Gene duplication provides raw material for the evolution of functional innovation. We recently developed a phylogenetic method that classifies evolutionary processes driving the retention of duplicate genes by quantifying divergence between their spatial gene expression profiles and that of their single-copy orthologous gene in a closely related sister species.Here, we apply our classification method to pairs of duplicate genes in eight mammalian genomes, using data from 11 tissues to construct spatial gene expression profiles. We find that young mammalian duplicates are often functionally conserved, and that expression divergence rapidly increases over evolutionary time. Moreover, expression divergence results in increased tissue specificity, with an overrepresentation of expression in male kidney, underrepresentation of expression in female liver, and strong underrepresentation of expression in testis. Thus, duplicate genes acquire a diversity of new tissue-specific functions outside of the testis, possibly contributing to the origin of a multitude of complex phenotypes during mammalian evolution.Drosophila duplicate genes. Due to the smaller effective population sizes of mammals relative to Drosophila, these analyses implicate natural selection in the functional evolution of duplicate genes.Our findings reveal that mammalian duplicate genes are initially functionally conserved, and then undergo rapid functional divergence over evolutionary time, acquiring diverse tissue-specific biological roles. These observations are in stark contrast to the much faster expression divergence and acquisition of broad housekeeping roles we previously observed in The online version of this article (doi:10.1186/s12862-015-0426-x) contains supplementary material, which is available to authorized users. Gene duplication produces copies of existing genes, which can diverge from their ancestral states and contribute to the evolution of novel phenotypes. A large proportion of mammalian genes arose via gene duplication , 2, manyDifferent processes may drive the long-term retention of duplicate genes: Parent and child copies may each maintain the function of their single-copy ancestral gene (conservation ); one coHomo sapiens), chimpanzee (Pan trogodytes), gorilla (Gorilla gorilla), orangutan (Pongo pygmaeus abelii), macaque (Macaca mulatta), mouse (Mus musculus), opossum (Monodelphis domestica), and platypus (Ornithorhynchus anatinus). Using synteny information from whole-genome alignments to determine orthologous genomic positions, and parsimony to infer gene acquisitions, we distinguished between parent and child copies and identified single-copy outgroup genes for each pair of duplicates , 9 cases of subfunctionalization, and 50 cases of specialization  between all single-copy genes in the two species, though using the median nonsynonymous sequence divergence rate (Ka) produced similar patterns , and neofunctionalized genes with highest expression levels in each tissue  to 1.79oobscura ). In ourimpanzee ) to 1.41impanzee ). Thus, Drosophila duplicates were neofunctionalized, and examination of evolutionary processes over shorter divergence times suggested that novel functions arise within a few million years of evolution [Ne) of Drosophila than of mammals [Drosophila [Drosophila duplicate genes. Even so, expression divergence of duplicate genes occurs much faster than that of single-copy genes in mammals. Thus, though natural selection may not be as efficient as in Drosophila, it still appears to play an important role in the functional divergence of duplicate genes in mammals.Contrary to our observation in mammalian duplicates, we found that most volution . This di mammals –30, whicosophila –33, and Ne is also thought to result in a higher prevalence of subfunctionalization [While small lization , this prlization , 35, whiDrosophila and mammals was that neofunctionalization primarily occurred in child copies in Drosophila [Drosophila and mammals. Under neutrality, most duplicate genes should be lost within the first few million years of evolution [Drosophila, many neofunctionalized child genes likely arose with or quickly acquired new beneficial functions that were retained by natural selection [Another difference between our findings in osophila , whereasvolution . In Droselection . In mammelection , 38. ThuDrosophila and mammals, neofunctionalized genes have tissue-specific functions. However, neofunctionalized Drosophila genes are primarily testis-specific [Drosophila, comparison of young and old duplicates supported the “out of the testis” hypothesis of new gene emergence, in which new genes arise with testis-specific functions and evolve broader functions over time [In both specific , whereasver time . Accordiver time , making Drosophila revealed that duplicate genes are primarily retained via neofunctionalization, and that they diverge even faster than in mammals, acquiring broad housekeeping functions. Thus, our current study highlights key differences in the retention of duplicate genes between mammals and Drosophila and, moreover, supports the hypothesis that positive selection drives the functional evolution of duplicate genes in both lineages.While gene duplication has long been hypothesized to play an important role in the evolution of novel phenotypes, the processes driving the retention of mammalian duplicate genes remained unclear. In this study, we utilized our previously developed classification method to identify the roles of different evolutionary processes in the retention of mammalian duplicate genes. We found that most mammalian duplicate genes are functionally conserved, and that they diverge rapidly over evolutionary time, acquiring a diversity of tissue-specific functions. In contrast, our previous study in Homo sapiens, Pan trogodytes, Gorilla gorilla, Pongo pygmaeus abelii, Macaca mulatta, Mus musculus, Monodelphis domestica, and Ornithorhynchus anatinus) and three outgroups  from the Ensembl database (release 74) at http://www.ensembl.org. We obtained lists of duplicate genes in each mammalian genome from the Ensembl database (release 74) at http://www.ensembl.org, from the Duplicated Genes Database (DGD) at http://www.dgd.genouest.org, and from protein BLAST searches [We downloaded protein sequences and annotation files for eight mammals  and UCSC Genome Bioinformatics (http://www.genome.ucsc.edu) databases and extracted syntenic regions in all genomes for each duplicate gene. We used parsimony to phylogenetically date the origin of each pair of duplicates. In particular, we inferred a duplication event that occurred after the divergence of two sister species if one sister contains two gene copies, while the other sister and all outgroups  contain a single-copy gene. Duplicates that are present in all species or that could not be resolved via parsimony  were removed from our analysis. For each pair, the gene copy aligned to outgroup genes in the whole-genome alignment was designated as the parent, and the copy that did not align to any regions of the outgroup genomes was considered the child. Because annotation of exons may be unreliable in many of the species used, we did not distinguish between DNA- and RNA-mediated duplication mechanisms. Orthologs for single-copy genes were also obtained via synteny and aligned with MACSE [Ka and Ks between orthologous pairs of single-copy genes.We downloaded whole-genome alignments from Ensembl , between expression profiles of child and outgroup copies , and between the combined parent–child expression profile and that of the outgroup copy . We next established baseline divergence levels for genes by calculating Euclidian distances between expression profiles of single-copy genes present in both sister species , and used these distances to set cutoffs for expression divergence in each pair of species . Two-sided t-tests were used to assess significance of slopes shown in Figs. U tests were used to compare distributions of tissue specificities of outgroup, parent, and child genes to those of single-copy genes shown in Fig. Fits of least-squares linear regression lines were tested with The data sets supporting the results of this article are included within the article and its additional files.


Juvenile Dermatomyositis (JDM) is a systemic, inflammatory, idiopathic disease, mainly affecting the skin and the muscles, starting before the age of 16 with an incidence around one case per million children. Some patients display typical features of JDM without skin involvement or even without muscle involvement, however both tissues are affected over time in most cases. Diagnosis criteria have been established by Bohan and Peter 35 years ago, based on the presence of typical skin rash and proximal muscle involvement. Other conditions have to be rulled out before making a diagnosis of JDM, such us other connective tissue diseases, polymyositis, infectious/post-infectious myositis, genetic diseases, metabolic or drug-induced myopathies. Unlike adult-onset dermatomyositis, JDM is exceptionnaly associated with a malignant disease.JDM may also affect several organs including the lungs and the digestive tract. In a subset of patients, glucose intolerance, lipodystrophia and/or calcinosis develop. Delay in treatment initiation or inadequate treatment may favour diffuse, debilitating calcinosis.JDM patients have to be referred to reference pediatric centers with a multidisciplinary team to properly assess disease activity, disease-related damage (including low bone density in most cases) and define the best treatment. Long-lasting corticosteroid therapy remains the gold standard, together with physiotherapy. Physiotherapists often play an important rule not only through their participation to patients care but also by assessing disease activity at each stage, using several tools including CMAS and MMT assessment of muscle strength. Other tools have been developed to assess extra-muscular disease and disease-related damage. Patients quality of life also needs to be properly assessed. Several collaborative efforts have been conducted to improve the way we assess patients health and response to treatment.Most patient respond to treatment, relapses are frequent but a complete disease remission is achieved in most cases before adulthood, with or without sequellae. Ongoing clinical trials are assessing the effect of several immunosuppressive and immunomodulatory drugs; preliminary results from a PRINTO international trial suggest that both methotrexate and cyclosporine may help controlling the disease, reducing the rate of early flares and may hence have a corticosteroid-sparing effect. On the short term, methotrexate seems better tolerated. Some preliminary data suggest that other drugs may also be of interest.None declared.


The growing attention that non-coding RNAs have attracted in the field of cancer research in recent years is undeniable. Whether investigated as prospective therapeutic targets or prognostic indicators or diagnostic biomarkers, the clinical relevance of these molecules is starting to emerge. In addition, identification of non-coding RNAs in a plethora of body fluids has further positioned these molecules as attractive non-invasive biomarkers. This review will first provide an overview of the synthetic cascade that leads to the production of the small non-coding RNAs microRNAs (miRNAs) and presents their strengths as biomarkers of disease. Our interest will next be directed at exploring the diagnostic utility of miRNAs in two types of cancer: the brain tumor glioblastoma multiforme (GBM) and breast cancer. Finally, we will discuss additional clinical implications associated with miRNA detection as well as introduce other non-coding RNAs that have generated recent interest in the cancer research community. Finally, we introduce examples of work that have assessed the usefulness of miRNAs in other, non-diagnostic, clinical applications as well as present additional non-coding RNAs with diagnostic relevance to cancer.Tremendous effort has been dedicated in recent years to elucidating the underlying functions of non-coding RNAs, including the small microRNAs (miRNAs), in numerous types of cancer. Several studies have characterized the roles played by miRNAs in primary tumors and have positioned these molecules as significant drivers of malignancy ,2,3. ImpMiRNA biogenesis usually starts with the transcription of miRNA genes by RNA polymerase II to generate a primary miRNA transcript termed pri-miRNA ,8. This There are multiple arguments that support the investigation of miRNAs as biomarkers for diseases. MiRNAs can notably be packaged into exosomes, small bioactive reservoirs secreted by cells, and subsequently regulate transcript targets of recipient cells . PreviouEGFR) gene is a frequent occurrence in primary GBMs as well as mutations of phosphatase and tensin homolog (PTEN) tumor suppressor gene [MGMT) promoter methylation status in elderly patients diagnosed with a GBM [Glioblastoma multiforme (GBM) is the most aggressive and frequently diagnosed primary brain tumor . This grsor gene ,40. Inteth a GBM .MiRNAs are appealing therapeutic targets and potential biomarkers of GBMs . DeregulWhile examples abound of modulated miRNAs in primary GBM tumors, miRNAs are also released by GBMs and can be subsequently isolated and quantified in various body fluid samples, thus positioning these molecules as circulating biomarkers of malignancy. A study revealed significant miR-128 upregulation and miR-342-3p downregulation in blood samples of GBM patients when compared with healthy individuals . SubsequBRCA1 gene, besides conferring a significant lifetime risk of breast cancer diagnosis [Breast cancer, unlike GBM, is at the opposite end of the cancer incidence being the most frequent carcinoma observed in women in the United States. It is also the cancer that ranks second on the list of estimated deaths per cancer types for the same gender . As for iagnosis , are alsiagnosis .Pioneering work performed in tumor samples collected from a cohort of 344 patients diagnosed with primary breast cancer revealed strong miR-21 expression . MiR-21 The former study also demonstrated elevated miR-221 and miR-222 expression in the triple-negative specimens. MiR-221/222 is upregulated in HER2-positive primary human breast cancer tissues and has been linked with tamoxifen resistance . MiR-221versus adjacent benign breast tissue [MiR-155 is also one of the first miRNAs to be reported as significantly deregulated in primary breast tumors . Severalt tissue  and subst tissue . MiR-206t tissue . The tumt tissue . Interest tissue  and thisAs for GBMs, miRNAs have also been identified in circulating samples of breast cancer patients and have been investigated further for their diagnostic potential . Early wSeveral miRNAs with diagnostic potential in GBMs and in breast cancer have been presented up to this point and a list of commonly deregulated miRNAs in these two types of cancer is presented in in vivo and is associated with poor overall patient survival [In addition and as alluded in this article, the clinical usefulness of miRNAs reach beyond their capabilities of diagnosing malignancy. Indeed, miRNAs have also been investigated as prognostic markers. Specific examples in brain tumors include miR-328 which is strongly expressed in glioma cells survival  as well survival . In breasurvival . A signasurvival . Examplesurvival . MiR-181survival . Other esurvival ,111,112.survival ,114. In survival . A studysurvival . While tin vivo models is not as vast as for the miRNAs, interesting work is starting to emerge in this research area. Two studies notably reported elevated HOX antisense intergenic RNA (HOTAIR) lncRNA levels in blood samples collected from cervical and colorectal cancer patients and correlated this observation with poor prognosis [In addition to miRNAs, it is important to mention that other non-coding RNAs such as long non-coding RNAs (lncRNAs) are appealing molecules to investigate for their diagnostic potential in different types of cancer. While the information available regarding lncRNAs as potential cancer biomarkers in human rognosis ,117. In rognosis . MEG3, arognosis . Early wrognosis  and a rerognosis .In conclusion, whether to monitor treatment response in GBMs or for early breast cancer detection, several examples exist that illustrate non-coding RNAs with diagnostic, prognostic and therapeutic response assessment potential. Deciphering the circulating miRNA footprint associated with these malignancies is undoubtedly of great clinical interest and tremendous progress has been made in this research area in recent years. Nevertheless, challenges remain before non-coding RNAs are leveraged as bona fide biomarkers in the two types of cancer explored in this review and further investigation is needed in this research field to unveil clinically relevant miRNA-based signatures.


The Golgi complex is decorated with so-called Golgin proteins that share a common feature: a large proportion of their amino acid sequences are predicted to form coiled-coil structures. The possible presence of extensive coiled coils implies that these proteins are highly elongated molecules that can extend a significant distance from the Golgi surface. This property would help them to capture or trap inbound transport vesicles and to tether Golgi mini-stacks together. This review will summarize our current understanding of coiled coil tethers that are needed for the receipt of transport vesicles at the trans Golgi network (TGN). How do long tethering proteins actually catch vesicles? Golgi-associated, coiled coil tethers contain numerous binding sites for small GTPases, SNARE proteins, and vesicle coat proteins. How are these interactions coordinated and are any or all of them important for the tethering process? Progress toward understanding these questions and remaining, unresolved mysteries will be discussed. Membrane trafficking involves the collection of cargo into transport vesicles, translocation of vesicles along cytoskeletal tracks, and tethering, docking, and fusion of vesicles at their target membranes. Tethering involves the initial capture of transport vesicle at a distance from the target membrane and the process by which the two membranes are brought together into close proximity. Tethering appears to be an important means to both enhance the efficiency and control the accuracy of subsequent, SNARE-mediated membrane fusion events Pfeffer, .At least 15 different, long, coiled-coil-containing proteins decorate the Golgi complex and have been nicknamed, Golgins , and require at least one small GTPase for their TGN recruitment. Figure At the TGN in human cells, the four Golgins, Golgin-245, Golgin-97, GCC185, and GCC88, are localized to at least three distinct subdomains  motif at its extreme N-terminus that binds liposomes of high membrane curvature (radius < 50 nm) but not flat membranes GMAP-210 is the only Golgin that contains an ALPS motif, thus other Golgins must tether vesicles by another mechanism. Several Golgi tethers interact with vesicle coat proteins .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.


DNA double strand breaks (DSBs) are the most dangerous form of DNA damage and need to be repaired efficiently in order to maintain genomic stability. The kinase ATM becomes activated upon recognition of DSBs and transduces this into cell cycle checkpoint activation and DNA repair. There are two main pathways by which DSBs can be repaired: Non-homologous end joining (NHEJ) and homologous recombination (HR). In NHEJ, the broken ends are minimally processed and after sequence-independent alignment, ligated back together often resulting in point mutations, small deletions and insertions. In the HR pathway, extensive DNA end resection produces single-stranded DNA, which subsequently invades a homologous DNA duplex that in turn templates new DNA synthesis and repair. Usually sister chromatids provide the repair template, thus HR is normally considered to be restricted to S and G2 phases of the cell cycle. The NHEJ versus HR pathway choice is also influenced by the genomic and nuclear location of DSBs . As nuclNucleoli, the sites of ribosome biogenesis, form around arrays of ribosomal gene (rDNA) repeats that are transcribed by the dedicated RNA polymerase I (pol I) transcription machinery. In humans, rDNA arrays, termed nucleolar organiser regions (NORs), are positioned on the short arms of the five acrocentric chromosomes. While rDNA repeats are localized within the nucleolar interior, NOR distal sequences, termed distal junction (DJ), are embedded in peri-nucleolar heterochromatin .Physarum, which has a recognition sequence once in each of the ~300 rDNA repeats and up to 13 sites elsewhere in the genome [In recent years, several groups have studied DSBs within the nucleolus using γ-irradiation and micro-irradiation. These studies have yielded conflicting results, ranging from complete ATM-dependent inhibition of nucleolar transcription to no observable effect on nucleolar function , 4. In oe genome .Introduction of I-PpoI into human cells causes ATM-dependent inhibition of pol I transcription followed by a reorganization of the nucleolar structure to form so-called caps at the nucleolar periphery. Using CRISPR/Cas9, we revealed that DSB introduced across the rDNA repeat elicit the same response. This movement of damaged rDNA to the nucleolar periphery facilitates repair. In contrast, DSBs introduced into DJ sequences do not exhibit this response. Nucleolar caps containing rDNA DSBs are highly enriched in activated ATM and components of the HR machinery. No enrichment of NHEJ factors is observed. These results reveal how genomic location and nuclear position influence repair choice .Surprisingly, HR factors could be observed in the majority of nucleolar caps, raising the possibility of repair by HR in G1 cells, where sister chromatids are absent. By exploiting the FUCCI system (fluorescence ubiquitination cell cycle indicator ), HR facin cis. HR templated in cis need not involve cross-overs with loss or gain of sequence. Indeed, previous evidence indicates that the Blooms helicase (BLM)-dependent branch migration pathway promotes non-crossover HR within rDNA arrays [These results strongly suggest that within repetitive rDNA arrays, DSBs can be repaired by HR independent of the cell cycle, probably templated by undamaged rDNA repeats A arrays .Future work will be needed to determine if repair is completed within G1 cells, and to assess the role of chromosomal context in maintaining the genomic stability of rDNA arrays.in vivo [Apart from advancing our understanding of the nucleolar response to DSBs, our recent work also presents a number of technical advances that may be of interest to other workers in the DNA damage field. Transfection of cells with synthetic mRNA is efficient in a wide variety of cell types, independent of cell cycle, and importantly, does not itself induce a DNA damage response. To our knowledge, this is the first use of the CRISPR/Cas9 system to probe DSB responses at specific chromosomal sites. Moreover, by using targeted introduction of DSBs in combination with the following techniques; antibody staining, FUCCI, EdU incorporation and FISH, repair processes at specific chromosomal sites can be studied in vivo .


The Clinical ProblemWith the increased use of advanced imaging techniques, incidental renal mass have become a very frequent finding .Approximately 13 to 27% of abdominal imaging studies incidentally identify a renal lesion , a fact that makes suspected renal cell carcinoma be diagnosed at an early stage  ( cm (4).Furthermore, the development of ablative techniques has widened the range of treatment options available to these patients and international consensus panels support other indications for ablative therapy for renal tumors  .Thermal ablation is performed by inserting needle applicators within the renal tumors to generate lethal temperatures to neoplastic tissues encompassed by ablation zone . Cryoablation and radiofrequency ablation are the most common methods .With refinements in probe size and design, a percutaneous image-guided approach may be preferable to a laparoscopic approach for thermal ablation, since procedure- associated morbidity would be lower  Figures-3.Historically, percutaneous ablation has been reserved for patients with small, exophytic tumors in the posterolateral kidney. However, the increased use of cryoablation and displacement techniques  e.g. hy.Cryoablation, rather than radiofrequency ablation, has shown significant promise in treating these larger more complex renal tumors .As the name implies, cryoablation relies on low temperatures to induce cell death. The process of cryoablation obeys the Joule- Thomson effect whereby expanding of some gases (e.g. argon) within a needle-like chamber (the cryoprobre) produces heat sink near the antenna tip that cools the probe to temperatures of –160ºC or colder . The celProximity of the tumor to the collecting system may represent a relative contraindication to cryoablation due to the risk of urothelial injury and ureteral strictures have been reported, particularly for tumors in the medial lower pole .Placement of a ureteral stent with retrograde warm saline irrigation of the collecting system and a very reliable identification of ureter during the ice ball monitoring may mitigate this risk .Freezing into calyceal structures or intrarenal pelvic collecting system did not cause any apparent strictures or vascular injuries in longer-term follow-up, similar to prior animal data . RelativCareful preprocedure cross-sectional imaging assessment of a candidate patient’s small renal mass is required to minimize complications and maximize therapeutic efficacy. A practical algorithm for procedure planning, ABLATE, has been proposed that takes into account the following tumor characteristics:A, axial tumor diameter; B, bowel proximity; L, location within the kidney; A, adjacency to the ureter; T, touching renal sinus fat; and E, endophytic or exophytic position .Of all tumor characteristics, the size of the renal mass is the most important factor in achieving local tumor control with ablation . This isEndophytic tumor position  can make ablation procedures more difficult and has been associated with increased local treatment failures. Gupta et al.  reportedUltrasound guidance, ultrasound-CT, or ultrasound-MRI fusion guidance or administration of IV contrast agent (iodinate for CT and microbubbles for ultrasound) may help with localizing endophytic tumors.Concerning to the location of the renal tumors, an important potential complication to consider before ablation is nerve injury, which can lead to postablative neuralgia and paresthesias. In the context of renal ablation planning, one should consider the position of the intercostal nerves, genitofemoral nerve, and lateral femoral cutaneous nerves. The ablation of posterior masses located close to major psoas muscle draws attention to the danger of damaging the genitofemoral nerve, resulting in chronic pain, tenderness, and diminished sensitivity within the skin area of the ipsilateral groin . DisplacLocal control rates approaching 100% for tumors smaller than 3 cm in select series, with very low tumor recurrence rates .Thompson et al recently compared the oncologic outcomes for cT1a tumors between RFA (n=180), PCA (n=187), and PN (n=1057). On univariable analysis, there was no difference in estimated 3 years disease-free survival between the three treatment modalities . Distant metastasis-free survival at 3 years, however, was better for PN (99%) and CA (100%) compared with RFA (93%) .Whitson et al.  comparedGiven that long-term studies have shown durable outcomes for nephron-sparing surgery, the American Urological Association guidelines assert that the most important disadvantage of any ablative technique is a higher local recurrence rate relative to that of partial nephrectomy . Howeverde novo tumors, becomes even more atractive the percutaneous approach in comparison to surgery.Furthermore, the possibility of repeated ablations in residual lesions or In addition, nephron-sparing surgery, including open partial nephrectomy and laparoscopic partial nephrectomy, can be more technically challenging and result in serious complications, such as excessive blood loss and urinary fistula formation .The other obvious advantages of ablation relative to extirpative surgery include reduced morbidity; better preservation of renal parenchymal volume, which correlates with overall renal function; faster recovery time; shorter or no inpatient hospitalization; and possibly being the only treatment option available to patients with serious comorbid conditions who are not considered surgical candidates.The cost to the health care system may also be reduced with increased use of noninvasive ablative techniques .Although nephrectomy  remains the reference standard for renal tumor treatment, physician acceptance and patient interest in percutaneous ablation are growing as intermediate and long-term outcome data become available.Percutaneous cryoablation of renal tumors under imaging guidance is a curative alternative technique with a low complication rate. The best indications are tumor smaller than 4 cm, even though larger tumors can be effectively treated in selected cases. Percutaneous cryoablation is effective for preserving renal function, even in patients with single kidney. Moreover, in cases of incomplete treatment, cryoablation can be repeated and does not preclude salvage surgery.So why not cryoablation as a first line therapy for a 2 cm complete endophitic tumor on the posterior side of the left kidney?


Malnourished children have increased risk of dying, with most deaths caused by infectious diseases. One mechanism behind this may be impaired immune function. However, this immune deficiency of malnutrition has not previously been systematically reviewed.To review the scientific literature about immune function in children with malnutrition.A systematic literature search was done in PubMed, and additional articles identified in reference lists and by correspondence with experts in the field. The inclusion criteria were studies investigating immune parameters in children aged 1–60 months, in relation to malnutrition, defined as wasting, underweight, stunting, or oedematous malnutrition.The literature search yielded 3402 articles, of which 245 met the inclusion criteria. Most were published between 1970 and 1990, and only 33 after 2003. Malnutrition is associated with impaired gut-barrier function, reduced exocrine secretion of protective substances, and low levels of plasma complement. Lymphatic tissue, particularly the thymus, undergoes atrophy, and delayed-type hypersensitivity responses are reduced. Levels of antibodies produced after vaccination are reduced in severely malnourished children, but intact in moderate malnutrition. Cytokine patterns are skewed towards a Th2-response. Other immune parameters seem intact or elevated: leukocyte and lymphocyte counts are unaffected, and levels of immunoglobulins, particularly immunoglobulin A, are high. The acute phase response appears intact, and sometimes present in the absence of clinical infection. Limitations to the studies include their observational and often cross-sectional design and frequent confounding by infections in the children studied.The immunological alterations associated with malnutrition in children may contribute to increased mortality. However, the underlying mechanisms are still inadequately understood, as well as why different types of malnutrition are associated with different immunological alterations. Better designed prospective studies are needed, based on current understanding of immunology and with state-of-the-art methods. Malnutrition in children is a global public health problem with wide implications. Malnourished children have increased risk of dying from infectious diseases, and it is estimated that malnutrition is the underlying cause of 45% of global deaths in children below 5 years of age Pneumocystis jirovecii and severe varicella has been reported in malnourished children Pneumocystis jirovecii pneumonia is not frequent in malnourished children not infected with HIV Since most infections and deaths in malnourished children occur in low-income settings, the organisms causing disease are rarely identified. Therefore, little is known about whether these differ from pathogens infecting well-nourished children, and whether malnourished children are susceptible to opportunistic infections. Although opportunistic infections like Non-immunological factors may also contribute to increased mortality in malnourished children: reduced muscle mass may impair respiratory work with lung infections This review considers childhood malnutrition in the sense of under-nutrition, causing growth failure or weight loss, or severe acute malnutrition, either oedematous, or non-oedematous.Growth failure caused by malnutrition has commonly been defined by low weight-for-age (underweight), length-for-age (stunting), or weight-for-length (wasting) Two forms of severe acute malnutrition in children exist: non-oedematous malnutrition, also known as marasmus, characterized by severe wasting and currently defined by weight-for-length z-score <−3 of the WHO growth standard, or MUAC <11,5 cm; and oedematous malnutrition defined by bilateral pitting oedema Figure 2A systematic literature search was carried out in PubMed using combinations of the search terms related to malnutrition and immune parameters. The full search strategy and the search terms used are described in Inclusion criteria were: studies presenting original clinical data regarding immune parameters in children, aged 1–60 months, where a comparison was made, either between malnourished and well-nourished children, or between malnourished children before and after nutritional rehabilitation. Exclusion criteria were studies of children with another primary diagnosis such as cancer, congenital heart disease or endocrine disease. Studies were accepted where children had co-morbid infections, since this is typically seen in malnourished children. Articles by RK Chandra were excluded, due to concerns about possible fraud The search was carried out in August 2013, and updated in December 2013. The search results were sorted by MJHR, based on titles, abstracts or full-text-articles. Additional literature was obtained from reference lists, text books and by personal communication with experts.For data retrieval, studies were sorted according to whether they investigated barrier function (skin and gut), innate immunity or acquired immune system, and listed in tables based on the specific immune parameter studied. Some studies were included in more than one table. The following data was extracted from each article: year and country, number and age range of malnourished and well-nourished participants, type of malnutrition and whether included children fulfilled WHOs current diagnostic criteria for severe acute malnutrition, whether infections were present, immune parameter studied, methods used, how the parameter was associated with malnutrition, and whether children with oedematous and non-oedematous malnutrition were differentially affected.Checklist S1).The results of the included articles were summarized for each immune parameter. Due to the heterogeneous nature of study designs, participants and outcomes, it was not meaningful to synthesize the results in a meta-analysis. The main potential bias was presence of infection. For this reason, presence and effect of infection was considered for each study as well as for each outcome. The PRISMA  guideline was followed, except for the items relating to meta-analysis . Another 49 articles were identified which, in addition to children 1–60 months old, also included older children. These studies were not included in the main analysis, but used in a sensitivity analysis in which all studies were included. The result of this additional analysis was essentially similar to the results obtained with studies only including children less than 60 month (results not shown). The studies were published between 1957–2014, mainly in the 1970s and 1980s. Only 33 studies were published after 2003  and the commensal bacterial flora Of the articles describing barrier function in malnourished children, six described skin structure and function, 21 described structure and permeability of intestinal mucosa, 19 protective factors in secretions and 11 the microbial flora colonizing mucosal surfaces.Skin barrier has mostly been studied in children with oedematous malnutrition, who may develop a characteristic dermatosis, characterized by hyper-pigmentation, cracking and scaling of the epidermis, resembling “peeling paint”, providing a potential entry port for pathogens table S1). Two articles describing histology reported atrophy of skin layers, but did not describe cutaneous immune cells Six articles assessed barrier and immune function of the skin in malnourished children (table S2). Autopsy-studies from as early as 1965 described a thin-walled intestine in malnourished children, and noted that “… the tissue paper intestine of kwashiorkor is well known to tropical pathologists.” The intestinal mucosa of malnourished children was described in 21 articles . Secretory IgA (sIgA) was investigated in 15 studies, of which 11 investigated saliva, urine, tears, nasal washings and duodenal fluid Nineteen articles were published on anti-microbial factors in secretions from malnourished children . All found malnourished children to host a different flora from well-nourished children. Their mouths and throats contained more yeast Microbes colonizing skin and mucosa may protect against infections by competing with pathogens, by producing specific antimicrobial substances, and by stimulating host immune function The colonic flora, containing the vast majority of commensal bacteria, was described by sequencing bacterial DNA from stool samples in four recent articles, which consistently found that the pattern of bacteria was different in malnourished and well-nourished children The innate immune system delivers an unspecific response relying on leukocytes , as well as soluble factors in blood (like acute phase proteins and the complement system) table S5). Most reported similar or higher numbers of total leukocytes in blood of malnourished children Thirty-eight articles described number and function of leukocytes of the innate immune system .Acute phase responses is induced by infection or trauma, and mediated by cytokines like IL-6 and TNF-α. It involve temporal suppression of acquired, and amplification of innate immune responses, with secretion of positive acute phase proteins (APP) like C-reactive protein (CRP), serum-amyloid-A (SAA), complement factors, α-1-acid-glycoprotein or ferritin Most studies found elevated positive APP in malnourished children with infections. This included CRP Three studies found elevated CRP in malnourished children without apparent infections Four articles described the acute phase response induced by a vaccine. Two reported a normal The complement system consists of plasma proteins secreted by the liver that, upon activation, react to recruit immune cells, opsonize and kill pathogens table S7). In 17 of 21 studies, levels of C3 were depressed in malnourished children Twenty-four articles described levels or in-vitro activity of complement proteins . All reported thymus atrophy in malnourished children, to an extent termed “nutritional thymectomy” Six articles reported autopsy studies of the thymo-lymphatic system in malnourished children, published between 1956 and 1988 table S9). Five of these studied children with severe malnutrition and found severe thymic atrophy Eight articles reported thymic size measured by ultrasound, in relation to nutritional status Six articles reported investigations of other lymphatic tissue. Four autopsy studies found atrophy of lymph nodes, spleen, tonsils, appendix and Peyer's patches, although not as pronounced as in the thymus. Histology revealed a reduction in germinal centres and depletion of lymphocytes from para-cortical areas table S10).Cellular immune function can be examined by dermal DTHR, the prototype of which is the Mantoux test. Intradermal application of substances like candida or phyto-hemaglutinin (PHA) are also used, as well as sensitizing skin with a local contact sensitizer such as 2-4-di-nitro-clorobenzene (DNCB). Twenty-one articles reported DTHR in relation to malnutrition . Of 16 articles, 13 reported similar or higher levels of lymphocytes in peripheral blood of malnourished children Fifty-eight articles reported either total numbers of lymphocytes or lymphocyte subsets in blood . Early studies identified T-lymphocytes as those forming rosettes with sheep red blood cells, while later studies used monoclonal antibodies to CD3. Using the rosette-method, 19 of 20 studies found lower levels of T-lymphocytes Numbers of T-lymphocytes were described in 29 articles . Nineteen of 27 studies found no difference in IgG antibodies or total γ-globulin between malnourished and well-nourished children Thirty-two articles described immunoglobulins in blood of malnourished children . The articles either reported sero-conversion rates, or antibody titre response. Studies assessing sero-conversion rates in children with severe malnutrition found mixed results: Six of 10 studies found reduced sero-conversion rates in children with severe malnutrition to typhoid Thirty-five articles described vaccination responses to a specific antigen .Cytokines are signal molecules acting locally between immune cells, and sometimes with systemic effects. Thirty-five articles described cytokines in malnourished children  Comparing cytokine pattern between children with oedematous and non-oedematous malnutrition, most found that the difference from well-nourished was greatest in children with oedematous malnutrition Leukotrienes (LT) are not strictly cytokines, but immune modulating molecules derived from long chain polyunsaturated fatty acids. Levels of LTC4 and LTE4 were higher, and LTB4 lower, in children with oedematous than with non-oedematous malnutrition, whose levels were similar to well-nourished We identified and reviewed 245 articles about immune function in malnourished children. Some general problems apply to many of the studies, mostly related to their observational design. For this reason they can only describe associations, not causalities.First, many studies were done in severely malnourished children from hospital settings, who were ill with infections, making it difficult to disentangle the immunological effect of malnutrition from the effect of infection. This problem has caused some to propose that there really is no immune impairment by malnutrition, and that all alterations seen are due to infections or underlying unknown immune deficiencies, which are also responsible for the poor growth Second, publication bias is a well-known problem, and may have occurred, particularly in older studies, where some small studies showed a dramatic effect.Third, studies used different diagnostic criteria for malnutrition, making it difficult to determine the children's degree of malnutrition as defined by present-day criteria. While children in 52 of the studies fulfilled WHOs present criteria for severe acute malnutrition, only two diagnosed children based on the new WHO growth reference. Those defined as severely malnourished based on old growth references would most likely also be classified as severely malnourished today, since the new WHO standard tend to classify more children as severely malnourished, while some children then defined as moderately malnourished would be classified as severely malnourished today. The studies including children based on weight-for-age probably included children with stunting and wasting, without differentiating between the two.Fourth, even using uniform criteria, malnourished children are a heterogeneous group. Anthropometric measurements are only crude markers of body composition, which - among other things - reflect nutrient deficiencies. It is unknown what specific nutrients were deficient, and to what extent infection contributed. Deficits in lean tissue and fat tissue are plausibly different physiologic conditions, and children appearing similarly malnourished may be so for entirely different reasons, with different immunological consequences. No articles have so far reported reliable measures of body composition, simultaneously with markers of immune function. Probably, the consequence of malnutrition on immune function may also depend on the pattern and load of infections. Although most studies were carried out in low-income settings with high infectious loads, a few were from middle- or high-income countries. This may also contribute to inconsistencies in the results.In spite of these limitations, common patterns emerge from the studies, summarized below Figure 5Total white blood cell and lymphocyte counts in peripheral blood are not decreased in malnourished children, and granulocytes are frequently elevated. Likewise, T-lymphocytes and CD4 counts appear normal in malnourished children, when measured by flow cytometry, the gold standard for characterizing cell subsets. Their levels seem to be determined more by infections than by nutritional state, and do not reflect the degree of malnutrition-related immune deficiency, as high infectious mortality is seen in malnourished children, despite unaffected white blood cell counts Malnourished children can mount an acute phase response to infections, with elevated CRP and low negative acute phase reactants, and this can also be seen in absence of clinical infection. Thus, based on available evidence, the acute phase response, if anything, seems exaggerated rather than diminished. Levels of IgM and IgG are normal or elevated in malnourished children. Secretory IgA is not consistently lower in duodenal fluid, and frequently elevated in urine.tropical sprue described in adults, and the term enteropathy of malnutrition has been replaced by the broader term environmental enteropathyThe gut mucosa is atrophied and permeable in malnourished children. This enteropathy also affects well-nourished children in poor communities, but probably most severely in malnourished children. The condition appears similar to Production of gastric acid and flow of saliva is reduced in malnourished children. Secretory IgA is also reduced in saliva, tears and nasal washings from children with severe, but not moderate malnutrition. The small bowel of malnourished children is often colonized with abundant bacteria, and their pattern of commensal flora is altered. Granulocytes kill ingested microorganisms less effectively. Levels of complement proteins are low in blood from malnourished children, particularly in children with oedematous malnutrition, and less in children with moderate malnutrition.Lymphatic tissue, particularly the thymus, undergoes atrophy in malnutrition in a dose-response fashion: thymic size depends on nutritional status even in milder degrees of malnutrition, and thymus size is a predictor of survival in children.DTHR is diminished in malnourished children. Lymphocytes of malnourished children are less responsive to stimulation with PHA, fewer are activated and more cells have markers of apoptosis. Plasma IgA is mostly elevated in malnourished children, particular in those with oedema. Children with severe, but not moderate, malnutrition mount a lower specific antibody response to vaccination, although for most children sufficient to obtain protection. The lower titres seen in malnourished may be due to a delay in vaccination response.Cytokines can be classified as those promoting a Th1 response of predominantly cellular immunity, and those promoting a Th2-response of humoral immunity The mechanisms behind these immunological alterations are still not adequately understood. Some explain it by lack of energy and building blocks to synthesize the proteins required Infections could obviously contribute to the changes seen, and interactions have been noted between infection and malnutrition in their respective effects on immune parameters Animal studies suggest hormonal factors to be involved in the immune profile of malnutrition. Leptin This hormonal profile is similar to that of an acute phase response, where thymus atrophy also occurs, acquired immunity is temporarily suppressed and innate immunity takes over In HIV infection, persisting subclinical inflammation and immune activation is frequently present, and may be partly responsible for immune deficiency and disease manifestations mammalian target of Raptomycin (mTOR), is present in most cells. It responds to concentrations of nutrients in the cell's surroundings, and to other signs of stress, such as hypoxia, enabling the cell to adapt its metabolism to locally available nutrients. Immune cells also use mTOR to regulate their state of activation. Nutrient availability may thereby determine whether an immune cell is activated The intracellular receptor, A research group working with animal models of malnutrition has proposed a theory called the “tolerance hypothesis” The pathogenesis of oedematous malnutrition is still unknown. Many immune parameters seem affected to a different degree in children with oedematous malnutrition, with higher levels of IgA, higher levels of abnormal antibodies like IgD, poorer vaccination responses and cytokines more skewed towards a Th2-response; their complement levels are lower, which may partly be caused by increased consumption of complement in-vivo. The pattern of leukotrienes is different in children with oedematous compared to non-oedematous malnutrition. This immunological profile resembles that seen in autoimmune diseases such as lupus erythematosus In spite of the prevalence of malnutrition, and its fatal consequences, scientific interest in the immune deficiency of malnutrition seems dwindling, and little research has been carried out on the topic during the last ten years. For this reason, most evidence on the subject relies on immunological methods used 30 to 40 years ago, many of which are no longer in use, and little research has been done with modern methods, and with the present understanding of immunology. Moreover, most studies have looked at isolated aspects of immune function, despite the fact that the parameters are interdependent, and the division into innate and adaptive immune function seems to be a simplification. Thus, our understanding of immune function in malnutrition is still very limited.This review illuminates the little that we know about the immunological alterations associated with malnutrition, and also points to significant gaps in our knowledge. Future well designed prospective cohort studies should examine how immune parameters are related to morbidity and mortality in malnourished children, with detailed characteristic of nutritional status, preferably body composition, of infections, enteropathy and of low-grade inflammation. When testing nutritional and medical interventions for malnutrition, immune parameters should be included as outcomes. Studies should investigate newer immunological parameters in malnutrition, like expression of innate pattern recognition receptors (as the Toll-like receptor), the lectin pathway of the complement system and mTOR expression and activity. It should be investigated whether a small thymus is associated with lower output of recent thymic-derived T-cells, and how it correlates with hormones like leptin, cortisol, insulin and Insulin Growth Factor-1. Innate and adaptive immune parameters should be assessed simultaneously, taking into account their dynamic interdependency. To understand whether malnutrition is indeed associated with active down-regulation of immune reactivity (as formulated in the “tolerance hypothesis”), the balance between regulatory T-lymphocytes and their counterparts, Th17 lymphocytes should be measured. Finally, prospective studies among children at risk should assess whether immune profiles differ in those who subsequently develop oedematous and non-oedematous malnutrition, and it should be investigated whether children with oedematous malnutrition have markers suggestive of auto-immune or inflammatory diseases. Such studies would reduce our current ignorance on the interplay between malnutrition and infectious diseases.Figure S1PRISMA Flow diagram showing study retrieval and selection.(DOCX)Click here for additional data file.Table S1Articles describing barrier and immune function of skin in malnourished children.(DOCX)Click here for additional data file.Table S2Articles describing intestinal function and mucosal structure in children with malnutrition.(DOCX)Click here for additional data file.Table S3Articles describing anti-microbial factors in mucosal secretions of malnourished children.(DOCX)Click here for additional data file.Table S4Articles describing commensal flora in children with malnutrition.(DOCX)Click here for additional data file.Table S5Articles describing function of innate immune cells: polymorph-nuclear cells and monocytes/macrophages in children with malnutrition.(DOCX)Click here for additional data file.Table S6Articles describing acute phase response in malnourished children.(DOCX)Click here for additional data file.Table S7Articles describing complement in malnourished children.(DOCX)Click here for additional data file.Table S8Articles describing thymus and other lymphatic tissue in autopsies of malnourished children.(DOCX)Click here for additional data file.Table S9Articles describing ultrasound scans of thymus in malnourished children.(DOCX)Click here for additional data file.Table S10Articles describing delayed type hypersensitivity response in children with malnutrition.(DOCX)Click here for additional data file.Table S11Articles describing lymphocyte subsets in children with malnutrition.(DOCX)Click here for additional data file.Table S12Articles describing antibody levels in children with malnutrition.(DOCX)Click here for additional data file.Table S13Articles describing humoral vaccination responses in children with malnutrition.(DOCX)Click here for additional data file.Table S14Articles describing cytokines in malnourished children.(DOCX)Click here for additional data file.Checklist S1PRISMA Checklist.(DOCX)Click here for additional data file.


Over the past decade mind and body practices, such as yoga and meditation, have raised interest in different scientific fields; in particular, the physiological mechanisms underlying the beneficial effects observed in meditators have been investigated. Neuroimaging studies have studied the effects of meditation on brain structure and function and findings have helped clarify the biological underpinnings of the positive effects of meditation practice and the possible integration of this technique in standard therapy. The large amount of data collected thus far allows drawing some conclusions about the neural effects of meditation practice. In the present study we used activation likelihood estimation (ALE) analysis to make a coordinate-based meta-analysis of neuroimaging data on the effects of meditation on brain structure and function. Results indicate that meditation leads to activation in brain areas involved in processing self-relevant information, self-regulation, focused problem-solving, adaptive behavior, and interoception. Results also show that meditation practice induces functional and structural brain modifications in expert meditators, especially in areas involved in self-referential processes such as self-awareness and self-regulation. These results demonstrate that a biological substrate underlies the positive pervasive effect of meditation practice and suggest that meditation techniques could be adopted in clinical populations and to prevent disease. Mind and body practices such as yoga, meditation, progressive relaxation, or guided imagery use mental and physical abilities to improve health and well-being. Over the past decade these practices have received increasing attention in different fields of study in which the physiological mechanisms underlying the beneficial effects observed in trained individuals have been investigated. Increased knowledge about the physiological effects of mind and body practices makes it possible to explore their therapeutic potential, identify adverse effects, and safely integrate these techniques into standard therapeutic approach.Meditation is a complex process aimed at self-regulating the body and mind and is often associated with psychological and neurophysiological modifications . MeditatMeditation practice has been found to promote well-being by fostering cognitive and emotional processes , 6. SpecInterestingly, the current literature suggests that meditation has a potential effect on age-related cognitive decline , 23, proThe effects of meditation on brain structure and function have received increasing attention in neuroimaging studies  and the number of published studies is steadily growing . SpecifiFunctional studies on the brain correlates of meditation have assessed neural activation during meditation by requiring participants to undergo fMRI scans during meditation tasks. These studies have reported increased activation in areas associated with attention, mind wandering, retrieval of episodic memories, and emotional processing during meditation [ditation . Specifiditation , parietaditation , middle ditation  has been functional brain modifications after meditation training have focused on functional and the metabolic changes after meditation training and/or in expert meditators compared with control participants. These studies adopted different paradigms  and structural (sMRI) changes in expert meditators. To pursue this aim we adopted a meta-analytic approach based on activation likelihood estimation (ALE) analysis, which allows performing coordinate-based meta-analyses of neuroimaging data .The database search on PubMed was performed using the following string: (((((((MRI) AND meditation) NOT Alzheimer's) NOT Parkinson's) NOT EEG) NOT MEG) NOT mild cognitive impairment). A total of 93 papers emerged. From this collection, we selected only papers that (1) included whole-brain analysis performed using magnetic resonance imaging (MRI), (2) provided coordinates of activation foci either in Montreal Neurological Institute (MNI) or in Talairach reference space, (3) studied young and healthy participants, (4) reported activation from group studies, (5) included meditators or required participants to perform a meditation task, and (6) used no pharmacological manipulation. We selected 57 papers: 42 reported fMRI studies and 15, sMRI studies. Out of the 42 fMRI studies, 5 were excluded because they did not provide coordinates of activation foci; out of the 15 papers on sMRI studies, 6 papers were excluded for the same reason and one paper was excluded because it did not include expert meditators.In line with the aims of the present meta-analysis, individual experimental studies from selected papers were divided according to three main axes: papers reporting (a) functional magnetic resonance imaging studies (fMRI) during meditation training, (b) functional magnetic resonance imaging studies (fMRI) that studied neural modifications after meditation training, and (c) structural MRI studies (sMRI). Note that the fMRI studies on neural modifications after meditation see  includedWe included 37 individual fMRI experimental studies on functional activations during meditation tasks (642 participants), 63 fMRI experimental studies see  on strucActivation likelihood estimation (ALE) analyzes the probability that a voxel will contain at least one of the activation foci; it is calculated at each voxel and results in a thresholded ALE map. In other words, ALE assesses the overlap between foci by modeling the probability distributions centered at the coordinates of each one .Our first aim was to provide a general picture of areas activated during meditation tasks. Thus, we carried out an ALE analysis of fMRI studies on functional activations during meditation tasks. Then, we performed two ALE analyses to determine whether meditation produces consistent modifications in brain structure and function. In the first analysis we included sMRI studies, and in the second analysis we included fMRI studies on neural modifications after meditation training.http://brainmap.org/) with MNI coordinates . Following Eickhoff et al.'s modified procedure [The ALE meta-analysis was performed using GingerALE 2.1.1  at the 0.05 level of significance . Moreover, a minimum cluster size of 200 mm3 was chosen. A cluster analysis was performed on the thresholded map.For the fMRI studies, the thresholded ALE map was computed using P values from the previous step and a cluster level correction at the 0.05 level of significance, with a minimum cluster size of 200 mm3. A cluster analysis was performed on the thresholded map.For the sMRI studies, the thresholded ALE map was computed using http://brainmap.org/) using Mricro (http://www.mccauslandcenter.sc.edu/mricro/index.html).The ALE results were registered on an MNI-normalized template , and middle occipital gyrus in the right hemisphere. We also found that left posterior cerebellum, specifically the declive, was more highly activated during meditation than the control condition .We found that meditation practice see  was assoWe found that meditation practice was associated with increased grey matter volume in the frontal lobe, at the level of the right anterior cingulate cortex and left middle and medial frontal gyrus. We also found increased grey matter volume in meditators at the level of the left precuneus and fusiform gyrus and the right thalamus .The main aim of the present study was to identify the neural network activated during meditation and to explore structural and functional brain modifications in expert meditators. We also aimed to explore the relationship between meditation practice and the neural mechanisms that allow maintaining the positive effects of meditation training. For this purpose we adopted ALE analysis, a technique used widely in coordinate-based meta-analyses of neuroimaging data . The res which brain areas were activated during mediation. We used ALE analysis to identify the neural networks activated during meditation tasks and carried out the coordinate-based meta-analysis on experimental studies that required participants to meditate during the fMRI scan, regardless of their previous experience. We found that a set of brain areas spanning from the occipital to the frontal lobes was more highly activated during the meditation condition than during the control condition. This network included areas involved in processing self-relevant information, such as the precuneus [The first question we tried to answer wasrecuneus , in procrecuneus , in interecuneus , in reorrecuneus , and in recuneus . It is nrecuneus .As previously described, meditation practice has been found to promote well-being by fostering cognitive and emotional functioning . Indeed,Results of the ALE analysis of sMRI studies showed increased grey matter volume in meditators compared to control groups in the right anterior cingulate cortex, left middle and medial frontal gyrus, left precuneus and fusiform gyrus, and right thalamus. It could be that the increased grey matter volume in the anterior cingulate cortex of meditators accounts for the improvement of specific abilities such as self-regulation, self-control, focused problem-solving, and adaptive behavioral responses under changing conditions , which aRegarding the differences among meditation techniques, as reported above, meditation practices can be grossly divided into two different approaches: focused attention meditation and open monitoring meditation. Anyway, most meditation approaches use both types of practices complementarily , 4 and iOverall, results of the present ALE analysis suggest that meditation practice induces functional and structural brain modifications, especially in areas involved in self-referential processes, including self-awareness and self-regulation , as well


Communication gaps impact the quality of patient care. Previous research has focused on communication barriers rather than seeking solutions. Our aim was to identify strategies for effective communication and decision-making about goals of care for medical interventions in serious illness, from the perspectives of hospital-based healthcare providers.A cross-sectional survey composed of closed- and open-ended questions about goals of care communication and decision-making was administered to healthcare providers in 13 centres in six Canadian provinces. We analyzed a portion of the open-ended survey questions, specifically (1) suggestions for overcoming barriers encountered in discussing goals of care, and (2) currently effective practices. Thematic content analysis was used to analyze responses to the open-ended questions.Of the 1,256 respondents to the larger survey, 468 responded to the open-ended questions (37 %), including 272 of 512 nurses (53 %), 153 of 484 internal medicine trainees (32 %), and 43 of 260 attending physicians (17 %). Responses to each of the two questions were similar, generating a common set of themes and subthemes. Effective strategies and ideas for improving communication and decision-making about goals of care clustered under five themes: patient and family factors, communication between healthcare providers and patients, interprofessional collaboration, education, and resources. Subthemes highlighted core elements of shared decision-making.Translating our findings into multifaceted interventions that consider patient and family factors, address knowledge gaps, optimize resource utilization, and facilitate communication and collaboration between patients, families and healthcare providers may improve communication and decision-making about goals of care.The online version of this article (doi:10.1186/s12904-015-0035-x) contains supplementary material, which is available to authorized users. With an aging population , planninHealthcare providers often do not discuss goals of care with seriously ill hospitalized patients , 4 or thAddressing the gap between the care provided and that desired, and providing high quality patient-centred EOL care will require improved communication and decision-making about goals of care. For seriously ill hospitalized patients, goals of care conversations include deliberation and decision-making about the use or non-use of life-sustaining treatments . While mThis cross-sectional study, conducted from September 2012 to March 2013, involved a self-administered questionnaire about effective goals of care communication and decision-making in relation to medical interventions desired in serious illness; it was distributed to hospital-based clinicians in general internal medical teaching units (MTUs) at 13 academic centres in six Canadian provinces . ResearcAll eligible attending physicians, residents, and nurses were invited to participate, with the exception of centres with more than 50 nurses, where computer generated random number lists selected a sample of 50 nurses. Eligible participants were: (i) MTU attending physicians; (ii) postgraduate internal medicine residents (visiting residents were excluded); (iii) nurses  employed full-time or part-time in a MTU at a participating centre. While many healthcare providers communicate with patients in hospital, we chose to focus upon nurses, residents and physicians for reasons of feasibility and because these providers are involved in the clinical care of every patient in a MTU while other providers are less frequently involved, depending on a patient’s needs.Details of questionnaire development and distribution have previously been described . Briefly“What specific suggestions do you have about ways to overcome these barriers [rated as very or extremely important in the preceding section] and make it easier for health care providers to talk with patients and their family members about goals of care?”“What is currently working well to promote communication and decision-making about goals of care between health care providers and patients and their family members?”In this paper, we report on free-text responses to two open-ended questions from the larger survey :We used descriptive statistics to summarize respondent demographics, computing the mean and standard deviation for continuous variables and proportions for categorical variables. To analyze free text responses, bilingual investigators first translated French responses into English. Some respondents only described barriers rather than providing strategies for improvement or strategies that were currently effective; these comments were excluded from analysis as they did not pertain to the research question for this paper. Thematic content analysis was used to analyze free text responses . Two of Questionnaires were returned by 1,256 of 1,617 eligible healthcare providers, with an overall response rate of 78 % for the larger survey that included both the open and closed-ended responses . A free-text response to Question 1 and/or Question 2 (the open-ended survey questions) was provided by 468 (37 %) of the 1,256 healthcare providers who responded to the larger survey , whereas others were specific to only one or two groups. The themes and subthemes are described below and summarized in Table Subthemes directly related to the patient and family included advanced illness, substitute decision-making, and philosophies about location of EOL care. Advanced illness was perceived as a trigger for healthcare providers to initiate goals of care discussions and help patients identify a substitute decision-maker. Nurses advocated for supporting family involvement in providing EOL care in the hospital and at home.Healthcare providers identified attention to the timing, content, process, and continuity of communication between healthcare providers, patients, and families as an important mechanism to improve the quality of goals of care discussions and decision-making.With respect to timing, all groups emphasized that early communication between healthcare providers, patients, and families was important. Early communication could facilitate building rapport, determine the family spokesperson and substitute decision-maker, identify advance directives, and help patients and family better understand diagnoses, treatment options, and prognosis in the first days of hospitalization. Many free-text question respondents felt that initiating goals of care discussions within the first twenty-four hours of hospital admission is important; some healthcare providers in each group mentioned that patients and families need time to make these complex decisions, while others emphasized that healthcare providers need to readdress goals of care as patient condition improves or worsens.Continuity of care within and across healthcare settings was identified as enabling difficult discussions. Specific strategies cited included involving community care physicians with longitudinal doctor-patient relationships in goals of care discussions, and consistent patient assignments in the hospital.In describing currently effective approaches and ideas for improving goals of care discussions, respondents noted several issues related to the content and process of communication. Content issues included eliciting and exploring patient and family experiences, values and preferences; providing guidance to patients and families around goals of care decision-making; and making a recommendation tailored to the patient’s medical condition, prognosis and values. Process considerations included building rapport; ensuring sufficient time for discussion; use of clear language; assessment of patient and family understanding; achieving a shared understanding; and respecting patient values and preferences. Being honest and realistic in discussing goals of care and prognosis was also emphasized.All groups, and nursing staff in particular, highlighted the effectiveness of a collaborative, interprofessional approach to discussing and determining goals of care. Responses clustered into three subthemes: consistency of interprofessional communication, role clarity, and documentation.Approaches to interprofessional collaboration included multidisciplinary family meetings and healthcare team meetings; nurses and residents specified the importance of attending physician involvement in these meetings. Nurses commented that dialogue between team members facilitated establishing plans of care, consistent messaging to patients and family, and coordinating services to achieve patient goals of care. Team support through reminders and communication between hospital and community healthcare providers was also described.The need for clarifying the role of both individual and groups of healthcare providers in discussing goals of care was also stressed. For example, should addressing goals of care be the role of nurses, residents, or attending physicians? Some nurses desired greater involvement in the process, and proposed expanding nursing roles in discussing and determining goals of care.All groups commented that documenting goals of care discussions and decisions would facilitate future healthcare provider awareness of the content and rationale for decisions made. A standardized form for documentation placed in a consistent section of the patient chart would enable consistency.Education of the public, patients and families, and healthcare providers about advance care planning and goals of care were core subthemes within the education theme. Suggested societal and patient/family education strategies included dialogue, informational brochures, and video clips of life support interventions accompanied by patient and family narratives of intensive care and chronic critical illness experiences.Helpful strategies to establish expectations that goals of care be addressed included hospital policies and increasing physician awareness of the importance of goals of care discussions. Residents mentioned undergraduate and postgraduate medical education initiatives improved their comfort with these discussions, along with experiential learning during clinical rotations. Respondents specified that courses and informal learning through role-modeling and feedback could improve knowledge and skills related to communication overall, cultural diversity, advance care planning, goals of care discussions, and palliative care. Residents specifically valued attending physicians' involvement in patient care in general, and goals of care discussions in particular.Each healthcare provider group identified the importance of directing resources towards facilitating goals of care discussions. Subthemes included document access, personnel, physical space, and organizational support. Residents in particular hoped for easier access to community practitioner documentation of advance care planning discussions and prognostication. Nursing staff and residents mentioned attending physician availability and sufficient time as conditions for effective goals of care discussions. All groups mentioned that higher healthcare provider to patient ratios would allow more time for family meetings and complex discussions. A facilitating role for consultants with expertise in EOL communication, and increased availability of interpreters were also described. Finally, nurses perceived a patient-centred focus at an organizational level as important, with policies and procedures that foster improved goals of care communication and decision-making.Through qualitative analysis of free text responses to open-ended questions in a national, multicentre survey, we have described hospital-based healthcare providers’ perspectives on strategies to improve goals of care discussions and decision-making. These strategies clustered into five overall themes: patient and family involvement, healthcare provider communication with patients and families, interprofessional communication, education, and resources.Our study focused on the positive, seeking to identify currently effective processes that can be further developed and expanded upon and ideas for improving current approaches, rather than focusing on negative experiences and challenges. Concepts mentioned as potential solutions by some participants were described by others as currently effective processes. Considering the multidisciplinary and multicentre nature of our study, this overlap may reflect different perspectives between individuals, or different practices between hospital units or institutions. A better understanding of this overlap could facilitate targeted interventions. For example, if one centre’s ideas for improvement are currently implemented elsewhere, sharing resources and experiences may both facilitate and expedite knowledge translation.The concept of shared decision-making can be identified within all themes described in our study. Previous research has reported that patients and families value shared decision-making in establishing goals of care , while gThis study represents a subset of data from a larger survey . The quaWhile the current study focused on healthcare provider experiences, the recent multicentre ACCEPT study explored patient and family perspectives on barriers and facilitators of advance care planning . NotablyThis alignment between patient and family needs and healthcare provider strategies for improving goals of care discussions is encouraging and may suggest that healthcare providers are sensitive to patient and family needs. However, if this was true, why do needs remain unmet? Perhaps the barriers outweigh the current potential for change. Change within the healthcare system is a complex process, influenced by individual and organizational readiness, perceived capacity to change, and contextual receptivity . In procTo impact patient care, insights about effective strategies for goals of care discussions will need to be translated into clinical practice. Our study describes the initial steps of knowledge creation – knowledge inquiry, identification of facilitators, and knowledge synthesis . CreativStrengths of our study include the multicentre data set involving multiple healthcare providers, with diversity in geography, language (French and English), and culture allowing generalizability across the Canadian context. Triangulation of data from nurses, residents and attending physicians adds additional rigour to our findings. Finally, open-ended questions elicited participant expression of ideas, affording a more elaborate description than can be obtained from closed-ended survey options, where investigator synthesis of pre-existing literature constrains findings.One of the limitations is in the nature of free-text data, where ideas cannot be explored, clarified, or expanded upon, as would be possible with interviews and focus groups. Another limitation is our focus on physicians and nurses in the hospital setting; community-based practitioners, social workers, counselors and spiritual care providers would likely identify different approaches and ideas. Their views, as well as a greater number of free-text responses from attending physicians in the study, may have allowed for a broader range of perspectives on the issues. Response to free-text questions was optional; we cannot discern from our data why physicians were less inclined to respond to these questions than nurses or residents. Potential reasons include fatigue at the end of a long questionnaire, lack of ideas, or the perception that their opinions were sufficiently expressed in the closed-ended section of the questionnaire. Finally, while we did not elicit patient or family perspectives, complementary patient and family data exist in the literature , 20.Overall, our findings suggest that enhancing patient and family involvement, communication between patients, families and healthcare providers, interprofessional collaboration, educational initiatives, and resource availability may improve discussions and decision-making about goals of care for medical interventions among seriously ill patients in hospital. Ongoing developments in these areas may be facilitated by a range of promising interventions, such as internet-based or video decision aids for advance care planning and goals of care determination, family meetings, teamwork training, societal and healthcare provider educational interventions, and quality improvement initiatives. Further consideration, tailored implementation, and evaluation of such interventions are warranted.


ERK1/2 MAP Kinases become activated in response to multiple intra- and extra-cellular stimuli through a signaling module composed of sequential tiers of cytoplasmic kinases. Scaffold proteins regulate ERK signals by connecting the different components of the module into a multi-enzymatic complex by which signal amplitude and duration are fine-tuned, and also provide signal fidelity by isolating this complex from external interferences. In addition, scaffold proteins play a central role as spatial regulators of ERKs signals. In this respect, depending on the subcellular localization from which the activating signals emanate, defined scaffolds specify which substrates are amenable to be phosphorylated. Recent evidence has unveiled direct interactions among different scaffold protein species. These scaffold-scaffold macro-complexes could constitute an additional level of regulation for ERK signals and may serve as nodes for the integration of incoming signals and the subsequent diversification of the outgoing signals with respect to substrate engagement. Exhaustive research efforts undertaken during the past decades, have positioned the signaling module mediated by Extracellular signal-Regulated Kinases 1 and 2 (ERKs) Mitogen-Activated Protein Kinases (MAPKs), among the best known signal transduction processes ever studied. ERKs signaling cascade encompasses sequential tiers, composed of sundry types of molecular intermediaries, which become activated in response to a broad panel of intra- and extra-cellular stimuli. The ERKs cascade is generally activated at its origin by GTPases of the RAS family that subsequently switch-on, by not fully understood mechanisms, an upstream echelon constituted by MAPKKKs of the RAF family. These, at their turn, convey signals downstream by phosphorylating/activating dual-specificity MAPKKs MEK 1 and 2, ultimately responsible for the phosphorylation and the unleashing of ERKs activity , Ste7 (MAPKK), and Ste11 (MAPKKK) and to increase their local concentration at the tips of mating projections, in response to mating pheromones .Finally, a pivotal concept in the scaffolds theory is that for any given scaffold there exists an optimal concentration that yields maximum signal efficiency, resulting in a bell-shaped MAPK activation kinetics. In this process, sub-optimal MAPK activation occurs both when there are not sufficient scaffolds to unite all of the available MAPKs, MAPKKs, and MAPKKKs, and also when an excessive scaffold concentration scatters MAPKs, MAPKKs, and MAPKKKs in incomplete, thereby unproductive, complexes. This phenomenon has been termed “combinatorial inhibition” and “prozone effect” . In most cases, the functional consequences of these modifications are completely unknown, but it is quite conceivable that, one way or another, they could have some bearing on their behavior as ERK pathway scaffolds. Indeed, it is well documented that the role played by KSR in the RAS-ERK pathway is regulated by diverse phosphorylation events  could interact in s Figure . In thisSuch interdependence among different scaffold species, could offer a plausible explanation for the dramatic consequences on ERK signaling, frequently observed when tampering with the expression levels of most scaffold proteins. If proven to be correct, associations among different scaffold proteins will add one further degree of regulation for an already tightly regulated cascade and could provide a novel means for manipulating ERK signals, even with therapeutic purposes.Both PC and BC have made substantial, direct, and intellectual contribution to the work, and approved it for publication.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.


It is common for individuals to engage in taxing cognitive activity for prolonged periods of time, resulting in cognitive fatigue that has the potential to produce significant effects in behaviour and decision making. We sought to examine whether cognitive fatigue modulates economic decision making.We employed a between-subject manipulation design, inducing fatigue through 60 to 90 minutes of taxing cognitive engagement against a control group that watched relaxing videos for a matched period of time. Both before and after the manipulation, participants engaged in two economic decision making tasks (one for gains and one for losses). The analyses focused on two areas of economic decision making—preferences and choice strategies. Uncertainty preferences (risk and ambiguity) were quantified as premium values, defined as the degree and direction in which participants alter the valuation of the gamble in comparison to the certain option. The strategies that each participant engaged in were quantified through a choice strategy metric, which contrasts the degree to which choice behaviour relies upon available satisficing or maximizing information. We separately examined these metrics for alterations within both the gains and losses domains, through the two choice tasks.The fatigue manipulation resulted in significantly greater levels of reported subjective fatigue, with correspondingly higher levels of reported effort during the cognitively taxing activity. Cognitive fatigue did not alter uncertainty preferences (risk or ambiguity) or informational strategies, in either the gains or losses domains. Rather, cognitive fatigue resulted in greater test-retest variability across most of our economic measures. These results indicate that cognitive fatigue destabilizes economic decision making, resulting in inconsistent preferences and informational strategies that may significantly reduce decision quality. Cognitive fatigue is a ubiquitous human condition, the result of sustained cognitive engagement that taxes our mental resources. The nature of work in our society is changing such that increasingly, work involves demanding cognitive activity, as opposed to physical exertion, with working hours no longer restricted by daylight. Demanding work schedules lead many people to experience cognitive fatigue on a daily basis, and have resulted in high burnout rates ,2. StudiWe sought to specify the impact of cognitive fatigue on economic decision making, with a focus on uncertainty preferences and strategy. Uncertainty refers to the absence of information about the eventual resolution of probabilistic events, such as in gambles. The two common forms of uncertainty are risk, which involves known probabilities , and ambiguity, which pertains to probabilities of outcomes that are unknown or cannot be estimated –15.Beyond economic preferences, we also examined what information the participant utilized to inform their choices . We contA priori, we hypothesized that cognitive fatigue would result in increased satisficing strategies, as fatigued participants opt for less effortful strategies over those requiring more mental resources. This was based on two directions of prior findings. First, studies suggest that cognitive fatigue results in compromised top-down control mechanisms with relative sparing of automatic processes . SecondlWe investigated the impact of cognitive fatigue on economic decision making, specifically alterations of uncertainty preferences and/or choice strategies. We employed two incentive-compatible economic decision making tasks, examining for alterations in economic decision making independently across both the gains and the losses domains. We utilized a between-subjects design, comparing participants that engaged in a cognitively taxing task (fatigue group) and those that watched relaxing videos for an equivalent period of time (control group). We found that cognitive fatigue did not produce a reliable shift in individual uncertainty preferences or choice strategies, contrary to our initial hypotheses. Rather, cognitive fatigue resulted in significantly greater test-retest variability across multiple measures. These results indicate that cognitive fatigue results in the destabilization of economic decision making, highlighting the dangers of fatigue.M = 22.3 years, SD = 1.74), recruited through advertisements at the National University of Singapore. Data were collected in 2 samples. In the initial sample, 44 participants took part in the study and were randomly assigned to a fatigue (N = 19) or control (N = 25) conditions. Examining the fatigue manipulation for these participants indicated that although the fatigue condition reported cognitive fatigue and significantly higher levels of cognitive effort expended, there was no decline in performance in the manipulation task. While this effect has been previously reported, we opted to enhance the cognitive fatigue manipulation to ensure that our manipulation was sufficient  and pWIN r2 ).As the choice strategy metric is a composite, and a lack of difference in the composite measure does not mean there is no change in the components, we also examined whether there were any significant differences in the use of rEV and pWIN information separately. Concurring with the composite measures, we found no significant effect of cognitive fatigue on either component  = 0.562, p = 0.576) and no alterations of the independent components  = 0.460, p = 0.647] and pWIN r2 ).The same pattern of results were found in the losses domain , with noPost-hoc, we noticed a pattern of reduced test-retest correlations within the fatigue group across the economic decision making measures. To quantify this, we used a Fisher’s r-to-z transformation to compare the pre- and post- correlations between fatigue and control groups for each metric .z = 3.10, p = 0.002). This effect was not found for the ambiguity premiums . The losses domain mirrored the results of the gains domain; risk premium test-retest correlations were significantly lower in the fatigue group than in the control group , with no difference for ambiguity premiums .Risk premium values in the gains domain showed significantly lower test-retest correlations in the fatigue group than the control group   .2, and the pWIN r2, in both the gains and losses domains. We applied a full-Bonferroni correction (p = 0.005) to account for these 10 additional tests on each of our measures of individual fatigue and individual effort. None of these factors indicated a relationship that survived the necessary multiple comparison correction.It is possible that individual changes in fatigue or effort could account for individual differences in the economic metrics. For example, while there is no mean shift in risk preference across groups, it is still possible that there could be a linear relationship between individual change in fatigue and individual change in risk preference. To specifically test for this post-hoc, across all participants, we correlated the change in fatigue and effort against our economic metrics of risk preference, ambiguity preference, choice strategy, the rEV r2, and the pWIN r2). Within each domain (gains or losses), we applied a full Bonferroni corrected threshold to p = 0.0125 for the 4 tests. None of the 8 correlations were significant.We also post-hoc examined whether individual levels self-reported fatigue or effort (RSME) relate to changes in our economic measures  and choice strategies in both the gains and losses domains. We found no mean shifts in preferences or strategies across either domain. Rather, post-hoc analyses revealed that cognitive fatigue resulted in decreases in test-retest stability across both risk preferences and choice strategies.We found no mean shifts of uncertainty preferences (risk or ambiguity) due to cognitive fatigue. Rather, cognitive fatigue reduced the stability of risk preferences. When fatigued, subjects were inconsistent in their risk preferences, demonstrating more variable risk attitudes.Multiple studies have suggested that suboptimal mental states are associated with increased intra-individual variability in performance measures such as reaction time –34. OnlyIn contrast to increased instability in risk preferences, we found no changes in ambiguity preferences. This finding appears to concur with neurobiological evidence that these two preferences may be dissociable . HoweverIn both the gains and losses domains, fatigue reduced the stability of choice strategy without leading to mean shifts . It is quite interesting that this result mirrors the alterations we found for risk preferences, as risk preferences and choice strategy are not correlated across our participants, and ostensibly measure different aspects of decision making.Of note, reduced effort in decision making is potentially an attractive explanation for how cognitive fatigue increased the noise in our choice strategy metric. However, such a motivational change would actually have presented as an increase in satisficing behaviour (decreased choice strategy)–exactly the shift that we initially hypothesized and did not find. Rather, participants continue to, on average, employ the same relative levels of satisficing and maximizing strategies in their choice behaviour, suggesting that they are maintaining their behavioural motivation in our incentive compatible tasks.Cognitive fatigue results in greater variability in both risk preferences and choice strategies, which will result in decreased consistency in the actual choices made. Insofar as choice quality can be simply judged based upon whether you would repeat your choice given the same options, these results indicate that cognitive fatigue reduces choice quality.In relation to economic theory, inconsistent preferences or strategies may lead to failures of transitivity  a key rule of rational choice behaviour . The impInterestingly, our results resemble the behavioural effects of ventromedial prefrontal cortex (vmPFC) damage, which leads to inconsistent or erratic preference judgements without a slowing of response times ,42. ThisParticipants in the fatigue group maintained a high level of performance on the N-back task despite reporting increasing levels of fatigue and indicating that high levels of mental effort were required to perform each subsequent block.This observed pattern of unchanged task performance under cognitive fatigue is not uncommon . SurprisIt is unclear how cognitive fatigue relates to other state alterations, such as sleep deprivation, aging, or ego-depletion. A fruitful area for future investigations would be to investigate how various state alterations may share common mechanisms.A priori, one may expect that there would be similarities between the effects of cognitive fatigue and sleep deprivation. In fact, there is a recent model of sleep deprivation effects, called ‘the state instability hypothesis’, whose name sounds strikingly similar to our found effects . This moOur cognitive fatigue manipulation involved 60 to 90 minutes of a strenuous task that engaged executive control. A similar methodology, although involving only ~10% of the manipulation duration, occurs in ego-depletion manipulations . In ego-Of specific interest for future studies will be examining how cognitive fatigue may dissociably alter sub-components of the cognitive processes engaged during economic decision making. As we find a general effect across economic decision making metrics, this suggests that cognitive fatigue is altering cognitive processes that are engaged across these economic metrics. Possible examples include interfering with working memory, preventing memory consolidation of prior choices, interfering with the application of preferences, or interfering directly with the comparison process.There is potentially great utility in examining and comparing how varied state modulations commonly and dissociably alter economic decision making. For example, in these same tasks, we recently showed that aging produces specific shifts of risk preferences and strategies in the losses domain, without altering these processes in the gains domain .The duration of our cognitive fatigue manipulation (60 to 90 minutes) was specifically aimed at examining a duration that is common in everyday life. It is unclear if longer durations of cognitive fatigue will result in specific shifts in economic preferences, such as enhanced/reduced risk aversion or reduced use of maximizing information. However, we saw no significant differences between the 5-blocks and 7-blocks cognitive fatigue groups .The results of this study show that cognitive fatigue results in destabilization of risk preferences and the informational strategies participants employ. This increased variability in choice behaviour can undermine the integrity of decisions, resulting in diminished choice behaviour quality.S1 Datafile(XLSX)Click here for additional data file.