Measurements of cytosolic Ca2+ report maximum concentrations of less than few micromolar, yet several cytosolic enzymes require concentrations of more than 20 μM Ca2+ to be activated. In this paper, we have resolved this apparent paradox by showing that the surface topology of cells represents an important and hitherto unrecognized feature for generating microdomains of high Ca2+ in cells. We show that whereas the standard modeling assumption of a smooth cell surface predicts only moderate localized effects, the more realistic “wrinkled” surface topology predicts that Ca2+ concentrations up to 80 μM can persist within the folds of membranes for significant times. This intra-wrinkle location may account for 5% of the total cell volume. Using different geometries of wrinkles, our simulations show that high Ca2+ microdomains will be generated most effectively by long narrow membrane wrinkles of similar dimensions to those found experimentally. This is a new concept which has not previously been considered, but which has ramifications as the intra-wrinkle location is also a strategic location at which Ca2+ acts as a regulator of the cortical cytoskeleton and plasma membrane expansion.It has long been speculated that cellular microdomains are important for many cellular processes, especially those involving Ca While the existence of high Ca2+ microdomains within cells has long been discussed 2+ can only exist transiently in limited cytosolic space within 100 nm off the open mouth of Ca2+ influx channels The ability to restrict enzyme activation to sub-domains within the cell is crucial for cell behaviour, such as migration, directed pseudopodia formation and cell polarization. Although calpains are known to be important in these activities 2+ signal which accompanies these events 2+ dependent protease calpain-1 2+ required for calpain-1 activation is at least 2 orders of magnitude higher than the resting level of cytosolic free Ca2+, i.e. 10–50 μM 2+ puffs can be observed within the bulk cytosol as Ca2+ is released from storage sites within a number of non-excitable cell types 2+ concentration reached is within the physiological range of 0.1–1 μM similar to that in the bulk cytosol during Ca2+ influx. Nevertheless, the existence of high Ca2+ microdomains near the plasma membrane has long been suspected 2+ concentrations 2+ is generated physiologically. Recently, it has been shown that TRPM2 channel opening is also activated by high micromolar cytosolic Ca2+ concentrations The surface of cells is rarely smooth and often appears wrinkled when viewed with sufficient resolution, such as scanning electron microscopy or atomic force microscopy 2+ can only exist transiently very near the open mouth of Ca2+ influx channels (within 100 nm). Therefore only molecules very close to open channels would sense such signals. Since it is speculated that high Ca2+ would be strategically important within wrinkles, we therefore sought to construct a model which included a wrinkled cell surface in order to establish whether the wrinkled topology had a significant influence on the near membrane Ca2+ concentration during Ca2+ influx. We show here that the wrinkled surface of cells provides a mechanism for generating high Ca2+ domains where the concentration of Ca2+ reaches tens of micromolar while the bulk cytosol remains sub-micromolar and that any cell with a wrinkled surface topology can have high Ca2+ microdomains due to this effect. These anatomical structures provide a hitherto unrecognised mechanism for restricting the activation of Ca2+ activated enzyme activity to near membrane microdomains within the cell.However, theoretical models 22+ influx, we created a 2D wrinkled surface segment  the boundary at the membrane was modelled with symmetry/insulation. For cytosolic free Ca2+ the boundary condition at the membrane surface is given by the flux:In order to generate a wrinkled cell surface to investigate the effect of Ca segment b with inwrinkles c. Althouwrinkles a, the mowrinkles c. Furtheed model d. The Caants see . At the 2+ across the membrane. The second term represents the pumping of Ca2+ out of the cell, while the third term represents the Ca2+-influx following stimulation of the cell. Although it is possible that some Ca2+ influx channels may be localized to surface projections such as sensory microvilli 2+ channels were distributed equally over the cell membrane. In this way, the model had not an in-built bias towards higher cytosolic free Ca2+ within the wrinkled areas of membrane.The first term in this expression represents the resting flux of Ca2.12+ influx, efflux, buffering and diffusion, we have modelled two cases; one of a smooth spherical cell surface, and the other for a more realistic topology with a “wrinkled” surface. The terms describing the flux of Ca2+ across the membrane is taken from neutrophils, because in these cells, surface wrinkles are of a particular interest and partly because many of the parameters required have been quantified in these cells. The model, however, employs the essential features of Ca2+ modelling from other models 2+ microdomains established.The parameters used in the equations and the initial conditions are listed in 2.22.2.12+ leak across the plasma membrane (Prest([Ca2+]ext − [Ca2+])) (Eq. Jefflux[Ca2+]/(Km + [Ca2+])) mediated by a Ca2+-ATPase (Eq. 2+ (Jefflux) was therefore calculated to balance influx resultant from a resting permeability coefficient for Ca2+ of 8 cm/s 2+ for the Ca2+-ATPase was set as constant Km = 1.5 μM. Although the affinity may increase following calmodulin activation The passive Ca+])) Eq.  is balanPase Eq.  .2.2.22+ in neutrophils. There is evidence to suggest that part of the influx is mediated by the non-selective cationic TRP channels, especially TRPM2 −1 during the first second of stimulation for a Ca2+ influx sufficiently large to generate the observed change in bulk cytoplasmic Ca2+ concentration 2+ channels is randomly distributed on the cell surface in the same way as Ca2+-ATPase. The effect of channel opening on cytosolic free Ca2+ (kopen) depends on the time at which there is an increased open probability, which is shown in A number of different stimuli generate a large increase in cytosolic Ca2+ influx in the model without wrinkles we use the functions shown in 2+ is the same. The additional influx is modelled using a built-in continuous function to simulate a step function. The extra influx is modelled as if it is independent on the extracellular Ca2+ concentration and we have normalized the influx such that the total Ca2+ influx is the same in the smooth surface and the wrinkled surface model. This implies that the influx pr membrane area is higher in the model without wrinkles.When we simulate the Ca2.2.32+ buffering results from binding of Ca2+ to both proteins and small molecules, which in neutrophils is equivalent to a total buffer concentration of 0.76 mM with an average Kd of 0.5 μM 2+ and the molecules that buffer Ca2+Cytosolic Ca2.2.4z-axis, a 3D surface is generated which has similarity to the wrinkled surface of a neutrophil. The same approach was used to generate the smooth cell model. The surface area of the wrinkled cell is 871 μm2, where 73% of the membrane is in the wrinkles. The smooth cell has a surface area of 314 μm2. The total length of the wrinkles is 446 μm as measured from where the wrinkle begins to protrude out of the cell membrane . They were simulated using Direct (Pardiso) solver, with relative and absolute tolerances of 1E−7 and 1E−8, respectively, and with the time step restricted to maximum 0.1 s. The models are available as a COMSOL model report .33.12+ concentration is proposed to be functionally important in both exocytsosis 2+ in these cells. The values and published sources of these parameters are given in 2+ influx channels on the surface of neutrophils is not known, but it can be estimated that there are at least 100–150 channels/cell (see above). Assuming a random distribution of the channels 70–100 of these are situated in the wrinkles. When simulated Ca2+ influx was run for a Ca2+ influx phase of 1 s period, the bulk cytosolic free Ca2+ rose after a delay of about 100 ms to a value of about 800 nM , shows a clear boundary approximately 1 μm within the cell  and with unlimited capacity and with no leakage, Ca2+ microdomains within the wrinkles would be elevated further. To compensate for near extracellular concentrations of Ca2+ in this model we have scaled the influx parameter Jstim with (1 − [Ca2+]/[Ca2+]ext) in all the simulations. If only a part of the membrane was covered the near membrane Ca2+ concentration in the wrinkles increased to around 0.45 mM  and 100 nm wide (base width), Ca2+ concentrations were even higher than in our previous model, reaching around 85 μM (2+ concentration was reduced but remained high at 40 μM (2+ was observed when the standard length (800 nm) wrinkles were widened to 200 nm, intra-wrinkle Ca2+ peaking at around 11 μM (2+ concentrations to 45 μM.Within surface wrinkles which were 1500 nm long (nd 85 μM a. When tat 40 μM b. A simind 11 μM c. ConverL1 + L2 = 300 nm) and 100 nm base width, the Ca2+ concentration is elevated at 5.2 μM  asσ is the electrical surface charge density, R is the gas constant, T is the absolute temperature and ɛ0 and ɛr are the absolute and relative permittivities, respectively.Although the Gouy–Chapman–Stern theory also provides explanation for elevated Caσ is between −0.02 C/m2 and −0.05 C/m2 the ψ0 was calculated. If the surface charge density is small, the potential will decay exponentially with distance from the membrane surface with the coefficient κ:ψ0 equals κ (around 1 nm from the surface of the membrane) the Ca2+ concentration will be increased by a factor of 2–4 with respect to the bulk concentration. Thus, the effect of the electrical surface potential is far weaker than that due to surface topology and not sufficient to explain the presence of the high Ca2+ concentrations. However, the electrical surface potential will increase the concentration of cations near the membrane and hence may work as a local cation buffer. If we assume that the surface charge density is −0.02 C/m2, then the membrane can at most bind 1.8 × 10−16 mole of positive charges or 9 × 10−17 mole of Ca2+ if we neglect the presence of Mg2+ and assume that no other cations interfere with the membrane. The intracellular buffer can, on the other hand, bind 4 × 10−16 mole Ca2+. Therefore, the mobile buffer still remains the dominant buffer, which binds at least 4 times more Ca2+ than the membrane.Setting the concentration of monovalent cations 2+ concentration slightly (2+ and their Kd is between 10 μM and 50 μM 2+ obtained with wrinkles that are 800 nm long (2+ became close to 0.1 mM (2+ domains in the micromolar range that can activate proteins which otherwise would show little if any activity in the cytosol. If the structures were much wider or a lot shorter the apparent wrinkle structure would be lost and Ca2+ domains would disappear.The wrinkles in this model are symmetrical structures and per se artificial, however, the real wrinkles also span the entire membrane and are connected in an almost similar geometric fashion . Differeslightly b. If the nm long  can easio 0.1 mM , which i2+ influx with a deterministic approach, which is based on the assumption that the individual behaviour of different molecules can be neglected due to the number of the species according to the law of large numbers. One result of the simulations is that it is very likely that there are at least 800 active channels in the plasma membrane, which corresponds to 0.9 channel per μm2. With the assumption of a homogeneous distribution of the channels this implies that there are at least 584 channels in the wrinkles or 1.3 channels per μm wrinkle. The wrinkles have a total volume of 0.219 fl, which corresponds to about 4% of the total cell volume. A concentration of Ca2+ ions of 100 nM corresponds to about 1300 free Ca2+ ions in the wrinkles and when the concentration is 25 μM there are 3.3 × 105 free Ca2+ ions in the wrinkles. Whether a system should be described as stochastic or deterministic depends on both the number of particles and the properties of the system as such. The number of free Ca2+ ions in the wrinkles following activation is around the deterministic limit as reported by Kummer et al. 2+ dynamics inside the wrinkles in single neutrophils have not yet been measured, but global cytosolic Ca2+ measurements of neutrophils suggests that there is a stochastic element We simulated the Ca2+ predicted by our simulations are not generated by assuming non-uniform distributions of Ca2+ channels, pumps, or Ca2+ release sites or by proposing new molecular properties for Ca2+ channels. The zones of high Ca2+ arise simply by including the known micro-anatomy of cell surfaces in the simulation. The work we report here has therefore highlighted the importance of including membrane surface topology when considering a model of chemical behaviour in cells.It is important to stress that the microdomains of high Ca


In the present work we have evaluated the status of the Ca2+-dependent proteolytic system in the gastrocnemius muscle of AH-130 tumour-bearing rats by assaying the activity of calpain as well as the levels of calpastatin, the natural calpain inhibitor, and of the 130 kDa Ca2+-ATPase, both of which are known calpain substrates. After tumour transplantation, total calpastatin activity progressively declined, while total calpain activity remained unchanged, resulting in a progressively increasing unbalance in the calpain/calpastatin ratio. A decrease was also observed for the 130 kDa plasma membrane form of Ca2+-ATPase, while there was no change in the level of the 90 kDa sarcoplasmic Ca2+-ATPase, which is resistant to the action of calpain. Decreased levels of both calpastatin and 130 kDa Ca2+-ATPase have been also detected in the heart of the tumour-bearers. These observations strongly suggest that Ca2+-dependent proteolysis was activated in the skeletal muscle and heart of tumour-bearing animals and raise the possibility that such activation may play a role in sparking off the muscle protein hypercatabolic response that characterizes cancer cachexia. © 2001 Cancer Research Campaignhttp://www.bjcancer.comCachexia is a syndrome characterized by profound tissue wasting that frequently complicates malignancies. In a cancer cachexia model we have shown that protein depletion in the skeletal muscle, which is a prominent feature of the syndrome, is mostly due to enhanced proteolysis. There is consensus on the views that the ubiquitin/proteasome pathway plays an important role in such metabolic response and that cytotoxic cytokines such as TNFα are involved in its triggering , yet the mechanisms by which the relevant extracellular signals are transduced into protein hypercatabolism are largely unknown. Moreover, little information is presently available as to the possible involvement in muscle protein waste of the Ca


While, the mechanism underlying stretch-induced Ca2+ release in smooth muscle is unknown.Hollow smooth muscle organs such as the bladder undergo significant changes in wall tension associated with filling and distension, with attendant changes in muscle tone. Our previous study indicated that stretch induces Ca2+ release. The probability and frequency of Ca2+ sparks induced by stretch were closely related to the extent of cell extension and the time that the stretch was maintained. Experiments in tissues and single myocytes indicated that mechanical stretch significantly increases the production of nitric oxide (NO) and the amplitude and duration of muscle contraction. Stretch- induced Ca2+ sparks and contractility increases were abrogated by the NO inhibitor L-NAME and were also absent in eNOS knockout mice. Furthermore, exposure of eNOS null mice to exogenously generated NO induced Ca2+ sparks. The soluble guanylyl cyclase inhibitor ODQ did not inhibit SICR, but this process was effectively blocked by the PI3 kinase inhibitors LY494002 and wortmannin; the phosphorylation of Akt and eNOS were up-regulated by 204±28.6% and 258±36.8% by stretch, respectively. Moreover, stretch significantly increased the eNOS protein expression level.We examined the transduction mechanism linking cell stretch to CaTaking together, these results suggest that stretch-induced Ca2+ release is NO dependent, resulting from the activation of PI3K/Akt pathway in smooth muscle. This stretch -induced Ca2+ release (SICR) process does not require an influx of extracellular Ca2+ ions, activation of ionic currents, or even a rise in [Ca2+]iIt has long been known that increases in passive tension evoke contraction of smooth muscle in arteries 2+ release by SR ryanodine receptors (RYR) 2+ release in the form of Ca2+ sparks in smooth muscle cells isolated from the urinary bladder 2+ release in smooth muscle is not known, however. In the present study we investigated the signaling pathways mediating stretch-induced Ca2+ release in smooth muscle. We report that smooth muscle stretch enhances NO production and that this increase correlates with augmented RYR -mediated Ca2+ sparks. The production of NO is both necessary and sufficient to trigger Ca2+ sparks, as stretch –induced Ca2+ sparks were not activated under conditions of NOS inhibition or in eNOS knockout smooth muscle, and exogenous NO restored stretch –induced Ca2+ release in eNOS knockout cells, indicating that, NO mediates stretch-induced Ca2+ release through activation of PI(3)K-Akt−endothelial NOS axis in smooth muscle.NO is a cellular second messenger that mediates numerous biological functions such as vasodilation, muscle contractility 2+-free solution. The fragments were then transferred into 1 mg/ml collagenase type II , and 100 µM Ca2+ solution, supplemented with 1 mg/ml bovine serum albumin. The tissue was incubated for 10 min, triturated with a wide-bore Pasteur pipette, and passed through 125-µm nylon mesh. Cells were concentrated by low speed centrifugation, washed with fresh medium, resuspended, and stored at 4°C.Mice including eNOS knockout mice that were obtained from the Jackson Laboratories  were anesthetized and euthanized in accordance with an approved laboratory animal protocol of Cornell University and Chinese Academy of Sciences. Single cells were prepared as described previously 2+ free solution, and cutting segments of the remaining muscle layer in 2–3 mm long strips, 100–200 µm in diameter, using a fine dissecting scissors. Tissue strips used for imaging were gently digested in 0.5 mg/ml collagenase type II with 1 mg/ml bovine serum albumin for 5 min at 32°C.Tissue segments were prepared by removing the fibrosal and mucosal layers from mouse bladders in ice-cold Ca2, 1.2 MgCl2, 10 HEPES, and 10 glucose . For the Ca2+-free extracellular solution, CaCl2 was omitted from the above solution or 3 mM EGTA and 1 mM CaCl2 was used to clamp free [Ca2+] at ∼100 nM, as indicated. Solutions were changed using a gravity perfusion system providing complete solution exchange within 30 s. Cell stretch was accomplished using two patch pipettes attached to separate manipulators (1) to a new length (L2) is reported as the percent increase in cell length, or (L2−L1)/L1. For experiments in tissue segments, the preparations were incubated with Fluo-4AM (10 µM) and 0.05% pluronic acid for 1 h. The strip was initially stretched by a few percent over slack length to approximate resting length. All experiments were conducted at room temperature. For CICR experiments the classical whole-cell voltage clamp technique was used. The intracellular solution was (mM): 130 CsCl, 1 MgCl2, 10 Hepes, 0.075 EGTA, 1 Mg-ATP .Single myocytes were incubated with 10 µM Fluo-4AM (Molecular Probes) for 10 min at room temperature in a recording chamber mounted on an inverted microscope  and perfused with physiological salt solution for 40 min at room temperature. The extracellular solution was (mM): 140 NaCl, 5.4 KCl, 1.8 CaClpulators . Pipette0 was obtained by averaging the fluorescence for each pixel (x dimension) for a period preceding activation of a Ca2+ spark, and the fluorescence of all pixels (F) was divided by F0.Fluo-4 fluorescence was recorded using a laser scanning confocal head  attached to an inverted microscope  with a plan-apo 60× water immersion objective lens. Cells were excited with 488 nm light from a krypton/argon laser and linescan or x-y images recorded using Fluoview software (Olympus). Linescans were obtained at an interval of 1.33 or 0.833 ms per line; x-y images (128 pixels×30 or 40 lines) were recorded at an average frame rate of 37.5-, 44-, or 57.3-ms intervals. Images were processed and analyzed using MATLAB 7.1 software (MathWorks). For linescan images, F2-Fe2+-NO was extracted by ethyl acetate, and then measured on an ESR spectrometer at room temperature. The process was carried out in a dark environment to avoid light -induced NO dissociation from the adduct.Mouse bladders were removed and dissected as described above, and the muscle segments incubated in 30 mM diethyldithiocarbamate (DETC), 3 mM ferrous sulfate, and 15 mM sodium dithionite for 25 min at room temperature. To test stretch dependence of NO formation, tissue segments were elongated by about 30% of initial length and the stretched and control segments homogenized. The adduct (DETC)Cells and tissues were incubated with the NO-sensitive indicator 4, 5-diaminofluorescein (DAF-2). Cells were loaded with DAF-2 by incubation with 5 µM for 30 min, and tissue segments with the same concentration for 60 min, at room temperature. NO fluorescence was detected using a laser scanning confocal head  attached to an inverted microscope .2 and 5% CO2, 37°C) and stretched to 0.2 g (as control) and 0.5 g (as stretch) of tension, and stabilized for 45 min before beginning experimental protocols. Contraction amplitude and frequency were analyzed.Contractility of isolated bladder smooth muscle segments was measured using a MyoMED myograph system . Strips were mounted in a tissue bath containing aerated PSS . The protein content was quantified using BCA reagent and10 µg total protein from slack and stretched smooth muscle tissues was processed for SDS-PAGE. The different lanes were equally transferred to a PVDF membrane, and then the membrane was processed for immunobloting using specific primary antibody against Akt , phosphorylated Akt , eNOS  and phosphorylated eNOS , respectively. At the end of the western-blot protocol, the specific signal was probed using a Horseradish peroxidase substrate (Millipore). The specific signals were quantified using densitometry with Image J software and the phosphorylation levels of Akt and eNOS were corrected for the total amount of respective protein from the same extracts.For the measurement of phosphorylation and expression level of Akt and eNOS, bladder smooth muscle tissues were subjected to 30∼50% stretch in oxygenated physiological solution at room temperature. At the end of the stretch protocol, the smooth muscle tissues were quickly homogenized and the total protein was extracted in a lysis buffer containing a mixture of phosphatase inhibitors  values. Significant differences were determined by the Student's t test. Data from three groups were compared by one-way, repeated measures ANOVA and significant differences between groups determined by the Student-Newman-Keuls (SNK) test for paired comparisons.Image processing and data analysis were done with custom software written in MATLAB. CaTo investigate the underlying mechanism of stretch-induced Ca2+ release in smooth muscle, we examined the properties of Ca2+ sparks at slack and stretched cell length. Stretch of single mouse bladder smooth muscle cells by 9% to 18% of the slack length triggered Ca2+ sparks and/or waves as previously reported 2+ release occurred in most of cells tested when cells were stretched by 18%  increased the probability of stretch -induced Ca2+ sparks further . When mouse bladder single cells were depolarized to −30 mV from the holding potential of −70 mV, Ca2+ sparks  occurred To further examine NO involvement in the stretch-induced Ca2+ release we next examined SICR in eNOS-deficient and strain –matched control (wildtype) mice. Myocytes were loaded with fluo4 and exposed to controlled stretch as described above. As shown in To directly determine the relationship between cell stretch and NO concentration, we used electron spin resonance (ESR) spin-trapping –based detection To determine the relationship between NO formation and contractile activity, the force production of detrusor tissue segments from wild type and eNOS deficient bladders was measured. 2+ release examined. As shown in 2+ sparks were not altered in myocytes exposed to ODQ 10 µM. Moreover, neither the PKG inhibitor KT5823, nor the cell-permeable cGMP analogue 8-Br-cGMP, affected Ca2+ sparks (data not shown), suggesting that NO formation did not promote Ca2+ release by activation of the classical GC/PKG pathway.We next examined whether stretch -induced formation of NO acts through the stimulation of guanylyl cyclase (GC) and cGMP/protein kinase G (PKG). Single bladder myocytes were co-incubated with Fluo-4 AM and the GC inhibitor 1-H-2+-spark frequency has been linked to a PI(3)K-dependent signaling pathway in cardiac myocytes 2+ sparks K inhibitor, wortmannin, was examined further. As shown in The stretch dependence of CaTo further prove the NO dependence of stretch-induced Ca2+ release via activation of PI(3)K/Akt axes in mooth muscle, we detected the phosphoryaltion of Akt and eNOS levels. As shown in Spatially and temporally restricted SR Ca2+ release events in the form of Ca2+ sparks play a crucial role in the regulation of sarcolemmal, Ca2+ activated ion channels 2+ release. NO has been shown to increase the activity of ryanodine receptors in cardiac These findings establish NO as a critical coupling molecule linking membrane tension to SR Ca2+ release via RyR2. The activation of ryanodine receptors through S-nitrosylation has been demonstrated extensively in vitroIt remains unclear how NO affects Ca


Under Funding, the grant number DE-FG02-01ER63221 was missing. The correct funding information reads: Funding. This research was supported by the Genomics:GTL program of the Office of Science (BER), US Department of Energy grants DE-FC02-02ER63446 and DE-FG02-01ER63221.


Although mentoring is acknowledged as a key to successful and satisfying careers in medicine, formal mentoring programs for medical students are lacking in most countries. Within the framework of planning a mentoring program for medical students at Zurich University, an investigation was carried out into what types of programs exist, what the objectives pursued by such programs are, and what effects are reported.A PubMed literature search was conducted for 2000 - 2008 using the following keywords or their combinations: mentoring, mentoring program, medical student, mentor, mentee, protégé, mentorship. Although a total of 438 publications were identified, only 25 papers met the selection criteria for structured programs and student mentoring surveys.The mentoring programs reported in 14 papers aim to provide career counseling, develop professionalism, increase students' interest in research, and support them in their personal growth. There are both one-to-one and group mentorships, established in the first two years of medical school and continuing through graduation. The personal student-faculty relationship is important in that it helps students to feel that they are benefiting from individual advice and encourages them to give more thought to their career choices. Other benefits are an increase in research productivity and improved medical school performance in general. Mentored students also rate their overall well-being as higher. - The 11 surveys address the requirements for being an effective mentor as well as a successful mentee. A mentor should empower and encourage the mentee, be a role model, build a professional network, and assist in the mentee's personal development. A mentee should set agendas, follow through, accept criticism, and be able to assess performance and the benefits derived from the mentoring relationship.Mentoring is obviously an important career advancement tool for medical students. In Europe, more mentoring programs should be developed, but would need to be rigorously assessed based on evidence of their value in terms of both their impact on the career paths of juniors and their benefit for the mentors. Medical schools could then be monitored with respect to the provision of mentorships as a quality characteristic. The mentor, who often (but not necessarily) works in the same organization or field as the mentee, achieves this by listening or talking in confidence to the mentee." Garmel [Mentoring was developed in the USA in the 1970s within large private-sector corporations to support junior staff. Since the 1990s, mentoring programs have been introduced in various medical professions, most frequently in the field of nursing. Formal mentoring programs for medical students and doctors, however, were not developed until the late 1990s ) is "A p" Garmel  describeadvisor is a faculty member who provides assistance in scheduling clinical electives and advice on residency applications; a role model is someone a student uses as a positive example of how to approach a career in medicine; a career mentor is someone who plays an active role in helping the student in his/her professional and personal development. Mentoring also comprises supporting a mentee in coping with stress and in establishing a satisfying work-life balance [Unlike coaching or counseling, mentoring is a cost-free career-promotion strategy based on a personal relationship in a professional context. Whereas a tutor, teacher/educator, coach, or supervisor mainly focuses on promoting and supporting a junior's professional skills, a mentor is an active partner in an ongoing relationship who helps a mentee to maximize his or her potential and to reach personal and professional goals . Coates  balance . Mentori balance ,8.Although several authors report that mentoring is a key to a successful and satisfying career in medicine ,9,10, thThe search strategy for this paper was set up to identify all scientific papers on mentoring programs for medical students. In order to distinguish between scientific and popular literature and between medicine and other professional fields, we decided to limit the search strategy to papers listed in PubMed for the time period 2000 - 2008. The search strategy included the following steps:mentoring, mentoring program, medical student, mentor, mentee, protégé, mentorship.(1) The PubMed online search dated December 2008 was conducted with the following keywords or combinations thereof: (2) Using this search strategy, we found a total of 438 articles, the titles and abstracts of which were reviewed. Papers that were easily identifiable as lying outside the scope of this study were excluded (n= 353). The remaining 85 papers were retained for the subsequent stage.(3) The full versions of these papers were reviewed separately by the first and senior author for final inclusion. All papers were written in English, but this was not a selection criterion. The following inclusion criteria were established: Mentoring is to be aimed at medical students; the aim of the mentoring is to support the professional and personal development of the mentee; the mentor is an experienced medical professional; mentoring is in the form of one-to-one mentoring or group mentoring. Only 25 papers met all of the inclusion criteria.(4) In the final stage, the full versions of these 25 papers were examined.mentoring programs, the publication data was compiled according to (a) author, year published and country; (b) goal of the program; (c) mentoring model; (d) participants; (e) program evaluation; (f) effects of the program.For mentoring for medical students in general, publications were compiled according to (a) author, year published and country; (b) aims of the article; (c) results; (d) conclusion.For articles referring to Of the 25 papers that met the four inclusion criteria established, 14 papers -24 descrEleven papers ,3,25-33 All 14 papers ,12-24 redifferent main goals: (1) to provide career counseling [The mentoring programs reported pursued unseling -17,21,24unseling ,19,22,23unseling ,13,18, aunseling ,20.Coates et al.  report oIn the online Professional Development Portfolio Program (PDP) described by Kalet et al. , mentoriKanter et al.  report oThe program reported by Dorrance et al.  aimed toSix of the programs offer one-to-one mentorships ,18,20,24Eight programs were evaluated by means of questionnaire surveys -18,22; sThe literature search revealed 11 papers reporting on mentoring for medical students in general: Keyser et al.  provide Five of the papers identified reported on the qualities required for being an effective mentor ,26,29,32Below, important aspects of the papers reviewed are discussed, addressing the issues of appreciation of mentoring, requirements for mentors and mentees, effects of mentoring programs, shortcomings, and suggestions for the design of future mentoring programs.It is striking that most papers originate in the USA, and few or no reports were searched from other countries using the described criteria and database. Mentoring for medical students is well established in some US medical faculties, and personal and financial resources are available for implementing these programs ,13,14. EExperience of mentoring programs in Switzerland has shown that faculty members and authorities often think that mentoring should be provided for advanced postgraduate trainees only , and shoMotivate, Empower and Encourage, Nurture self-confidence, Teach by example, Offer wise counsel and Raise the performance bar'. Only a few authors [Most conceptual and survey papers focus on the qualities required to become an effective mentor ,28,29,32 authors  point to authors -19,21-24 authors , being pEvidence from the reviewed papers shows that three factors are important for effective mentoring programs. Firstly, for students pursuing an academic career, a one-to-one mentorship with an advanced scientist involving the junior in his/her research proves most effective. Secondly, the mentor must serve as both a professional and personal role model. Thirdly, provision of career counseling by mentors leads to juniors' making an earlier choice in terms of specialty and career.It has to be said, however, that most of the evaluation studies on the effects of student mentoring programs are not based on validated questionnaires. Consequently, there is only weak evidence that mentoring is important for career success, as pointed out in Sambunjak et al.'s review . Future A further problem emerges from the studies reviewed. Some aspects of the mentoring programs mentioned appear to overlap with tutoring, counseling and coaching systems. Moreover, the difference between advisor, role model and career mentor, as described by Coates et al. , is not There is an absence of studies into cost-effectiveness. If we compare the cost of conducting a mentoring program with the benefits to students of earlier career choice, better performance, and higher research productivity, the expense seems to be more than warranted. The mentors do their job without any financial incentives. The costs arising relate to program leaders, the holding of workshops, and some social events.No data is available in terms of whether mentoring could also help students out of medical school if they are obviously not cut out to be physicians. Admittedly, it must be borne in mind that entrance tests and interviews as well as selective exams in the first year of medical school increase the probability that a majority of students will fit the profile of a medical professional.Furthermore, the negative effects of mentoring are not reported in the studies. As noted in the Resident Mentoring Program at Zurich University Hospital , mentoriIn our view, a useful and feasible model for a student mentoring program could be designed using tiers, as reported by Kosoko-Lasaki et al. : youngerCompared to our review on formal mentoring programs for medical students and physicians , the preMentoring is obviously an important career advancement tool, which would benefit from early implementation at medical school. Mentorships must be goal-oriented and rigorously evaluated in terms of the positive outcomes for mentees as well as for mentors. Once the effects of mentoring are more clearly documented, mentoring will receive more appreciation.The authors declare that they have no competing interests.EF has conducted the PubMed literature search, reviewed the papers and compiled the publication data together with MS. BBF reviewed the papers separately. BBF drafted the manuscript, which was critically revised by the other authors. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6920/10/32/prepub


Leukotrienes (LTs) are potent biological proinflammatory mediators. LTC4, LTD4, and LTE4 are more frequently involved in chronic inflammatory responses and exert their actions binding to a cysteinyl-LT 1 (CysLT1) receptor and a cysteinyl-LT 2 (CysLT2) receptor. LTs receptor antagonists available for clinical use demonstrate high-affinity binding to the CysLT1 receptor. In this paper the employment of anti-LTs in allergic cutaneous diseases is analyzed showing that several studies have recently reported a beneficial effects of these agents (montelukast and zafirlukast as well as zileuton) for the treatment of some allergic cutaneous related diseases-like chronic urticaria and atopic eczema although their proper application remains to be established. Several studies have recently reported a beneficial effects of leukotriene (LT) receptor antagonists (montelukast and zafirlukast) as well as zileuton, a 5-lipoxygenase inhibitor, for the treatment of some allergic cutaneous related diseases-like chronic urticaria and atopic eczema , 2, althAlthough histamine is considered the principal mediator of immediate allergic responses, other factors  prolong the inflammatory process in the so-called late phase response of allergic reaction  thus cau Leukotrienes (LTs) are a class of potent biological pro-inflammatory mediators derived from arachidonic acid through the 5-lipoxygenase pathway divided into two groups according to their chemical structure: those with a sulphur linkage or cysteinyl LTs: LTC4, LTD4, LTE4 are more frequently involved in chronic inflammatory responses and exert their actions through the binding to two types of activating receptors: a cysteinyl-LT 1 (CysLT1) receptor and a cysteinyl-LT 2 (CysLT2) receptor [in situ. Eosinophils, basophils and mast cells are the most important sources of cysteinyl-LTs and epidermal cells are able to transform neutrophil-derived LTA4 into LTB4 and LTC4 . Thus th Surely Cys-LTs play a role in promoting and maintaining the allergic inflammatory response in cutaneous disease as atopic dermatitis and chronic urticaria as well as in asthma through their active effects on chemotaxis, vasodilatation and oedema, LTs are, in fact, potent spasmogenic and chemotactic agents that increase capillary and small vessels permeability. When LTs are injected into human skin, they cause wheal and flare reactions , with an There is evidence of enhanced LT production in the pathogenesis of AD. Patients with AD have activated circulating basophils and increased basophil and neutrophil releasability of LT-C4 compared with healthy subjects , 11, whi Actually, three LTs receptor antagonists are available for clinical use, montelukast, used in patients older than 6 years, and zafirlukast, approved for adolescent and adult subjects. Montelukast, Zafirlukast and Pranlukast  are LT rThe EEACI/GA²LEN/EDF guideline for the management of urticaria is a consensus reached during panel discussion at the Second International Consensus Meeting on Urticaria, Urticaria 2004 , joint i First approach is avoidance, elimination or treatment of eliciting stimulus or cause. This is the best way since identification of the cause allows successful treatment; however, it may not be possible in all cases. It includes elimination of medicaments, physical stimuli, eradication of infectious agents and treatment of inflammatory processes, and also removal of autoantibodies to the high-affinity IgE receptor. It is applicable in patients with IgE-mediated or physical urticaria. Second approach is inhibition of mast cell mediator release and nowadays the most commonly used drugs inhibiting mast cell release are corticosteroids. Other drugs with inhibiting activity on mast cells are, for example cyclosporin A and PUVA therapy. In the light of these considerations, even the LTs receptor antagonists could play a their role in the treatment of chronic urticaria , 16.  The chronic urticaria shows different complex aspects in its pathogenesis: approximately 45% of patients with chronic urticaria have an IgG autoantibody directed to the alphasubunit of the high-affinity IgE receptor leading to cutaneous mast cell and basophil activation , but it  At last the third approach to urticaria is based on therapy to target organ, for example, antihistamines, according to EEACI/GA²LEN/EDF guideline , but chr Based on the important role of LTs in the pathophysiologic mechanisms of allergic inflammation, antiLT receptors, montelukast and zafirlukast, have recently been used, either as monotherapy or in combination with H1-receptor antagonists, to treat different forms of urticaria .  The results of several studies have indicated a positive therapeutic effect of antiLTs in such different conditions as chronic urticaria,  and by other nonsteroidal antiinflammatory drugs), cold urticaria, urticaria related to food additives, chronic autoimmune urticaria, steroid-dependent urticaria, delayed-pressure urticaria, chronic idiopathic urticaria and, finally, dermographism , 22–25.  A single study reported that pranlukast may provoke urticaria in patients with ASA-induced urticaria; however, this molecule is not available in Europe . plus desloratadine, patients in the third harm were treated with desloratadine and, finally, the fourth harm received desloratadine with placebo. This study showed that the therapeutic regimen based on the association of monteleukast and desloratadine was effective in controlling symptoms of urticaria, even though the second drug proved more efficacious than the LTs antagonist [A better therapeutic effect of montelukast versus cetirizine or placebo has been demonstrated by Pacor et al. in a double-blind, placebo-controlled trial in patients suffering from chronic urticaria related to food additive and/or ASA intolerance . The samtagonist . In lightagonist . Bagensttagonist .  According to Nettis et al. positive and better results in terms of improvement of symptoms were obtained with a treatment based on montelukast alone, compared with fexofenadine in patients suffering from chronic idiopathic urticaria; in the same patients these AA. also demonstrated a reduction of wheal performing the autologous serum test after montelukast treatment . In another randomized, double-blind, placebo-controlled study on patients with mild chronic urticaria, Nettis also demonstrated that the concomitant administration of desloratadine and montelukast provides a significant improvement in overall urticaria conditions, compared with placebo and desloratadine alone . EffectiNorris and Sullivan, studying LTs and cytokines in steroid-dependent urticaria, found that 60% of patients enrolled in the study manifested a significant improvement of their severe symptoms taking zafirlukast in combination with antihistamines .Sanada et al. confirmed the effectiveness of montelukast in chronic urticaria unresponsive to the antihistamine treatment and, at variance from other observations, they did not reported differences between patients with positive skin reactions to autologous sera and/or those with ASA hypersensitivity. While critical factors were represented by age and duration of symptoms, whereby young patients having a illness for short duration, were more responsive to the treatment with montelukast . Asero showed a nearly total remission of the disease in the half of twelve patients with unremitting, steroid-dependent urticaria, after treatment with montelukast 10 mg once a day or zafirlukast 20 mg twice a day. Therefore, according to Asero and on the basis of the safety, tolerability and low cost, LT receptor antagonists should be administered in all patients with steroid-dependent chronic urticaria, unresponsive to other therapies .  Reimers et al. by realizing a double-blind, placebo-controlled, cross-over study with 20 mg daily of zafirlukast, demonstrated the ineffectiveness of antiLTs in the treatment of chronic urticaria, concluding that LTs have no significant role in the aetiology of this disease. However, evaluating their results, it is important to consider that they observed in 19 cases (41.3%) a resolution of chronic urticaria that was interpreted as a spontaneous remission, on the basis of the high variability of the course of chronic urticaria . plus montelukast versus loratadine monotherapy, administered for two weeks, the authors reported a more marked improvement of this form of urticaria in patients treated with montelukast combined with loratadine than loratadine alone. They described a negative result of the rechallenge test in 80% of patients enrolled to the combined therapy versus 20% of subjects that received loratadine monotherapy [plus montelukast, improved urticaria in respect to the baseline assessment. However, the combination of antihistamine with antiLTs resulted more efficacious in the suppression of symptoms and the wheal with dermographometer challenge test [Regarding to this aspect, Nettis et al. observed that the remission or improvement of the urticaria induced by antiLTs therapy could not be considered as a spontaneous remission because the excellent results were recorded after 3 weeks of active treatment . Furtherotherapy . In anotnge test .A successful treatment of delayed pressure urticaria with montelukast has been reported also by Berkun and Shalit . He descFinally, since Asero has suggested a common link among chronic urticaria, NSAIDs cutaneous hypersensitivity and alterations of coagulative cascade , 41 someLTs are also involved in the pathogenesis of other atopic skin disorders. In particular, atopic dermatitis or Atopic Eczema Dermatitis Syndrome (AEDS), similarly to the different forms of urticaria, are associated with infiltration and activation of mast cells and consequent release of vasoactive and pro-inflammatory mediators at the cutaneous level. This mechanism is indirectly supported by the results of some studies which revealed the effectiveness of LTs receptor antagonists in the treatment of AEDS. In this connection, Nettis and colleagues showed, in a placebo-controlled study, that a 6-week treatment with montelukast was effective in inducing a moderate reduction of cutaneous inflammation, as evaluated by the SCORAD index, in 20 adults suffering from moderate to severe AEDS . Positive effects of therapy with antiLTs in AEDS have also been reported by other authors. Yanase and David-Bajar showed a statistically significant improvement of the clinical manifestations of atopic dermatitis of moderate severity in adult patients receiving 10 mg of montelukast for 8 weeks . In the  By contrast, some other interventional studies in atopic eczema were not able to show any superiority of treatments based on LTs antagonist in respect to other treatment options or placebo. In particular, Capello et al. compared two treatment regimens based on (a) 10 mg once a day of montelukast and (b) a combined regimen , in 32 adult patients with moderate-to-severe atopic dermatitis; no difference in the reduction of the SCORAD index in the two treatment groups was reported . MoreoveIn conclusion, experimental data suggest that LTs are involved in the allergic inflammation, even though their precise pathogenetic role has not been elucidated . These f


Leucocytes from normal individuals and from patients with acute myeloid leukaemia (AML) in remission receiving active immunotherapy with allogeneic AML blasts (AML-I) were cultured for 6 days with AML-I blasts, Burkitt's lymphoma cells (BL) or lymphoblastoid cells (LCL). The leucocytes were then tested for cell-mediated cytotoxicity (CMC) against 51Cr-labelled AML-I, BL or LCL target cells. There was no substantial difference in the CMC of leucocytes from patients and normals cultured without stimulation, and tested against AML-I, BL or LCL targets. Patient's leucocytes stimulated in vitro with AML-I had a greater frequency of positive CMC responses against AML-I, BL and LCL than normal individuals. The results suggest that co-cultivation of leucocytes with AML-I blasts reactivates memory cytotoxic leucocytes in AML patients receiving immunotherapy and that this test may be useful in measuring the effectiveness of immunotherapy.


Such ‘uncontrolled’ Ca2+ sparks were suggested to act as dystrophic signals. They may be related to mechanosensitive pathways but the mechanisms are elusive. Also, it is not known whether truncated dystrophins can correct the dystrophic disinhibition.In dystrophic skeletal muscle, osmotic stimuli somehow relieve inhibitory control of dihydropyridine receptors (DHPR) on spontaneous sarcoplasmic reticulum elementary Ca2+ concentrations amplified osmotic ECRE responses. The eliminated ECRE suppression in intact osmotically stressed mdx fibers was completely and reversibly resuscitated by streptomycine (200 µM), spider peptide GsMTx-4 (5 µM) and Gd3+ (20 µM) that block unspecific, specific cationic and Ca2+ selective mechanosensitive channels (MsC), respectively. ECRE morphology was not substantially altered by membrane stress. During hyperosmotic challenge, membrane potentials were polarised and a putative depolarisation through aberrant MsC negligible excluding direct activation of ECRE through tubular depolarisation.We recorded ECRE activity in single intact fibers from adult wt, mdx and mini-dystrophin expressing mice (MinD) under resting isotonic conditions and following hyper-/hypo-osmolar external shock using confocal microscopy and imaging techniques. Isotonic ECRE frequencies were small in wt and MinD fibers, but were markedly increased in mdx fibers. Osmotic challenge dramatically increased ECRE activity in mdx fibers. Sustained osmotic challenge induced marked exponential ECRE activity adaptation that was three times faster in mdx compared to wt and MinD fibers. Rising external Ca2+ homeostasis. MsC blockers should have considerable therapeutic potential if more muscle specific compounds can be found.Dystrophin suppresses spontaneous ECRE activity by control of mechanosensitive pathways which are suggested to interact with the inhibitory DHPR loop to the ryanodine receptor. MsC-related disinhibition prevails in dystrophic muscle and can be resuscitated by transgenic mini-dystrophin expression. Our results have important implications for the pathophysiology of DMD where abnormal MsC in dystrophic muscle confer disruption of microdomain Ca Duchenne muscular dystrophy (DMD) is a common inherited muscle disease. Death usually occurs early in adulthood from respiratory or cardiac failure. Different mutations in the dystrophin gene result in the complete absence of the 427 kDa protein dystrophin 2+ regulation 2+ levels have been controversial 2+ homeostasis is disturbed 2+ influx through altered mechanosensitive channels 2+ dependent proteolysis Dystrophin plays a pivotal role in protecting the sarcolemma from mechanical, e.g. osmotic, stress 2+ release events (ECRE) from sarcoplasmic reticulum (SR) in mammalian skeletal muscle were first demonstrated by our group in ‘skinned’ fibers skinned’ fibers 2+ release were not resolved. Also, the authors performed their osmotic challenge experiments in hypertonic solutions containing unphysiologically high Ca2+ concentrations of 50 mM Elementary CaFrom the results of our present study, we propose a model in which altered MsC in dystrophic muscle modify the otherwise suppressed coupling between DHPR and RYR in intact resting fibers under isotonic conditions and even more so in response to membrane stress.2-atmosphere, interossei muscles were quickly dissected. Killing of the animals and tissue preparation were according to guidelines of the local Animal Care Committee. Single fibers were obtained by mild enzymatical treatment in isotonic Ringer's solution that contained (mM): NaCl 140, KCl 5, CaCl2 2, MgCl2 1, Hepes 10, pH 7.4. Hypertonic 50 mM Ca2+ containing solution was composed as given by Wang et al. 2 50, MgCl2 1, Hepes 10, pH 7.4. As we consider its Ca2+ concentration as unphysiologically high, 300 mM sucrose was added to Ringer's solution . A similar solution was previously used to record L-type Ca2+ currents in the strains 2+, ∼120 mosM) and contained (mM): NaCl 70, KCl 4, CaCl2 2, MgCl2 1, Hepes 10, ph 7.4. Ca2+-free solutions were made without addition of CaCl2 and additionally contained 1 mM EGTA. To keep free Mg2+ concentration roughly constant, Mg2+ was increased to 2 mM in these solutions. Streptomycine and Gd3+  and GsMTx-4  were added from stocks to the desired concentrations. All experiments were performed at room temperature (22–24°C).C57/SVJ129 wild-type (wt), mdx mice and mini-dystrophin (MinD) mice referring to a transgenic strain with the mdx background (CVBA3') that stably expresses a Δexon17-48 truncated mouse dystrophin of 228 kDa molecular weight 2+ dye Fluo-4-AM (10 µM) in isotonic Ringer's solution for 20 min. Single fibers were transferred to the experimental chamber and mounted on the stage of a laser-scanning confocal microscope  equipped with a 20 mW Ar+/Kr+ ion laser . Excess external dye ester was washed out by exchanging the solution three times using a fast custom-built perfusion system that allowed complete solution exchange within 3 s. Intracellular, de-esterified Fluo-4 was excited at 488 nm and emission collected using a >515 nm low-pass filter. PMT voltage was between 630 V and 680 V between experiments. XYT image series were acquired at a sampling rate of 0.9 Hz with a 512×512 pixels resolution using a ×40 water immersion objective . XYT stacks contained up to 200 consecutive images . XT line-scans were recorded at random line positions within the fibers. Repetitive acquisition time was 2 ms/line at a 512×1024 resolution, thus obtaining a full line-scan within two seconds. Different zoom factors between ×3 and ×7 were used and the adjacent voxel sizes used for calculation of Ca2+ spark dimensions.Single fibers were stained with the fast high-affinity Ca2+ release events in the intact single fibers were recorded in isotonic Ringer's solution. Subsequently, XYT series and several XT line-scans were recorded (control). Then, fibers were either subjected to hypo- or hyperosmolar stress by quickly exchanging the isotonic Ringer solution to the appropriate solution of given osmolarity (see above). The solution exchange was performed while recording an XYT series to follow the time course of osmolarity effect on ECRE frequency. After a steady-state was reached, the solution was changed back to isotonic Ringer's solution to test for reversibility of the observed osmolarity induced changes. During solution exchange, movement artefacts of the single fibers were frequently observed. As the solution exchange was complete within 3 s, this corresponded to two subsequent images in the recording sequence only. Therefore, in the corresponding recordings, the images containing movement artefacts were discarded from ECRE analysis, as they gave abrupt changes in ECRE frequency resulting from false positive events during the fiber movement. In a subset of experiments evaluating the contribution from extracellular Ca2+ to the ECRE frequency during osmotic stress, ECRE were first recorded in isotonic Ca2+ containing Ringer's solution, then solution exchanged to hypo-/hyperosmolar Ca2+ free solution while recording and, finally, back to isotonic Ca2+ containing Ringer's solution. To test the involvement of mechanosensitive channels to the osmotic stress induced changes in ECRE, different blockers of mechanosensitive channels (MsC) were used: streptomycine (200 µM), an unspecific blocker of MsC 3+ (20 µM) a blocker of more Ca2+ sensitive MsC 2+ containing solution and either drug applied after spark frequency increased in response to the osmotic stimulus. This was suitable to assess the blocking potency of the drugs on osmotic stress-induced ECRE frequencies. (ii) ECRE were recorded in Ringer's solution that was then pre-incubated with either streptomycine or Gd3+ and solution was then exchanged to hypo-/hyperosmolar Ca2+ containing external solution also containing the drug. This was suitable to test whether both drugs prevented uncontrolled spark activity in mdx fibers by osmotic stress. Both experimental approaches were carried out in 2 mM Ca2+ containing solutions. In some experiments, (i) was performed in mdx fibers applying GsMTx-4 after osmotic challenge under Ca2+-free external conditions throughout.The following experimental procedures were carried out. First, elementary Ca0+1.5 σ). A second run for background estimate was then performed on this set of images with probable ECRE removed and all raw images were finally divided by this improved mean image, yielding normalized F/F0 images of a series. The á trous implementation contains the cubic B-spline as scaling function and a discrete low-pass filter . Additionally, ECRE frequency was normalized with respect to fiber cross-sectional area  using the voxel size at a given zoom factor. For osmotic challenge, up to ∼380 individual fibers were scanned and analysed for each strain and condition. The imaging algorithm yielded the overall average SF or SSF for a given XYT series. To quantify the time course and kinetics of ECRE frequency changes upon solution exchange, the SF or SSF were analysed on a frame by frame basis. The apparent SF and SSF values are obviously larger than those obtained for the whole time series, simply due to averaging. ECRE kinetics were analysed in 15 randomly selected fibers from each strain under conditions of solution exchange to either hypertonic or hypotonic 2 mM Ca2+ containing external medium (experiments without blockers). The time-to-peak (TTP) from the exchange of solution to the maximum of SSF induced by hypertonic or hypotonic medium and the mean apparent peak SSF values (SSFmax) were analysed. Under maintained osmotic challenge, apparent SSF following osmotic shock showed adaptation with spontaneous decay of SSF. This decay was fitted by a single exponential with a time constant τdec for the decay kinetics. In experiments testing different MsC blockers, XYT series from all fibers were analysed on a frame-by-frame basis extracting the apparent SSFmax values and aligned so that blocker applications were at the same time point for all fibres tested per blocker and condition.XYT and XT images were background normalized and denoised using the á trous implementation of the two-dimensional undecimated discrete wavelet transform (DWT) as previously described http://rsb.info.nih.gov/ij/). Specifically, LUTs and a median filter (2.0 pixels) were applied for visualization purposes only.In line scan images (XT), the ECRE morphology was determined as given by v Wegner et al. 2+). An equilibration time of 30 min was allowed before commencing potential recordings. Resting membrane potentials were recorded using a GeneClamp 500 amplifier . Sharp microelectrodes were pulled from borosilicate capillaries using a horizontal puller  and filled with 3 M KCl. Microelectrodes were mounted on a pipette holder and connected to the headstage of the amplifier. The headstage was held by a manual micromanipulator to allow fine movements of the pipette tip. Tip resistances were between 5 MΩ and 10 MΩ. The approach using exposed muscles ensured recordings to be made as close as possible to the physiological condition without further unspecific damage during dissection and subsequent enzymatic treatment. The latter may otherwise result in a larger proportion of depolarised fibres, thus skewing the real membrane potential distributions especially in the more fragile mdx muscle. After offsetting the potential readings (0 mV), the muscle was slowly impaled with the tip of the microelectrode until a negative potential deflection was noticed. The most negative value during this deflection was taken as the resting membrane potential of an individually impaled single fiber. The pipette was then pulled back and impalement was repeatedly performed after random repositioning of the electrode over the muscle flap. Impalements with a potential drift >5 mV after withdrawal of the electrodes were discarded. In experiments assessing the contribution of mechanosensitive channels to the resting membrane potentials, the specific MsC blocker GsMTx-4, a peptide contained in the venom of the Chilean Rose Tarantula, Grammostola spatulata, was used. The peptide was added at a concentration of 5 µM to the isotonic or hypertonic recording solution before the challenge of the contralateral muscle from the animal of which the corresponding recordings were done in the absence of GsMTx-4 that day.Whole interossei muscles from wt and mdx mice were exposed from the paw and placed in a recording chamber containing either isotonic or hypertonic (300 mM sucrose) Ringer's solution (2 mM Ca2+) in intact single wt, mdx and MinD fibers, as shown in ECRE recordings under physiological isotonic conditions  resulted in ‘uncontrolled’ spark activity in mdx fibers 2+ concentrations at physiological levels (i.e. 2 mM). Under such conditions, there was a clear increase in ECRE activity following osmotic challenge  and following osmotic challenge in Ca2+-free hypertonic solution at the time points indicated. The marked increase in SF and SSF was confirmed in up to 100 single mdx fibers, as shown in 2+ parallels a similar, albeit somewhat smaller, increase as compared to the presence of 2 mM external Ca2+ and, thus, rules out the necessity of (but a regulation by) external Ca2+ to markedly increase ECRE activity in mdx fibers.We next reasoned whether external Ca2+), followed by addition of either streptomycine (200 µM) or Gd3+ (20 µM). Note that their mean values SSFmax, shown in max values were omitted from the plot, thus, producing some discontinuity  ECRE activity increased again but with a rate lower than that compared to the increase before blocker application. To test whether the blockers could prevent the osmotic-induced increase in ECRE activity, osmotic shock was initiated after pre-incubation of either blocker in isotonic solution and maintained in the non-isotonic solution  was used. 2+ containing or Ca2+-free environment. GsMTx-4 potently terminated the increased ECRE activity even if external Ca2+ was absent. Averaged SSF values (inset in 2+ free isotonic starting conditions (inset) highlighting Ca2+ influx already being present in mdx fibers under isotonic conditions As seen before ; Fig. 4,inset in  were muc50: 0.73 (hypertonic), 0.58 (hypotonic) and 0.53 (isotonic)).To clarify whether ECRE morphology was altered by osmotic challenge in intact mdx fibers, line-scans (XT) were recorded . The inc2+ waves could be detected that spread forth and backwards on a seconds time scale  and hypotonic 2 mM Ca2+ containing solution .In some mdx fibers , global Came scale  and seemme scale . Such CaS II–III loop could be directly or indirectly relieved or alternatively, the RyR1 be activated via depolarisation of the tubular membrane following cation influx through the MsC. In dystrophic muscle, a higher resting [Na+] has been described m were measured in mdx and wt muscle under isotonic and hypertonic conditions, either without or in the presence of the specific MsC blocker GsMTx-4 (5 µM). To obtain recordings that as closely as possible reflect the physiological condition of the muscle fibers, recordings were made in whole muscles exposed from the paw bathed in the appropriate solution. We chose not to record from enzymatically isolated single fibers because the enzymatic treatment may already induce some unspecific damage to the membrane that might readily increase Na+ influx unrelated to MsC. m as negative deflections (m was −67 mV under isotonic conditions and significantly more negative under hypertonic conditions (−72 mV), as would be expected for this tonicity increase m was only marginal. In mdx fibers under isotonic conditions, Em was significantly more depolarised at ∼−57 mV. However, MsC activity only accounted for about 4 mV of this depolarisation (Em∼−61 mV in the presence of 5 µM GsMTx-4). Interestingly, under hypertonic conditions, Em was more negative at −61 mV with a similar further repolarisation in the presence of GsMTx-4 as seen under isotonic conditions. This rules out a major contribution from MsC induced depolarisation accounting for a direct activation of the RyR1 by the DHPR as the cause for the markedly increased ECRE frequencies seen during osmotic challenge in mdx fibers.In mdx fibers, the increased ECRE frequencies under isotonic and, in particular, after osmotic challenge seem to be a direct consequence of aberrant MsC activity due to the lack of dystrophin. The modulation of the DHPR inhibition on the RyR1 via the α1lections . In the ‘uncontrolled’ spark activity in mdx fibers in 50 mM Ca2+ containing hypertonic solutions 2+), there were also some striking differences. First, spontaneous ECRE activity was present in single intact fibers already under isotonic resting conditions, albeit at much lower frequency than following osmotic challenge. This is a very robust finding from several hundred single fibers and contrasts with the finding from Wang et al. 2+ spark activity’ under resting isotonic conditions in an ungiven number of fibers. Present, albeit very low, spark activity was previously documented in native muscle after having been observed in abundance in the embryonic form with a postnatal decline −4 µm2, as compared to ∼0.15*10−4 µm2 in the present study, 2+ containing medium (2 mM Ca2+). It was less prominent under hypotonic challenge in wt and MinD fibers, but similar to hypertonic challenge in mdx fibers. The absolute increase showed a clear dependence on external Ca2+, e.g. 1.5-fold smaller SSF in mdx fibers in Ca2+-free hypertonic solution  in uncoupling spontaneous ECRE suppression in osmotically stressed intact single mdx muscle fibers. Our study is the first to confirm some previous results on solution . Interes2+ transients were abolished in patch-clamp experiments using MsC blockers 3+-sensitive MsC Ca2+ channels (MsCa) has been shown to disrupt Ca2+ homeostasis in resting mdx EDL fibers 2+ influx through leak channels and MsCa 2+ selective MsCa are involved in the control of ECRE inhibition in healthy muscle. So far, there are some speculations about the molecular nature of the channels involved (see below). However, detailed identification and their pharmacological and physiological properties regarding ion sensitivity and drug response in skeletal muscle are still elusive.Changes in tubular wall tension are likely to activate mechanosensitive channels 2+ sparks in a very limited set of single mdx fibers  are very similar to the values given by Wang et al. 2+ containing solution  that may be modulated by MsCa. Studies in healthy mammalian muscle largely excluded a significant involvement of CICR during ec-coupling + influx through MsC could also be responsible for a direct voltage-induced activation of the RYR1, as a greater rise in intracellular [Na+]i was found in mdx muscle fibers in response to stretch compared with wt fibers + concentration was found in young (12–16 week old) mdx flexor digitorum brevis muscle fibers, as a result of altered gating properties of Nav1.4 channels +. Also, the resting membrane potentials given by Hirn et al. + levels secondary to the experimental protocol and not primary to the absence of dystrophin m values m was already about 10 mV more depolarised in isotonic resting mdx fibres. However, even this substantial difference might not fully explain the already twofold increased spark frequencies that we saw in the isotonic mdx fibers. In intact muscle, SR Ca2+ release via the ryanodine channel is initiated by the conformational coupling between the DHPR and the RyR1 upon depolarisation that is reflected by DHPR charge movements. However, in a study on intact single fibers, no difference in the sigmoidal voltage distribution of charge movements was observed between wt and mdx muscle m values observed here, the membrane potential – charge movement curve should still be at the lower knee of the activation curve 2+ release transients for potentials up to around −55 mV in wt and mdx fibers m values in mdx fibres under isotonic conditions were still subthreshold for RyR1 opening. Nevertheless, even if there was some activation of RyR1 openings in mdx fibers under isotonic resting conditions, depolarisation clearly cannot account for the marked ECRE frequency increase in mdx fibers subjected to osmotic shock. Similar to a previous study in amphibian muscle, Em values became more negative when increasing the external osmolarity twofold, in accordance with the Nernst-equation 2+ concentration resulting in hindered SR Ca2+ release 3+ and streptomycine in osmotically challenged mdx fibers under 2 mM external Ca2+ conditions  to the initiation of spontaneous ECRE following osmotic shock in intact wt muscle fibers that could be traced to the activation of NADPH oxidase , as compared to the faster TTP values in our study . Possible candidates for this MsCa channel may be related to the TRP family, as the Gd3+ sensitive growth-factor related channel (GRC) is translocated to the sarcolemma by mechanical stress in mdx muscle 2+ influx. Likewise, TRPV2 was shown to belong to osmotically sensitive cation channels in murine aortic myocytes 2+ leak channels in dystrophic fibers Caase NOX, . These e10–30 s, . Thus, s2+ wave activity. These Ca2+ waves are of interest, as they only appeared in mdx fibres after osmotic challenge at a prevalence of ∼7%. From recordings such as shown in 2+ waves observed in polarised intact frog muscle fibers following hypertonic challenge 2+ induced Ca2+ release (CICR). In mammalian muscle, however, CICR does not occur (see above). When analysing our line-scan recordings, we consistently saw a flat front on the Ca2+ sparks (not propagating) that is consistent with a concerted opening of channel arrays rather than recruitment by CICR 2+ release through RYR1 in dystrophic skeletal muscle where the inhibitory control of spontaneous ECRE by DHPR 2+ release (SOICR), as previously suggested to regularly occur in cardiac muscle RYR2 2+ content in mdx muscle are available. SR Ca2+ reloading and SR Ca2+ leak were not different in EDL fibers from wt and mdx mice 2+ load was estimated to be roughly 40% decreased in mdx diaphragm muscle 2+ load in dystrophic muscle fibers.Apart from controlling ECRE, we show that the disruption of MsC/MsCa control in mdx muscle also induced global Ca2+ binding site 2+ concentration, additional Ca2+ influx through mechanosensitive channels would modulate peak ECRE frequencies by the mechanism suggested by Martins et al. From the discussion above, our results track down the possible mechanisms involved in osmotically induced spontaneous ECRE activity in dystrophin-deficient muscle to a direct interaction of MsC with the inhibitory DHPR-RyR1 loop. We suggest a model  in which2+ homeostasis and ec-coupling How do our results obtained from the mdx animal model translate to the situation in human DMD muscle? The mdx mouse has been confirmed in many studies to be a suitable model to unravel various aspects of the pathophysiological mechanisms in dystrophin-deficient muscle regarding membrane fragility, contractility, inflammatory pathways, Ca+ overload, and partly restored related reductions in force development 3+ that is also used in some contrast-agents, has the potential to induce nephrogenic systemic fibrosis One clinically relevant interpretation of our study would also be that blockers of mechanosensitive channels might prove beneficial on the phenotype in dystrophic muscle if more muscle-selective compounds could be found. The MsC blockers used in our and other studies are mainly used as tools to study cellular reactions on altered channel availability in acute single cell 2+ transients in mdx fibers 2+ handling and supports the ongoing research for suitable gene transfer strategies in DMD. Another implication of our study is that suitable mechanosensitive channel blockers might be useful in a potential drug therapy setting in DMD.In summary, our study closes the gap from altered MsC activity Movie S12+ wave activity in the mdx fibre shown in 2+ wave is initiated at the one fibre end and spreads with a velocity of approximately 25 µm/s.Movie of the Ca(0.54 MB MOV)Click here for additional data file.


AML-ETO fusion was positive and trisomy of 4 was confirmed with WCP probes. Trisomy 4 in AML with t is a rare numerical abnormality. Here we present such case of patient which may constitute a distinctive subtype.t is the most frequently observed karyotypic abnormality associated with acute myeloid leukemia (AML), specifically in FAB-M2. Short-term unstimulated bone marrow (BM) and peripheral blood lymphocyte culture showed 47,XX, +4,t in all metaphase plates; and interphase and metaphase results of  M2 was the most frequent subtype in this series (9 out of 21 cases).[t8;21) is the most frequently observed karyotypic abnormality associated with acute myeloid leukemia (AML), specifically in FAB M2. Trisomy 4 as the sole anomaly is a rare chromosomal abnormality associated with a specific subtype of primary acute non lymphocytic leukemia (ANLL) and secondary (treatment-related) ANLL with myelomonocytic morphology; it has been found with the same frequencies in the M1, M2, and M4 FAB phenotypes.[1 cases). Associat9/L, polymorphs 7%, lymphocytes 14%, myelocytes 4%, blast cells 75%, and Platelet count 0.0046 × 109/L. Peripheral blood smear showed presence of blasts with Auer rods. Bone marrow report revealed hypercellular marrow with marked depletion of all the normal marrow precursor cells. Few blasts showed presence of Auer rods in cytoplasm, Sudan Black-B was positive, and periodic acid Schiff (PAS) was negative, M:E ratio was altered, and megakaryocytes were not seen, lymphocytes 8%, eosinophills 1%, polymorphs 3%, band cells 2%, metamyelocytes 1%, myelocyte 8%, promyelocytes 3%, and blast cells 72%. Final diagnosis based on morphological and cytochemistry findings was AML with M2 subtype as per French-American-British classification. Constitutional nature of trisomy 4 could not be ruled out. After 1 month of sample received, the patient was lost to follow up.A 60-year-old female with complaints of general weakness was registered at Gujarat Cancer and Research Institute in May 2007. Peripheral blood report was hemoglobin concentration 7.8 g/dL, White Blood Cell count 0.03 × 102 incubator. Following overnight incubation in presence of Colcemid (10 µL/8 mL of culture) the cultures were exposed to hypotonic solution (0.075 mol/L KCl) and fixed with methanol:acetic acid (3:1). The slides were prepared by air-dry method and stained with GTG-banding. Twenty metaphases were analyzed and karyograms were prepared using the Cytovision computer-assisted karyotyping system . The karyotypes were described according to the International System for Human Cytogenetics Nomenclature 2005.[A G-banded chromosome study was performed using standard cytogenetic protocol. Briefly, unstimulated cultures of bone marrow aspirate were set up in RPMI-1640 medium supplemented with 20% newborn calf serum, l-glutamine, and antibiotics (penicillin and streptomycin). The cells were cultured for 24 and 48 h in 5% COAML-ETO dual-color dual-fusion probe was used to determine the AML-ETO fusion status to confirm the diagnosis of AML-M2 subtype. Whole chromosome paint for chromosome 4 with spectrum orange was applied to confirm trisomy and/or cryptic rearrangements of chromosome 4.FISH procedure was performed on interphase and metaphase cells following the manufacturer's  guidelines. The LSI Classical chromosome analysis detected an abnormal female chromosome complement. Karyotyping results revealed trisomy of 4 with t in all metaphase plates in 15 metaphases .AML-ETO fusion positive sample [The interphase and metaphase FISH results were one green, one orange, and two green-orange or yellow fusion signals indicating e sample . The whoe sample .Trisomy 4 is a rare nonrandom cytogenetic abnormality found in association with AML. The Mitelman database for chromosomal aberrations in cancer queried for trisomy 4 with t in AML showed only two cases.Gains or losses of chromosomes are frequent findings in AML, more common being monosomy 7 and trisomy 8. Most of such changes are not restricted to any specific FAB types of AML, are often also associated with secondary AML and AML with pre-existing myelodysplasia, or during clonal progression of AML. Rarely gain of chromosome 4 or chromosomes 10 are reported as the sole abnormality in AML.Some morphologic subtypes of AML are associated with specific chromosomal abnormalities. Most of these abnormalities are chromosomal translocations which amplify or activate chimeric genes situated near the breakpoints of translocations. Trisomy 4 occurs in AML with frequency of <1% and a strong association with the presence of double minutes has been described. Double minutes were not observed in our patient.et al, have also suggested that acquisition of trisomy 8 leading to MYC locus amplification might underline the molecular mechanisms for the clonal progression of chronic myeloid leukemia. A direct link between chromosomal gain, oncogenes amplification, and leukemogeneisis has yet to be established.[CD56 expression in AML is reported in granulocytic sarcoma and multidrug resistance, and is known to confer poor prognosis in AML-M2 with t8;21) and acute promyelocytic leukemia. It has been suggested that there might exist a dosage effect of certain genes resulting in growth advantage of malignant cells. In the case of AML with trisomy 4 and double minutes, V-myc myelocytomatosis viral oncogene homolog (avian) amplification is a common finding. Jennings ablished.8;21 and aOur patient was lost to follow up hence the CD56 status was unclear. Therefore, relation between CD56 expression and trisomy 4 needs further investigation. While development of AML with trisomy 4 secondary to chemo or radiotherapy has also been suggested, our patient had no history of long-term medication, radiotherapy, or any relevant occupational exposure. In conclusion, based on the morphological, cytochemistry, and clinical features the present case of AML-M2 is a rare case in terms of cytogenetic results. Even though trisomy 4 is likely to be a secondary event after t translocation, the presence of this additional numberical aberration may define a distinctive subtype. Follow up of more such cases over a period of time is required to know the possible prognostic effect of this cytogenetic entity.


Mentoring is important throughout a physician's career and has been noted to be particularly important during residency training. Other studies suggest that women may experience difficulty in finding mentors.This study explored gender-specific differences in residents’ mentoring experiences.The authors conducted two focus groups at the University of Pittsburgh in July, 2004. One group was composed of 12 female residents; the other was composed of nine male residents. Discussions were audiotaped and transcribed. Two investigators coded the transcripts and identified emerging themes.Residents of both genders cited multiple barriers to mentoring. Men′s strategies for finding mentors were more numerous than women′s and included identifying mentors through research, similar interests, friendship, and networking. Female strategies were limited and included identifying mentors through “word of mouth” and work experiences. Women described more passive approaches for finding a mentor than men.Female residents may lack strategies and initiatives for finding mentors. Residency programs should create opportunities for residents to develop mentoring relationships, with special attention paid to gender differences. Mentoring is important throughout a physician's career, but may be especially critical at times of professional transitions, such as residency.1689Women, in particular, are thought to benefit substantially from mentoring. Women with mentors report greater job satisfaction, are more likely to be promoted, and spend more time engaged in scholarly activities.10Female residents may experience problems with stress and confidence, and are less likely to establish a solid career network when compared to their male counterparts.4134Formal mentoring programs, which assign a faculty mentor to a resident, ensure that all residents are mentored; however, these relationships may be less effective than those formed spontaneously.316Gender differences in initiating mentoring relationships may result from a number of factors. A lack of women in leadership positions may result in fewer potential mentors for women.Mentoring relationships are nuanced interactions which are ideally explored using qualitative research methods. Few qualitative studies of residents’ mentoring relationships exist in the literature; a recent systematic review specifically excluded qualitative studies. The purpose of our study was to understand gender differences in residents’ experiences with mentoring. We asked female and male residents to describe the barriers they encounter in finding a mentor and the strategies they use to overcome these barriers. Using residents’ own words, we can gain a better understanding of the most effective methods for ensuring that all residents have access to mentoring.Study Design - This study used a grounded theory approach to explore residents’ experiences with mentoring during residency. We elicited residents’ personal accounts about their experiences with mentoring during residency training using focus group interviews. Our study used focus groups because we felt that residents would be more comfortable talking about this topic in a group setting, and that the exchange of shared experiences would facilitate thoughtful and rich discussion. The study was reviewed and approved by the Institutional Review Board at the University of Pittsburgh prior to the start of the study.Sampling - We collaborated with the program directors of six core training programs at the University of Pittsburgh Medical Center to recruit residents in Internal Medicine, Pediatrics, General Surgery, Family Medicine, Psychiatry, and Obstetrics and Gynecology for two focus groups, one with male residents and one with female residents. We chose to recruit from these specialties because they each have accredited programs which train a large number of residents every year.Our goal was to recruit a total of 24 residents . We used a combination of stratified random and quota sampling strategies to achieve equal representation by gender across the specialties and levels of training. Program directors for each specialty provided the names of 366 current trainees for the 2004–2005 academic year; these names were further stratified according to training level. From these names, the PI selected 54 men and 54 women to invite for participation in the study; estimating that 20% of invitations would be accepted. Names were randomly selected from within each stratum. Potential subjects were invited to participate through a personalized email invitation; this invitation described the purpose of the study, the dates and times of the focus groups, and compensation. Residents responded to this invitation by emailing the PI directly. The PI then phoned each of the respondents, described the study in further detail, and confirmed the resident's interest. A second reminder phone call was made just prior to each focus group. We conducted the focus groups after regular work hours at a central site on the University of Pittsburgh Medical Center campus. Residents were compensated with $25 for their study participation.Data collection - Each participant completed a brief anonymous questionnaire at the beginning of the focus group session. This questionnaire collected data on age, race, training level, future career plans, and current involvement in a mentoring relationship. A trained moderator conducted each focus group, and an observer took notes. Each focus group was audiotaped and lasted 1.5 hours. We asked each group three questions to stimulate discussion regarding their experiences with mentoring during residency and their strategies for finding a mentor. These questions were reviewed by local experts in qualitative analysis at the University of Pittsburgh, and were thought to be sufficiently open-ended and general to stimulate discussion. These questions were: “What do you think of when you hear the word ‘mentor’?”; “What have your mentoring experiences during residency been like?”; “How do you find a mentor who is helpful to you?”Data analysis- Each session was transcribed verbatim and both the moderator and observer reviewed each transcript to make sure it matched their recollection of the discussion. In our analysis, we used a variety of methods to corroborate our interpretation of the data; these steps helped to ensure consistency and prevent bias. Two coders independently analyzed each transcript and assigned codes to individual words, phrases, and sentences. They then met and compared their coding. There were no differences in interpretation of the data; differences in the application of specific coding terms to a particular phrase or section were discussed and resolved by consensus. The list of codes was iterative; a final coding scheme was developed from the list. This final coding scheme was applied to the transcripts and thematic trends were identified. Additional methods to corroborate our findings included the following: 1) review of analysis among a larger study group; 2) review of analysis by focus group participants; 3) review of analysis by a former residency program director, a current residency program director, a local expert in resident education, and a local expert in mentoring; and 4) peer review in formal work-in progress sessions and invited presentations. These reviewers found the themes we presented to be plausible based on their own experiences.Participants – Among the 108 residents who were contacted, 30 residents  volunteered for the focus groups. The first 12 female residents who responded to the email invitation were recruited for participation in the study. Three of the 12 male residents who volunteered did not come to the scheduled focus group and thus did not participate in the study; thus, the total sample included 21 residents (12 women and 9 men). Specialties represented included Internal Medicine, Pediatrics, General Surgery, Family Medicine, and Obstetrics/Gynecology; years of training ranged from post-graduate year 1 to post-graduate year 7. Nine of the female residents and five of the male residents reported that their residency had a mentoring program. More detailed information regarding the composition of each group and the characteristics of our study participants is provided in the Table What the residents said - Residents had participated in mentoring relationships by being assigned a mentor through their residency program, or by finding a mentor on their own. Residents of both genders expressed similar views regarding assigned mentoring and barriers to establishing mentoring relationships. However, male and female residents articulated different approaches and strategies for finding a mentor. In the following section, individual quotes are italicized; when known, the specialty of the respondent is listed in parentheses.Residents’ perceptions about assigned mentoring - Residents did not view assigned mentors as “true” mentors. Assigned mentors were perceived as advisors, and these relationships were typically transient and impersonal: “But I think very often that first person everyone gets assigned to in residency is any advisor who may change.” ; “They…have a list of 30 other people they're advising.” {male, Internal Medicine}.Sometimes maybe assigned things can work as a launching block.” .Trainees had few expectations for assigned relationships, and seldom anticipated that assigned faculty would become genuine mentors. At best, assigned mentoring relationships served as a springboard for identifying other, more fruitful relationships: “I think it's better if somebody gives you guidance as to how to go about finding a mentor.” .At worst, such relationships could be considered an obstacle, delaying the resident from developing a relationship that really worked. Residents agreed that the best mentoring relationship were “found” but noted that this process was challenging; they welcomed assistance in identifying faculty mentors: “Barriers to establishing mentoring relationships - Residents perceived multiple barriers to establishing mentoring relationships. Busy call schedules and multiple clinical responsibilities often took priority for residents, and identifying a mentor necessarily assumed less importance. Residents had to be dedicated to the process of finding a mentor, because no “official” time was allocated for this process: “How can you find (mentors) except by bum luck or true motivation in finding the spare time and by the grace of God ‘having the day off’ or the half hour for lunch.” .I think particularly at the academic level, there are a lot of people who are really great at what they do, but don't have a lot of time for being a mentor and so to have a mentor who… serves as a great role model and is doing a lot of really impressive things is great, but not if you can't ever talk to them or be around them.”. Even faculty mentors who had committed to the relationship made residents feel that they were being squeezed in. One resident described a meeting with her mentor: “Once was in the cafeteria for ten minutes because we couldn't connect… And it was kind of like ‘So how are things going?’ It was like, how can I tell you my whole intern year, my experience from emotional to physical? I talk fast, but not that fast.”.A perceived lack of interested and accessible faculty mentors represented another barrier for residents trying to establish mentoring relationships. Faculty members’ busy schedules and multiple responsibilities often rendered potential mentors unapproachable: “I went to (a university) where it seemed like they lived and breathed mentoring…And then I cam here and it was the total opposite. It didn't seem as if anyone was interested.” . Residents expressed a sense of abandonment and isolation, and noted they were receiving little assistance from faculty: “So now I'm kind of like doing it on my own. And I figured it out, but it's kind of like…the process was done without any kind of help.” .Trainees felt that residency programs did little to foster effective mentoring relationships. Residents commented that they were “on their own” in seeking a mentor, and that programs failed to cultivate an atmosphere of mentoring: “Residents’ strategies for finding a mentor - Male and female residents employed distinctly different strategies for finding a mentor. Men's strategies were more numerous than women's, and included identifying mentors through:Research ;Similar interests ;Fiendship ;“And sometimes the role of the mentor is just teaming you up with someone who is a better mentor for you.”);networking .Female strategies were limited and included identifying mentors through:Word of mouth ;Work experiences .If you strike up a conversation and happen to strike an interest, follow it up. Don't let it go by. Seize the moment.”. If a mentoring relationship was not successful, male residents took steps to identify a new mentor: “They set us up with a faculty advisor, but he was an anesthesiologist. I'm like, that's great, but how about some surgeon friend. He gave me three names and I went and met this guy.” . Male residents surrounded themselves with a “pool of eligible mentors,” and then chose among those one who would be most helpful for their career focus. Similarly, they worked to maintain the relationship once it was established by combining mentoring with other activities: “So it's good if you can do two things at the same time, you know. That can help deal with the restraint of time.”.Men reported strategies indicating they took initiative in developing mentoring relationships and actively sought out mentors: “They will offer sometimes too, which is really nice because then you know they're approachable because they approached you.”. A few of the female residents were willing to approach a mentor if they identified one with similar career goals: “So I kind of got to know one of the gastroenterologists and he's kind of been mentoring me a little bit along the way.” .In contrast, women described more passive approaches to finding a mentor. Women worried about being a “bother” to faculty mentors, and seemed concerned that faculty may not be interested in, or willing, to help them. Most were reluctant to initiate contact with mentors, preferring instead that the mentor show interest first: “I have never gone to someone and said, ‘Do you want to be my mentor?” . Even when women recognized the need for career advice from a mentor, many were hesitant to approach one: “It sort of felt like it was up to me to really find someone that was more in tune to what I want to do. [interviewer: And have you done that?] Not really yet.” . Female residents expected mentors to continue to “prove” their interest once the relationship had developed. Mentors should be the ones to arrange meetings, initiate communication, and follow-up with the resident: “I think it's important for a mentor to be the one to say ‘let's get together’.” .For many female residents, mentoring relationships that developed usually resulted from chance circumstances, such as being paired with an interested faculty member on a clinical rotation. In those situations, female residents were more comfortable letting the relationship evolve, rather than formally asking for mentoring: “In this qualitative pilot study, we found that residents face a variety of challenges in forming mentoring relationships. Although these barriers were perceived equally by male and female residents, there were gender-specific differences in residents’ methods for overcoming these barriers. As compared to male residents, we found that female residents reported fewer strategies for finding a mentor and were more passive in their approach to forming mentoring relationships.Studies of female residents have demonstrated that they are less likely to identify mentors, and receive mentoring, compared to their male colleagues. 181920172117Women in our study were reluctant to seek out faculty, and assumed that mentors would approach them. This may in part be due to socialized behaviors, in which women are taught to assume more traditionally passive roles in initiating relationships.22Many residency programs have recognized the challenges trainees face in finding mentors, and have developed assigned mentoring programs.3Our study has limitations that deserve mention. This descriptive study used qualitative methods and purposive sampling, and its results cannot be generalized to all residents. Due to funding constraints, we were also limited to two focus groups. We also recognize that thematic saturation may not have been achieved with two focus groups. Although we corroborated all emerging themes through member-checking and expert review, a larger number of focus groups might provide additional information. Despite including residents from a variety of specialties and training levels, our study design and size limited any comparisons that we could make based on these characteristics. Finally, we did not explore faculty mentors’ views about resident mentoring. Further study is needed to investigate any perceived gender differences in mentoring from the perspectives of faculty mentors.The information obtained in this study has important implications. Training programs should acknowledge the importance of resident mentoring and create an environment that prioritizes it for both residents and faculty. Special attention should be paid to the needs of female residents, who may need guidance to successfully find or use mentors. Women residents should be educated about the importance of mentorship, given various strategies to initiate contact with potential mentors, and encouraged to actively seek and maintain their mentoring relationships. Potential mentors should recognize that they may need to approach women and offer mentorship. Additionally, programs may need to consider creative approaches to ensuring mentoring of residents. One possibility is to combine an assigned mentor/selected mentor approach by providing an assigned mentor for the first year with the expectation that this mentor assist the resident in identifying and establishing a relationship with a mentor more appropriate to the resident's interests and career goals. This mixed approach may be potentially useful in assisting female residents find good mentors. Further research is needed to obtain a richer understanding of gender differences in residents’ mentoring relationships; knowledge gained from such studies could inform the development of residency mentoring programs specifically tailored to meet all residents’ needs.