We report a theoretical study on the cyclic stretch-induced reorientation of spindle-shaped cells. Specifically, by taking into account the evolution of sub-cellular structures like the contractile stress fibers and adhesive receptor-ligand clusters, we develop a mechanochemical model to describe the dynamics of cell realignment in response to cyclically stretched substrates. Our main hypothesis is that cells tend to orient in the direction where the formation of stress fibers is energetically most favorable. We show that, when subjected to cyclic stretch, the final alignment of cells reflects the competition between the elevated force within stress fibers that accelerates their disassembly and the disruption of cell-substrate adhesion as well, and an effectively increased substrate rigidity that promotes more stable focal adhesions. Our model predictions are consistent with various observations like the substrate rigidity dependent formation of stable adhesions and the stretching frequency, as well as stretching amplitude, dependence of cell realignment. This theory also provides a simple explanation on the regulation of protein Rho in the formation of stretch-induced stress fibers in cells. There exists mounting evidence that biological cells have the remarkable ability to sense and react to mechanical cues, although the exact nature of the underlying mechanisms is still largely unknown. For example, it has been shown that strong cell adhesion on extracellular matrix (ECM) cannot be formed when the matrix is softer than a threshold value Given the fact that many organs and tissues, such as heart and artery wall, are subjected to cyclic deformation in physiological conditions, intensive efforts have been spent to investigate why and how cells respond to cyclic stretch, in hopes of shedding light on how processes like angiogenesis take place, as well as finding ways to control or cure various diseases associated with blood vessels and heart in the future. Indeed, several intriguing observations on how cell reorientation is tightly regulated by stretching frequency and amplitude have been reported The striking similarity of various cell types responding to cyclically stretched substrates seems to support the hypothesis that cell realignment shares a common physical mechanism. Theoretically, Wang Despite all these efforts, several fundamental questions remain unsettled. For example, as evident from the above discussions, the issues of cyclic stretch-induced cell reorientation and how substrate rigidity regulates the behaviors of cell-substrate adhesion were often treated separately in existing studies. Moreover, a limitation of many aforementioned models concerning cell alignment is that no remodeling or evolution of sub-cellular structures has been taken into account. For example, cell reorientation is explained by invoking an effective free-energy, regulated by substrate rigidity, to reflect cells' tendency to achieve “tensional homeostasis” Aiming to address these issues, we have conducted a theoretical study on how and why cells reorient themselves in response to cyclic stretch. Specifically, we develop a coupled mechanochemical model to simulate the temporal response of cell realignment over cyclically stretched substrates, accounting for the dynamic evolutions of both stress fibers and adhesion clusters. The main hypothesis here is that cells tend to orient in the direction where the maximum density of stress fibers, and hence the strongest cell-substrate attachment, can be achieved. We show that, when subjected to a waveform stretch, the final alignment of cells represents a competition between the elevated force within stress fibers that leads to their disassembly, as well as the disruption of cell-substrate adhesion, and the stretch-induced stiffening of the elastomeric substrate which promotes more stable focal adhesions. Our model is capable of explaining a variety of observations like the dependence of cell realignment on stretching frequency, stretching amplitude and Rho regulation.Consider a spindle-shaped cell adhered to an elastomeric membrane functionally coated with extracellular matrix molecules. The membrane is subjected to a cyclic tensile strain he poles . These FAs mentioned earlier, cell realignment involves nucleation and development of both FAs and SFs . Here, wG represents the energy reduction  associated with the formation of a single ligand-receptor bond, G is defined as energy reduction. K is the effective spring constant of the bond-substrate system and Treating filament , with  bK. In other words, the bond is expected to displace by a distance F is applied (K can be expressed asNote that the combined stiffness of the bond-substrate system is represented by the effective spring constant  applied . A simplIt is well-known that forces can be generated within SFs due to the activities of associated myosin motor proteins. In addition, forces can also arise from the fact that SFs respond viscoelastically when subjected to external stretching For those cells in some orientations that cannot successfully develop long-term stability in FAs and SFs, they will lose contact with the substrate after a short while and then undergo rotational diffusion at random to explore other orientations where formation of new focal adhesions and polymerization of new stress fibers are possible. We assume that 2/s. In an alternative description of equivalence, a cell will hop by an angleIn modeling rotational diffusion at random of whole-cell, the mean-square angular deviation in elapsed time We proceed by normalizing the physical parameters as c and d are all estimated to be of the order of 0.1, following the observations that the time for the disassembly/elongation of SFs is of the order of minutes The time needed for the formation of single receptor-ligand bonds, characterized by the bond association rate a, dimensionless energy in units of The energy reduction by forming a single bond is around 5–10 a and Let's first neglect any externally applied stretch, correspondingly By choosing initial conditions as When subjected to a cyclic stretch 10% in amplitude and 1 Hz in frequency, or tion at  . Howeverrt while . Basicalrt while , which eIf we denote Alternatively, when the stretching frequency is fixed at 1 Hz, We have also carried out calculations for the case of substrates with linear response, i.e. constant substrate modulus irrespective of stretch amplitude, which are compared to the aforementioned results where strain stiffening is present (lignment , which ilignment , the twos linear . Hence, Jungbauer et al. conducted the first detailed experimental investigation of the temporal reorientation of multiple cells in response to cyclic substrate stretch of various amplitude and frequency The characteristic time for the dynamic reorientation is frequency-dependent and is within a range from 1 to 5 hours, indicated by real-time track of the order parameter;For dependence of stretching amplitude, the characteristic time of cell reorientation increases linearly with reducing amplitude, and no apparent cell reorientation is observed when the amplitude of stretching is reduced to below ∼1–2%;The orientation change of multiple cells occurs faster at higher frequencies, and a threshold frequency, ∼0.01 Hz for rat embryonic fibroblasts and ∼0.1 Hz for human dermal fibroblasts, is found below which no significant cell reorientation occurs;More interestingly, a biphasic relation is found between the characteristic time of cell reorientation and stretching frequency for both cell types, with a generic threshold frequency ∼1 Hz separating the two phases.To quantitatively compare our model to the experimental results, the order parameterThe characteristic time for cell reorientation to occur is largely determined by the parameter Cell reorientation is almost unnoticeable when the amplitude of stretching is reduced to below 2%, referring to Cell reorientation is found to occur faster but the steady state value of the order parameter is smaller in magnitude at a lower stretching frequency of 0.2 Hz , consistS decay is 26,000 for 1.0 Hz and 19,000 for 0.2 Hz, differing by ∼40% but not as significant as the observation of the biphasic dependence of time required to achieve steady state as a function of frequency Fitted There have been studies focusing on the effects of transverse deformation in substrates on cell reorientation when the substrates are stretched in the axial direction ormation , with th present .c that appears in As pointed out earlier, it has been shown that inhibition of Rho or its effector proteins almost completely blocks the formation of SFs. However, a 10% cyclic stretch at 1 Hz causes the reappearance of SFs more along the stretching direction, having much lower SF density compared to that in normal cells We must point out that the treatment adopted here to represent the effect of Rho inhibition is extremely simple given the complexity of cell biology. Essentially, what we have demonstrated is that by adjusting one single parameter according to well-known unique features of Rho-inhibited cells in comparison to normal ones, our model is capable of explaining how and why these cells respond to cyclic stretch in certain manners, as observed in experiments In this paper, we have reported a detailed quantitative examination on how and why various cell types reorient in response to cyclically stretched substrates. Specifically, by taking into account the dynamic evolutions of sub-cellular structures such as stress fibers and receptor-ligand bond clusters, a mechanochemical modeling framework has been developed to predict the preferred alignment of cells under stretch. Our main hypothesis is that cells tend to orient in the direction where the maximum density of stress fibers, and hence the strongest cell-substrate attachment, can be achieved. We show that, for a cell-substrate system subjected to cyclic stretch, the final alignment of cells represents the competitive coupling between stress fiber assembly/disassembly, focal adhesion growth/disruption, substrate stiffening and whole-cell rotation. Our model is capable of explaining a broad range of observations like the absence of stable FAs or SFs on sufficiently soft substrates as well as the stretching frequency and amplitude dependent realignment of cells. In addition, this theory provides a possible explanation on the stretch-induced reappearance of SFs in Rho-inhibited cells.Several important aspects of the problem have been neglected in the present study, which certainly warrant future investigation. For one thing, the role of myosin motors is represented by the generation of a constant force in the contracting filaments in contact with SFs' viscoelastic response, which certainly might be an oversimplification of the real system of filament-myosin architecture. A more realistic formulation should explicitly take into account the activities of filament-myosin assembly during the time-varying stretch as well as its implications on the evolution of SFs. For another, one of our hypotheses is that cells tend to orient in the direction where the maximum densities of ligand-receptor bond and actin contracting filament are achieved. In other words, our results reflect this preferred criterion of final cell alignment but have done little to address the question of how cells actually conform themselves to achieve so, which is certainly an important and interesting task to pursue in the future. In addition, the diffusion and possible self-aggregation of adhesion molecules in FAs have been neglected in our analysis. However, it is commonly believed that the recruiting and clustering processes of these proteins, such as integrins, are important for the formation of stable focal adhesions.Despite all the limitations mentioned above, we feel that our simple model successfully establishes a connection between the evolutions of sub-cellular structures, like adhesion clusters and stress fibers, and various mechanical factors such as substrate rigidity, SF viscoelasticity and imposed stretch. The validity of the key assumptions made in our formulation, like the viscoelastic response of SFs and the strain stiffening of polymeric substrates, has been well confirmed by existing studies. The fact that the predictions from our model compare favorably to a variety of experimental observations suggests that the main physics of the problem under investigation may have been captured by this formulation, which we believe can serve as a theoretical framework to motivate future studies on cell reorientation, where further verification of the main mechanisms and more realistic features can be added.


Subperiosteal hematoma of the orbit is one of the rare lesions that cause exophthalmos after craniomaxillofacial trauma. Presently, there is no consensus for how to treat this disease. Although some reports have suggested a conservative type of therapy, others have recommended surgical treatments be done during the early stages.This case report provides details on the clinical course of a 9-year-old girl with subperiosteal hematoma of the orbit. In this particular patient, a rare case of ipsilateral subfrontal extradural hematoma was also observed. Due to our performing the surgical intervention during the subacute stage, functional complications as well as cosmetic problems were avoided.Our results demonstrate that surgical treatments for subperiosteal hematoma of the orbit should be delayed until it can be confirmed that a patient has no other complications. On the other hand, once it has been confirmed that the patient has no other existing problems, immediate surgical therapy with a small skin incision followed by the setting of a drain is recommended in order to achieve an early resolution and avoid complications. The differential diagnosis of proptosis after craniomaxillofacial trauma includes orbital fracture, cavernous fistula, orbital emphysema, retrobulbar hematoma and orbital abscess. Subperiosteal hemangioma of the orbit, which may cause proptosis, is a rare injury. In some cases, the lesion shows diplopia, malposition of the eye, ophthalmoplegia and blepharohematoma. In the literature, there are both reports that suggest using a wait-and-see treatment with the expectation that natural extinction will occur, while others suggest that an early recovery can be attained by employing surgical therapy during the early stages.In this report, we present the case of a 9-year-old girl with subperiosteal hematoma of the right orbit who also exhibited extradural hematoma in the frontal cranial fossa. The symptoms observed included diplopia, proptosis of the right eye and hypoophthalmia. After the patient underwent surgical treatment during the subacute stage, all of her symptoms disappeared and she completely recovered.A 9-year-old girl fell from her school’s morning assembly platform and knocked her head against the ground. Although she did not lose consciousness, she immediately noticed diplopia with right proptosis. She was examined on the same day at the Department of Ophthalmology at a nearby hospital, and underwent computed tomography (CT) of her head. After noting she had a subfrontal extradural hematoma on the right side with an ipsilateral subperiosteal hematoma in her orbit, she was admitted to the Department of Neurosurgery for further observation.The patient was discharged 3 days after her admission, as follow-up CT indicated that neither the extradural hematoma nor the subperiosteal hematoma worsened during this time. However, since the patient showed no improvement of her right exophthalmos with diplopia, she was brought to our institution 9 days after the initial injury.At the time of her first visit to our department, her right side upper and lower palpebrae were still edematous, with non-pulsatile proptosis Figure . HorizonAlthough plain roentgenograms indicated there was no sign of fracture, CT imaging found a right subfrontal extradural hematoma and exophthalmos of her right eye, with a low-density area also noted in the ipsilateral orbit Figure . MagnetiAlthough the patient’s exophthalmos, disturbance of upward gaze and diplopia did not improve, there were also no changes in her central nervous system symptoms. After a neurosurgeon examined the patient, surgical treatment was prescribed and performed 14 days after her injury. An incision line was made just above the upper lateral border of her right eyebrow. After incision of the periosteum on the upper border of the orbit, the subperiosteal space was dissected towards the apex of the orbital cone Figure . There wDue to continuation of the clear red discharge that was observed during irrigation of the area, a drain was placed within the subperiosteal space at the end of the procedure. After the surgery, there was improvement of both the exophthalmos and the malposition of the right ocular globe, which was in line with the lack of disturbance noted for the forced duction test.A Hess coordimetry chart was used to record the patient’s condition during a 4-week period after the operation. During this time, the patient showed improvement of her diplopia  trauma, 2) vascular lesion, 3) hematological disease, and 4) idiopathic [ trauma,  vascular hematolo idiopathThe common symptoms of subperiosteal hematoma in the orbit during the acute phase are exophthalmos, dislocation of the ocular globe, disturbance of the eye movement and diplopia, along with an occasional association with pain ,6. The pOn the other hand, subperiosteal hematomas during the chronic phase have been shown to cause irreversible changes involving organization, calcification, pseudoaneurysms, or blood cysts. Therefore, all of the changes could potentially lead to exophthalmos, intraocular high pressure, corneal injury or visual acuity deficits ,8.The differential diagnosis of the subperiosteal hematoma of the orbit includes fracture of the orbit, internal jugular-cavernous sinus fistula and emphysema of the orbit, all of which can cause posttraumatic exophthalmos and disturbance of the eye movement. In order to make a diagnosis of subperiosteal hematoma in the orbit, it is recommended that CT or MRI be used to confirm images of distended hematoma attached to the wall of the orbit ,9,10.Many published studies in the literature have recommended that surgical treatments be performed for subperiosteal hematoma in the orbit, with some reporting that these treatments have resulted in a rapid recovery without sequelae . In contIn the current case, neither the proptosis, the disturbance of the ocular movement, nor the diplopia showed any improvement at 10 days after the injury. Therefore, after considering the patient’s age, the current operation was performed in an attempt to avoid any possible sequelae . HoweverAlthough in this case the CT images did not indicate any signs of expansion in the subperiosteal hematoma in the orbit, we observed fresh bleeding in the backflow while irrigating the area during the surgical procedure. However, is quite likely that simply aspirating the area could have lead to this recurrence of the hematoma, as a small amount of bleeding was found around the drain on the day after the operation. Our current findings support performing surgical removal of hematomas through a small skin incision followed by the setting of a drain during the subacute stage in cases where early complications have prevented surgical treatment of subperiosteal hematomas in the orbit of younger patients. This strategy is both simple and safe, and provides a solution for patients` early complaints without the occurrence of late sequelae.We report on the clinical course of a very rare case of subperiosteal hematoma with ipsilateral frontal epidural hematoma. After ensuring there were no complications in the patient’s general status, we performed surgical treatment during the subacute phase, and the subsequent prognosis has been good. Therefore, in cases of subperiosteal hematoma in the orbit, once it has been confirmed that the patient has no other existing problems, immediate surgical therapy with a small skin incision followed by the setting of a drain is recommended in order to achieve an early resolution and avoid complications.Written informed consent was obtained from the patient and her parent for publication of this case report and all of the accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.CT, computed tomography; MRI, magnetic resonance imaging.The authors declare that they have no competing interests.TM and MK have made substantial contributions to the acquisition of the clinical data and analysis of the previous reports in the literature. TM and JM have made decisions regarding the patient and were responsible for all treatments performed in this case. YY and KY were involved in drafting this manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2415/12/18/prepubFigure S1. "Look-up" position at the first visit and after 2 years of treatment. Proptosis of the right eye is observed at the first visit (A). Two years after the initial treatment, proptosis of the right eye has improved (B).Click here for fileFigure S2. Hess coordimetry. Hess coordimetry corresponds to the movement of the right eye during the patient’s first visit (A). Clinical findings also show the disturbance of the upward gaze of the right eye  shows a low intensity area in the right upper orbit (A). A T2-weighted image (T2WI) shows a high intensity area in the same portion. A high intensity area is found in the frontal cranial space, which suggests subfrontal extradural hematoma (B).Click here for fileFigure S5. Follow-up CT images. These images show there is no remnant hematoma in the right orbit. The white line in this slice corresponds to the drain (A). There is neither a sign of recurrence of the hematoma nor any deviation of the eye noted in these CT images that were taken 6 months after the surgery (B).Click here for fileFigure S6. Eye movement observed at 2 years after the initial surgical treatment. These photos show no sign of eye movement disturbance, which is compatible with the Hess coordimetry results (Additional file Click here for file


An accurate diagnosis of melanomas at an early stage correlates directly with a better prognosis. However the incidence of melanoma is still increasing along with the number of related deaths. Melanoma cells grow extremely fast, with the result that many patients present after metastasis has occurred, too late for effective treatment. This paper describes the changes in the fibre diffraction patterns of skin that indicate the presence of a melanoma. Identification of these changes would provide an alternative early low-cost, reliable diagnostic test which could be conducted on a regular basis in local radiology facilities using rotating anode X-ray generators or as a mass screening test using suitable small angle x-ray beam-lines at synchrotrons. Melanomas are the most potentially lethal of all the skin cancers. Although major changes have been made in the surgical procedures and in the therapy for melanoma, the incidence of this skin cancer is still increasing. These changes eliminate the need for grafts thereby greatly reducing hospital and recovery time. However they do not appear to improve the survival rate ,2. DiagnThis use of x-rays is completely different from the familiar standard shadow patterns from routine medical examinations where the full wavelength spectrum of hard x-rays [energy range 20 to 120 keV (0.10 to 0.010 nm wavelength)] are shot through the body area of concern, hence the accepted metonymical use of the word x-ray to refer to a radiographic image so produced. Because bones absorb more energy than the soft tissue, the latent image on the film shows white areas for the bones, dark areas for the less dense tissue, but the pattern is clearly identifiable to the viewer.In contrast, x-ray diffraction patterns bear no visual resemblance to the sample. Such patterns are produced by the scattering of a finely focussed monochromatic x-ray beam from the electron density distribution within the samples. Hard x-ray wavelengths are approximately the same length as the distances between atoms, (carbon-carbon and carbon-oxygen bond-lengths are ~0.15 nm), a requirement for the production of diffraction patterns, and are therefore ideal for diffraction studies of materials. The specific wavelength for the monochromatic beam produced by a rotating anode is determined by the material composition of the anode. Synchrotron x-radiation has many advantages such as vastly increased flux and brilliance, and the choice of a specific wavelength for an experiment, the latter is simply selected using a monochromator. Time for exposures on a synchrotron is greatly reduced from those required when using rotating anodes (days/hours to minutes/seconds) as a result of the increased flux.Using such a beam the molecular structure of any crystalline material can be determined. The atoms in a crystalline material are arranged in regular lattices. For such crystals, the interference/diffraction patterns obtained are made up of a series of spots, as the crystal acts like a 3-D diffraction grating. The positions and the intensities of these spots can be measured and then using standard computational procedures the molecular structure can be determined. Since the atoms of any given crystalline substance are arranged in a pattern specific to that substance, any crystal of a particular substance will always give the same diffraction pattern, regardless of the location or X-ray source used. These comments apply equally to any crystals. The quality of the pattern will vary only with the quality of the beam and the selection and loading of the crystallised sample. Any change in the chemical composition of the substance, even the addition or subtraction of one atom, will cause a change in the pattern observed as the molecular structure is altered.et al. [Similar comments apply to natural pseudo-crystalline materials. A comparison of the excellent pattern obtained by Fraser and McCrae  using a et al.  using a et al. ,6, or hoet al. .Many reports of the changes in x-ray diffraction patterns of human collagenous tissue which correlate with diseases of the particular tissues have been reported over the last 40 years. These include changes in tendon with insulin dependent diabetes , changesIn order to obtain diffraction patterns of any tissue, the molecules must be arranged in an ordered parallel array, as for example the arrangement of collagen in tendon or keratin in hair. Good patterns from other tissues can be obtained by careful selection of the sample, such as the collagen aligned along the ducts in the breast. For better patterns of skin therefore, only the collagen plaques from the dermal layer are used, the epithelial and epidermal layers are scraped off as all such amorphous tissue contributes only to noise.The collagen plaques in the dermal layer of the skin are made up of a two dimensional crimped arrangement of heterogeneous helices containing collagen I, collagen III, and collagen V. When stretched these molecules preferentially align in the direction of the stretch. This alignment is never complete and therefore the meridional reflections are not as discrete as those of tendon but are smeared into arcs. The strong inner peaks become circles, shown in The specific changes that appear in skin for patients with breast cancer or prostate cancer ,14 appeaThese changes only indicate the presence of the particular carcinoma. The actual location must then be determined by routine procedures. However, since the changes are specific to the different carcinomas, the type of carcinoma is clearly defined for each sample. No alternative external easily harvested tissue has been found for the diagnosis of melanoma . Whilst A full description of the normal pattern for skin collagen ,19 and tTo determine the margin of this change in relation to the site of the tumour, a set of blinded samples supplied for our study included two samples of skin from each patient. The first was taken from the area adjacent to the tumour. The second was taken from an area remote from the tumour, outside the accepted excision margin. The results of this study are given in To verify this result, a sector was cut from the circular section of tissue (radius 4 cm) removed with a melanoma, with the point nearest the tumour. Studies were made at 2 mm intervals along three radii of this segment. Although these results are based on very few samples, the findings here are consistent with the results obtained with other cancers where changes in skin have been consistently identified in areas remote from the cancer site . If thisOur studies have also confirmed that if the patient has more than one type of cancer, the specific changes associated with each type will be present in the final diffraction pattern. An example of this is given in A total of 52 skin samples taken from melanoma patients were included in the 296 blinded skin samples that have been examined in this ongoing research project. The age range for these samples was between 18 and 90. These have included 211 controls. Some of these skin samples were obtained from routine surgery or from necropsies and these were typically small sections, 1mmx5mm approximately. Alternatively, 3 mm full punch biopsies of skin were collected with the consent of the patient. Ethics approval for all experiments and procedures has been granted at all collection points. Immediately after excision, the samples were placed in physiological saline and stored at −20 °C until required. Before mounting in the cells, which have been specifically designed to maintain 100% humidity throughout the experiment, the skin samples were gently scraped to remove the epithelial and epidermal layers to expose the dermal layer, at all time maintaining 100% humidity by keeping the sample submerged in the buffer. Small sections are removed from the collagen plaques and sutures are attached to both ends to assist with mounting to the experimental cells, Fig 6. These cells have the capability to stretch samples slightly to remove the natural crimp in the collagen fibres. The collagen is aligned preferentially in the direction of the applied stretch thus giving rise to the meridional  pattern, The skin samples produced excellent diffraction patterns. All samples yielded patterns which varied only in the equatorial direction over the age range of samples used in this study. Diffraction experiments were conducting using the Rotating Anode X-ray generator at the Rex Vowels Low Angle Diffraction Laboratory, UNSW, and at Rigaku Corporation Japan with sample to detector distances of 200 mm and 1000 mm. Synchrotron studies were carried out on the BL15A Photon Factory, Tsukuba, Japan, at the BioCAT Facility, the Advanced Photon Source (APS), Argonne, USA and at the SAXS-WAXS Beam line at the Australian Synchrotron, Melbourne. Sample to detector distances at the synchrotrons varied between 400 mm and 1300 mm at the various facilities. Calibration was achieved by comparison with diffraction patterns from moist rat-tail tendon or by comparison with a silver behenate sample (first order spacing: 5.838 nm) at the synchrotrons. The synchrotron fibre diffraction intensity distributions were recorded on FUJI BASIII imaging plates at the Photon Factory and at BioCAT and the data was extracted by electronic scan. A MAR165 detector was used on-line on BioCAT and at the Australian Synchrotron to aid in the alignment of the sample and also for data collection. Initial analyses were carried out using a combination of MATLAB-R-2007 and ProcessFITS or SAX15ID. This was followed by a high precision analysis of the two dimensional data using the astronomy packages IRAF (1986) and SAO Image (1991). These Smithsonian packages have been designed for viewing stars over a wide range of intensities from the very weak to the very strong by continually limiting the intensity range under observation. They are, therefore, perfectly designed for handling the wide dynamic range of intensities obtained from the imaging plates and the MAR165. In addition, intensity/position plots (implots) can be displayed and printed out along any designated line. This is especially important as changes associated with Grade 1 tumours can be extremely weak and not apparent using other computer programs. Depending on the quality of the data, different methods available in both IRAF and MATLAB were used in the analysis. These included curve fitting which was extended to second order derivative plots to locate ill defined or weak peaks [Our results to date indicate that diffraction studies of skin could provide a diagnostic test for melanoma. Only one test is required whether one or more types of cancer are present as the specific change for each cancer present will be identifiable on the x-ray. As an added advantage all such skin diagnostic tests could be routinely studied at a reasonable time-rate and personnel cost in local radiology units, thus removing the need for transportation to remote facilities. The specimens can be harvested at local facilities, since samples do not deteriorate with correct storage. Tests are continuing but at this stage insufficient samples have been studied to obtain statistically reliable specificities and sensitivities but our results would indicate that such tests would provide early, low-cost, yet reliable tests using this easily accessible biological tissue.


Autophagy is a housekeeping survival mechanism with a protective function against stress conditions. However, when stress severity or duration increases, it may promote cell death. Paradoxically, autophagy favors cancer development, since cancer cells could enhance their proliferation potential (thus becoming able to resist anticancer therapy) thanks to the energetic supply provided by organelle degradation typically driven by autophagy following a stepwise pathway. The main actors of the autophagic machinery as well as the features shared with apoptosis will be described. Special attention will be paid to the effects of autophagy manipulation. On the whole, the knowledge of the molecular bases of autophagy has encouraged many attempts to modulate autophagy in order to identify new tools for elaborating an efficient action plan against cancer. In fact, the existence of more than 20 ongoing clinical trials based on inhibition/stimulation of autophagy  supportsAs a further matter of debate, although the development of autophagy-based anticancer strategies is promising, this approach has to be carefully examined with respect to undesirable effects on non-cancer cells . On the The aim of this review is to delineate the impact of the so complex and amazing autophagic process on cancer cell survival. In this respect, the investigation of the role played by autophagy in the complex network of cell death(s) as well as the in-depth examination of its intricate connection with apoptosis, could help in understanding when and how autophagy switches from a survival to a death function . Taking


AbstractAgonum viridicupreum due to climate change in the western Palaearctic. Species’ records were obtained from extensive literature sources as well as from collections, and consistent amateur entomologists’ recordings. Within the general geographic range of the species, we analyzed in detail two parts of both, the northern and southern distribution range boundaries: (1 and 2) north-western Germany (leading or high-latitude edge), (3) Israel and (4) southern Italy (rear or low-latitude edge). Temporal changes in the occurrence data of the species indicated a northward shift of the leading edge of a minimum of 100 km within the last 50 to 100 years. In contrast, according to the data gathered, the rear edge has not changed during the last decades. Further studies are PageBreakneeded in order to fully understand the underlying mechanisms of the different behaviour of leading and rear range edges of Agonum viridicupreum in the current context of global change. Despite our incomplete understanding, chronosequences of the occurrence of the given species have the potential to optimize climate niche modelling to predict trends in the distribution range in the future.We investigated the extent of poleward shifts in the distribution range of  For about 250 years, man has released radiatively active gases and particles in substantial amounts into the atmosphere. As one of the consequences, the global mean near surface temperature has increased, a phenomenon commonly referred to as ‘global warming’ or ‘climate change’. Deduced mainly from instrumental observations initiated around 1860, the observed climate change can be attributed to a large extent to human activities, which influence not only global temperature, but also pH-values of the oceans, precipitation and the general hydrological cycles on Earth .For many animal and plant species, theoretical analyses on the climate determination of the species’ occurrence have predicted a general poleward shift and (in mountain areas) an uphill shift of the given distribution areas and populations, respectively, as a response to climate change. In agreement with theory, numerous range shifts have been documented in the last years. Examples are known from vascular plants, birds, and many insects such as butterflies, dragonflies and damselflies . CarabidStenolophus mixtus in Scandinavia , as their patterns of geographic distribution are strongly determined by climatic factors  edges of their distribution areas (literature cited above). Changes occurring at the leading edge are interesting, especially in the framework of dispersal biology, and they enable us to understand many population biological processes ; 1989. IAgonum viridicupreum as our study object because it fulfils many preconditions of a suitable model species to assess potential margin shifts: The specimens can be easily found in the field, they are fully winged and fly actively, and the species’ habitat preferences are well-known. Furthermore, the specimens are nicely coloured, stimulating many amateur entomologists to record the species, and, therefore, allowing suitable faunistic data from large parts of its distribution area. Moreover, the species is not restricted to habitats that are influenced or even destroyed by other drivers of global change, nor have been altered simultaneously by the temperature increase in the last decades  is a macropterous and thermophilous species restricted to open, wet habitats such as meadows, fens and rain ponds. The day-active beetle prefers sun-exposed muddy sites where it can be easily detected by its green-bronze-coloured surface. Due to its occurrence in floodplain areas (with high probPageBreakability of diversion), the dispersal of individuals is not only determined by the species’ ability to fly. Specimens can be transported downstream by flooding events into areas where the species might not be able to establish autochthonous populations , the beetle lives in wet habitats, mostly close to winter or rain ponds . In southern Italy  the species lives in river bank habitats around Agonum fulgidicolle Erichson, 1841, an allopatric sibling taxon of Agonum viridicupreum  to determine the general distribution area of the study species  and of AThe situation of faunistic recordings is sufficient for one region at the northern distribution edge (north-western Germany) and for two regions at the southern distribution edge .– North-western Germany has been studied by numerous amateur entomologists who have greatly contributed to our knowledge on the geographic distribution of carabid beetles. We therefore analyzed the changes in the species’ distribution separately for (a) West Lower Saxony (west of river Weser) and for (b) East Lower Saxony (east of river Weser), Hamburg, and Schleswig Holstein. For these regions records from three periods  were summed up to document tendencies in the numbers of catches.– For Israel, the first records date from the 1920s . We therefore distinguished only two periods of collecting: before 1980 and after 1980.PageBreak– For southern Italy  there are scarce historical records (before 1980). However, after 1980, intensive ecological surveys were carried out on populations in several sites of the Crati river valley .Agonum fulgidicolle ). For these areas we compiled numerous faunistic records mostly published in local journals  a part of the leading edge , and (3 and 4) the only continental areas at the rear edge that are not limited by the sea or by the presence of journals . At the We surveyed climatological literature and compiled information about recent climate changes in the three regions north-western Germany, Israel and southern Italy . We focussed only on changes in temperature and precipitation, the main factors influencing the ground beetles’ biology and distribution.North-West Germany – warmer springs with wetter winters and drier summerstemperature by about 0.8°C has been observed. Also for western Europe the measurements show a warming trend. For Germany during the 20th century a mean temperature rise of about 1.0°C PageBreakwas reported by Over the last 150 years a considerable increase of the global mean precipitation trends for Germany have been reported to be about 8.5%  multidecadal periods (30 winters in a row) were from 1951 to 1980 (1973 to 2002) with 5 mm (-15 mm) departures from the climatological average (1960 to 1990). Interestingly, in the overall wettest winters Calabria was drier than the climatological mean (10 to 20 mm) and in the overall driest winter series Calabria was about 10 to 20 mm wetter than the 61 to 90 average.Overall it can be said that the most southern part of Italy and especially Calabria has become drier over the last decades. While the linear trend in annual bout 40% . A compabout 40% . LuterbaAgonum viridicupreum occurs around the Mediterranean Sea (with a distribution gap in north-eastern Africa). The northern edge of the distribution area runs from the Netherlands through northern Germany and Poland , and reaches also the north-western parts of the considered area. Interpreting the old records from the floodplain area of the river Elbe as autochthonous populations leads us to think that the species’ geographic range has experienced a northward shift of about 100 km during the last century. Even if these records were not seen as autochthonous populations, the shift would have spanned over about 200 km .Agonum viridicupreum from Israel for the first time, and the former documented records were taken in the 1920s (TAU). In this region, the beetle is abundant at many rain or winter ponds . So, it is very likely that the late discovery of the species at its south-eastern distribution edge would be a consequence of the poor carabidological exploration of the country. Since Bodenheimer’s time, numerous new records of the species have been reported, also during the last years  there are scarce historical records . Also in the eastern part of Lower Saxony the species has spread northwards a minimum distance of 100 km and it reaches the centre of Schleswig-Holstein today. These results allow the assumption that a further temperature rise will make the species’ occurrence in Denmark highly probable in the near future.Today, in the western part of Lower Saxony, Agonum viridicupreum has not changed within the last decades. Indeed, there are still populations with numerous individuals south of Tel Aviv, which is close to the southern limit of the PageBreakMediterranean climate. Consequently, in Israel we expect the beetle to occur in most of the regions characterized by Mediterranean climate. In southern Italy  Agonum viridicupreum shows a stable rear edge north of the 39th parallel, with permanent populations in the Crati Valley in the Cosenza province, in areas marked by Submediterranean or Mediterranean climate.Unlike the northern distribution edge, the southern range margin (rear edge) of intrinsic factors would imply that distinct genetic make-ups of the populations from the opposite edges of the distribution range are likely  do not support the assumption of the species declining in the Levant.In accordance with numerous other authors e.g. , we intee likely . Howeverrear one  – a possextrinsic factors may be acting differently at the leading and rear edge of the distribution range of Agonum viridicupreum. Our compilation of recent climate trends, however, reveals generally rising temperatures in all regions under study. In contrast, less consistent changes in precipitation can be observed. The populations in both, Calabria and the Levant will have to deal with a reduction in mean annual precipitation. In the face of the virtual exclusive occurrence of the species close to rain ponds in the southern edge of its distribution, it seems likely that the southern populations are limited by the given ground water tables which predominantly result from the annual amount of precipitation (mainly in the winter months).On the contrary, PageBreakIn the Levant, larval development takes place during winter and early spring, as revealed by numerous tenerals, even at higher altitudes, e.g. 900 m a.s.l., in the Golan Heights, in April and May . In contrast, the northern populations in Central Europe are unlikely to be limited by the amount of precipitation, but rather by temperatures during the species’ activity period. In fact, in this region, larval development takes place during summer and tenerals occur in late summer and autumn  scientific discourse (This study is the first one that investigates simultaneously the possible shifts of the northern and southern margins of a carabid species’ distribution due to climate change. Undoubtedly, at present we are not able to fully understand the underlying mechanisms of the different behaviours of the leading and rear boundaries of the geographic range of iscourse , can be


Many cancers are aneuploid. However, the precise role that chromosomal instability plays in the development of cancer and in the response of tumours to treatment is still hotly debated. Here, to explore this question from a theoretical standpoint we have developed an agent-based model of tissue homeostasis in which to test the likely effects of whole chromosome mis-segregation during cancer development. In stochastic simulations, chromosome mis-segregation events at cell division lead to the generation of a diverse population of aneuploid clones that over time exhibit hyperplastic growth. Significantly, the course of cancer evolution depends on genetic linkage, as the structure of chromosomes lost or gained through mis-segregation events and the level of genetic instability function in tandem to determine the trajectory of cancer evolution. As a result, simulated cancers differ in their level of genetic stability and in their growth rates. We used this system to investigate the consequences of these differences in tumour heterogeneity for anti-cancer therapies based on surgery and anti-mitotic drugs that selectively target proliferating cells. As expected, simulated treatments induce a transient delay in tumour growth, and reveal a significant difference in the efficacy of different therapy regimes in treating genetically stable and unstable tumours. These data support clinical observations in which a poor prognosis is correlated with a high level of chromosome mis-segregation. However, stochastic simulations run in parallel also exhibit a wide range of behaviours, and the response of individual simulations  to anti-cancer therapy prove extremely variable. The model therefore highlights the difficulties of predicting the outcome of a given anti-cancer treatment, even in cases in which it is possible to determine the genotype of the entire set of cells within the developing tumour. Cells with a wide range of structural and numerical defects in chromosomes are found in many types of cancers. Whether these changes contribute directly to the evolution of cancer or are just a by-product of carcinogenesis itself, however, is a question that has puzzled cancer researchers for more than a century. Although there is strong experimental evidence for changes in chromosomal copy number (aneuploidy) and chromosome mis-segregation playing a central role in the way cancer evolves Computational modelling has recently become a practical approach for the study of such emergent behaviours and complex phenomenon To address whether chromosome missegregation plays an important role in the development and progression of a cancer we developed a simple model of tissue homeostasis in which to study cancer evolution. To focus our analysis on this poorly understood phenomenon we chose to disregard other types of mutations . For this, individual cells were modelled, each equipped with a genetically defined genome, as agents in a computational simulation (see Methods). We then represent the tissue as a linear array of individual cells, where daughter cells are spatially introduced adjacent to the mother cell of origin. The simulated tissue initially exhibits homeostatic behaviour, as the result of balanced rates of cell proliferation and cell death. These behaviours were modelled as stochastic processes that are regulated at a genetic level, based upon the properties of known proto-oncogenes and tumour suppressor genes Apoptosis regulatory genes are an abstraction of tumour suppressor genes such as p53cell division regulatory genes provide an abstraction of proto-oncogenes such as Ras Each cell in the system has a simulated genome composed of three kinds of genes. chromosome segregation regulatory genes, which model genes controlling the fidelity of cell division such as BUB1 In addition, the model contains a finite rate of chromosome mis-segregation during cell division, which generates variation amongst the cell population. This level of genetic variation depends on the action of Because the exact role, location and linkage of the key genes regulating cell growth, death and chromosome segregation in real human chromosomes remains unknown The evolutionary dynamics in our model are then determined by the gene expression of the individual cells and the global behaviour that emerges through cell death, proliferation and mis-segregation over time. Focusing on the genotypes that emerge throughout the simulation, we denote the initial state as : corresponding to 2 functional copies of each gene . As expected, the plot of the total number of cells across the simulations of Distribution A revealed increasing variability in the genetic make-up of individual cells over time as the result of chromosome mis-segregation induced genetic drift; similar to that which might be seen in an ageing homeostatic tissue. Although this variation makes the statistical analysis challenging, an invariant behaviour can be observed for each configuration; best visualized by broom plots in For Gene Distribution B, the gradual accumulation of chromosome mis-segregation events leads to a breakdown in homeostatic behaviour, giving rise to uncontrolled proliferation . Once thSimulations of Gene Distribution C displayed over-proliferative behaviour, similar to that of Gene Distribution B . On a clTo test the effects of leaving genes unlinked, a fourth genetic distribution was investigated by modifying the model to accommodate a third chromosome. This system exhibited all three behaviours obtained previously in stochastic simulations: prolonged homeostasis (as in Distribution A), unregulated growth driven by loss of tumour suppressors (Distribution B) or by oncogene activation (Distribution C), We also observed three kinds of chromosome segregation event: up-regulated (Distribution B), down-regulated (Distribution C) and neutral. This control experiment shows how linkage between genes serves to limit the common evolutionary paths exhibited by the system.In patients, tumours composed of cells that are chromosomally unstable have been associated with a poor prognosis Scenario i: Surgical Treatment. The simulation of tumour removal was implemented by retaining the first 100 connected cells in the linked list and removing the rest of the connected component of 900 cells in a single time-step. Since the tumour rapidly emerged from a homeostatic population of 200 cells, the vast majority of these represent cells related to cells in the tumour. Scenario ii: Chemotherapy: To simulate the effects of chemotherapy, we implemented an algorithm that killed all the cells that attempted cell division in the nine consecutive time steps following tumour detection. Scenario iii: Combination Therapy. As in common in the clinic we combined therapies by implementing surgery followed by nine rounds of chemotherapy.Surgery was modelled to mirror the clinical intervention. Thus, it was implemented when the population of cells has broken through the homeostatic limit of 200 cells, and grown to reach 1000 cells. At this point, the population is made up of descendants of many of the cells present in the initial population used to seed the simulation, but is dominated by a small number of related but genetically heterogeneous aggressive cell clones, as in human cancers When we then examined the recovery following therapy, results proved highly variable and depended on the nature of the cells that survived . Though Next we explored the role of genetic linkage in the course of tumour relapse following surgery. For this analysis, as a measure of the types of lesion driving tumour formation and relapse, we compared the ratio of the average number of Apoptosis Genes to the average number of Division Genes in simulations shown in . When thHaving carried out an analysis of the effects of surgery, we next simulated chemotherapy in the model (Scenario ii). Chemotherapy was implemented in consecutive rounds, as done in the clinic using a treatment such as taxanes, to specifically target dividing cells When examining the effect of genetic linkage on the recovery following chemotherapy we found that the relapse time was again faster for cell populations with Gene Distribution C. Thus, Gene Distribution B relapsed on average at 21.95 time steps (std. 4.89), while tumours recurred in Gene Distribution C at an average of 18.30 time steps (std. 3.42), as seen in Finally, a combination of the two therapies (Scenario iii) yielded an overall better prognosis for the two gene distributions than surgery or chemotherapy alone. After this combined therapy there were on average 36.09 (std. 8.56) cells left for Distribution B and 36.29 (std. 7.99) cells for Distribution C. Again, the results indicate that Gene Distribution B still has a significantly better prognosis (p = 0.008) than Gene Distribution C: Gene Distribution B had an average relapse of 46.55 (std. 10.06), while Gene Distribution C had an average relapse of 43.09 (std. 9.44). These results can be compared across scenarios in the form of histograms in Tumours have been recognised as aneuploid for over a century For this purpose, we focused on modelling individual cells and their genomes in a homeostatic tissue whose behaviour is determined by a balance of cell death and cell proliferation within the context of a constrained environment. When events of chromosome mis-segregation are introduced, however, the dynamics of the system change in such a way that new, interesting complex behaviours emerge, which can be used to shed some light on the basic principles of aneuploidy in tumourigenesis. In simulations, chromosome mis-segregation events generate novel genotypes that promote unregulated cellular proliferation and impair cellular death, driving cancer development. Importantly, this analysis also revealed that the location of these genes across chromosomes plays a key role in determining the system’s behaviour and in shaping the genetic structure of the tumour population In our model we observe two distinct pathways for evolution towards oncogenesis that have a direct impact on the tumour’s response to treatments. In the first case, dominant proliferating clones within the tumour exhibit a relatively stable state of aneuploidy driven by the acquisition of genes that ensure the fidelity of chromosome segregation along with division genes that encourage proliferation. In the second, selection for the loss of the chromosome segregation regulatory genes together with the loss of tumour suppressors results in tumours that continually generate increasing levels of heterogeneity and ever-more malignant subclones. This latter pathway exhibited a more rapid expansion, suggesting that chromosomally unstable tumours are inherently more aggressive.In this analysis we also explored the effects of 3 different types of simulated treatment in each case: surgery, chemotherapy and combination therapy. When comparing surgery and chemotherapy, it is important to note that the way in which the two therapies are implemented has important implications for interpreting the relapse data. First, when studying the effects of surgery, it was necessary to compare the genetic make up of cells in the simulation immediately before and after surgery. To do so, we compared the genetic make up of the 1000 cells in the time-step prior to the intervention, with the average of 100 cells in the time step following the intervention. The abrupt change in cell numbers generates a shift in make-up of the population, which leads to a marked increase in apparent population heterogeneity immediately following treatment. By contrast, when the chemotherapy treatment is implemented in a population reaching 1000 cells, the intervention leads to a variable decrease in population size with each time step that depends on the proliferation rate. On average this yielded a 16% decrease in the population size . This caused a smoother transition that depends on the precise impact of the treatment on the population.These differences are reflected in the larger variation across simulations in the response to surgery compared to chemotherapy , which tNevertheless, clear differences in the trajectory of relapse were seen following surgery and chemotherapy. A comparison between the ratio of the average number of Apoptosis Genes to the average number of Division Genes following surgery  and follUnder the chemotherapy scenario, the treatment also leads to an average reduction in the oncogenic load, as the result of the selective killing of dividing cells. This therapy, does not however offer relief from the steady loss of genes regulating apoptosis. As a result, the ratio of apoptosis genes and division genes remained, on average, nearly constant under these conditions, as can be seen by a slight change in the slope of their ratios after chemotherapy . It seemIt is also possible to use these simulations to examine the chances of remission. Following both surgery and chemotherapy, the most successful individual cases were accompanied by the recovery of genotypes with active tumour suppression. If tumour suppression is not recovered, the treatment fails. Thus, if a large proportion of cells retain functional tumour suppressors at the time of therapeutic intervention, it is possible to recover less aggressive genotypes through treatment, leading to a better prognosis. Conversely, if tumour suppressor function is compromised prior to treatment in the bulk of the population, e.g. through field cancerization, the intervention can lead to an evolutionary bottleneck that selects for the rapid expansion of the most malignant cells in the population. Thus, while the speed of relapse in aggressive cases is dominated by the action of oncogenes that drive proliferation, a sustained recovery following treatment depends largely on tumour suppression.These results suggest that targeting chromosomally unstable cells may be an important part of future cancer therapies. It has been suggested that chromosomal instability may also play a role later in generating the genetic diversity required for cancer cells to survive the trials of invasion and metastasis, something that we have not explored here. Only through the tracing of clear evolutionary pathways in real cancers will it be possible to understand the different roles that these complex mutations have throughout the process of carcinogenesis and thus help us to develop better treatments. Future work will be needed to assess how scrambling of the genome may combine with mis-segregation events to drive the evolution of chromosomes that have specific complements of genes.In sum, in exploring the evolutionary pathway of cancer clones in tumour development our model shows the interplay between aneuploidy and tumour therapies. Future research will need to build on such models to being them closer to reality; to study the role of aneuploidy on more advanced kinds of tumours, and to simulate other kinds of cancer treatments.All cells are ordered in a linked list, and each cell has a simulated genome composed of three kinds of genes. Each of the three genes code for corresponding actions at a cellular level, inspired by biological systems and known cancer genes Tumour Suppressors- Apoptosis Regulatory GenesProto-oncogenes- Cell Division Regulatory GenesGenetic stability- Chromosome Segregation Regulatory GenesThe homeostatic constraints in the model were abstracted from real biological systems, where the overall goal of homeostasis is to maintain the tissue’s relative constant size and shape Inspired by the processes in biological cellular behaviour through which homeostasis is maintained in organisms, the algorithm is as follows:An initial population of 100 cells is created, each with diploid chromosomes. Each initial genome was equipped with 2 copies of each type of gene, grouped into chromosomes according to the gene distribution type A, B or C. The normal carrying capacity of the tissue is fixed at 200 cells.For each time step, the total number of cells is measured and is not updated until the next time step.If a measurement of the total number of cells is greater than the tissue’s carrying capacity, then the probability of cell death is calculated. The probability of death depends on the number of available copies of the apoptosis regulatory genes, If the cell has not died, it has a chance to divide. The probability of division depends on the number of available copies of the division regulatory genes, If dividing, a new cell is introduced in the linked list, spatially adjacent to the mother cell , and theThe update rules are applied synchronously to all the cells in each time step. Also, it is important to note that the probabilities are conditional . For instance, the probability of chromosome missegregation seems to be the largest, but because it can only happen conditional to the probability of division, it is in reality low.To investigate the properties and the dynamics of the system, and specifically the role that chromosome segregation regulatory genes have, four genome distributions were considered: Three reported here and a fourth distribution that considers every gene to be uncoupled as a control experiment. The parameter settings were determined through a series of preliminary experiments, in order to ensure that the behaviour of the system was both biologically plausible and computationally feasible. Simulations were carried out with the following initial parameters:Initial population: 100 cellsHomeostatic size of the tissue: 200 cellsSimulation end time: when reaching 7000 cells or reaching 300 time steps = 0.045, Scenario i: Surgery: Once the size of the population had reached 1000 cells, a segment of the linked list that contains 900 cells is selected and deleted, leaving a residual segment of 100 . ScenariThere is an important difference between the two treatments. When carrying out a surgical procedure, the cancer is removed along with some surrounding tissue at the tumour margin whose extent depends on the location and the extent of tumour invasion For the analysis of the simulations, the emergent genotypes were assessed. By quantifying the number of chromosomes that a cell has at a given time, a genotype state Cell Division Regulatory Genes, Apoptosis Regulatory Genes and Chromosome Segregation Regulatory Genes respectively. The initial genotype consists of two functional copies of each chromosome: genotype state .Where Colours in the RGB model are defined by three components . BecauseFor the statistical tests we used an unpaired t-test to determine if the means of the results in our two sets of experiments (Configuration B and C) are significantly different in key aspects of simulated treatments.Our null hypothesis is that the observed response of the two configurations to treatments is due to chance. The alternative hypothesis is that the observed response to treatments depends on the configuration. For these tests, we have assumed a two-tailed distribution and equal variance.Figure S1Actions within the linked lists of cells.A. When dividing, a new cell is introduced in the linked list of cells, spatially adjacent to the mother cell. B. During surgery, a segment of the linked list that contains 900 cells is selected and deleted, leaving a residual segment of 100 cells. C. During chemotherapy, all the cells that attempt cell division in the nine consecutive time steps after the list has reached 1000 cells are deleted.(TIFF)Click here for additional data file.


Performing music is a multimodal experience involving the visual, auditory, and somatosensory modalities as well as the motor system. Therefore, musical training is an excellent model to study multimodal brain plasticity. Indeed, we have previously shown that short-term piano practice increase the magnetoencephalographic (MEG) response to melodic material in novice players. Here we investigate the impact of piano training using a rhythmic-focused exercise on responses to rhythmic musical material. Musical training with non musicians was conducted over a period of two weeks. One group  learned to play a piano sequence with a distinct musical rhythm, another group  listened to, and evaluated the rhythmic accuracy of the performances of the SA-group. Training-induced cortical plasticity was evaluated using MEG, comparing the mismatch negativity (MMN) in response to occasional rhythmic deviants in a repeating rhythm pattern before and after training. The SA-group showed a significantly greater enlargement of MMN and P2 to deviants after training compared to the A- group. The training-induced increase of the rhythm MMN was bilaterally expressed in contrast to our previous finding where the MMN for deviants in the pitch domain showed a larger right than left increase. The results indicate that when auditory experience is strictly controlled during training, involvement of the sensorimotor system and perhaps increased attentional recources that are needed in producing rhythms lead to more robust plastic changes in the auditory cortex compared to when rhythms are simply attended to in the auditory domain in the absence of motor production. Musicians and non musicians exhibit structural and functional differences in a wide range of brain areas Enjoyment of music relates to familiarity with musical genres that help the listener to form and develop perceptual expectations for musical events. In that sense pitch, harmony, timbre and rhythm establish a musical predictive template that produces musical expectations Musical pitch expectations can be quickly formed by short-term musical piano training that shapes the brain activation within the auditory cortex In the same way that the chord structure of a musical piece shapes expectations about upcoming melodic events, the temporal structure of a musical piece induces anticipation of rhythmic events in the listener. A number of studies indicate that interactions between the auditory and motor systems may be particularly strong when rhythm is involved To investigate this hypothesis we measured the MMN to rhythmic deviants before and after sensorimotor-auditory or auditory musical training. Musical training strengthens expectations for musical events, which is reflected in the auditory system as better performance on discrimination of tonal frequencies Twenty-four non musicians  between 24 and 38 years of age participated in the study. Participants had no formal musical training, except for their compulsory music lessons at school. The data of four subjects had to be excluded because of the very low signal-to-noise ratio (insufficiently pronounced MMN). Thus 20 subjects were included in the analyses. Subjects were all right-handed as assessed by the Edinburgh Handedness Inventory The musical stimuli for the MEG measurement before and after training comprised six-tone piano sequences generated in a realistic piano timbre with a digital audio workstation . The seqThe first 16 measures of an exercise from a piano workbook for beginners The SA-group was instructed in how to play the piano exercise. The piano sequence was demonstrated by the experimenter at the beginning of the first training session. Training sessions were scheduled on 8 days within two weeks, each session lasting 30 minutes. A computer recorded the keystrokes of the subjects during the training through a MIDI connection. The MIDI data of the SA-group provided the stimuli for the training of the A-group. This included also the first training sessions when piano performance of the SA-group was still poor. Consequently, musical exposure of the SA- and A-group was identical. Each participant of the A-group was paired with a participant of the SA-group and listened to all the training session of that subject. Prior to training the A-group received a short introduction to the correct piano exercise. As in the SA-group, training sessions for the A-group were scheduled on 8 days within two weeks. During auditory training subjects of the A-group were seated in front of the piano. However, they received no visual information regarding the keys that had been pressed. Subjects in the auditory group were instructed to press the right foot-pedal whenever they noticed that the rhythm was played incorrect. This task ensured that subjects of the auditory group listened thoroughly and participated actively in the experiment.To evaluate the effect of the training on a behavioral level, all subjects participated in an auditory discrimination test before and after the two-weeks of training. For this test we extracted the first two measures of the piano exercise and recorded them via MIDI connection to a computer. Thus we obtained a sequence that contained 8 notes in the melody part and two accompanying intervals on the first beat of each measure. During the behavioral test this sequence or temporally altered sequences were presented. In temporally altered sequences a randomly chosen note of the melody was played earlier or later than expected by 10, 20, 30, 40 or 50 ms. These temporal offsets were chosen through pilot testing, which had revealed that a 50 ms time shift is easy to detect even for non musicians, whereas a 10 ms or even 20 ms time shift is very hard to detect. Sequences with temporal errors were presented randomly interleaved with correct sequences and subjects responded by pressing the left-foot pedal of the piano whenever they detected a temporal advance or delay of a note. If they did not detect a temporal error they had to press the right foot-pedal to start the next trial. The test contained 243 trials and lasted about 20 minutes. The first three trials were correct sequences to accustom the subject to the task. The remaining 240 trials contained 120 error sequences and 120 correct sequences. Each time shift was thus repeated 12 times during the test.The auditory MMN responses were measured from all participants before and after training. Training induced plasticity was evaluated by comparing the MMN differences before and after training between the SA and A-groups.Magnetic field responses were recorded with a 275-channel whole-cortex magnetometer system . The MEG signals were low-pass filtered at 150 Hz and sampled at a rate of 600 Hz. For each individual subject, epochs of 2 s for the standard and of 1.9 s for the deviant stimulus beginning 0.2 s before the last tone of the stimulus and ending 0.4 s after stimulus offset were extracted from the continous data set. The total recording time was 35 min. The recordings were performed in a magnetically shielded and acoustically silent room. The subjects were in an upright position, seated as comfortably as possible while ensuring that they did not move during the measurement. Three localization coils, that were fixed to the nasion and the entrances of both ear canals, were used to check the subject's head position at the beginning and end of each recording block. Subjects were instructed to move and blink as little as possible, to stay relaxed but awake during the measurement, and to pay no attention to the sound stimuli. Alertness and compliance were verified by video monitoring. To control for confounding changes in attention and vigilance, subjects watched a soundless movie of their choice, which was projected on a screen placed in front of them.The recorded magnetic field data were averaged separately for the standard and the deviant stimuli. Epochs contaminated by muscle or eye blink artifacts containing field amplitudes greater than 3 pT in any MEG channel were automatically rejected by the averaging procedure. The MMN was expected to be elicited in the deviant sequences after the onset of the sixth tone. Therefore, standard and deviant datasets were temporally aligned to the onset of the sixth tone of the sequence and subtracted to generate difference waveform data sets, representing the MMN.Although the alignment ensures that the onset time and duration of the sixth tone of the standard and the deviant were identical in both sequences there is still the possibility that the fifth tone, which is shorter in the deviant than in the standard sequence, provides additional MEG components that interfere with the subtraction procedure. Since the fifth tone of the deviant sequence is of shorter duration than the standard one, its corresponding N1 response will be closer to the onset of the sixth tone and this N1 component could then be mistakenly interpreted in the deviant-standard difference waveform as an MMN component For the MMN source analysis a baseline correction was performed relying on the 100 ms time interval prior to the onset of the piano tone sequences. Then, the source analysis model of two equivalent current dipoles (ECD) (one in each hemisphere) was applied to the MMN component identified in the data between 120 to 180 ms after tone onset. The two spatiotemporal dipoles, defined by their dipole moment, orientation, and spatial coordinates, were fitted simultaneously to the MMN derived from the difference waveforms for both hemispheres and for each recorded dataset before and after training. The source space projection method Prior to the training procedure participants of both groups listened to a correct version of the piano exercise. The subjects of the SA-group started in their first training session to play the upper line of the piece with the right hand only. The left hand was added in later training sessions after subjects were able to play the first part with the right hand correctly. After finishing the first line of the piano piece the same procedure was applied for the second line. The employment of the left and right hand was reversed, however, since the melody in the second part of the piece was in the left hand. The transfer of the melody to the left hand was difficult for most of the subjects, but was eventually mastered by all participants. Due to differences in learning progress among subjects, the different steps during the training process  were performed at different times during the training of each individual subject. At the end of the training, after 8 sessions, all subjects of the SA-group were able to play the piece within an acceptable speed with few mistakes. However, two subjects only reached successful performance of the second line with a reduced accompaniment in the right hand. Instead of playing the complete intervals, they simply played a single accompanying tone in each measure.The Performance on the behavioral test was evaluated by computing the detection rate for the error trials of each absolute time shift . Positive and negative time shifts were analyzed together. The data from one subject in the A-group  and two subjects from the SA-group (due to misunderstanding the task) had to be excluded, so that overall the data of 9 subjects of the A-group and 8 subjects of the SA-group were analyzed.The detection rates were fitted with a Weibull function and the 75% detection threshold was determined. A 2×2 mixed model ANOVA with factors group and pretraining/posttraining revealed a significant interaction of group x pretraining/postraining,  = 5.098; p = 0.039), demonstrating that rhythmic discrimination ability improved more strongly in the SA than in the A-group. On average, the detection threshold in the SA-group improved by 9 ms . No threThe MEG data showed a clear MMN dipolar pattern in most of the individual subjects, which justified the use of a single equivalent current dipole model for the cortical source analysis of the data. Group averages of the pre/post-training differences of the individual MMN source strength peak amplitudes are shown in the bar plots of A further observation was a difference in source strength between pre- and post training in the SA-group at about 230 ms. This indicates an increase in the P2 wave of the slow auditory evoked response to the deviant stimuli after training. The group averages of the pre-training/post-training differences of the individual source strength amplitudes of the P2 difference between deviant and standard were therefore tested for statistical significance . A mixedRhythm-focussed sensorimotor-auditory training in non musicians results in representational changes in the auditory cortices. The SA-group that had received sensorimotor-auditory piano training showed a significant post-training enhancement of the MMN to temporal deviants in rhythmic sequences. The A-group that had received only auditory training showed no significant training effect on MMN. This is consistent with the behavioral finding that thresholds for detecting temporal errors only improved in the SA-group but not in the A-group. However, both groups showed significant enhancement of the P2 component between deviant and standard after training, although the enhancement was larger in the SA-group, indicating that even the auditory-only training led to some plastic changes in auditory cortex. Previous studies indicate that the P2 component is larger in skilled musicians Training and test stimuli were not identical, indicating that the multimodal effects of the training generalized. Because MMN and P2 are generated primarily in auditory cortices Several studies show different brain responses in musicians and non musicians to pitch-, melodic- or harmonic-based deviants Our finding of superior learning with multimodal training is in line with other evidence that the brain is very sensitive to relations across modalities. The interaction and integration of different sensory modalities is especially important when playing a musical instrument. The multimodal effects that we obversed likely involved both somatosensory and motor interactions with auditory processing. Previously, Schulz et al. As far as the importance of the motor aspect of our training protocol, the concept of a strong link between the auditory and motor systems has a long history In addition, the results of the present study indicate that the interaction between auditory and motor areas is bidirectional, suggesting that movement can affect auditory processing. Phillips-Silver & Trainor Since the mismatch negativity is mainly generated in the auditory cortex, the mismatch paradigm is not suited to directly investigate response changes in motor related areas to musical stimuli after musical training. A different experimental design would be needed to demonstrate directly this connection. However, we suggest that auditory-motor interaction is bidirectional because the auditory input was identical for both groups, the only difference was motor-execution with associated attentional mechanisms in the SA- group. We therefore reason that auditory-motor interactions are likely involved in the generation of the increased mismatch negativity after sensorimotor training.Whereas many studies show larger MMN in the right compared to left hemisphere, the present results showed no difference in hemispheric involvement. It is possible that MMN tends to be right-hemiphere dominant for pitch-based discriminations, but not for duration-based discrminations. Indeed, our previous training study involving melodic chord sequences showed a greater plasticity effect in the right hemisphere whereas the present rhythm training study showed plastic changes of similar magnitude in both hemispheres. The strong involvement of the right hemispheric auditory cortex in the melody study, and the relatively well-pronounced involvement of the left auditory cortex in the rhythm study are consistent with data showing preferential encoding of spectral information on the right and temporal encoding on the left It has also been suggested that musical expertise could lead to a higher degree of analytical processing, which is believed to favor left hemispheric mechanisms Playing the piano is a motivating and demanding task. It is thus conceivable that the participants in the SA-group were more motivated or more engaged in their task than the participants of the A-group. The stronger MMN plasticity in the SA-group, therefore, might originate also from a stronger involvement of motivation or attention in this group. Attention, and other top-down modulatory signals, can increase plasticity effects in the auditory cortex We conclude that sensorimotor-auditory training of rhythmic material can increase the neural responses to a temporal mismatch in non musicians. The response increase was achieved after only eight training sessions. The enhancement of cortical activity was based on new musical material, suggesting strong generalization effects of sensorimotor-auditory training. Since cortical activity was enhanced to a lesser degree in the control group after auditory only training, we conclude that rhythm-focussed multimodal piano training has causal effects on auditory cortex plasticity. Together with our previous study


Sinusitis of dental origin is a relatively frequent entity, and odontomas are considered to be the most common odontogenic tumors of the oral cavity. Eruption and infection of odontomas are extremely rare. Here, we report an interesting case where odontoma was found in the wake of the maxillary sinusitis onset. Although sinusitis of dental origin is a relatively frequent entity , maxillaOn the other hand, the occurrence of complex odontomas is not considered to be rare in the jaws –11. MostA 12-year-and-7-month-old female pediatric patient presented with suspicious left odontogenic maxillary sinusitis and no noteworthy personal history. She complained of persistent nasal congestion and tenderness in the left molars. As a disease history, she had pain and swelling of the left cervical lymph node for one month. Symptoms were improved by intravenous infusion of antibiotics by a pediatrician who pointed out the left maxillary sinusitis by CT imaging. On suspecting odontogenic maxillary sinusitis, the patient was referred to our hospital. There was no sign of eruption of the second upper left molar; however, there was tenderness in a considerable part of the gum. Normal X-ray of the skull showed an opaque image of teeth surrounding the impaction of the second molars in the upper left maxillary sinus area . ComputeThe presence of an odontoma in the sinus is exceptional . FurtherOdontomas are the most common odontogenic tumors –13, 15 aEruption and infection of odontoma are uncommon –12, 16,