Incidence of neurologic manifestations associated with Zika virus infection has been increasing. In 2016, neuropsychological and cognitive changes developed in an adolescent after travel to a Zika virus–endemic area. Single-photon emission computed tomography and neuropsychological testing raised the possibility that Zika virus infection may lead to neuropsychiatric and cognitive symptoms. Although the full clinical spectrum of complications associated with Zika virus disease remains unclear, evidence of an association between Zika virus infection and diseases of the nervous system is growing  results positive for Zika virus. Serologic results were positive for Epstein-Barr virus, consistent with the patient’s history of infection 15 months earlier  Table 1.Symptoms failed to improve, and 13 days after symptom onset, the patient was hospitalized for further work-up. Fourteen days after symptom onset, magnetic resonance imaging of the brain with contrast showed no abnormalities. Serum was positive for dengue virus IgG and IgM  Table 2.After discharge, the patient’s symptoms of regression, disinhibition, and cognitive impairment persisted. Seven weeks after symptom onset, single-photon emission computed tomography revealed mildly heterogeneous cerebral cortical perfusion, with focal moderate-to-severe hypoperfusion in the inferior left frontal region  Figure 2Although we cannot entirely rule out Epstein-Barr virus as a possible trigger, the length of time between previous infection and onset of neuropsychological symptoms would be unusual. In addition, although it is impossible to exclude contributions of coinfection from other mosquitoborne viruses , given the Zika virus positivity on RT-PCR, the patient’s condition met criteria for definitive Zika virus infection and the CSF IgM titer was consistent with CNS involvement of Zika virus. The changes on single-photon emission computed tomographs and neuropsychological test scores raise the possibility that Zika virus infection may trigger neuropsychiatric and cognitive symptoms. Although we cannot prove that the patient’s symptoms were related to Zika virus, clinicians should be aware of this potential association and the value of closely monitoring patients with Zika virus infection.Outpatient and inpatient laboratory results, neuropsychological test results, photograph of rash, and single-photon emission computed tomography and magnetic resonance images for an adolescent returning from a Zika virus–endemic area, 2016.


Influenza virus infections pose a significant threat to public health due to annual seasonal epidemics and occasional pandemics. Influenza is also associated with significant economic losses in animal production. The most effective way to prevent influenza infections is through vaccination. Current vaccine programs rely heavily on the vaccine's ability to stimulate neutralizing antibody responses to the hemagglutinin (HA) protein. One of the biggest challenges to an effective vaccination program lies on the fact that influenza viruses are ever-changing, leading to antigenic drift that results in escape from earlier immune responses. Efforts toward overcoming these challenges aim at improving the strength and/or breadth of the immune response. Novel vaccine technologies, the so-called universal vaccines, focus on stimulating better cross-protection against many or all influenza strains. However, vaccine platforms or manufacturing technologies being tested to improve vaccine efficacy are heterogeneous between different species and/or either tailored for epidemic or pandemic influenza. Here, we discuss current vaccines to protect humans and animals against influenza, highlighting challenges faced to effective and uniform novel vaccination strategies and approaches. Type A (IAV) and type B (IBV) influenza viruses are responsible for yearly epidemics of respiratory disease in humans. IAVs infect a broad range of avian and mammalian species, including humans. IBVs are considered primarily a human pathogen. According to the World Health Organization (WHO), ~300,000 deaths occur annually worldwide associated with seasonal influenza infections . Despite many advances in terms of vaccine manufacturing and production, the technology available for influenza vaccines differs little from its origins and continue to face significant shortcomings about availability and/or efficacy. In this review, we will discuss the use of vaccines to prevent and control influenza in animals and humans. We will highlight challenges faced to establish an effective and standardized vaccination program and discuss new approaches that are being tested to address these issues.Vaccination remains the most efficient and cost-effective means to prevent and control influenza in human and animal populations. Vaccines rely on the effective stimulation of the immune response against the virus, mostly against the surface glycoprotein hemagglutinin (HA), the primary immunogen of influenza viruses. Influenza vaccines were a high priority for the U.S. military since the Spanish influenza pandemic of 1918–1919 when 1 in every 67 soldiers died from influenza-related infections. However, it was not until 1946 that influenza vaccines became available for the general population  segments that encode for at least 10 viral proteins . Once in Galliformes, IAVs of the H5 and H7 subtypes can become HPAIVs due to accumulation of basic amino acids in the cleavage site of the HA0 precursor protein. IBVs are usually restricted to humans, and two antigenically distinct lineages circulate, the Victoria-like and the Yamagata-like lineages.The HA is a type I glycoprotein present as homotrimers where each monomer consists of two di-sulfide-linked HA1 and HA2 subunits after cleavage of the HA0 precursor. The HA ectodomain also consists of a globular head domain, and the stalk or stem domain, which are responsible for receptor binding and membrane fusion, respectively  formulations . However, two antigenically distinct lineages of influenza B viruses have circulated globally since the mid 1980's with limited cross-protection between them  through colostrum to their litters, in two to three doses before farrowing  or virus neutralizing (VN) assays. HI-titers ≥1:40 are considered protective and the accepted standards used by regulatory agencies  mediate the cellular arm of the adaptive immune response against influenza and play an important role in viral clearance and cross-protective immunity. Virus-specific CD8+ T cells recognize influenza peptides presented by major histocompatibility complex class I (MHC-I) molecules present on the surface of infected cells, resulting in cell lysis. These CTLs are mainly directed against the relatively conserved internal proteins NP, M1, and the P-complex subunits, which confer a high degree of cross-reactive immune response to various influenza strains in humans and swine  monitors seasonal viruses' evolution by using data obtained year-round from more than 130 national influenza centers located in more than 100 collaborating countries. Meetings are held twice a year to select vaccine strains for the following influenza season. The meetings take place in February for the Northern Hemisphere and in September for the Southern Hemisphere, at least 6 months before their respective influenza seasons. Based on antigenic data from ferret antisera, paired serologic analysis of human samples, and virologic surveillance data, the GISRS recommends that vaccine strains are either maintained or updated to offer more effective protection. Usually following WHO recommendations, each country and its regulatory agencies decide about the influenza vaccines licensed in their territory. In general, each vaccine strain in the vaccine requires an update every 2–3 years, with at least one strain updated each year.Except for vaccine strain selection against equine influenza, for systematic vaccine strain selection or updates is lacking for other animal production systems. A panel of specialists coordinated by the World Organization for Animal Health (OIE) meets annually to analyze surveillance and epidemiological data related to worldwide circulation of IAVs in horses. Genetic sequences and antigenic data are used for recommendations of suitable vaccine strains for inclusion in commercial equine influenza vaccines Paillot, .The U.S. Department of Agriculture (USDA) established a national swine IAV surveillance system in 2009. Similarly, efforts are also being made to intensify surveillance in European countries through the European Surveillance Network for Influenza in Pigs (ESNIP) . This network aims to help recognized emerging strains, provide training and advice, to increase collaboration and data sharing within the scientific community, and to help promote animal influenza research and development.More recently, the OIE along with the Food and Agriculture Organization of the United Nations (FAO) launched a worldwide collaborative network of experts on animal influenza (OFFLU) to support the veterinary community to better manage and reduce the risk of IAVs of animal origin to animal and public health . For humans and horses there are also live-attenuated influenza vaccines (LAIV) available Table . RegardlInactivated vaccines are the most commonly used products for influenza prevention due to the large history of manufacturing practices associated with relatively low production costs, safety, and effectiveness. There are four types of inactivated vaccines: whole inactivated virus vaccines (WIV), split virus vaccines, subunit vaccines, and virosomal influenza vaccines  injection, although intradermal (i.d.) formulations are also available. Current guidelines recommend that children 6 months old to <9 years old receive an extra dose administered 4 weeks after the first vaccination. Efficacy of existing vaccines is highly dependent on the age group, with much lower efficacy in children, the elderly, and adults with underlying conditions.Most swine and poultry influenza vaccines used in the field are inactivated whole virus products combined with potent oil-in-water adjuvants delivered by the i.m. route  are available in several countries for use in humans and horses. Experimental LAIVs are effective in swine and poultry Table . LAIVs hLAIVs for human use were independently obtained by the U.S. and Russia in the 1960's after serial passage in eggs resulting in viruses with cold-adapted, temperature-sensitive mutations that prevented their growth at temperatures higher than 35°C and thus restricting virus replication to the nasal cavity  was licensed in the U.S. in 2003. A quadrivalent version of FluMist received approval in 2012. Overall, adjusted vaccine effectiveness for LAIV against any influenza has been consistent with IIV (~50–60%), showing better effectiveness to some components than others in comparison to IIV  is the only live-attenuated vaccine approved for use in animals. The vaccine has a cold adapted and temperature sensitive virus derived from the wild-type A/eq/Kentucky/1/91 (H3N8) EIV strain and has been proven safe and efficacious for use in horses in the U.S. , monkey kidney cells (Vero), and human embryonic retinal cells (PER.C6)  induced the highest antibody titers compared to other compounds . Interestingly, protection of poultry against a H7N3 strain was not statistically different among various adjuvanted vaccine formulations  to induce cross-reactive antibodies, and/or target internal proteins like NP and M1 to induce cross-protective T-cell response  that protected them against lethal homologous  technology, which uses consensus sequences to design vaccines that represent multiple circulating strains. A COBRA VLP-based H1 vaccine showed broad cross-reactivity to multiple H1 strains and protected mice against H1N1pdm09 challenge either in a monovalent cocktail or in prime-boost regimens  has been extensively explored as a “universal”antigen candidate for vaccines. Anti-M2e antibodies do not neutralize the virus, instead these antibodies are thought to prevent viral budding, mediate killing of infected cells by NK cells or macrophages through ADCC, Fc-opsonization, or complement activation  predominantly target conserved internal influenza virus proteins, such as the NP and M1, and, therefore, generate cross-reactive immune responses to influenza viruses of different subtypes  showed remarkable stability over multiple passages and in different IAV backbones of avian-, human-, and swine-origin. This strategy is safe and effective resulting in protection against low- and high-pathogenic influenza strains in chickens and against swine-origin IAVs in swine  by using splicing mechanisms  are a non-replicating safe approach for different species that eliminates the risk of virus recombination. Vector vaccines can mimic the natural infection when delivered through the intranasal route, and the antigens of interest are expressed in the native conformation, which results in higher antibody specificity. Additionally, this approach can be rapidly produced, is fully scalable and does not require the use of eggs. Not all virus vectors can be used in common for all species, but the concept of vectored-vaccine production tends to be similar between different vector systems. Modified vaccinia virus Ankara  is a highly attenuated strain of vaccinia virus that originated from growth selection on chicken embryo fibroblasts (CEF), and is one of the most commonly used vectors for human vaccines. Goodman et al.  showed iAlthough replication-defective vectors can mediate potent protection against influenza virus infection, replication-competent vectors are considerably more potent since they can amplify transgene expression by several-fold. A single-cycle adenovirus (Ad) vector vaccine expressing PR8 HA resulted in markedly higher HA expression and HI titers in cotton rats than a non-replicating vector, and increased protection against homologous infection -based vaccines, another variation of the nucleic acid-based technology, offers safety advantages in comparison to DNA-based vaccines since it harbors only components required for protein expression, is rapidly degraded and does not interact with the host genome. An intradermal mRNA vaccine encoding the full-length PR8 H1 protected young and old mice from lethal challenge with H1N1, H3N2, and H5N1 viruses, while the same mRNA vaccine encoding HA, NA and NP protected pigs from disease and reduced virus shedding after homologous challenge  and was shown to be safe and immunogenic in healthy adults and the elderly, but not in children  allows for expression of large quantities of HA and/or NA Virus-like particles (VLPs) are recombinant virus particles formed solely by viral structural proteins that do not have any genomic component. Although these are non-replicating particles, they retain the morphology of the virus and, therefore, the antigenicity and can activate innate immunity. VLPs can be produced in several expression systems, including baculovirus-, insect cell-, bacteria-, or plant-derived systems  could greatly improve vaccine production as such approach could create standardized regulations for vaccine licensing for different hosts and streamlined vaccine production processes. Such uniform approach is not necessarily novel. One could argue that production of inactivated vaccines follows similar paths irrespective of the targeted species. Thus, “universal vaccine platforms” for “universal vaccines” would ultimately be a great complement to drastically reduce the disease burden caused by influenza viruses in people and animals.DR and DP have contributed in the conceptualization of the ideas, drafting the manuscript, critical revision of the manuscript, and final approval.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.


The ageing population continues to suffer from its primary killer, cardiovascular disease (CVD). Despite recent advances in interventional medicinal and surgical therapies towards the end of the 20th century, the epidemic of cardiovascular disease has not been halted. Yet, rather than receding globally, the burden of CVD has risen to become a top cause of morbidity and mortality worldwide. Most CVD arises from thrombotic rupture of an atherosclerotic plaque, the pathologic thickening of coronary and carotid artery segments and subsequent distal ischemia in heart or brain. In fact, one-fifth of deaths are directly attributable to thrombotic rupture of a vulnerable plaque. Atherosclerotic lesion formation is caused by a concert of interactions between circulating leukocytes and platelets, interacting with the endothelial barrier, signalling into the arterial wall by the release of cytokines and extracellular vesicles (EVs). Both platelet- and cell-derived EVs represent a novel mechanism of cellular communication, particularly by the transport and transfer of cargo and by reprogramming of the recipient cell. These interactions result in phenotypic switching of vascular smooth muscle cells (VSMCs) causing migration and proliferation, and subsequent secretion of EVs. Loss of VSMCs attracts perivascular Mesenchymal Stem Cells (MSCs) from the adventitia, which are a source of VSMCs and contribute to repair after vascular injury. However, continuous stress stimuli eventually switch phenotype of cells into osteochondrogenic VSMCs facilitating vascular calcification. Although Virchow’s triad is over 100 years old, it is a reality that is accurate today. It can be briefly summarised as changes in the composition of blood (platelet EVs), alterations in the vessel wall  and disruption of blood flow (atherothrombosis). In this paper, we review the latest relevant advances in the identification of extracellular vesicle pathways as well as VSMCs and pericyte/MSC phenotypic switching, underlying vascular calcification. Extracellular vesicles (EVs) are currently considered as important physiological players. Their secretion represents a universally active cellular function in all living organisms from bacteria to humans . EVs areEVs from platelets were first described in 1967 by Peter Wolf from human blood samples, where they were originally referred to as “platelet dust” . FurtherOn the other hand, platelet EVs are known to accumulate in platelet concentrates . TransfuBesides the involvement of platelet-derived EVs in the coagulation process, evidence also points towards a role in immune- and inflammation-related processes. For example, platelet-derived EVs have been shown to influence vascular cells  and leukocytes, thereby changing their phenotype and function. EVs are considered to play an important role in cell-cell communication, their membrane-enclosed content, small size and repertoire of surface receptors facilitate long distance transport within bodily fluids , 8, 34. in vitro experiments. The importance of leukocyte-endothelium signalling in pathophysiological conditions of the vasculature is already well known. More recently the role of platelet EVs in these processes has been demonstrated. In vitro experimentation has shown that shear stress-activated platelet EVs facilitate the interaction between monocytes and endothelial cells. This is facilitated by increases in inflammatory cytokine levels and cell adhesion molecules on both cell types , vascular cells  and their direct microenvironment . EV-medill types . Anotherll types . Platelell types . It has ll types . In thisll types .Interestingly, more than 700 miRNAs have been found to be stored in platelets . Moreove3CR1 on platelets with CX3CL1 on VSMCs  are the most abundant cell source of the vasculature. Unlike most cells, VSMCs arise from several lineages . They arWhile in a contractile state, VSMCs contract and relax to enable blood flow around the body. In this contractile state, they express highly VSMC-specific markers for contractility such as SM-αA, calponin and SM22α. These cells have low motility, hence decreased cellular migration is observed, as well as decreased levels of proliferation and a reduced production of extracellular matrices. This enables the blood vessels to maintain elasticity allowing proper function and delivery of blood supply to the anatomy. When synthetic, VSMCs exhibit a marked decrease in expression for VSMC-specific contractility markers, but express more highly markers for matrix metalloproteinase, collagenase, osteopontin and an increase in production of EVs. Phenotype switching enables VSMCs to maintain blood flow as well as support the vascular niche. During vessel repair, migration and proliferation of VSMCs is necessary. Additionally, increases in expression of growth factors such as PDGF, TGF and VEGF, as well as an increased production of ECM is required to reconstruct vasculature following injury.Terminal differentiation of VSMCs is not a definitive end and it is possible to switch between phenotypes depending on the demand of the vascular niche. Contractile VSMCs are generally referred to as quiescent differentiated cells, whereas the synthetic state is associated with plasticity and appropriately referred to by some as dedifferentiated VSMCs. Several pathologies of the vasculature are associated with VSMC phenotype switching such as atherosclerosis, restenosis, aneurysm and calcification –87. Vascin vitro maintenance of human VSMCs in either synthetic or contractile phenotypes have been identified, the precise mechanisms that enable VSMC phenotype switching remains unknown  patients implicates that SMPD3 is a potential novel therapeutic target to inhibit EV genesis and thus vascular calcification.Today, it is appreciated by many that the role of EVs released by VSMCs is significant in VSMC phenotype switching and calcification. VSMCs have been found to release a variety of EVs when in either synthetic or osteogenic phenotype. A group of VSMC EVs have been identified and somewhat characterised by tetraspanin markers CD9, CD63 and CD81 . Furtherin vivo observation of CD63 co-localisation with calcification in calcified CKD vessels   . AnnexinPropagation of calcification via vitamin K-antagonist (VKA) treatment led to the identification of matrix Gla-protein (MGP) as a potent calcification inhibitor . Twenty in vitro. Sortilin 1 contributes to vascular calcification via tissue nonspecific alkaline phosphatase (TNAP) although regulation of TNAP expression within VSMC calcification remains poorly understood. Notably, Sortilin 1 has been identified as a driver in generation of osteoblasts, while TNAP expression was unaffected. This eludes to a potential novel mechanism in the production of VSMC-derived calcifying EVs, which might be a unique mechanism in vascular calcification , switch to a dedifferentiated synthetic phenotype and later become osteoblast like cells , 137. GeThe perivascular niche houses pericytes, which are present at intervals along microcapilaries and pericyte-like cells are also located in the adventitia of large arteries. Peault and coworkers were the first to show that many pericytes are MSCs , 140. Ho+/CD34+/PDGFRβ+ cells that reside in the adventitia of arteries possess a differentiation capacity towards smooth muscle cells and osteoblasts in vitro  and then underwent a phenotypic switching with loss of contractile VSMC markers and acquisition of synthetic VSMC markers such as Tropomyosin alpha 4 (TPM4) and non-muscle myosin heave chain 2b (nmMHC2b) (+-derived cells acquired nuclear expression of the transcription factor Runx2 indicating differentiation into osteoblast-like cells. Imaging experiments after injection of a fluorochrome conjugated bisphosphonate (Osteosense) indicated that calcified areas showed intense accumulation of tdTomato+ cells. Importantly, genetic ablation of Gli1+ cells in triple transgenic Gli1CreER; iDTR; ApoEKO mice before onset of CKD completely abolished vascular calcification in both the intima and media. Thus, clearly demonstrating that adventitial Gli1+ cells are important progenitors of synthetic VSMCs and osteoblast-like cells in the vascular wall. Adventitial Gli1+ cells can be considered an important therapeutic target in vascular calcification. Interestingly, we observed a Shh domain in the adventitia of human arteries where endothelial cells of vasa vasorum stained positive for Shh, whereas surrounding cells showed Gli1 expression. Further staining experiments in calcified human arteries showed intense Gli1 expression around calcified intima and media areas.The role of adventitial Gli1ng aging . ImportanmMHC2b) . Importa+ population is a homogenous progenitor population or whether several subpopulations exist. Single-cell qPCR analysis of sorted Gli1+ cells for reported markers of adventitial progenitors indicates heterogeneity with three distinct subpopulations (unpublished data). Thus, it will be important to differentiate between these subpopulations. Next, it remains elusive as to whether Gli1-expressing cells of human arteries may also secrete EVs and thereby contribute to vascular calcification. Finally, while our data clearly indicates migration and differentiation of Gli1+ cells during acute injury repair and chronic disease progression, the underlying molecular pathways that activate migration and differentiation of the progenitor population remain obscure. It is currently not known whether EVs are involved in migration and differentiation of MSCs. Further, we must await as to whether adventitial Gli1+ cells produce EVs or interact with EVs from other cells such as platelets, immune cells and VSMCs. Experiments in myelofibrosis demonstrated that a malignant hematopoietic clone in particular megakaryocytes can activate Gli1+ cells to leave their niche partly by CXCL4 release (+ cells. Further studies are needed to answer whether EVs are involved in Gli1+ cell recruitment and differentiation.While these data clearly demonstrate an important role of adventitial MSC-like cells in vascular calcification there are still various open questions that need to be answered . It is u release . Thus, iThe biology of vascular disease and calcification is complex and still poorly understood with respect to cause and consequence of players and pathways, and thus complexity increases continuously by ongoing elucidation of novel players and pathways. A subset of EVs act as mediators of cell-induced extracellular matrix calcification in the pathogenesis of cardiovascular disease. On the other hand, it has become evident that platelet and cellular vesicles play an important role in cellular communication. Whether such communication from platelet EVs can transpire through endothelial cells to the media and adventitia remains unknown, and so a role for EV crosstalk in calcification remains open with promise.LS wrote the manuscript, supervised writing process and was responsible for the final version; AA wrote the manuscript; DK wrote the manuscript; MH wrote the manuscript; RRK wrote the manuscript; RK wrote the manuscript and was responsible for the final version.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.


Over the last decades, the importance of cerebellar processing for cortical functions has been acknowledged and consensus was reached on the strict functional and structural cortico-cerebellar interrelations. From an anatomical point of view strictly contralateral interconnections link the cerebellum to the cerebral cortex mainly through the middle and superior cerebellar peduncle. Diffusion MRI (dMRI) based tractography has already been applied to address cortico-cerebellar-cortical loops in healthy subjects and to detect diffusivity alteration patterns in patients with neurodegenerative pathologies of the cerebellum. In the present study we used dMRI-based tractography to determine the degree and pattern of pathological changes of cerebellar white matter microstructure in patients with focal cerebellar lesions. Diffusion imaging and high-resolution volumes were obtained in patients with left cerebellar lesions and in normal controls. Middle cerebellar peduncles and superior cerebellar peduncles were reconstructed by multi fiber diffusion tractography. From each tract, measures of microscopic damage were assessed, and despite the presence of unilateral lesions, bilateral diffusivity differences in white matter tracts were found comparing patients with normal controls. Consistently, bilateral alterations were also evidenced in specific brain regions linked to the cerebellum and involved in higher-level functions. This could be in line with the evidence that in the presence of unilateral cerebellar lesions, different cognitive functions can be affected and they are not strictly linked to the side of the cerebellar lesion. Written informed consent was obtained from each subject before study initiation.2, slice thickness = 3 mm); 2) fast-FLAIR ; 3) 3D T1-weighted Modified Driven Equilibrium Fourier Transform (MDEFT) scan ; 4) diffusion weighted Spin-Echo Echo Planar Imaging (SE EPI) along 61 non-collinear directions . TSE scans were reviewed to exclude the presence of macroscopic brain abnormalities.All subjects underwent an MRI examination at 3T , including the following acquisitions: 1) dual-echo turbo spin echo [TSE]  and anatomically localized with reference to the SUIT atlas.For each patient, a detailed assessment of the macroscopic cerebellar lesion was performed on high-resolution T1-weighted images. The cerebellum was normalized separately to the Spatially Unbiased Atlas Template of the cerebellum and brainstem (SUIT) . Each leDue to the fact that the cerebellum is notorious for having pulsatile artifacts from nearby pulsating blood vessels, we first examined visually the raw diffusion data  for any such degradation and found the data quality not to be noticeably affected by this kind of artifacts.Correction for eddy currents and small head movements was done on DTI volumes by means of affine registration to the first non-diffusion weighted volume using FSL . After bMCP and SCP were reconstructed using tractography based on two multi-fiber models implemented in Camino . Q-Ball X2 = 3,01, df = 1, p = 0.08), sex was also set as covariate.A voxel-wise analysis was performed in order to compare diffusivity white matter changes between patients and healthy controls, restricting the comparison to the voxels of the MCP and SCP, based on the average tract masks obtained as described above and similarly to . T-contrhttp://www.fil.ion.ucl.ac.uk/spm/), Wellcome Trust Centre for Neuroimaging, Institute of Neurology, University College London, UK) [T1 volumes were segmented into grey matter (GM) maps and registered to MNI space by means of the “New Segment” and “DARTEL” routines in SPM8  . VBM staT-contrasts were evaluated with voxel significance set at p < 0.0001 and corrected for family-wise error (FWE) at cluster level with significance level chosen for p < 0.05.The cerebellar lesion distribution is summarized, for each patient, in Specifically, cerebellar lesion distribution for each patient is illustrated in Additionally, the overall white matter difference was assessed between patients and controls. As showed by the t-test analysis, no differences between groups were detected in terms of total cerebral white matter volume (p = .99).MCP and SCP were successfully reconstructed in patients and HS See . Fig 3 sNone of the considered T1 volume scans were affected by macroscopic artifacts as assessed by visual examination. Comparisons were performed between patient group and HS. Compared to HS, patients showed a widespread pattern of regional GM loss, which involved both cerebral hemispheres. Specifically, a pattern of GM loss was found to affect the contralateral thalamus, caudate, orbitofrontal and paracingulate cortices. Additionally, ipsilateral caudate and putamen were found to show a pattern of GM loss. These results are illustrated in In the present study we aimed at reconstructing MCP and SCP and describing the pattern of white matter alterations associated with unilateral cerebellar lesions. We performed diffusion-based tractography to assess the sub-voxel organization/disruption of these tracts. Previous DTI studies have identified and isolated the cerebellar projections to prefrontal and parietal cortices in healthy subjects –22, and Different arguments can be put forward to explain this result. Firstly considering the extensive literature, cited above, indicating that a unilateral cerebellar lesion may functionally and structurally affect cerebral cortex bilaterally, it might be hypothesized that the bilateral cortical impairment may rebound on the functionality of the lesion-free cerebellum inducing the observed myelin peduncle damages.On the other hand, resting state functional MRI (fMRI) studies widely also demonstrated functional coherence between the two cerebellar hemispheres –52 and dHowever, another possibility has to be considered. Bilateral microstructural alterations in the peduncles might be related to closeness to the ischemic lesion. Indeed, a different grade of structural alterations in perilesional chronic ischemic areas is a well-known phenomenon . The DTIOverall, although the precise mechanism inducing bilateral abnormalities in MCP and SCP is still unclear, its presence questions the interpretation of functional and structural alterations observed in the cerebral cortex after unilateral damage of the cerebellar efferents.A limitation of the study that needs to be discussed is that, due to the small sample of patients recruited, direct correlation between GM changes, WM structural alterations and cognitive performances was not attempted. However, it should be considered that the strict inclusion criteria clearly affected the recruitment rate. In spite of these limitations, present results highly support the hypothesis of a bilateral cerebello-cortical functional whose confirmation will require further studies involving larger populations of patients. This will also allow to explore the relationship between brain characteristics and clinical outcomes of patients, as well as to better establish the direct causality between GM changes and WM structural alterations. To conclude, present findings indicate that, in face of a unilateral cerebellar lesion, bilateral changes in the cerebellar peduncles microstructure can be observed. Consistently, cerebral GM reduction can be found without lateralization. Altogether, these structural observations may provide important insights into understanding cerebro-cerebellar interaction in health and disease. This latter aspect is of particular value considering the increasing interest of cerebellar neuromodulation to treat different CNS disease –59.S1 File(ZIP)Click here for additional data file.S2 File(ZIP)Click here for additional data file.


Spontaneous abortion is one of the most important complications of pregnancy with short and long adverse psychological effects on women. This study assesses the implications of a spontaneous abortion history has on women’s psychiatric symptoms and pregnancy distress in subsequent pregnancy less than one years after spontaneous abortion.A case-control study was conducted on pregnant women of Babol city from September 2014 to May 2015. In this study, 100 pregnant women with spontaneous abortion history during a year ago and 100 pregnant women without spontaneous abortion history were enrolled. All the participants in two groups completed the Symptom Checklist-90-Revised (SCL-90-R), and pregnancy Distress Questionnaire (PDQ).Women with spontaneous abortion history had significantly higher mean of many subscales of SCL-90  more than women without spontaneous abortion history. Also, women with spontaneous abortion history had significantly higher mean of two subscales of PDQ concerns about birth and the baby, concerns about emotions and relationships) and total PDQ more than women without spontaneous abortion history.Pregnant women with less than a year after spontaneous abortion history are at risk of psychiatric symptoms and pregnancy distress more than controls. This study supports those implications for planning the post spontaneous abortion psychological care for women, especially women who wanted to be pregnant during 12 month after spontaneous abortion. Spontaneous abortion is the unexpected loss of pregnancy before the 20th week. It occurs in 12-30% of all confirmed pregnancies.7The symptoms of anxiety and depression, which occur after spontaneous abortion, may also extend into a subsequent pregnancy.10Although during the last decade there has been a fast growing literature on the emotional impact of spontaneous abortion elevated rates of psychological morbidity in the early months following the pregnancy loss, few studies assessed the adverse effects of prior spontaneous abortion on increasing levels of psychiatric symptoms on subsequent pregnancy. A study reported that women with a spontaneous abortion history had significantly higher pregnancy-specific anxiety at trimester one than women with no spontaneous abortion history.11As adverse effects of prior spontaneous abortion on increasing levels of psychiatric symptoms on subsequent pregnancy less than 12 months is controversial in previous studies,This cross-sectional (case-control) study was conducted between September 2014 to May 2015 in pregnant women of seven private and public health prenatal clinics of Babol city. Available sampling was utilized to recruit the pregnant women based on their eligibility.Eligible women who were more than 18 years old, with more than five years of education, and willingn to participate in the study were invited to enter the study. Socio-demographic variables and pregnancy information were obtained during the interview. Subjects were considered the spontaneous abortion history group (case) via self-reported history of spontaneous abortion during a prior pregnancy which terminated less than a year ago. One hundred patients diagnosed with spontaneous abortion history and uncomplicated pregnancy agreed to participate in the study. A control group, consisting of 100 patients with no history of spontaneous abortion, no uncomplicated pregnancy was selected from the patients admitted to other outpatient clinics during the study period. Two hundred pregnant women participated in the study.The researcher distributed the questionnaires to the respondents during prenatal visit. All the participants in two groups completed the Symptom Checklist-90-Revised (SCL-90-R), and pregnancy Distress Questionnaire (PDQ). Ethical approval was granted by the Medical Education Ethic of Committee at Islamic Azad University.The psychological symptoms were assessed with the widely-used Symptom Checklist-90-Revised (SCL-90-R), a self-rating inventory with 9 clinical scales for somatization, interpersonal sensitiveness, obsessive-compulsiveness, hostility, phobic anxiety, paranoid ideation, depression, anxiety and psychoticism. Global Severity Index (GSI) were considered to be the measures of overall psychological symptoms.The PDQ  is a reliable and valid measure of pregnancy-specific stress in assessing pregnancy-specific stress . The PDQ is a 12-item scale that provides three subscales: concerns about birth and the baby, concerns about weight/body image, and concerns about emotions and relationships. Each item is rated on a 5-point Likert scale ranging from 0  to 4 (extremely). PDQ has a possible range of 0–48 for a total score, 0–24 for birth concerns, and 0–12 for both body concerns and relationship concerns.12Two groups of subjects were compared in demographic status using Chi square test. Two Multivariate Analysis of Variance Models (MONOVAs) was used to compare the mean of all variables between two groups.There was no difference between pregnant women with miscarriage history and without miscarriage history in characteristic baseline. Our data support the conclusion that pregnancy distress was higher in women with spontaneous abortion history than controls. A study confirmed that perinatal loss was associated with an increased likelihood of several mental disorders.11Pregnant women with spontaneous abortion history reported higher psychiatric symptoms and pregnancy distress than women without spontaneous abortion history. First, because spontaneous abortion may also put in doubt a woman’s sense of self-worthiness. Second, women who are pregnant after spontaneous abortion can have the feeling of lost control. Third, approximately 50% of women who have spontaneous abortion may conceive within a year following their loss. Studies have emphasized that the effects of spontaneous abortion on women’s anxiety levels during a subsequent pregnancy are of great interest.20First, the cross-sectional nature of our study prevents any conclusion regarding causality. Second, this study did not assess psychiatric disorders in terms of clinical significance, but only in relation to specific pregnancy concerns that women are experiencing, via utilization of the SCL-90 and PDQ.In summary, this study has suggested that pregnant women with less than a year after spontaneous abortion history are at risk of psychiatric symptoms  and pregnancy distress (concerns about birth and the baby and concerns about emotions and relationships) more than controls. This study supports those interventions for planning the post spontaneous abortion psychological care for women, especially women who wanted to be pregnant during 12 month after spontaneous abortion.


Human peripheral blood mononuclear cells (PBMCs) are the key drivers of the immune responses. These cells undergo activation, proliferation and differentiation into various subsets. During these processes they initiate metabolic reprogramming, which is coordinated by specific gene and protein activities. PBMCs as a model system have been widely used to study metabolic and autoimmune diseases. Herein we review various omics and systems-based approaches such as transcriptomics, epigenomics, proteomics, and metabolomics as applied to PBMCs, particularly T helper subsets, that unveiled disease markers and the underlying mechanisms. We also discuss and emphasize several aspects of T cell metabolic modeling in healthy and disease states using genome-scale metabolic models. PBMCs are comprised of several classes of immune cells, including T cells (~70%), B cells (~15%), monocytes (~5%), dendritic cells (~1%) and natural killer (NK) cells (~10%)  metabolism. Plasma metabolomics validated the identity of glycoceramide as a marker of emphysema  were also found to be altered , while functional analysis inspired by these transcriptomes suggested differences in the control of cell cycle regulation  obtained from 197 subjects were performed to assess the impact of cis-genetic and epigenetic factors. The major outcome of this study was the identification of 345 molecular trait QTLs (quantitative trait loci) which co-localized with immune disease specific loci  to identify protein biomarker(s) associated with specific pathophysiological processes, and (b) to characterize different subsets of immune cells based on their proteomes. Recently, comparative proteomics using tandem mass spectrometry (MS) was applied to PBMC samples obtained from kidney biopsies of 40 kidney allograft recipients, either with healthy transplants or those suffering acute rejection. A total of 344 proteins were identified, cataloged and mapped to 2905 proteoforms  based profiling of cell surface proteome was used to identify differentially expressed proteins between human Th1 and Th2 cells. Among the differentially expressed proteins, BST2  and TRIM (T cell receptor interacting molecule) were found to be significantly differently regulated  was used to identify 42 IL-12 regulated genes, among which 22 were up- and 20 were down-regulated. Functional characterization of the up-regulated proteins helped to identify a multifunctional cytokine macrophage migration inhibitory factor and a novel IL-12 target gene  based targeted metabolomics was used to quantify glucose derived metabolites in PBMCs of healthy controls, schizophrenia and major depressions. Most of these metabolites were found to be significantly altered particularly in schizophrenic subjects. In addition, ribose 5-phosphate showed a high diagnostic performance for first-episode drug-naïve schizophrenia subjects  in PBMC phospholipids were positively correlated with phagocytosis by neutrophils and monocytes, neutrophil oxidative burst, lymphocyte proliferation, and interferon-γ production. The study also suggested that variations in the fatty acid composition of PBMCs phospholipids might induce subtle variations in immune cell functions as seen in healthy individuals  is a constraint-based mathematical modeling approach that integrates biochemical, genetic and genomic informations within a computational framework  was built with a vision to integrate and analyze biological datasets      algorithm uses cell specific protein abundances to generate genome-scale active metabolic networks  that includes semi-automatically reconstructed GEMs of 773 human gut bacteria . The reconstruction can accommodate metagenomics or 16S rRNA sequencing datasets that can be used to study metabolic diversities among microbial communities  was designed to study the effect of diet on microbial communities , i.e., reactions marked by significant and coordinated changes in the surrounding metabolites following the environmental/genetic perturbations. By combining transcriptome data, it is possible to infer whether the reactions are hierarchically or metabolically regulated  that modulate immune responses. They could also be used to identify resident microbe(s) which perform specialized metabolic functions. Moreover, GEMs can provide mechanistic overview of substrate allocation, microbe-microbe competition for resources and microbe-assisted modulation of the host immune responses. Modeling metabolic interactions among cells- and tissue-specific GEMs using a cellular compartment and/or metabolic intermediates have been previously possible (Bordbar et al., While GEMs mechanistically link metabolic genotypes and phenotypes, at the same time they handle multitude of constraints and variables which could in turn enhance uncertainty of predictions. Therefore, clear standards for GEM reconstruction, solver integration and usability has to be decided prior to the modeling (Orth et al., As indicated in this review, transcriptome, proteome, epigenome and signaling of PBMCs and Th subsets, have been well studied. In comparison, the metabolism of Th subsets and its underlying regulations is so far poorly studied. It is known that metabolism of circulating T cell undergoes dramatic changes under the environmental stress which drives the immunity (Gerriets and Rathmell, PS: drafted the manuscript; EK and MO: provided critical comments and edits to the manuscript; All authors approved the final version of 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.


Endoscopic submucosal dissection (ESD) has been widely accepted as an effective, minimally invasive treatment for superficial esophageal cancers. However, esophageal stricture often occurs in patients with large mucosal defects after ESD. In this review, we discuss various approaches recently researched to prevent esophageal strictures after ESD. These approaches can be classified as pharmacological treatments, esophageal stent treatments, and tissue engineering approaches. Most of the preventive approaches still have their limitations and require further research. With the improvement of current therapies, ESD can be more widely utilized as a minimally invasive treatment with minimal complications. Currently, endoscopic submucosal dissection (ESD) has been widely accepted as an effective, minimally invasive treatment for superficial esophageal cancers, including esophageal squamous cell carcinoma and Barrett's adenocarcinoma –3. CompaEsophageal stricture after ESD can be structurally divided into two categories: (1) a simple stricture, meaning that the stricture is short, focal, and not angulated and has a diameter that will allow the endoscope to pass, and 2) a complex stricture, meaning that the stricture is long (>2 cm), irregular, and angulated or has a severely narrow diameter . In clin a compleSteroids can inhibit inflammation and fibrosis. The endoscopic intralesional injection of triamcinolone acetonide (TA) has been used for the treatment of benign and malignant esophageal strictures , 16. HasAn improper endoscopic intralesional injection carries the risk of bleeding and myofiber atrophy because TA must be injected in the submucosa. Therefore, Mori et al. changed the application method of TA by a prospective study . Twenty The oral administration of steroids has also been widely researched to prevent esophageal stricture due to their anti-inflammatory effects. Yamaguchi et al. reported the effectiveness for stricture prevention in the study of 41 patients who underwent more than three-quarters of circumference circular ESD . Twenty-Some antifibrotic drugs have been studied in the prevention of esophageal stricture. Antifibrotic drugs inhibit the proliferation of fibrous scars. 5-Fluorouracil (5-FU) is an antineoplastic drug. 5-FU combined with TA was researched in regard to the reduction of strictures that occur after subepiglottic surgery . Mizutanβ1), prostaglandin-E2 (PGE2), and interleukin-1 (IL-1), which reduces collagen synthesis and fibrosis [Tranilast is an antiallergic drug that can inhibit the release of transforming growth factor-beta  is a neurotoxin that inhibits the deposition of collagen fibers and improves hypertrophic scars . Wen et Metallic esophageal stents function to expand the esophagus. Metallic esophageal stents were initially researched for the interventional treatment of esophageal fistulas and esophageal strictures caused by malignant esophageal neoplasms , 39. TheSome researchers attempted to use biodegradable stents to treat benign esophageal strictures . ComparePolyglycolic acid (PGA) sheets have been used in implantation surgeries to reinforce sutures . In receExtracellular matrix (ECM) scaffolds can support the growth of epithelial cells, are compatible with perivascular stem cells, and promote wound recovery and esophageal structure remodeling –63. The Cell transplantation is applied to esophageal mucosal defects, inducing early reepithelization and promoting scarless wound healing. The efficacy of implantation of autologous keratinocytes or adipose stromal cells was proven in several animal model studies –70. HoweA new approach using endoscopic transplantation of cultured autologous cell sheets overcame the limitations of cell migration and low viability rates of transplanted cells. The cell sheets are fabricated on a temperature-responsive culture surface and can be easily harvested. The sheets can be transplanted to the target sites without the use of sutures or adhesives . The traIn brief, we reviewed recent publications on the prevention of esophageal stricture after ESD, all of which inevitably have their own limitations and cannot be widely accepted in clinical application . The steIn recent years, many innovative therapies have shown appreciable feasibility, but they still require controlled clinical research to confirm effectiveness. Some single case reports lack consensus, needing more evidence to sufficiently confirm safety. After the improvement of current therapies, ESD can be more widely utilized as a minimally invasive treatment with minimal complications.


The ability to generate probabilistic expectancies regarding when and where sensory stimuli will occur, is critical to derive timely and accurate inferences about updating contexts. However, the existence of specialized neural networks for inferring predictive relationships between events is still debated. Using graph theoretical analysis applied to structural connectivity data, we tested the extent of brain connectivity properties associated with spatio-temporal predictive performance across 29 healthy subjects. Participants detected visual targets appearing at one out of three locations after one out of three intervals; expectations about stimulus location  or onset  were induced by valid or invalid symbolic cues. Connectivity matrices and centrality/segregation measures, expressing the relative importance of, and the local interactions among specific cerebral areas respect to the behavior under investigation, were calculated from whole-brain tractography and cortico-subcortical parcellation.Results: Response preparedness to cued stimuli relied on different structural connectivity networks for the temporal and spatial domains. Significant covariance was observed between centrality measures of regions within a subcortical-fronto-parietal-occipital network -comprising the left putamen, the right caudate nucleus, the left frontal operculum, the right inferior parietal cortex, the right paracentral lobule and the right superior occipital cortex-, and the ability to respond after a short cue-target delay suggesting that the local connectedness of such nodes plays a central role when the source of temporal expectation is explicit. When the potential for functional segregation was tested, we found highly clustered structural connectivity across the right superior, the left middle inferior frontal gyrus and the left caudate nucleus as related to explicit temporal orienting. Conversely, when the interaction between explicit and implicit temporal orienting processes was considered at the long interval, we found that explicit processes were related to centrality measures of the bilateral inferior parietal lobule. Degree centrality of the same region in the left hemisphere covaried with behavioral measures indexing the process of attentional re-orienting. These results represent a crucial step forward the ordinary predictive processing description, as we identified the patterns of connectivity characterizing the brain organization associated with the ability to generate and update temporal expectancies in case of contextual violations. The brain is capable of allocating attention dynamically on the basis of expectations concerning events occurrence, and this ability is critical for goal-directed behavior in a changeable environment. Indeed, real world information is often dynamic and imprecise, and the ability to derive timely and accurate inferences about changing contexts is crucial, given that prospective action control bears the potential for a successful interaction with the environment.Behavioral findings  evidenceAccordingly, computational approaches  have conThat’s why defining the neural basis of predictive coding is a major challenge in psychiatry and neuroscience. Conversely, functional Magnetic Resonance Imaging (fMRI) investigations on predictive timing in healthy subjects  have impPharmacological and fMRI evidence suggest that explicit and implicit processes of temporal expectations are functionally dissociable. Indeed, ketamine selectively impaired the ability to use internal estimates of time to make predictions , while pHowever, as far as we know, the neural substrates of the attentional re-orienting in case of invalid trials and of the explicit forms of temporal expectations have been directly compared in one investigation only .In recent years, advances in network neuroscience and graph theory proved to be effective tools in mapping neural connectomics  and implicit  predictive timing using centrality and segregation metrics derived from complex network analysis of diffusion tensor imaging (DTI) data. Specifically, we aimed at identifying the connectivity patterns underlying predictive behavior in terms of local connectedness   and locaTwenty-nine healthy volunteers  were enrolled in the present study. Participants were recruited within the local community through advertising, and consecutively assessed at IRCCS Santa Lucia Foundation in Rome. All participants were screened for current or past diagnosis of any DSM-5 Axis I or II disorder using the SCID-5 Research Version edition  and SCIDAll participants were right-handed, with normal or corrected-to-normal vision. They gave written informed consent to participate after the procedures had been fully explained. The study was approved and carried out in accordance with the guidelines of the IRCCS Santa Lucia Foundation Ethics Committee.∗1200 resolution 1280∗768 pixels, frame rate 60 Hz).Subjects were seated comfortably in a quiet room facing a Toshiba computer screen .Predictive timing was investigated with an adaptation of the ion task , which mStimuli presentation and collection of behavioral responses were controlled using e-Prime 2.0 software and a Serial Response Box Model 200a  images were acquired with the following parameters: TR/TE = 10000/76 ms, b = 1,000 s/mm2, 60 slices, slice thickness = 2 mm, FOV = 224 × 224, acquisition matrix = 112 × 112, resulting in data acquired with a 2 mm3 isotropic resolution. A high-resolution T1-weighted whole-brain structural scan was also acquired (1 mm3 isotropic).MRI data were collected at 3T (Philips Achieva) using a thirty-two channel receive-only head RF coil. A diffusion-weighted spin-echo echo-planar imaging sequence was used to acquire high angular resolution diffusion weighted images (HARDI) . One hunData were preprocessed and analyzed in ExploreDTI v4.8.6 . Data weTensor estimation was performed using a non-linear least square method . Whole-bThe automated atlas labeling (AAL) atlas  was regispatialValidity as the difference between RTs at spatial valid trials and RTs at neutral trials; (2) spatialInvalidity as the difference between RTs at spatial invalid trials and RTs at spatial valid trials. In order to further explore the temporal validity/invalidity effects unmasked by the foreperiod effect, 4 indices were calculated as follows: (1) shortTemporal Validity as the difference between RTs at the short interval for early cue (V540) and RTs at the short interval when no cue was provided (N540); (2) lonTemporal Validityg as the difference between RTs at the long delay for late cue (V1620) and RTs at the long delay when no cue was provided (N1620); (3) shortTemporal Invalidity as the difference in RTs at the short interval for late cue (I540) and RTs at the short interval for early cue (V540); (4) longTemporal Invalidity as the difference between RTs at the long interval for early cue (I1620) and RTs at the long interval for late cue (V1620).Reaction times to target stimuli faster than 100 ms were considered anticipatory and removed from the analysis. The benefit of temporal and spatial cueing on performance  was analyzed in one repeated measures ANOVA, with cue type  as the within-subjects factor. In order to examine the costs of invalid cueing and the potential interaction with the foreperiod length, another repeated measures ANOVA with cue type , validity  and foreperiod  as within-subjects factors was performed. A Bonferroni correction for multiple comparisons was applied. The benefits and costs of spatial cuing were explored by calculating 2 indices: (1) spatial, Invalidityspatial, Temporal Validityshort, Temporal Validitylong, Temporal Invalidityshort, Temporal Invaliditylong)Validity and network topology measures from the eighty-eight selected brain regions were included in the analyses, by investigating reciprocal dependencies. In order to look at relationships without inflating the risk of a type I error (of erroneously concluding that a significant correlation is present) (F > 4 to enter) was chosen. Indeed, the forward stepwise procedure starts with no variables in the model and it tries out the variables one by one, including them if they are statistically significant, thus identifying the best set of predictors that gives the biggest improvement to the model. Simple linear regressions were preliminarily performed to include in subsequent multiple regressions analyses only variables significantly (p < 0.05) related to the behavioral measures considered.Behavioral indices (present) , and to All statistics were performed on StatView statistical software.F = 5.597; p = 0.0061] confirmed that targets appearing in a predictable location or moment in time are detected faster than uncued ones. Post hoc analyses with paired t-test evidenced a stronger effect for the spatial  as opposed to temporal  cueing, indicating that the latter did not significantly affect performance. The second analysis concerning temporal and spatial invalidity costs showed a significant effect of cue type, validity, and foreperiod , a significant cue × validity interaction , a significant validity × foreperiod interaction  and a significant three way interaction between cue type, validity and foreperiod . Post hoc analyses (Bonferroni test) confirmed that for the temporal domain, RTs at the short interval after an early cue were not different from RTs for the medium interval at the medium cue, but significantly slower than RTs for the long interval at the late cue (due to the interaction with the foreperiod effect) [p = 0.0002]. Concurrently, RTs at the short interval for the late cue were slower from RTs at the medium interval after an invalid cue (either early or late) [p < 0.0001] and from RTs at the long interval after an early cue [p < 0.0001], while no difference was observed between the latter two variables. Behavioral performance at the medium interval was therefore discharged from the further indices computation and only RTs at the short and long delay considered (see Table 1).A main effect of cue type  . Further, for the longTemporal Validity effect the same analysis revealed that 67% of total variance was explained by right supramarginal (r = -0.29), left inferior parietal (r = -0.39), left fusiform (r = 0.51) and right insula (r = 0.24) nodes’ degree centrality variability .The covariance between the behavioral benefit of knowing in advance when the target will occur and the centrality of nodes within the whole structural connectivity network was investigated. Statistical analyses were separately performed for both short, Temporal InvaliditylongTemporal Invalidity). Positive and negative correlations between the two indices and nodes centrality of a cortical-subcortical network (see Table 2) were found. Results from stepwise regressions showed that 40% of total variance observed in the shortTemporal Invalidity effect was explained by left putamen (r = 0.44), right paracentral lobule (r = 0.4) and right caudate nucleus (r = 0.32) nodes’ degree centrality variability . Conversely, in the case of the longTemporal Invalidity effect, results from the stepwise regression evidenced a negative relationship between the ability to re-orient attention to a later time point and degree centrality of the left inferior parietal lobe .The covariance between the behavioral cost of receiving incoherent information about the target temporal occurrence and the centrality of nodes within the whole structural connectivity network was also investigated. Again, in the temporal invalid condition, two different variables were analyzed as to explore implicit temporal orienting processes  and the negative one with centrality of the post-central gyrus (r = -0.4)  could explain 40% of the total covariance.The analyses applied to subjects’ ability to orient attention in space evidenced that centrality of two regions was positively and negatively correlated with RTs to stimuli appearing in a predictable location. Results from the stepwise regression evidenced that the observed positive covariance between the spatial)Invalidity was found to positively covary with degree centrality of one region. However, the stepwise regression model produced no significant result .The cost of spatial invalidity on performance  and the prevalence of clustered connectivity around individual nodes was investigated. Simple linear regressions results showed significant negative correlations between the shortTemporal Validity effect and clustering coefficient of nodes within a cortical network (see Table 3). Conversely, significant positive correlations were found between the longTemporal Validity effect and clustering coefficient of nodes within a cortical-subcortical network (see Table 3). The stepwise regression model showed a specific negative relationship between the shortTemporal Validity effect and the clustering coefficient of the right superior frontal cortex, indicating that its’ connectedness with topological neighbors nodes explained 18% of the total variance . Moreover, significant results from stepwise regression analyses indicates that 75% of total variance observed in the longTemporal Validity effect was explained by right putamen (r = 0.5), left paracentral lobule (r = 0.6) and right amygdala nodes’ clustering coefficient (r = 0.6) . The same analysis applied to the two temporal invalidity indices showed significant correlations between the latters and the clustering coefficient of a set of cortical nodes and in a single subcortical region (see Table 3). In the subsequent stepwise regressions, we found that the 50% of total covariance between the whole sub-network’s clustering coefficient and RTs indexing the shortTemporal Invalidity effect  was explained by the left middle frontal gyrus (r = 0.6) and the left caudate nucleus (r = 0.4). Conversely, when temporal expectations had to be updated to a later time point , the clustering coefficient of the right posterior cingulate gyrus (r = -0.4), the right middle temporal pole (r = -0.4) and the left superior temporal pole (r = 0.4) explained the 40% of the observed covariance .The covariance between the four temporal indices . However, such correlation was no longer significant in the stepwise regression model.In case of predictable targets, the clustering coefficient of a single area was found to covary with the ability to orient attention in space . Results from the stepwise regression evidenced that 30% of the total covariance between the whole sub-network’s clustering coefficient and the cueing cost for spatial invalid trials  was explained by the left paracentral lobule’s (r = 0.4) and right putamen’s (r = 0.4) clustering coefficient (Table 3).Conversely, results evidenced positive correlations between the In the present study, we investigated the covariance between performance on a spatio-temporal predictive task and topological measures of complex brain networks. Degree centrality  and clustering coefficient  were computed  and corrTo date, although relevant behavioral  and fMRIHere we found that response preparedness to cued stimuli relies on different structural connectivity networks for the temporal and spatial domains. Specifically, while the use of temporal informative cues was found to covary with centrality and segregation measures within a distributed cortical-subcortical network, its spatial counterpart was related to clustered connectivity around few brain regions. Particularly, the behavioral advantage of being validly cued to a certain spatial location was found to covary with local connectedness  of a region in the right parietal lobe  more anterior relative to previous fMRI reports see . AlthougAs for temporal orienting, significant covariance was observed between centrality measures  of regions within a subcortical-fronto-parieto-occipital network -comprising the left putamen, the right caudate nucleus, the left frontal operculum, the right inferior parietal cortex, the right paracentral lobule and the right superior occipital cortex-, and the ability to respond after a short cue-target delay suggesting that the local connectedness of such nodes plays a central role when the source of temporal expectation is explicit . When loInterestingly, different connectivity clusters were observed when the automatic shifting of attention (related to unexpected premature targets) was separated from the voluntary top–down control of attentional orienting  since clustering coefficient of the left middle frontal gyrus and caudate nucleus covaried with the former process, while the latter was related to the right superior frontal gyrus clustering coefficient.These findings expand prior results  by definAlthough a large corpus of research has implicated complex and distributed networks -including prefrontal, premotor, parietal and insula cortices and comprising also the striatum -, in temIn case of premature targets -target appears at the short interval after a late cue- a bottom–up, automatic grabbing of attention has been previously described and linked to the activity of the posterior extrastriate visual cortex . Here weWhen the interaction between explicit and implicit temporal orienting processes was considered at the long interval, we found that explicit processes  were related to centrality measures of the bilateral inferior parietal lobule. Degree centrality of the same region in the left hemisphere covaried with behavioral measures  indexing the process of attentional re-orienting. These results confirm the key role of the left parietal cortex in instantiating the behavioral benefits of temporal predictability, whether temporal information is conveyed by explicit predictive cues or by the probabilistic information associated with the passage of time . Denselyconnectopathy in SZ. Indeed, neuroimaging investigations evidenced connectivity deficits in patients with SZ, with positron emission tomography (The selectivity of our results, indicating segregated structural connectivity networks for temporal and non-temporal stimuli dimensions, but also for explicit and implicit temporal orienting provides empirical support to the notion that complex networks theory enhances behavioral driven neuroimaging data analysis of predictive timing. Indeed, actual evidence on the neural underpinnings of temporal orienting is currently restricted to task-based fMRI experiments in which the core of the process circuitry is blurred by the recruitment of additional areas related to ancillary cognitive/sensory processes. Conversely, a task-independent structural neuroimaging approach within a network-based modular framework provides quantitative information on anatomical brain connectivity, at both global and regional levels, thus unraveling the lasting patterns of relations among brain areas for supporting the process under investigation . We alsomography , fMRI (Fmography  and diffmography . Here wemography  . Such pmography , and redmography . Moreovemography  that parmography .Before concluding, few limitations have to be acknowledged. First, the relatively small sample size might reduce the impact of our findings and rescale the assertions here discussed. However, we intended to provide preliminary evidence of the neural architecture subtending the ability to optimize prospective motor behavior as a function of informative cues. In fact, complex and distributed networks have been thus far implicated in predictive timing, while aberrant connectivity has been suggested to underlie failures in predictive coding . In addiFP conceived the experimental design, supervised the data analysis, contributed to results interpretation, and wrote the paper. VC conceived the experimental design, collected the data, performed the behavioral data analysis, contributed to results interpretation, and wrote the paper. GS supervised the data analysis, contributed to results interpretation, and revised the paper. TG conceived the experimental design, performed the MRI data analysis, contributed to results interpretation, and wrote the paper. DV conceived the experimental design, collected the data, performed the MRI data analysis, contributed to results interpretation, and revised the paper.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. The reviewer MC and handling Editor declared their shared affiliation.


Patients on renal replacement therapy are often unaware of their underlying condition and hence suffer from so-called end-stage renal disease (ESRD) of unknown origin. However, an exact diagnosis is not only important for better estimating the prognosis, but also when preparing for kidney transplantation. Whilst patients with FSGS without a confirmed genetic cause have a high recurrence rate in the transplanted organ, patients with a mutation generally exhibit no recurrence and have a good prognosis. Furthermore, renal biopsy, which may be helpful for differential diagnosis, is usually contraindicated in end-stage kidneys. We here present the case of familial ESRD of unknown origin, which could be resolved by targeted genetic testing prior to planning of kidney transplantation.INF2 . The same mutation could be detected in the index patient’s mother (ESRD at age 50) and three brothers with normal serum-creatinine but mid or low range proteinuria.A 32-year-old female with ESRD and nephrotic range proteinuria was admitted to our clinic. Family-history revealed that both mother and maternal grandmother had ESRD of unknown origin. As renal biopsy was impossible due to atrophic kidneys, we performed mutation analysis of genes known for dominant forms of FSGS and found a novel heterozygous mutation of Genetic testing is warranted in families with ESRD of unknown origin and may provide a robust diagnosis even without kidney biopsy. It will help detecting relatives at risk who have to be excluded from potential kidney donation and who may benefit from timely initiation of protective measures in order to slow down disease progression. INF2 [INF2-gene mutations were identified in both FSGS and Charcot-Marie-Tooth disease, a hereditary motor and sensory neuropathy that is characterized by peripheral nerval demyelination [INF2 gene associated with FSGS have been found within exons coding for its highly conserved diaphanous-inhibitory domain (DID), which serves as a regulator for polymerization and depolymerization of actin filaments [INF2-mutations do usually not manifest before adolescence or early adulthood and age at ESRD can vary significantly (published data range 12–70 years) [Unfortunately, ignorance of the underlying renal condition is a common phenomenon among patients on renal replacement therapy. This is often due to late clinical presentation at stages of advanced disease, when important diagnostic tools, such as kidney biopsy, are no longer possible for diminished organ size and consecutive risk of bleeding. Focal segmental glomerulosclerosis (FSGS) is such an underlying, potentially underdiagnosed, condition. FSGS is derived from its histological appearance upon kidney biopsy and represents a strictly descriptive term that comprises heterogeneous disorders which altogether represent a major cause of nephrotic syndrome, progressive renal failure, and end-stage renal disease (ESRD) . It is cINF2 , which eINF2 . INF2-gelination . Virtualilaments . In cont0 years) –8.2), was admitted to our nephrology department. Except for edema of the legs, physical examination showed no further abnormalities, in particular, no indication of a sensorimotor or other neurological disorder. Both kidneys presented atrophic on ultrasonography  and impaired renal function  was unremarkable. Direct sequencing of inverted formin 2 (INF2), however, showed a heterozygous nucleotide exchange from thymine to cytosine (c.485 T > C) in exon 3, resulting in the substitution of a highly conserved leucine to proline at amino acid position 162 (p.Leu162Pro)  in a family with ESRD of previously unknown etiology. As in virtually all patients with FSGS due to mutated INF2, the detected mutation is located within the first exons and results in an amino acid change within the functionally important N-terminal DID [INF2-mutation was found in heterozygous state , the resulting dysfunction of INF2 is responsible for a deranged structure of the cytoskeleton, leading to an abnormal distribution of podocin and nephrin as important components of the podocytic slit membrane . DisturbIn conclusion, we strongly suggest genetic testing in younger patients with a positive family history but ESRD of unknown origin: i) targeted genetic testing based on clinical suspicion provides a reasonable likelihood of detecting the underlying condition even when kidney biopsy is contraindicated, ii) knowledge of the causative renal disorder is highly informative for risk-assessment and planning of kidney transplantation , iii) consecutive detection of family members at risk will help to exclude potential living kidney donors within the family, iv) timely initiation of reno-protective measures will help affected family members to slow down disease progression and protract or even avoid ESRD.