The large majority of structural MRI studies of major depressive disorder (MDD) investigated volumetric changes in chronic medicated patients in whom course of illness and treatment effects may impact anatomic measurements. Further, in few studies, separate measurements of cortical thickness and surface area have been performed that reflect different neurobiological processes regulated by different genetic mechanisms. In the present study, we investigated both cortical thickness and surface area in first-episode, treatment-naïve, mid-life MDD to elucidate the core pathophysiology of this disease and its early impact on the brain. We observed increased cortical thickness in the right hemisphere, including medial orbitofrontal gyrus, pars opercularis, rostral middle frontal gyrus and supramarginal gyrus. Increased thickness of rostral middle frontal gyrus was negatively related with depression severity on the Hamilton Depression Rating Scale. Furthermore, MDD patients showed significantly increased associations in cortical thickness measurements among areas where increased cortical thickness was observed. Analysis of pial area revealed a trend toward increased surface area in the left parahippocampal gyrus in MDD. To permit comparison of our data with those of previous gray matter volume studies, voxel-based morphometry was performed. That analysis revealed significantly increased gray matter volume in left paracentral lobule, left superior frontal gyrus, bilateral cuneus and thalamus which form limbic-cortico–striato–pallido–thalamic loops. These changes in first-episode, treatment-naïve, mid-life MDD patients may reflect an active illness-related cortical change close to illness onset, and thus potentially provide important new insight into the early neurobiology of the disorder. Investigations of brain morphology may help to clarify the impact of this disorder on the brain. Most previous structural studies of brain anatomy in MDD investigated volumetric changes in chronic medicated patients. These studies most commonly reported reduced gray matter volume in regions including anterior cingulate,3 orbitofrontal cortex,4 dorsal anterolateral and ventrolateral prefrontal cortex, precuneus and inferior parietal lobule.3 Two meta-analyses reported smaller volumes of the basal ganglia, thalamus, hippocampus, anterior cingulate and orbitofrontal cortex in MDD.6 In addition, three studies9 measured cortical thickness in elderly MDD patients and one of these studies9 found significantly decreased cortical thickness. Other studies12 reported reduced cortical thickness in depressed patients with type 2 diabetes patients10 and MDD with high risk for suicide11 when compared with healthy controls and depressed patients at lower risk for suicide respectively. Another study found significantly thinner posterior cingulate cortex in non-remitters with MDD than in remitters.12Though major depressive disorder (MDD) ranks as the leading cause of years lived with disability among all diseases,15 Pediatric MDD patients early in the course of illness and with positive family history have been reported to have significantly larger left prefrontal cortical volumes than those with a negative family history and controls.16 This inconsistency regarding reports of increased and decreased regional volumes in MDD is potentially due to factors such as field strength of the MR scanner, co-morbid illness, heterogeneity and chronicity of patients, medication effects, as well as factors relating to unique aspects of childhood and late-onset depression.Although the majority of studies reported reduced cortical volumes, some studies have reported volume increases in precuneus, cingulate gyrus, middle frontal gyrus and angular gyrus in adult patients.18 In particular, one recent study by van Eijndhoven et al.19 investigated cortical thickness in first episode depression by studying 20 medication-naive currently depressed patients and 20 medication-free recovered MDD patients. They reported increased cortical thickness in the temporal pole, the caudal anterior and posterior cingulate cortex, and rostral dorsolateral prefrontal cortex (DLPFC) in the first episode medication-free patient group, and reduced cortical thickness in the medial orbitofrontal gyrus. Notably in that study, analysis was performed on the combined data of both the medication-naïve currently depressed patients and the medication-free recovered MDD patients relative to controls. Thus the results may be influenced by prior treatment.19Few studies have examined anatomical changes in first-episode, drug-naïve adult MDD patients which may be important for elucidating the core pathophysiology of this illness independent from these potential confounds and for evaluating the trajectory of its early impact on the brain in longitudinal studies.20 Previous studies23 have found that cortical volume is driven mostly by cortical surface area rather than cortical thickness. Advanced automated analytic techniques such as FreeSurfer (http://surfer.nmr.mgh.harvard.edu) now make possible the examination of cortical thickness and area in vivo using high spatial resolution structural MRI. Independent measurements of these aspects of cortical anatomy are important as cortical thickness reflects the size, density and arrangement of neurons, neuroglia and nerve fibers,24 and thus its measurement could provide important and relatively unique information about disease-specific neuroanatomical changes. For instance, the regional thinning of cortex can reflect reduced dendritic arborization or changing myelination at the gray/white matter interface25 within specific brain systems. Postmortem studies in MDD have reported modest thinning of the cortical ribbon and/or layer-specific decreases in cell density due to cell loss and cell atrophy in localized regions, including the rostral and caudal orbitofrontal and DLPFC.27 However, postmortem cytoarchitectural measurements have limitations, including tissue fixation artifacts and limited antemortem characterization, and they typically are done with chronic patients typically with a protracted course of illness.In addition to those factors, cortical gray matter volume measures combine influences of cortical thickness and surface area, features which are believed to be influenced by different genetic factors regulating sulcal patterning and the thickness of the cortical mantle itself.31 across the brain can provide information about changes in distributed brain circuitry. Regional changes of anatomical connection patterns of the human cerebral cortex are reflected in parallel changes in cortical thickness measurements as proposed by He et al.33 This has been examined in other neuropsychiatric disorders,35 but not yet in MDD. Correlational analysis of cortical thickness measurements from structural MRI data rests on the assumption that positive correlations indicate connectivity, as axonally connected regions are believed to have common trophic and maturational influences.37 In clinical studies, such correlational analyses thus have the potential to map networks that may undergo common pathological processes. Our previous studies of a smaller independent sample of treatment-naïve, mid-life MDD revealed both regional and functional connectivity alterations within cerebral circuitry.39 Thus, in the present study we hypothesized that alterations in regional cortical thickness and in the relationships of thickness patterns across the brain would exist within cortico-cortical circuits in first-episode, treatment-naïve, mid-life MDD patients.In addition to the regional measurement of cortical thickness, correlational analysis of the pattern of cortical thicknessForty-six first-episode, drug-naïve, mid-life MDD patients and 46 healthy controls were recruited at the Department of Psychiatry at the West China Hospital of Sichuan University . The dia2 and data matrix=256 × 256.MR scanning was carried out on a 3.0 T MR scanner . Subjects were fitted with soft ear plugs, positioned comfortably in the coil and instructed to relax and remain still. Head motion was minimized with foam pads. High resolution three-dimensional T1-weighted images were acquired employing a spoiled gradient recalled sequence with TR/TE=8.5/3.4 ms, flip angle=12°, 156 axial slices with thickness=1 mm, axial FOV=24 × 24 cmhttp://surfer.nmr.mgh.harvard.edu/, vision 4.5.0), which uses automated surface reconstruction, transformation and high-resolution inter-subject alignment procedures to measure the thickness of the entire cortex.44 In brief, the procedure involves segmentation of white matter, tessellation of gray/white matter junctions, inflation of the folded surface tessellation patterns and automatic correction of topological defects in the resulting manifold. The thickness of each subject's cortical ribbon was computed on a uniform grid with 1 mm spacing across both cortical hemispheres, with thickness defined as the shortest distance between the gray/white and pial surface models,40 providing sub-millimeter resolution. Surface maps were generated following registration of all subjects' cortical reconstructions to a common average surface and then smoothed using a surface-based Gaussian kernel of 20 mm full width half-maximum.Constructions of cortical surface were developed from three-dimensional spoiled gradient recalled images using FreeSurfer software  to z values to test for significant differences in correlations between subject groups.Cortical thickness maps from patients and controls were compared using a general linear model with age and sex as covariates. As all images were aligned to a common surface template, we did not use intracranial volume as covariates. The regions that showed group differences in cortical thickness after FDR  correction were extracted in common space for each individual. In exploratory analyses, partial correlations using age and sex as covariates were computed to examine relationships between the mean cortical thickness in regions with altered thickness and clinical characteristics, including duration (weeks) and severity of depression. Partial correlations of cortical thickness among brain regions with group differences were obtained for each subject group using age and sex as covariates. Testing for differences of correlation coefficients between patients and controls was performed using Snedecor's method,http://www.fil.ion.ucl.ac.uk/spm) and the VBM8 (Voxel-Based Morphometry 8) toolbox (http://dbm.neuro.uni-jena.de/vbm8). Details of the analytic method are presented in Analysis of pial area and volume were also performed. The analysis of pial area was vertex based using FreeSurfer software. The volume analysis was performed by using SPM8 software (P<0.05 after FDR correction) in right frontoparietal regions including medial orbitofrontal gyrus =5.67), pars opercularis =4.81), rostral middle frontal gyrus =3.82) and supramarginal gyrus =3.98). No region with significantly decreased cortical thickness was found in MDD patients . Detailed results of analyses with pial surface measurements are provided in Analysis of pial area revealed a trend toward increased surface area in left parahippocampal gyrus in MDD  analytic method. We observed increased volume in left paracentral lobule, left superior frontal gyrus, bilateral cuneus and thalamus after FDR correction in MDD patients.r=−0.34, P=0.020). A similar trend was observed between mean cortical thickness of right supramarginal gyrus and the HDRS   . There wP<0.05) suggesting a generalized effect of influences impacting regional cortical thickness. Controls showed no significant correlation among these areas except for a positive correlation between right medial orbitofrontal gyrus and right rostral middle frontal gyrus . Significantly greater correlation coefficients for interregional cortical thickness were found in patients relative to controls in all areas except for the associations of the right rostral middle frontal gyrus with right supramarginal gyrus and right medial orbitofrontal gyrus with right supramarginal gyrus -6 and lead to cellular hypertrophy, astrocyte proliferation, process extension and interdigitation, which could increase cortical thickness.47 Furthermore, the activated astrocytes could promote neuronal survival by producing neurotrophic factors that promote recovery in central nervous system function. In other words, astrocyte activation could increase cortical thickness and produce increased levels of neurotrophic factors which could provide a neuronal protection effect and attenuate symptoms. Thus, the inverse relationship of increased thickness of right rostral middle frontal gyrus with lower HDRS scores in the MDD patients may represent a stronger neuronal protection effect by the activated astrocytes. Another study recently reported increased cortical thickness48 in right medial orbitofrontal cortex in individuals at increased risk for MDD, raising the possibility that the effect we observed close to illness onset may precede and be related to the pathophysiology of early illness manifestations. Illness-related physiological hyperfunction (higher metabolism and blood flow) also might through various mechanisms increase cortical thickness. These processes may be particularly relevant early in the course of illness, whereas the reduced gray matter volume15 and decreased cortical thickness9 in chronic MDD may be due to neurotoxic effect of recurrent or chronic depression that might occur over the longer course of illness progression.49 The exact mechanism for increased regional cortical thickness in MDD remains to be elucidated, hence further research is needed to clarify the cause, course and consequences of increased regional cortical thickness early in the course of MDD. Further, the extent to which the alterations we observed represent pre-existing risk factors or early effects of illness, and whether it is differentially seen in a subgroup of patients, remain to be determined.Though the reasons for the increased thickness of neocortex in MDD are currently unclear, one possible account for this effect is that it may be related to an inflammatory response, representing a compensatory effect in the early stage of depression.50 In contrast to our finding of increased cortical thickness in the right  medial orbitofrontal gyrus, van Eijndhoven et al.19 reported reduced thickness in left  medial orbitofrontal cortex. Although the areas are somewhat different, van Eijndhoven et al.19 combined data of 20 medication-naive and 20 medication-free recovered MDD patients in their analyses, so that previous treatment might account for some differences between our studies. Discrepancies between these study findings might also be attributed to factors such as differences in sample size, subject handedness, ethnic background and illness duration. Notably, the illness duration on average is 17.4 weeks in present study, but 28.4 weeks on average for medication-naïve patients and 86.4 weeks for medication-free patients with an average duration of 66.8 weeks of medication treatment in the van Eijndhoven et al. study.19 In addition, the studies differed in MR field strength, as van Eijndhoven et al. utilized a 1.5T MR scanner whereas we used a 3.0T scanner in the present study. Nevertheless, the observation of abnormalities in both the studies suggest a critical role of medial orbitofrontal cortex in the early developmental course of the depression, and the difference in direction of effect across studies suggest that the course of illness or treatment may significantly impact the integrity of this region.The increased cortical thickness observed in the present study was primarily found in prefrontal cortex, suggesting a particularly important role of this brain region in MDD. This observation parallels previous volumetric studies of prefrontal cortex in MDD, especially of orbital and medial prefrontal regions and DLPFC, in which abnormalities have been widely reported.51 and thus abnormalities in this region might be related to symptoms of anhedonia and apathy in MDD patients. Pars opercularis, another region with increased cortical thickness in our study, anatomically is in the ventrolateral prefrontal cortex and interacts with orbital prefrontal systems. Abnormalities in this region in depression are believed to be related to symptoms such as apathy, psychomotor slowing and impaired performance on tasks of attention and executive function.52 Rostral middle frontal gyrus, part of DLPFC, has an essential role in mood regulation, working memory and problem solving.53 Abnormalities of DLPFC have been broadly reported in post-mortem,26 neuroanatomical,50 functional neuroimaging56 and neuropsychological studies57 in MDD patients. Consistent with our study, increased cortical thickness in right rostral middle frontal gyrus was also observed by van Eijndhoven et al.19 in first-episode, medication-free MDD patients.According to recent human fMRI studies, medial orbitofrontal cortex contributes to the processing of reward and other hedonic qualities of life experience,58 Abnormalities in these two interconnected cortical circuits are believed to have critical roles in disturbances of affect regulation in patients with mood disorders.58 Considering our observation of greater cortical thickness in medial orbitofrontal cortex, pars opercularis and rostral middle frontal gyrus in first-episode, treatment-naïve, mid-life MDD patients, together with prior postmortem studies in MDD that have demonstrated modest thinning in orbitofrontal and dorsolateral prefrontal cortical regions,27 it may be that the cortices of these areas may have increased thickness during episodes of illness or close to illness onset whereas showing a declining thickness over the course of multiple episodes that could contribute to the persistent disability and reduced treatment response seen in some chronic patients. Such a process over the course of illness could contribute to the smaller orbitofrontal and dorsolateral prefrontal volume as seen in morphometric MRI studies in patients with recurrent MDD.59 Consistent with this possibility, a longitudinal VBM study found that gray matter reductions in DLPFC and medial orbitofrontal cortex progressively worsened in patients with chronic recurrent depression.60Medial orbitofrontal cortex and pars opercularis, which had increased cortical thickness in the present study, are part of medial prefrontal and orbital neural networks respectively. The rostral middle frontal gyrus shares many of its connections with the medial prefrontal network.62 and that changes in the parietal lobe may contribute to some neuropsychological63 and sensorimotor coordination problems in MDD patients.61Another interesting finding in the current study was the increased cortical thickness in right supramarginal gyrus which has been less commonly reported in MDD patients. Supramarginal and angular gyri constitute the inferior parietal lobule, which is involved in visual perception and visuomotor control. Previous studies suggest that these processes may be altered in depression,The analysis of pial area revealed a trend toward increased surface area in the left parahippocampal gyrus. This is of interest because this region is an important source of input to the hippocampus, which, as many lines of work suggest, is impaired in depression. Although it is broadly consistent with cortical thickness measurements in showing increased rather than decreased regional brain size, it is notable for being in a different region from those showing alterations in cortical thickness see . As theset al.,32 brain areas are often anatomically connected in relation to the correlations in their cortical thickness. Lerch et al.,64 taking Brodmann Area 44 as a seed region, reported patterns of anatomical correlations across the cerebral cortex that were strikingly similar to tractography maps obtained from diffusion tensor imaging. In contrast to the earlier observations of decreased functional connectivity within prefrontal-limbic-thalamic circuitry,39 present study showed increased correlations of cortical thickness across the regions with increased cortical thickness. Therefore, although the increased correlations of cortical thickness across brain regions in MDD patients may represent a consistent local effect across multiple cortical regions such as an inflammatory response, it is also possible that it may represent abnormal or compensatory cortico-cortical connections across multiple regions in parallel. In the latter case, the synchronous increases in cortical thickness across brain areas may reflect a close functional connection of the two networks whose alteration may be relevant for the pathogenesis of MDD. In either case, the enhanced correlations of changes in thickness of the right supramarginal gyrus with the three prefrontal regions in MDD patients suggest a wide scale process. Related findings from resting-state fMRI66 and diffusion tensor imaging67 studies also have reported altered neural circuitry/connectivity alterations in first-episode MDD patients, and these may be relevant for the mechanistic understanding of the cortical thickness findings of the present study.Another interesting finding from the present study is the increased correlations of regional cortical thickness measurements across the brain in MDD patients relative to healthy controls. According to a recent study by He 59 Longitudinal clinical studies and studies of ‘at risk' individuals are needed to address these issues and questions about the longer-term course and clinical implications of our findings. Third, as the analyses of interregional anatomical connectivity were based on a correlational analysis of cortical thickness, it is not clear whether the altered correlations represent real alterations in functional connections or simply parallel effects of illness pathophysiology on different brain regions.Several potential limitations of this study should be noted. First, since our measurements of cortical thickness were restricted to the cortical mantle, we cannot assess the thickness of regions such as thalamus, hippocampus, amygdala, striatum, and accumbens, which are critical to MDD. Second, because this study was cross-sectional, our findings cannot address the question of whether the structural differences that we report close to illness onset reflect altered brain maturation that developed over time because of gene–environment interactions, cellular, molecular and epigenetic forms of plasticity over the years before illness onset, or pathophysiological changes associated with the acute onset of depression.In conclusion, the present study complements and extends previous neuroimaging studies of MDD by demonstrating increased cortical thickness of prefrontal and parietal cortex in a large sample of first-episode, treatment-naïve, mid-life adult MDD patients mainly in regions where functional alterations have been demonstrated previously. As these findings of increased thickness vary notably from studies of chronic patients that suggest volume loss, future longitudinal imaging studies are needed to determine whether there are dynamic changes in cortical thickness, and regional changes of anatomical connection patterns and other aspects of brain anatomy over the course of MDD, to clarify the maturational and episode-related nature of these alterations of brain anatomy, their potential progression over the course of illness in MDD patients, and the potential impact of therapeutic intervention on these processes.


Lymphoma may affect the ureter in cases of retroperitoneal involvement. We present a case of an adolescent male found to have non-Hodgkin lymphoma initially presenting as ureteral stricture evident on imaging. He was treated and responded to multiagent chemotherapy with resolution of both the lymphoma and the ureteral stricture. Although rare, non-Hodgkin lymphoma should be included in the differential diagnosis of pediatric patients with noncalculous, idiopathic ureteral strictures. While the initial clinical presentation of non-Hodgkin lymphoma (NHL) varies based on lymphoma type and location, diffuse large B-cell lymphoma (DLBCL) may present with a rapid worsening of focal symptoms from nodal or extranodal disease, consistent with other diffuse lymphomas . We presA 16-year-old male was referred with subacute-onset right flank pain with hematuria. Initial CT scan without contrast  obtainedLymphomas are the third most frequent cancer in children, with DLBCL being the most common subtype of NHL in adolescents . In the Lymphomas directly involving the urinary tract are rare, although a compressive effect secondary to retroperitoneal lymphadenopathy is present more often than invasion or primary extranodal organ involvement. Ureteral obstruction exists in multiple reports with variable incidence of less than 1% to 14% and only a minority of patients experiencing symptoms of obstruction . When diLymphomas arising in the genitourinary tract account for less than 5% of extranodal lymphomas . While cWe report an interesting case of NHL presenting as a ureteral stricture in an adolescent male. Although rare, NHL should be included in the differential diagnosis of the adolescent patient with noncalculous, idiopathic ureteral stricture.


Retinal infarction and scalp necrosis are described as unusual but devastating complications of giant-cell arteritis. We report a patient with this rare complication and emphasize the importance of timely diagnosis and treatment of giant-cell arteritis. A previously healthy 70-year-old smoking female presented with 10-day history of loss of vision and scalp-tenderness. The event was diagnosed as amaurosis fugax. Fundoscopy indicated a pale oedematous papilla and a temporal peripapillary lozenge-shaped whitish patch on the retina, indicating optic nerve and cilioretinal artery infarction . Her ESRAn endarterectomy was performed and prednisolone was withdrawn postoperatively as the carotid stenosis was assessed as the cause of patient's symptoms. Within the following two months the patient's vision deteriorated and she developed a bilateral scalp necrosis . A seconGCA is a chronic granulomatous vasculitis of unknown etiology. The prevalence of the disease increase with age. The diagnosis of CGA can be based on clinical findings such as headache, scalp tenderness, jaw claudication, temporal artery abnormalities, elevated erythrocyte sedimentation rate, and biopsy of temporal artery showing granulomatous process , 2. Retinal infarction and scalp necrosis are described as unusual but devastating complications to inadequately treated GCA. The present case illustrates that the vasculitis in GCA can affect several extracranial arteries resulting in cilioretinal artery and disc infarction as well as scalp necrosis  3–5]. T–5. T3–5]Cilioretinal artery and disc infarction can occur in the context of carotid disease , 5. Both


Drusen are important risk factor for neovascular age-related macular degeneration (AMD) and have a dynamic nature as they can enlarge, newly form, or disappear over time. There have been few reports on drusen regression or choroidal neovascularization (CNV) development after macular hole surgery. We report, to our knowledge, the first case of both drusen regression and subsequent CNV development within 7 months of successful macular hole surgery.A 73-year-old woman presented with a stage 3 full-thickness macular hole and large, confluent soft macular drusen in the right eye and a neovascular age-related macular degeneration (AMD) in the fellow eye. Four months after the successful macular hole surgery, significant regression of drusen was seen, especially in the temporal area to the fovea. Three months later, the patient developed CNV and her best-corrected visual acuity decreased to 20/100, despite further regression of macular drusen.Macular hole patients with macular soft drusen need to be carefully followed up after surgery for possible drusen regression and CNV development. Large, soft drusen are associated with a greater risk for developing advanced age-related macular degeneration (AMD), including choroidal neovascularization (CNV) and geographic atrophy . Drusen 6 gas tamponade in the right eye. The fluid at the base of macular hole was not aspirated during fluid-air exchange to avoid damage to underlying retinal pigment epithelium (RPE). After a week of prone positioning, OCT showed a closed macular hole. Four months after the surgery, fundus showed drusen regression, especially in the temporal area to the fovea, and the BCVA improved to 20/50  showed a stage 3 full-thickness macular hole and large, confluent soft drusen in the right eye and a neovascular AMD in the left eye, for which she had undergone four sessions of intravitreal ranibizumab injections in other hospital Figure A,B. Her 0 Figure C. Howeve0 Figure D. Her feThis case illustrated the regression soft macular drusen and subsequent development of CNV after successful macular hole surgery, which may have acted as an exogenous stimuli for the regression of drusen and the development of advanced AMD. Spontaneous regression of drusen is well documented, but drusen regression does not necessarily indicate beneficial consequence since the development of CNV or geographic atrophy can ensue . It onlyThe mechanism of drusen disappearance after macular hole surgery is uncertain. Holz et al. proposed several possibilities including phagocytosis of drusen material by macrophages or multinucleated giant cells, phagocytosis by newly proliferated RPE, and reactive RPE changes to exogenous stimuli resulting in phagocytosis of drusen material . This seIn the present case, vitrectomy, ILM peeling, and gas tamponade were performed to close macular hole. ILM peeling may not be a significant factor associated with drusen regression; Holz et al. did not attempt to peel ILM during his macular hole surgery, whereas Dithmar et al. did, but both noted the disappearance of drusen after surgery ,6. OtherThere have been few case reports on CNV development after macular hole surgery -10. Age-Careful follow-up is warranted in macular hole patients with soft drusen since drusen regression and CNV development may occur after macular hole surgery.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.


Ruxolitinib, a JAK1 and JAK2 inhibitor drug, has recently been approved for the treatment of patients with high- or intermediate-risk myelofibrosis with symptomatic splenomegaly. Ruxolitinib is the first clinically useful targeted therapy in Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). The aim of this paper is to indicate pharmacobiological aspects of ruxolitinib within the potential context of MPNs. Pharmacobiological assessments, in addition to knowledge of the risk profile for ruxolitinib in MPNs, are required. We propose hypotheses based on our experience in a splenectomized MPN patient with hyperproliferative bone marrow and moderate fibrosis receiving ruxolitinib. We believe that a true clinical development approach for this drug should include pharmacobiological assessments for ruxolitinib in addition to the disease risk profile of MPNs. Ruxolitinib, a JAK1 and JAK2 inhibitor drug, has recently been approved for the treatment of patients with high- or intermediate-risk myelofibrosis (MF) with symptomatic splenomegaly . ClinicaRuxolitinib is a “JAK-STAT signaling pathway inhibitor” targeted drug with predictable pharmacobiological actions. The main function of the JAK-STAT signaling pathway is cellular proliferation in health and disease. Ruxolitinib should thus be considered as an “anti-proliferative” medicine ,5,6,7. RThe aim of this paper is to indicate pharmacobiological aspects of ruxolitinib within the potential context of MPNs. Pharmacobiological assessments, in addition to clarification of the risk profile  for ruxoA 64-year-old female patient with elevated blood counts was evaluated in our hematology unit. Medical history revealed systemic hypertension for 35 years and the diagnosis of polycythemia vera (PV) 20 years earlier. In 1994, the patient underwent total gastrectomy and splenectomy in order to cure gastric cancer. The JAK2V617F mutation was also detected in due course. The patient was treated by phlebotomy only until 2003, and then hydroxyurea plus phlebotomy until 2008 to control the disease. At that time, the patient had acute respiratory failure due to hyperviscosity (Plt count over 4 million per mm3 and white blood cell (WBC) count of about 50,000 per mm3), deep vein thrombosis, gastrointestinal bleeding, nasal bleeding, and hydroxyurea-induced skin lesions. After emergency treatment with leukapheresis and Ara-C infusions, an effort was made to control the patient’s thrombocytosis with aa combination of hydroxyurea plus anagrelide. In the following years, the patient had several severe attacks due to hyperleukocytosis  and extreme thrombocytosis (Plt count reaching 2 million per mm3) with hyperproliferative bone marrow  requirinOur patient represents a model of the ideal MPN population in which ruxolitinib should be administered, with hyperproliferative bone marrow with or without fibrosis and peripheral cytosis and organomegaly. Informed consent was obtained.Patients who have hyperproliferative bone marrow in any lineage  plus/minus fibrosis with or without peripheral cytosis , and splenomegaly plus peripheral cytosis ; all of the ruxolitinib arm of the RESPONSE trial patients; all ruxolitinib-receiving prefibrotic primary myelofibrosis  patients; all ruxolitinib-receiving patients splenectomized for any reason; and any MPN patient to whom ruxolitinib was already administered within other trials  on compassionate use could be registered and independently evaluated in the context of a ‘Ruxolitinib Pan-MPN Trial’. In this specific MPN patient population (Ruxolitinib Pan-MPN Registry), some critical clinical/laboratory evaluations including effects of ruxolitinib on white blood cell counts (control versus leukopenia) and leukostasis/infections, on hematocrit levels (control versus anemia) and hyperviscosity, on platelet levels (control versus thrombocytopenia) and leukostasis/thrombosis/hemorrhage, on hepatic enlargement and complications of portal hypertension (particularly after splenectomy), and on hyperproliferative bone marrow neoplastic cellular proliferation and fibrosis would be assessed. MPN disease risk categories of this specific MPN patient population should be detected, as well as the established clinically important ruxolitinib effects (reduction in spleen size and MPN symptoms). A Ruxolitinib Pan-MPN Phase II study should be performed if the Step 1 Ruxolitinib Pan-MPN Registry reveals that ruxolitinib can control hyperproliferative bone marrow , fibrosis and/or peripheral cytosis , cytosis-related acute/sub-acute complications , or hepatic enlargement and complications of portal hypertension (particularly after splenectomy), or can decrease spleen size in PV and normalize bone marrow architecture in MPNs in the long term in a study population whose main inclusion criteria are any MPN patient with hyperproliferative bone marrow in any lineage AND bone marrow fibrosis AND splenomegaly AND peripheral cytosis in at least one lineage , and all of the MPN patients with PMF (WHO 2008). These proposals will be tested as hypotheses on efficacy. Hypotheses on safety, including that the degree of ruxolitinib-induced anemia, leukopenia, thrombocytopenia, and related complications is lower in hyperproliferative MPN and that the tolerability and adherence of ruxolitinib with proper dosage and duration is enhanced in patients with hyperproliferative MPNs, will also be tested. The development of any drug from bench side to clinic is very difficult and expensive. Therefore, proper scientific strategy is absolutely necessary during the design of clinical studies. Ruxolitinib is the first clinically useful targeted therapy in Ph -MPNs. We think that a true clinical development approach for this drug should include pharmacobiological assessments for ruxolitinib in addition to the disease risk profile of MPNs.


Tracking and predicting motor outcomes is important in determining effective stroke rehabilitation strategies. Diffusion tensor imaging (DTI) allows for evaluation of the underlying structural integrity of brain white matter tracts and may serve as a potential biomarker for tracking and predicting motor recovery. In this study, we examined the longitudinal relationship between DTI measures of the posterior limb of the internal capsule (PLIC) and upper-limb motor outcomes in 13 stroke patients (median 20-month post-stroke) who completed up to 15 sessions of intervention using brain–computer interface (BCI) technology. Patients’ upper-limb motor outcomes and PLIC DTI measures including fractional anisotropy (FA), axial diffusivity (AD), radial diffusivity (RD), and mean diffusivity (MD) were assessed longitudinally at four time points: pre-, mid-, immediately post- and 1-month-post intervention. DTI measures and ratios of each DTI measure comparing the ipsilesional and contralesional PLIC were correlated with patients’ motor outcomes to examine the relationship between structural integrity of the PLIC and patients’ motor recovery. We found that lower diffusivity and higher FA values of the ipsilesional PLIC were significantly correlated with better upper-limb motor function. Baseline DTI ratios were significantly correlated with motor outcomes measured immediately post and 1-month-post BCI interventions. A few patients achieved improvements in motor recovery meeting the minimum clinically important difference (MCID). These findings suggest that upper-limb motor recovery in stroke patients receiving BCI interventions relates to the microstructural status of the PLIC. Lower diffusivity and higher FA measures of the ipsilesional PLIC contribute toward better motor recovery in the stroke-affected upper-limb. DTI-derived measures may be a clinically useful biomarker in tracking and predicting motor recovery in stroke patients receiving BCI interventions. One of the most common deficits following stroke is upper-limb motor impairment, which can have a significant impact on disability and health . RehabilWe have recently proposed that FA is a valuable measure for examining the microstructural integrity of the PLIC and a promising biomarker in tracking and predicting motor functional recovery in stroke patients receiving BCI interventions . In thisTable 1.Thirteen patients with stroke were included into this study. This study was approved by the University of Wisconsin-Madison’s Institutional Review Board. All patients provided written informed consent. Standard clinical MRI was utilized to assess damage to PLIC by a neuroradiologist (VP). Ten of the 13 patients showed damage to the PLIC due to stroke. Patients CI003 with a minor left frontal lobe infarct, CI010 with right occipital stroke and CT003 with a right pontine infarct did not show damage to the PLIC. Patient CT004 with a left middle cerebral artery (MCA) territory infarct showed minimal damage to the PLIC. A summary of patient characteristics is given in Diffusion tensor imaging data and motor outcome assessments were acquired at four time points: before the start of intervention , at the midpoint of intervention , upon completion of intervention phase , and 1 month following the last session of BCI intervention . A detailed description of the study design and the procedure of BCI intervention is elaborated in references ,b. Each Patient inclusion criteria include: (1) ages 18 years and above; (2) no known neurologic, psychiatric or developmental disability; (3) persistent upper-extremity motor impairment resulting from ischemic or hemorrhagic stroke. Exclusion criteria include: (1) contraindications for MRI; (2) allergy to electrode gel, surgical tape and metals that would be used in BCI intervention; (3) under treatment for infectious disease or having apparent oral lesions or inflammation.Table 2.Each patient was assessed for clinical stroke severity in addition to neurological examination at four time points throughout the BCI intervention. The overall neurological deficit and severity of motor paresis was evaluated using the National Institute of Health Stroke Scale NIHSS; . Upper-l2, voxel size = 1 × 1 × 2 mm3, 75 axial slices with no gap between slices, flip angle = 90°, 56 gradient encoded directions, b value = 1000 s/mm2.DTI data was acquired on a 3 T whole-body MRI scanner  with an 8-channel head coil. MR diffusion imaging parameters were: single-shot echo planar imaging (EPI), TR = 9000 ms, TE = 66.2 ms, single average (NEX = 1), field of view (FOV) = 256 × 256 mmFigure 1); (6) estimation of diffusivity for each hemispheric PLIC; (7) calculation of ratios of DTI measures  between the ipsilesional and contralesional PLIC as rValue = Valueipsi/ Valuecontra  for regression analysis . The threshold for statistical significance was set to analysis . GEEs taTable 2. The ARAT scores varied from zero, indicating no ability to perform the task, to maximum of 57, indicating unimpaired performance. The SIS-hand function measure varied widely, with a value of zero indicating a patient reporting no ability to use the impaired hand, and higher positive values indicating decreasing levels of difficulty using the impaired hand in daily activities such as carrying heavy objects, turning a doorknob, opening a can or jar, tying a shoe lace and picking up a dime. Although group performance on the ARAT and SIS-hand function measures was not significantly different over the course of BCI intervention , 7 of 13 patients reported improved hand function based on their SIS-hand function scores measured at 1-month-post intervention. Three of these seven patients  also achieved improvements meeting the minimum clinically important difference (MCID) of SIS-hand function .The mean values of AD, RD, and MD were higher on the ipsilesional PLIC than on the contralesional side, while FA was lower in the ipsilesional PLIC than in the controlesional PLIC . A secondary statistical analysis, the GEE analysis, was further computed to account for the dependence of repeated measurements from each patient at different time points. These analyses confirmed that the relationships observed between DTI and objective motor outcome measures  remained statistically significant (Table 3). We performed the same analyses to estimate the correlations between baseline DTI measures and motor outcomes. Only baseline PLIC FA was shown to be significantly correlated with motor outcome measures .To assess the relationship between PLIC DTI and motor outcome measures, Spearman’s rank correlation coefficients were first computed for all time points of the longitudinal data acquired from all patients. This correlation analysis showed that higher ARAT scores and higher SIS-hand function scores, indicating better performance, were significantly correlated with lower diffusivity ratios  but higher FA ratio  in the ipsilesional PLIC . Diffusivity ratios  outperformed diffusivity measures  in the regression model . FA remained the best predictor of motor recovery among these DTI measures . In addition, we examined whether baseline DTI measures of the ipsilesional PLIC were correlated with motor outcomes evaluated immediately post-intervention and observed very similar findings .In the regression model, baseline DTI measures of the ipsilesional PLIC correlated with ARAT and SIS-hand function scores tested 1-month-post intervention . This phenomenon has been suggested as a characteristic of chronic white matter degeneration . More specifically, higher ARAT scores, indicating better upper-limb functioning, and higher SIS-hand function scores, indicating greater satisfaction with hand use, were significantly correlated with lower diffusivity ratio but higher FA ratio in ipsilesional PLIC. Given our observation that ipsilesional PLIC had increased diffusivity and decreased FA compared with the contralesional side, these findings suggest that the more the DTI measures of the ipsilesional PLIC resembled those observed in the contralesional PLIC, the greater the potential for functional recovery in stroke-affected limb.In additional to its role as a structural integrity indicator, DTI measures linearly correlate with motor impairment and motor function , 2013. OFigures 3 and 4). This might be due to the fact that diffusivity ratios provided a normalized directional diffusivity measure by taking into account the underlying changes in contralesional side during stroke recovery. Furthermore, each diffusivity ratio correlated with motor outcomes which might indicate white matter remodeling in the ipsilesional and contralesional hemispheres during the rehabilitation period. Interestingly, both FA and rFA had a significant and strong correlation with motor outcomes. FA itself is a normalized scalar index proportional to the standard deviation of three eigenvalues, while pure diffusivity measures are combinations of eigenvalues and not normalized . Similar findings were also observed for baseline DTI measures and motor outcomes assessed immediately post-intervention . This supports the potential clinical utility of DTI measures to predict upper-limb motor functional recovery of stroke patients receiving BCI and other motor recovery interventions.One of the most important findings of this study is the predictive value of DTI measures to functional motor recovery following BCI facilitated FES of the impaired arm. Baseline DTI measures, especially the ratio of each DTI measure comparing the ipsilesional and contralesional PLIC, significantly predicted motor outcomes assessed 1-month-post intervention (n = 13) and the heterogeneity of stroke patients were the primary limitations of this study. Lesion location varied in these patients. Seven of 13 patients had left hemisphere stroke and six of them had right hemisphere stroke. Within the 13 patients, three patients had subcortical stroke. A wide range of time since stroke onset was another limitation. Four patients were severely impaired and exhibited no or little improvement in functional recovery as assessed by clinical behavioral performance. These patients were minimally able to perform the designed intervention tasks, resulting in a floor effect in outcome measurements. While changes in DTI measurements were observed across time, a Kruskal–Wallis test did not show these changes to be significant. There are many factors that may result in the absence of significant changes in DTI measures across time such as the small sample size, high between-patient variance in terms of age, stroke severity and duration of stroke, and the sensitivity of the current DTI technique used in the study. Future studies will target a more homogeneous subtype of stroke patients.Small sample size .There are other factors that could be useful for predicting outcomes such as age, duration of stroke, stroke severity. In the current study, we are specifically interested in the value of PLIC DTI measures in predicting motor outcomes. We performed additional analyses using age, duration of stroke  and stroke severity  as covariates, and found that ipsilesional FA, RD, and MD, age, months since stroke and motor arm scores of NIHSS baseline measures and interaction between any two of these parameters all significantly correlated with ARAT measures . We compared motor outcome performances between the intervention (nine patients) and the control group (seven patients) who did not receive BCI intervention and found significant improvements only at mid-intervention. We also examined the relationship between FA and motor outcomes at individual time points at pre-, mid-, immediately post, and 1-month-post intervention. We observed changes in regression slope and correlation coefficients between FA and motor outcome measures across intervention and especially at immediately post intervention, suggesting that BCI intervention may have an effect on motor recovery compared to pre-intervention. These results can be seen in the Supplementary Data. It is worth noting that these results were preliminary with a small sample size and the analysis was done under the assumption of a linear relationship between FA and motor outcome measures. Additional data is needed for further validation.In this study, we investigated the relationship between DTI and motor outcome measures in order to track and predict motor functional recovery in a group of stroke patients with persistent upper extremity impairment receiving BCI intervention facilitated with FES of the impaired arm. Given the previously established significance of PLIC involvement in motor recovery, this study evaluated stroke-induced changes in structural integrity of the PLIC using DTI measures and also investigated if these changes were related to motor recovery after a course of BCI interventions. We observed that ratio of each DTI measure was significantly correlated with motor outcomes, with lower diffusivity and higher FA measures of the ipsilesional PLIC correlating with better motor recovery. Interestingly, we also observed that baseline PLIC DTI measures assessed pre-intervention significantly correlated with motor outcomes measured immediately post and 1-month-post intervention, suggesting a predictive value of PLIC DTI in motor recovery, especially in stroke patients receiving BCI-FES interventions.JS: Assisted with patient recruitment, data collection, data analysis, and writing.VN: Assisted with patient recruitment, data collection, and data analysis.BY: Assisted with patient recruitment and data collection.LW, ZN, and AR: Assisted with data collection.MT: Provided part of system hardware and expertise.DE: Assisted with study design and outcome measure selection and interpretation.KC: Assisted with patient recruitment.JAS: Assisted with study design and patient recruitment.JW: This author is one of two lead PI’s on this project and supervised the technical and engineering aspects of the work.VP: This author is one of two lead PI’s on this project and supervised the neuroimaging aspects of this work.There is one patentpending on the closed-loop neurofeedback device used for the BCI-facilitated intervention administered in this study . This patent was filed jointly by the two lead investigators Justin C. Williams and Vivek Prabhakaran. Otherwise, the authors have no conflicts of interest to report, as this research was conducted in the absence of commercial and financial relationships that might compromise the integrity of the results reported herein.


Background: Manufacturers’ recommendations on cleaning of tracheostomy tubes focus on general warning information and non-specific manual cleaning procedures. The aim of this experimental study was to evaluate different reprocessing methods and to determine the mechanical integrity and functionality of tracheostomy tubes following reprocessing.Methods: Sixteen cuffed or un-cuffed tracheostomy tubes obtained from hospital in-patients were reprocessed using one of the following reprocessing methods: a) manual brushing and rinsing with tap water, b) manual brushing followed by disinfection with a glutaraldehyde solution, c) manual brushing followed machine-based cleaning in a dishwasher, and d) manual brushing followed by ultrasound cleaning in a commercially available ultrasound device. Microbial burden of the tubes before and after reprocessing was assessed by measurement of microbial colony-forming units per mL (CFU/mL) of rinsing fluid. After cleaning, tracheostomy tubes were investigated for loss of functionality. Findings: Manual brushing and rinsing with tap water reduced microbial colonization in average by 102 CFU/mL, but with poor reproducibility and reliability. Complete microbial reduction was achieved only with additional chemical or machine-based thermal disinfection. Ultrasound sonification yielded no further microbial reduction after manual brushing. Conclusion: Manual brushing alone will not result in complete eradication of microorganism colonising cuffed or un-cuffed tracheostomy tubes. However, manual cleaning followed by chemical or thermal disinfection may be regarded as safe and reproducible reprocessing method. If a machine-based reprocessing method is used for cuffed tubes, the cuffs’ ventilation hose must be secured in a safe position prior to thermal disinfection. During the past decades, advances in material and functionality of tracheostomy tubes have allowed improved patient care . Cuffed Therefore, depending on the manufacturer’s recommendations on maintenance and wear time, cuffed tracheostomy tubes usually may need to be replaced after 3–7 days. Un-cuffed tubes may have a maximum wear time of less than 4 weeks. Polymer tracheostomy tubes are single-patients devices; cuffed tracheostomy tubes are frequently marketed as single-use medical devices by the manufacturer, however, un-cuffed tracheostomy tubes may need to be cleaned daily by the patients themselves or by healthcare workers in order to prevent biofilm formation and to maintain the tube’s functionality.Manufacturers’ recommendations for un-cuffed tracheostomy tubes frequently derive from general cleaning guidelines for parts of respiratory circuits, but have not thoroughly been validated for individual tracheostomy tubes. The recommended cleaning and maintenance methods usually comprise manual brushing and rinsing of the disassembled tube parts under tap water followed by disinfection using antimicrobial compounds such as glutaraldehydes, and have been adopted by nursing best practice statements . For cufAside of physical and chemical considerations, reprocessing of tracheostomy tubes in hospital settings should be time- and cost-effective, and must conform to quality management policies. Compared to conditions at home care, hospital settings require short reprocessing hands-on times, complete quality control and strict infection control assurance in order to prevent transmission of pathogens from still unprocessed to already processed tracheostomy tubes during reprocessing. These practical requirements are met best by adoption of automated cleaning and disinfection processes, which have to be implemented and validated with specific attention to polymer material sensitivity. Therefore, the aim of this experimental study was to evaluate the antimicrobial efficacy of manual brushing and flushing on worn tracheostomy tubes alone, and the added effect of either disinfection using a glutaraldehyde solution, a thermal disinfection using a dishwasher, or ultrasound cleaning in a commercially available ultrasound cleaning device. The mechanical integrity and functionality of tracheostomy tubes following various reprocessing methods was also assessed.®, Kerpen, Germany and Teleflex Medical®, Kernen, Germany) were obtained from hospitalised in-patients directly after use. Un-cuffed tubes were changed and collected after minimum of 1 day of use, cuffed tubes after a minimum of 3 days of use . For reprocessing methods B and C (below), instead of 0.9% saline solution validated neutralizers were used. Microbial concentration of the rinsing solution was determined by using the standard microbiological serial dilution method and plating on different culture agars: Columbia 5% agar, Columbia CNA agar (selective to Gram-positive bacteria), McConkey agar (selective for Gram-negative bacteria), and Sabouraud agar (selective for yeast). These agars allow identification of a broad microbiological spectrum, which is frequently encountered in oral cavities and the respiratory tract. Colony-forming units (CFUs) were counted and morphologically identified after 24 h incubation at 37°C. The effectiveness of the applied reprocessing procedures in terms of microbial reduction was tested. This study examined four different reprocessing methods: Method A – Manual cleaning. Manual brushing with a new tube brush followed by rinsing with tap water until a macroscopically clean result was achieved.Method B – Manual pre-cleaning and additional chemical disinfection: Manual pre-cleaning and complete submersion of the cleansed tubes in a commercially available and by the manufacturer recommended disinfection solution  for 1 hour at room temperature, followed by final rinsing with tap water.Method C – Manual pre-cleaning followed by a machine-based thermal disinfection in a dishwasher: After manual cleaning, tubes were placed vertically in a dishwasher , peak process temperature: 65° C, standard washing program (48 min) without additional cleaning agents.Method D – Manual pre-cleaning followed by ultrasound sonification: Ultrasound cleaning in a commercially available ultrasound household device for cleaning jewellery and optical instruments , final rinsing with tap water. Cleaned and reprocessed tracheostomy tubes were examined for visible signs of material alteration including colour changes, deformation, or other causes for loss of tube function. Cuff integrity was tested by inflation at 25 mmHg of air and documentation of the maintenance of pressure during 20 minutes with a cuff pressure manometer .Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa and Candida spp. were identified as most frequent pathogens colonising the inner lumen of tracheostomy tubes. The microbial burden of the rinsing solution before cleaning ranged between 101 CFU/mL and 106 CFU/mL, mean: 9×104 CFU/mL. Neither quality nor quantity of the microbial colonisation differed between un-cuffed and cuffed tracheostomy tubes. 1 and 104 CFU/mL, with a mean microbial reduction of 2 log10. However, the reliability and reproducibility of the cleaning efficiency was poor. Only manual cleaning with additional disinfection in a glutaraldehyde-based solution achieved a complete microbial elimination in all tested samples. Additional machine based thermal cleaning using a dishwasher reduced the microbial burden to less than 101 CFU/mL, however, starting from an already initial low microbial bio-burden after manual pre-cleaning. Surprisingly, ultrasound sonification using a household ultrasound-device following a final rinse with tap water did not improve the results of a previously conducted manual pre-cleaning. Detailed results of the microbial colonisation of polymer tracheostomy tubes before and after application of the investigated reprocessing methods are summarised in Table 2 In all 16 tested tubes, manual brushing achieved microbial reduction ranging between 10No signs of material alteration were found on any of the tested tracheostomy tubes. Cuff functionality after the single reprocessing regiments remained intact in all but one cuffed tubes. In this instance, improper fixation in the machine resulted in cutting off the cuff’s ventilation hose by a dishwasher rotor arm. Tracheostomy tubes are semi-critical A single-patient medical devices, which allow securing the patient’s airway, and, depending of the functionality, may also assist the patient’s phonetic capability. However, because of accumulation of large deposits of mucus and debris, as well as microbial colonisation and consecutive formation of biofilm Figure 2 , tracheoIndeed, despite poor quality and low level of detail, reprocessing of tracheostomy tubes is briefly highlighted in most manufacturers’ instructions of use Table 1 , and an In this study, we investigated the microbial colonisation of tracheostomy tubes after 1 to 3 days of wear, and explored the microbial reduction capacity of a manual cleaning method such as manual brushing and rinsing with tap water, and the additional effect of glutaraldehyde-based disinfection, cleaning and thermal disinfection in a dishwasher, and sonification by use of an ultrasound device. With particular regard to homecare, only easily obtainable and commercially available procedures and devices were included in the study. With exception of the ultrasound sonification device, all used brushes and disinfectants are frequently available from the tube manufacturers themselves. Although the used thermal dishwasher was a medical-grade professional device, today, most household dishwashers also provide programs at 65°C, hence, allowing repeating the investigated procedure at home.10 microbial reduction was achieved. This level of reduction may be too low to prevent long-term damage to the tube caused by Candida albicans colonisation or in situations where the patients’ nasopharyngeal region is colonised by potentially pathogenic organisms, such as methicillin-resistant Staphylococcus aureus (MRSA) strains. Hence, brushing and flushing tubes with water alone seems not to be a reprocessing method, which may be recommended, even not for common household use. This procedure needs to be augmented by additional support. Indeed, Björling et al. [The results of this study demonstrated that manual cleaning with a tube brush and rinsing with tap water resulted in poor reliability and low reproducibility of microbial reduction. Even with good manual dexterity and sufficient time, only an average of 2 logg et al.  have demManual cleaning together with complete immersion of the pre-cleaned tube in a glutaraldehyde-based disinfection solution always achieved constant results with almost complete elimination of microbial colonisation. Considering the broad and highly reliable antimicrobial efficacy of glutaraldehydes , this fiCleaning and thermal disinfection in a dishwasher seems to be an elegant and automated alternative, which is more environmental friend than chemical disinfection. Only in one instance, less than 10 CFUs were found on the inside of a tracheostomy tube after withdrawal from the dishwasher  of the Austrian National Bank. The authors wish to thank Mrs Maria Stadler for excellent laboratory support.


A colorectal tumor is not an isolated entity growing in a restricted location of the body. The patient’s gut environment constitutes the framework where the tumor evolves and this relationship promotes and includes a complex and tight correlation of the tumor with inflammation, blood vessels formation, nutrition, and gut microbiome composition. The tumor influence in the environment could both promote an anti-tumor or a pro-tumor response.A set of 98 paired adjacent mucosa and tumor tissues from colorectal cancer (CRC) patients and 50 colon mucosa from healthy donors  were included in this work. RNA extracted from each sample was hybridized in Affymetrix chips Human Genome U219. Functional relationships between genes were inferred by means of systems biology using both transcriptional regulation networks  and protein-protein interaction networks (BIANA software).Here we report a transcriptomic analysis revealing a number of genes activated in adjacent mucosa from CRC patients, not activated in mucosa from healthy donors. A functional analysis of these genes suggested that this active reaction of the adjacent mucosa was related to the presence of the tumor. Transcriptional and protein-interaction networks were used to further elucidate this response of normal gut in front of the tumor, revealing a crosstalk between proteins secreted by the tumor and receptors activated in the adjacent colon tissue; and vice versa. Remarkably, Slit family of proteins activated ROBO receptors in tumor whereas tumor-secreted proteins transduced a cellular signal finally activating AP-1 in adjacent tissue.The systems-level approach provides new insights into the micro-ecology of colorectal tumorogenesis. Disrupting this intricate molecular network of cell-cell communication and pro-inflammatory microenvironment could be a therapeutic target in CRC patients. Colorectal cancer (CRC) is a complex disease in which many genes, proteins, and molecular processes are implicated. Proteins do not work independently in a tumor cell, but are organized into co-regulated units or pathways that perform a common biological function . RelevanBesides, a colorectal tumor is not an isolated entity growing in a restricted location of the body. An active communication exists not only between different cell communities within the tumor bulk but also between the tumor and the non-tumor distant mucosa. Hence, the patient’s gut environment constitutes the framework where the tumor evolves and this relationship promotes and includes a complex and tight correlation with inflammation, blood vessels formation, nutrition and gut microbiome composition . ConsequField cancerization or the field-effect is a theory first described by Slaughter et al. in oral carcinoma . In the Understanding the complex ways in which cancer cells interact with their surroundings, both locally in the tumor organ and systemically in the body as a whole has implications for effective cancer prevention and therapy. In contrast to the gene-centric view, a systems biology approach  can be useful to depict a global view of the cancer disease not only as a tumor cell but as an intricate systemic disease .In this study, mRNA expression from paired tumor (T) and adjacent mucosa from CRC patients (A) and mRNA from mucosa healthy donors (H) were measured using microarrays. The inclusion of samples from healthy subjects has allowed us assessing whether adjacent mucosa from colon cancer patients differs from healthy donors’ mucosa possibly due to the tumor presence. Indeed, a number of differentially expressed genes (DEG) were found between these two entities (A vs. H). Considering their level of expression in tumor tissues, these DEGs were classified as “Tumor-like”, “Trend” or “Adjacent-specific” (A vs. T) patterns. To explain the mechanisms that regulate these patterns of differential expression, networks mimicking transcription regulation were used to search for those transcription factors directly influencing DEG. Then, a systems biology approach using PPIN was applied to describe a crosstalk between cytokines and other proteins secreted by the tumor and receptors activated in the adjacent colon tissue; and vice versa, providing new insights into the micro-ecology of colorectal tumorigenesis. Finally, relevant cytokines and receptors up-regulated in tumor tissue were identified comparing T vs. H expression Figure . FurtherA principal component analysis (PCA) was done to explore the variability of the transcriptomic data from our 246 samples , 260/230 ratio and plate. In addition, a functional analysis including differentially expressed genes between these two clusters did not show specific functions but essentially those described as characteristic of adjacent mucosa. These results suggest that the smaller cluster of adjacent samples was just an extreme phenotype of these samples. Interestingly, this pattern was also observed in the validation dataset  but none was found associated to healthy mucosa phenotype  suggesting that DEG are not only performing common functions but also co-regulated in a sub-transcriptional network not active in healthy mucosa samples. Out of 895 DEG, 60 (13%) were transcription factors (TF), and random re-sampling of genes among the complete dataset revealed that DEG were significantly enriched in TF . Among these 60 TF, 35 were specific of the adjacent mucosa transcriptional network.To further explore this hypothesis, transcriptional networks were inferred and compared using gene expression data of adjacent and healthy mucosa  and the proportion of targets in our DEG list. This rank suggested sub-networks specifically active in adjacent mucosa tissue. TF with higher rank were more specific of adjacent mucosa, and showed higher values of eccentricity  and lower values of closeness centrality Table .Fosb and Jun) ranked first in the TF list. The AP-1 subunits Fos, Junb, Mafb and Atf3 also appeared in the list. Previous GSEA analysis also had revealed as most significant motive “Genes with promoter regions  around transcription start site containing the motif TGACTCANNSKN which matches annotation for JUN”  corresponded to secreted (n = 111) or membrane (n = 213) genes. In addition, 442 genes (250 secreted and 192 membrane) over-expressed in tumors were included in the analysis. A level 0 (only direct interactions) protein-protein interaction network was retrieved using the 766 up-regulated secreted/membrane genes in adjacent and tumor samples as input. The resulting network included 291 nodes connected by 596 interactions, the majority of them integrated in a giant component , extracellular-matrix components  or molecules related to invasion like SPARC linked with receptors such as integrins or complement receptors . As a result, a strong physical interaction between these two cellular components (the extracellular one and the nuclear one) was found. Twenty-one membrane receptors (out of 22) interact with each other through linker proteins to transduce a cellular signal across the extracellular matrix and membrane, finally activating TF belonging to the AP-1 complex  remodeling, organ morphogenesis, and cell adhesion. Remodeling of the ECM network though controlled proteolysis regulates tissue tension, generate pathways for migration, and release ECM protein fragments to direct normal developmental processes such as branching morphogenesis . CollageOne important finding is that DEG are enriched in transcription factors. This indicates the existence of a transcriptional program driving the altered expression pattern observed in adjacent mucosa. A loop including members of the AP-1 family of transcription factors emerged as the most significant one in the analysis. Interestingly, these TF are over-expressed in both adjacent mucosa and tumor tissue. AP-1 members homo or hetero dimerize to assemble the activator protein 1 (AP-1). AP-1 transcription factor acts synergistically with SMAD3/SMAD4 component and is implicated in the regulation of a variety of cellular processes including proliferation and survival, differentiation, growth, apoptosis, cell migration, and inflammation ,20. TopoWe hypothesized that cytokines and other signaling proteins secreted by the tumor activate membrane receptors of adjacent mucosa cells that initiate this transcription factor activity. Tumor-secreted growth factors act as paracrine agents distorting the normal tissue homeostasis. In turn, tumors are both maintained or attacked by signals from the surrounding microenvironment inducing stromal reaction, angiogenesis and inflammatory responses. To gain insight into the molecular mechanisms underlying this phenomenon, a bi-tissue PPIN analysis strategy was performed to extract patterns of receptor activation in both directions from adjacent mucosa to tumor and vice-versa. Robo genes appeared as the most recurrently receptors activated in tumor membrane by Slit family of proteins. Slits have been implicated in regulating a variety of life activities, such as axon guidance, neuronal migration, neuronal morphological differentiation, tumor metastasis, angiogenesis and heart morphogenesis . SeveralRELN), a glycoprotein that plays an important role in neuronal migration through the activation of lipoproteins receptors such as LRP8  around transcription start site containing the motif TGACTCANNSKN which matches annotation for JUN: jun oncogene”. Red and blue bar stands for adjacent and healthy mucosa, respectively. Figure S5. Protein-protein interaction network showing the axis membrane receptors – AP-1 transcription factors, activated in adjacent mucosa. Seed proteins are colored in green (transcription factors) or brown (membrane receptors), and highlighted in grey. Inferred interacting proteins are colored in light purple.GSEA representative results. Red and blue bar stands for adjacent and healthy mucosa, respectively. Click here for fileGene expression levels of the 895 DEG between adjacent and mucosa samples, in independent public datasets GSE38713 and GSE23878. Only 825 out of 895 genes were found in the validation serie microarray.Click here for fileList of significant functions stratified by pattern.Click here for fileCrosstalk network functional analysis.Click here for fileOrigin of proteins implicated in the crosstalk which are secreted by the tumor or located in tumor membrane.Click here for fileBaseline characteristics of healthy donors and CRC patients.Click here for file


DNA damage response (DDR) is a complex biological system activated by different types of DNA damage. Mutations in certain components of the DDR machinery can lead to genomic instability disorders that culminate in tissue degeneration, premature aging, and various types of cancers. Intriguingly, malfunctioning DDR plays a role in the etiology of late onset brain degenerative disorders such as Parkinson’s, Alzheimer’s, and Huntington’s diseases. For many years, brain degenerative disorders were thought to result from aberrant neural death. Here we discuss the evidence that supports our novel hypothesis that brain degenerative diseases involve dysfunction of glial cells . Impairment in the functionality of glial cells results in pathological neuro-glial interactions that, in turn, generate a “hostile” environment that impairs the functionality of neuronal cells. These events can lead to systematic neural demise on a scale that appears to be proportional to the severity of the neurological deficit.The  The most serious threat to genome stability is damage inflicted on DNA molecules The relationship between genome stability and human health is highlighted by the genome instability syndromes. Patients with these syndromes are typically characterized by progressive degeneration of specific tissues, chromosomal instability, cancer predisposition, and increased sensitivity to DNA damaging agents ATM gene encoding ataxia-telangiectasia mutated) MRE11) ATR) NBS1) e.g., microcephaly, mental retardation, genomic instability, and hematological malignancies). Patients with each of these diseases have neurological defects, suggesting that the DDR is important in neurogenesis and neurodegeneration.Mutations in key DDR molecules are implicated in human genomic instability syndromes ATM gene supports the “neuron doctrine” thought to underlie neurodegenerative diseases. Cellularly, A-T is characterized by cerebellar degeneration of various cell types, premature senescence of fibroblasts, chromosomal instability, and hypersensitivity to DNA-damaging agents, particularly those that induce DSBs Malfunctioning DDR is implicated in many human brain degenerative diseases (BDDs) N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) that mediates synaptic vesicle fusion with the cell membrane during the release of neurotransmitters, while synapsin-I is an abundant neuronal phosphoprotein that is associated with synaptic vesicles ATM deficiency is a representative of genomic instability disorders that severely affect brain functionality. Thus, we will focus on ATM deficiency and its effects on neuronal and glial cell functionality. Neurons contain significant levels of ATM in the cytoplasm It is highly likely that information coding in the brain is not performed at the level of single neurons but rather at the level of neural-glial networks ATM−/− mice of different ages In a quest to understand the role of ATM in the activity of Purkinje cells, Chiesa et al. conducted morphological and electrophysiological analyses of Purkinje cells from ATM-deficient mice, severe deficiencies are found in the retina of ATM−/− mice, which affect retinal neurons, vascular system, and retinal electrical activity. Retinal neurons are terminally differentiated and have the highest metabolic activity of all neurons in the central nervous system (CNS) ATM-deficient cells ATM−/− mice, which progressed to significant functional deficits in the older mice. Taken together, these findings suggest that hampered vascularization and astrocyte–endothelial cell interactions in the CNS have an important role in the etiology of A-T and that vascular abnormalities likely aggravate brain degeneration.In addition to cerebellar defects in NBS1 in the CNS (NBS1-Δ-CNS) leads to severe cerebellar atrophy characterized by reduced number of Purkinje cells Brain functionality is highly dependent on astrocytes. Malfunctioning astrocytes can cause many brain diseases 9 mm−3Until very recently, brain function was thought to be mainly dependent on neuronal cells. The “neuron doctrine”, which has governed modern brain research since the late 19th century During the past 20 years, knowledge regarding the appearance, physiological properties, and functions of glial cells has grown tremendously For many years BDDs have been thought to be primarily associated with dysfunction or death of neural cells. However, emerging evidence points to dysfunction in neuron-glia communications as playing a large role Astroglial cells function under both normal and pathological conditions. Under normal physiological conditions, astroglial cells control water movement and distribution, as well as buffer the concentrations of extracellular potassium ions. They are capable of regulating the release of neurotrophic factors as well as extracellular concentrations of neurotransmitters released from neighboring synapses. One of the most important roles of glial cells is the neutralization of ROS generated in the vicinity of various types of cells including neurons. Astrocytes are important for metabolic support of adjacent neurons. They do so by absorbing glucose from blood vessels that they contact and converting it to lactate, which is then provided to neurons . The fun2+. Increased intracellular Ca2+ levels can activate calcium-dependent cysteine proteases , thereby causing neural demise + concentrations promotes further over-excitation of neuronal cells Astrocytes are also involved in various aspects of synaptogenesis, synaptic maturation, neurotransmitter homeostasis, brain microcirculation, brain metabolism, as well as control over formation and maintenance of the blood–brain barrier Whereas neuronal cells are highly differentiated and post-mitotic cells, mature astrocytes in the brain are capable of cell proliferation Rad51 expression using RNAi is associated with increased cell death following ionizing radiation (IR) treatment In vitro and in vivo observations revealed that compared to somatic stem cells, ATM activation and its downstream DDR factors such p53, checkpoint kinase 2 (CHK2), and ATR are strongly inhibited in astrocytes compared to somatic stem cells. The inhibition of the DDR machinery involves a stable transcriptional repression of key DDR genes as mentioned above. The astroglial cells examined in this investigation are non-proliferative mature terminally-differentiated cells, mimicking the state of most of adult astroglial cells. Interestingly, astrocytes retain the expression of NHEJ genes and are proficient in DNA repair. It seems that upon DNA damage induced by IR, DNA-dependent protein kinase DNA-PK, a PI3K-like kinase, is responsible for γH2AX signal in astrocytes Unlike mature and fully-differentiated astrocytes, glioblastoma cells retain their ability to activate HR repair. There is a dose-dependent increase of Rad51 focus formation in response to the induction of DSBs in glioblastoma cells. Down-regulation of FOXO3a, the main FOXO transcription factor isoform expressed in astrocytes, is responsible for the cellular response to oxidative stress. This is achieved by regulating the expression of specific genes, including those genes encoding factors involved in DNA repair and glutamine metabolism such as glutamine synthetase. Immunohistochemical analyses of human samples from the population-based neuropathology cohort of the Medical Research Council’s Cognitive Function and Ageing Study have shown that the FOXO3a nuclear retention significantly correlates with DDR and with glutamine synthetase expression by astrocytes Using a variety of cellular, molecular, and genomic methodologies, Yong et al. 3) resulting in PIP2. PTEN is involved in the regulation of the cell cycle, preventing cells from growing and dividing too rapidly PTEN-null astrocytes to N-methyl-N′-nitro-N-nitrosoguanidine, a functional analog of temozolomide, results in the accumulation of DSBs due to inefficient DNA repair and apoptosis PTEN deletion also compromises HR repair. It is possible that PTEN inactivation increases astrocyte proliferation and that the HR repair pathway is activated in proliferating astrocytes, although it is inactivated in non-dividing astrocytes. The compromised HR repair in PTEN-deficient astrocytes can be explained by reduced expression Rad51 paralogs.Phosphatase and tensin homolog (PTEN) acts as a tumor suppressor ATM−/− cerebellar neural-glial networks as revealed by GFAP staining; the number of branches originating from the cell bodies is also significantly lower in astrocytes of ATM−/− mice in vivo. Similarly, morphological alterations in astrocytes are found in ATM-deficient retinas and optic nerves. Reduced arborization of the retina and optic nerve is associated with reduced retinal functionality as shown by electroretinographic examination Glial fibrillary acidic protein (GFAP) is an astrocyte-specific marker. Compared to wild type, there is a less complex cell arborization in In the year 1919, Rio Hortega described a new type of glial cell, the microglia Alkylating agents, such as methylnitrosourea (MNU), severely affect mitochondrial DNA (mtDNA). In response to MNU, there are no cell-specific differences in initial mtDNA damage repair in different glial cells including microglia, astrocytes, and oligodendrocytes. However, compared to astrocytes, mtDNA repair in oligodendrocyte progenitors, oligodendrocytes, and microglia was significantly reduced. A variety of xenobiotics can lead to mitochondrial perturbations, which would culminate in apoptosis. Whereas MNU-treated oligodendrocytes display DNA laddering and other characteristics of apoptosis, no signs of apoptosis are detected in astrocytes exposed to MNU Carcinogenesis and neurodegeneration can be caused by ROS lesions that lead to the formation of 8-oxoG. Severe striatal neurodegeneration is detected in mice lacking genes encoding 7,8-dihydro-8-oxoG triphosphatase (MTH1) that prevents the incorporation of 8-oxo-2′-deoxyguanosine triphosphate to the DNA and/or OGG1 that excises 8-oxoG. In contrast, mice deficient in mutY homolog (MUTYH) and/or OGG1 are resistant to neurodegeneration induced by oxidative stress. Together, these findings indicate that MUTYH, a glycosylase involved in oxidative DDR, promotes neurodegeneration, whereas OGG1 and MTH1 are protective. Accumulation of 8-oxoG in neuronal mtDNA leads to calpain-dependent neuronal loss. This process is associated with delayed 8-oxoG nuclear accumulation in microglial cells leading to PARP-dependent activation of apoptosis-inducing factor and exacerbated microgliosis. Taken together, these findings show accumulation of 8-oxoG in the genomes of neurons and microglia results in neurodegeneration Ercc1) mutant mice, which display DNA repair deficiencies, serve as a good model for accelerated aging. These mutant mice show the accelerated aging in various tissues including the brain. Hallmarks of microglial priming such as cytokine expression and phagocytosis can be detected in the Ercc1 mutant mice in response to peripheral lipopolysaccharide. Progressive priming response of microglial cells is also observed in mice with Ercc1 deleted in forebrain neurons, in association with phenotypic alterations Alterations in microglia have been suggested to contribute to age-related CNS deterioration. Enhanced sensitivity to various inflammatory stimuli is among the most prominent age-related changes in microglial cells, which is referred to as priming. During microglial priming, microglia multiply and adopt an activated state in response to neurodegeneration and abnormally folded proteins In human Alzheimer’s disease patients, elevated S-adenosylhomocysteine (SAH) is found in plasma and cerebral tissue, which is inversely correlated with the cognitive ability of the patients (+)-Catechin, a flavanol antioxidant extracted from Acacia catechu NBS1 locus lead to the mental retardation, microcephaly, radiation sensitivity, chromosomal instability, immunodeficiency, and cancer predisposition experience by patients with NBS NBS1 – null mice, making it difficult to study the physiological functions of NBS1 during development Hypomorphic mutations in the NBS1 deletion specifically in astrocytes would affect the DDR and thereby cause cerebellar pathology and behavior. We generated the GFAP-Cre-lox mouse strain with down-regulated expression of the NBS1 specifically in astrocytes . These NBS1-GFAP-Δ astrocytes displayed reduced functionality as well as malfunctioning DDR in response to X-ray irradiation or the radiomimetic agent neocarzinostatin. Compared to wild-type cells, expression of BDNF and glutamine synthetase (GS) is reduced in the NBS1-deficient astrocytes. Interestingly, there is no evidence of altered cerebellar morphology in 10 month-old NBS1-GFAP-Δ mice, which seem to have normal behavior. Therefore, the observed malfunction DDR may not be sufficient to cause pathological effects. It is also possible that NBS1 deletion in one cell population is not sufficient to cause cerebellar pathology .We hypothesized that CD11b encodes an integrin expressed specifically in microglia CD11b expression is significantly reduced in NBS1-CNS-Δ cerebella, together with reduced levels of cysteine–cysteine chemokine ligand 2 (CCL2), the chemokine primarily responsible for attracting CD11b-positive cells and mediating the innate immune response in the CNS CCR2-deficiency accelerates the severity of BDDs and markedly impairs microglial cell accumulation in the mouse brain How does malfunctioning DDR affect glial cell functionality and thereby network dynamics? Glial cells are key players in neuronal homeostasis through the following processes, including metabolic support + ion buffering and water distribution can also affect neural activity, influencing network dynamics. The excitatory neurotransmitter glutamate and the supply of glutamine to surrounding neural cells are also important for network functionality. Our results clearly demonstrate that NBS1 deletion reduces the secretion of neurotrophic factors and reduces expression of glutamine synthetase, the enzyme responsible for conversion of glutamate to glutamine Network dynamics is heavily dependent on network topology, the connections among neural cells, the interactions between neurons and glial cells, and on the mode of synaptic transmission. Malfunctioning DDR severely affects the glial cell functionality, and malfunctioning astrocytes can alter synaptic activity, thereby affecting the dynamics of neural-glial networks. Alterations in KThe brain is the most complicated tissue in higher organisms, responsible for control of vital processes and also at the center of motor functions, cognition, and personality. Loss of brain functions through trauma or aging exacts an enormous human toll on patients and families. Cases of age-related dementia have increased as average life spans have reached record highs. Moreover, advances in modern medicine have enabled us to replace or repair almost all body organs except the brain. To ensure that brain functionality can be maintained in old age, it is critically important to gain a better understanding of the cellular and molecular processes involved in the brain dysfunction that can result from BDDs .For many years the neuronal doctrine has dominated the field of brain research, but it is now clear that various types of glial cells are critical to brain function. Like neurons, the glial cell population contains a vast variety of cells, each with a different role. Glial cells are important in the most rudimentary synaptic processes and in the regulation of complex brain networks and circuitries. Some glial cells provide the structure necessary for CNS development, others provide nutrients to support neurons, and others appear essential to activities such as the formation of synaptic connections and pruning. Some glial cells undertake immune system functions, such as the destruction of invading pathogens. Such an important role is performed by the microglial cells that invade the brain during embryonic development.As mentioned in this review, much of the gliobiology research has focused upon astrocytes and microglia, which have very broad portfolios of responsibilities and contributions. These two populations of glial cells form highly-complex connections, which are disrupted during brain pathologies. Structurally, astrocytes are very complex and their activation has been reported in a variety of brain diseases and injuries. The specific functions of astrocytes under physiological as well as in pathological conditions are still not completely understood. This is due in part to the enormous heterogeneity of glial cells starting from stem cells and ending in mature highly polarized and differentiated cells. Naturally, this makes the comparison of individual studies rather difficult A common denominator in various BDDs is malfunctioning of DDR pathways. Here we present evidence that impaired DDR severely affects the functionality of glial cells and underlies the symptoms of certain degenerative diseases of the brain. Based on a wealth of data, we suggest that glial cells play a major role in brain aging as well as in the etiology of early onset genomic instability disorders and late onset BDDs.The authors declare that they have no competing interests.