Detrimental changes in the composition and function of rotator cuff (RC) muscles are hallmarks of RC disease progression. Previous studies have demonstrated both atrophic and degenerative muscle loss in advanced RC disease. However, the relationship between gene expression and RC muscle pathology remains poorly defined, in large part due to a lack of studies correlating gene expression to tissue composition. Therefore, the purpose of this study was to determine how tissue composition relates to gene expression in muscle biopsies from patients undergoing reverse shoulder arthroplasty (RSA). Gene expression related to myogenesis, atrophy and cell death, adipogenesis and metabolism, inflammation, and fibrosis was measured in 40 RC muscle biopsies, including 31 biopsies from reverse shoulder arthroplasty (RSA) cases that had available histology data and 9 control biopsies from patients with intact RC tendons. After normalization to reference genes, linear regression was used to identify relationships between gene expression and tissue composition. Hierarchical clustering and principal component analysis (PCA) identified unique clusters, and fold-change analysis was used to determine significant differences in expression between clusters. We found that gene expression profiles were largely dependent on muscle presence, with muscle fraction being the only histological parameter that was significantly correlated to gene expression by linear regression. Similarly, samples with histologically-confirmed muscle distinctly segregated from samples without muscle. However, two sub-groups within the muscle-containing RSA biopsies suggest distinct phases of disease, with one group expressing markers of both atrophy and regeneration, and another group not significantly different from either control biopsies or biopsies lacking muscle. In conclusion, this study provides context for the interpretation of gene expression in heterogeneous and degenerating muscle, and provides further evidence for distinct stages of RC disease in humans. The progressive and irreversible loss of rotator cuff (RC) muscle that occurs in RC disease is a vexing clinical challenge. DespiteTwo previous studies of human RC muscle gene expression showed that when compared to small tears, large or massive tears generally exhibit depressed expression of key myogenic, adipogenic, and fibrotic genes along with high myostatin expression, 8, suggBeyond the technical limitations of previous studies, we placed an emphasis on patients with advanced RC disease in this study, as these patients typically have the most severe muscle loss and the poorest outcomes among patients with cuff tears. We were particularly interested in genes and pathways involved in muscle atrophy and regeneration along with adipogenic and fibrotic genes, given the apparent irreversibility of muscle loss and fat and fibrotic tissue accumulation following chronic, massive RC tear. By providing insight into the relationship between gene expression and tissue composition, we hope to provide some perspective and context for previous studies while offering insight into the biological processes that govern the latter stages of RC disease.Twenty-three patients undergoing reverse total shoulder arthroplasty (RSA) were consented for RC muscle biopsy. All biopsies were performed with informed written consent under the approval of the UC San Diego IRB (study #090829). In order to specifically target muscle, obvious regions of tendon and fat were avoided and only samples that macroscopically appeared vascular and organized in fascicular structures were retained  with TRIZOL (Ambion). RNeasy spin columns (Qiagen) were used to extract RNA using the manufacturer’s protocol. One microgram of complimentary DNA (cDNA) was reverse transcribed with iScript cDNA Synthesis kits (Biorad). Quantitative PCR was carried out on custom plates on a BioRad CFX384 Touch qPCR analyzer for a panel of 42 genes associated with myogenic, atrophic, adipogenic, fibrotic and inflammatory pathways , with cyRPS18 and ACTB used as reference genes). Note that a maximum Ct of 39 was applied to all genes of interest to allow for statistical comparisons, and that lower values indicate higher expression in this method[Raw cycle-threshold values  were obtained from all samples and read into a qPCR expression set using the R Bioconductor package HTqPC, and were normalized using the delta-Ct normalization method to obtain gene expression values .To determine the effect of tissue composition on muscle gene expression, linear regression of normalized gene expression and previously measured and reported histological parameters was implUnsupervised hierarchical clustering using Euclidean distance was then applied to the normalized expression values to determine the ability of gene expression patterns to differentiate muscle-containing samples from those without muscle, and to identify potential sub-clustering of muscle-containing samples. Where appropriate, compositional parameters were compared using a two-tailed t-test (α < 0.05) to determine significant differences in average composition of the sub-group versus the remaining biopsy pool. Additionally, principle component analysis (PCA) was performed on the normalized gene expression values using the R package prcomp, in ordet-test[Differential expression values (delta-delta-Ct) were calt-test. Based oAs discussed in our previous publication, only 8/Hierarchical cluster analysis of normalized expression values  resulted in two primary clusters, with all muscle-containing RSA samples contained in one primary cluster that also included 5/9 intact samples . Within The strong effect of muscle presence and relative expression of muscle-related genes was observed in PCA as well. PCA analysis showed a high degree of separation between muscle-containing RSA samples and RSA biopsies without muscle . While tDifferential expression analysis within the RSA biopsy pool showed that all but three of the analyzed genes (93%) were differentially expressed in samples with muscle compared to those without, with the majority of genes up-regulated in muscle containing samples . In starThese contrasting results highlight the need for a more detailed segregation and comparison of samples. Compared to INTACT, the HIGH group red in  demonstrFinally, fold-change expression was calculated between each sub-group . Compared to LOW group, the HIGH group demonstrated significantly increased expression of both pro- and anti-myogenic genes, with a similar but higher magnitude pattern observed between the HIGH and NO-MUSCLE groups . This paThis study highlights the importance of understanding tissue composition, and in particular muscle content, when evaluating gene expression in human muscle biopsies taken from patients with chronic, degenerative disease. Even when screened at the time of surgery and harvested to avoid non-muscle tissue, biopsies are highly heterogeneous and often do not contain any muscle fibers. This poThe first broad aim of this study, to determine whether and to what extent the composition of a biopsy predicts its measured gene expression, demonstrated mixed results. Perhaps unsurprisingly, linear regression did demonstrate significant predictive capacity of muscle content for 10/17 genes expected to be expressed either solely or primarily by muscle fibers or muscle stem cells. However, the same analysis did not find any significant relationship between fat or fibrous tissue content and any gene family, suggesting that gene expression levels are not necessarily reflective of underlying tissue composition, as is often assumed. At a high level, this suggests that even when accounting for tissue composition, the mechanical and biochemical milieu of the torn RC effect gene expression in multiple cell and tissue types relevant to RC disease progression independdo not contain any muscle, and therefore would not be expected to express muscle-specific myogenic genes. Indeed, the lack of a significant signal in the pooled RSA analysis appears to be due to high variability across RSA samples, and it is clear that any conclusions reached from this particular analysis, without the context of histology would, at best, be right for the wrong (or at least incomplete) reasons.When taken as a single pool, there are no significant differences in the expression profile between RSA and INTACT biopsies. Alone, this finding could either be interpreted as RSA biopsies lacking a distinct gene expression signature compared to ‘healthy’ muscle, or as the INTACT biopsies possessing a ‘pathologic’ expression pattern . With a more liberal approach, the trends (though insignificant) toward diminished signaling from muscle-related genes and pro-myogenic genes in RSA biopsies could be interpreted as diminished muscle maintenance overall, and indeed would align with previous studies, 8. On tA clearer picture begins to emerge when biopsy composition is taken into account. Despite no significant difference in muscle fraction, muscle-containing biopsies segregate into two distinct groups (HIGH and LOW). Compared to INTACT, the HIGH group demonstrated increased expression of both atrophic/anti-myogenic and, to a lesser extent, pro-myogenic genes. This suggests that the HIGH biopsies are still responding to the atrophy pressure of mechanical unloading, and areIn contrast, the LOW group did not have any differentially expressed genes compared to INTACT, though myogenic gene expression trended downward. To better understand this finding, we looked to the sub-group comparisons. Surprisingly, the low-expression muscle group did not differentially express any genes compared to either the high fat or no muscle groups, but had increased expression of inflammatory and fibrotic markers compared to the high-expression muscle group. Two competing explanations may account for this finding. One possibility is that this group is more ‘terminally degenerated’, evidenced by its similarity to the high-fat and no-muscle sub-groups. In this case, the similarity of expression to the INTACT group is indicative of pathology—the muscle is mechanically unloaded and contains degenerating fibers, but is not expressing the atrophic genes that are expressed in otherwise healthy unloaded muscle, 29, 30,Together, the segregation and interpretation of expression results for the HIGH and LOW groups provide further evidence for the existence of a biological spectrum of RC disease, though Despite the fact that histological parameters other than muscle content are not linear predictors of gene expression, which may be explained by the chronicity of disease, 39 relaA major remaining limitation of this work lies in our inability to histologically characterize our INTACT samples. Because the INTACT patients did have sufficient shoulder symptoms to warrant surgery, it is possible that these samples are not representative of normal, healthy muscle. Supporting this, 44% of INTACT biopsies were excluded from the differential expression analysis based on cluster analysis, though it is important to note that trends in gene expression fold-change were not altered when normalized to the entire pool of INTACT samples. In the same vein, the relatively poor predictive ability of histological parameters outside of muscle content remains a limitation, and points to one of the overarching limitations of studying clinical samples, which is an inherent inability to resolve the time course of disease in the majority of patients. To adeqAs a group, pooled RSA muscle biopsies provide little insight into gene expression patterns in RC disease, with only trends toward down-regulation of muscle-related genes and up-regulation of a limited number of inflammatory and adipogenic genes. But when muscle content determined via histology is considered, much of the variation in RSA gene expression can be explained—samples containing muscle fibers are unanimously separated from samples without muscle, and further segregate into two categories: one with increased expression of both myogenic and atrophic genes indicative of a responsive muscle undergoing active muscle turnover, 43, andS1 FileThis file contains all of the raw PCR and histological data for this paper. The ‘Raw Cq Data’ tab contains Cq data, where blank cells represent undetected genes which were set to the maximum Ct of 39 in order to perform statistical analysis. All histology data used for regression can be found in the ‘Histology Data’ tab.(XLSX)Click here for additional data file.


The regenerative capacity of adult human tissues and organs is limited, but recent developments have seen the advent of promising new technologies for regenerative therapy. The human heart is of particular interest for regenerative medicine, as cardiac tissue damage is repaired by the formation of rigid scar tissue, which causes inevitable structural changes and progressive functional decline leading to heart failure. Cardiac regenerative medicine aims to prevent scar formation or replace existing scars to halt or reverse adverse remodeling and therapeutic approaches include the use of biomaterials, gene therapies, delivery of growth factors, and (stem) cell therapies. Regenerative therapies, however, face significant obstacles in a hostile microenvironment. While the early immune response to a myocardial infarct is essential to ensure tissue integrity and to avoid fatal cardiac rupture, excessive activation of endogenous repair mechanisms may lead to ongoing inflammation, fibrosis, and sustained autoimmune-mediated tissue damage. Anti-cardiac autoreactivity of the adaptive immune system has been suggested to be involved in structural remodeling, functional decline, and the development of heart failure. It is, therefore, crucial to first understand the endogenous response to cardiac tissue damage and how to restore immune tolerance to cardiac tissue, before additional regenerative therapies can achieve their full potential. Importantly, post MI patients present elevated levels of anti-cardiac auto-antibodies,2 consistent with the involvement of the adaptive immune system and indicating an aberrant activation by self-antigens causing anti-cardiac autoimmunity. The notion that the development and progression of heart failure is promoted by an underlying autoimmune-inflammatory condition, maintaining persistent low levels of tissue damage that sustain cardiomyocyte loss, fibrosis, and pathological ventricular remodeling is recently gaining support.Heart failure after an acute myocardial infarct (MI) remains a significant cause of mortality and a major clinical challenge. An infarct induces severe tissue damage and the immune system is crucial in orchestrating the various steps of the post MI healing process. A tightly controlled interplay between immune cell populations is responsible for removing cellular debris, restoring tissue integrity to prevent fatal cardiac rupture, and then quickly resolve inflammation. An appropriate balance between inflammatory and regenerative mechanisms is essential, as excessive early inflammation may cause collateral damage to healthy tissue and is detrimental to healing. While beneficial vs. detrimental effects of early innate immune populations, including neutrophils and macrophages, have received considerable attention in recent years,We propose that restoring cardiac immune tolerance by immunomodulatory interventions post MI is essential to pave the way for more efficient outcomes of regenerative therapies.3 However, endogenous repair mechanisms gradually replace necrotic cardiac tissue by a non-contractile scar, which may lead to ventricular remodeling. This is due to the overstretching of surviving cardiomyocytes to maintain cardiac output, leading to thinning of the ventricular wall and development of heart failure.5 Current management of heart failure relies on beta-blockers, diuretics, and vasodilators such as angiotensin converting enzyme inhibitors. Surgical options are to change the shape of the left ventricle or implant assist devices such as pacemakers or defibrillators.6 However, these treatments do not completely stop progression of disease and cannot restore heart function, presenting an urgent clinical need for curative treatment options that target underlying mechanisms. Current experimental approaches to treat heart failure largely focus on cardiomyocyte deficiency and aim to replace lost cardiomyocytes in the infarcted area. Efforts to increase cardiomyocyte numbers target the limited proliferative capacity of pre-existing cardiomyocytes or aim to differentiate endogenous or delivered progenitor cells into cardiomyocytes.7 Recent advances have been reviewed extensively9 and include (1) biomaterial-based approaches,10 (2) gene therapy using plasmid DNA for gene transfer11 or silencing/short hairpin RNA, microRNA, and long non-coding RNA for gene modulation,13 (3) exogenous administration of growth factors such as neuregulin14 or fibroblast growth factor,15 and (4) cell therapies using mesenchymal stromal cells (MSCs),18 cardiac and bone marrow-derived c-kit+ cells,20 cardiosphere cells,22 and pluripotent stem cell-derived cardiac cells.23 While promising results have been obtained in experimental pre-clinical models, many of these approaches still face severe obstacles concerning efficacy of production and delivery, as well as immunological rejection. In particular though, they all share the common hurdle of a hostile inflammatory microenvironment and so far benefits observed in clinical trials are disappointing or inconsistent.23Recent advances in the clinical response to MI have significantly reduced acute mortality.24 Tight regulatory networks are in place to restrain immune responses to self-antigens and to downregulate a response once an initial insult is cleared to minimize inflammatory damage. If these regulatory networks fail, chronic inflammatory conditions may develop, including allergies and autoimmunity. Autoimmune disease is a highly diverse group of conditions, classified according to main target organs and manifestations. While a certain level of immunological autoreactivity is normal and generally kept in check, an environmental trigger in a genetically susceptible individual can overwhelm regulatory mechanisms and result in immune-mediated tissue destruction.The immune system is a sophisticated system of cells and effector molecules essential to protect the host from pathogens and ensure tissue integrity. Two fundamental features to ensure these roles are the ability to discriminate between the body’s own (self) and foreign (non-self) agents and to react quickly if there are signs of “danger” usually associated with tissue damage, including the release of intracellular molecules  such as high-mobility group box-1, heat shock proteins, nucleic acids, and mitochondrial molecules.25 The release of previously sequestered antigens from necrotic cells progressively diversifies the autoreactive lymphocyte repertoire in a process termed epitope spreading, which is well established as a crucial contributor to chronic autoimmune tissue destruction.26 An infarct causes severe tissue trauma, with necrotic heart tissue releasing vast amounts of DAMPs together with cardiac proteins. In these highly inflammatory circumstances, the ectopically encountered cardiac antigens are recognized by autoreactive lymphocyte clones and can induce autoimmunity. These antigens include α-myosin heavy chain, a prominent example of an abundant cardiac protein that is commonly targeted by post MI anti-cardiac autoreactivity.27 Subsequent immune-mediated tissue injury provides an ongoing supply of the same auto-antigens, culminating in persistent immune autoreactivity  patients have a worse outcome post MI. Knowing which factors predispose a post MI patient to detrimental autoreactivity and rapid progression to heart failure may allow earlier and more successful intervention.Although the field is still in its infancy, there is accumulating evidence that cardiac regeneration is hampered by excessive activation of the immune system and that the progression of ischemic damage to adverse remodeling and heart failure in particular is exacerbated by an ongoing adaptive immune response. Current experimental therapies rely on the assumption that the damaging incident—the infarct—has passed, and it is now time to start repairing the myocardium. Unfortunately, this might be a flawed assumption. While acute early inflammation may be resolved, adaptive autoreactivity will linger, may cause persistent low-level tissue damage, even in previously healthy areas, and may counteract any beneficial effects of regenerative therapies.The ideal post MI treatment to prevent progression to heart failure should therefore aim to induce immune tolerance to cardiac antigens as a priority. This will prevent the development of pathological adaptive immune autoreactivity, and thus provide other therapies with an environment truly permissive of regeneration.Data sharing not applicable to this article, as no data sets were generated or analyzed during the current study.


Autism is not correlated with any neuropathological hallmark as the brain of autistic individuals lack defined lesions. However, previous investigations have reported cortical heterotopias and local distortion of the cytoarchitecture of the neocortex in some cases of autism.Our patient was a 40-year-old white woman diagnosed at an early age with autism and mental retardation. Pencil fibers were present within the prefrontal cortex (Brodmann area 47) and its composition resembled that of the underlying white matter region. Pencil fibers encompassed most of the extent of the cortical grey matter and were populated by oligodendrocytes, astrocytes, and microglial cells, but not by neurons.Here we report a new cytoarchitectural abnormality that has not been previously described in autism. Future pathological examinations should keep in mind the potential presence of pencil fibers within the prefrontal cortex of cases with autism. Autism spectrum disorders (ASD) are defined by a pattern of qualitative abnormalities in reciprocal social interaction, communication, and repetitive interest and behavior. Altered functioning in several areas of the brain underlies the social and cognitive phenotype in autism. Regions of the brain in which alterations have been identified include the prefrontal cerebral cortex. Autism is accompanied by altered patterns of connectivity in the adult brain and impairments in brain development including cell generation and migration during prenatal cortical development –3. HowevWe report the case of a 40-year-old white woman diagnosed with autism whose clinical features included qualitative abnormalities in reciprocal social interaction and communication, and restricted repetitive and stereotyped patterns of behavior together with mental retardation, as is common in autism . Her cliet al. that reported on the temporal cortex  and the orbitofrontal cortex (BA11) [We analyzed prefrontal tissue from areas BA45, 46, 47, and 9. No obvious modification in cell size or density was observed. Of these areas, only BA47 presented with abnormal white matter islands extending into the cortical grey matter. The rest of the brain tissue from this patient was distributed to other research groups. We are not aware of any additional report of pathology in this case. Publications reporting on this case included that by McKavanagh Our patient presented with abnormal white matter extensions and islands into the cortical grey matter in BA47. These white matter regions resembled the pencil fibers of the striatum  and therThe formation of cortical pencil fibers in this patient most likely arose from abnormal cortical development rather than from a neurodegenerative process. We did not detect any atypical microscopic feature common to neurodegenerative disorders within the pencil fibers such as neuritic plaques and neurofibrillary tangles, dysplastic neurons and/or balloon cells. Therefore, we conclude that pencil fibers in this patient were most likely the product of altered prenatal cortical development linked to autism, rather than the later manifested pathological consequence of PKAN.The presence of pencil fibers within the grey matter of the cerebral cortex disrupts cortical cytoarchitecture, occupying a position in the cortex that would otherwise be populated by excitatory and inhibitory neurons and glial cells. The abnormal laminar cytoarchitecture and cortical pencil fibers could represent a form of cortical dysplasia resulting from defects in cellular migration during prenatal cortical development. Defects in the radial migration of excitatory cortical neurons can produce defects as observed here. In addition, defects in the tangential migration of inhibitory cortical interneurons could also impair the regional position of these cells and disrupt proper formation of the cortex. Dysregulated patterns of progenitor cell division  could also modify the final laminar destination of neurons –11. AlteOverall, we show a new cytoarchitectural abnormality, cortical pencil fibers, not previously described in the cerebral cortex of an autistic individual. Future pathological examinations should keep in mind the potential presence of pencil fibers within the prefrontal cortex of cases with autism.ADI-R, Autism Diagnostic Interview - Revised; ASD, autism spectrum disorders; ATP, Autism Tissue Program; BA, Brodmann area; GFAP, glial fibrillary acidic protein; Iba1, ionized calcium-binding adapter molecule 1; MRI, magnetic resonance imaging; Olig2, oligodendrocyte transcription factor 2; PKAN, pantothenate kinase-associated neurodegeneration; S100, protein of low molecular weight characterized by two calcium-binding sites that have helix-loop-helix (“EF-hand type”) conformation; SMI312, axonal neurofilament 312; SMI32, neurofilament H non-phosphorylated 132; Sox10, SRY (sex determining region Y)-box 10


Spinturnix across their three European bat hosts  during the bats’ autumn mating season. Mites mainly reproduce in host maternity colonies in summer, but as these colonies are closed, opportunities for inter-colony transmission are limited to host interactions during the autumn mating season. The three investigated hosts differ considerably in their social system, most notably in maternity colony size, mating system, and degree of male summer aggregation. We observed marked differences in parasite infection during the autumn mating period between the species, closely mirroring the predictions made based on the social systems of the hosts. Increased host aggregation sizes in summer yielded higher overall parasite prevalence and intensity, both in male and female hosts. Moreover, parasite levels in male hosts differentially increased throughout the autumn mating season in concordance with the degree of contact with female hosts afforded by the different mating systems of the hosts. Critically, the observed host-specific differences have important consequences for parasite population structure and will thus affect the coevolutionary dynamics between the interacting species. Therefore, in order to accurately characterize host-parasite dynamics in hosts with complex social systems, a holistic approach that investigates parasite infection and transmission across all periods is warranted.For non-mobile parasites living on social hosts, infection dynamics are strongly influenced by host life history and social system. We explore the impact of host social systems on parasite population dynamics by comparing the infection intensity and transmission opportunities of three mite species of the genus The online version of this article (doi:10.1007/s00436-016-5323-8) contains supplementary material, which is available to authorized users. Host-parasite interactions are omnipresent in biological communities  on three common European bat species at swarming sites during the autumn mating period. The three bat species, Daubenton’s bat (Myotis daubentonii), the greater mouse-eared bat (Myotis myotis), and Natterer’s bat (Myotis nattereri), all follow the same annual cycle described above, but differ substantially in social system, as summarized in Table In this context, we explore the transmission and temporal infection dynamics of a common genus of ectoparasite , thereby excluding recaptures. During our study, bats of ten other species were also caught (n = 214); a summary of these captures  is given in the Online Resource  based on the level of ossification of the epiphyseal joints . As we intended to compare the temporal infection dynamics of male hosts to those of females, adult female hosts were used as the intercept for all models, and all other classes were evaluated relative to these estimates. To assess model output and to evaluate differences among host classes, we used the sim function  models were overdispersed, both with and without day as an additional fixed effect, they were also analyzed using a zero-truncated distribution . Approximately one third of the bats caught were identified as young of year .A total of 1598 bats of the three target species were caught, and 2528 mites were collected from them , this pattern does not hold for the males of these species and across the other bat species also captured at the same sites  were broadly similar between the investigated species. Overall mite prevalence and intensity levels at the beginning of the autumn mating season were already lower than peak infection levels observed in host summer colonies  for one or several days . This should increase the overall parasite persistence within the local meta-population, making regional extinctions (see M. nattereri discussion below) far less likely. Additionally, it effectively increases the amount of transmission of mites during the bats’ mating season, as mites no longer need to transfer exclusively to female hosts, but can additionally persist on male hosts. Therefore, despite lower overall levels of mite infection than in M. myotis, mite (genetic) population structure is likely to be similarly well mixed and temporally stable across the mite meta-population. Interestingly, mites have previously been shown to have a preference for female hosts over male hosts in M. daubentonii ESM 2Raw data supporting the findings presented in this manuscript. (PDF 1045 kb)Below is the link to the electronic supplementary material.


Onset of chronic periodontitis is associated with an aberrant polymicrobial community, termed dysbiosis. Findings regarding its etiology obtained using high-throughput sequencing technique suggested that dysbiosis holds a conserved metabolic signature as an emergent property. The purpose of this study was to identify robust biomarkers for periodontal inflammation severity. Furthermore, we investigated disease-associated metabolic signatures of periodontal microbiota using a salivary metabolomics approach. Whole saliva samples were obtained from adult subjects before and after removal of supragingival plaque (debridement). Periodontal inflamed surface area (PISA) was employed as an indicator of periodontal inflammatory status. Based on multivariate analyses using pre-debridement salivary metabolomics data, we found that metabolites associated with higher PISA included cadaverine and hydrocinnamate, while uric acid and ethanolamine were associated with lower PISA. Next, we focused on dental plaque metabolic byproducts by selecting salivary metabolites significantly decreased following debridement. Metabolite set enrichment analysis revealed that polyamine metabolism, arginine and proline metabolism, butyric acid metabolism, and lysine degradation were distinctive metabolic signatures of dental plaque in the high PISA group, which may be related to the metabolic signatures of disease-associated communities. Collectively, our findings identified potential biomarkers of periodontal inflammatory status and also provide insight into metabolic signatures of dysbiotic communities. Dental plaque is known to possess a distinct fluidic component, termed dental plaque fluid, that can be separated by centrifugation of a plaque sampleIn addition, next generation sequencing techniques have been used to describe the composition of the periodontal polymicrobial community in great depth and previous studies have revealed the presence of an imbalance in relative abundance of members of the community associated with periodontitis, termed dysbiosis, in affected patients9We speculated that metabolic signatures associated with the dysbiotic community can be explained by periodontal inflammation severity and, in particular, distinct signatures of metabolic byproducts from dental plaque can be observed in patients with high PISA values. Thus, the second aim of this study was to examine metabolic byproducts from dental plaque present in saliva by comparing the salivary metabolome between before and after debridement, and also investigate how these metabolites are impacted by differences in PISA.p < 0.01), and the definition of periodontitis provided by the Centers for Diseases Control and Prevention (CDC) and American Academy of Periodontology (AAP)p < 0.01). Modified plaque index  also showed correlations with PISA , PESA , and CDC-AAP case definitions . Meanwhile, CDC-AAP case definitions had a significantly positive correlation with age .A total of 50 subjects, with a median age of 45.0 years . Of the clinical parameters tested, PISA was shown to be the most reliable for explaining the variation of salivary metabolic profile, suggesting that the saliva metabolome is strongly influenced by periodontal inflammatory status  regression analyses, where metabolome information was employed as an explanatory variant to evaluate each clinical parameter as a response variant. Eventually, OPLS model quality was assessed through interpretation of model parameters  using the salivary metabolic profile as an explanatory variant. Briefly, PISA represents the bleeding (inflamed) surface area of pocket epithelium, while PESA is the surface area of both healthy and inflamed pocket epitheliaHow to cite this article: Sakanaka, A. et al. Distinct signatures of dental plaque metabolic byproducts dictated by periodontal inflammatory status. Sci. Rep.7, 42818; doi: 10.1038/srep42818 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Malaria risk can vary markedly between households in the same village, or between villages, but the determinants of this “micro-epidemiological” variation in malaria risk remain poorly understood. This study aimed to identify factors that explain fine-scale variation in malaria risk across settings and improve definitions and methods for malaria micro-epidemiology.A systematic review of studies that examined risk factors for variation in malaria infection between individuals, households, clusters, hotspots, or villages in any malaria-endemic setting was conducted. Four databases were searched for studies published up until 6th October 2015. Crude and adjusted effect estimates for risk factors for malaria infection were combined in random effects meta-analyses. Bias was assessed using the Newcastle–Ottawa Quality Assessment Scale.From 743 retrieved records, 51 studies were selected, representing populations comprising over 160,000 individuals in 21 countries, in high- and low-endemicity settings. Sixty-five risk factors were identified and meta-analyses were conducted for 11 risk factors. Most studies focused on environmental factors, especially increasing distance from a breeding site . Individual bed net use was protective , but not household bed net ownership. Increasing household size  and household crowding  were associated with malaria infection. Health seeking behaviour, medical history and genetic traits were less frequently studied. Only six studies examined whether individual-level risk factors explained differences in malaria risk at village or hotspot level, and five studies reported different risk factors at different levels of analysis. The risk of bias varied from low to high in individual studies. Insufficient reporting and comparability of measurements limited the number of meta-analyses conducted.Several variables associated with individual-level malaria infection were identified, but there was limited evidence that these factors explain variation in malaria risk at village or hotspot level. Social, population and other factors may confound estimates of environmental risk factors, yet these variables are not included in many studies. A structured framework of malaria risk factors is proposed to improve study design and quality of evidence in future micro-epidemiological studies.The online version of this article (doi:10.1186/s12936-017-1792-1) contains supplementary material, which is available to authorized users. Heterogeneity in malaria risk at fine spatial scales is well recognized and factors that may contribute to this fine-scale heterogeneity were described nearly 30 years ago, and include genetic, social and environmental factors affecting exposure and response to infection . As malaSeveral studies have recently described and analysed micro-epidemiological variation in malaria risk at different endemicity levels , 4–7, coThe aims of this study are to identify factors that explain micro-epidemiological variation in risk, and to contribute to the development of theory and methods in the field of malaria micro-epidemiology.A protocol for this review was prepared but not registered because it does not concern an intervention and at present, systematic reviews of risk factors in observational studies are not eligible for registration with the PROSPERO, Cochrane or Campbell systematic review registries. This review is reported according to PRISMA guidelines .Plasmodium infection frequency between households or other sub-village groupings within villages, or between neighbouring villages or other similar socio-spatial aggregations such as urban neighbourhoods, agricultural settlements and health centre catchment areas.A working definition of ‘micro-epidemiology’ was developed based on a preliminary review of the literature, guiding the selection of studies. ‘Micro-epidemiology’ was considered to encompass studies assessing variation in measures of Plasmodium transmission occurs were included, except studies of sporadic imported malaria cases, or limited outbreaks of autochthonous malaria transmission following an imported case in settings that are otherwise malaria-free.Observational studies in any setting where human Plasmodium infection in a person, which is parasitologically or serologically confirmed. This outcome definition differs somewhat from the revised standard World Health Organization definition of malaria case, which is based on current infection only [Plasmodium infection prevalence in cross-sectional studies, particularly in low-endemicity settings [The primary outcome was defined as current or recent ion only , as studsettings .Plasmodium infection at micro-spatial scales. Studies were excluded if they did not present any risk factor analyses for Plasmodium infection.No restriction was applied to the types of risk factors included in studies, as the aim was to canvas the scope of risk factors that potentially explain variation in  and  and )The primary information source for this study was the PubMed database, and ISI Web of Knowledge, LILACS and Google Scholar were used as secondary databases. The search strategy below was used to retrieve titles and abstracts of potentially relevant studies in PubMed. The search strategy was constructed using the PubMed advanced search builder and run on 6th October 2015, without date restriction. An additional search was run excluding MeSH terms for the years 2014 and 2015 to allow for retrieval of articles that have not yet been indexed. The review was restricted to studies published in English. In all databases, additional searches for ‘malaria small area studies’ and ‘malaria local variation’ did not yield additional relevant papers beyond those already identified. Additionally, reference lists of key articles were checked for additional studies.Two authors independently screened titles and abstracts, selected articles for full-text review, and made the final article selection. The final list of articles selected were compared, and in case of uncertainty or disagreement about whether a record was eligible for inclusion in the review, it was discussed amongst the two reviewers until consensus was reached.All retrieved citations were exported into an Endnote X7 library. Titles, abstracts and the selected full-text articles were reviewed in Endnote, and data were extracted into a piloted, pre-specified table in Microsoft Excel for studies that met the inclusion criteria. The following items were collected: study population and location; malaria species; vector(s); study design; sample size ; time period of study; spatial scale of study; malaria prevalence/incidence; malaria case detection method ; malaria diagnostic; risk factors (see below for classification scheme); and, analytical methods. From the results of each study, risk factors reported to be significantly associated with malaria risk as defined in each study  were collected, including effect estimates and 95% confidence intervals where presented. For descriptive and qualitative studies, significance was broadly defined as the authors attributing observed heterogeneity in malaria infection to a risk factor based on presented data, such as site maps , frequency tables or qualitative findings, but these studies were not incorporated into meta-analyses.Malaria risk factors were initially extracted as reported, and then grouped into variables. For example, reported items such as used a bed net last night, owns a bed net, long-lasting insecticidal net (LLIN) or insecticide-treated net (ITN) use were grouped as ‘bed net use/ownership’, with distinctions made between whether a variable was measured at individual, household or other level. From these initial groupings, risk factors were further classified using the following classification scheme that was developed a priori and refined upon record review and extraction.Personal characteristics such age, gender, ethnicity, socio-economic status, migrant status, which influence malaria risk by modifying or acting through factors described below.all variables describing social patterns and behaviours that may directly or indirectly modify exposure to biting vectors, such as bed net use and outdoor activities.Variables measuring relative exposure to biting mosquitoes related to physical or landscape features such as proximity to vector breeding sites, landscape features and weather and climate conditions.Human host and genetic factors related to development of parasitaemia and clinical disease once exposed to an infectious bite, such as immune status, co-infections and genetic traits.Plasmodium populations, including household malaria cases or residence in hotspot, as well as prevalence of drug-resistant strains. Household and village population size were also included because here they affect exposure to Plasmodium populations as a function of the number and density of available human hosts.Variables measuring exposure to local Variables related to seeking testing and treatment for malaria, including knowledge and perceptions of malaria illness, access to and availability of malaria control programmes, provider and treatment preferences.Risk of bias in individual studies was assessed using the Newcastle–Ottawa scale for assessing quality of nonrandomized studies in meta-analyses . The quaIncluded studies were described in terms of study setting, study design, spatial area, endemicity, and level of analysis . Frequency tables of risk factors were produced, contrasting frequency with which a risk factor was studied to the frequency that significant associations  for each factor were reported.Results from individual studies were combined in meta-analyses to estimate the magnitude of effect sizes and heterogeneity of effects across studies. Several methods were used to generate effect estimates that were not presented in the required form for meta-analysis in the individual publications, which are fully described in the Additional file Plasmodium or vector species, study setting, and various other sources of heterogeneity were explored qualitatively but there were too few studies per variable to stratify on study design or risk measure. All effect estimates were assumed to estimate the odds ratio, as this was the most commonly calculated measure. Heterogeneity between study estimates included in meta-analysis was assessed using the I2 statistic.Heterogeneity by relative risk measure , To synthesize the descriptive and quantitative findings, a conceptual framework was developed for the relationships between risk factors for which there is evidence of association with malaria infection risk in different settings.Risk of bias across studies in meta-analyses was considered high because the search strategy was systematic with respect to study design only, not individual variables, and furthermore, many studies did not present effect estimates (including unadjusted or adjusted) for variables reported to be non-significant. Statistical tests of significance for pooled effect estimates are not presented because the high risk of bias limits meaningful interpretation of p values, and the effect estimates and confidence intervals should be considered indicative rather than conclusive.Some 717 records were retrieved across the database searches and 25 additional titles were retrieved from article reference lists. 121 articles were selected for full-text review, including five that were identified from reference list screening  were conducted in rural settings including coastal, forest/forest fringe, highland, and large agricultural settlement sites. There were six studies conducted in urban or peri-urban settings and one study that contrasted a peri-urban to a nearby rural setting . There wia Table .Table 1C2, with a median of 38 km2. Many studies were conducted within one or a few neighbouring villages or sub-village clusters, with the largest study conducted in 109 villages in a 145 km2 district.The spatial scale of study sites ranged from <1 to 1188 kmThere were 12 studies that examined risk factors for aggregated malaria infections or risk of infection, including six studies that investigated risk factors for malaria risk at village level , 14–19, Most but not all studies included basic demographic variables such as age, gender and a measure of income or wealth, and often occupation and education level Table . Age wasMost studies 32) included at least one social or behavioural risk factor, mostly individual bed net ownership or use 21 studies) and/or household bed net ownership or use (11 studies) , residence in an identified hotspot (four of five studies based on three datasets), or malaria prevalence in adjoining localities (two of two studies). Of the population-related factors, village population size was associated with increased malaria risk in four of four studies . Household size (four studies) or household crowding (four studies) was associated with increased malaria risk in unadjusted but not adjusted estimates , 24 studies at moderate risk of bias (scored six or seven stars) and eight studies at high risk of bias (scored two to five stars). Variation in bias scores between studies mainly related to adjustment for confounders  and lack of description of participation rates. The bias assessment is presented in full in the Additional file Most studies examined individual and household-level characteristics as risk factors for individual-level malaria infection and separately described aggregated variation in malaria, typically through detection of spatial clusters of malaria based on household of residence. A small number of studies explicitly analysed whether individual-level risk factors for malaria explain variation in risk between villages or other units. In a high-endemicity setting in Ghana , there wIn this review, several factors that were associated with variation in malaria risk at fine spatial scales were identified. To synthesize the descriptive and quantitative pooled results, a conceptual causal framework for micro-epidemiological studies of malaria is proposed Fig.  that incgroups of individuals, clustered in socio-spatial aggregations such as households, sub-village clusters, villages or urban zones. Although several studies linked the purpose of micro-epidemiological analysis to more efficient identification and targeting of malaria hotspots, this review demonstrates that there is limited evidence that variation in malaria risk at household, sub-village cluster or higher-level units can be fully explained by individual-level risk factors. Therefore, explaining local variation requires that analyses at the level of individual-level risk factors must be related to higher-level units at which heterogeneity in malaria risk occurs, including analysis of how risk factors interact or reinforce each other in the local context to potentiate malaria transmission.This review presents the first attempt to systematically identify risk factors that explain local variation in malaria risk. Several risk factors were identified that are consistently associated with malaria risk at fine spatial scales, including individual bed net use, presence of open eaves in housing construction, proximity to vector breeding sites, household size and crowding, and distance to a health facility. In the studies screened and included in this review, no clear description of what micro-epidemiology should entail was found. It is proposed that micro-epidemiology should aim to explain local variation, where ‘local’ implies a transmission network (or a component of it) that is characteristic of a defined socio-spatial aggregation (such as a village) and ‘variation’ describes heterogeneity in malaria risk between Through this review, several issues were identified that should be considered when planning micro-epidemiological studies, including challenges associated with small sample sizes, units of analysis, and interdisciplinary approaches.The minimum number of villages or other socio-spatial aggregations to include in micro-epidemiological studies remains unclear. It is proposed that the number of higher-level units that are included should reflect the underlying transmission network; that is, if variation within a village is hypothesized to relate to variation in malaria risk between contiguous villages in the area, then sufficient units should be included to allow between-village differences to be explored. One study  showed hAdditionally, small sample sizes may limit statistical power to detect important risk factors, and effect sizes may be consistently over-estimated , which cThe studies included in this review analysed aggregated malaria risk mostly through spatial analyses, in which the registered domicile address is taken as the primary spatial unit for assessing spatial clustering and measuring household-based risk factors for malaria infection. This approach implicitly assumes that malaria transmission is occurring in the vicinity of the village-based household. However in several settings, especially South-East Asia and Latin America, occupation-related mobility and multiple residence systems are associated with malaria risk –35. In aPlasmodium pathogens were only considered in African study settings. As these factors were consistently associated with malaria infection, micro-epidemiological malaria studies in other settings should consider including more genetic and clinical characteristics, as these characteristics may explain some heterogeneity in malaria infection that has different implications for intervention strategies. The role of health services and health systems was rarely considered; only two studies explicitly measured variation in access to malaria control programs, but no studies considered the effectiveness and acceptability of malaria control and other health care problems as a source of micro-epidemiological variation in malaria risk. Similarly, there is scope for more detailed research on how specific local socioeconomic conditions modify malaria risk, and the pathways through which this occurs, which goes beyond simple descriptions of individual or household-level socioeconomic status.An interdisciplinary research design is integral to micro-epidemiology, as the lack of inclusion of data from different disciplines contributes to unmeasured confounding. For example, estimates of the effect of proximity to breeding sites in individual studies, the most frequently studied risk factor for malaria infection, were in some cases substantially attenuated after adjustment , or not Only one study  used a mSeveral sources of bias limit the strength of the evidence on risk factors underlying micro-epidemiological patterns in malaria risk across different settings. As there has been no consistent use of the term ‘micro-epidemiology’ or other terms to describe studies that analyse variation in malaria risk within or between sub-village clusters or villages, defining a search strategy was not straightforward, and some potentially relevant studies may have been missed. Future reviews on this topic could consider a broader use of keywords. The choice of malaria diagnostic  may impact the observed variation in malaria risk, as risk factors for asymptomatic carriage may differ from those for clinical cases particularly in high-endemicity settings. Passive case detection, compared to active case detection or population screening, may introduce confounding by health seeking behavior and miss groups of individuals at higher risk but with less access to care.Plasmodium species, vectors, at-risk populations and study design, but this limits the utility of the meta-analysis results. For example, keeping animals in or near the house was not associated with malaria risk overall, but as this varies substantially with vector and host species, as well as extent of urbanization, this risk factor may be important in specific settings. In this review, studies that measured serological as well as parasitological outcomes were pooled, as there is evidence that seroprevalence of anti-malarial antibodies may be a more stable marker of recent malaria risk at micro-epidemiological scales [Across studies, there was a substantial risk of bias, given that studies frequently excluded effect estimates for variables with reportedly non-significant associations with malaria risk. Most meta-analysis estimates presented are calculated using unadjusted findings. Furthermore, there were insufficient studies to conduct meta-analyses stratified on different l scales . Howeverin context rather than comparing isolated risk factors for individual-level infection would allow us to understand how different risk factors combine to produce variation in malaria risk at aggregated rather than just individual level. The conceptual framework proposed in this review attempts to identify and structure relevant risk factors that were frequently associated with malaria risk in micro-epidemiological studies, which will contribute to progress in theorization and assist in planning of future studies. Further research is required to fully operationalise the concept of micro-epidemiology and incorporate it into discussions of malaria elimination strategies.Conceptual recognition of the relevance of micro-epidemiology for malaria control is not new; as expressed by Hackett in 1937, “everything about malaria is so molded by local conditions that it becomes a thousand epidemiological puzzles” . HoweverAdditional file 1. Meta-analysis forest plots. This file includes detailed methods and forest plots for all meta-analyses conducted, as well as additional information on why meta-analysis could not be conducted for some risk factors.Additional file 2. Risk of bias in studies included in systematic review. This table provides a detailed bias assessment and score across the three domains of the Newcastle–Ottawa Quality Assessment Scale.


The present study aimed to undertake a review of available evidence assessing whether time-lapse imaging (TLI) has favorable outcomes for embryo incubation and selection compared with conventional methods in clinical in vitro fertilization (IVF).Using PubMed, EMBASE, Cochrane library and ClinicalTrial.gov up to February 2017 to search for randomized controlled trials (RCTs) comparing TLI versus conventional methods. Both studies randomized women and oocytes were included. For studies randomized women, the primary outcomes were live birth and ongoing pregnancy, the secondary outcomes were clinical pregnancy and miscarriage; for studies randomized oocytes, the primary outcome was blastocyst rate, the secondary outcome was good quality embryo on Day 2/3. Subgroup analysis was conducted based on different incubation and embryo selection between groups.Ten RCTs were included, four randomized oocytes and six randomized women. For oocyte-based review, the pool-analysis observed no significant difference between TLI group and control group for blastocyst rate . The quality of evidence was moderate for all outcomes in oocyte-based review. For woman-based review, only one study provided live birth rate , the pooled result showed no significant difference in ongoing pregnancy rate  between two groups. The quality of the evidence was low or very low for all outcomes in woman-based review.Currently there is insufficient evidence to support that TLI is superior to conventional methods for human embryo incubation and selection. In consideration of the limitations and flaws of included studies, more well designed RCTs are still in need to comprehensively evaluate the effectiveness of clinical TLI use. Cultivation of fertilized oocytes and the subsequent selection of embryos is a vital step in assisted reproductive technology. Since the inception of clinical in vitro fertilization (IVF), most laboratories have routinely selected embryos on the basis of morphological characteristics. This method requires moving embryos outside the controlled environment of the incubator for microscopic examination. However, owing to the potentially deleterious effect of exposure embryos to undesirable changes in culture environment when evaluated, inspection of embryo morphology is limited to snapshots at a few discrete time points , reducing the amount of information that could be obtained and important developmental events might be missed. Conventional embryo selection methods are commonly associated with relatively low clinical pregnancy rate of approximately 30% per transfer . NowadayTime-lapse imaging (TLI) is an emerging technology, clinical available TLI systems allow continuous observation of embryo development without removal from controlled and stable incubator condition. TLI systems may improve outcomes of IVF cycles over conventional methods due to several potential advantages. First, decreased frequency of handling and exposure of embryos to suboptimal conditions eliminates the risks of stress from temperature changes, high oxygen exposures and pH changes in the culture medium and thus provides improved culture conditions. Second, by serial imaging, more information on embryo development is obtained. TLI allows embryologists to assess the quality of embryos by tracking the timing of events and length of different intervals in embryo development , adds another dimension to embryo selecting and grading. Until now, a number of studies have been conducted regarding whether morphokinetic variables are correlated with important outcomes such as blastocyst formation–8, implaAs stated, the potential benefit of clinical TLI may be due to improved incubation or improved embryo selection or both, the relative contribution of each variable needs to be acknowledged. Available RCTs detecting the effectiveness of TLI so far can be divided in to 3 types: studies comparing TLI incubation to conventional incubation, studies assessing the overall impact of TLI to conventional methods and studies focusing on the potential effect of TLI morphokinetic monitoring and embryo selection. A few meta-analyses and systematic reviews evaluating TLI have been published including a Cochrane meta-analysis–23. HoweOn the other hand, distinct light exposure, heat due to motion, friction of moving parts, presence of magnetic fields, sheer stress of moving culture dishes and lubricants related to TLI systems may bring harmful effect on embryo quality. Considering the conflicting results of studies comparing TLI incubation to conventional incubation, the safety of TLI systems needs to reconfirm–28. BesiRandomized controlled trials (RCTs) that compared TLI with conventional methods were considered eligible. We only included true RCTs, quasi- or pseudo- randomized trials were excluded. Both studies that randomized oocytes and studies that randomized women were included, however, these studies were analyzed separately as a woman-based review and an oocyte-based review.A systematic search of PubMed, EMBASE, Cochrane library and ClinicalTrials.gov up to February 2017 was conducted. The following terms were used, adjusting for each database as necessary: (time-lapse OR “time lapse” OR EmbryoScope OR EEVA OR “Primo Vision” OR “live cell imaging” OR “closed system”) AND (“in vitro fertilization” OR IVF OR “introcytoplasmic sperm injection” OR ICSI OR “assisted reproductive technology” OR “assisted reproduction” OR ART OR embryo OR blastocyst). Limitation categorical terms were used: human, clinical trials. Additionally, the reference lists of included studies and related reviews were also manually-searched to identify potentially relevant studies. There was no limitation regarding the language, publication date or publication status.Two reviewers (CMH and WSY) independently examined the searching results of possible relevant trials and full-texts were obtained when necessary. Disagreements were solved by group discussion. We contacted the original authors by e-mail when necessary.All authors independently extracted data from each included study using a standardized data-extraction form. If necessary, we contacted the authors to retrieve missing data. In the case of duplicate publications with accumulating numbers of patients or increased lengths of follow-up, we included the most recent or complete data for analysis.Related information was sought for the following variables: first author’s name, year of publication, country, incubation and embryo selection of two groups (TLI group and control group), period of enrollment, type of TLI system, number of women included, number of oocytes included, inclusion and exclusion criteria for women, fertilization method, number of embryos transferred, day for transfer, fresh or frozen cycles, oocyte source, culture parameters, sample size calculation, implantation rate, embryo assessment time usage, economical burden and extra place needed in laboratory. For woman-based review, the primary outcomes were ongoing pregnancy and live birth, the secondary outcomes were clinical pregnancy and miscarriage. For oocyte-based review, the primary outcome was blastocyst formation and the secondary outcome was good quality embryo on Day 2/3. For the definition of good quality embryos, any classification criteria was accepted.Two reviewers (CMH and WSY) independently assessed the quality of included RCTs according to the Cochrane Collaboration’s Handbook. The risTo minimize the potential impact of reporting bias, we attempted to perform a comprehensive search for eligible studies, avoiding language restriction, broadening eligibility to conference proceedings and grey literatures, and maintaining alertness for duplication of data.The overall quality of the body of evidence was assessed in a structured way using the GRADE criteria. This acRelative risk (RR) with corresponding 95% confidence intervals (CIs) was used to assess the overall effects for discontinuous data.2 statistic. A I2 value of ≥50% was considered to indicate substantial heterogeneity. Any observed heterogeneity was taken into account in the interpretation of the estimates. Subgroup analysis was conducted based on different incubation and embryo selection between groups: TLI incubation + conventional selection vs. conventional incubation and selection, TLI incubation and selection vs. conventional incubation and selection and TLI incubation and selection vs. TLI incubation + conventional selection.All analyses were conducted with Review Manager Version 5.3  in a random-effect model since the interventions varied among included studies. Statistical heterogeneity among the study results was examined by the P value and I2593 no duplicate records were identified in the initial electronic search, and two records were retrieved by manual-search of the reference list of relevant studies. After reading titles and abstracts, 2578 records clearly did not meet the eligible criteria, and 17 records were selected to undergo further full-text review. Among these studies, five were excluded because they were not randomized, 31–34; Main characteristics of included studies are shown in For oocyte-based review, three studies used EmbryoScope (Vitrolife) as the incubation system for embryos randomized to TLI group, 25, 28,For woman-based review, five studies used EmbryoScope for TLI incubation and one used PrimoVision (Vitrolife), 40. CulOnly one study provided data on the time of embryo assessment and found that TLI doubled the time up to Day 3 comparing to conventional assessment. Data ofRisk of bias judgment of all included studies is shown in Except one study (categorized to oocyte-based review), every sSummary of results are presented in Table 3Two studies presented data on blastocyst formation, 25, andLive birth was only reported by one study comparing TLI incubation and embryo selection to conventional incubation and embryo selection , 39. TheSince all four studies categorizing to the oocyte-based review compared TLI incubation + conventional selection to conventional incubation and selection, subgroup analysis was only conducted for woman-based review.2 N/A, one study, including 329 women) or clinical pregnancy rate . However, data from one study showed that miscarriage rate was significantly increased for patients randomized to the TLI group [Two studies reported TLI incubation and conventional selection vs. conventional incubation and selection, and no significant difference was observed between TLI incubation and conventional incubation for ongoing pregnancy rate .2 = 0, three studies, including 1039 women) than conventional incubation + conventional selection. There were no significant differences between the two groups in clinical pregnancy rate  and miscarriage rate .There were three studies including TLI incubation and selection vs. conventional incubation and selection, and TLI incubation and TLI selection showed significant higher ongoing pregnancy rate .Only one study reported TLI incubation and selection vs. TLI incubation and conventional selection. However, the only included study did not provide data on ongoing pregnancy, live birth or miscarriage. No signResults of pooled analysis of the woman-based review and subgroup analysis were shown in The present systematic review and meta-analysis shows that clinical TLI may have the potential to improve IVF outcomes but currently there is insufficient evidence to support regular TLI use when considering ongoing pregnancy rates and blastocyst formation rates. A systematic review published by Cochrane collaboration summarizing no difference between TLI and conventional methods included the same three studies, the litConsidering the four studies that compared TLI incubation to conventional incubation and randomized oocytes, embryos incubated in TLI systems were removed out for assessment in two studies, 25, thaConsidering the two studies that compared TLI incubation to conventional incubation and randomized women, the pool-analysis showed no significant difference in clinical pregnancy rate between the two groups. Only one study provided ongoing pregnancy and found no significant effect of TLI incubation compared to conventional incubation on this outcome, but theConsidering the studies that compared TLI incubation and embryo selection to conventional incubation and embryo selection, we observed very low quality evidence that the undisturbed culture condition and continuous embryo monitoring of clinical TLI is related with higher ongoing pregnancy rate, clinical pregnancy rate and lower miscarriage rate, but the differences of clinical pregnancy rate and miscarriage rate between the two groups were not statistically significant. Live birth was only reported by one study in a follow-up analysis and was found to be significantly higher in TLI group, 39. TheOnly one RCT used TLI incubation for both groups and selected embryos with the addition of morphokinetic parameters to standard morphological assessment in TLI group. This stEight out of the ten included studies used EmbryoScope as TLI system, one study used PrimoVision and one used SANYO In vitro Live Cell Imaging Incubation System. Currently, several TLI systems are commercially available, the most widely used are EmbryoScope, Primo Vision and Eeva . These systems differ in many ways including illumination, image capture and embryo culture. EmbryoScope is an incubator with integrated time-lapse imaging system using bright-field illumination, in which embryos are cultured individually in multi-well dishes. A tray holding the culture dishes is under constant movement to bring embryos one by one into the field of view of the inbuilt microscope at each image acquisition. Unlike EmbryoScope, Primo Vision (bright-field illumination) and Eeva (dark-field illumination) are microscopes that fit into standard incubators and facilitate group culture, both systems monitor all embryos at the same time without moving culture dishes, so these two systems provide more undisturbed environment than EmbryoScope concerning culture dish movement, heat and volatile organic compounds released from lubricants and electromagnetic effect. Based on the results of this review, no negative effect of EmbryoScope compared with conventional incubator is confirmed. Safety of Primo Vision and Eeva have been confirmed by some observational studies published in recent years–33, 43. Compared with improved incubation, more promising benefit of TLI is the ability to combine evaluation of morphology and morphokinetic characteristics and therefore better identifying the most viable embryos for transfer. Four included studies used new algorithms based on morphology and morphokinetic parameters to select embryo for TLI group: two studies used a hierarchical classification described by Meseguer et al, one stuSeveral limitations and flaws of this review are worth mentioning. Limited data are available regarding advantages and disadvantages of clinical TLI. Live birth and neonatal outcomes were only reported by one study with severe risk of bias; data of embryo assessment time was only provided by one study and this study did not mention the TLI training status of embryologists which may seriously affect the time usage; the extra expense for TLI use and extra space need for TLI equipment was not mentioned in any of the included studies. The overall quality of evidence of all outcomes was considered to be moderate, low or very low, so inference based on these results should be made with caution. Confounding factors varied among included studies including day of transfer, number of embryos transferred, fresh or frozen embryo use, oocyte source, TLI type and especially, patient population. Eight out of 10 included studies investigated average-prognosis, good-prognosis and best-prognosis (egg donors) patients, only one small study randomized 31 poor-prognosis patients. So whetThis analysis can help guide the clinical use of TLI. But more well designed RCTs are still required for providing sufficient evidence on the effectiveness of TLI. RCTs focusing on the effect of TLI on specific group of patients, the effectiveness comparison among different types of TLI systems, the potential disadvantages of this technology and the applicability of developed morphokinetic algorithms are currently very scarce.S1 FilePRISMA checklist.doc.(DOC)Click here for additional data file.


First described in the 1950s, tissue tranglutaminase , is a unique multifunctional member of the transglutaminase family with effects that extend beyond its enzymatic actions. A recently published study demonstrates an important role for endogenous tTG in the pathogenesis of pressure overload-induced heart failure, suggesting crosslinking actions that promote diastolic dysfunction, and protective matrix-preserving effects that prevent chamber dilation [Ca2+-dependent transamidation, the best characterized enzymatic function of tTG, modifies proteins by crosslinking their reactive carboxamide side chains to primary amines . In vitrtTG is constitutively expressed in normal mammalian myocardium, and is upregulated in injured and failing hearts . In a reCardiac pressure overload activates mechanosensitive signaling pathways in all myocardial cells. In response to biomechanical stress, cardiomyocytes activate integrins, transducing downstream mitogen-activated protein kinase cascades, that may be involved in induction of tTG transcription . MechanoConsidering its ubiquitous expression, its secretion and deposition in the activated extracellular matrix, and its broad functional repertoire, tTG may have a wide range of cellular targets in heart failure Fig. . PublishIn our study, use of a mouse line with global loss of tTG precludes conclusions regarding the in vivo role of specific cellular responses in mediating the effects of tTG loss. Thus, our experiments did not explain the cellular basis for the strikingly increased mortality in tTG null mice undergoing pressure overload protocols. Accentuated dilative remodeling due to loss of protective matrix-preserving effects may be in part responsible for increased mortality in pressure-overloaded tTG knockout animals; however, the severity of systolic dysfunction could not account for the observed effect on survival. Whether endogenous tTG protects the myocardium from arrhythmogenesis or defective impulse conduction is unknown.On the other hand, the observed effects on matrix remodeling and fibrosis may reflect important actions of tTG on fibroblasts, or effects on the fibrogenic phenotype of immune cells (such as macrophages). Our in vitro findings suggested that tTG restrains fibroblast proliferative capacity and promotes a matrix-preserving program in cardiac fibroblasts by inducing tissue inhibitor of metalloproteinases (TIMP)1 synthesis.Although tTG expression is markedly induced in the remodeling myocardium, whether increased protein levels are associated with accentuated enzymatic activity remains unclear. In normal tissues, tTG activity is tightly regulated. In the cytoplasm, low Ca2+ concentrations prevent intracellular transamidation activity, and GTP/GDP act as allosteric inhibitors, reducing accessibility of the catalytic domain, and preventing inappropriate intracellular tTG activation. In the pressure-overloaded heart, increased calcium concentrations may co-operate with pro-inflammatory cytokines, triggering intracellular tTG activation. In the cardiac interstitium, high levels of Ca2+ may stimulate tTG-mediated transamidase activity, promoting crosslinking of extracellular matrix proteins and leading to formation of a protease-resistant matrix. The crosslinking actions of tTG may also serve to localize and activate TGF-β, further accentuating fibrosis. However, interstitial activation of tTG may be transient due to release of mediators that inactivate tTG, such as reactive oxygen and nitric oxide . Thus, tNon-enzymatic functions of tTG may play an important role in activation of cardiac interstitial cells and in the pathogenesis of myocardial fibrosis. Through binding with both fibronectin and heparin sulfate proteoglycans, tTG may function as a molecular bridge between the matrix and the cells, activating syndecan-4-mediated signaling . tTG mayOur recently published findings suggest that tTG is a critical stress-induced signal in the failing heart, exerting both profibrotic effects that increase ventricular stiffness and protective actions that preserve extracellular matrix integrity, preventing chamber dilation. Considering the potential translational significance of tTG-mediated actions in heart failure, study of the molecular basis for the effects of tTG is an important priority. Future experiments should focus on several different directions. First, dissection of the enzymatic and non-enzymatic actions of tTG in the remodeling myocardium and identification of the main substrates is important in order to design safe and effective therapeutic strategies. Second, investigation of the cellular targets of tTG in the remodeling heart is needed in order to identify specific cellular responses that could be targeted in human patients. Third, characterization of the molecular signals interacting with tTG and understanding of their role in mediating specific cellular responses can provide critical information on specific mechanisms of action with direct translational implications. Fourth, systematic analysis of changes in myocardial tTG expression in human failing hearts may provide insights into the role of tTG in specific heart failure subpopulations. Considering the heterogeneity of human heart failure, identification of patients with prominent fibrosis and excessive activation of myocardial tTG-mediated matrix crosslinking may mark a subpopulation that may benefit from attenuation of enzymatic tTG actions.


The recognition of invading pathogens and endogenous molecules from damaged tissues by toll-like receptors (TLRs) triggers protective self-defense mechanisms. However, excessive TLR activation disrupts the immune homeostasis by sustained pro-inflammatory cytokines and chemokines production and consequently contributes to the development of many inflammatory and autoimmune diseases, such as systemic lupus erythematosus (SLE), infection-associated sepsis, atherosclerosis, and asthma. Therefore, inhibitors/antagonists targeting TLR signals may be beneficial to treat these disorders. In this article, we first briefly summarize the pathophysiological role of TLRs in the inflammatory diseases. We then focus on reviewing the current knowledge in both preclinical and clinical studies of various TLR antagonists/inhibitors for the prevention and treatment of inflammatory diseases. These compounds range from conventional small molecules to therapeutic biologics and nanodevices. In particular, nanodevices are emerging as a new class of potent TLR inhibitors for their unique properties in desired bio-distribution, sustained circulation, and preferred pharmacodynamic and pharmacokinetic profiles. More interestingly, the inhibitory activity of these nanodevices can be regulated through precise nano-functionalization, making them the next generation therapeutics or “nano-drugs.” Although, significant efforts have been made in developing different kinds of new TLR inhibitors/antagonists, only limited numbers of them have undergone clinical trials, and none have been approved for clinical uses to date. Nevertheless, these findings and continuous studies of TLR inhibition highlight the pharmacological regulation of TLR signaling, especially on multiple TLR pathways, as future promising therapeutic strategy for various inflammatory and autoimmune diseases. Toll-like receptors (TLRs) are a family of pattern recognition receptors (PRRs) that can recognize and respond to a unique repertoire of distinct molecules referred to as pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns . Accordingly, novel nanodevices have emerged with bio-activities to potently regulate TLR signaling, aiming for inhibiting disease-associated TLR activation.Despite many efforts have been put in developing (bio)molecular inhibitors targeting TLR signaling pathways, unfortunately, very few compounds are currently available for clinical uses. Therefore, it is important to search for novel TLR inhibitors that can be utilized both as therapeutic agents and as experimental reagents for dissecting innate immune molecular pathways. Manipulation of immune responsiveness using nanodevices provides a new strategy to treat human diseases, because nanotechnology offers many beneficial features for therapeutics development, including cell penetrating and targeting capabilities, desired biodistribution, improved pharmacokinetics, enhanced therapeutic activity, better biocompatibility, and In this review, we first briefly summarize the pathophysiological role of TLRs in many diseases of acute and chronic inflammation as well as autoimmunity. These diseases include SLE in autoimmunity, infection-induced sepsis for acute inflammation, vascular system disorders across both acute and chronic inflammation, and other inflammation-associated diseases such as psoriasis, gastrointestinal cancer, asthma, and chronic obstructive pulmonary disease  is a complex autoimmune disease characterized by the loss of tolerance to self-nuclear antigens and the production of autoantibodies, leading to the attack of the immune system on the self-components and healthy tissues. Recent evidences have suggested a close relationship between the endosomal TLR activation and the onset of SLE  by TLRs expressed on innate immune cells  and endothelial cells Miller, ; TLR areIn the case of hypertension, TLR4 has been well-documented to mediate aberrant immune and inflammation in vasculature, kidneys and autonomic nervous system -α and heat shock proteins  from keratinocytes can upregulate TLR5 and TLR9  greatly increase the risk of having gastrointestinal malignancies .Although, evolutionarily TLRs are essential elements in innate immune system and play a critical role in the host defensive mechanism against invading pathogens, overactivation of TLRs is inevitably involved in the pathogenesis of many inflammatory diseases. Thus, inhibition of TLR signaling pathways has been predicted to be an effective therapeutic strategy to suppress unwanted, disease-associated inflammatory responses. In general, TLR inhibition can be achieved by two major strategies: (1) blocking the binding of TLR ligands to the receptor; 2) interfering the intracellular signaling pathways to stop the signal transduction  are synthetic or naturally derived chemical agents with the activity to inhibit TLR signal transduction. Their amphipathic property and small size allow them to cross cell membranes and act on specific intracellular adapter proteins or compartments along the TLR signaling pathways. They usually have good bioavailability but poor specificity and targeting capability. The ease of manufacturing and handling of SMIs makes them popular drug candidates in pharmaceutical industries Table . In the In addition to autoimmune diseases, these SMIs of TLR7/8/9 are also under investigation in other diseases associated with uncontrolled acute or chronic inflammation. For example, the antimalarial drug CQ has shown its clinical potential in treating severe sepsis, especially in reducing the risk of sepsis-induced acute renal failure  is an anti-sepsis SMI that targets TLR4 signaling pathways , and statins are among the early developed drugs with newly discovered inhibitory activity on TLR2 and TLR4 signaling. For example, the ARB family valsartan can decrease proinflammatory cytokines release and infarct size by inhibiting TLR4 signaling  injury animal models  to block the binding of ligands to the specific TLRs Table . One proBacillus subtilis  or lethally challenged with ex vivo and in vivo, and prevent flu-like symptoms upon LPS administration. Recent data demonstrated that NI-0101 could also block rheumatoid arthritis (RA) synovial fluids-induced pro-inflammatory cytokine production in monocytes isolated from RA patients  complex . Advancing from the earlier encouraging prototypes, IRS-661 (TLR7 specific) and IRS-869 (TLR9 specific), a dual-function targeting both TLR7 and TLR9 antagonist, IRS-954 was developed for the treatment of SLE  has made some success into clinical trials . The compound IMO-3100 is also a TLR7/9 dual antagonist. In preclinical studies, IMO-3100 could significantly reduce the expression of inflammatory genes, such as IL-17A, β-defensin, CXCL1, and keratin 16 in a mice model of IL-23-induced psoriasis  2088 is a potent TLR9 antagonist. In spontaneously hypertensive rats, IHN-ODN 2088 treatment showed positive effects on systolic blood pressure reduction , is probably the most exciting but also disappointing clinical development of TLR4 antagonist (by Eisai Research Institute of Boston Inc.)  may not be enough to stop the overwhelming inflammation in severe sepsis. To ensure future success in developing new TLR inhibitors for inflammatory diseases like sepsis, perhaps new inhibitory strategy on multiple TLR pathways should be considered; and clearly in the clinical trials, patients should be carefully selected and examined based on appropriate criteria with well-defined biomarkers to obtain the fair evaluation and interpretation of the therapeutic efficacy of the tested drugs.Apart from treating sepsis, eritoran could still be therapeutically beneficial for other inflammatory diseases. In fact, one study has shown that eritoran could prevent influenza-induced death in a mouse model, where it improved the clinical symptoms, inhibited cytokine production , and reduced viral titers  are small (~22 nucleotides), endogenous non-coding RNAs with post-transcriptional regulatory functions to finely tune gene expression Bartel, . They biTill now, there are about 20 miRNAs identified to be involved in the regulation of TLR signaling pathways  expression, serving as a negative regulator of the inflammatory response -like nanoparticle (HDL-like NP), was developed as a TLR4 antagonist by sequestering LPS  have caught much attention as emerging nanotheranostics in nanomedicine, owing to their ease of fabrication and functionalization, chemical stability, excellent photothermal physical properties, and good biocompatibility , can also recognize LPS and induce the secretion of inflammatory mediators in macrophages  formulated L-asparaginase  and liposome formulated doxorubicin . Furthermore, several inorganic nanoparticles have been approved by FDA as diagnostic imaging agents. For instance, the iron oxide nanoparticles  have been used in medical magnetic resonance imaging (MRI). Recent records of clinically used nanoparticles are summarized in two comprehensive reviews in 2016 (Anselmo and Mitragotri, TLRs are very important PRRs in the first-line defense system of our bodies. They can recognize both invading pathogens and endogenous danger molecules released from dying cells and damaged tissues, and play a key role in linking innate and adaptive immunity. Despite their well-known function in sensing pathogens and mounting defense mechanisms, overactivation of TLRs can ultimately lead to disruption of immune homeostasis, and thus increase the risk for inflammatory diseases and autoimmune disorders, such as SLE, RA, atherosclerosis, stoke, IR-induced injury, COPD, asthma, gastrointestinal inflammation, and even cancer. Evidences have shown the involvement of TLRs in the development and progression of these diseases. Accordingly, antagonists/inhibitors targeting TLR signaling pathways have emerged as novel therapeutics to treat these diseases. Although, this therapeutic strategy has been well-accepted experimentally, there are only limited numbers of TLR inhibitors/antagonists that have undergone clinical trials to date; many of them failed during the trials, and none have been approved for clinical uses. Lessons have been learned from these failed developments. The efficacy, safety, and stability of the new therapeutic agents, as well as whether these therapies could impair local immune defenses should be considered comprehensively in the clinical development. More importantly, given the facts that pathogen infection or danger signals often activate multiple TLRs, and that the failure of TLR4 specific antagonist/inhibitor in the clinical trials despite its success in preclinical studies, developing more potent antagonists/inhibitors that target multiple TLR signaling pathways perhaps could shine the way to facilitate the translation of such a promising strategy into clinical uses in the future. Certainly, nano-devices will play an important role in the development of next generation TLR inhibitors.QL and HY proposed and designed the scope of the review. WG and HY wrote the manuscript. YX and QL critically revised and commented on the manuscript. All the authors read and approved the final manuscript and agreed to be accountable for the accuracy and integrity of any part of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cytomegalovirus (CMV) is a ubiquitous infection that causes disease in congenitally infected children and immunocompromised patients. Although nearly all CMV infections remain latent and asymptomatic in immunologically normal individuals, numerous studies have found that systemic viral reactivation is common in immunocompetent critically ill adults, as measured by detection of CMV in the blood (viremia). Furthermore, CMV viremia is strongly correlated with adverse outcomes in the adult intensive care unit (ICU), including prolonged stay, duration of mechanical ventilation, and death. Increasing evidence, including from a randomized clinical trial of antiviral treatment, suggests that these effects of CMV may be causal. Therefore, interventions targeting CMV might improve outcomes in adult ICU patients. CMV may have an even greater impact on critically ill children, particularly in low and middle income countries (LMIC), where CMV is regularly acquired in early childhood, and where inpatient morbidity and mortality are inordinately high. However, to date, there are few data regarding the clinical relevance of CMV infection or viremia in immunocompetent critically ill children. We propose that CMV infection should be studied as a potential modifiable cause of disease in critically ill children, and that these studies be conducted in LMIC. Below, we briefly review the role of CMV in immunologically normal critically ill adults and children, outline age-dependent differences in CMV infection that may influence ICU outcomes, and describe an agenda for future research. Cytomegalovirus (CMV) is a double-stranded DNA herpesvirus that infects most of the world’s population , 2. Natuin utero  is rare in immunologically normal individuals , 13, 14.There have been more than 30 studies of immunologically normal, critically ill adult patients demonstrating an association between systemic CMV reactivation and worse outcomes in intensive care units (ICUs), as summarized in recent reviews –25. In tCritical illness may impair immune control of CMV replication normally mediated by cytotoxic T cells and natural killer (NK) cells –41. AdulExposure of latently infected cells to a pro-inflammatory environment contributes to CMV reactivation . Thus, iAdverse ICU outcomes often result from uncontrolled inflammation and/or impaired immunity . CMV infCytomegalovirus replication in tissues of immunocompromised individuals is a well-recognized cause of end-organ damage, including pneumonitis, hepatitis, and colitis , 16–18. In transplant recipients, CMV is associated with the so-called “indirect” effects on the immune system, including increased risk of bacterial and fungal infections , 58, 59.Randomized clinical trials exploring the effect of CMV treatment on outcomes in adult ICU patients are ongoing, and have the ability to determine the causality of CMV in these associations , 62. A rSubsequently, a phase 2, randomized controlled multicenter trial of prophylactic ganciclovir or valganciclovir versus placebo in immunocompetent CMV seropositive adult ICU patients with respiratory failure and severe sepsis or trauma was published . The stuThere are qualitative and quantitative age-dependent differences in the immune responses of young children that impair the control of many viral infections, including CMV –69. ChilThere are very few data available about the frequency and effects of active CMV infection in critically ill, immunocompetent children. We performed a pilot study of CMV infection in children admitted to the ICU of BC Children’s Hospital in Vancouver, Canada (unpublished data). Among the 27 children enrolled, the median age was 4 years (range 4 months–17 years). Only one child was CMV seropositive, and none were found to have viremia. This is consistent with the relatively low CMV seroprevalence among children in most high-income countries –77. By cWell-designed cohort studies are needed to understand the role of CMV in immunocompetent critically ill children, to estimate the potential benefits, and to inform the design of appropriate pediatric treatment trials. Given the differences between children and adults in the natural history and outcomes of CMV infection, extrapolation from adult studies is inappropriate. As an example, in the adult studies conducted to date, the clinical impact of CMV has been ascribed to reactivation and dissemination of chronic infection –25. HoweQuantitative CMV DNA PCR assays can reliably detect ≤50 international units/mL and have largely replaced older methods of detection , 82. BecThe risk of CMV disease is correlated to the level of viremia in many clinical scenarios. For example, in HIV-infected individuals and transplant recipients, CMV viral load is proportionally associated with progression of disease , 87. HowStudying the impact of CMV infection on critically ill immunocompetent children is anticipated to be both easier and more clinically relevant in low- and middle-income countries (LMIC), where CMV infection is holoendemic and mortality and morbidity rates from sepsis are often dismal. In LMIC, nearly all children acquire CMV infection within the first few years of life . By contIn addition to the relatively low rates of CMV infection in North American and European children, the excellent outcomes of pediatric ICU patients there hinder the ability to detect a clinically important impact of CMV infection on critically ill children –96. FurtImportantly, appropriate studies in children require the incorporation of pediatric-specific illness severity scores and outcome measures Table . MortaliStrong evidence supports the association between CMV viremia and adverse outcomes in adult ICU patients –25, and The pilot study Reactivation of Cytomegalovirus in Pediatrics (ReCIPe) for the prevalence of CMV viremia among pediatric critical care patients in BC Children’s Hospital was approved by BCCHR Ethics Board.RA: literature review and writing; SG: reviewing, editing, writing, and knowledge check; JS: reviewing statistics and literature, editing, and writing; SM: checking literature and ICU data.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.