reduced in T2D and with mtCaMKII inhibition A. To stuWe also assessed the mitochondrial membrane potential and found that VSMC mitochondria from T2D mice were more depolarized compared to those from normoglycemic mice, which provides a mechanistic explanation for the decrease in Ca2+ entry E. To eliminate the possibility that differences in the number of mitochondria per cell contribute to the variances in mitochondrial Ca2+ transients, we evaluated the ratios of mitochondrial to nuclear DNA using ND1/HK2 and Cox1/HK2. We found no significant differences between cells from T2D mice with and without mtCaMKIIN expression . In summDecreased mitochondrial Ca2+ entry, potentially due to depolarization of the mitochondrial membrane potential, as well as ER dysfunction with decreased levels of SERCA and increased expression of IP3R favoring Ca2+ release, increase baseline [Ca2+]cyto. Inhibition of mitochondrial Ca2+ entry by mtCaMKIIN further exacerbates cytosolic Ca2+ overload and diminishes dynamic Ca2+ transients following PDGF stimulation.To identify cytosolic signaling events responsible for driving cell proliferation in relation to [Ca2+]cyto, we conducted a screening of canonical growth pathways, including MAP kinases. However, we observed a significant increase in the phosphorylation of Erk1/2 in VSMCs from T2D mice compared to normoglycemic mice A. These We confirmed that Erk1/2 activation contributes to accelerated proliferation in T2D. Higher concentrations of the Erk1/2 inhibitor U0126 were required to reduce proliferation in T2D VSMCs compared to normoglycemic mice to levels below control conditions without PDGF C,D. To dTo investigate the involvement of cytosolic CaMKII, a Ca2+-dependent upstream regulator of Erk1/2, we examined its autophosphorylation at Thr287. As expected based on increased [Ca2+]cyto, we observed enhanced autophosphorylation of CaMKII at baseline in whole cell lysates of VSMCs from T2D mice A. The exTo examine the functional implications of cytosolic CaMKII, we inhibited its activity by adenovirus-mediated expression of the untargeted inhibitor peptide CaMKIIN in the cytosol. This intervention reduced the baseline activation of CaMKII, MEK, and Erk1/2, restored the activation of the signaling pathway by PDGF C, and deWe next examined the occurrence of transient Ca2+ leakage from mitochondria through the mitochondrial transition pore (mPTP) in T2D VSMCs, as previously reported in mitochondria from diabetic cardiomyocytes ,33. We cTo further investigate the impact of mPTP on [Ca2+]cyto, we measured [Ca2+]cyto after adding the mitochondrial uncoupler FCCP in the presence and absence of the mPTP inhibitor ER-000444793 B–E. We ePretreatment with ER-000444793 significantly increased Ca2+ release after FCCP, indicating the occurrence of mPTP leakage in T2D VSMCs. Moreover, treatment with ER-000444793 reduced baseline [Ca2+]cyto F. Lastly, we investigated the effect of mPTP opening on cell proliferation by conducting cell counts in the presence of ER-000444793 G. In T2DOur study aimed to identify molecular mechanisms underlying VSMC proliferation in type 2 diabetes (T2D). While growth factors promote cell proliferation by increasing [Ca2+]cyto and transients ,14, the Here, we propose that in T2D, altered mitochondrial Ca2+ handling increases [Ca2+]cyto and drives excessive VSMC proliferation. In support of this notion, we observed reduced Ca2+ entry into mitochondria and lower [Ca2+]mito that inversely correlated with high [Ca2+]cyto. We proposed a decrease in the mitochondrial membrane potential as a mechanism for decreased Ca2+ entry into mitochondria in T2D. Increased metabolic supplies with high glucose or fatty acid flux to mitochondria lead to the production of mitochondrial oxygen radicals that can lower the mitochondrial membrane potential . BlockinConversely, we saw evidence of leakage of Ca2+ through the mPTP, and blocking mPTP reduced both [Ca2+]cyto and VSMC proliferation in T2D. Thus, in T2D, both decreased Ca2+ entry into the mitochondrial matrix and leakage through mPTP increased [Ca2+]cyto.The mPTP is a supramolecular complex at the interface of the inner and outer mitochondrial membranes . DespiteIncreased [Ca2+]cyto affects numerous functions in VSMCs, including contraction and vascular tone, and regulates cell cycle progression through activation of cytosolic CaMKII. While high cytosolic CaMKII activity has been reported in cardiac myocytes under hyperglycemia and various diabetes models ,52,53, oOur study has several limitations. While we concentrate on how mitochondrial dysfunction in T2D alters [Ca2+]cyto and on cytosolic signaling pathways, mitochondria also modulate VSMC proliferation through the production of ATP, precursors of macromolecules and intracellular redox homeostasis as well as enhanced glycolysis and fatty acid metabolism. Reprogramming of glycolytic pathways and lipid metabolism have recently been identified as drivers of phenotypic switching of VSMCs from a differentiated contractile to a de-differentiated proliferative VSMC and neointima formation . MoreoveStudies on VSMC proliferation as a driver of neointimal hyperplasia after balloon angioplasty have mainly focused on normoglycemic conditions ,62,63,64All animal procedures were approved by the University of Iowa Institutional Animal Care and Use Committee. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH).Studies conducted with protocol number: 0051189 and Approval date: 7 June 2020.Mice of the C57BL/6 background that express tamoxifen-inducible Cre recombinase  were obtained from Jackson Laboratories . HA-tagged mtCaMKIIN mice were generated by cloning a cDNA that encodes an HA-tagged form of the CaMKII inhibitor peptide CaMKIIN (HA-CaMKIIN) fused to the mitochondria-targeting Cox8-palmitoylation sequence into a construct that contains the CX-1 promoter and a floxed eGFP sequence. Double-transgenic mice expressing mtCaMKIIN specifically in VSMCs (denoted as “mtCaMKIIN mice”) were generated by crossing SM-CreERT2 mice with HA-tagged mtCaMKIIN mice . Eight-wSM-Cre mice treated with tamoxifen were used as controls for SM-mtCaMKIIN mice. Correct recombination and mtCaMKIIN expression were previously established . BecausAt 8–12 weeks of age and SM-mtCaMKIIN mice and littermate control mice were fed a high-fat diet . After 8 weeks, streptozotocin (STZ)  was administered by i.p. injections as two separate doses, per published protocols ,65. Norm2). Cells were routinely tested for mycoplasma contamination.Primary aortic VSMCs were isolated from mtCaMKIIN, and control mice by enzymatic digestion. The aortas were incubated in elastase (to remove adventitia) and cut into rings (about 1 mm in length) that were subsequently incubated in 2 mg/mL collagenase type II  for 2 h. The digested pieces were plated in DMEM with 450 mg/dL glucose, 1% penicillin/streptomycin, MEM nonessential amino acids, MEM Vitamin and 8 mmol/L HEPES) supplemented with 20% fetal bovine serum (FBS) and 0.1% fungizone. The cells were cultured in DMEM with 10% FBS at 37 °C in a humidified incubator (95% air and 5% COAdenoviruses expressing mtCaMKIIN (Ad-mtCaMKIIN), untargeted CaMKIIN (Ad-CaMKIIN), or mito-Pericam (Ad-mtPericam), as well as empty vector (Ad-control), were generated by the Viral Vector Core Facility at the University of Iowa. Mitochondrial calcium content was measured in isolated mitochondria using a Cayman Chemical Calcium Assay kit (Item No. 701220). Readings were normalized to protein concentration.Cells were loaded with Fura-2 acetoxymethyl ester (Fura-2AM) by incubation with 2 µM Fura-2AM in HBSS for 20 min at room temperature, then washed twice and incubated at 37 °C for 5 min for de-esterification. Cells were excited alternatively at 340 and 380 nm. Intensity of the fluorescence signal was acquired at 510 nm. Real-time shifts in the Fura-2AM fluorescence ratio were recorded before, during, and after acute addition of PDGF (20 ng/mL) using a Nikon Eclipse Ti2 inverted light microscope. Peak amplitude was calculated by subtracting the baseline fluorescence ratio from the peak fluorescence ratio. The area under the curve (AUC) was determined using GraphPad Prism and normalized by subtracting the AUC at baseline. Summary data represent the average differences in the basal and peak increases in [Ca2+]cyto.The free [Ca2+]cyto was calculated from Fura-2AM fluorescence using the equation [Ca2+]cyto = (R − Rmin)/(Rmax − R) · Kd · β, where R is the basal ratio of fluorescence at 340 nm to 380 nm, Kd is the Fura-2AM dissociation constant (145 nM), and β is the difference in Fura fluorescence intensity in Ca2+-free vs. Ca2+-saturated media.Ratiometric Ca2+ measurements in mitochondria were performed using adenovirus-delivered mtPericam (Ad-mtPericam), a fluorescent Ca2+ indicator protein with a COX IIIV targeting sequence. VSMCs were infected with Ad-mtPericam 48 h prior to analysis. Ratiometric fluorescent imaging of mtPericam via a Nikon Eclipse Ti2 microscope was used to determine the intensity of the fluorescence signal, with excitation at 415 nm and 480 nm, with emission at 510 nm. Peak amplitude and AUC were determined as described for Fura-2 AM.Cepia ER plasmid was purchased from Addgene (#58215). The CEPIA1er protein was excited at 543 nm, and emitted fluorescence was measured at wavelengths of 580 nm. VSMCs were transfected in a Nucleofector I device  using the Basic Nucleofector Kit for Primary Mammalian Smooth Muscle Cells  and following the manufacturer’s protocol. 50,000 cells were transfected in the presence of 1 μg of plasmid DNA, plated onto 35-mm glass bottom microwell dishes (MatTek Corporation), and grown for 48 h before experiments were performed.Calcium Green-5N (or Fura-2) was used to monitor cytosolic Ca2+ in permeabilized VSMCs. Signal was recorded in a 96-well plate. Total volume of reaction was 100 µL with 50 µL of cell suspension  and 50 μL of respiration buffer ) supplemented with 5 μM thapsigargin, 0.005% digitonin, and 1 μM Calcium Green-5N (or Fura-2) . Fluorescence was monitored at 485 nm excitation and at 535 nm emission. Following baseline measurement, CaCl2 (10 μM Ca2+ at 30 °C) was added sequentially every 4 min for 5 times until Ca2+ uptake ceased.Genomic DNA was isolated from approximately 500,000 VSMCs using the DNeasy Blood & Tissue extraction kit . DNA samples were treated with RNase and subsequently quantified using the Power SYBR Green Master Mix real-time PCR kit, with 100 ng genomic DNA per reaction . Mitochondrial DNA copy number was calculated using the 2−ΔCt formula, where Δ = Ctmito − Ctnuclear, for selected mitochondrial  and nuclear  genes.The mitochondrial membrane potential was measured using the tetramethylrhodamine methyl ester  and Mitotracker Green  fluorescent probes according to the manufacturers’ protocols. Images were taken at baseline and 15 min after FCCP treatment , using an LSM 510 confocal microscope at a magnification of 40× . Image analysis was performed using NIH ImageJ. All images were taken at the same time and using the same imaging settings. Data are presented as fold change over control with reference to the completely depolarized state.g for 10 min at 4 °C. Mitochondrial fractions were prepared in MSEM buffer , with homogenization performed in cold MSEM buffer in a Potter-Elvehjem glass Teflon homogenizer (50 strokes). Nuclei and cell debris were pelleted by centrifugation at 600× g for 5 min at 4 °C. Mitochondria were separated from the cytosolic fraction by centrifugation at 8000× g for 10 min at 4 °C. Protein concentrations were determined using the Pierce™ BCA protein assay (Thermo Scientific).Whole cells were lysed in RIPA buffer  supplemented with both protease inhibitors  and phosphatase inhibitors . Lysates were sonicated and debris was pelleted by centrifugation at 10,000× For immunoprecipitation assay, cells were lysed in Pierce IP Lysis Buffer . For each condition, 300 µg of proteins were used and incubated with primary antibody overnight at 4 °C degrees, and then with magnetic Dynabeads Protein G  for 1 h at room temperature. Dynabeads with precipitated proteins were washed 3 times with DynaMag-2 (Invitrogen) and eluted with LDS buffer .Equivalent amounts of 5–15 μg of protein for gel loading  were separated by SDS/PAGE on 4–20% Tris/glycine precast gels  and transferred to PVDF membranes (BioRad). Membranes were blocked in 5% BSA and incubated overnight at 4 °C with primary antibodies. Blots were washed 3 times for 10 min with 0.05% Tween-20 in TBS, incubated for 1 h at room temperature with the respective secondary antibodies, and then washed again. The blots were then developed using ECL chemiluminescent substrate  according to the manufacturer’s instructions.VSMCs were cultured in 12-well plates at 5000 cells per well in DMEM containing 10% FBS. At 24 h after plating they were treated with PDGF (20 ng/mL), and at 72 h after they were trypsinized and triplicate samples were counted using a Beckman Coulter Z1 cell counter.VSMCs were plated in a 96-well plate with a density of 1000 cells/well in 72 h before performing the MTT assay. After attaching, cells underwent PDGF treatment (20 ng/mL) and BAPTA treatment (1 µM) for 48 h. MTT stock solution was made in PBS at 5 mg/mL. 10 µL of MTT stock solution was added into each well with 90 µL of media in. After 2 h of incubation media was aspirated, cells washed with PBS once and intracellular MTT formazan crystals were dissolved in 50 µL of DMSO. Color intensity (absorbance) was measured on a microtiter plate reader at 570 nm light wavelength. p-value of <0.05 was considered significant.Data are expressed as mean ± SEM and were analyzed using the GraphPad Prism 9.0 software. All data sets were analyzed for normality and equal variance. The Kruskal–Wallis test with Dunn’s post hoc test was used to assess data sets where normal distribution could not be assumed. Student’s T-test and one-way ANOVA followed by Tukey’s multiple comparison test were used for data sets with normal distributions. Two-way ANOVA followed by Tukey’s multiple comparison test was used for grouped data sets. A Here, we present several key findings: VSMCs from T2D mice exhibited excessive proliferation and elevated [Ca2+]cyto. T2D decreased mitochondrial Ca2+ transients in response to PDGF administration compared to normoglycemic conditions. Moreover, we observed Ca2+ leakage through the mPTP as well as for ER Ca2+ depletion. Activation of Erk1/2 and its upstream regulators was enhanced in VSMCs from T2D mice, driven by increased [Ca2+]cyto. Inhibition of mitochondrial Ca2+ entry through mtCaMKIIN increased [Ca2+]cyto, leading to exaggerated Ca2+ imbalance and baseline Erk1/2 hyperactivation. This inhibited further PDGF-induced Erk1/2 activation and cell proliferation. Inhibition of cytosolic Ca2+-dependent signaling reduced excessive Erk1/2 activation and decreased VSMC proliferation. Furthermore, blocking mPTP reduced [Ca2+]cyto and proliferation in VSMCs from T2D compared to normoglycemic mice. Thus, in T2D, a condition with elevated [Ca2+]cyto, both lowering of [Ca2+]cyto by mPTP inhibition as well as further increasing it by MCU inhibition can reduce cell proliferation, suggesting a “sweet spot” or “goldilocks effect” for [Ca2+]cyto to promote proliferation. These findings provide novel insights into the role of altered Ca2+ handling and mitochondrial dysfunction in VSMC proliferation in T2D.


Pak1 deficiency in the cardiac atria have not been well explored. In this study, Pak1 cardiac-conditional knock-out (cKO) mice were studied under baseline and adrenergic challenge conditions. Pak1 cKO mice show atrial arrhythmias including atrial fibrillation (AF) in vivo, detected during anaesthetized electrocardiography without evidence of interstitial fibrosis upon Masson's trichrome staining. Optical mapping of left atrial preparations from Pak1 cKO mice revealed a higher incidence of Ca2+ and action potential alternans under isoprenaline challenge and differences in baseline action potential and calcium transient characteristics. Type-2 ryanodine receptor (RyR2) channels from Pak1 cKO hearts had a higher open probability than those from wild-type. Reverse transcription-quantitative polymerase chain reaction and Western blotting indicated that pCamkIIδ and RyR2 are highly phosphorylated at baseline in the atria of Pak1 cKO mice, while the expression of Slc8a2 and Slc8a3 as a Na+–Ca2+ exchanger, controlling the influx of Ca2+ from outside of the cell and efflux of Na+ from the cytoplasm, are augmented. Chromatin immunoprecipitation study showed that pCreb1 interacts with Slc8a2 and Slc8a3. Our study thus demonstrates that deficiency of Pak1 promotes atrial arrhythmogenesis under adrenergic stress, probably through post-translational and transcriptional modifications of key molecules that are critical to Ca2+ homeostasis.P21-activated kinase 1 (Pak1) signalling plays a vital and overall protective role in the heart. However, the phenotypes of This article is part of the theme issue ‘The heartbeat: its molecular basis and physiological mechanisms’. Abnormal ‘phosphorylation states' of Ca2+-handling proteins occurs in several disease conditions including AF  approximately 50 mM) on the trans  side of the bilayer at 21°C. The luminal chamber was voltage-clamped at ground. The free [Ca2+] and pH of the solutions were measured using a Ca2+ electrode  and a Ross-type pH electrode  as previously described i under Ang II stress at single cell level.Our electrophysiological studies in intact animals and isolated atrial independently implicated out mice  that sho2+ alternans recorded in LA in particular when they are acutely exposed to isoprenaline stress in our study suggests the alteration of Ca2+ homeostasis that could be linked to either abnormal SR Ca2+ release or decrease in kinetics of SERCA2A and NCX currents and an increase in diastolic Ca2+ levels in as we found in Pak1 cKO mouse ventricular cardiomyocytes under baseline conditions [2+ reuptake into the SR via SERCA2A inhibition using the antagonist thapsigargin was demonstrated to increase the susceptibility to cellular Ca2+ alternans [et al. [Canditions . Upon islternans . Additio [et al.  have sho [et al. .2+ refilling time in Pak1 cKO ventricular myocytes when compared to WT littermates. This observation along with slow NCX and SERCA2A kinetics, may imply longer cytosolic Ca2+ transients. In fact, ventricular myocyte experimentation using total Pak1 KO mice have reported longer Ca2+ transient recovery time in KO than in WT mice [Pak1 cKO mice under baseline conditions in the present study.We previously  have als WT mice . The two2+ sensitivity of RyR2 channels from PAK1 cKO mice. This increase in RyR2 activity might have resulted from the higher phosphorylation levels at S2808 and S2814 detected in the PAK1 cKO heart preparations since enhanced phosphorylation at these sites has been linked to RyR2 dysregulation and increased RyR2-mediated cytosolic Ca2+ leak [2+ would probably impact on the consistency of local SR Ca2+-release events, providing an explanation of the cellular basis for an increase in arrhythmogenesis and abnormal Ca2+ transient observed in Pak1 cKO mice.Our data on RyR2 single-channel recordings revealed that channels derived from PAK1 cKO hearts exhibit a markedly higher Po than channels from control hearts, suggesting an increase in cytosolic Caa2+ leak –39. The Pak1 cKO mice with alterations of the activity of RyR2 and CamKII and expression of Serca2a and Ncx genes that are essential to Ca2+ homeostasis and electrophysiological stability in the atria. These changes may reflect multiple regulatory mechanisms that PAK1 is involved in Ca2+ handling proteins activity and expression at both post-translational and transcriptional levels. Firstly, in our previous study, we have established the action of PAK1 on the phosphatase PP2A and the resulting balance between kinase and phosphatase activity controlling LTCC and IK activity in sinoatrial node pacemaker cells [Pak1 cKO mouse atrial tissue. Admittedly, such mechanisms require more investigations in the future, while the downregulated SERCA2a and upregulated Slc8a2 and Slc8a3 transcripts in Pak1 cKO atrial tissue may reflect a complicated transcriptional regulation but important role of Pak1 as a regulator of cardiac SERCA2a and Slc8a2 and Slc8a3 through transcriptional mechanisms, which is consistent with our previous finding in the ventricular tissue [Our molecular studies then associated PAK deficiency in er cells ; An impoer cells . As a coer cells . Thus Par tissue .2+ homestasis in the atria through regulation of calcium handling proteins RyR2, Serca2a and NCX via either post-translational and transcriptional mechanisms, thus providing new insights into atrial Ca2+ signalling regulatory mechanism that has an implication in developing new therapeutic strategies for atrial tachyarrhythmias.In conclusion, our study demonstrates for the first time, to our knowledge, a key regulatory role of Pak1 for maintaining electrophysiological stability and Ca


Dear Editor,AML is a heterogenous disease characterized by distinct clinical courses and prognoses based on genomic, epigenomic and transcriptomic profiles , 2. Thern = 671), GSE6891 [n = 461) and GSE106291 [n = 250), we demonstrated the prognostic power of the 571-gene panel in classifying AML into clinically relevant subtypes.In this study, we performed an integrative clustering (iCluster) , 10 anal GSE6891  (n = 461SE106291  (n = 250p = 0.039)  and RUNX1 (27%); the iSubtype 2 was characterized by CK and high-frequency mutation of CEBPA (20%); while the iSubtypes 3 and 4 were characterized by normal karyotype (NK), deficiency of TP53/CEBPA/RUNX1 mutations, and abundance of FLT3/NPM1/DNMT3A mutations, with the iSubtype 4 having higher mutation rates in the three genes than the iSubtype 3 . At the epigenomic level  Fig. . At the vel Fig. . At tranvel Fig. , mRNA, tvel Fig. .Fig. 1InIn comparison to the ELN2022 classification [There were 571 common genes identified as the subtype-drivers in the methylation and transcriptomic datasets and these genes formed three major clusters c1-3 and their overall expression patterns were negatively correlated Fig. . For exap = 0.7), implying that using copy number data alone is not sufficient to stratify AML into clinically meaningful subgroups. By integrating the other omics data, iCluster further divided the CK-AML into iSubtypes 1 and 2, and the NK-AML into iSubtypes 3 and 4, respectively. The iSubtype 1 had an inferior OS than the iSubtype 2, which might be due to its much higher mutation rate of TP53 (30% in 1 vs. 4% in 2). These observations are consistent with the reports that a subgroup of CK-AML/MDS (myelodysplastic syndromes) with mutated TP53 (mTP53) had a worse prognosis than the subgroup with CK alone [WNT beta catenin, TGF beta, KRAS, NOTCH, hedgehog, IL2_STAT5), and cellular component organization . Additionally, CTLA4 and PDL1 had a significantly higher expression in the iSubtype 1 compared to the other iSubtypes, which could be potential targets for anti-CTLA4 and anti-PDL1 therapies  and NK-AML (iSubtype 3 + 4). However, the OS was not significantly different between iSubtype 1 + 2 and iSubtype 3 + 4 , signaling , proliferation  and metabolism  ; negative regulation of T cell receptor signaling: LGALS3, PTPRJ, LAPTM5) had an elevated expression in the iSubtype 4 (Supplementary Fig. It is still a challenge to classify NK-AML into subgroups for prognosis and target therapy. By iCluster analysis, the NK-AML iSubtypes 3 and 4 were distinguished by Currently, diagnosis and management of AML are heavily dependent on genetics-based risk classification such as ELN2022. Overall, the risk classifications of AML by iCluster, ELN2022 and cytogenetics were comparable (Supplementary Fig. Supplementary MaterialsSupplementary Table 1Supplementary Table 2Supplementary Table 3


Urticaria is a common disease that can affect individuals of all age groups, with approximately one-quarter of the population experiencing it at least once in their lifetime. Lesions characterized by erythema and itchy hives can appear anywhere on the body. These can vary in size ranging from millimeters to centimeters, and typically clear within 24 h. About 40% of patients with urticaria have accompanying angioedema, which involves localized deep tissue swelling. Urticaria usually occurs spontaneously and is classified into acute and chronic forms, with the latter referring to a condition that lasts for more than 6 weeks. The prevalence of chronic urticaria in the general population ranges from 0.5% to 5%, and it can either be inducible or spontaneous. The most common form of pediatric urticaria is acute and is usually self-limiting. However, a broad differential diagnosis should be considered in children with urticaria, particularly if they also have accompanying systemic complaints. Differential diagnoses of pediatric urticaria include chronic spontaneous urticaria, chronic inducible urticaria, serum sickness-like reaction, urticarial vasculitis, and mast cell disorders. Conditions that can mimic urticaria, including but not limited to cryopyrinopathies, hyper IgD syndrome, Periodic Fever, Aphthous Stomatitis, Pharyngitis and Adenitis (PFAPA), Tumor Necrosis Factor Receptor Associated Periodic Syndrome (TRAPs), and Schnitzler syndrome should also be considered. The many faces of pediatric urticaria can be both easy and confusing. A pragmatic approach relies on clinical foresight and understanding the various forms of urticaria and their potential mimickers. This approach can pave the way for an accurate and optimized diagnostic approach in children with urticaria. The lesions can vary in size from millimetric to centimeters. Angioedema, a localized deep-tissue swelling, is associated with urticaria in 40% of patients and resolves within hours to days. Urticaria and angioedema are usually accompanied by an itching and burning sensation, respectively , 2.In children, acute urticaria is the most commonly observed form of the condition, although chronic spontaneous urticaria (CSU) and chronic inducible urticaria (CındU) are also frequently seen, with a prevalence comparable to that in adults . While dUrticaria is a prevalent and time-consuming condition encountered in pediatric practice. While it is generally diagnosed and managed promptly, atypical presentations and clinical courses can pose challenges. The primary objective of this review article was to provide a pragmatic approach and comprehensive summary of urticaria and its mimickers, thereby aiding clinicians in accurate diagnosis and effective management.2.2.1.Anaphylaxis is a severe and potentially life-threatening reaction that usually occurs within seconds or minutes of exposure to an allergen. Several distinguishing characteristics can aid in the differentiation between anaphylaxis and urticaria. Anaphylaxis typically manifests with a sudden onset, occurring within minutes to hours after exposure to an allergen, whereas urticaria tends to develop more gradually and can persist for longer. Anaphylaxis often co-exists with urticaria, but is characterized by the presence of additional systemic symptoms. These symptoms can affect various organ systems, including the gastrointestinal, respiratory, cardiovascular, and neurological systems. Urticaria typically resolves within a few hours to several days, whereas anaphylaxis can have a shorter duration but may persist for longer if not promptly treated .Anaphylaxis affects multiple systems in the body and can progress rapidly if left untreated. The signs and symptoms of anaphylaxis can vary, but mostly (>80%) include skin reactions, such as urticaria, angioedema, pruritis, flushing, or pallor. Gastrointestinal symptoms include nausea, vomiting, abdominal pain, or diarrhea. Morbidity and mortality are high if there is respiratory or cardiovascular system involvement . Respira2.2.Exercise-induced anaphylaxis is characterized by the development of anaphylaxis, including hives, dyspnea, hypotension, and systemic reactions, triggered explicitly by physical activity or exercise. Many patients notice a tingling, itching, or burning sensation on the skin 10–25 min after exercise before hives appear. The macules may coalesce to form large areas of erythema . It is cIn exercise-induced anaphylaxis, the appearance of urticaria usually coincides with or shortly follows the onset of physical activity. Urticarial lesions are often widespread, affecting various areas of the body. They can occur on the skin surface exposed to friction during exercise, such as the thighs, trunk, arms, and neck. Urticaria in exercise-induced anaphylaxis is accompanied by intense itching, which may be a notable symptom during physical activity. The individual hives are typically larger in size (often >1 cm in diameter) and may coalesce to form larger areas of swollen and red skin. The hives may persist for variable durations, ranging from minutes to hours after the exercise has ceased .It is important to consider the sudden onset of other systemic symptoms, such as respiratory distress, gastrointestinal symptoms, and cardiovascular changes, as these can help differentiate this condition from isolated urticaria.In some cases, exercise alone can trigger anaphylaxis, whereas in others, the combination of exercise and specific triggers, such as certain foods or medications, is necessary. Food-dependent exercise-induced anaphylaxis (FDEIA) is an exercise-related allergy after food intake. The most commonly reported triggering foods are wheat, celery, grapes, nuts, peaches, eggs, rice, tomatoes, seafood, and cheese . Choline2.3.Urticaria is the most prevalent form of pediatric urticaria, affecting up to 25% of the pediatric population . It can A study evaluating urticaria ICD codes suggested that the most common causes of urticaria after infection are drugs, foods, insect bites, pollen, blood products, and vaccination . The rat2.4.2.4.1.Chronic spontaneous urticaria, previously named chronic idiopathic urticaria, is a long-lasting form characterized by hives/wheals that occur spontaneously without an identifiable trigger . UrticarSeveral factors are known to be associated with the development or exacerbation of chronic urticaria including autoimmune factors, physical stimuli , infections, drugs such as non-steroidal anti-inflammatory drugs (NSAIDs), antibiotics , and antihypertensives , food and food additives , allergens  and emotional stress. CSU also appears to be one of the leading risk factors for hypersensitivity to NSAIDs in childhood , 19, 20.The prevalence of pediatric CSU is 0.8%, which is a comparable rate to that of adults . CSU doeIn about half of cases, CSU may resolve within 5 years, but recurrence is common , 24, 25.Chronic spontaneous urticaria is thought to involve an abnormal immune response, although the exact underlying mechanisms are not fully understood . It may 2.4.2.Chronic inducible urticaria, previously known as physical urticaria, is a form of chronic urticaria where certain stimuli specifically trigger hives . Unlike Managing CIndU involves identifying and avoiding specific triggers whenever possible. Antihistamines are typically the first-line treatment to help alleviate symptoms and reduce the frequency and severity of hives , 46.2.5.Contact urticaria and urticarial contact dermatitis are two distinct skin conditions, which although sharing some similarities, exhibit differences in their characteristics.Contact urticaria typically manifests as the development of hives and itching at the point of contact with an allergenic or irritating substance. These symptoms arise swiftly, often within minutes of exposure to the triggering agent. Common culprits encompass foods, latex, specific chemicals, plants, and insect bites. The manifestations of contact urticaria typically subside within a few hours once the triggering substance is removed .Urticarial Contact Dermatitis also presents with hives and itching at the site of contact with an allergen or irritant. The visual appearance of the rash resembles that of contact urticaria. However, in urticarial contact dermatitis, the rash emerges relatively rapidly, akin to contact urticaria, but can also occur several hours to a day following exposure. Urticarial contact dermatitis stems from allergic or irritant reactions upon skin contact with particular substances, including certain chemicals, plants (such as poison ivy or poison oak), or metals  , 49. UnlIn children, the most frequent causes of contact urticaria are foods, which are particularly prevalent in children with atopic dermatitis. Food-associated contact urticaria typically results from immunological reactions . Neverth2.6.Mast Cell Activation Syndrome (MCAS) is a condition characterized by the abnormal activation and release of mast cell mediators throughout the body. It is considered a subset of mastocytosis, but unlike mastocytosis, MCAS does not involve a significant increase in mast cell numbers . The symUnlike urticaria, MCAS has a wide range of systemic symptoms that go beyond just skin manifestations. In addition, the diagnosis of MCAS includes evaluation of the levels of mast cell mediators such as tryptase during both asymptomatic and symptomatic episodes .3.3.1.Serum sickness-like reaction, also known as drug-induced SSLR, is a type III hypersensitivity reaction that can occur in response to certain medications. It resembles serum sickness, but the term “serum sickness-like” is used because it typically occurs without the exposure of serum or its components . In SSLRSerum sickness-like reaction can occur in a broad age range from infancy to adolescence. In the study of Magana et al. , which i3.2.Kawasaki disease primarily affects children under the age of five and is characterized by the involvement of medium-sized blood vessels, including the coronary arteries , 59. The3.3.Papular urticaria results from a chronic, recurrent hypersensitivity reaction that often develops late after insect bites , 63. The3.4.Urticaria multiforme, previously referred to as “acute annular urticaria” , 66, desDiagnostic criteria for urticaria multiforme include: (i) Annular to polycyclic urticarial lesions with transient ecchymotic skin changes, (ii) Individual lesions lasting less than 24 h, (iii) Presence of angioedema or acral edema, (iv) Dermatographism, (v) Increased levels of acute phase reactants, (vi) Favorable response to antihistamines . Unlike 3.5.Juvenile idiopathic arthritis (JIA) is an inflammatory disease that occurs before the age of 16 years, and is characterized by arthritis lasting at least 6 weeks. It is the most common form of arthritis in childhood .Cutaneous involvements are non-fixed erythematous rashes. The rash associated with systemic-onset JIA typically appears early in the course of the disease, often before arthritis develops. It is usually one of the first noticeable signs, together with systemic symptoms such as fever, fatigue, and other general malaise. The rash in systemic-onset JIA is characterized by salmon-pink or light red spots, and it is often described as “evanescent” because the rash comes and goes, changing its location and appearance. The rash can be transient, with individual spots lasting for a few hours to a few days and then disappearing. New spots may appear while others fade away, leading to a migratory pattern .Studies have shown that IL-6 plays a role in the pathogenesis of the disease, and therefore, tociluzumab, an anti-IL-6 receptor antibody, is beneficial in the treatment of the disease .3.6.Mastocytosis is a rare disorder characterized by the abnormal accumulation and activation of mast cells in various tissues throughout the body , 72. CutSystemic mastocytosis is a form that involves the proliferation of mast cells in various organs and tissues throughout the body, including the bone marrow, gastrointestinal tract, liver, spleen, and lymph nodes. Symptoms can vary widely and may include flushing, itching, abdominal pain, diarrhea, low blood pressure, and symptoms associated with allergic reactions .The diagnosis of mastocytosis involves a combination of clinical evaluation, physical examination, skin biopsies, and laboratory tests. Specialized testing, such as bone marrow biopsy and genetic analysis, may be required to assess the extent and subtype of the condition .3.7.Urticarial vasculitis is a rare condition that combines both urticaria and vasculitis. It is characterized by recurrent episodes of urticaria lasting longer than 24 h which leave behind purpura, a bruise-like discoloration , 77. The3.8.Autoinflammatory urticarial syndromes, also known as “autoinflammatory diseases with urticarial rash”, constitute a group of rare genetic disorders characterized by recurrent episodes of urticarial rashes accompanied by systemic inflammation. These syndromes are classified within the broader category of autoinflammatory diseases, distinct from autoimmune diseases due to dysregulated innate immune responses instead of abnormal adaptive immune responses.The hallmark of these syndromes is urticarial rashes presenting as red, itchy, raised patches on the skin with transient presence from hours to days. Lesions usually last up to 24 h and appear as flat erythematous swellings, mostly distributed on the trunk and/or extremities and which do not respond to H1- antihistamines. Often the lesions may be painful and may not be accompanied by itching.Patients with autoinflammatory urticarial syndromes experience not only skin involvement but also episodes of systemic inflammation. This inflammation can affect various organs and tissues, leading to symptoms such as fever, joint pain, swelling, and occasionally, organ-specific manifestations such as abdominal or chest pain .The primary cause of Autoinflammatory Urticarial Syndromes lies in genetic mutations impacting innate immune system proteins that regulate inflammation and immune responses. The genetic basis varies depending on the specific syndrome, with several genes implicated in different cases .While certain triggers, such as infections, stress, or environmental factors, may exacerbate or provoke rash and inflammation episodes in some cases, they are not universally necessary for symptom development.The age of onset and duration of symptoms can vary depending on the specific syndrome, with some presenting early in infancy or childhood, while others develop later in life .Autoinflammatory urticarial syndromes encompass different diseases, each with a distinct genetic basis, clinical features, and management considerations.3.8.1.Cryopyrin-Associated Periodic Syndrome (CAPS) is a rare autoinflammatory disorde, which belongs to a group of conditions known as hereditary periodic fevers. It is caused by mutations in the NLRP3 gene encoding cryopirin, which leads to the overproduction of interleukin-1 beta (IL-1β). There are three subtypes of CAPS, which vary in severity .Familial Cold Autoinflammatory Syndrome (FCAS) is the mildest form of CAPS and is characterized by recurrent episodes of rash, fever, joint pain, and malaise triggered by exposure to cold temperatures. The symptoms typically resolve spontaneously within 24 to 48 h .Muckle-Wells Syndrome (MWS) is an intermediate form of CAPS characterized by recurrent episodes of rash, fever, joint pain, conjunctivitis, and hearing loss. In addition, patients may experience systemic symptoms such as headache, abdominal pain, and fatigue. The symptoms usually last longer than those of FCAS .Neonatal-Onset Multisystem Inflammatory Disease (NOMID) or Chronic Infantile Neurological, Cutaneous, Articular Syndrome (CINCA) is the most severe form of CAPS. It typically presents in infancy and is characterized by persistent inflammation affecting multiple organs, including the skin, joints, eyes, and central nervous system. Patients may experience chronic rash, arthritis, meningitis, hearing loss, developmental delays, and other neurological abnormalities .When the clinical presentation shows the early onset of recurrent urticaria without itching, triggered by cold, fever, constant musculoskeletal pain, fatigue, and listlessness, CAPS should be considered in the differential diagnosis . The dia3.8.2.TNF Receptor-Associated Periodic Syndrome is a rare autoinflammatory disorder characterized by recurrent inflammation episodes affecting various body parts . It is cIndividuals with TRAPS experience recurrent episodes of fever that last from 1 to 3 weeks. The fever is often accompanied by fatigue, malaise, and increased levels of acute-phase reactants. TRAPS can cause various cutaneous symptoms, including rash, hyperemia, edema, and urticaria . These sIn some cases, TRAPS can involve other organs, such as the eyes, heart, and lungs. Uveitis is a common complication and can lead to permanent vision problems . DiagnosTreatment for TRAPS focuses on managing acute episodes and preventing long-term complications. Non-steroidal anti-inflammatory drugs (NSAIDs) and corticosteroids may be used to control symptoms during episodes. Biological medications targeting tumor necrosis factor or interleukin-1 may be considered for individuals with severe or refractory symptoms .3.8.3.Mevalonate Kinase Deficiency, Hyper IgD syndrome, is a rare autosomal recessive metabolic disease. It is due to mutations in the MVK gene, which leads to a decrease in the activity of the enzyme mevalonate kinase preventing the conversion of mevalonic acid to phosphomevalonic acid. This results in the accumulation of mevalonic acid and a deficiency in isoprenoid end products. All these changes cause recurrent inflammation .Individuals with Mevalonate Kinase Deficiency experience recurrent episodes of high fever that typically last 3 to 7 days. These febrile episodes can occur at irregular intervals, ranging from weeks to months. The fever episodes often start in early childhood, and together with fever, patients may experience various other symptoms during episodes, such as abdominal pain, diarrhea, arthritis/arthralgia, headache, lymph node enlargement, and skin rash. Cutaneous manifestations include maculopapular rashes, vasculitis, oral aphthous ulcers, and occasionally erythema nodosum . MacrophThe diagnosis of Mevalonate Kinase Deficiency is based on the combination of clinical evaluation, genetic testing to identify mutations in the MVK gene, and laboratory tests to assess elevated IgD levels during fever episodes.Treatment focuses on managing the symptoms and preventing complications. Medications such as non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, and anti-interleukin-1 (IL-1) neutralizing agents may control inflammation during episodes.3.8.4.Periodic fever, Aphthous stomatitis, Pharyngitis and Cervical Adenitis syndrome is a rare autoinflammatory disorder primarily affecting children. It is characterized by recurrent episodes of high fever (39°C) accompanied by aphthous stomatitis, pharyngitis, and cervical lymphadenopathy . These pAphthae can be seen in the oral and genital mucosa. PFAPA can also be confused with Behçet's disease .The exact cause of PFAPA syndrome is unknown, but it is believed to involve immune system dysregulation. Diagnosis is typically based on the pattern of recurrent fever episodes and the presence of associated symptoms. Laboratory tests are usually performed to rule out other causes of periodic fever syndromes. Most children with PFAPA syndrome eventually outgrow the condition by adolescence.3.8.5.Schnitzler Syndrome is a rare autoinflammatory disorder, and the typical age range of patients with this condition is between 40 and 70 years old. It is more commonly diagnosed in middle-aged to older adults, although cases outside this age range have also been reported, albeit infrequently .Schnitzler syndrome is characterized by the presence of recurrent fever, chronic urticarial, skeletal and muscular pain, fatigue, and monoclonal gammopathy . IndividThe exact cause of Schnitzler syndrome has not been clarified as yet, but it is considered to be an acquired disorder.Diagnosing Schnitzler syndrome requires a combination of clinical evaluation, the presence of specific symptoms, and laboratory investigations to confirm the presence of monoclonal gammopathy. Laboratory examinations include measurement of levels of acute phase reactants, monoclonal IgM/IgG, serum amyloid A (SAA), S100, A8, A12, serum immune fixation, and skin biopsy .3.8.6.Adult-onset Still's Disease is a rare systemic autoinflammatory disease that is difficult to diagnose. The onset of the disease usually occurs between the ages of 16–82 years . EnvironHepatosplenomegaly and lymphadenopathy are common physical examination findings . The dia3.9.Wells syndrome, also known as eosinophilic cellulitis, eosinophilic dermatitis, or hyper-eosinophilic syndrome, is a rare skin disorder characterized by recurrent episodes of skin inflammation with prominent eosinophilic infiltration . The epiAfter excluding causes of hypereosinophilia, further investigations should be performed regarding organ involvement, and genetic analysis can clarify the diagnosis .3.10.Bullous pemphigoid is a chronic autoimmune blistering disorder that primarily affects the elderly, but it has been reported in a wide range of age groups. It generally starts with a non-specific itchy rash and may develop an urticarial appearance over time  Figure . Lesions3.11.Frey syndrome, also known as auriculotemporal syndrome, is a condition characterized by abnormal flushing of the skin on the face, typically triggered by eating certain foods . Frey sy4.Urticaria in childhood is a condition that impairs the quality of life of parents and children and can cause serious anxiety when it appears, does not improve, continues, or recurs. Since urticaria plaque is both the result of many different conditions and can mimic different skin lesions, a wide range of diseases should always be considered in the differential diagnosis. The range and extent of the diseases to be considered in the differential diagnosis can be confusing. A pragmatic approach relies on clinical foresight and knowledge of different clinical situations and their mimics, so that often a simple diagnostic approach can be performed in children without the need for extensive diagnostic work-up.


Angiomyolipoma (AML) is classified as perivascular epithelioid cell neoplasm (PEComas) and is commonly seen in the kidney. AML is a solid mesenchymal neoplasm rarely encountered at the extrarenal site. Extrarenal AML is infrequently seen in the female genital tract. Four cases of AML of the cervix have been reported in the literature to our knowledge. We report a case of a 44-year-old female patient who presented with complaints of “lower abdominal pressure” and a history of post-coital bleeding and human papillomavirus (HPV) infection. A cyst in the uterine cervix was found incidentally on computerized tomography (CT) scan of the abdomen and pelvis. The patient underwent a loop electrosurgical excision procedure. The histologic and immunohistochemical features of the cervical biopsy favored the diagnosis of AML. The patient underwent a laparoscopic hysterectomy with bilateral salpingectomy. Grossly, a 4 cm white soft-to-firm mass was identified within the anterior lip of the cervix. Microscopy of the mass showed smooth muscle proliferation with prominent blood vessels, and scant mature adipose tissue trapped in between the smooth muscle bundles. Immunohistochemical stains showed smooth muscle actin (SMA) and desmin highlighting the smooth muscle component of AML. The histology and immunohistochemistry of the cervical mass in the surgical specimen were identical to the biopsy specimen and a diagnosis of AML was made.  Angiomyolipoma (AML) is a benign, solid, mesenchymal tumor and is one of the perivascular epithelioid cell neoplasms (PEComas) -2. It isUsually, AML occurs sporadically but an association with tuberous sclerosis has been reported . AngiomyA 44-year-old female patient presented with complaints of “lower abdominal pressure” and a history of post-coital bleeding and Human papillomavirus (HPV) infection. A computerized tomography (CT) scan of the abdomen and pelvis revealed an incidental finding of a cyst in the uterine cervix. The patient underwent a loop electrosurgical excision procedure (LEEP), and a biopsy from the “cervical lesion at 12 o clock” was collected. The histologic and immunohistochemical features of the biopsy specimen helped to make a diagnosis of AML. The patient underwent a laparoscopic hysterectomy with bilateral salpingectomy. Gross examination revealed a 4 x 3 x 2 cm white soft-to-firm mass present within the anterior lip of the cervix . The histomorphology and immunohistochemical profile of the lesion in the surgical specimen was identical to the biopsy specimen. The diagnosis of AML of the uterine cervix was confirmed.Angiomyolipoma (AML) is a benign tumor that predominantly arises in the kidney and rarely as an extrarenal tumor. The distinctive histological features of AML, characterized by mature adipose tissue, tortuous thick-walled blood vessels, and smooth muscle cells arranged in sheets or other patterns, were described several decades before the term "angiomyolipoma" was coined by Morgan et al. in 1951 .AML is composed of haphazardly arranged mature adipose tissue, smooth muscle cells, and tortuous, thickened blood vessels. AML can occur in two forms: sporadic  and hereditary (with no gender predilection). AML is commonly associated with tuberous sclerosis (TSC), and renal AML is the most common manifestation of renal disease in patients with TSC. Mutations in TSC1 and TSC2 genes increase the risk of developing renal AML -13. AssoDifferential diagnoses include lipoleiomyoma and vascular leiomyoma with the fat component. Lipoleiomyomas commonly occur in postmenopausal women aged 50-60 years and are rarely found in the cervix . The patWhile the kidney is the most common location for AML, extrarenal AMLs are extremely rare. Only four cases of AML of the uterine cervix have been reported .Angiomyolipoma occurring in the uterine cervix is very rare. To date, only four cases have been reported. Angiomyolipoma should be considered as one of the differential diagnoses in the case of lower abdominal mass and dysfunctional uterine bleeding. A careful and detailed histologic investigation is necessary to alleviate the concerns of both the patient and the surgeon. Reporting this extremely rare and interesting case will definitely help both pathologists and clinicians to diagnose and treat this benign condition.


The pro-episkevic approach directly derives from a mechanistic model based on the RI nucleoshuttling of the ATM protein (RIANS) and contributes to increase the number of DSB managed by NHEJ, the most predominant DSB repair and signaling pathway in mammalians. Here, three radioresistant and three radiosensitive human fibroblast cell lines were pretreated with antioxidative agents (N-acetylcysteine or amifostine) or to two pro-episkevic agents (zoledronate or pravastatin or both (ZOPRA)) before X-ray irradiation. The fate of the RI DSB was analyzed by using γH2AX and pATM immunofluorescence. While amifostine pretreatment appeared to be the most efficient antioxidative process, ZOPRA shows the most powerful radiation protection, suggesting that the pro-episkevic strategy may be an alternative to the antioxidative one. Additional investigations are needed to develop some new drugs that may elicit both antioxidative and pro-episkevic properties and to quantify the radiation protection action of both types of drugs applied concomitantly.The radiation protection strategy with chemical agents has long been based on an antioxidative approach consisting in reducing the number of radical oxygen and nitrogen species responsible for the formation of the radiation-induced (RI) DNA damage, notably the DNA double-strand breaks (DSB), whose subset participates in the RI lethal effect as unrepairable damage. Conversely, a DSB repair-stimulating strategy that may be called the “pro-episkevic” approach (from the ancient Greek  Military operations under radiological/nuclear threat  could result in numerous cases of acute radiation syndrome. Targeting fallout may be more realistic and could offer opportunities for innovative radioprotectors use. As an example, nuclear power plant security during military operations has been a concern for some years in NATO strategy, and recent operations in Ukraine (Tchernobyl area and Zaporizhia power plant) clearly illustrate some of these various scenarios. In such context, maintaining the freedom of action of forces may result in deliberate or accidental limited irradiation of soldiers up to 750 mGy .-Space radiation represents the most hazardous factor of space exploration . For the-Radioprotective agents are useful in the medical context, notably in anticancer radiotherapy (RT) to specifically protect healthy tissues surrounding the tumor ,3,10,11.Exposure to ionizing radiation (IR) causes a large spectrum of DNA damage and cellular deaths and affects individuals by increasing risks of radiation-induced (RI) tissue/organ injuries (radiosensitivity), cancers (radiosusceptibility) and/or aging (radiodegeneration) . Since tIn addition to amifostine, a number of antioxidants such as glutathione, vitamin E, selenium or N-acetylcysteine (NAC) have been used concomitantly with chemo- and radiotherapy ,3,24. Amantioxidative approach, based on the reduction in the ROS levels that produce RI DNA damage. Consistently, this approach results in decreasing the number of DNA breaks, notably that of DNA double-strand breaks (DSB), the key damage of the cell killing [stimulating DSB repair than reducing the number of the RI DSB. Such a strategy has been named the pro-episkevic approach (from the ancient Greek επισκευε (episkeve), meaning “repair”). This term was first mentioned in the PhD thesis dissertation of M.L.F presented in 2017 at the University of Lyon, France [unrecognized DSB remain unrepaired and participate in cell death, or else, they are misrepaired by error-prone recombination-like pathways and participate in cell transformation and cancer [To date, the great majority of the studies about radioprotectors follow the same  killing ,29. An a, France . The pro, France ,32,33,34, France ,36,37;  , supplemented with 20% fetal calf serum, penicillin and streptomycin. The origin and the radiobiological features of the RIANS-normal radioresistant AG1521, 1BR3 and 149BR controls are published elsewhere [All the skin fibroblasts tested were untransformed to avoid any bias linked to genomic stability in the plateau phase of growth (95–99% in G0/G1) to overcome any cell cycle and homologous recombination effects. Skin fibroblasts were routinely cultured at 37 °C in 5% COlsewhere ,44,45. Tlsewhere . The surlsewhere . Brieflylsewhere ,42.With regard to the antioxidative drugs, N-acetyl-L-cysteine (NAC) and amifostine (2-(3-Aminopropyl)aminoethyl phosphorothioate, WR2721, Ethyol) were purchased from Sigma-Aldrich . Cells were incubated with these two last drugs separately for 24 h at 37 °C at 10–300 mM NAC or at 24–96 mM for amifostine. The choice of these drug concentrations was motivated by preliminary experiments based on the assessment of γH2AX foci when drug was applied alone to radioresistant fibroblasts. All the conditions leading to the production of more than 2 γH2AX foci per cell were excluded. With regard to the pro-episkevic drugs, cells were incubated with either 1 µM pravastatin  for 24 h at 37 °C alone or 1 µM zoledronate  for 12 h at 37 °C alone (to permit intercomparisons) or together (ZOPRA treatment) . The cho−1) at the anticancer Center Léon-Bérard  [Irradiations were performed with a 6 MeV X-ray medical irradiator (SL 15 Philips) (dose rate: 6 Gy.min France) ,45. In aser139γH2AX antibody  was used at 1:800. The monoclonal anti-mouse anti-ser1981pATM (#ab2888) from Abcam  was used at 1:100. Incubations with anti-mouse fluorescein (FITC) and rhodamine (TRITC) secondary antibodies were performed at 1:100 at 37 °C for 20 min. Slides were mounted in 4′,6′ Diamidino-2-Phenyl-indole (DAPI)-stained Vectashield , and foci were examined with Olympus fluorescence microscope. The foci scoring procedure applied here received the certification agreement of CE mark and ISO-13485 quality management system norms. Our foci scoring procedure also developed some features that are protected in the frame of the Soleau Envelop and patents  [γH2AX foci reveal only the presence of DSB recognized by the NHEJ pathway: vis-à-vis the DSB induced “physically” by IR, the proportion of DSB recognized by NHEJ is maximal in radioresistant cells, while the number of γH2AX foci may be lower than the total number of DSB induced in radiosensitive cells, notably in those eliciting a delayed RIANS [The immunofluorescence protocol was described elsewhere ,48. Brieed RIANS ,39,42.Statistical significance between data points was verified with the one-way ANOVA test. Statistical analysis was performed by using Kaleidagraph v4 .p < 0.001, p < 0.01 and p < 0.05 for the 09CLB, 11CLB and 15 CLB fibroblasts, respectively) (p < 0.05). These findings suggest that millimolar concentrations of NAC may decrease the number of spontaneous DSB  A. Intereeous DSB A.p > 0.8) [p < 0.01), according to previous data [and recognized by NHEJ. However, the lowest average number of γH2AX foci reached with radiosensitive cells was not found significantly different from that reached by radioresistant controls (p > 0.1)    B.p > 0.5) (p > 0.3). Interestingly, this last conclusion was also reached with the 09CLB and 15CLB radiosensitive fibroblasts (p > 0.3), even if the γH2AX foci values were significantly higher than those found with the radioresistant controls (p < 0.01). In the radiosensitive 11CLB fibroblasts, the NAC pretreatment was found to decrease significantly the number of γH2AX foci assessed 24 h post irradiation (p < 0.01)  C. The NA < 0.01) C. Altoge < 0.01) .p > 0.2)  A. Converp > 0.2) A.p > 0.4). Interestingly, this value was found significantly lower than that obtained with the NAC pretreatment (p < 0.001). These findings support that amifostine results in significantly decreasing the number of DSB induced by IR and recognized by NHEJ  B. The lo by NHEJ B.p > 0.5) (p < 0.04) from 26 mM and in the 11CLB cell lines (p < 0.05) at 96 mM amifostine (p < 0.03). Altogether, these data suggest that amifostine pretreatment provides a radioprotective effect on spontaneous DSB, a reduction in the number of DSB recognized by NHEJ in a larger extent than with NAC pretreatment and a significant reduction in the remaining unrepaired DSB  C. Converifostine C. In theired DSB .The findings described above showed that the pretreatment with NAC or amifostine antioxidative agents consisted in decreasing the number of γH2AX foci, likely because they contribute to reduce the ROS and therefore the number of DSB induced by IR in general. Such reduction is likely to be performed chemically, i.e., independently of any DSB repair and signaling pathway recognizing the DSB induced by IR. Conversely, pro-episkevic drugs such as zoledronate and pravastatin accelerate the RIANS and therefore increase the number of DSB recognized by NHEJ . Hence, p < 0.03) (p > 0.8). The extent of such a decrease is the same as that found with the pretreatment with amifostine (p > 0.1). In radiosensitive cells, the picture changed, since more DSB recognized by NHEJ are expected: 0.5 ± 0.1 γH2AX foci vs. 0.3 ± 0.1 γH2AX foci per cell were found on average with the pro-episkevic drugs and the antioxidative drugs, respectively (p < 0.01). Altogether, these data suggest that zoledronate, pravastatin and ZOPRA may significantly reduce the number of spontaneous DSB but not to the same extent as antioxidative agents such as NAC or amifostine . In the radioresistant controls pretreated with zoledronate and pravastatin separately or concomitantly, the number of γH2AX foci assessed before irradiation was found to be significantly lower than that observed in the non-pre-treated cells ( < 0.03) A. The saifostine A.p > 0.8), in agreement with the hypothesis that all the RI DSB are recognized by NHEJ in radioresistant cells (p < 0.08). The ZOPRA combination provided the largest increase by reaching the values of the radioresistant controls for the 09CLB and 11CLB cell lines. Such a tendency was drastically different from the reduction in the number of γH2AX foci assessed 10 min post irradiation observed with antioxidative agents, (p > 0.5 with NAC and p > 0.8 with amifostine). These findings suggested that the pro-episkevic drugs increase the number of DSB recognized by NHEJ among the DSB physically induced by IR, while the antioxidative drugs decrease the number of all the DSB physically induced by IR . By contrast, in radiosensitive cells, the number of γH2AX foci assessed 24 h post irradiation decreased significantly with pro-episkevic drugs (p < 0.03 with NAC and p < 0.04 with amifostine) and reached its lowest values with ZOPRA pretreatment . These findings suggested that, even if NAC and amifostine may reduce the DSB physically induced by IR, they do not reduce the severity of the DSB remaining  and lower reduction with NAC and amifostine pretreatment (p < 0.04 by comparison with untreated conditions)  B.ATM-mutated hyper-radiosensitive cell lines (p > 0.8)  . The sig2+ ions, particularly produced after exposure to IR [An exposure to IR triggers the formation of a large spectrum of reactive oxygen and nitrogen species . The natre to IR . These vre to IR . Conversre to IR . Seker ere to IR . Despitere to IR . HoweverFinally, with regard to the concentrations used, while it must be stressed that the drug concentrations used in ZOPRA treatment correspond to the posology applied in standard antiosteoporosis and anticholesterolemiant treatments , the quePravastatin is currently used as countermeasure of hypercholesterolemia and has showed anti-inflammatory properties associated with the inhibition of Rho and Rho-associated kinases involved in a number of RI tissue injuries . PravastIn parallel, bisphosphonates in general, and zoledronate in particular, are currently used in treatment of osteoporosis and of cancer linked to hypercalcemia . Bisphos2+ ions strongly suggests an important role in the regulation of the pores of the nuclear membranes [A combined action of statins and aminobisphosphonates leads to the inhibition of both farnesylation and geranylgeranylation of progerin and prelamin A proteins, which are notably found cumulated in the nuclear membrane of progeroid cells . Interesembranes . Hence, -DSB and ATM monomers induction: Immediately after irradiation, cytoplasmic ATM dimers dissociate in ATM monomers in a linearly dose-dependent manner; in parallel, in the nucleus, the RI DNA damage, and notably, the RI DSB, are induced in a dose-dependent manner as well . During -ATM monomers diffusion and DSB recognition: The RI ATM monomers diffuse in the nucleus. The pro-episkevic approach may result in accelerating and facilitating the diffusion of ATM monomers in the nucleus. Consequently, the number of pATM monomers that diffuse in the nucleus increases ,48,49,50-DSB repair: In the case of the normal-RIANS quiescent cells, the pro-episkevic approach has no significant effect, while the antioxidative approach that leads to decrease the number of DSB does not reduce the proportion of unrepairable DSB. In the case of delayed-RIANS quiescent cells, the pro-episkevic approach contributes to increase the subset of DSB repaired by NHEJ, which decreases the RI lethal effect, while the antioxidative approach is not efficient enough to make the unrepaired DSB disappear.As evoked in the Introduction, the RIANS model is based on the occurrence of some important molecular steps :-DSB andS as a function of dose D as follows:α and β are adjustable parameters to be determined [The linear–quadratic (LQ) model is an empirical formula that links the cell survival to the IR dose. Since the 1970s, such a formula has been extensively used by radiobiologists and radiation oncologists to describe the individual response to IR . The LQ termined .In 2016, the RIANS model provided a coherent interpretation of the two LQ parameters, α and β, by defining two types of RI and lethal DSB : (1) The-antioxidative approach consisting in reducing the oxidization process of the RI water radiolysis through an efficient reduction in ROS:⚬The number of DSB induced by IR decreases significantly;⚬Our findings confirm the efficiency of amifostine as one of the most efficient antioxidative drugs.The radiation protection strategy has long been based on an -pro-episkevic approach, directly deriving from the RIANS model, aims at stimulating repair and contributes to increase the number of DSB managed by NHEJ—the most predominant DSB repair and signaling pathway in mammalians: ⚬The combination of pravastatin and zoledronate (ZOPRA) shows a significant pro-episkevic property that appears to be more efficient than amifostine;⚬In the case of genetic syndromes associated with the loss of function of some genes essential for DSB repair, the effect of ZOPRA appears to be limited.The In this study, we aimed to investigate and compare the molecular features of two different approaches of radioprotection. These investigations were performed, as a first step, with quiescent human fibroblasts and in standard conditions of a current RT session. Altogether, our findings suggest the following:Additional investigations are needed to develop some new drugs that may elicit both antioxidative and pro-episkevic properties and to quantify the radiation protection action of both types of drugs applied together, whether on quiescent or proliferating cells. The question of the innocuity of long-term treatments should also be examined.


Nonlinear silicon photonics has a high compatibility with CMOS technology and therefore is particularly attractive for various purposes and applications. Second harmonic generation (SHG) in silicon nanowires (NWs) is widely studied for its high sensitivity to structural changes, low-cost fabrication, and efficient tunability of photonic properties. In this study, we report a fabrication and SHG study of Si nanowire/siloxane flexible membranes. The proposed highly transparent flexible membranes revealed a strong nonlinear response, which was enhanced via activation by an infrared laser beam. The vertical arrays of several nanometer-thin Si NWs effectively generate the SH signal after being exposed to femtosecond infrared laser irradiation in the spectral range of 800–1020 nm. The stable enhancement of SHG induced by laser exposure can be attributed to the functional modifications of the Si NW surface, which can be used for the development of efficient nonlinear platforms based on silicon. This study delivers a valuable contribution to the advancement of optical devices based on silicon and presents novel design and fabrication methods for infrared converters. Highly developed and widely used semiconductors with crystalline centrosymmetry, such as silicon ,2 and gehotonics . Second hotonics ,5,6 is thotonics ,8,9,10 ahotonics  and phothotonics  have prohotonics ,14.Arrays of silicon NWs have THG  in the NIn this study, we fabricate Si NWs/siloxane membranes and present the study of SHG via IR femtosecond laser irradiation, for the first time, investigated in the proposed structure design. We observe a stable laser-induced enhancement of SHG, which is attributed to functional modifications of the Si NW surface. The proposed approach has the potential to aid in the development of efficient nonlinear platforms based on silicon nanostructures .6 and O2 with a pressure of 5 mTorr was used for dry etching. The process was performed at a temperature of −150 °C to achieve over passivation  direction oriented normally to the membrane surface. Two-dimensional cross-sections of RSM cuts were analyzed to determine the presence of any other crystalline phases. Two orthogonal RSM cross-sections (ΔQ = ±0.05 Å−1) were plotted to illustrate the Si lattice orientation in relation to the PDMS membrane plane. Si zone axis and in the (hk3)Si reciprocal space plane, respectively, obtained for the as-formed sample.However, even the best developed peeling technique cannot provide a complete transfer of a 5–7 cmThus, we conclude that the ICP etching retains the initial substrate crystalline structure, ensuring it is unchanged, so the Si NWs have a monocrystalline silicon structure with a 001 orientation along the NW axis. This orientation coincides with the orientation of the Si(001) substrate on the surface on which the SiNWs were etched.It should be noted that mechanical peeling of the Si NW/PDMS membranes from the Si wafer with a razor blade disrupts the initial ordering of Si NWs in the array  nm for several bright spots in the measured SHG map are presented in The confocal SHG mapping setup also provided a 3D visualization of the SHG in the studied area, constructed from the stack of 2D maps measured with a consequent focal plane shift . This 3DThe 3D visualization also confirmed that the SHG signal is very sensitive to the pump laser focal plane. As shown in However, we did not perform a systematic study of the SHG dependency on the pump laser beam focal plane position. All the presented results were achieved at optimal condition, i.e., when the focal plane is adjusted to the maximum SHG signal. The resonant pump laser beam focus can be further increased for the SiNW array of a higher NW diameter (above 400–600 nm), when the incident light can tangle with the self-modes of the NW cylinder resonator. In this case, the direct aiming of the pump laser focal plane at the plane of SiNW top facets is critical for the resonance SHG conversion.The SHG–pump power measurements were performed at the optimal wavelength of 920 nm. This revealed an unexpected phenomenon in the form of a dramatic increase in the SHG signal at a specific threshold pump power. Namely, the SHG signal abruptly increases when the pump power reaches the specific value, which is the same for all tested regions of the studied SiNW/PDMS membrane samples. After this ‘activation,’ the studied area continues to demonstrate the enhanced SHG signal even below the threshold pump power, i.e., when the SHG signal acquisition is repeated at the previously measured pump power value below the threshold. This phenomenon can be called the activation effect, and it can be clearly seen from the SHG map presented in To study this phenomenon in detail, the SHG–pump power study was performed in a repetitive manner. First, the original sample area was mapped at the pump power rising from the minimal value  to the maximum value of the pump power range (defined in advance as the highest pump power before the structure degradation due to the overheating). Second, the same area was tested at the descending pump power at the same power range. Finally, the first run was repeated, i.e., the pump power was increased again from the minimal to the maximal value but at the already exposed area. The acquired data were processed to draw curves for selected bright spots corresponding to the most efficient individual Si NWs a–c and tAs can be expected, the slope value for the initial measurements at the virgin area, when the activation effect occurs, is significantly different from the characteristic SHG value of 2. Indeed, it varies from 3 to 3.6 for the individual NWs, and is about 2.1–2.2 for the averaged signal. Meanwhile, the SHG-to-pump slope for the repeated measurements after the first run is very close to 2 for both the individual Si NWs and the array . This leWe speculate that this transition process can be explained either by the structural  transformations in the Si NWs induced by the pump power -Si transforms in polycrystalline-Si  or surfaTaking into account the centrosymmetric structure of Si . At staSi NWs represent thin silicon material nanoscale crystals with a high surface-to-volume ratio. Therefore, the well-known Si surface modifications induced by the intense laser beam irradiation can dramatically affect the SHG process. The surface oxidation or other material process under femtosecond laser irradiation is expected, and it can modify the near-surface SiNW material at the depth of several tens of nanometers . Thus, tχnph(2) and Considering the transition effect, the dependence of SHG signal changes according to the following expression x or SiC formed under fs-laser irradiation) can trap light-excited carriers, changing the height of surface potential barrier in Si NW. At the same time, it is well known that the effect of laser-induced oxidation of NWs [Moreover, due to the transition process, n of NWs ,35 leadsThe transition is finished after the initial SHG-to-pump measurements; thus, the exposed area generates a normal SH signal characterized by the typical slope value of 2.We have demonstrated an enhanced generation of the visual second harmonic by the proposed Si NW/PDMS membranes. The SHG can be attributed to the Si NW near-surface mc-Si material, and it is facilitated by the intrinsic electric field by the EFISH mechanism. In the experiment, we observed the effect of fs-laser irradiation, leading to an abrupt and permanent increase in the SHG signal, which we associate with the restructuring of the surface material and EFISH due to the charging of the near-surface electronic trap states that is proven by SHG-to-pump slopes of around 3.5.As a result, the proposed Si NW/PDMS membrane, due to the laser activation effect, can serve as infrared-to-visual range converters and would be in demand for the advanced optical schemes where efficient, semitransparent, permanent, flexible, and non-disturbing control of the laser beam is of high importance, as well as in the general field of CMOS-integrated Si-based photonic circuits.Si NW/PDMS membranes are promising materials for studying the nonlinear optical effects at nanoscales, and could have a variety of uses in sensor applications due to the large surface-to-volume ratio of Si NWs. Si NWs can be used for nonlinear light manipulation in future integrated optoelectronics (CMOS-integrated Si-based photonic circuits).


This article was migrated. The article was marked as recommended.Mentoring in medicine is not a new concept. However in recent years it has been sadly neglected and consigned to the wings of the medical education theatre. In this age of disparate clinical teams, disillusionment and shift working, the NHS needs to proactively nurture and develop junior doctors to support them on their career path. In this paper, we argue that effective mentoring is key to achieving this goal.Context and methods: The researcher is a Core Medical Trainee (CMT) and set up a near-peer mentoring programme between Foundation Year (FY) doctors and CMTs. A focus group was conducted and inductive thematic analysis was used to analyse the resulting transcript. Thematic maps demonstrating the benefits and barriers to mentoring were produced.Results: Results demonstrated that mentoring was useful for mentees’ personal development in terms of networking opportunities, pastoral support and the sharing of experiences. Positivity and camaraderie were key to an effective mentoring partnership, whilst rota clashes posed a significant barrier. Dangers identified included mentors adhering too tightly to a ‘mentoring formula’ and encouraging mentees to become ‘carbon copies’ of themselves.Conclusions: This study has provided a valuable insight into the benefits of mentoring for junior doctors. Recommendations include promotion and active creation of formal mentoring programmes, and integration of formal mentoring training into the CMT curriculum. Mentoring is not a new concept. Indeed, it has been a key component of a medical education from the advent of the profession and the days of apprenticeship . The oriThe traditional concept of mentorship is based on a hierarchical dyadic relationship whereby “a senior faculty member counsels and instructs a junior protégé” , pp.1578Mentoring is a topic that has enjoyed increasing attention in the medical education sphere recently . The vasIn today’s economic climate, with the NHS stretched to breaking point, mentoring could provide a strategy for building morale, increasing retention, in a financially favourable manner. Although training of mentors admittedly requires some resources, we strongly believe that this investment pays handsome dividends.What are the perceived benefits of near-peer (NP) mentoring for Foundation Year (FY) doctors?’A literature review was conducted in the preliminary stage of this project, which demonstrated the value of mentoring in career and personal development. Although some disadvantages were acknowledged, the vast majority of the literature supports mentoring as an important development tool. There is limited literature focusing on the mentor perspective and comparing formal and informal mentoring programmes within medicine. In attempting to address this gap in the literature, this paper will address the following question: ‘In answering the research question proposed, the following sub-questions will be explored:•What are the benefits and challenges of formal versus informal mentoring?•How does the mentoring format  impact upon the mentoring relationship?•How accessible is mentoring to junior doctors currently?The researcher is a second year CMT and set up a mentoring programme between FY doctors and CMTs in September 2015. The research project took place at a busy District General Hospital (DGH) where there are 88 FY doctors and 34 CMTs. The study population was made up of the 29 FY doctors who adopted the role of mentee in the mentoring programme.All 29 FY mentees were invited to participate in the focus group via email. A total of four to eight focus group participants was aimed for, and a variety of times and locations were offered to maximise participation.A Bricolage approach to analysis was employed to generate meaning and draw conclusions. Asrecursive  in line with the British Educational Research Association ethical guidelines for educational research . PatientThe final focus group comprised five mentees from the mentoring programme, all female, aged between 24 and 27. Four of the five mentees had experienced some form of mentoring prior to enrolling in the programme. The focus group participants had all met each other in the working environment prior to the focus group; it was hoped that this would make for a relaxed focus group dynamic. The focus group lasted 50 minutes.The pre-formed themes were generated from the literature review. There were 14 themes identified which broadly fit into one of the four following overarching themes: prevalence of mentoring; barriers to mentoring; requirements for effective mentoring; and current thoughts regarding the state of mentoring today.Generally, the focus group found the mentoring scheme to be beneficial and useful for their personal development. One participant had not had a successful mentoring relationship with the allocated mentor as they had been unable to find a mutually appropriate time to meet: rota clashes were described as one of the significant barriers to effective mentoring. Despite this, the mentee had experienced informal mentoring which had been useful.The key attributes of successful mentors were discussed, as were the strengths and weaknesses of informal and formal mentoring schemes. The desire and need for mentoring for junior doctors was reiterated on several occasions, and described as particularly pertinent given the unsettlement regarding the 2016 junior doctor contract  and the The focus group recognised a growing culture for mentoring. They identified improved accessibility secondary to the use of various modes of mentoring . Formal training for mentors gave those junior doctors the tools needed to identify potential mentors for themselves, whilst the precipitation of a ‘mentoring culture’ meant that mentees were also encouraged to become mentors. However, there was still a call for increased availability of formal mentoring programmes. Although the existence of informal mentoring was recognised, the need for the correct ‘mentoring formula’ with engagement, enthusiasm, and compatibility between both mentor and mentee was noted in order to secure successful mentoring partnerships.A condensed thematic map with the key findings from the study was produced . The posThe benefits and challenges of formal and informal mentoring generated lively focus group discussion. There were advantages and disadvantages identified for each, with rota issues a significant barrier to effective formal mentoring. The overall preference in the focus group was for informal mentoring. This was deemed to be even more effective when the mentor had access to ‘mentoring education’ - essentially, instruction in how to mentor effectively. However, this training was typically available only with formalmentoring programmes. A combination of formal and informal mentoring was therefore deemed to be most effective.The focus group afforded greater prominence to the presence of positivity and camaraderie in the mentoring partnership as opposed to highlighting a particularly effective mode of mentoring . Peer mentors were deemed to be particularly approachable and helpful for the purposes of networking. However, the potentially limited experience of a peer was also acknowledged.Informal mentoring was judged to be infinitely accessible, especially in the provision of role models. Formal mentoring was less accessible, although the idea of virtual mentoring was suggested as a potential solution to this. The concept of precipitationwas introduced as helping to amplify the accessibility of mentoring for junior doctors.The small study size must be acknowledged. Although the target of between four and eight focus group participants was met, the reliability of the data may have been improved through running multiple focus groups, with cross-referencing between data sets to improve both validity and reliability. The potential for self-selection bias must be considered, as interestingly, the focus group participants were all female. This was not representative of the study participants where only 57%  were female. This limits the generalisability of results. It would be interesting to see whether male participation in the focus group would have changed the focus group dynamic and hence the issues discussed and opinions presented. The question as to why no males volunteered to participate in the focus group also invites further investigation: would they have been more inclined to participate in a questionnaire rather than a focus group? Or was it just by chance that no males were available on the dates suggested for the study? Perhaps they did not find mentoring useful and therefore did not want to participate in the focus group? This is all supposition, but further work would certainly involve investigating the views of males on the mentoring programme.The issue of researcher-related bias has already been considered. One approach to tackle this would be for the researcher to use a reflexive journal, which ‘sensitises the interviewer to his or her prejudices and subjectivities, while more fully informing the researcher of the impact of these influences on the credibility of the research outcomes’ , p.4. ThGiven the phenomenological nature of this study, it has limited generalisability. The study took place at one particular hospital in relation to one specific mentoring scheme over the course of one year. It would be interesting to see whether similar results would be achieved in subsequent years with different cohorts of mentors and mentees. Despite this, the findings of this study are extremely significant for the mentoring scheme in question and the conclusions are still worth considering within the wider context of mentoring for junior doctors.In conclusion, the focus group considered their mentoring experience to be mostly positive. Although some challenges were discussed in terms of initiating mentoring, all focus group participants felt they had benefitted from formal mentoring, informal mentoring, or a combination of these.The greatest pitfall voiced in the focus group was to be wary of a ‘carbon-copy danger’: essentially mentors avoiding encouraging mentees to just follow in their own footsteps. Mentoring was acknowledged as a very individual process with mentors and mentees having varying educational and development needs.The current availability of mentoring was deemed insufficient for junior doctors. There was a call for more formal mentoring programmes with the hope that this would encourage a growing mentoring culture with the subsequent precipitation of more informal mentoring.Positive motivation was judged essential to a successful mentoring partnership. A mentoring education was considered useful for mentors in their mentoring role and additionally in providing them with the ‘finding tools’ to identify their own mentors, helping promote a ‘bidirectional mentoring cycle’.This paper has sought to demonstrate the wide-ranging benefits of mentoring for junior doctors, as well as some of the potential pitfalls to avoid. Given our findings, we would suggest promotion and active creation of formal mentoring programmes, integration of formal mentoring training into the CMT curriculum, and improved access to resources to facilitate self-mentoring and virtual mentoring.•A ‘mentor’ is an experienced, empathetic person who guides another individual (the mentee) through their personal and professional development .•Mentoring is currently an underutilised resource, despite its potential to benefit junior doctors’ personal and professional development.•Barriers to effective formal mentoring include incompatible rotas whilst dangers include encouraging the creation of a ‘carbon copy’ of the mentor.•Formal mentoring programmes with appropriate mentor training can precipitate informal mentoring which can be particularly beneficial.Dr Lucy Havard completed a Masters in Medical Education in September 2017. She is now working as a Medical Registrar having completed Core Medical Training in London. Her medical education interests comprise mentoring and near-peer teaching.Mr Tom Baker Completed a Masters in Teaching and Learning in 2013. He is now working as Head of Education Programmes at the Royal College of Physicians of London. He is also programme director for the RCP/UCL MSc in Medical Education. His interests are mentoring, assessment, leadership, and curriculum design. This review has been migrated. The reviewer awarded 4 stars out of 5 As someone who has a very close educational relationship to junior doctors and their development, I enjoyed reading this well written and researched paper. many of the authors findings from their research and their small study are not surprising , but the clarity that they have presented the does highlight the difficulties experienced by the junior doctors, which in turn, I feel, do affect quality of care.I will not expand on my co-reviewers excellent comments, many of which I agree with. Only to suggest that this paper is written by all involved in the mentoring of our junior colleagues.Reviewer Expertise:NANo decision status is available This review has been migrated. The reviewer awarded 3 stars out of 5 Thank you for inviting me to review this article. It is good to see an article investigating the topic of mentoring from the student perspective. At my medical school, we have a long history of using formal mentoring groups with our undergraduate medical students and we are in the process of evaluating their functioning and outcomes. Therefore, this article is a timely read that I will share with my colleagues.While reading this article, I had several thoughts about how it might be improved. The authors may wish to follow up on the points below or consider them for when they construct their next article for publication.Please consider omitting the abbreviations CMT and FY in such a short article. It increases the reading difficulty for the reader. Please do not assume that an international reader is familiar with the names and functions of ‘Core Medical Trainee’ and ‘foundation year doctor’ roles in the UK medical profession. For example, in my country, we do not used the same designations, therefore, I was wondering who is more senior? The reader had to make an inference when the role of foundation year doctor was described in the study as a mentee.In the introduction the authors state that they have performed a literature review. It would be helpful to know more the limited literature found about mentoring. This enhancement would further strengthen the warrant for the study.In the methods section, the bricolage approach was well described so that another researcher could conduct a literature review and arrive at similar conclusions, however, the themes generated from the literature review and later used guided the analysis of the data are not stated explicitly in this section. For a complete description of the data collection, the authors might consider including the questions that were asked to the focus group. I’m assuming that the focus group questions were related to the three sub-questions presented at the end of the introduction section.Since this is a qualitative study, I am wondering about the use of the terms reliability and validity. With qualitative methods, these concepts often manifest themselves in how clearly the researcher explains and justifies the steps taken to design the study and to collect and interpret the data. These features are often described in terms such as credibility, trustworthiness, representativeness, and robustness. Running the focus group again with a different set of mentees might result in different themes emerging if the composition of the group was not selected to consist of people that represent the different possible views held by individuals in the cohort. As noted by the authors in the discussion section, this difference is often why more than one focus group is conducted. In this study, the researchers may be looking for ‘saturation’, when no new themes emerge with new data being collected : Sage Publications Ltd). Please consider adding a reference to Appendix 1 in the methods section. I found the heading “Results/Analysis” a little confusing. Please consider describing the methods used to conduct the data analysis before the results are presented. In the results section, I am wondering if the pre-formed themes could have been introduced to the reader earlier. The results section might be strengthened if the themes that emerged from the analysis were reported with representative quotations to support the interpretation of the data. In the discussion section, please consider revising the following sentence: “There were advantages and disadvantages identified for each (Is a noun missing here?), with rota issues a significant barrier to effective formal mentoring”. In the limitations of the study section, the generalisability of the study’s findings is discussed twice. The authors might consider consolidating the issues about generalisability or perhaps focusing this part of the discussion on how the findings were representative of the cohort that was sampled. In the conclusion section, I appreciated the well-written summary of the main points. I found the take home messages very engaging and think they will work well to invite a skim reader to read the article in more depth.Reviewer Expertise:NANo decision status is available