Tnf (Tnf-/-) in an acute TNBS colitis model to investigate the role of TNF in colitis and how its presence or absence affects the colonic microbiota. As expected, Tnf-/- had less severe inflammation than WT mice. Microbiome analysis revealed significant Tnf dependent-differences in alpha and beta diversity. There were also notable differences in many species that were also primarily Tnf dependent. Taken together, our data indicates that TNF contributes significantly to the inflammation and microbiotal alterations in that occur in IBD.Inflammatory bowel disease (IBD) is associated with prolonged, excess secretions of Tumor Necrosis Factor (TNF). Many patients with IBD have successful management of IBD symptoms by blocking TNF secretion or signaling. However, some patients are non-responsive to this therapy, eventually become refractory to therapy, or Alterations in the microbiota that are associated with the lack of TNF could be a contributing cause of this therapeutic insufficiency seen in some patients. Here we use wildtype (WT) and mice lacking  Inflammatory bowel disease (IBD) consists of two inflammatory disorders, Crohn’s disease (CD) and Ulcerative Colitis (UC), both of which are characterized by lifelong and repeated bouts of inflammation and healing in the gastrointestinal (GI) tract . The patEscherichia-Shigella group [The human gut is the largest reservoir of microbes in the body. Many studies have indicated that the gut microbiota plays an active and integral role in maintaining host health ,16. The la group ,26. In hTNF is a pleiotropic cytokine, considered to be a master regulator of cytokine production. This cytokine is elevated in both the serum and mucosa of IBD patients –30. The Tnf or lack Tnf in order to determine how TNF suppression correlates with alterations in the microbiota and decreased inflammation. Our long term goal is to identify specific microbial communities that can be manipulated, in addition to TNF suppression, to increase the effectiveness of the anti-TNF therapeutic protocol.Despite intense study, the underlying etiology and mechanism(s) of pathogenesis of CD remain to be fully elucidated. Altered microbiota and excessive inflammation are two major components that contribute to the development and progression of CD and studies detailing changes in the microbiome that occur as disease progresses from acute colitis to chronic colitis are limited. We hypothesize that neutralization of TNF induces alterations in the intestinal microbiota that correlate with reduced inflammation. In this study, we use the trinotrobenzene sulfonic acid (TNBS) colitis mouse model of CD ,39 to evTnf gene (Tnf-/- mice) [Tnf-/-) and treatment (TNBS vs SHAM).All colitis experiments with animals in this study were approved by the Purdue Animal Care and Use Committee. Six to eight week old, female B6.129S mice with a homozygous deletion of the /- mice)  and contet al. with minor modifications [Induction of TNBS colitis was performed according to the protocol of Wirtz ications . Five miμm sections, and stained with hematoxylin and eosin (H&E) for histological analysis via light microscopy. The degree of inflammation in cross sections of the colon was assessed semi-quantitatively by an experienced pathologist, blinded to treatment allocation, as previously described [Body weights were recorded every other day and were reported as percentage of weight loss from initial body weight. Necropsy was performed 3 days post IR injection and tissues were harvested. At necropsy, mice were euthanized by an overdose of CO2 followed by cervical dislocation. The colons were removed, opened and washed with PBS. The colons were cut in half longitudinally and 4 centimeters of one-half of the colon was fixed in 10% buffered formalin for histological examination. The remaining one-half of the colon was divided into two, 2-cm segments, which were snap frozen in liquid nitrogen and stored at -80°C. Fixed sections of colonic tissues were embedded in paraffin, cut into 6-escribed  with minTotal genomic DNA was extracted from each fecal sample (50 mg) using the Fast DNA Soil Spin kit  according to the manufacturer’s instructions. DNA concentration was determined using a NanoDrop 3300  fluorospectrometer and quality was assessed using a NanoDrop 1000 spectrometry (260/280 OD ratio) as well as agarose gel electrophoresis.MiSeq Illumina 2x250 paired end sequencing was used to determine fecal bacterial community composition in the first and last fecal samples collected from each mouse during the acute testing period. Primers that amplify the V3–V4 region of the 16S rRNA gene (forward TAC GGR AGG CAG CAG and reverse CTA CCR GGG TAT CTA ATC C primers) were used to take advantage of primer accuracy and coverage of phylogenetic information that has been determined for short sequencing reads ,44. MultPrimer tags and low quality sequence reads were first removed using Illumina software then paired end reads were merged using Panda software . Sequenchttp://folk.uio.no/ohammer/past/index.html). Significant differences in beta diversity between communities were determined using PERMANOVA a non-parametric multivariate statistics [Comparisons of histological scores were statistically analyzed using the nonparametric Mann-Whitney U test. All data in bar graph or dot plot formats are expressed as mean ± S.E.M. Differences were considered statistically significant at P <0.05. For the microbiome data, Krustral Wallis analysis  was used for an overall comparison of average proportions of each taxa level  in mice fecal samples collected on day 0 (pre-colitis) and day 10 (post-colitis) of the experiments. Mann-Whitney U test was used to determine significant differences between treatments of day 10 samples. It was also used to compare day 0 and day 10 samples within treatments. The change in bacterial genera proportions was calculated by subtracting day 0 values from day 10) and significant differences were determined using the Mann-Whitney U test. All basic statistics were performed using the Paleontological Statistics package version 3.01  sequences. Based on these numbers, all results reported herein use a subset of 30,330 sequences for each sample. More than 95% of the sequences in all mice at days 0 and 10 were classified into two phyla, Bacteroidetes and Firmicutes . Other pwn genus . Of thesTnf-/- mice for all the alpha diversity measures evaluated, the richness indices ChaoI (WT 1029.9 ± 130.5 vs Tnf-/- 879.1 ± 136.8) and observed species (WT 497.2 ±46.4 vs Tnf-/- 418.1 ± 42.2), and phylogenetic diversity (PD) whole-tree measure (WT 21.8 ± 1.4 vs Tnf-/- 19.0 ± 1.5) ; these differences can be determined using a variety of algorithms for distance measures . All theTnf-/- and WT mice prior to colitis. At the phylum level significant differences were found between the Bacteroidetes (WT 8.6% ± 4.2% vs Tnf-/- 20.7% ± 16.1%) and Firmicutes (WT 89.1% ± 4.6% vs Tnf-/- 76.4% ± 15.8%) (n = 20 per genotype). Examination at the genus level indicated that within these phyla at day 0 there were three genera, Ruminococcus, Turicibacter and Anaerovorax, and five groups that have yet to be assigned to a genus that differed significantly (p < 0.05) (Lactobacillus decreased significantly from about 15% at day 0 to about 5% at day 10 in TNBS treated WT mice (Tnf-/- mice significant differences in four taxa after TNBS and four other taxa after SHAM treatment but know of these taxa could be classified to any known genera (Turicibacter after TNBS treatment. In the Tnf-/-mice only the “Other” Ruminococcaceae group differed significantly (p = 0.01), -0.53 ± 1.28% (SHAM) vs 0.58 ± 0.52% (TNBS).Comparisons of taxa proportions identified a number of groups that were possibly contributing to the differences in the community alpha and beta diversity measures. Significant differences were found between  < 0.05) . There w < 0.05) . The rel WT mice . In the n genera . There wn genera , most noTnf-/- mice on day 0, confirming that Tnf-/- mice weigh less than WT mice at day 0. The arrows along the upper y-axis indicate there is correspondence between the microbial communities of SHAM and TNBS treated Tnf-/-mice on day 10. Since colitis scores were taken only on day 10, another CCA was used to determine correspondence with day 10 microbial communities samples  indicated that the variation in bacterial community composition, treatment and some mouse traits had significant correlations. Factors that significantly corresponded (p<0.05) with the microbial communities were mice genotype, starting weight and ending weights . The rel samples . DiffereIn this study, we evaluated how TNF ablation affects acute colitis, and how colitis correlates with TNF dependent alterations in the microbiota. As expected, we found that TNF promotes acute TNBS colitis, and CCA indicated colitis scores corresponded with alterations in the colonic microbiota. The decreased inflammation in TNF- mice is consistent with previous reports ,54. SimiTnf-/- mice did not decrease alpha diversity significantly after 10 days but WT mice developed more severe colitis. Other studies have found a decrease in alpha diversity associated with IBD in people [Tnf-/- mice than in WT mice before any treatment but Tnf-/- mice developed less severe colitis. It is possible that the gene mutation is sufficient for colitis protection and the microbiota are not playing a major role in the pathology or the necessary microbiota are still present for protection. In contrast, compared to WT mice alpha diversity was higher in mice lacking the Tata Element Modulatory Factor (TMF/ARA160) and the microbiota were playing a role in protection from chronic colitis [Tnf-/- mice have chronic protection from colitis and despite the lower diversity if the microbiota play a role in protection.TNBS treatment of WT or n people  but a10- TMF/ARA1 and the Tnf-/- mice are generally smaller than their WT counterparts and, alpha and beta diversity comparisons using CCA indicated that the smaller size corresponded with differences in the microbial communities. Further studies are needed to determine if the differences in microbiota community composition are a contributing cause of the lower body weight of Tnf-/- mice or if other factors associated with TNF ablation causes lower body weight, which then leads to the difference in the colonic communities. The lower weight and lower microbial diversity observed in the Tnf-/- mice may contribute to more detrimental pathology when the disease is chronic or cause secondary health problems.Interestingly, Tnf-/- mice. This differs from a study that used anti-TNF-α to inhibit TNF production in mice in which the Firmicutes to Bacteroidetes ratio decrease but no information was given if this difference was significant [Tnf-/- mice but the roles of many of them are currently not known. However, there have been reports on Ruminococcus, Turicibacter, and unclassified S24–7. Ruminococcus is believed to be a beneficial microbe that can ferment resistant starches [Tnf-/- compared to WT mice potentially contributing to protection from colitis. There was a significantly greater proportion of Turicibacter in WT compared to Tnf-/- mice both prior to and after colitis induction, suggesting that its presence may be related to TNF expression. Little is known about the role of Turicibacter in IBD. However, one study found an increase in Turicibacter in mice with depletion of CD8+ T cells [Tnf-/- mice compared to WT mice. Recently, this group of bacteria has been found to be associated with remission of colitis in mice [Tnf-/- mice, which develop less colitis than WT mice. A bacterium that may be playing a protective role in WT mice is Lactobacillus. There was a significant decrease in Lactobacillus in WT mice after TNBS treatment. This genus is known to be “beneficial” and is extensively used as a probiotic in a variety of diseases, including gastrointestinal diseases [Beta diversity comparisons revealed that the most significant difference in communities was due to mouse genotype. At the phylum level this difference was likely due to the higher Firmicutes to Bacteroidetes ratio in WT vs nificant . This sustarches . It was  T cells , while a T cells . These s in mice . This madiseases . The decWe describe here for the first time, the effects of TNF ablation on acute TNBS colitis and the microbiota. Our data demonstrate a pathogenic role for TNF in acute TNBS colitis and importantly, we show for the first time how inflammation (and TNF production) is associated with significant differences in the microbiota. These microbiota data support our general conclusion that the strongest factor contributing to the microbiome alterations, and subsequently, inflammation, in this model is TNF production. These findings suggest that combinatory therapy including inhibition of TNF and the target altering specific microbial communities (e.g. via probiotic therapy) may prove to be a beneficial therapeutic approach for IBD patients.S1 Figp<0.001) using perMANOVA, but additional analysis using PERMDISP indicates dispersion is significant, mainly due to the relatively tight cluster of WT-SHAM F0 samples compared to greater dispersion of the other groups.Significant differences between these groups ((TIFF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


The NLRP3 inflammasome is a protein complex responsible for caspase-1 dependent maturation of the pro-inflammatory cytokines IL-1β and IL-18. Gain of function missense mutations in NLRP3 cause the disease spectrum known as cryopyrin-associated periodic syndromes (CAPS).To elucidate systemic autoinflammatory disease mechanisms involved NLRP3 inflammasomeopathies other than IL-1β and IL-18.L351P/+/CreL/Il1b-/-/Il18-/-Nlrp3mice (-/-Il18-/-FCAS Il1b), L351P/+/CreL/Nlrp3 Casp1-/-, (FCAS Casp1-/-),A350V/+/CreL (MWS)Nlrp3 and A350V/+/CreL/Tnf-/- (MWS Tnf-/-)Nlrp3 were generated in which Cre-mediated expression is limited to the myeloid cell lineage.Knock-in -/-Il18-/-FCAS Il1bmice survived and grew normally until adulthood however, investigation of mice at > 6 months of age showed marked splenomegaly and elevated numbers of white blood cells as compared to FCAS Casp1-/-mice and non-mutant -/-Il18-/-Il1blittermates, suggesting a caspase-1 dependent phenotype independent of IL-1β and IL-18. To examine other potential inflammatory mediators, non-lethal in vivo LPS (5 ug/g) stimulation of FCAS Il1b-/-Il18-/-mice revealed significantly elevated levels of serum TNF at both 2 and 6 hours post induction when compared to FCAS Casp1-/-mice and non-mutant -/-Il18-/-Il1bcontrols. To further investigate the role of TNF in Nlrp3 inflammasomeopathies, MWS mice (which die within two weeks of birth) were bred on a TNF knockout background. MWS Tnf-/- pups were indistinguishable from non-mutant controls with all animals surviving to adulthood with normalization of both body and spleen/body weight comparisons. Serum analysis of -/-MWS Tnf pups showed attenuation of NLRP3 inflammsome related cytokines when compared to intact MWS pups. The skin of intact MWS pups exhibited strong neutrophilic and inflammatory macrophage infiltrations, which were normalized in MWS Tnf-/- animals. Interestingly, +/-MWS Tnf  pups also showed an intermediate protective effect on all of the aforementioned comparisons. To determine if TNF ablation could be recapitulated with therapeutic intervention, MWS pups were treated with recombinant soluble TNF receptor (Etanercept 400ug/g sc EOD). Treatment provided similar phenotypic rescue and extended survival for an average of 22 days after cessation of treatment. Adult -/-MWS Tnf mice at > 6 months of age were found to have splenomegaly and elevated numbers of white blood cells when compared to non-mutant -/-Tnf littermates, implicating a role for other inflammatory mechanisms as mice age.Nearly all TNF plays an unexpected and significant role in murine inflammasomeopathies, which may have therapeutic implications for CAPS patients with incomplete responses to IL-1 targeted therapies.


Imaging of the brain with computerized tomography (CT) or magnetic resonance imaging (MRI) is crucial to support the diagnosis of normal pressure hydrocephalus (NPH). The aim of this study was to construct a radiological scale, composed of morphological signs of NPH, and compare with an existing scale for clinical NPH symptoms developed by Hellström et al.In a prospective population-based study of the prevalence of NPH, 91 individuals , mean age 74 years (range 66-92 years), underwent CT of the brain and neurological examination with assessment of clinical symptoms. A radiological scale consisting of eight radiological parameters was developed and correlated with the clinical NPH scale score to yield a reliable diagnostic tool. Two independent radiologists, blinded to clinical data, visually assessed and performed all measurements of the parameters Evans index >0.3, callosal angle <90°, narrow high convexity sulci, focally dilated sulci, dilated Sylvian fissures, focal bulging of ventricular roof, dilated temporal horns, and periventricular hyperintensities. After conversion into points, the parameters were summarized yielding a score ranging from 0 to 10 points where higher scores indicates more severe radiological changes.Mean clinical NPH score was 83 . Mean NPH radscale was 2 . There was a significant correlation  between the new NPH radscale and clinical NPH symptoms as assessed by the NPH symptom scale.The new NPH radscale seems to be a promising tool for diagnosing NPH.


We present a systematic review on International Classification of Functioning, Disability and Health (ICF) used in the Nordic countries from 2001 through 2013, describing and quantifying the development in utilization of ICF, and describe the extent to which the different components of the ICF have been used. A search was conducted in EMBASE, MEDLINE and PsycInfo. Papers from Nordic countries were included if ICF was mentioned in title or abstract. Papers were assigned to one of eight categories covering the wide rehabilitation area; furthermore, area of focus was assigned. Use of ICF components and intervention were coded in papers categorized as “clinical and/or rehabilitation contexts” or “non-clinical contexts”. One hundred seventy papers were included, of these 99 papers were from the categories “clinical and/or rehabilitation contexts” or “non-clinical contexts”. Forty-two percent of the 170 included papers were published in the period 2011 - 2013. There was an increase in ICF-relevant papers from 2001 to 2013, especially in the categories “clinical and/or rehabilitation contexts” and “non-clinical contexts”. The most represented focus areas were neurology, musculoskeletal, and work-related areas. All five or at least four ICF components were mentioned in the results or discussions in most papers, and activity was most frequently mentioned. Has the mission been successful, does the International Classification of Functioning, Disability and Health (ICF) provide a common language? The ICF was developed by the World Health Organization (WHO) and formally endorsed in 2001 as a framework to document and evaluate functioning and disability .The overall aim of the ICF is to provide a common language of functioning in order to facilitate data comparisons and a systematic coding scheme for health information systems . The ICFSince 2001, the ICF has become widely accepted both as a theoretical framework and as a useful clinical practice tool used across different health and social disciplines. In 2009, Jelsma published a literature survey on papers published until 2007 . Thus, tThere is no consensus in reporting ICF results in the literature, and the quality of the ICF papers within “clinical practice and/or rehabilitation contexts” and “non-clinical contexts” varies a lot. A group under the WHO Family of International Classifications (WHO-FIC) Network - the Functioning and Disability Reference Group (FDRG) - has discussed criteria to evaluate literature related to ICF . These cTo set the threshold criteria for good quality papers, and in order to explore the quality of papers from the categories “clinical and/or rehabilitation contexts” and “non-clinical contexts”, it is necessary to select a sample of papers between the many hundreds published from 2001 to 2013. Cerniauskaite et al in fact found 173 papers from “clinical and/or rehabilitation contexts”, and this figure would be tripled if papers from 2001 to 2013 were included. Thus, it seems reasonable to apply selection criteria to reduce the scope of the study; therefore we have applied a geographic selection criterion. The Nordic countries  are not only geographically close to each other, but also largely similar in terms of their welfare systems, although it is acknowledged that no two welfare states are quite alike. In the Nordic health and welfare systems, strong state intervention, and wide universal welfare schemes are among the key traits. In the Nordic countries, the recommendations on ICF have followed international trends, making the region adequately representative as a focus for study [The objective of this study was to present a systematic review on ICF used in the Nordic countries from its release in 2001 through 2013, describing and quantifying the development in utilization of ICF within the categories “clinical and/or rehabilitation contexts” and “non-clinical context”. Furthermore, the objective was to describe the extent to which the different components of the ICF have been used.A systematic literature search was conducted using three electronic databases, EMBASE, MEDLINE and PsycInfo, in June 2013 and again in May 2014. The search strategy used by Cerniauskaite et al  was refiPapers were eligible for inclusion if they met the following inclusion criteria: date of publication from 2001 up to and including 2013; ICF or International Classification of Functioning, Disability and Health mentioned in the title or abstract; the language of the abstract was English; and if the first author was affiliated with a Nordic country. Peer reviewed original and review papers were included, excluding book chapters and theses. Papers were excluded if they only mentioned ICIDH or ICIDH-2, if they were on ICF-CY only, or if they were on persons aged < 18 years only. No additional papers were included. The inclusion was performed by five teams of reviewers; each record was read by two reviewers. In case of disagreement, the record was discussed in the research group until consensus was reached.Full texts of all papers included in this review were read independently by two researchers from the research group and assigned to one of six categories, created by Cerniauskaite et al : 1) concFurthermore, an area of focus was assigned to all papers. Area of focus was generated from medical specialities and expanded with relevant areas such as “elderly” and “mixed population”. Editorials papers on the ICF framework were coded “not relevant” as they do not cover a single area of focus. All papers categorized as “clinical and/or rehabilitation contexts” or “non-clinical contexts” were reread with focus on the use of the components  mentioned in results or discussion and whether the paper represented an intervention study. Intervention was defined as “a treatment, whether for preventative or therapeutic reasons, an assessment or diagnostic tool or some other type of service or condition to which a patient might be exposed” . Papers In case of disagreement amongst the two reviewers, one of the leaders of the research group (TM or ML) read and categorized the study blinded for previous coding. In case of further disagreement, the final decision was taken after a discussion of the issue.Totally 2,388 papers were found, after removing duplicates, 1,623 papers were assessed for eligibility, and finally 170 papers were included in the review. Information on publication year and the eight categories is reported in Disability and Rehabilitation and Journal of Rehabilitation Medicine published the majority of papers (35 (20.6%) and 17 (10.0%), respectively) and 64 papers (37.6%) were published in journals that just published one of the papers. Three authors were first authors of four of the included papers: Alguren, Paltamaa and Roe. Seven authors were first in three papers, 15 authors were first on two papers, and 107 authors were represented with one paper each.The distribution between the five Nordic countries was diverse as more than half of the published papers were Swedish (55.9%), 42 (24.7%) papers from Norway, 24 (14.1%) papers from Finland, nine (5.3%) papers from Denmark and no papers from Iceland .In 102 papers (60.0%), a quantitative research method was used, 27 (15.9%) papers used qualitative research methods, and both methods were used in four (2.4%) of the papers; in 37 (21.8%) papers, this was not coded as the papers were reviews or editorials (29 papers (17.1%)) or “not relevant” to code (eight papers (4.7%)); the latter were papers describing development of ICF and ICF-related instruments.Of the 170 selected papers, 99 were included in the two categories “clinical and/or rehabilitation contexts” and “non-clinical context”.The publication years of the 99 papers follow the same pattern as the total 170 included papers . A markeDisability and Rehabilitation was the most frequently seen journal, publishing 24 (24.2%) of the papers. Journal of Rehabilitation Medicine published 11 (11.1%); 36 papers (36.4%) were published in journals that just published one paper each.Four authors were first authors in three papers: Alguren , PaltamaThe distribution between the five Nordic countries followed the distribution of the total 170 included papers . Table 3The most frequently reported area of focus was neurology 27 (27.3%), followed by work-related papers 17 (17.2%), and musculoskeletal 15 (15.2%). Area of focus was not relevant in one (1.0%) paper as it was on ICF as a general matter. In 73 papers (73.7%), a quantitative research method was used, 22 (22.2%) papers used qualitative research methods, and both methods were used in three (3.0%) of the papers; in one (1.0%) paper, this was not coded as the paper was “not relevant” to code because it described the development of an ICF-related instrument. Analysis on the five ICF components was performed: all five components were mentioned in results and/or discussion in 27 (27.3%) of the papers, whereas 11 (11.1%) papers mentioned none of the five components . The comIntervention was featured in 15 (15.1%) of the 99 papers in the category “clinical and/or rehabilitation contexts” . One papTwenty-one of the included papers were reviews , 41-60, The present review’s objective was to quantify the development in utilization of ICF in the categories “clinical and/or rehabilitation contexts” and “non-clinical context”, and we found that this group was the largest with around 60% of all papers and there was a marked increase in 2005 - 2006 and after 2010.Thus, this review confirms that the international trend of increased use of ICF is present in the Nordic countries too. Throughout the period under study (2001 - 2013), there has been a steady rise in the number of published ICF-related papers reflecting an increase in scientific papers in general. The results from the present review are in close accordance with the former review , althougThe present review shows that the use of ICF is increasing, but there is a marked difference between the five Nordic countries. Norway, Finland and Denmark have almost the same population size, while the population of Sweden is twice as big. Norway and Sweden have the same rate of papers per inhabitant, Finland half that, Denmark just one-fifth, and no papers were included from Iceland, the smallest country.This difference in number of papers might be explained by the fact that there has been heightened focus on rehabilitation in Norway by the government and the municipalities, and Sweden has a substantially higher number of academic therapists and nurses. Denmark has no medical speciality in rehabilitation and only few academic therapists and nurses; this might reduce the focus on the bio-psycho-social approach and thereby on the use of ICF.The majority of the papers are published primarily in two rehabilitation journals which are promoting the ICF, and more than one-third is from other journals publishing only one paper. Although it is not possible to draw a firm conclusion from this review, these results do not support the goal for ICF to be the common language in function and disability.When discussing the dissemination of ICF in clinical and rehabilitation research settings, one also has to consider the fact that ICF might not always be the right tool. The substantial core sets can prove too extensive in some types of data collection settings, and in cases where assessment of progression is a primary aim, ICF might not be the first choice. In other words, promoting ICF as the primary tool for evaluation of research in clinical and rehabilitation settings should be done recognizing such facts. One of the key elements in modern rehabilitation and the bio-psycho-social mind set is the context’s impact on function. The results from this systematic review do not fully support this, as we found the contextual factors were the least used components. It is also worth noting that a quarter of the papers mention just two or fewer components, and that only a quarter mentioned all five components. If the understanding of functioning as described in the ICF were to be fully implemented, more papers would have used all five components in at least the discussion of their results.Regarding the distribution of categories, the present review found similar results to Cerniauskaite et al , whereasThis review confirms the use of ICF in rehabilitation literature in the geographic selection “The Nordic countries”.There is a marked difference in the use of ICF in different diagnosis and areas of focus.The five ICF components are not systematically assessed in all ICF papers. However, at least four ICF components were mentioned in result or discussion in most papers, with the activity component being the most frequently reported.One of the aims of the ICF was to provide a common language in rehabilitation. This aim is still not quite met and this review concludes: “There is still a long way to go before ICF is widely implemented in rehabilitation and clinical settings”.This study was subject to four main limitations. First, the literature search included three databases: EMBASE, MEDLINE and PsycInfo, whilst other databases such as SCOPUS and CINAHL were excluded with potentially other papers. However, the methods used were largely based on the former review by Cerniauskaite et al , and as In conclusion, this review systemically searched the ICF literature in the five Nordic countries with emphasis on the categories “clinical and/or rehabilitation contexts” and “non-clinical contexts” and found an increase in papers during the study period, especially in these categories. Sweden, Norway and Finland accounted for most papers, and in all papers the most represented focus areas were neurology, musculoskeletal, and work-related. Finally, in most papers, all five or at least four ICF components were mentioned in the result or discussion, with the activity component as the most frequently reported.The answer to the question whether ICF provides a common language is mostly no. There is still a long way to go, before ICF is widely implemented in rehabilitation and clinical settings. The question is, whether the ICF, besides being a framework, also works as a classification system that supports standardized identification and description of health and health-related function. Further studies should be conducted in order to answer this question.


Background: Eukaryotes typically encode a range of ISWI chromatin remodeling complexes with different functions.Results: We have identified and analyzed three novel ISWI partners in the early branching eukaryote Trypanosoma brucei.Conclusion:T. brucei appears to have a single major ISWI complex.Significance: This unusually simple ISWI configuration could be a consequence of the relative lack of transcriptional regulation in this ancient eukaryote. Trypanosoma brucei, which causes African sleeping sickness, TbISWI down-regulates RNA polymerase I (Pol I)-transcribed variant surface glycoprotein (VSG) gene expression sites (ESs), which are monoallelically expressed. Here, we use tandem affinity purification to determine the interacting partners of TbISWI. We identify three proteins that do not show significant homology with known ISWI-associated partners. Surprisingly, one of these is nucleoplasmin-like protein (NLP), which we had previously shown to play a role in ES control. In addition, we identify two novel ISWI partners, regulator of chromosome condensation 1-like protein (RCCP) and phenylalanine/tyrosine-rich protein (FYRP), both containing protein motifs typically found on chromatin proteins. Knockdown of RCCP or FYRP in bloodstream form T. brucei results in derepression of silent variant surface glycoprotein ESs, as had previously been shown for TbISWI and NLP. All four proteins are expressed and interact with each other in both major life cycle stages and show similar distributions at Pol I-transcribed loci. They are also found at Pol II strand switch regions as determined with ChIP. ISWI, NLP, RCCP, and FYRP therefore appear to form a single major ISWI complex in T. brucei (TbIC). This reduced complexity of ISWI regulation and the presence of novel ISWI partners highlights the early divergence of trypanosomes in evolution.ISWI chromatin remodelers are highly conserved in eukaryotes and are important for the assembly and spacing of nucleosomes, thereby controlling transcription initiation and elongation. ISWI is typically associated with different subunits, forming specialized complexes with discrete functions. In the unicellular parasite  Eukaryotes package their genomic DNA into chromatin, whereby DNA is wrapped around octamers of histones forming nucleosomes. This allows the compaction of extensive stretches of DNA into the restricted space of the nucleus as well as being a major factor in controlling DNA access. For example, the exact phasing or degree of compaction of nucleosomes can either block or expose promoter sequences to recognition by the transcriptional machinery , 2. Chro3–de novo assembly of nucleosomes in concert with core histone chaperones , which in combination with different subunits, form a total of four different complexes , each containing ISWI bound to various combinations of nine different subunits , at least seven different ISWI complexes have a similar broad range of functions, including facilitating DNA repair , 26, acttromeres –31.Trypanosoma brucei is a unicellular eukaryote and causative agent of African sleeping sickness   , 45. TheT. brucei. Putative Pol II transcription initiation sites have a simple structure lacking canonical Pol II promoter elements  containing Pol II promoters and terminators  and the procyclic form (PF) present in the tsetse fly insect vector. Surprisingly, these ISWI-interacting proteins include the nucleoplasmin-like protein (NLP), which we have previously shown to have a similar role to TbISWI in down-regulating ESs , TbISWI (GeneDB: Tb927.2.1810) and NLP (GeneDB: Tb927.10.5450) were tagged at the C terminus with a Protein C-tobacco etch virus (TEV) protease site-Protein A (PTP) epitope  in PF T.onstruct .in situ at the endogenous locus at the C terminus using either a triple Myc epitope or a triple HA epitope using either the pMoTAG42M or pMoTAG4H construct  or FYRP (GeneDB: Tb927.7.1060) were integrated into the T. brucei minichromosomes to generate either T. brucei T3-RCCP or T. brucei T3-FYRP. The BF T. brucei 90-13 cell line  in a Poly-Prep chromatography column (Bio-Rad) with protease inhibitors for 3.5 h. The beads were washed, and the Protein A portion of the tag was removed by the addition of AcTEV protease (Invitrogen). The TEV eluate was incubated with anti-Protein C matrix (Roche Applied Science) in a fresh Poly-Prep column overnight at 4 °C. The anti-Protein C matrix was washed, and the final TAP-purified material was eluted with EGTA. The purified product was concentrated using a vacuum concentrator and StrataClean resin (Stratagene) before separation under denaturing or nondenaturing conditions on 4–15% SDS-polyacrylamide or polyacrylamide gels (Bio-Rad). Bands were visualized with Imperial Protein Stain (Thermo Scientific) or Silver Stain (Thermo Scientific), excised as specified, and subjected to analysis by liquid chromatography-tandem mass spectrometry .The C termini of TbISWI and NLP were tagged with the PTP epitope , and TAPm sucrose, 20 mml-glutamic acid, 20 mm HEPES-KOH (pH 7.7), 3 mm MgCl2, 1 mm DTT, 150 mm KCl, 0.1% Nonidet P-40) and incubated with either monoclonal anti-HA  or anti-Myc  antibodies or no antibody for 2 h at 4 °C. Crude extract (100 μl) was added to the columns with the immobilized antibodies and incubated for 2 h at 4 °C. Washes were carried out with ice-cold wash buffer . Purified proteins were eluted into boiling SDS-PAGE loading buffer, boiled for 5 min, and centrifuged at 1000 × g for 7 min. The supernatant was removed, and 15 μl was loaded onto either 8 or 10% polyacrylamide gels.For co-immunoprecipitation (co-IP) analysis, TbISWI, NLP, RCCP, and FYRP were tagged at the C terminus with either a triple Myc epitope using the pMoTAG43M vector  or with T. brucei T3, T3-FYRP, and T3-RCCP cell lines, and cells were harvested at different time points, washed once in PSG buffer, and fixed in 2% paraformaldehyde. These cell lines contain an eGFP reporter gene inserted behind the promoter of a silent VSG221 expression site. Fluorescence of the cells was monitored in the FL-1 channel using a BD FACSCalibur (BD Biosciences). CellQuest software (BD Biosciences) was used to calculate the average of 100,000 events (BD Biosciences). The -fold ES derepression was calculated by dividing the average FL-1 fluorescence of RNAi-induced populations at each time point by the average FL-1 fluorescence of uninduced populations. Three independent experiments were performed with each cell line with the S.D. values shown with error bars.RNAi was induced in the BF T. brucei T3-FYRP and T3-RCCP cell lines were used for quantitative RT-PCR. RNAi was induced against FYRP or RCCP, and total RNA was isolated at various time points using the RNeasy kit (Qiagen). RNA was treated with DNase using the TURBO DNA-free kit (Ambion), and reverse transcription was carried out using the Omniscript RT kit (Qiagen) with random hexamer primers (Promega). qPCR was performed on a 7500 Fast Real-Time PCR system (Applied Biosystems) using Brilliant II SYBR Green (Agilent Technologies). We used primers that our bioinformatic analyses indicated would recognize single copy sequences within the T. brucei genome (results not shown), and the reaction conditions for each primer pair were individually optimized. Control reactions without RT were performed using DNase-treated RNA for each time point. Transcript levels were normalized to levels for γ-tubulin transcripts and plotted as -fold increase with respect to the 0 h time point. Three independent experiments were performed, with S.D. values shown with error bars.The BF 5 cells/μl. This was incubated at 100 °C for 10 min before loading onto 6 or 10% SDS-polyacrylamide gels. Gels were blotted onto Hybond-P membrane (Amersham Biosciences) and probed with rabbit polyclonal antibodies against Protein A, BiP (gift of Jay Bangs)  , TbISWI y Bangs) , NLP (55y Bangs) , and RCCT. brucei 221GP1(VO2+) cell line  was performed as described previously . HA-taggell line  were immstigated . StatistT. brucei genome with the sequences of ISWI partners in other organisms but were unsuccessful in detecting potential homologues in T. brucei. In order to identify TbISWI-interacting proteins, we used a TAP tagging approach with a PTP epitope tag . The second allele of TbISWI was knocked out (ISWI-sKO) without resulting in a growth defect, demonstrating that PTP-tagged TbISWI is fully functional (A).In general, in eukaryotes, ISWI is present in one or more functional complexes . We perftope tag . We geneus locus A. The senctional A.B and C). Briefly, a crude protein extract was first purified by IgG affinity chromatography, and the TEV protease was used to cleave off the Protein A portion of the PTP tag. Subsequently, the TEV protease eluate underwent anti-Protein C affinity purification, and the final purified products were eluted with EGTA. The concentrated proteins, along with fractions obtained throughout the purification procedure, were separated by SDS-PAGE under denaturing conditions (B and C).This cell line was used for the TAP tagging procedure , B and Cnditions , B and CB) to show that the purification was successful. The same samples were separated by SDS-PAGE and stained with Coomassie, where a range of bands were easily detectable in the final eluate (C). These bands were excised, and the associated proteins were identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). As expected, TbISWI-P was recovered in the EGTA eluate (C) (108 unique peptide hits) as well as histones H2A, H2B, and H4. Because ISWI is a nucleosome remodeler, this interaction with histones is not unexpected. However, three additional potential TbISWI partners were discovered with molecular masses of ∼110, 70, and 55 kDa, with 70, 32, and 26 unique peptide hits, respectively.We monitored the enrichment of TbISWI-PTP by Western blot using an anti-TbISWI antibody B to showl eluate C. These A eluate C (108 unT. brucei (A) (eGFP reporter gene inserted immediately downstream of a silent ES promoter (A). The human RCC1 protein is a cell cycle regulator that binds chromatin and acts as a guanine nucleotide exchange factor for Ran GTPase  was the NLP, which contains an AT-hook and a nucleoplasmin-like domain and is essential in BF . brucei A 55). W. WT. brupromoter A. The hun GTPase .A). FYR domains are poorly characterized but have been found in chromatin-associated proteins, including histone methyltransferases, such as trithorax (The 54-kDa TbISWI-interacting protein was named FYRP (Tb927.7.1060) because it has an N-terminal phenylalanine/tyrosine-rich (FYR) domain A. FYR dorithorax , 68. BecB). TbISWI is orthologous to ISWI in S. cerevisiae and SMARCA1/SNF2L in Homo sapiens. This protein is very highly conserved across eukaryotes, with orthologs present in all species analyzed from a wide range of lineages. In contrast, TbISWI-interacting proteins appear to be restricted to the Kinetoplastida. NLP and FYRP homologs are only identifiable in this lineage. RCCP is an RCC1 repeat domain-containing protein, of which there are several members in trypanosomes and most other eukaryotes. However, the RCCP paralog itself is specific to the Kinetoplastida (B).We next investigated whether TbISWI and its interacting partners were conserved in a range of different eukaryotic species B. TbISWIplastida B.T. brucei? Typically, in different eukaryotes, ISWI is a component of a number of functional ISWI complexes, with discrete roles depending on the composition of the subunits . Similarly, the same experiment was performed with TAP affinity-purified NLP-PTP (B). The visible bands were excised and analyzed by mass spectrometry. A predominant major band was seen in both cases, corresponding to either TbISWI or NLP complexed with each other and with RCCP.To elucidate whether TbISWI forms one complex or multiple subcomplexes, TbISWI-PTP and its co-purified components from the TAP affinity purification experiments were separated under nondenaturing conditions and silver-stained A. Simila NLP-PTP B. The viT. brucei ISWI complex (TbIC) (C). However, we cannot exclude the presence of additional minor subcomplexes composed of just some of the TbISWI complex subunits.FYRP was detected in both experiments, albeit below the threshold score of 80, indicating weak association with this complex. However, based on its score in the initial TbISWI and NLP TAP tagging experiments and the extensive co-IP experiments, we are confident that FYRP is a true member of the TbISWI complex. Additional minor bands observed below the main band contain different stoichiometries of complex partners indicating possible different degradation states of a single complex. These data therefore indicate that there is a single major x (TbIC) C. HoweveT. brucei VSGT3-expressing reporter cell line where eGFP had been inserted immediately downstream of the promoter of the inactive VSG221 ES (eGFP in the silent VSG221 ES after 72 h as monitored in the FL-1 channel using flow cytometry (C).We have previously established that both TbISWI and NLP play a role in ES silencing , 55. We SG221 ES . RNAi waSG221 ES . Only a ytometry C.E). Here too, although the induction of RNAi resulted in only a minor reduction in cell growth , there was 26–61-fold derepression of the silent VSG221 ES.We performed a similar analysis of the role of FYRP . The FYRFig. 7T. brucei. However, to investigate this further, we determined the genomic localization of the four potential components using ChIP experiments. ChIP was performed in different BF cell lines expressing either HA-tagged TbISWI, HA-NLP, or HA-FYRP, using a monoclonal anti-HA antibody. Multiple attempts of ChIP using HA epitope-tagged RCCP proved unsuccessful, indicating a possible lack of accessibility of the HA epitope to antibodies when the ISWI complex is in association with DNA. We therefore used a rabbit polyclonal antibody against RCCP in the RCCP ChIP experiments.The native gels and the co-IP experiments suggested that there is a single predominant TbISWI complex (TbIC) in A). TbISWI and NLP are relatively depleted within Pol I transcription units but enriched at non-transcribed regions (B). In the case of FYRP, the statistical significance of this differential localization was extremely significant (p < 0.001) . In the case of RCCP, although there was a trend, this was not statistically significant. Similarly, at the Pol I-transcribed procyclin loci (C), TbISWI and NLP are relatively enriched upstream compared with within the transcription units (p = 0.01–0.05 (primer pairs a versus primer pairs b or c) in both cases (D).We first investigated the localization of the TbISWI complex components at the RNA Pol I-transcribed rDNA loci A. TbISWI regions , 55. Thi regions B. In thelin loci C, TbISWIon units , 55. Thith cases D.T. brucei are polycistronic. Pol II transcription initiates in SSRs, where two opposing transcription units diverge, and terminates where they converge. TbISWI was proposed to be enriched at these SSRs and particularly in the regions around divergent SSRs containing promoters  in both BF and PF T. brucei. Using a number of different experimental methods, we show that all of the TbIC subunits are expressed and interact with each other in both trypanosome life cycle stages. The previously characterized nucleoplasmin-like protein NLP was found to be a member of this TbIC complex. This unexpected discovery explains the observation that knockdown of either TbISWI or NLP leads to similar phenotypes, including the derepression of VSG ESs. In addition, using TAP affinity purification with either TbISWI or NLP, we identify two novel and previously uncharacterized TbIC components: RCCP and FYRP. Neither of these ISWI partners is a homologue of known ISWI partners in other eukaryotes. However, both proteins contain amino acid sequence motifs indicating a possible interaction with chromatin.In eukaryotes, the ISWI chromatin remodeler is typically present in a variety of different complexes with distinct functions, depending on exactly which subunits ISWI is partnered up with. Here, we have characterized TbISWI and its interacting partners in Xenopus, ISWI is a RanGTP-dependent microtubule-associated protein required for chromosome segregation . In addition, ChIP experiments showed a trend for localization of FYRP with other TbIC members on similar regions of genomic DNA. Similarly, knockdown of FYRP also led to comparable derepression of silent VSG ESs as observed after knockdown of the other TbIC subunits. Our data therefore indicate that FYRP could have a weak or transient interaction with other complex members, making it prone to disassociation during protein affinity purification.One possibility that could explain our data is that in T. brucei? Both the TbISWI and NLP affinity purification experiments identified each other as well as the RCCP and FYRP subunits. In addition, as mentioned above, co-IP experiments in both life cycle stages show that all four components interact with each other, and ChIP experiments indicate that all four proteins associate with similar regions of genomic DNA. Therefore, all of the available evidence, using a variety of different experimental approaches, would argue that a single predominant TbISWI complex is present in the early branching eukaryote T. brucei. As expected for subunits participating in the same complex, knockdown of each of these TbIC subunits leads to VSG ES derepression. However, these experiments do not rule out the presence of minor TbISWI complexes containing a subset of the subunits.Is there indeed only one ISWI complex in S. cerevisiae.Chromatin remodelers, including ISWI complexes, are extremely difficult to analyze using ChIP . This maS. cerevisiae, ISWI is important for regulation of Pol II transcription, and ISWI variants are found both within Pol II gene bodies and at both promoters and terminators. The Isw1 variant has different functions, depending on which Ioc subunits it is partnered up with (In  up with , 17. Isw up with . In cont up with . The Isw up with . This reT. brucei, we found a trend for TbISWI and the NLP, RCCP, and FYRP subunits binding at both divergent and convergent Pol II strand switch regions; however, these data supporting four proteins being relatively enriched in these regions were not statistically significant. This relative simplicity of ISWI complex architecture could be a consequence of the lack of control of Pol II expression in T. brucei at the level of either transcription initiation or elongation (In ongation .T. brucei, all of the TbIC components are located at the rDNA, particularly in the non-transcribed spacers. This is also the case at the Pol I-transcribed procyclin loci and the ESs (T. brucei.In most eukaryotes, Pol I exclusively transcribes the rDNA arrays, of which typically about half are transcriptionally silent . ISWI va the ESs , althoug the ESs . BecauseT. brucei, which is a very early branching eukaryote, although we cannot rule out the presence of relatively minor subcomplexes. The apparent presence of all TbIC components at a range of different genomic loci, including Pol II SSRs, as well as at different Pol I loci argues that the predominant TbIC complex could be multifunctional. Chromatin remodeling enzymes appear to have arisen soon after the origin of the eukaryotic lineage, and as eukaryotic genomes expanded in size and complexity, there was an increasing need for a larger array of specialized chromatin remodeling factors (T. brucei appears to have a relatively reduced set of these chromatin remodelers, coupled with a greatly reduced set of Pol II transcription factors (T. brucei evolved, large amounts of gene loss occurred as a consequence of the lack of the need for intricate control systems as the organism relied on constitutive transcription by Pol II. We show that the major T. brucei TbIC complex contains novel subunits compared with other non kinetoplastid eukaryotes.All of our experimental evidence therefore points to a single ISWI-containing complex in  factors . In comm factors , 86. PosThe challenge for us now is to understand the role of these unique chromatin remodelers in the maintenance of genome architecture in these ancient eukaryotes. In addition, hopefully, increased knowledge of the role that these divergent chromatin remodelers play in transcriptional control, including that of the VSG expression sites, will allow us to disrupt this process, thereby leading to new forms of antiparasitic therapies.T. S., M. N., A. L., and G. R. designed the study. G. R. and S. R. wrote the paper. B. W. performed bioinformatic analyses. T. S., M. N., S. R., A. L., K. W., M. K., S. W., and J. W. performed the experiments. All authors analyzed the results and approved the final version of the manuscript.


Familial spastic paraplegia (FSP) is a heterogeneous group of disorders characterized primarily by progressive lower limb spasticity and weakness. More than 50 disease loci have been described with different modes of inheritance. Recently, we described a novel missense mutation  in the plasma membrane calcium ATPase  gene in a Chinese family with autosomal dominant FSP. Further to this finding, here we describe the functional effect of this mutation.fura-2 fluorescent dye with confocal microscopy in human SH-SY5Y neuroblastoma cells overexpressing either wild-type or R268Q mutant PMCA4.As PMCA4 removes cytosolic calcium, we measured transient changes and the time-dependent decay of cytosolic calcium level as visualized by using Overexpressing both wild-type and R268Q PMCA4 significantly reduced maximum calcium surge after KCl-induced depolarization as compared with vector control cells. However, cells overexpressing mutant PMCA4 protein demonstrated significantly higher level of calcium surge when compared with wild-type. Furthermore, the steady-state cytosolic calcium concentration in these mutant cells remained markedly higher than the wild-type after SERCA inhibition by thapsigargin.Our result showed that p.R268Q mutation in PMCA4 resulted in functional changes in calcium homeostasis in human neuronal cells. This suggests that calcium dysregulation may be associated with the pathogenesis of FSP. Familial spastic paraplegia (FSP) is a clinically and genetically heterogeneous group of diseases characterized by progressive lower limb spasticity and weakness, and is classified according to phenotype, mode of inheritance, and the mutated gene . Positive clones containing either PMCA4b or mut-PMCA4b were confirmed with sequencing.Full-length human PMCA4 isoform b (Protein ID: ENSP00000350310) cDNA was amplified from total RNA extracted from HEK-293 cells. The gene-specific primers used were as followed: PMCA4b-forward, 5′-ATCGGATCCATGACGAACCCATCAGACC, and PMCA4b-reverse, 5′-ATCGCGGCCGCTCAAACTGATGTCTCTAGGCTCT. A mutated mega-primer, prepared using PMCA4b-forward and PMCA4b-M-reverse, 5′-CATCTGGCCAGAACCTT, along with PMCA4b-reverse was used to amplify the mutant form of PMCA4b  using full-length wild-type PMCA4b amplicon as template. The PCR products were then subcloned into mammalian expression vector, pcDNA3.1(+), and transformed into μg/mL penicillin-streptomycin  at 37°C in humidified 5% CO2 atmosphere. Expression plasmids containing transcript of either wild-type or mutant PMCA4 were transfected into SH-SY5Y cells using Lipofectamine2000 (Invitrogen). Twenty-four hours after transfection, cells were selected using geneticin (G-418) at 250 μg/mL until resistant clones were obtained.Human neuroblastoma SH-SY5Y cells  were cultured in DMEM-F12 supplemented with 10% FBS, 2 mmol/L glutamine and 100 260/280 ratio of extracted RNA was kept above 1.95 to ensure purity and integrity. Reverse transcription was performed using total RNA by AccuScript High Fidelity 1st Strand cDNA synthesis kit . Amplification reactions were performed using Power SYBR Green PCR Master Mix (Applied Biosystems) according to manufacturer's protocol. The primers for real-time PCR: PMCA4b-forward: GCAATACCTACCCGATCCCTG; PMCA4b-reverse: ACCGCATTGTTGTTTGTATTGG. GAPDH expression was used to normalize for mRNA loading. Each RNA sample was run in triplicate and repeated in at least three independent treatments.Total ATP2B4 mRNA expression was determined by quantitative RT-PCR. Total RNA was isolated and digested with DNase I as previously described  were used to detect the proteins, respectively. Relative expression level of target proteins was determined by the band intensity as measured using densitometry.Overexpression of PMCA4 was confirmed using SDS-PAGE western blotting. Equal amount of total cell lysates extracted in standard RIPA were subjected to polyacrylamide gel electrophoresis, followed by standard western blotting procedures. Antibodies against total PMCA4  and 5 cells/well) were plated in 24-well plates coated with 100 or 10 μg/ml poly-L-lysine  with DMEM/F12 at 37°C overnight for adherence. To determine the effects of PMCA4 mutation on the intracellular calcium transient, cells stably overexpressing either wild-type or mutant PMCA4 were labeled with 5 μmol/L fura2-AM fluorescent dye (Invitrogen) in serum-free DMEM for 20 min in darkness at room temperature. After subsequent washes with Gey's balanced salt solution (GBSS), cells were incubated in GBSS for a further 10 min at 37°C to allow de-etherification of AM esters. Labeled cells were put on the stage of an Olympus inverted epifluorescence microscope  and visualized using a 20 × objective. Images were collected by a CCD camera every 2 sec and analyzed using Olympus Cell R Imaging System  at room temperature with excitation of 340 and 380 nm, and emission was measured at 510 nm. Ratio (R) of F340/F380 was obtained after subtracting background noises. Cells were allowed to stabilize for 20 sec and baseline ratios (Ro) were obtained prior to addition of potassium chloride  to induce membrane depolarization, or incubation with the SERCA inhibitor, thapsigargin . The measurement continued for 4 min after addition of KCl or TG. The relative changes in intracellular calcium levels [Ca2+]i, indicated as normalized ratio (R/Ro), were plotted against time. Intracellular concentration of calcium was calculated using the general formula, [Ca2+]i = Kd * Q(R-Rmin)/(Rmax-R). (Kd =183 nmol/L). Kd is the dissociation constant for Ca2+ binding to the indicator, R is the fluorescence intensity ratio between emission at 340 and 380 nm with fura-2. Q is the ratio of minimum (Fmin) to maximum (Fmax) intensity at OD380. Fmax was determined by exposing cells to 1.5 mmol/L Ca2+ and 5 μmol/L ionomycin, and Fmin was determined by the addition of 4 mmol/L EGTA and 5 μmol/L ionomycin to cells.Human SH-SY5Y cells . Differences were considered significant at a level of P < 0.05.Statistical analyses were carried out using the Prism . Data are expressed as mean ± standard error mean (SEM) from at least three independent experiments. Statistical significance among individual groups was calculated by the Student's PMCA4, we overexpressed either wild-type or mutant PMCA4 protein in human SH-SY5Y cells and assessed changes in intracellular calcium transient. Overexpression of either wild-type or mutant PMCA4 was confirmed by western blotting using antibodies against PMCA4 and β-actin  : 769.0 ± 16.1 μmol/L; PMCA4(R268Q): 843.9 ± 20.6 μmol/L)   of calcium surge between cells overexpressing WT (t1/2 = 23.06) and mutant PMCA4 (t1/2 = 22.26),  based on extra sum-of-squares F test in one phase decay analysis. To further elucidate the contribution of PMCA4 to the calcium transient, we performed additional experiments to measure the steady state [Ca2+]i after incubation with SERCA inhibitor, thapsigargin    between neuronal excitation in cells overexpressing mutant PMCA4 may result in subsequent activation of various cell death pathways, for example, Ca2+-dependent synthases and proteases to damage cytoskeleton, membrane, and DNA leading to excitotoxicity and neuronal death (Gleichmann and Mattson In this study, we showed that the R268Q mutation of the PMCA4 gene had functional implication and resulted in increased maximum KCl-induced calcium transient. Even though the absolute difference of the two peak ratios between WT and mutant PMCA4 appeared to be small at 74 Taken together, we believe that the R268Q mutation in PMCA4 caused neuronal deficits associated with FSP. This is the first report to demonstrate a PMCA4 mutation which caused functional changes in calcium extrusion to be associated with autosomal dominant FSP, indicating that calcium dysregulation may be involved in the pathogenesis of spastic paraplegia. The detailed pathogenic mechanisms of how impairment in neuronal calcium flux can directly cause the disease phenotype in FSP require further studies.


Peste des petits ruminants (PPR) is an acute, febrile, viral disease of small ruminants that has a significant economic impact. For many viral diseases, vaccination with virus-like particles (VLPs) has shown considerable promise as a prophylactic approach; however, the processes of assembly and release of peste des petits ruminants virus (PPRV) VLPs are not well characterized, and their immunogenicity in the host is unknown. In this study, VLPs of PPRV were generated in a baculovirus system through simultaneous expression of PPRV matrix (M) protein and hemaglutin in (H) or fusion (F) protein. The released VLPs showed morphology similar to that of the native virus particles. Subcutaneous injection of these VLPs  into mice and goats elicited PPRV-specific IgG production, increased the levels of virus neutralizing antibodies, and promoted lymphocyte proliferation. Without adjuvants, the immune response induced by the PPRV-H VLPs was comparable to that obtained using equivalent amounts of PPRV vaccine. Thus, our results demonstrated that VLPs containing PPRV M protein and H or F protein are potential “differentiating infected from vaccinated animals” (DIVA) vaccine candidates for the surveillance and eradication of PPR. Peste des petits ruminants (PPR) is a highly contagious and economically important viral disease of domestic and some wild small ruminants, and in particular, of goats and sheep. It is notifiable to the Office International des Epizooties (OIE). Clinically, the disease is characterized by severe pyrexia, oculonasal discharges, necrotizing and erosive stomatitis, enteritis, and pneumonia. It was first described in the Ivory Coast, West Africa, but has now become wide-spread in sub-Saharan Africa, Arabia, the Middle East, Southwest Asia, India, and other countriesMorbillivirusThe causative agent, PPR virus (PPRV), is a member of the family Paramyxoviridae and the genus For the control of PPR, four attenuated vaccines are available commercially. In endemic areas, the virus is currently controlled using the Nigeria 75/1 strain (Nig75/1), which has been attenuated by serial passage in Vero cells. In India, three other live attenuated vaccines are currently licensed for use: Sungri 96, Arasur 87, and Coimbatore 97 Virus-like particles (VLPs) recently emerged as alternatives to subunit vaccines; these have the advantage that they mimic the organization and conformation of the native virus capsid and envelope, but lack the viral genome To date, the assembly and release of PPRV VLPs have not been well characterized, and their immunogenicity remains unknown. It is known that the PPRV glycoproteins H and F contain neutralizing antibody epitopes and several T cell epitopes In this study, two types of VLPs for PPRV (PPRV-H and PPRV-F)were developed, using viral M protein co-expressed with H or F protein. Mice and goats immunized subcutaneously with these PPRV VLPs developed both humoral and cellular immune responses.2, and were used for PPRV propagation and titration. Spodoptera frugiperda Sf21 insect cells were maintained in Sf-900II insect serum-free medium  as monolayer cultures or in suspension cultures maintained on temperate orbital shakers (120rpm) at 28°C.PPRV vaccine strain (Nig75/1) was obtained from the China Institute of Veterinary Drug Control, Beijing, China. Vero cells  were cultured in Dulbecco's modified Eagle medium  containing 10% fetal bovine serum  at 37°C with 5% COPPRV RNA was extracted using Trizol LS . Reverse transcription(RT) and polymerase chain reaction (PCR) were performed on extracted viral RNA with gene-specific oligonucleotide primers that hadXhoI–KpnIDNA fragment, downstream of the p10 promoter within the pFastBacDual bacmid transfer vector that had been digested with XhoI and KpnI. The resulting baculovirus transfer plasmid containing the PPRV M gene was designated p-M.The transfer plasmids were generated using the pFastBac Dual vector , which contains two promoters. The M gene was cloned, as a1-kbBamHI–HindIII DNA fragment, into the BamHI/HindIII sites downstream of the Autographacalifornica multiple nuclear polyhedrosis virus (AcMNPV) polyhedron(PH) promoter in p-M. The resulting plasmid, p-MH, encoded the M and H genes downstream from the p10 and PH promoters, respectively.A bacmid transfer vector expressing both the M and H genes was prepared by cloning the H gene, as a 1.8-kb BamHI–XbaI DNA fragment, downstream of the PH promoter, into p-M , which contained the AcMNPV baculovirus genome. The recombinant bacmids were identified by PCR using M13 primers together with gene-specific primers  viral stock. Cells were continuously passaged in order to amplify baculovirus stock with the highest viral titer. The recombinant baculovirus stock was titrated by agarose plaque assay on Sf21 insect cell cultures in 6-well plates.Recombinant bacmids were produced by site-specific transpositioning after transformation of bacmid transfer plasmids containing the PPRV genes into  primers . The rec6 cells per mL, with rBVs expressing PPRV H and M proteins, at a multiplicity of infection (MOI)of 4. Similarly, PPRV-F VLPs were produced by infecting Sf21 insect cells with rBVs expressing the PPRVF and M proteins. Cell culture supernatants were harvested 3 days post-infection, cleared by low-speed centrifugation to remove cells, and VLPs in the supernatants were then pelleted by ultracentrifugation using a SorvallT1250 rotor . The resulting pellets were dissolved in 2 mL of phosphate-buffered saline solution , and were then further purified through a discontinuous sucrose gradient  and sedimented by ultracentrifugation in a SorvallTH641 swinging bucket rotor . As a control, we used the PPRV Nig75/1 virus. The visible opalescent bands in the sucrose gradient were collected, diluted in PBS, and pelleted by centrifugation in a Sorvall T1250 rotor  to remove the sucrose. The sedimented particles were resuspended in PBS and stored at 4°C. The concentration of the VLPs was determined using a bicinchoninic acid protein assay kit .To produce PPRV-H VLPs, Sf21 insect cells were infected in a 200-mL volume, at a cell density of 2×10To detect the protein reactivity of VLPs, purified VLPs were boiled for 5 min in reducing Laemmli sample buffer and resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred to a polyvinylidene difluoride membrane by electroblotting . The membrane was blocked with 5% dried non-fat milk in PBST (0.05% Tween-20 in PBS), overnight at 4°C, and then incubated with goat serum against PPRV (diluted 1∶200 in blocking buffer), for 1 h at 37°C. The membrane was washed three times with PBST, for 5 min each time, and then incubated with horseradish peroxidase (HRP)-conjugated rabbit anti-goat IgG antibody . Following several washes with PBST, the specific bands were detected using the substrate 3,3′-diaminobenzidine tetrahydrochloride  for 5 min at room temperature. The reaction was stopped with water and membranes were photographed.To examine budding of VLPs, Sf21 insect cells that had been infected with rBVs expressing the M and H/F proteins were fixed with 0.25% glutaraldehyde and 1% osmium tetraoxide, dehydrated with ethanol, and then embedded in SPI-PON 812 resin . Thin sections were stained with lead citrate and uranyl acetate and then observed by electron microscopy The presence and morphology of the purified VLPs and PPRV were evaluated by TEM of negatively stained preparations. A carbon-coated grid was set on a 20-µL sample drop for 10 min, and washed with deionized water. Washed samples were negatively stained with 1% uranyl acetate at pH 4.5. Excess stain was removed using filter paper, and the grids were observed at magnifications ranging from 60,000× to 200,000× on a transmission electron microscope  operating at 80 kV.The designs of the animal experiments are shown in For mouse experiments, female inbred BALB/c mice , aged 6 to 8 weeks, were randomly divided into seven groups (n = 10 for each group). Mice were immunized subcutaneously (s.c.) with different amounts  of PPRV VLPs or PPRV Nig75/1diluted in 100µL of PBS. All groups received a booster injection of the original dosage at 4 weeks after the primary immunization. No adjuvant was used for any of the mice. Blood samples were collected by retro-orbital plexus puncture, at various time-points. After the blood samples were allowed to clot and then centrifuged, serum samples were collected and stored at −20°Cuntil required for antibody titration.5.5 TCID50), as positive control. Group 4 goats were inoculated subcutaneously with 1 mL PBS as negative control. Booster vaccination was performed using the same dose, at 3 weeks after primary immunization. Serum samples were collected at various time-points post-immunization, and stored at −20°C until analyzed.For goat experiments, 20 outbred goats (6–24 months of age) were randomly divided into four groups of five each, and housed separately. None of the goats had detectable levels (titers less than1∶5) of PPRV neutralizing antibodies. Groups 1 and 2 were vaccinated subcutaneously with 300 µg PPRV-H or PPRV-F VLPs diluted in 1 mL of PBS, respectively. Group 3 goats were inoculated with 1 mL PPRV Nig75/1 values were measured at a wavelength of 450 nm using an ELISA microplate reader . The mean titers were calculated from log10 of the highest serum dilution that yielded a two-fold higher OD value compared to that of sera from PBS-vaccinated mice.Sera from immunized and control mice were analyzed for PPRV-specific antibodies  by indirect ELISA. Briefly, 96-well flat-bottomed plates were coated with 100µLof purified PPRV whole-virus antigen at a concentration of 2 µg/mL per well in 0.1 M carbonate/bicarbonate coating buffer (pH 9.6),overnight at 4°C, and then the plates were blocked with 5% skim milk in PBST, for 2 h at 37°C. Serum samples, serially diluted in PBST, were added to the wells, in duplicate, and incubated at 37°C for 1 h. After washing with PBST three times, each well received 100µL of a 1/4000-diluted HRP-labeled goat anti-mouse IgG, IgG1, and IgG2a  and incubated at 37°C for 1 h. Finally, after washing with PBST three times, 100 µL of freshly prepared tetramethylbenzidine  solution was added to each well and color was allowed to develop for 10 min, before stopping the reaction with 100µL of 2 M H50/mL) in a 96-well cell culture plate and incubated at 37°C for 1 h; then, 50µL of Vero cell suspension was added to each well and the cells were cultured at 37°C in the presence of 5% CO2.A series of control wells for virus and uninfected cells were also included. Cells were observed for a cytopathic effect (CPE) by microscope at day 8 after incubation, and the VNA titer was calculated. VNA titer was defined as the highest dilution of the test serum at which the CPE was inhibited by at least 50%.All serum samples from immunized mice and goats were analyzed for the detection of PPRV-specific virus neutralization antibody (VNA) titers using a micro-neutralization assay, according to the 2013 OIE terrestrial manual6cells per well in RPMI1640 medium  containing 10% FBS. Splenocytes were stimulated in vitro with 50µL of UV-inactivated PPRV Nig75/1(104 TCID50). Concanavalin A  was used as positive control, and medium was used as negative control. After 3 days' incubation at 37°C in a 5% CO2atmosphere, the proliferative response was determined using a 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay, using the Cell Titer 96 Aqueous One Solution Cell Proliferation kit , following the manufacturer's instructions. The lymphocyte proliferation rate was quantified using the stimulation index (SI), which was calculated as the ratio of the OD490 of the stimulated cells to the OD490 of the negative controls.Three mice from different groups were sacrificed at 7 days after booster immunization and splenocyte suspensions were prepared as previously described5 cells per well) were plated in 96-well plates and stimulated with inactivated PPRV Nig75/1 for 72 h at 37°C under 5% CO2. Unstimulated cells, cultured in medium alone, were used as negative control. The proliferative response was determined by MTS assay, as described above.Goats were bled via jugular vein puncture at week 2 after the booster injection. Peripheral blood mononuclear cells were prepared as previously described 5 cells per well in a 96-well ELISpot plate, which had been coated with IFN-γ. One hundred microliters of inactivated PPRV Nig75/1was added as stimulus. The cultures were then incubated in a 37°C humidified incubator, with 5% CO2, for 16 h, and then spots were detected as per the manufacturer's instructions .Splenocytes from inoculated mice were obtained and plated at a density of 2×10The data were analyzed with one-way ANOVA in GraphPad Prism version 5.00 ; p-values less than 0.05 were considered statistically significant.7 and 4.7×107PFU/ml, respectively, at the third passage.The open reading frames of PPRV M, H, and F genes from PPRV strain Nig75/1 were amplified by RT-PCR and sub-cloned into pFastBacDual vectors .The insePPRV-H VLPs and PPRV-F VLPs were produced and purified as described in the Materials and Methods section. The VLPs bands were mainly distributed between 30% and 60%sucrose in the gradient after ultracentrifugation . ElectroIncorporation of the H or F proteins, as well as M protein, into VLPs was confirmed by western blotting using anti-PPRV antibodies, by the presence of 37 kDa, 70 kDa, and 70 kDa bands, corresponding to the M, H, and F proteins, respectively, on the blots . The sizThe specific antibody responses of mice immunized with PPRV-H VLPs or PPRV-F VLPs were determined by indirect ELISA. The levels of total PPRV-specific IgG in the serum after primary and booster vaccinations were determined; the results are shown in PPRV-specific serum antibodies of IgG1 and IgG2a were also evaluated by ELISA. These results demonstrated that mice immunized with PPRV-H VLPs and PPRV using a lower dose of antigen (10 µg) induced high IgG1 serum antibody titers(1∶1000 to 1∶2400), and lower specific IgG2a serum antibody titers(1∶200 to 1∶250) after the first immunization. In contrast, the levels of IgG1 antibody responses were lower(1∶200),and the IgG2a responses were higher (1∶700),in mice immunized with PPRV-F VLPs relative to those observed in mice immunized with PPRV-H VLPs or PPRV. The levels of IgG1 and IgG2a from all three immunized groups of mice increased after the booster immunization . SimilarTaken together, the data indicated that immunization with PPRV-H VLPs induced predominantly specific IgG1 serum antibodies, favoring the stimulation of a T-helper class 2(Th-2) immune response against PPRV, while immunization with PPRV-F VLPs induced a stronger Th-1 immune response, despite the use of a lower immunization dose.Since the neutralizing activity against PPRV is an indicator of functional antibodies that confer protective immunity, we investigated whether mice and goats immunized with PPRV VLPs could produce neutralizing antibodies, using a micro-neutralization assay. Serially diluted sera from various time-points post-immunization were complement-inactivated, and incubated with live PPRV Nig75/1; the CPE assays were conducted in Vero cells. As shown in Taken together, the data of the mouse and goat vaccination experiments showed that serum VNA levels were significantly higher in the PPRV-H VLPs-immunized group than in the PPRV-F VLPs-immunized group, and were comparable to that of the whole virus-immunized group. No neutralization activity against PPRV was detected when using pre-immune serum or in the sera of experimental animals injected with PBS.To investigate the cell-mediated immune responses induced by VLPs, the lymphocyte proliferation response of mice was analyzed after the booster immunization. As shown in We also evaluated the mounting of PPRV-specific T-cell responses in vaccinated mice. Splenocytes, obtained at 7days after the booster immunization, were cultured in the presence of inactivated PPRV, and IFN-γ production was measured by ELISPOT assays . Both PPAltogether, these data demonstrated that inoculation with PPRV-H VLPs and PPRV-F VLPs can generate specific T-cells that recognize the PPRV H and F proteins in vivo.The minimum molecular requirement for efficient assembly and release of PPRV virions is still unknown. In this study, we found that the major structural genes of PPRV can be efficiently expressed in recombinant baculovirus-infected Sf21 insect cells and that these genes can then support the production of PPRV VLPs. The expression of M protein was necessary and sufficient for the formation of VLPs (data not shown); the additional expression of the H or F glycoproteins allowed formation of budding particles with the typical morphology and size of PPRV and related paramyxoviruses. A recent study demonstrated that PPRV spikeless VLPs could be formed by co-expression of the M and N proteins in insect cells; however, expression of M protein alone in insect cells did not result in VLP formation, as assessed by TEM The baculovirus expression vector–insect cell system has been utilized extensively for VLP production, and particularly for production of enveloped VLP vaccines In this study, we chose the pFastBacDual (Invitrogen) baculovirus transfer vector, which contains two promoters, so that insect cells infected with a single recombinant bicistronic baculovirus could express two proteins concurrently. The co-expression strategy is particularly important for the efficient assembly of multi-protein complexes, where simultaneous expression of multiple viral proteins is essentialThe main limitation of the baculovirus expression vector–insect cell system is the co-production of recombinant baculoviruses (enveloped viruses) during the infection process; thus, when developing PPRV VLP vaccines, the challenging task is the purification of VLPs from the recombinant baculoviruses, which share a similar density and enveloped structure For many viruses, recombinant VLPs have been shown to yield promising vaccines, because of their robust immunogenicity, elicitation of protective neutralizing antibodies , and their safety profiles . In this study, the effectiveness of PPRV VLPs as an immunogen was demonstrated in mice and goats. The results indicated that high specific antibody titers against PPRV could be induced by both PPRV-H VLPs and PPRV-F VLPs, which were as high as those resulting from immunization with PPRVNig75/1. The VNA titers elicited by PPRV-H VLPs were much higher than those induced by PPRV-F VLPs, and the VNA titers of PPRV-H VLPs were as high as the responses to the attenuated virus vaccine. Previous reports have shown that the H protein of PPRV is a more potent inducer of high VNA titers than the F proteinWe further showed that subcutaneous immunization of mice with PPRV-H VLPs at a dose of 10 µg, in the absence of adjuvant, induced strong humoral immune responses. This immunogenicity is related to the structural features of the VLPs, which are particulate structures that contain a dense and repetitive pattern of epitopes on their surfaces. In contrast, monovalent, non-particulate antigens do not always reflect the native structure of the protein, and the related antibody responses are not always specific for physiologically relevant epitopes. Consequently, administration of these vaccines often requires use of strong adjuvants, as well as large and frequent doses of the antigen. In contrast, VLP-based vaccines are strongly immunogenic, often reducing or eliminating the need for exogenous adjuvantsTo our knowledge, this is the first report that demonstrated the construction of PPRV VLPs consisting of immunogenic proteins and that assessed their immunogenicity in two animal models, viz., mice and goats. Taken together, the results of our study suggested that PPRV VLPs can be generated in insect cells simultaneously expressing the PPRV M protein with H or F protein. The VLPs had a morphology and size similar to those of native virus particles. Humoral and cellular immunity against PPRV were efficiently induced in mice and goats vaccinated with these PPRV VLPs, without the need for adjuvants. Thus, PPRV-H VLPs or PPRV-F VLPs may be promising vaccine candidates for PPR, and DIVA tests may prove of great benefit in future control programs for this disease.