Transient ischemic attack (TIA) was originally defined as self-resolving focal cerebral ischemia with symptoms lasting <24 h. The newer definition also added the limitation that there should be no evidence of acute brain tissue infarction, to recognize that acute injury to the brain can result from ischemia of <24-h duration. However, several recent findings suggest that having a TIA correlates with deficits that can persist far beyond the resolution of clinical symptoms, even in the absence of imaging evidence of ischemic tissue injury. These deficits may be the result of subtle perturbations to brain structure and/or function that are not easily appreciated using the standard clinical and imaging tools that are currently employed in practice. Here, we will discuss evidence that suggests that TIA may lead to lasting changes to the structure and function of the brain. Currently it can be argued that the approach to investigating transient ischemic attack (TIA) and minor stroke is a compromise between timeliness of investigation and accuracy of diagnosis. Diagnostic accuracy hinges on identification of tissue injury, i.e., infarction. However, the tools that can be used to achieve this degree of accuracy, e.g., magnetic resonance imaging (MRI), are not always readily available at the point of care. This dilemma is also linked to the evolving definition of TIA and stroke, which has changed over the past few decades to accommodate both clinical utility and biological significance. The initial description of TIA as a transient neurological deficit of suspected vascular origin lasting <24 h has been used widely throughout epidemiological studies and for pragmatic clinical decision-making. As such, it has been a useful construct that could be applied in any clinical setting requiring nothing more than the clinician’s judgment from the patient’s history and examination findings, facilitating rapid diagnosis. However, this definition has been abandoned in light of recent imaging data that demonstrates that about 50% of these patients have evidence of infarction on diffusion-weighted MRI . In some cases, FLAIR will be positive for a clinically-relevant lesion after 30 days even though DWI was negative <24 h after symptom onset  can now identify injury to white matter (WM) tracts  is a valuable tool for assessing changes in brain metabolism or connectivity related to tasks or at rest. In people diagnosed with TIA without T2- or FLAIR (3.0-Tesla) lesions, fMRI has shown reduced resting state network connectivity and reduced dorsolateral prefrontal cortex regional homogeneity for 1 month after symptom resolution . Three common TMS metrics are utilized: cortical silent period (SP), EMG suppression after a single high-intensity TMS pulse; intracortical inhibition (ICI), motor-evoked potential reduction elicited by a low-intensity TMS pulse if it is followed by a higher-intensity one; and intracortical facilitation (ICF), motor-evoked potential increase elicited by high-intensity TMS pulse if it is followed by a lower-intensity one. People diagnosed with TIA have demonstrated both increased ICF and reduced ICI in the affected hemisphere compared to the unaffected hemisphere, without evidence of structural MRI lesions  TIA (Pendlebury et al., The problem of imprecision in tradition clinical examinations could be addressed with the use of novel technologies. Robotics provide a means by which to investigate behaviors in a more sensitive way than is possible with standard clinical tools and allows the behavioral correlates of neurological injury to be investigated. Recently, a cohort of people who had a first-ever TIA was assessed using the KINARM robot (Simmatis et al., The current approach to TIA and minor stroke investigation is focused appropriately on immediate threats to health such as recurrent stroke. However, these approaches offer limited insight on how the consequences of transient cerebral ischemia evolve for the patient. Additionally, the ability to identify tissue injury rapidly remains incomplete. The original definition of symptom resolution in <24 h did not account for structural injury to the brain. DWI has vastly improved our capacity to characterize TIA and minor stroke pathophysiology; however, limitations in availability and sensitivity of this and other MRI-based tools should drive the adoption of supplementary diagnostic techniques. Research on tools such as MEG, EEG, robotics, and various MRI-based tools to study brain function after TIA and minor stroke is still a “work in progress,” and presently has no prognostic value. However, with further refinement, these tools could be eventually used to supplement DWI and provide additional diagnostic/prognostic insight. Importantly, there is increasing recognition that there are other consequences to TIA and minor stroke beyond the presence or absence of DWI lesions. A more comprehensive approach incorporating clinical history, imaging, tests of brain function, and associated behavioral consequences after cerebral ischemia could help in the timely and accurate identification of individuals who are likely to suffer recurrent stroke or functional decline.LS was the primary researcher and writer of this manuscript. SS and AJ assisted with writing and editing the manuscript.SS is the co-founder and Chief Scientific Officer of BKIN Technologies Ltd., the company that commercializes the KINARM robot that is discussed in this article. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Acalculia is an acquired disorder in calculation abilities, usually associated with left posterior parietal damage. Two types of acalculic disorders are usually distinguished: (1) primary acalculia or anarithmetia, where the patient presents a loss of numerical concepts , and (2) secondary acalculia due to a different disturbance in cognition and affecting mathematical abilities. Secondary acalculias are associated with aphasia, alexia, agraphia, executive function disorders, or visuospatial difficulties. This paper is a proposal for clinical intervention to rehabilitation of acquired primary and secondary acalculias. It is important to emphasize that rehabilitation is based in both targeting restitution of the original impaired function and reorganization of functions employing other less or unimpaired cognitive functions , 22.Research about acalculia rehabilitation has been limited . FrequenIn a review of the research devoted to the rehabilitation of acalculia published from 1980 to 2007, only 7 papers were found, all of them single case studies . RegardlIn one of the few studies analyzing the evolution of acalculia, Caporali et al.  reportedFew studies have analyzed the brain changes associated with acalculia recovery . Claros-Major strategies used in the rehabilitation of primary and secondary acalculias will be presented. However, frequently cognitive rehabilitation procedures have been implemented in single cases; few studies have included large groups of acalculia patients.Primary acalculia is found associated with left posterior parietal lesions , usuallyIt has been observed that anarithmetia usually appears simultaneously with the so-called semantic aphasia , 33, 34;According to Tsvetkova , the iniNoteworthily, primary acalculia can be observed without language disturbances, but its correlation with aphasia is significant . The rehIn conclusion, rehabilitation of the patient with primary acalculia should begin by relearning the basic concepts of number. Later, the patient should move to the acquisition of more sophisticated concepts that imply the relationship between numbers and finally to solving arithmetical operations and numerical problems.Patients with acalculia due to a language defect  and receiving language therapy usually present a significant improvement in their calculation abilities in a parallel way to the language recovery . In otheSometimes brain-damaged patients can mentally solve mathematical operations, but they fail in recognizing numbers due to visuoperceptual impairments. The intervention in those cases should be directed to recover the visual perception . PerceptThe technique known as “number reconstruction”  is basedPatients with pure alexia—alexia without agraphia—have difficulties for the visual integration of stimuli (deficit that is known as simultanagnosia) and defects in visual-motor coordination (known as optic ataxia). Consequently, the intervention should include exercises that require the visual exploration of space. Frequently used tasks included in rehabilitation programs suppose a progressive increase in their level of difficulty, beginning with simple movements such as reaching objects, followed by tasks that require more complex movements such as copying figures in two dimensions to conclude with the copy of three-dimensional designs . TraininWhen there is a visual exploration defect (ocular apraxia) impairing the ability to normally scan quantities, practicing eye-tracking exercises can help to overcome this defect. Rosselli et al.  describePatients with frontal damage due to diverse conditions, such as head injuries and tumors, usually present executive function disturbances. These patients present particularly significant difficulties in solving mathematical problems. Written calculations are easier than mental calculations. Attentional difficulties significantly contribute to the calculation difficulties. Tsvetkova  suggesteSpatial acalculia is usually observed in cases or right hemisphere damage associated with spatial neglect . SpatialRosselli and Ardila  describeBenavides-Varela et al.  reportedIn primary acalculia, the fundamental therapeutic goal is to reconstruct the understanding of the numerical system. Patients with primary acalculia seemingly do not understand how the numerical system is organizedIn aphasic acalculia, numerical impairment and recovery in general parallelize aphasia impairment and recoveryWhen acalculia is due to visuoperceptual disturbances, the fundamental aim of therapy is to recover visual perceptionIn frontal dysexecutive acalculia, providing the patient with external support  and attentional control strategies  may result in improving the numerical defectNeglect represents probably not the only but the fundamental defect responsible for the written calculation defects observed in patients with right hemisphere damage. Some simple strategies, such as using clear marks to indicate where to begin to read, can successfully improve the calculation defectsThe available information about the cognitive rehabilitation of acquired calculation disturbances is limited. However, some general conclusions can be drawn.


Deep vein thrombosis (DVT) is a major origin of morbidity and mortality. While DVT has long been considered as blood coagulation disorder, several recent lines of evidence demonstrate that immune cells and inflammatory processes are involved in DVT initiation. Here, we discuss these mechanisms, in particular, the role of immune cells in endothelial activation, and the immune cascades leading to expression of adhesion receptors on endothelial cells. We analyze the specific recruitment and functional roles of different immune cells, such as mast cells and leukocytes, in DVT. Importantly, we also speculate how immune modulation could be used for DVT prevention with a lower risk of bleeding complications than conventional therapeutic approaches. Local endothelial activation, and the release of Weibel–Palade bodies present in endothelial cells, are instrumental for deep vein thrombosis (DVT) initiation. In addition to the vascular system, the immune system is emerging as a pivotal player in the pathophysiology of DVT, potentially opening new prophylactic approaches.Mast cell activation and degranulation are crucial for the local inflammatory response resulting in DVT. Mast cells may potentially link hypoxia and endothelial activation.Neutrophil extracellular traps play an important role in DVT by providing a scaffold for adhering platelets and red blood cells and promoting thrombin generation and fibrin deposition.Platelets are an important source of damage-associated molecular patterns facilitating further leukocyte recruitment and promoting venous thrombosis. VTE is the third most common cardiovascular pathology by its prevalence after myocardial infarction and stroke Blood clotting is based on a protein polymer called fibrin, produced by cleavage of its precursor, fibrinogen, by the protease thrombin  [4]. ThrBecause of the known roles of these factors in clot formation, the current paradigm of DVT prophylaxis focuses predominantly on the coagulation system, by targeting thrombin , active FXa  or vitamin K-dependent clotting factors  blood flow stagnancy and hypoxia; (ii) activation of the endothelium; and (iii) blood cell recruitment leading to activation of blood coagulation and thrombus development . We outlDVT develops in the valvular pockets of the veins Animal models recapitulating complete Blood inside the vein is the only source of oxygen for the venous wall. Thus, diminished supply with new portions of blood creates local hypoxia in the vein. Hypoxia is considered the major pathogenetic mechanism linking blood flow stagnancy with following processes in the vessel wall leading to thrombosis. Hamer and co-authors directly demonstrated in dogs and patients that blood oxygenation in venous valve pockets quickly falls once the flow becomes static and returns back to original values when pulsatile  flow is applied Endothelial activation and exocytosis of WPBs, central events in DVT initiation, are rapidly (within 1–6 h) induced by stenosis of the IVC Hypoxia results in the synthesis of reactive oxygen species (ROS), which activate the endothelium, inducing the expression of adhesion receptors and recruitment and extravasation of leukocytes In addition to cell recruitment, activated endothelium enhances blood coagulation and exerts suppressed anticoagulant function, thus contributing to thrombus formation It has recently become clear that endothelial activation and cell recruitment, the critical events in DVT initiation likely induced by local hypoxia, require an intermediary: MCs. MCs are a part of the innate immune system that largely reside in tissues and are present in the vicinity of blood vessels in vitro-differentiated MCs into MC-deficient animals restored thrombosis, suggesting that it is the lack of MCs that prevents DVT. Thus, the net effect of MC degranulation in this model is the exacerbation of venous thrombosis, suggesting that the activity of MC-originated proinflammatory factors outweighs the activity of antithrombotic factors. Absence of MCs completely prevents thrombosis, suggesting that MCs are absolutely required  for its initiation.Using the stenosis DVT model in mice, we have recently demonstrated that two strains of MC-deficient mice are completely protected against DVT In addition to protection from DVT, MC deficiency is accompanied by reduced cell recruitment at the venous wall after stenosis application 2O2 has been shown to mediate increased susceptibility to DVT in aged mice, whereas mice overexpressing an antioxidant, glutathione peroxidase 1, are protected against the prothrombotic effect of H2O2Mechanisms of MC activation by hypoxia remain obscure, but oxidative stress and ROS appear to play a role in this process. ROS can be both the cause and the consequence of MC activation because, on the one hand, inhibition of MC activation downregulates ROS production and, on the other hand, antioxidants prevent MC degranulation Mastocytosis, which is associated with abnormally high numbers of MCs, is accompanied by bleeding symptoms in a small proportion of patients Restriction of venous blood flow induces rapid leukocyte recruitment. After 1 h of IVC stenosis, leukocytes start to roll along and adhere to the venous endothelium, and after 5–6 h, leukocytes carpet the entire endothelial surface. Neutrophils account for more than 80% of adherent leukocytes and monocytes represent the remainder The involvement of neutrophils in DVT was discovered several decades ago Upon recruitment to the venous wall, neutrophils undergo activation and release their nuclear material, forming a web-like extracellular structures designated as neutrophil extracellular traps (NETs). These are composed of DNA, histones, secretory granule constituents, and other components implicated in antimicrobial defense In vitro experiments have also demonstrated that NETs stimulate platelet adhesion and aggregation at a venous shear rate and induce thrombocytopenia in vivo, with both effects being abolished by histone inactivation The mechanisms by which NETs may contribute to venous thrombosis have become an important area of research. It has been shown that various adhesion proteins, including VWF, fibrinogen, and fibronectin, may bind to DNA/histone strings so that NETs become a scaffold for adhering platelets and red blood cells independent of the fibrin network in vitro that while NETs components, DNA and histones, potentiate thrombin generation and blood clotting, NETs, as a biological entity, are unable to do so in vivo) that NETs might need a certain degree of degradation to acquire procoagulant activity. Thus, NETs could represent an important mechanistic link between neutrophil accrual and venous thrombogenesis.Another mechanism of the prothrombotic effect of NETs is potentiation of the coagulant cascade and reduction of anticoagulant activity. NETs can bind FXII and provide a scaffold for FXII activation Monocytes and, to a lesser extent, neutrophils recruited to the venous wall serve as a principal source of TF; the major initiator of the extrinsic coagulation pathway and fibrin deposition. In the stenosis model of DVT, deletion of TF in myeloid leukocytes completely prevents thrombus formation without affecting leukocyte recruitment The prothrombotic function of leukocytes is negatively regulated by signaling via TLR-9. It has been shown that lack of this pattern-recognition receptor is associated with larger venous thrombi and increased levels of NETosis, necrosis, and apoptosis markers in the stasis, but not stenosis, model of DVT in mice Platelets are recruited to the venous wall shortly after blood flow restriction and play an important role in DVT as platelet depletion substantially reduces thrombosis Platelet recruitment to the venous thrombus is mediated by binding of platelet receptor GPIbα to VWF exposed on the endothelial surface. Indeed, deficiency in either GPIbα extracellular domain The analysis of signal transduction pathways in platelets following recruitment to the venous wall has shown a role for mechanistic target of rapamycin complex 1 (mTORC1), a rapamycin-sensitive protein complex consisting of mTOR, Raptor, and mLST8  Besides procoagulant activity , with endothelial receptor scavenger receptor-BI (SR-BI). This interaction diminishes endothelial activation and WPB release in an endothelial NO synthase (eNOS)-dependent manner Mechanisms of DVT initiation represent a cascade of events virtually identical to the local inflammatory response recently designated as immunothrombosis Although the precise mechanisms of how the immune-system-related cells and molecules are implicated in DVT may differ, some of them converge on local inflammation in the venous wall. Thus, focusing on this common denominator, reduction of endothelial activation, release of WPBs and local cell recruitment could be a promising strategy ameliorating venous thrombosis. For example, NO is one of the most potent inhibitors of WPB liberation Outstanding QuestionsIs local hypoxia a leading factor exacerbating DVT? Are there (if any) other causes?What are the mechanisms of hypoxia-driven local inflammation?What mechanisms mediate mast cell activation and degranulation under flow stagnancy conditions? What mediators released by mast cells trigger DVT?What therapeutic interventions targeting the immune system and local inflammation will be most efficient against DVT?Based on combination of targeting the immune response with conventional methods of tackling DVT, it is tempting to develop a personalized approach of DVT prevention in different predisposing conditions .In conclusion, DVT develops as a form of immunothrombosis with a particularly important role of local inflammation at the stage of thrombosis initiation. Targeting inflammatory pathways is less likely to cause bleeding complications than inhibition of blood coagulation mechanisms. Thus, it may be considered as an alternative and safer approach for the prevention of DVT in at-risk populations and further research in this area may provide important new therapeutic options (See Outstanding Questions).


Furthermore, we aimed to determine whether the [18F]-fluorodeoxyglucose. Thereafter, a positron emission tomography/computed tomography scan of the lower limb was acquired. Three-dimensional muscle contours of 78 (= 39x2) muscles of the left and right lower limb were semi-automatically determined from magnetic resonance imaging scans. After non-rigid registration, those muscle contours were used to extract [18F]-fluorodeoxyglucose uptake from the positron emission tomography scans.Ten healthy participants walked on a treadmill at self-selected comfortable walking speed for a total of 90 minutes, 60 minutes before and 30 minutes after intravenous injection of 50 MBq [18F]-fluorodeoxyglucose distribution was not distributed normally; most voxels had a relatively low standardized uptake value, and a minority of voxels had a relatively high standardized uptake value. The voxels with higher [18F]-fluorodeoxyglucose uptake were distributed heterogeneously; they were clustered in virtually all muscles.Large asymmetries were observed in the lower leg muscles  and in the gluteus minimus (30% asymmetry) and gluteus medius (15% asymmetry), whereas the uptake in the thighs was relatively symmetrical between the limbs (<6% asymmetry). These were not related to limb-dominance nor to inter-limb differences in muscle volume. The [The findings in this study challenge the common assumption of symmetry in muscle activity between the limbs in healthy subjects. The clustering of voxels with high uptake suggests that even in this prolonged repetitive task, different spatial regions of muscles contribute differently to walking than others. Walking involves a complex integration of muscular contractions with the goal of maintaining stance stability and forward progression. Walking in healthy persons has traditionally been assumed to be a symmetrical motion, either from assumption or for convenience of data collection and analysis . This asTo assess muscle activation patterns, researchers typically utilize surface electromyography. This method is convenient and noninvasive, but has disadvantages such as a limited number of muscles that can be measured simultaneously, the need for a reference contraction if muscles are to be compared with each other, inability to measure deep-lying muscles and deep-lying aspects of superficial muscles, and confounding factors such as crosstalk and adipose tissue , 9. In m18F]-fluorodeoxyglucose (FDG) is a technique that can be used to simultaneously assess glucose uptake in entire muscles during activity in both limbs, as well as region-specific differences in glucose uptake within muscles. It thus offers several advantages for assessing walking asymmetry and within-muscle activity compared to other techniques such as surface electromyography: it can measure all muscles at the same time, it can measure deep-lying muscles that would otherwise be difficult or impossible to access unless needle electrodes are inserted, and it is not hindered by crosstalk from other muscles or adipose tissue. The FDG-PET technique might, therefore, expand our understanding of asymmetries in walking and intra-muscular inhomogeneity in activation. This technique is based on the principle that contracting muscles take up glucose (and FDG) from the blood to replenish their expended energy. Although muscle cells can use various substrates , the plasma glucose uptake is likely constant in a low intensity exercise such as walking -fluorodeoxyglucose uptake between muscles in the dominant and the non-dominant lower limb, as well as to determine heterogeneities in [18F]-fluorodeoxyglucose uptake within muscles during walking, using three-dimensional magnetic resonance imaging-based whole-muscle segmentations. Large inter-limb differences in uptake were found in the lower leg muscles and in the gluteus medius and gluteus minimus (differences up to 98% were found), whereas the uptake in the thighs was relatively symmetrical between the limbs. Thus, our first hypothesis was rejected. [18F]-fluorodeoxyglucose uptake was not systematically higher in the dominant or the non-dominant limb in any muscle, including when inter-limb muscle volume differences were taken into account. The skew of the standardized uptake values was to the right almost exclusively, indicating that most voxels had a relatively low standardized uptake value, and that a minority of voxels had a relatively high standardized uptake value. Thus, our second hypothesis was confirmed. Finally, rather than being homogeneously distributed in the muscle, the voxels with high standardized uptake value tended to cluster in virtually all muscles, the clustering being strongest in the adductor magnus, the ankle plantar flexors, and the gluteus medius and minimus. Thus, our third hypothesis was rejected.This study aimed to determine asymmetries in [18F]-fluorodeoxyglucose uptake between the limbs in several muscles was unexpected. According to traditional assumptions and a substantial body of literature  in either the dominant or in the non-dominant lower limb across subjects, which was not observed. Finally, a fourth explanation is that a very active muscle could be compensating for reduced activity in another muscle that has a similar functional role. For example, high activity in one of the plantar flexors (e.g. soleus) could be accompanied by a lower activity in the other plantar flexors . Such a trend was not observed; subjects who had a high absolute uptake value in the soleus generally also had a high uptake in the medial and lateral gastrocnemius, and vice versa. Hence, it remains difficult to explain the large asymmetries, and this warrants further research using other methods  in conjunction with the [18F]-fluorodeoxyglucose-positron emission tomography to examine the heterogeneity of glucose uptake in the quadriceps during exercise at different intensity levels -fluorodeoxyglucose were clustered instead of being distributed homogeneously throughout the muscle, especially in the adductor magnus, the ankle plantar flexors, and the gluteus medius and minimus. This suggests that most of the work during walking is performed by limited regions within muscles. Since comfortable level walking is an aerobic activity, and since it is known that the different types of muscle fibers are organized in regions of different composition rather than being homogeneously distributed [18F]-fluorodeoxyglucose-positron emission tomography -fluorodeoxyglucose uptake in the muscles was very variable. Typically, three or four subjects could be grouped into one category of regional uptake  due to varying levels of resting metabolism within muscles. Extending the protocol was not allowed by the medical ethical board at our hospital due to ethical constraints regarding the radiation dose to these healthy subjects. Furthermore, our measurement protocol did not include gait analysis. This would have allowed us to test our visual observation that the healthy subjects walked symmetrically. Third, our walking time was long (90 minutes), which has two disadvantages. One is in transitioning from research on healthy subjects to clinical applications, as patients may change their gait during the prolonged walking task due to pain or fatigue in certain muscles. The other disadvantage is that [18F]-fluorodeoxyglucose-positron emission tomography is a cumulative measurement that provides no temporal information. Therefore, activation of muscles and different regions within each muscle may have shifted during the task, which makes interpretation of the asymmetry and region-specific uptake results difficult. It is possible that the subjects activated their muscles more symmetrically at the onset of walking, and later shifted their strategy to (some) more asymmetrical muscle activations, or vice versa. Future work could focus on examining region-specific [18F]-fluorodeoxyglucose uptake in a simpler, more controlled task, or it could focus on combining [18F]-fluorodeoxyglucose-positron emission tomography with multi-channel electromyography, such that changes in motor unit recruitment and asymmetry can be tracked over time and coupled with the [18F]-fluorodeoxyglucose-positron emission tomography results.This study has some limitations. First, the inter-subject variation in standardized uptake values was high in some muscles, and the current sample size was too small to attribute this to any particular characteristic of these healthy subjects such as age, body mass index or general fitness level. However, high inter-subject variability in muscle activation during walking has been shown to exist with techniques such as electromyography as well –47. Seco18F]-fluorodeoxyglucose uptake in both lower limbs after walking showed that muscle activity (in terms of glucose uptake) was symmetrical in the thigh muscles, whereas it was highly asymmetric in the lower leg and gluteal muscles in many subjects. These findings challenge the common assumption of symmetry between the limbs in healthy subjects. Practically all of the inter-subject differences disappeared when the results were averaged across subjects, which underlines that averaging individual subject data stemming from both limbs may preclude asymmetries from being noticed or reported. Studies that have the goal of validating muscle activation levels using subject-specific musculoskeletal models, will need to be cautious that muscle activity for the most active muscles during the activity  needs to be validated for each subject and each limb individually. As for the [18F]-fluorodeoxyglucose distribution within muscles, there was a majority of voxels that had a standardized uptake value that was lower than the mean standardized uptake value, and there was a minority of voxels with a relatively high standardized uptake value in almost all muscles. Those ‘hot’ voxels tended to cluster rather than being spread homogeneously throughout the muscle, but those clusters were not located in consistent locations in a given muscle across subjects. The inconsistent locations of ‘hot’ clusters highlights the added value of performing a three-dimensional analysis of uptake rather than examining uptake in only a single slice for each muscle. Future research could focus on revealing the relation of active versus resting [18F]-fluorodeoxyglucose uptake levels, on measuring kinematics and kinetics while the exercise is performed, and on combining the cumulative [18F]-fluorodeoxyglucose-positron emission tomography measurement with a temporal-sensitive method such as multi-channel electromyography. When applied in patients, FDG-PET offers the perspective of being able to show which muscles and which parts of muscles are activated less than ‘normal’, and which are more active and might be compensating for other muscles or for underlying pathology. This would be useful information that would be difficult to obtain through other methods. It could have benefit, for example, in studying patients who have undergone joint replacement, who have had an anterior cruciate ligament reconstruction, or who are suffering from neurological disease. Using [18F]-fluorodeoxyglucose-positron emission tomography to measure muscle activity remains an exciting new technique, which has the potential to further improve our understanding of muscle contributions during exercise in many ways, including in detecting differences in muscle activity levels between subjects, between limbs, and between regions within muscles.The volumetric analysis of [


Until recently employment prospects of older workers have been rather poor. In recent years, however, the duration of unemployment among older workers has diminished, meaning that older workers are more frequently hired by employers. Changing employment prospects of older workers are no doubt framed by a decrease in overall unemployment. The aim of this paper, however, is to shed light on the emergence of new inclusive mechanisms by answering three interrelated research questions: (1) where are the job openings for unemployed seniors? (2) How have unemployed seniors been recruited? (3) Why do companies hire older workers. Using Denmark as a test case findings show that job openings are rather frequent in branches with tight labor markets and that characteristics of companies and management are important; for instance, the older the average age of management the higher the inclination to hire unemployed older workers. Findings furthermore show that mouth-to-mouth recommendation and the internet are the most used recruitment channels, while the public employment service is less used. Finally, findings show that qualification, stability and experience are the most reported reasons as to why employers hire unemployed older workers are. The paper is based on a survey with 2,525 valid respondents, response rate: 25.


Cardiovascular disease is one of the most prominent serious conditions whose incidence is linked to abnormal sleep patterns. While it is completely intuitive that lack of sleep can predispose one to illness, the molecular mechanisms that are responsible for such pathological consequences have not been well explored. In a recent manuscript published in Nature, McAlpine and colleagues describe the impact of sleep fragmentation on murine hematopoiesis and how the resulting impact on myeloid biology may comprise a causal molecular link with atherosclerosis.Apoe−/−), resulting in: poor lipoprotein clearance; accumulation of cholesterol in the blood; and development of atherosclerotic plaques.2 When Apoe−/− mice were subjected to sleep fragmentation, they developed larger plaques that contained a significantly higher proportion of myeloid cells compared to controls, suggesting that this would be a valid model to explore the etiology of this disease. Analysis of blood and bone marrow in both Apoe−/− and wild-type mice demonstrated increased myeloid-biased hematopoiesis as a result of sleep deprivation, which correlated with the observation that leukocyte numbers are elevated in humans subject to sleep deficit. Since the hypothalamus is responsible for the production of several sleep-regulating proteins, its function was assessed in mice subject to sleep fragmentation, revealing decreased expression of the neuropeptide hypocretin. In humans, low serum levels of hypocretin are associated with increased risk of heart disease and obesity, suggesting that this could be a major mediator of the link between sleep interruption and heart disease. Importantly, analysis of hematopoiesis in hypocretin knock-out mice (Hcrt−/−) revealed a similar overproduction of monocytes and neutrophils and bone marrow myeloid progenitor expansion to that observed in sleep-fragmented mice, indicating a causal relationship. Transplantation of wild-type bone marrow into Hcrt−/− mice also resulted in heightened hematopoiesis, reinforcing the hypothesis that compromised production of hypocretin in the hypothalamus can promote abnormal hematopoiesis in the bone marrow. In line with this data, combination of the Hcrt−/− and the Apoe−/− genotypes resulted in larger atherosclerotic lesions with greater levels of leukocyte recruitment than observed in control mice with the single Apoe−/− mutation.In order to interrogate potential mechanistic links between cardiovascular disease and sleep deprivation, this study first made use of an atherosclerosis-prone genetic mouse model that harbors a targeted deletion of the lipid transporter apolipoprotein E  compared to analogous cells from wild-type mice, implying a potential mediator of increased myelopoiesis in the setting of decreased hypothalamic hypocretin. In line with this observation, sleep fragmentation was also found to result in elevated production of CSF1 in the bone marrow and transplantation of Hcrt1−/− bone marrow cells into wild-type recipients demonstrated increased myelopoiesis along with elevated levels of CSF1.To delve into the molecular mechanism that links these processes, the authors next used a mouse GFP knock-in reporter line at the locus of the hypocretin receptor, Ldlr−/−).3 These Ldlr−/− mice were transplanted with either wild-type or Csf1−/− bone marrow and were then subject to sleep fragmentation. Recipients of wild-type bone marrow demonstrated increased myelopoiesis and elevated bone marrow levels of CSF1 and developed large atherosclerotic plaques. In contrast, mice receiving the Csf1/−− graft had a reduction in all of these properties, establishing that bone marrow production of CSF1 is a critical facilitator of both atherosclerosis and increased myelopoiesis as a result of sleep fragmentation. Secondly, the authors performed a second type of rescue experiment in order to firmly establish hypocretin as a mediator of this phenotype. Thus, Apoe−/− mice were subject to sleep fragmentation as before, but were implanted with mini-osmotic pumps to deliver hypocretin into the periphery for a period of 8 weeks. Such mice had lower levels of circulating myeloid cells, decreased myeloproliferation and CSF1 in the bone marrow, and developed smaller atherosclerotic plaques (Fig. Finally, the authors sought to complete the mechanistic link between sleep fragmentation, decreased hypothalamic hypocretin, increased production of CSF1 by bone marrow pre-neutrophils, and the generation of atherosclerotic plaques. Firstly, they used a different atherosclerosis-prone mouse model, which harbors a deletion of the low-density lipoprotein receptor (Taken together, this study provides novel molecular insight into the process via which sleep disruption can lead to cardiovascular disease as a result of dysregulation of hematopoiesis. Clearly, these findings are of potential translational relevance, both in terms of identifying biomarkers that might predict risk of cardiovascular disease in individuals with a history of chronic sleep disruption, and in terms of identifying potential novel targets that might facilitate therapeutic intervention in this process. One outstanding mechanistic link relates to whether the increased myelopoiesis observed in this model is, in itself, sufficient to facilitate the enhanced formation of atherosclerotic plaques. Alternatively, it remains to be seen whether the functional properties of CSF1-activated monocytes and neutrophils are altered in a way as to make them more prone to be recruited to such lesions and to more effectively contribute towards plaque formation. If so, it may be that this mechanism is more broadly applicable to the pathology of other diseases, such as those that are mediated by an excessive inflammatory innate immune response. It will also be interesting to determine whether chronic pharmacological induction of pseudo-normal sleep patterns by chronic ingestion of “sleeping tablets”, is also associated with a similar atheroscleric mechanism.


Concussions are difficult to diagnose and symptoms may not appear immediately. As an accurate initial diagnosis has profound implications for the clinical management, there is an unmet need for better diagnostic tools. Fluid biomarkers for CNS injury may represent such tools. These markers are often proteins, peptides or other molecules with selective or high expression in the brain, which can be measured in the cerebrospinal fluid or blood as they leak out or get secreted into the biofluid in response to the injury. Here, we review the literature on fluid markers of neuronal, axonal and astroglial injury and response mechanisms to diagnose CNS injury upon head impact and to determine when the injurious process has resolved. Severe traumatic brain injury (sTBI) is easily diagnosed by clinical examination and standard neuroimaging techniques. However, mild TBI (mTBI) or concussion (the two terms are used interchangeably in the literature), defined as a head trauma resulting in brief loss of consciousness and/or alteration of mental state , is muchA chronic syndrome, likely related to repetitive concussions, has not received much attention until recently. However, in 1928, Martland published a case series describing symptoms such as slowed movement, tremor, confusion and speech problems in a group of former boxers, which he called ‘the punch drunk syndrome’ . In 1973Neuronal dysfunction in concussion is at least partly caused by direct damage to axons and other structures in the CNS. Approximately 15% of concussion patients suffer persisting cognitive dysfunction ,11 and dIt is important to remember that head injury and concussion are not synonyms , which may be a problem in epidemiological studies. It is also clear that different sports and other risk activities have different types of contact and, therefore, probably different types of lesion. What are the damaging types of lesion and what types of damage are reversible and what are not reversible? How long does it take to recover from any particular type of damage? What are the outcomes of playing long-term professional sports today, when everything is faster, the players are heavier and the protective gear for the head appears less effective than protective gear for other parts of the body?We now need systematic research on injury mechanisms in concussion and how they relate to CTE. Fluid biomarkers in TBI may relate directly to brain injury, released principally from neurons, glia or blood vessels or to response mechanisms such as microglial and astrocytic migration and activation, which may be protective or deleterious. Most individuals with concussion do not develop CTE. However, in some individuals there may be a transition from an injury/recovery pattern to a persistent, progressive process representing the initiation of a neurodegenerative process and a transition to the earliest phases of CTE. We hypothesize that this may relate to the extent, frequency and cumulative effect of brain injury together with variation in individual vulnerability to injury. It is possible that intrinsic  risk factors for Alzheimer's, Parkinson's or tauopathies such as progressive supranuclear palsy will also be risk factors for the development of CTE. Identification of individuals in the earliest phases of CTE is of crucial importance in terms of instituting appropriate advice on subsequent exposure, and in selecting individuals for future trials of disease-modifying therapies. Fluid biomarkers have the potential therefore to help to define: the severity of TBI; adaptive and recovery processes following TBI; and the transition between a normal injury/recovery pattern and a currently irreversible and inexorable CTE process. In this review, we start by discussing the different fluids that may be used as samples in which concussion biomarkers may be measured. We thereafter discuss technical aspects of the biomarker analysis. We then review the literature published so far on candidate biomarkers for concussion. Finally, we discuss the special case of CTE and emphasize the need for biomarker development for this disease entity.Cerebrospinal fluid (CSF) is a clear fluid that surrounds the brain, provides mechanical support and helps clear metabolites from the brain parenchyma together with direct transport across the blood–brain barrier, clearance via the glymphatic system and clearance via the recently discovered meningeal lymphatic vessels ,16. The The other major biofluid for the measurement of concussion markers is blood (serum or plasma). Blood is more accessible than CSF but most CNS-enriched markers are present in blood at very low concentrations that necessitate the employment of ultrasensitive techniques that can measure in the femtomolar range . The blood–brain barrier also poses a challenge in the analysis of CNS injury markers in blood. Normally, the blood–brain barrier restricts the release of CNS-enriched proteins and peptides into the bloodstream and, for some molecules, specific transport mechanisms exist. In concussion, there may be a transient opening of the blood–brain barrier, which could increase the blood concentrations of CNS-enriched molecules, also in the absence of direct injury to the structures they are thought to represent. It may thus be difficult to tell to what extent a peak in the blood concentration of such a biomarker reflects CNS injury or blood-brain barrier damage/dysfunction. Reliable blood biomarkers for blood–brain biomarker integrity relatable to CNS injury markers would be a major contribution to the field . FurtheIt is possible that some CNS-derived proteins are eventually excreted into body fluids other than CSF and blood. The presence of the axonal protein tau in saliva has been demonstrated using mass spectrometry . The samMost fluid markers of CNS injury are proteins or protein fragments that can be measured using immunochemical or MS-based techniques (or combinations thereof).ELISA has become established as a standard method for the measurement of proteins in biofluids. The general principle is that a capture antibody directed against one epitope on the target analyte is immobilized on a surface, whereafter sample and labeled detector antibody  are added sequentially between blocking and washing steps to remove unspecific signal. Capture and detector antibodies are in molar excess so that most of the target analyte is captured in a sandwich between the antibody pair. Many ELISAs and ELISA-like techniques can reach lower limits of quantification of 10–100 pg/ml, but measuring even lower concentrations, as is needed for most brain-specific proteins in the blood, is a challenge. Auto-antibodies against the target analyte or heterophilic antibodies  that may react with the antibodies in the assay may block epitopes or bridge the capture and detector antibodies , giving falsely low or high signals. Interference from heterophilic antibodies may be blocked using polyclonal mouse IgG or commercially available blockers.To allow for ultrasensitive measurement, two new techniques have entered the market: Erenna and Simoa . The magMS-based explorative proteomics has been applied to discover novel biomarkers in complex samples such as CSF and plasma for many years. More recently, however, antibody-independent selected or parallel reaction monitoring (SRM or PRM)-based MS techniques have been developed for the quantitative measurement of proteins and protein fragments in a manner that is stable enough to allow for use on large sample series and in clinical laboratory practice . SRM-basCandidate fluid markers of acute mild TBI are summarized in Axonal injury in concussion can be identified and monitored using CSF levels of the intra-axonal proteins neurofilament light (NF-L) and tau ,27, measTau is primarily expressed in thin unmyelinated axons and may respond more to cortical contusions than rotational brain injuries. However, CSF concentrations of tau change after both concussive and subconcussive head blows in a manner similar to NF-L but with a lower amplitude in the changes .Studies in TBI models and on human brain tissue samples, as well as in brain interstitial fluid have demonstrated that APP accumulates in neurons and axons after brain trauma with axonal damage and that there is release of amyloid β  into brain interstitial fluid with plaque formation around damaged axons . In spitet al. used conventional ELISA for detection of NF-L in serum of TBI patients, showing increased serum concentrations in patients with the most severe neurotrauma [In regard to blood biomarkers for concussion, recent data show promising results for tau, when measured on the ultrasensitive Simoa platform discussed above. A tau assay has been established on this platform and the first pilot studies have shown: a strong correlation between serum tau concentrations and neurological outcome in resuscitated cardiac arrest patients , increasrotrauma . The anarotrauma , which iAnother promising blood marker of concussion is the 1176 residue N-terminal fragment of α-spectrin, termed SNTF, which is a protein that accumulates preferentially in damaged axons . SNTF isOther candidate blood biomarkers for brain injury in concussion include NSE, UCHL1, as well as the astroglia-enriched S100B and GFAP . ClinicaCTE is a neurodegenerative disease associated with repetitive head trauma . Althougin vivo markers of the molecular pathology in CTE, preliminary findings from positron emission tomography (PET) scanning using PET ligands for brain tau in retired national football league players have recently been reported; five retired players were compared with five matched controls without a concussion history and displayed higher overall signals of tau deposition in their brains [In regards to r brains . Amyloidr brains .No data on fluid markers of tau and Aβ pathology have been presented so far but clinical biomarker studies with longitudinal follow-up of patients with chronic or progressive symptoms after TBI, for example, the Diagnosing and Evaluating Traumatic Encephalopathy using Clinical Tests (DETECT) study , are in The primary goal of biomarker research in TBI is not to develop techniques to identify moderate to severe brain injury, as such already exist, but rather to identify molecular changes in mTBI/concussion to indicate if the brain was injured by a head injury and to monitor the recovery process. Most current research suggests that the risk of long-term symptoms following concussion is highest in individuals who have received repetitive concussions before the brain has recovered properly . IdentifSeveral promising CSF and blood markers of concussion exist but the field is not yet mature enough to rank them according to diagnostic accuracy. Their potential predictive value in regard to time to complete recovery and risk of incomplete recovery/CTE needs to be established. Further, we need more knowledge on release and clearance mechanisms of the candidate biomarkers and how their blood concentrations relate to blood–brain barrier dysfunction. Another outstanding research question is if additional information could be gained by combining different markers with each other, for example, by quantifying the extent of both the axonal injury and the inflammatory response using different biomarker combinations. All this should be relevant not only for sports-related concussions but also for the many concussions that are not sports related. Information gained using biomarkers for TBI could potentially be used to identify at-risk cases most appropriate for enrollment in clinical research studies and therapeutic trials.Concussion is difficult to diagnose clinically.Objective biomarker tests for brain injury in suspected concussion would be an important diagnostic tool.Such biomarker tests could also help to tell when the injurious process has resolved.Several cerebrospinal tests for brain injury in concussion are at hand.Ultrasensitive assays have made it possible to measure CNS-specific proteins in the blood.


IntroductionDisproportionately large communicating fourth ventricle (DLCFV) is an unusual presentation of communicating hydrocephalus, in which patients with hydrocephalus have a disproportionately enlarged fourth ventricle in the absence of obstructive pathology. We present six cases of DLCFV which, to date, is the largest series of this relatively rare condition. We highlight the significance of diagnosis and its differentiation from trapped fourth ventricle (TFV) and discuss the nuances for optimal management of DLCFV.MethodsRetrospective case series of consecutive patients with DLCFV, managed by the senior author (LT) over a 10-year period.ResultsSix cases were identified, five of whom had previous posterior fossa surgery and one with previous encephalitis. All patients presented with cerebellar signs, the initial group had unsuccessful initial management with typical cerebrospinal fluid (CSF) diversion. Consistent symptom resolution was achieved by the application of negative CSF pressures via external ventricular drainage (EVD), maintained with subsequent ventriculopleural shunt (VPL), valveless lumbopleural shunt (LPS) or valveless ventriculoperitoneal shunt (VPS), or proceeding directly to a low-pressure system.ConclusionsDLCFV is a diagnosis characterised by cerebellar dysfunction, with or without cranial nerve palsies, often in the setting of previous posterior fossa pathology. Optimal management relies on knowledge of this unique diagnostic entity, and use of an EVD at negative pressures to confirm symptomatic and radiological improvement prior to definitive treatment. Communicating hydrocephalus can occur as a result of a number of different pathological processes and is usually characterised by proportionate enlargement of the lateral, third and fourth ventricles . A subgrThe majority of the aforementioned patients present with cerebellar signs (ataxia and nystagmus) and cranial nerve palsies, and often without the traditional signs and symptoms of raised intracranial pressure . Treatment is often difficult and patients either undergo multiple shunt revisions or require additional procedures prior to symptom resolution , 11, 13.Effective management depends on early identification of the condition. It is easy to mistake this entity for a more common communicating hydrocephalus or an isolated fourth ventricle. This is likely to lead to treatment failure or an unnecessary attempt to cannulate the fourth ventricle. Our case series demonstrates that this is a distinct entity characterised by a low-pressure state in patients who are likely to have altered compliance in the posterior fossa, either through disease or previous intervention.We present a retrospective case series of DLCFV, outlining the aetiology, clinical and radiological findings and outcomes for these patients. We present a novel treatment paradigm for this condition, which results in consistent radiological and symptomatic improvement, without a need for multiple surgical interventions and shunt revisions.Consecutive cases of DLCFV managed by the senior author (LT) over the last 10 years (2006-2015) were retrospectively identified  or lumbar drain (LD) was employed. Symptomatic improvement and corresponding resolution of fourth ventricular enlargement occurred only when the drain was set below 0 cm HDefinitive treatment, therefore, sought to establish a low-pressure system capable of maintaining negative pressures. This was in the form of a valveless ventriculoperitoneal shunt , a ventriculopleural shunt (two patients) or a lumboperitoneal shunt .Clinical and radiological outcomeAll patients improved symptomatically following low-pressure cerebrospinal fluid (CSF) diversion, with complete resolution of ataxia, nystagmus and somnolence. No patients required further revision or intervention after low-pressure CSF diversion.Figure We present six cases of DLCFV which, to date, is the largest series of this relatively rare condition. Optimal management was achieved through negative pressure CSF diversion. We highlight the potential pitfalls, salient points and pearls in the management of DLCFV.Presentation of DLCFVIn our case series, DLCFV occurred in patients with previous posterior fossa pathology and there were no ‘idiopathic’ cases observed. The preceding posterior fossa disease or intervention suggests that the aetiology is likely to involve an inflammatory process resulting in altered compliance of the fourth ventricle. All patients with DLCFV characteristically present with cerebellar symptoms of nausea, ataxia and often had nystagmus. DLCFV was often associated with particularly patulous foramen of Lushka on magnetic resonance imaging (MRI), and importantly, all patients had communicating hydrocephalus with a patent aqueduct of Sylvius.DLCFV versus the trapped fourth ventricleClinically, DLCFV and syndrome of the isolated (or trapped) fourth ventricle (TFV) present in a similar fashion. Distinguishing DLCFV from TFV is a critically important. TFV is a form of non-communicating hydrocephalus for which the treatment is very different; it may require a separate shunt into the fourth ventricle and/or additional open/endoscopic procedures to ensure the fourth ventricle communicates with either the third ventricle and/or the subarachnoid space . If applManagement of DLCFV2O. An initial very low pressure setting may encourage return of the ventricles to normal size, required due to pressure-volume hysteresis curve [If there is doubt over the initial diagnosis of DLCFV, confirmatory testing may be performed through a temporary EVD. Symptomatic and radiological improvement should be experienced when the drain is set at progressively lower pressures; sometimes as low as -20 cm His curve . In addiis curve , and in In our experience, DLCFV does not respond to VPS. This may be because our initial shunt management was with a programmable system incorporating an anti-siphon device that would have been unable to generate the low CSF pressure required to resolve the condition, as demonstrated by progressive lowering of EVD drainage pressure. Successful management required the establishment of a low-pressure system (often negative pressure system), for example via a ventriculopleural shunt that is able to generate negative pressures during inspiration.Finally, we caution against attributing the initial lack of response to shunting to shunt failure. Such patients are at risk of being exposed to multiple and unnecessary VPS revisions if early low-pressure CSF diversion is not considered.Literature reviewSince the initial reports by Zimmerman et al. from the USA , DLCFV hManagement of the patients in the literature was varied, with most patients improving with standard VPS. Few studies reported the type of shunt and valve used and therefore, the pressure dynamics of those systems are unknown. It is important to consider that a standard differential pressure valve without an antisiphon device will generate negative pressures in the standing patient that might be sufficient to manage a proportion of these patients.The use of additional shunts, such as fourth ventricular-peritoneal shunts, creates increasingly complex systems with increased risk of shunt failure and need for further operative interventions. In addition, the literature is limited by a heterogeneous definition of DLCFV; some authors report the phenomenon of DLCFV in cases where there is clear fourth ventricular outlet obstruction with a patent aqueduct and no ‘disproportionate enlargement’ of the fourth ventricle, which, in our opinion, is a separate entity of obstructive hydrocephalus and should be managed differently, such as with endoscopic third ventriculostomy , 14. TheStudy strengths and limitationsWhilst this study is limited by the small numbers, this is a rare condition in adults and this series of six patients is the largest on DCLFV to date. The complete and persistent resolution of symptoms after negative pressure CSF diversion was a consistent finding in a heterogeneous group of patients.This case series of six cases of DLCFV illustrates the importance of awareness of this condition in directing definitive management. Traditional VPS, even with a valve set at low pressures, may not result in symptomatic improvement and therefore, a systematic approach using temporary CSF diversion via an EVD to assess the pressures required for fourth ventricular collapse and corresponding symptomatic improvement prior to definitive treatment with either a vVPS, an LPS or ventriculopleural shunt seems to be the most pragmatic approach to these patients.


The ever increasing proportion of aged people in modern societies is leading to a substantial increase in the number of people affected by dementia, and Alzheimer’s Disease (AD) in particular, which is the most common cause for dementia. Throughout the course of the last decades several different compounds have been tested to stop or slow disease progression with limited success, which is giving rise to a strong interest toward the early stages of the disease. Alzheimer’s disease has an extended an insidious preclinical stage in which brain pathology accumulates slowly until clinical symptoms are observable in prodromal stages and in dementia. For this reason, the scientific community is focusing into investigating early signs of AD which could lead to the development of validated biomarkers. While some CSF and PET biomarkers have already been introduced in the clinical practice, the use of non-invasive measures of brain function as early biomarkers is still under investigation. However, the electrophysiological mechanisms and the early functional alterations underlying preclinical Alzheimer’s Disease is still scarcely studied. This work aims to briefly review the most relevant findings in the field of electrophysiological brain changes as measured by magnetoencephalography (MEG). MEG has proven its utility in some clinical areas. However, although its clinical relevance in dementia is still limited, a growing number of studies highlighted its sensitivity in these preclinical stages. Studies focusing on different analytical approaches will be reviewed. Furthermore, their potential applications to establish early diagnosis and determine subsequent progression to dementia are discussed. Dementia is a condition usually affecting people aged older than 65 years that causes major cognitive dysfunction and loss of independence. Recent studies have demonstrated an incidence reduction over the past decades  associatMild cognitive impairment (MCI) was first described in an attempt to fill the diagnostic gap between a healthy state without cognitive impairment and dementia, previously undiagnosed . This inCurrent validated biomarkers for early AD detection include the presence of tau pathology (CSF or PET-Tau), amyloid pathology (CSF Aβ42 or PET-amyloid) and gray matter atrophy. However, as highlighted by the International Working Group, structural and metabolic changes, occurring many years before the onset of clinical symptoms, should induce functional changes which can be well captured with electrophysiological techniques . In thisThe aim of this work is to briefly summarize the state-of-the-art in the study of the preclinical and prodromal stages of AD with MEG. While specific findings regarding later stages of the disease, or from different neuroimaging modalities are briefly touched for the sake of contextualization, it is out of the scope of this work to summarize the vast literature in those fields  estimates the statistical dependence between two time series of brain activity. The evaluation of FC in brain imaging is motivated by the assumption that successful cognitive processing results from distributed and coordinated activity between distant brain regions. FC disruption in AD patients is a well-established finding in different neuroimaging modalities . HoweverAlzheimer’s Disease is considered a disconnection syndrome , since FInterestingly, a significant number of studies also reported synchronization increases over certain areas in early AD. A recent study  showed tStudies in even earlier stages , highlight the existence of hyper-synchronization affecting posterior brain regions, such as the connections between precuneus and inferior parietal lobules . This inSynchronization abnormalities in the preclinical stages during task performance have been scarcely characterized with MEG. While two studies reported overall increased FC in MCI, particularly over interhemispheric frontal sensors , anotherSome useful packages, already mentioned, to calculate FC in neurophysiological datasets are Brainstorm and Fieldtrip. Furthermore, HERMES is a dedicated toolbox for FC analyses .Graph theory offers a promising framework to study how brain networks are organized in normal and pathological conditions. AD patients in particular, show a tendency toward random structure of these networks . AlthougNetwork topology has been also studied in MCI during task performance. Overall, results reflect a tendency toward more random and less efficient networks in patients compared to controls in sensor analyses . This stGraph theory studies have paid special attention to the evolution of those regions with a more central role in the network structure, the so-called hubs. Hubs are known to be particularly affected in AD patients . MEG stuThere is a variety of software freely available specifically devoted to network analyses, such as toolboxes for Matlab for dynamic networks , Gephi , Brain CEarly electrophysiological alterations represent a sensitive biomarker to distinguish between MCI patients and controls . FurtherIn the same vein, FC metrics have demonstrated great utility in predicting AD risk. Hyper-synchronization over parieto-occipital regions during a memory task was related to AD progression in a follow-up study . AccordiAll the above-mentioned evidence endorse the potential relevance of MEG as an early biomarker of AD burden, highlighting its potential to predict future dementia. However, literature is still scarce in comparison with other neuroimaging modalities, hence, more studies with larger populations and validation of the potential biomarkers identified in different samples is a mandatory step in the coming future. Furthermore, no study to date carried a systematic comparison of the different metrics available and their respective ability to discriminate between healthy volunteers and patients, which should be a future step in the literature. Regardless, power analysis have the most abundant and consistent body of literature, probably partly due to their simplicity and relatively easy implementation. FC results are also a very promising tool for the coming future as a biomarker, since they could detect brain alterations potentially more specific than power alterations. Network and entropy analyses in AD count with a smaller and less consistent background to date, which could be due to the difficulty and complexity of the analyses leading to heterogeneous methodology implementation. There are still limitations for MEG as potential AD biomarker, such as the scarce number of MEG systems available worldwide, which limits its development. However, findings relating MEG alterations with current biomarkers such as CSF, amyloid-PET, brain atrophy, hypometabolism and genetic profiles, indicate its high sensitivity to reveal the functional impairment due to underlying AD pathology.DL-S reviewed literature and wrote the paper. NS wrote the paper. FM designed the study and reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Macrophages were assumed to have a moderate life span and lack self-renewing potential, but recent discoveries challenge this dogma and suggest a potentially important role of these cells as long-lived HIV reservoirs. This, combined with new HIV infection animal models, has led to a resurgence of interest in monocyte/macrophage reservoirs. Infection of non-human primates with myeloid-tropic SIV implicates monocyte/macrophage activation and infection in the brain with neurocognitive disorders, and infection of myeloid-only humanized mouse models are consistent with the potential of the monocyte/macrophage reservoir to sustain infection and be a source of rebound viremia following cART cessation. An increased resistance to HIV-induced cytopathic effects and a reduced susceptibility to some antiretroviral drugs implies macrophages may be relevant to residual replication under cART and to rebound viremia. With a reappraisal of monocyte circulation dynamics, and the development of techniques to differentiate between self-renewing tissue-resident, and monocyte-derived macrophages in different tissues, a new framework exists to contextualize and evaluate the significance and relevance of the monocyte/macrophage HIV reservoir. In this review, we discuss recent developments in monocyte and macrophage biology and appraise current and emerging techniques to quantify the reservoir. We discuss how this knowledge influences our evaluation of the myeloid HIV reservoir, the implications for HIV pathogenesis in both viremic and virologically-suppressed PLWH and the need to address the myeloid reservoir in future treatment and cure strategies.In people living with HIV (PLWH) who are failing or unable to access combination antiretroviral therapy (cART), monocytes and macrophages are important drivers of pathogenesis and progression to AIDS. The relevance of the monocyte/macrophage reservoir in PLWH receiving cART is debatable as  Monocytes and macrophages are significant mediators of inflammation, and dysregulation of their inflammatory functions either by direct or bystander mechanisms during HIV infection is a key driver of comorbidities with an inflammatory etiology in PLWH. The significance of macrophage infection in viremic individuals is well established: HIV species within individuals become increasingly macrophage-tropic with disease progression  which expresses both M1 (IL-1R) and M2 (CD206) markers. Mi-polarized macrophages are enriched in HIV+ individuals and preferentially infected by HIV  to study the impact of cART on reservoirs in specific cell types using tissues not readily available from human subjects. Moreover, viral DNA and RNA can be detected simultaneously using Scope technology allowing detection of potentially latent HIV DNA+RNA- cells  of virologically suppressed individuals  but the in situ and distinguish genuine macrophage infection from phagocytosis of infected T cells  model has been invaluable for studying HIV replication dynamics in macrophages. Similar to HIV infection in humans, SIV targets macrophages in addition to CD4+ T cells , 136 and T cells . Using D T cells . This maThe SIV macaque model has also been used to study disease pathologies such as SIV encephalitis (SIVE). Several SIVE NHP models have been developed [reviewed in ], and arMonocyte/macrophage targeted therapeutics have also been evaluated in the SIV model as an approach to minimize neurodegenerative disorders and cardiovascular disease. Campbell et al. investigated the effect of anti-α4 integrin blocking antibody , which prevents monocyte and lymphocyte trafficking into the brain and gut in SIV infected macaques. Early administration during acute infection blocked CNS infection and macrophage accumulation, and administration of the antibody during chronic infection  led to stabilization of neuronal injury , which sin vivo in humans since human macrophage turnover in this model was estimated to be 1.05 days, which is far shorter than the estimated half-lives of normal tissue and MDM, and T cell interactions may be necessary for the persistence of the HIV macrophage reservoir. Nevertheless, data from these models are consistent with persistence and relevance of this reservoir, and are noteworthy due to limited in vivo data from people living with HIV.Humanized mouse models have also allowed targeted studies of the monocyte/macrophage reservoir alone, or in combination with T cells, and its potential contribution in the context of cART suppression or disease states like HIV-associated encephalitis . AraingaIn the MoM model, HIV infected mice have a greater accumulation of human macrophages in the brain compared to non-infected mice. HIV infection of human macrophages resident in the brain of these mice was demonstrated by immunohistochemistry for HIV capsid protein p24 and by detection of HIV RNA using qRT-PCR on RNA from isolated macrophages . This unOther mouse models have been used to evaluate the contribution of the monocyte/macrophage reservoir to HIV associated pathologies, specifically HIVE. In early studies, intracranial injection of HIV-infected human macrophages or microglia into SCID mice were used to produce a SCID-HIVE model  which efin vivo modified qVOA systems allowing for long-term viral outgrowth detection to better capture reactivation of the latent reservoir. These systems involve the xenografting of cells from HIV+ individuals into immune-modified mice and detecting virus in plasma following an incubation period to detect low frequency reactivation of latent proviruses. Two types of mouse-based qVOAs, reviewed by Schmitt and Akkina  into irradiated NSG mice. The mVOA, developed by Metcalf Pate et al. involves injecting immunodeficient NSG mice with large numbers of PBMC or CD4+ T cells, combined with antibody mediated CD8 depletion and, in some cases, CD3 activation   . These c factor) , suggestCurrent cure research is focused on T cells, the primary HIV reservoir. Cure strategies are varied and include CRISPR based gene therapy, vaccines to boost anti-HIV immune response, broadly neutralizing antibody immunotherapy approaches to cure, and the “shock and kill” strategy, in which the latent reservoir is targeted by reversing latency under continued suppressive cART and triggering cell death . This stThe development of new animal models and HIV detection techniques coupled with greater understanding of the scope of monocyte and macrophage biology has significant implications on how HIV myeloid reservoir research is appraised. The circulation of monocytes and trafficking through tissues, including through lymph nodes which are tissue reservoirs of HIV, represents a new avenue by which monocytes can become infected and contribute to HIV persistence. Moreover, the existence of long-lived macrophage populations which can maintain a HIV reservoir via homeostatic cell division is analogous to memory T cell reservoirs and indicates the potential for tissue macrophages to be a viable HIV reservoir. With this greater understanding of how a myeloid reservoir could be seeded and maintained, coupled with mouse studies indicating that the myeloid reservoir can sustain infection and serve as the source of rebound viremia independently of T cell infection, there has been a resurgence in interest in the myeloid reservoir as a legitimate barrier to HIV cure. Current data suggests that the monocyte/macrophage reservoir is very small in PLWH and in non-human primates with cART suppression and is likely to be a minor contributor to viral rebound compared to the latent T cell reservoir. Nevertheless, the persistent monocyte/macrophage reservoir may be relevant in HIV+ individuals on cART through contribution to pathologies but also as a potential viral source which should not be ignored in attempts to cure or treat HIV.MW wrote the article. AJ and AH conceived the topic and contributed to the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Plants produce a plethora of specialized metabolites to defend themselves against pathogens and insects, to attract pollinators and to communicate with other organisms. Many of these are also applied in the clinic and in agriculture. Genes encoding the enzymes that drive the biosynthesis of these metabolites are sometimes physically grouped on the chromosome, in regions called biosynthetic gene clusters (BGCs). Several algorithms have been developed to identify plant BGCs, but a large percentage of predicted gene clusters upon further inspection do not show coexpression or do not encode a single functional biosynthetic pathway. Hence, further prioritization is needed.Oryza sativa and a genome-wide association study (GWAS) of Arabidopsis thaliana. In these data, we identified several links for which the enzyme content of the BGCs matches well with the chemical features observed in the metabolite structure, suggesting that this method can effectively guide bioprospecting of plant BGCs.Here, we introduce a strategy to systematically evaluate potential functions of predicted BGCs by superimposing their locations on metabolite quantitative trait loci (mQTLs). We show the feasibility of such an approach by integrating automated BGC prediction with mQTL datasets originating from a recombinant inbred line (RIL) population of The online version of this article (10.1186/s13104-019-4222-3) contains supplementary material, which is available to authorized users. Plant specialized metabolism is the source of hundreds of thousands of natural products. These molecules play key roles in plant development and ecology as, e.g., defense agents and signals, and are broadly applied as medicines, dyes, flavorings and cosmetics. With the sequencing of hundreds of plant genomes, genome mining has become a new strategy to uncover the biosynthetic pathways towards known molecules of interest as well as to identify pathways towards novel compounds . The recbona fide BGCs; in such cases, multiple enzyme-coding genes—while located adjacently on the chromosome and therefore triggering BGC prediction—do not in fact encode subsequent catalytic steps in one and the same pathway, and their transcription is not co-regulated. Indeed, Wisecaver et al. reported limited overlap between BGCs and coexpression modules they obtained from large-scale transcriptomics data ). Indeed, six mQTLs mapped to different genes in this BGC, and two of these mQTLs mapped specifically to the PEN1 and PEN2 oxidosqualene cyclase-encoding genes. The masses of the metabolites connected to this mQTL represent yet unknown compounds, and further research  is needed to confirm whether these masses belong to arabidiol/baruol derivatives or unrelated metabolites due to, e.g., downstream effects.Of the four experimentally characterized BGCs in in roots –25; hencWe also observed cases in which predicted BGCs may be regarded as putative false positives, in the sense that they do not encode enzyme-coding genes that are likely to function within the same pathway. E.g., a methoxyglucobrassicin mQTL was associated with the cytochrome P450 gene CYP81F2  within bona fide BGCs by finding cases in which multiple mQTLs overlap with the same predicted BGC  and are likely to represent biosynthetically connected molecules, based on e.g. having defined mass differences between them.Finally and perhaps most interestingly, 23 predicted gene clusters showed only overlap with mQTLs of metabolites that have not been annotated yet, showing a clear potential for discovery of novel enzymes that can be tested through synthetic biology approaches to identify novel chemistry. Among such predicted gene clusters, one may identify likely Arabidopsis mQTLs for enzyme families known to be involved in biosynthetic pathways using all profile Hidden Markov Models from plantiSMASH [Of course, it is also possible to look for overlap of mQTLs with enzyme-coding genes in a non-BGC-centric fashion. By means of example, we scanned all ntiSMASH  that arentiSMASH , 28.Our results confirm that indeed (genes in) predicted BGCs can be meaningfully linked to mQTLs explaining variation in their metabolic products by GWAS and RIL-based studies, allowing the prioritization of BGCs for further analysis and the generation of new hypotheses about the functions of these predicted BGCs. Altogether, we conclude that the prediction of BGCs in plant genomes for bioprospecting can become a more powerful tool for discovery when complemented not only with coexpression analysis but also with unsupervised metabolomics linked to genetic variation. Most importantly, this allows identifying predicted BGCs that overlap with genomic loci associated with the abundance of unknown molecules, which would constitute candidates for further investigation. At the same time, this makes it possible to distinguish these from genomic loci involved in the biosynthesis of well-known molecules whose biosynthetic pathways are known not to be clustered.Thus, the metabolomics-based systems genetics approach described here has the potential to become an important technology for the systematic genome-wide assessment of biosynthetic genes that can be prioritized for heterologous expression using the latest synthetic biology methodologies .Although we were able to predict several links between BGCs and metabolites, we did not have the resources to experimentally validate these links through mutagenesis or heterologous expression. It is possible that some mQTLs in fact represent indirect effects. Also, the RIL data from rice resulted in relatively broad mQTL regions, in which other genes may be hidden that could be causative of the metabolic differences underlying the QTLs.Additionally, the metabolomics datasets were limited to shoots and leaves, while many key metabolites may be specifically produced in roots. Using a larger number of relevant conditions in the future (including root metabolomics and samples from biotic or abiotic stress treatments) will make it possible to connect metabolites to gene clusters and gene cluster-like genomic loci on larger scales.Arabidopsis metabolomics study applied here, we did not generate dedicated metabolite fragmentation data, such as MS/MS and MSn spectra [Finally, in the  spectra , making  spectra –32 will Additional file 1. List of predicted BGCs, list of mQTLs, list of BGCs with mQTLs, list of matches for mQTL genes with profile Hidden Markov Models related to scaffold biosynthesis, overview of LCMS clusters.Additional file 2: Figure S1. Coexpression analysis of two dioxygenase-coding genes and two dirigent-enzyme-coding genes on rice chromosome 1. Figure S2. Coexpression analysis of the kaempferitrin-associated predicted BGC in Arabidopsis thaliana.


Oncogenic activation of RAS isoforms leads tumor initiation and progression in many types of cancers and is gaining increasing interest as target for novel therapeutic strategies. In sharp contrast with other types of cancer, the importance of RAS in breast tumorigenesis has long been undermined by the low frequency of its oncogenic mutation in human breast lesions. Nevertheless, a wealth of studies over the last years have revealed how the engagement of RAS function might be mandatory downstream varied oncogenic alterations for the progression, metastatic dissemination, and therapy resistance in breast cancers. We review herein the major studies over the last three decades which have explored the controversial role of RAS proteins and their mutation status in breast tumorigenesis and have contributed to reveal their role as supporting actors, instead of as primary cause, in breast cancer. Harvey rat sarcoma viral oncogene homolog (HRAS), NRAS, encoded by neuroblastoma RAS viral (v-ras) oncogene homolog (NRAS), and KRAS4A and KRAS4B, alternative splice variants of the Kristen rat sarcoma viral oncogene homolog (KRAS). RAS proteins function as binary switches that cycle between an active (“on”) GDP-bound to an inactive (“off”) GTP-bound state depending on the activation status of the upstream receptors. The switch between “on” and “off” states is modulated by the complementary action of enzymes that promote either the GDP to GTP exchange  or the conversion back to GDP-bound form . The multiplicity of GTPases and GAPs allows the function of RAS to be finely regulated depending on the variety of extracellular and intracellular signal inputs. RAS proteins activate a hierarchical cascade of intersecting pathways which modulate biological functions such as cell proliferation, apoptosis, motility, metabolism, immune evasion. Dysregulation of RAS function is largely associated with tumorigenesis. This may rely either on genomic mutations which alter the RAS-intrinsic structure or on alteration of RAS regulating factors, which enhances RAS expression and activity.RAS G-proteins mediate the signal transduction through the transmembrane receptors. In humans, there are four highly homologous ≈21 KDa RAS isoforms: HRAS, encoded by the This review is an effort to recapitulate more than 30 years of studies on RAS oncogenes and breast cancer, with the aim to reconcile two apparently conflicting evidences arisen by these studies: (1) experimental studies on cancer cells and murine models have demonstrated that RAS oncogenes and their mutations have a strong potential in breast cancer initiation and progression as it does in other type of cancers; (2) clinical studies have demonstrated that, actually, the incidence of tumorigenic RAS mutations in human breast cancers is marginal, in sharp contrast with other types of cancer.in vitro and could provide them with the ability to induce tumors in mice , and estrogen-insensitivity –18. Singin vivo tumorigenesis  is a heterogeneous group of tumors defined on the basis of their negativity for Estrogen receptor, Progesterone Receptor and HER2. They account for ≈15% of breast tumors and are statistically associated to poor prognosis. TNBC phenotype and clinical outcome partially overlap those of the basal-like breast cancer subtype previously identified on the basis of the gene expression profiling , 53, altOther than being a potent mediator of tumor transformation and progression, RAS might also confer resistance to therapies in breast cancer . RAS indThe role of RAS in breast tumorigenesis and resistance to therapies provides the rationale to assess RAS as target in breast cancer treatment. Three decades of studies contributed to rise the notion that RAS oncogenes are “undruggable,” due to its conformational architecture, which lacks of pockets to facilitate the binding of small inhibitors, and its picomolar affinity for the nucleotide substrate. However, recent technologies and approaches have renewed the challenge to thwart cancer by targeting RAS directly or through its downstream signaling pathways. Direct approaches currently under investigation are addressed to enhance GTP hydrolytic activity of RAS, to inhibit its nucleotide exchange function or to prevent its interaction with downstream effectors , 92. TheA reliable alternative approach consists in blocking the RAS downstream pathways . As for in vitro, the marginal incidence of RAS mutations in clinics does not support a primary role of RAS proteins in breast tumor etiology. Nevertheless, a wealth of studies over many years have demonstrated the importance of RAS function in the progression, metastatic dissemination and therapy resistance in breast cancers, regardless the molecular trigger they are initiated by, thus contributing to draw for RAS proteins a crucial role as supporting actors in breast tumorigenesis.Decades of studies have contributed to unveil the primary role of RAS oncogenes in leading tumor initiation in many types of cancers. For reasons that are still unknown, breast cancer is not amongst them. Although oncogenic RAS is able to transform mammary cancer cell lines MG: conceptualization, writing, and financial support.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.