Primary central nervous system lymphomas (PCNSL) have a dramatically increased prevalence among persons living with AIDS and are known to be associated with human Epstein Barr virus (EBV) infection. Previous work suggests that in some cases, co-infection with other viruses may be important for PCNSL pathogenesis. Viral transcription in tumor samples can be measured using next generation transcriptome sequencing. We demonstrate the ability of transcriptome sequencing to identify viruses, characterize viral expression, and identify viral variants by sequencing four archived AIDS-related PCNSL tissue samples and analyzing raw sequencing reads. EBV was detected in all four PCNSL samples and cytomegalovirus (CMV), JC polyomavirus (JCV), and HIV were also discovered, consistent with clinical diagnoses. CMV was found to express three long non-coding RNAs recently reported as expressed during active infection. Single nucleotide variants were observed in each of the viruses observed and three indels were found in CMV. No viruses were found in several control tumor types including 32 diffuse large B-cell lymphoma samples. This study demonstrates the ability of next generation transcriptome sequencing to accurately identify viruses, including DNA viruses, in solid human cancer tissue samples. Primary central nervous system lymphomas (PCNSL) accounted for 7% of all AIDS cancers in the early HAART era and have a roughly 1000-fold increased prevalence in persons living with AIDS a priori assumptions regarding gene sequences or a sample’s viral population. Previous experimental approaches for identifying viruses in host samples relied on PCR or microarrays to identify viral sequences. PCR-based approaches are confounded by the need to clone viruses or design primers for genomes that can be highly polymorphic or poorly characterized Next generation transcriptome sequencing offers the ability to detect viruses with few Here we have performed highly parallel transcriptome sequencing of four AIDS-related PCNSL tissue samples and built upon previous analysis methods to identify expected and unexpected viruses and characterize viral gene expression. We were able to identify EBV in all four PCNSL samples, consistent with previous studies that have reported finding EBV in effectively all AIDS-related PCNSL patients 1 mm bores were extracted from HIV-positive PCNSL post-mortem brain tumor samples from two 37 year old non-Hispanic white cases (PCNSL4 and PCNSL2) and two Hispanic cases, aged 37 and 41 (PCNSL1 and PCNSL3 respectively), from the California NeuroAIDS Tissue Network (CNTN) + cells were then purified by magnetic bead separation . These cells were transduced with lentiviral vectors, FACS sorted using human-specific CD34 and CD38 antibodies and a lineage cocktail directly into RLT buffer  as described previously To serve as a positive control a cord blood sample was purchased from AllCells . Peripheral blood mononuclear cells (PBMC) were extracted from peripheral blood following Ficoll density centrifugation and CD34Resected tumor from a 75-year-old man with a 30-pack-year history of smoking and an incidentally found squamous cell carcinoma was xenografted into NSG mice and harvested after palpable tumor growth was observed as previously described Two CLL samples were obtained from the CLL Consortium. Sample CLL1 is from a Caucasian female age 70 with indolent disease while CLL2 is from a Caucasian male age 63 with aggressive disease. CLL1 was found to have chr13 deletion in 72% of cells and CLL2 had trisomy 12 in 66% of cells according to FISH.RNA was isolated from samples with the QIAGEN RNeasy kit and analyzed on the Bioanalyzer . Whole transcriptome libraries were prepared following “SOLiD Total RNA-Seq kit protocol” . Total RNA samples were depleted using “RiboMinus Eukaryote Kit for RNA-Seq” . One PCNSL total RNA sample (PCNSL3) was non-depleted due to low amount of starting material (∼1.2 ug). The total RNA or rRNA-depleted total RNA were fragmented by RNAse III and cleaned up using RiboMinus concentration Module . RNA fragments were hybridized and ligated to SOLiD adaptor mix and reverse transcription performed. Purified cDNA  was amplified 15 cycles using SOLiD 3′ and SOLID 5′ primers. Amplified DNA were purified using PureLink PCR Micro kit , QC by Bioanalyzer 2100 DNA 1000 kit , and quantified by Qubit . The whole transcriptome libraries were used for making SOLiD templated beads following SOLiD4 System Templated Bead Preparation Guide. 50 bp fragment sequencing was performed on the ABI SOLiD3 platform generating between 472 and 525 million raw reads per sample for PCNSL samples and similar amounts for other samples.http://cgap.nci.nih.gov/cgci.html) [GenBank:phs000235]. Sequencing methods and information are available through the Short Read Archive.32 diffuse large B-cell lymphoma transcriptomes were sequenced on the Illumina GAII as part of the Cancer Genome Characterization Initiative  using BWA  to select for reads that were likely of viral origin and aligned reads were sorted with SAMtools Raw sequencing reads were converted from csfasta to csfastq files using solid2fastq  and aligned against a viral database consisting of 3,906 complete viral genomes (ftp://ftp.ncbi.nih.gov/pub/UniVec/) or a human database consisting of the hg19 genome plus RefSeq, Ensembl, and UCSC genes were removed using SeqClean  to enrich for informative reads.Low complexity reads, polysequences, and reads that aligned to either a vector database  to provide contigs that would align to viruses uniquely and with high significance. These contigs were aligned against the NCBI nucleotide (nt) sequence database using blastn (default parameters) et al. which calculates E-values that indicate which viruses and strains are likely present E-value and highest alignment score on a per sample basis were used to calculate a virus table for each sample. This table reported for each virus a total E-value that was calculated by multiplying the E-values of all unique matches for that virus . To aid in intraspecies differentiation, a total score for each virus was calculated by summing the blastn scores of unique matches with the scores of multiple matches  divided by the different number of viruses the hit aligns to The blastn matches were processed using the analysis script from Isakov ftp://ftp.ncbi.nih.gov/refseq/release/viral/) and counting the number of reads that overlapped each gene (intersectBed -c) After identifying viruses present in each sample, the viral reads were realigned using BWA to a reference consisting of only EBV, JCV, CMV, and HIV for visualization, variant calling, and expression estimation. For variant calling, duplicate reads were removed using Picard tools and pileup files were generated from reads with mapping quality greater than 30 using Samtools. Variants were considered significant if the total coverage of the site and the variant was greater than five and the variant allele was present at a frequency greater than 10%. Read coverage for EBV genes was calculated by intersecting the realigned reads with the NCBI viral gene annotation  under approval by the University of California San Diego Human Research Protections Program. The NSCLC patient provided written informed consent following UCSD Institutional Review Board approved protocols and the mouse experimental procedures were approved by the Pfizer Global Research and Development Institutional Animal Care and Use Committee as previously described This research was submitted to the University of California San Diego Human Research Protections Program and was found to be exempt since the tissue is post-mortem. The CLL patient was consented by the CLL Research Consortium . Samples were obtained from two 37-year-old non-Hispanic white patients, PCNSL2 and PCNSL4, and two Hispanic patients, PCNSL1 and PCNSL3, aged 37 and 41 respectively [SRA:SRP017364]. The four patients had clinical diagnoses consistent with heavy viral load including hepatitis, microglial nodule encephalitis, and progressive multifocal leukoencephalopathy in addition to PCNSL . An aver present . No viru present . Notablyet al.) for EBV varied between samples, reflecting differing numbers of reads mapping to the virus in each sample. The discovery of the EBER non-coding RNA’s (ncRNA) in all four PCNSL samples serves as a positive control for the viral discovery method used here since EBV is known to be associated with PCNSL and the EBER ncRNAs are known to be transcribed in large quantities (∼107 transcripts per cell) in EBV-transformed cells during latent infection We identified EBV expression in all four PCNSL samples by identifying reads that mapped to Epstein-Barr encoded RNA (EBER) 1 and EBER 2 . The totEBV is known to express 10 genes during lymphoproliferation and at least three genes during latent infection, including EBER 1, EBER 2, and latent membrane protein 2A (LMP2A) The relatively high read coverage across the EBV genome in PCNSL2 allowed us to examine the sequences for single nucleotide variants (SNV’s) relative to the EBV reference. We identified 37 SNVs, of which 24 were in protein coding genes and 13 were located in intergenic regions. There are a similar number of putative variants in the PCNSL sample from PCNSL1, but these variants were not deemed significant due to the lower read coverage across the EBV genome. There are two EBV types present in the NCBI viral database used here; the BLAST scores indicate that the viruses in each sample are more similar to EBV type 2 (NC_009334.1) because more contigs map uniquely to this reference sequence. This categorization may not be accurate, however, because EBV types 1 and 2 are normally distinguished by investigating the sequence of the EBNA-2 and EBNA-3 genes which was not possible here due to the lack of read coverage for these genes. While an individual can be infected with multiple EBV strains or both subtypes, PCNSL2 is likely infected with a single strain because the 37 variants observed have 100% alternate allele frequency We identified JC polyomavirus (JCV) in the sample from PCNSL3 by BLASTing contigs assembled from the sequencing reads against the viral database. Investigating the alignment of the reads to the viral database showed reads covering nearly the entire JCV genome  [45]. ThE<0.01) after Velvet assembly and BLASTing against the nucleotide database.The other three patients  were not diagnosed with PML, so we did not expect to find evidence of an active JCV infection. Interestingly, PCNSL1’s sample did have 18 unique reads that mapped to JCV and the samples from PCNSL4 and PCNSL2 had four and one reads that mapped to JCV, respectively. However, none of the samples besides that of PCNSL3 reached significance for JCV Click here for additional data file.


Next we examined mouth swabs and feces. We did not find a difference in the MyD88/TRIF−/− mice. However, we did observe a significant shift in the microbial composition in the feces and mouths of Rag1−/− mice. Thus, we conclude that the adaptive immune system modulates the microbial composition at mucosal surfaces in the steady-state but LC, adaptive immunity, and MyD88-dependent innate responses do not affect the skin microbiome revealing a major distinction between barrier sites.Barrier surfaces, such as the intestinal lining and the skin, are colonized by a diverse community of commensal microorganisms. Although commensal microorganisms clearly impact the host immune system, whether the immune system also shapes the commensal community is poorly understood. We used 16S rDNA deep sequencing to test whether mice with specific immune defects have an altered commensal microflora. Initially, skin swabs were obtained from wild-type and Langerhans Cell (LC) deficient mice. Despite the intimate contacts that LC make with the upper epidermis, no significant differences were observed in microbial community composition. Similarly, the skin of MyD88/TRIF Epithelial surfaces of the body are colonized by a complex and diverse microbiota that varies between individuals, between tissues site and even within a single individual Candidatus svagella, are common commensals of epithelial lining of the gut and are required for differentiation of T helper 17 (TH17) cells and also induce luminal secretion of IgA Bacteroidetes species strains induce IL-10 production and promote T-regulatory cell development Staphylococcus aureus is strongly associated with flares of atopic dermatitis Staphylococcus epidermidis enhances protection from cutaneous infection via increased expression of antimicrobial peptides In the intestines, it is now well documented that the presence of a microbiota as well as the presence/absence of specific commensal microorganisms influences development of the intestinal immune system. Segmented filamentous bacteria (SFB), stratum corneum, have an altered cutaneous microbiome −/− mice which have an intact intestinal microbiome, the ability of host immunity to shape the microbiome at barrier surfaces in the absence of barrier disruption has not been explored The microbiota can also be affected by the host. This has been best demonstrated during intestinal inflammation. Mice with either chemically induced colitis or genetic mutations, like NLRP6 or TRUC mice that produce colitis, have a greatly altered intestinal microbiome stratum corneumNumerous immune cells reside in the skin. Langerhans cells are antigen-presenting cells that reside in the epidermis where they are able to acquire antigen from the epidermis and −/−) and innate immunity (MyD88/TRIF−/−).To determine whether components of the immune system actively control the composition of commensal communities at barrier surfaces, we used 16S rDNA deep sequencing to determine the microbial community composition of the skin, mouth, and intestines in mice with isolated defects in intraepithelial dendritic cells (Langerhans cells), adaptive immunity (Rag1To test whether the absence of Langerhans Cells (LC) in the epidermis affects the composition of bacterial commensal communities on the skin, we obtained skin swabs from the ears of huLangerin-DTA mice DNA isolated from the swabs was PCR amplified using primers that span the V6 hypervariable region of the bacterial 16S ribosomal DNA (rDNA) gene. The amplicons were sequenced, screened and quality sorted and clustered into operational taxonomic units (OTUs) at a cutoff of 90%. The number of OTUs per sample ranged between 414 and 572. Taxonomy was assigned to OTU consensus sequences and the unweighted UniFrac distances were calculated between all sample pairs Firmicutes, Proteobacteria, Actinobacteria, and low amounts of Bacteroidetes were observed on the skin which is consistent with prior reports The Shannon diversity index, a measure of microbial diversity, was similar between the two groups . As expe−/− and control Rag1−/+ mice. Wild-type C57BL/6 mice (littermate controls from huLangerin-DTA matings) were bred with Rag1−/− mice. The resulting heterozygous F1 mice were backcrossed with Rag1−/− mice. Cohorts were generated using Rag1+/− and Rag1−/− littermates. As above, ear skin of these cohorts were swabbed for sequencing. As an additional control, ear skin swabs from the parental homozygous Rag1−/− used to establish the cohorts were also obtained.To determine whether skin resident T cells  or humoral responses have the capacity to alter the commensal communities of the skin, we compared the microbiome from ear swabs of Rag1−/− and Rag1+/− mice displayed similar phyla distribution and clustered together by PCoA with no significant differences   highlighting the importance of vertical transmission in determining commensal community structure. Thus, despite the ability to detect differences in commensal community between independently housed animals, we did not observe any alteration in the microbial community composition in Rag1−/− vs. Rag1+/− mice indicating that cells of the adaptive immune system do not affect the skin microbiome. Interestingly, the observed phyla in Rag1−/− and Rag1+/− mice were dominated by Proteobacteria and Bacteroidetes with fewer Firmicutes and Actinobacteria. This is distinct from the distribution of phyla seen in huLangerin-DTA cohorts and closely resembled the phyla seen in the parental Rag1−/− mice used to establish the cohort  . Shannon= 0.348) . Notablye cohort . Thus, t−/− mice with that of MyD88/TRIF+/− controls. We employed the same breeding strategy used for Rag1−/− mice to generate cohorts of MyD88/TRIF−/− mice and MyD88/TRIF+/− littermate controls. Microbiomes from MyD88/TRIF−/− and MyD88/TRIF+/− mice clustered together by PCoA and were not significantly different  (Bacteroidetes were somewhat increased compared with the Rag1−/− and huLangerin-DTA cohorts in this independently maintained mouse line with but was similar between MyD88/TRIF−/− and MyD88/TRIF+/− controls (To determine whether engagement of Toll-like receptors (TLR) and the inflammatory mediators IL-1, IL-18, IL-33 and IL-36α affect skin commensal microbial communities, we compared the skin microbiome of MyD88/TRIF= 0.348) . The div= 0.348) . The procontrols . Thus, t−/−, MyD88/TRIF−/−, and control mice used above. As expected, PCoA analysis of OTUs pooled from all mouse genotypes revealed strong clustering based on site and highly significant differences in microbiome composition were detected   . Bacteroin feces . As was in feces , the disin feces  and commin feces  were sim−/− vs. Rag1+/− mice and a significantly altered community composition   . This wa−/− mice . Concomiobserved .−/− and MyD88/TRIF+/− mice, ANOSIM analysis was not significant (p = 0.63), and in PCoA visualization OTUs samples from both genotypes clustered together   . The tax−/− mice . Thus, t−/−), and mice in which all cells are unable to respond to TLR-agonists as well as the pro-inflammatory cytokines IL-1, IL-18, IL-33, and IL-36α (MyD88/TRIF−/−). These data demonstrate that recognition of bacterial products via TLRs is not an obligate component determining the composition of the skin microbiome under steady-state conditions. The generation of active IL-1β and IL-18 resulting from inflammasome activation is also redundant. These data also demonstrate that local immune cells such as epidermal LC and dendritic epidermal T cells (DETC) as well as dermal T cell populations that could potentially sense the presence of commensal bacteria through other pathways are also redundant. The presence of skin commensal microorganisms regulates and enhances cutaneous immune responses Here we report that the overall composition of the microbiome on the skin of mice is not significantly affected by the loss of Langerhans cells, MyD88/TRIF signaling or the adaptive immune system. The diversity of commensal bacteria on the skin was similar in mice with a constitutive absence of epidermal Langerhans cells (huLangerin-DTA), mice that lack all B and T cells . All mice used for experiments were housed under specific pathogen-free conditions. The huLangerin-DTA mice were bred heterozygous and animals from the same litters and cages were used for experiments. To generate comparable Rag1Three types of samples were obtained over the course of the study. Skin and mouth samples were taken by vigorously swabbing the ear or oral cavity of the mouse, respectively. Swabs  were soaked in a buffer solution of sterile PBS 1% TritonX before use. After sampling, the swabs were digested with 25 U/µl achromopeptidase  for 30 minutes to lyse cells. Buffer soaked but unused swabs were also digested as controls. Stool samples were obtained via the collection of fecal pellets from the colon, from which DNA was extracted using a MOBIO PowerSoil DNA extraction kit , according to the manufacturer's instructions. Approximately 0.25 to 0.50 grams of fecal material was used as input material for extraction. All DNA samples were stored frozen at −20°C until amplified.The V6 hypervariable region of the bacterial 16S rDNA was amplified for sequencing in this study. In the initial portion of the study, focusing on huLangerin-DTA mice, a single set of V6 primers was used Three sequencing runs were performed for this study. For each sequencing run, equimolar aliquots of the purified amplicon library from each sample were pooled to give ∼1 µg of DNA in a 100 µl total volume. Final pooled DNA concentration was measured by Quant-IT PicoGreen quantitation . Amplicon size analysis was done using an Agilent DNA 1000 chip and a 2100 BioAnalyzer  to ensure no undesired products were in the pool. The pooled samples were then submitted frozen to the sequencing center for library preparation and sequencing on the Illumina HiSeq 2000 platform with 25% Phi-X added to increase sequence diversity . The first sequencing run (8 pooled samples) was conducted by the National Center for Genome Resources  and the second and third sequencing runs were conducted by the University of Minnesota Biomedical Genomics Center . The first sequencing run yielded in a total of 6.8 million reads with a post filter average of 357,666 reads per sample. The second run yielded in 47 million reads with a post filter average of 408,316 reads per sample and the third run yielded 108 million reads and a post filter average of 1.2 million reads per sample. Each sequencing run represented an independent set of experimental samples. Data from different runs were not compared to avoid confounding results with any potential variation between primer sets or sequencing facilities.Sequence data were processed and analyzed using MOTHUR Prior to analysis, a random subset of sequences were chosen from each sample to match the lowest number among all samples in order to balance sampling effort and ensure comparable diversity measures. In the first and second sequencing runs, 41,500 sequences were selected, and 100,000 were selected in the third. Sequences were then clustered into OTUs at a cutoff of 90% similarity to avoid overestimating diversity For beta diversity, similarities in community compostions among samples were calculated using the unweighted UniFrac distance metric on the Fast UniFrac website Sequence data are available in the NCBI Short Read Archive as BioProject PRJNA226001.Figure S1Commensal diversity varies by husbandry but not genotype. Shannon diversity indices of microbiomes from litter mate controls (LMC), huLangerin-DTA (DTA), Rag1+/−,Rag1−/−, MyD88/TRIF+/− and MyD88/TRIF−/− obtained from (a) ear skin swabs (b) fecal pellets and (c) oral swabs. No statistical differences between littermates within cohorts of individual experiments have been detected.(TIF)Click here for additional data file.Figure S2−/− mice.Family composition of feces and oral microbiome is altered in Rag1 Bacterial families showing the greatest difference between Rag1+/− (n = 9) and Rag1−/− (n = 8) mice is shown based on analysis of OTUs observed in the feces (a) or mouth (b). The percentage of total sequences from each sample belonging to the indicated family, significance and percent difference are shown.(TIFF)Click here for additional data file.


LRRTM1 rs6733871 and a number of polymorphisms in PCSK6 and different aspects of handedness assessed with the Edinburgh handedness inventory in a sample of unrelated healthy adults (n = 1113). An intronic 33bp variable-number tandem repeat (VNTR) polymorphism in PCSK6 (rs10523972) shows a significant association  with a handedness category comparison (P = 0.0005) and degree of handedness (P = 0.001). These results provide further evidence for the role of PCSK6 as candidate for involvement in the biological mechanisms that underlie the establishment of normal brain lateralization and thus handedness and support the assumption that the degree of handedness, instead the direction, may be the more appropriate indicator of cerebral organization.Although the left and right human cerebral hemispheres differ both functionally and anatomically, the mechanisms that underlie the establishment of these hemispheric specializations, as well as their physiological and behavioral implications, remain largely unknown. Since cerebral asymmetry is strongly correlated with handedness, and handedness is assumed to be influenced by a number of genetic and environmental factors, we performed an association study of  In addition to the haplotype specific markers, imprinting effects were also observed for the non-synonymous SNP rs6733871 that leads to an amino acid change (N330S) in LRRTM1. Furthermore, using the quantitative transmission disequilibrium test (QTDT), association between variation in LRRTM1 rs6733871 and handedness in a well-characterized dyslexia sample of German descent was observed Handedness is the most obvious manifestation of cerebral lateralization in humans, with more than 90% preferring to use their right hand PCSK6  which is known to have regulatory function during anterior CNS patterning and left-right axis formation PCSK6 SNPs, rs9806256 and rs11855415. RD individuals carrying the minor allele showed significantly greater relative right-hand skill as compared with the frequent wild-type allele. Replication in independent cohorts of individuals with RD revealed the same effect for the minor rs11855415 allele, while the same allele showed a significant trend towards reduced hand skill in the general population Recently, a genome-wide association study for a quantitative measure of relative handskill in individuals with reading disability (RD) revealed association with SNPs in LRRTM1 rs6733871 and a number of polymorphisms in PCSK6 and different aspects of handedness assessed with the Edinburgh handedness inventory in a sample of 1113 unrelated healthy Caucasian adults. The results show a significant association with an intronic 33bp variable-number tandem repeat (VNTR) polymorphism in PCSK6 (rs10523972) and handedness.Here we report an association study of LRRTM1 SNP rs6733871 and a number of SNPs in PCSK6 for association with different aspects of handedness  in response to 10 questions . All responses were converted to a laterality quotient (LQ) using the formula (R − L)/(R+L)×100. LQ scores thus ranged from −100 for strong left-handedness to +100 for strong right-handedness. Individuals were assigned to one of six categories of handedness: 1) consistent left , 2) inconsistent left , 3) ambidextrous with a tendency towards left , 4) ambidextrous with a tendency towards right ,5) inconsistent right , or 6) consistent right .By using data from 1113 unrelated individuals, we tested the ndedness . Each invs. inconsistent) and affiliation to one of the six categories of handedness for association with the LRRTM1 and PCSK6 one SNP rendered p-values below p<0.0025, the adjusted significance level for multiple comparisons  with each other. Rs10523972 is an intronic 33bp variable-number tandem repeat (VNTR) polymorphism in PCSK6 with eight different alleles (4–11 copies); of which 6 and 9 copies were most frequently observed (20.4% and 70.2%). Dichotomizing these alleles into short (≤6 repeats) and long (≥9 repeats) alleles reveals significant associations for the handedness category comparison  and the degree of handedness .When testing LQ, handedness direction , degree of handedness  than in individuals homozygous for the long allele (50.52%). The findings for individuals homozygous for the short allele should be interpreted carefully, since this group is very small  indicates a difference in the number of participants with consistent and non-consistent handedness. To further investigate this effect, we compared the distribution of consistent and non-consistent handedness for the different allele combinations against a uniform 50/50 distribution, resembling the overall distribution of consistent or inconsistent handed participants . This analysis revealed that heterozygous participants were more prone to inconsistent hand preference  report a significant association with the minor allele of rs11855415 and increased relative right-hand skill in individuals with RD. Yet, the minor allele showed a significant trend towards reduced laterality of hand skill in the general population PCSK6 with handedness. No supportive evidence was obtained for a possible association of the non-synonymous LRTTM1 SNP rs6733871. However, neither the three-marker haplotype nor parental effects were analyzed. The finding that tandem repeat variation in PCSK6 (rs10523972) shows a significant association with the degree of handedness could be explained by altered physiological and biological changes associated with this polymorphism. For example, VNTRs in regulating sequences can affect gene expression by altering the number of transcription factor binding sites or introducing changes in spacing between critical promoter elements http://genome.ucsc.edu/), the region containing the PCSK6 VNTR sequence clusters multiple transcription factor binding sites and is enriched for histone H3 lysine 27 acetylation (H3K27ac), a reliable chromatin marker of active , PCSK6 plays an important role in the pathway for left-right asymmetry and has also been described to be a direct target of the transcription factor FOXP2, whose gene was the first one known to be involved in the development of speech and language Our data show a statistical trend supporting earlier findings for an association of f active  [20], [2LRRTM1 might be explained by different study designs, including the participant populations and definition of handedness. Individual differences in handedness were often reduced on the direction of hand preference, left versus right-handers. Yet, evidence has accumulated that the degree (consistent versus inconsistent) of hand preference probably reflects a more critical dimension on which the handedness groups differ The lack of replication in our sample of the reported association with PCSK6 variation and the degree of handedness in the general population. Together with previously published data on a PCSK6 association and reduced variability in relative hand skill a healthy cohort PCSK6s likely role as candidate for involvement in the biological mechanisms that underlie the establishment of normal brain lateralization and thus handedness. However, independent replication in larger cohorts is needed in order to further validate the present results.In summary, the results of our study provide evidence for an association of i.e. how consistently a person prefers to use one versus the other hand over a wide variety of tasks may be the more appropriate indicator of cerebral organization. Future association studies should therefore take into consideration the different measures to define handedness. Understanding the complex genetic contribution to handedness could be crucial in order to advance our knowledge of normal brain functioning, but also the understanding of neurodevelopmental and psychiatric diseases.Furthermore, our data support the assumption that the degree of handedness, The study was approved by the ethics committee, Ruhr-University Bochum. All participants gave written informed consent and were treated in accordance with the declaration of Helsinki.In total, 1113 genetically unrelated, healthy adult participants of Caucasian descent for at least two generations participated in the present study (655 women and 458 men). The participants had a mean age of 32.97 (SEM = 0.55) and none of them had a history of any neurological or psychiatric diseases. 895 of the participants were young adults (mostly university students), that were recruited for the present study, while the remaining 218 participants came from a sample of healthy elderly volunteers. All participants were native German speakers. No participants are included in the cohort that had been forced to write with the right hand in school, although they actually would have preferred to use the left.Handedness was assessed using the Edinburgh handedness inventory LRRTM1 rs6733871, PCSK6 rs11855415, rs9806256) or from the UCSC genome browser, with particular attention to SNPs in conserved sequences (rs1947942) or SNPs that disrupt putative transcription factor binding sites (rs10523972). Genotyping of four SNPs  was conducted by polymerase chain reaction (PCR) and differential enzymatic analysis with the PCR restriction fragment length polymorphism method. Genotyping of the PCSK6 VNTR, rs10523972, was performed on the Beckman Coulter CEQ8000 8-capillary system using ’Fragment Analysis Module’ software . The genotypes were confirmed by sequence analysis. Oligonucleotides were designed using Primer Express 2.0 Software (Applied Biosystems). Further details of methodology and primer sequences are available upon request.For non-invasive sampling, exfoliated cells were brushed from the oral mucosa of the participants. DNA isolation was performed with QIAamp DNA mini Kit (50) . SNPs were chosen for genotyping by selecting SNPs from the literature  = 0.0025 (Bonferroni correction), a highly conservative significance threshold. All statistics were calculated using IBM SPSS Statistics 20.The statistical analyses were performed assuming a co-dominant effect for each polymorphism. Thus, all three genotypes for each polymorphism were analyzed separately. Since the LQ is an interval-scaled variable, it was analyzed parametrically using univariate analyses of variance (ANOVAs). Greenhouse-Geisser correction was applied when appropriate. Nominal-scaled variables  were analyzed non-parametrically using the Kruskal-Wallis Test. In order to account for multiple comparisons, an effect was considered significant if the Figure S1Plot of z-standardized LQ score  distribution (y-axis) for each genotype of rs11855415 (x-axis) in 1113 genetically unrelated, healthy adult participants of Caucasian descent.(JPG)Click here for additional data file.Figure S2Plot of z-standardized LQ score  distribution (y-axis) for the dichotomized genotypes of rs10523972 (x-axis) in 1113 genetically unrelated, healthy adult participants of Caucasian descent.(JPG)Click here for additional data file.Figure S3Plot of the absolute value of the LQ score (y-axis) for each genotype of rs11855415 (x-axis) in 1113 genetically unrelated, healthy adult participants of Caucasian descent.(JPG)Click here for additional data file.Figure S4Plot of the absolute value of the LQ score (y-axis) for the dichotomized genotypes of rs10523972 (x-axis) in 1113 genetically unrelated, healthy adult participants of Caucasian descent.(JPG)Click here for additional data file.


Obesity is a major cause of morbidity and mortality resulting in pathologic changes in virtually every organ system. Although the cardiovascular system has been a focus of intense study, the effects of obesity on the lymphatic system remain essentially unknown. The purpose of this study was to identify the pathologic consequences of diet induced obesity (DIO) on the lymphatic system.Adult male wild-type or RAG C57B6-6J mice were fed a high fat (60%) or normal chow diet for 8–10 weeks followed by analysis of lymphatic transport capacity. In addition, we assessed migration of dendritic cells (DCs) to local lymph nodes, lymph node architecture, and lymph node cellular make up.High fat diet resulted in obesity in both wild-type and RAG mice and significantly impaired lymphatic fluid transport and lymph node uptake; interestingly, obese wild-type but not obese RAG mice had significantly impaired migration of DCs to the peripheral lymph nodes. Obesity also resulted in significant changes in the macro and microscopic anatomy of lymph nodes as reflected by a marked decrease in size of inguinal lymph nodes (3.4-fold), decreased number of lymph node lymphatics (1.6-fold), loss of follicular pattern of B cells, and dysregulation of CCL21 expression gradients. Finally, obesity resulted in a significant decrease in the number of lymph node T cells and increased number of B cells and macrophages.Obesity has significant negative effects on lymphatic transport, DC cell migration, and lymph node architecture. Loss of T and B cell inflammatory reactions does not protect from impaired lymphatic fluid transport but preserves DC migration capacity. Future studies are needed to determine how the interplay between diet, obesity, and the lymphatic system modulate systemic complications of obesity. Obesity results in pathologic changes in virtually every organ system and is a leading cause of morbidity and mortality. It is estimated that nearly 1 in 4 Americans are obese with the incidence rapidly rising in adults and pediatric populations The hypothesis that obesity or dietary changes can cause pathological changes in the lymphatic system is supported by several studies. For example, recent reports have shown that obese patients have significantly impaired tissue clearance of macromolecules The purpose of this study was to identify the pathologic consequences of diet induced obesity (DIO) on the lymphatic system. We show that C57B6-6J mice fed a high fat diet not only become obese over a relatively short period of time, but that these mice also have markedly decreased lymphatic fluid flow and impaired migration of dendritic cells (DCs) to draining lymph nodes. In addition, obese mice have significantly smaller peripheral lymph nodes with loss of normal T and B cell architecture, and loss of gradients of lymph node CCL21 expression. Because previous studies have shown that metabolic syndrome secondary to obesity is dependent on T and B cell inflammation ad libitum for 8–10 weeks. Age-matched control male wild-type or RAG mice were maintained on a normal chow diet  for the same period of time. At the conclusion of the experiment, animals were weighed using a digital scale , and a 12-hour fasting blood draw was performed from the retro-orbital sinus. Serum glucose, cholesterol, and triglyceride levels were analyzed using standard assays .All experimental protocols were reviewed and approved by the IACUC committee at Memorial Sloan-Kettering Cancer Center. Male C57BL/6J and RAG mice based on the same genetic background (B6.129S7-Rag1tm1Mom/J) were purchased from Jackson Laboratories  and maintained in a temperature and light controlled environment. Previous studies have shown that a diet high in fat  in these animals reliably results in moderate obesity as compared to animals fed a normal chow or low fat diet and that this model is a useful analog to diet induced (rather than genetic) obesity in humans We used multiple methods to analyze lymphatic transport in order to enable us to visualize dermal lymphatic vessels and to analyze lymph node transport. Microlymphangiography was performed to visualize lymphatic vessels and to analyze the rate of lymphatic fluid transport using our previously published methods To confirm our analysis with microlymphangiography and to better analyze three-dimensional structural changes in the lymphatic system, we used a technique recently described by our group using Positron Emission Tomography (PET) and Computed Tomography (CT) imaging 99m (Tc99) labeled sulfur colloid was injected in the distal hind limb  and subsequent popliteal lymph node uptake was assessed using an X-SPECT camera  for 90 minutes. Region-of-interest analysis was performed to derive decay-adjusted activity using ASIPro software . All experiments were performed in a minimum of 5 animals per group.We have previously shown that lymphoscintigraphy can be used to accurately analyze lymph node uptake after peripheral injection and is therefore a means of quantifying lymphatic transport to the local lymph node drainage basin. We therefore used a modification of this technique to analyze popliteal lymph node uptake in DIO or control mice Back sections were harvested from obese mice and control mice utilizing 5 mm punch biopsy kits . Specimens were briefly fixed in 4% paraformaldehyde , placed in 70% alcohol and subsequently paraffin-embedded. Tail sections were harvested for cross-sectional cuts and longitudinal cuts, briefly fixed in PFA and subjected to EDTA (Sigma-Aldrich) for 72 hours for decalcification, after which point sections were placed in 70% alcohol and subsequently paraffin-embedded. We performed immunohistochemistry on tissue sections to localize the expression of CD45 (Rat anti-mouse CD45 mAb (Clone 30-F11), R&D Systems, Minneapolis, MN), LYVE-1 , and Podoplanin  utilizing a DAB-based development system . Additionally, to co-localize CD45 and LYVE-1 expression, tissue sections were stained for CD45 utilizing a DAB-based development followed by florescent LYVE-1 staining. Images were then overlaid to localize inflammatory cell populations in relation to lymphatic vessels.2) as previously described Inguinal lymph nodes were harvested and briefly fixed in 4% PFA and embedded in Optimal Cutting Temperature compound . Tissue sections were then prepared at a thickness of 10 microns using a cryostat  and mounted on Fisher Plus Slides . Immunofluorescent staining of lymph nodes was performed using our previous methods +, CD3+, CD4+), cytotoxic T cells , B cells , and macrophages . We blocked endogenous Fc receptor binding using Fc block (CD16/CD32) and optimized cytometer settings using splenocytes. Flow cytometry was performed using an LSR II flow cytometer with BD FACSDiva software and data was analyzed using FlowJo Software . We used 5–7 animals per group for this analysis.In order to determine how DIO regulates the populations of cells in lymph nodes, we performed flow cytometric analysis on inguinal lymph nodes. Single cell suspensions were prepared from inguinal lymph nodes and analyzed using fluorophore-conjugated flow cytometry optimized mouse monoclonal antibodies  to identify T-helper cells  with green pixels (LYVE-1) was calculated using Metamorph™.Ear whole mounts were performed as described by Nitschke et al in order to determine how DIO regulates migration of dendritic cells (DCs) from the periphery to draining lymph node basins + cells  according to the manufacturer's recommendations. Isolated cells were resuspended in PBS and injected into the right hindpaw (106 cells per injection) of experimental or control animals, and 36 hours later the right popliteal lymph nodes were harvested and analyzed to determine the number of migrating DCs (GFP+/CD11c+/MHC-IIhigh) using flow cytometry  and Flowjo software . Each experiment was repeated in 4–5 animals per group.We used a modification of the methods outlined by Lim +/CD11c+/MHC-IIhigh) by flow cytometry . The experiment was repeated in 4 animals per group.We used a well-described FITC painting assay in order to analyze baseline rates of DC migration in wild-type and RAG mice The Student’s T-test was used to compare differences between 2 groups while comparison of multiple groups was performed using analysis of variance with post hoc tests (Tukey Kramer) to compare differences between individual groups. Pearson’s coefficient was used to determine the correlation between groups. Data are presented as mean ± standard deviation unless otherwise noted with p<0.05 considered significant.As expected, feeding male C57BL/6J mice a high fat diet for 8–10 weeks resulted in a modest (33%), though significant increase in body weight as compared to control mice .We next used microlymphangiography in order to visualize dermal lymphatic vessels and analyze lymphatic fluid flow. Interestingly, we found that lymphatic transport of FITC-dextran labeled colloid to the proximal portion of the tail in obese mice was not only markedly delayed , but also of lower intensity as compared to controls suggesting that both the uptake and transport of interstitial fluid in obese mice is decreased relative to controls . In addition, similar to wild-type mice, obese RAG mice had modest, though significant increases in total serum cholesterol as compared to controls (p = 0.02); however, triglycerides and glucose were relatively unchanged .In line with previous reports, RAG mice fed a HFD (obese RAG) weighed on average 15 g more than control mice fed a normal chow diet .Interestingly, these studies demonstrated baseline differences between wild-type and RAG mice in Tc+ mice, enriched this population for dendritic cells (DC) using magnetic selection , and injected them into the hind paw of obese or lean wild-type and RAG mice. Analysis of popliteal lymph nodes for GFP+ DCs (i.e. MCHIIhigh/CD11chigh/GFP+) 36 hours after injection demonstrated that obese wild-type mice had markedly decreased DC migration to the lymph node from the injection site as compared with their lean controls  suggesting that higher baseline lymphatic transport rates of RAG mice is likely not the mechanism by which DC migration potential is preserved in obese RAG mice.We next used FITC skin painting to determine if the higher baseline rates of lymph node TCTaken together, the findings in this portion of our study suggest that T and B cell inflammatory reactions in obese animals are not necessary for impaired lymph node interstitial fluid uptake but that these immune responses do have a negative effect on migration of DCs to the lymph node.; p<0.0001). In contrast, inguinal lymph nodes harvested from obese RAG mice were nearly 1.7 fold (p = 0.009) larger than their respective controls although the baseline size of the lymph nodes in the lean control RAG mice was significantly smaller than lean control wild-type mice.Given that we found that obese mice had diminished lymphatic flow and DC migration to regional lymph nodes, we next sought to determine if these effects translate to structural changes in lymph nodes. Consistent with previous reports on mesenteric lymph nodes in obese mice, + lymphatic vessels (1.6 fold) in their lymph nodes . In contrast, obese RAG mice had similar numbers of lymph node lymphatic vessels as compared to RAG mice fed a NCD. Also consistent with lymphatic dysfunction, we found that obese wild-type mice had mildly dilated (2.3 fold) lymph node lymphatics as compared to controls  in mice results in significant impairment of the lymphatic system as reflected by decreased lymphatic flow, changes in lymph node architecture, and impaired dendritic cell migration. We chose the DIO model rather than genetic models of obesity (e.g. db/db or ob/ob mice) since the cause of obesity in the vast majority of patients is dietary excess rather than genetic abnormalities. Therefore, the DIO model will be useful in future studies since altering dietary fats or pharmacologic interventions will enable us to determine if the negative consequences of obesity on the lymphatic system are reversible. In addition, the DIO model is valuable for our analysis since changes in serum glucose levels in these mice, in contrast to genetic models of obesity, are mild Using a variety of techniques we found that DIO results in impaired lymphatic transport in dermal lymphatics and drainage to the regional lymph nodes. We specifically used multiple imaging modalities (some of which utilize radioactive tracers) to minimize visualization artifacts and confirm that our findings were not secondary to changes in the tissue thickness or conductivity due to obesity. For example, we used not only microlymphangiography, but also technetium 99 lymphoscintigraphy and FDG-PET imaging. Importantly, it is highly unlikely that the depth of penetration would be altered in radioactive tracers. In addition, the fact that all these different modalities had very similar patterns of lymphatic transport and were confirmatory of our findings with microlymphaniography was highly encouraging and suggests strongly that the changes we noted were not merely an artifact but rather a biologic phenomenon.et al reported decreased lymphatic washout from subcutaneous adipose depot of obese patients et al recently reported that massively obese patients have impaired lymphatic drainage by lymphoscintigraphy and spontaneous onset of lymphedema −/− mice) demonstrating that high fat diets in these animals results in similar acquired defects of the lymphatic system −/− mice based on the same background (C57B6). Thus, while Lim, et al reported total serum cholesterol levels ranging between 1000–2000 mg/dl in APO E−/− mice fed a normal chow or high fat diet, respectively, they only found significant decreases in lymphatic conductance in animals fed a high fat diet (i.e. cholesterol of 2000mg/dl) suggesting that the effect of serum cholesterol on lymphatic function is dose dependent. In our study, mice fed a high fat diet had a total serum cholesterol level of 200mg/dl (10 fold lower than APO E−/− mice) thus making it unlikely that deficits in lymphatic drainage were a consequence of mildly increased serum cholesterol. This concept suggests that pharmacological interventions designed to decrease serum cholesterol may also be minimally helpful in obese patients who do not have genetic abnormalities in cholesterol metabolism.Our findings are supported by recent clinical studies in obese patients. For example, in a study comparing obese and lean patients, Arngrim 99 is therefore important and suggests that lymphatic transport defects that develop in response to HFD induced obesity are independent of T cell or B cell inflammation. However, it is likely that the effects of obesity-induced T/B cell inflammation on the pathological responses of the lymphatic system are complex. This hypothesis is based on previous findings demonstrating that T cell inflammation is necessary for, and precedes macrophage inflammation, of visceral fat We used the DIO model in RAG mice to test the hypothesis that T and B cell inflammation resulting from obesity contributes to lymphatic dysfunction. This hypothesis was based on recent literature demonstrating that T cells have important anti-lymphangiogenic roles and can regulate lymphatic function. For example, Kataru et al demonstrated that T cell inflammation in lymph nodes and resultant expression of interferon gamma significantly inhibited inflammatory lymph node lymphangiogenesis et al who found that DIO results in decreased size of mesenteric lymph nodes and reductions in the number of T cells et al found that DIO also resulted in decreased number of B cells in the mesenteric lymph nodes and hypothesized that T/B cell apoptosis is induced in obesity possibly as a result of exposure to free fatty acids and resultant oxidative stress.An interesting finding of our study was that obese mice had significantly smaller lymph nodes, abnormal cellular distribution, decreased relative numbers of T cells, and increased relative numbers of B cells and macrophages as compared with controls. These findings are supported by those of Kim et al using K14-VEGF-R3-Ig mice et al reported markedly decreased lymph node size and abnormal architecture (B cells scattered among T cells rather than arranged in a follicular pattern) and suggested that lymphatic fluid flow to the lymph node has important regulatory effects on lymph node homeostasis. In addition, similar to our study, the authors reported that the expression patterns of CCL21 was abnormal in K14-VEGF-R3-Ig mice. Taken together, our findings and those of Thomas et al support the hypothesis that lymphatic flow to the lymph node is a critical regulator of lymph node architecture and morphology. This hypothesis is further supported by previous studies demonstrating that the expression of CCL21 by lymph node reticular cells is regulated by fluid shear and lymph node interstitial flow Our findings of decreased lymph node size and architecture are also supported by recent work reported by Thomas −/− mice fed a HFD, Lim et al also found similar impairments in DC trafficking et al defined HFD as chow containing 21% milk fat and NCD as 5% milk fat. This diet seemingly did not appear to induce significant subcutaneous fat deposition or obesity, although differences in weight were not noted. In addition, in our study we utilized a strain of C57B6 mice (6/J) that are more prone to diet induced obesity where as the subtype used by Lim et al was not reported (other than C57B6). Thus, some of the reported differences likely reflect strain differences as have been previously reported We found that obesity in wild-type animals markedly decreased dendritic cell migration to regional lymph nodes. Interestingly, using APO E99 lymphoscintigraphy demonstrated that the total lymph node uptake was markedly higher in RAG mice (both lean and obese) as compared to wild-type animals. This finding suggests that T and B cell function, both in normal and obese animals, may regulate lymphatic fluid transport and lymph node uptake. This hypothesis is supported by our finding that, in contrast to obese wild-type animals, lymph node lymphangiogenesis was not decreased in obese RAG mice. This hypothesis is also supported by recent mice studies demonstrating that T cells negatively regulate lymphangiogenesis during inflammation and wound repair Although obesity caused impairments in dendritic cell migration in obese wild-type mice, DC migration was relatively preserved in obese RAG mice. This preservation of immune function is likely not due to baseline differences in the rates of DC migration in wild-type or RAG mice since we found no differences using an FITC painting assay. However, analysis of lymphatic transport using TcThe finding that obese RAG mice have preserved DC migration capacity suggests that T and B cell inflammatory reactions secondary to obesity may play a role in this response, however, future experiments are needed to further dissect this observation.In conclusion, we have shown that diet induced obesity has significant negative effects on the lymphatic system regulating lymphatic transport, lymph node architecture, and dendritic cell migration. These changes may be in part due to T or B cell inflammation induced by obesity. These findings are important since the lymphatic system is an important regulator of both cardiovascular fluid transport and inflammatory responses suggesting that diet induced changes in the lymphatic system may modulate the pathology of obesity.File S1Contains: Figure A: Mean weights of control and obese RAG mice. Figure B: Mean 12 hour fasting serum cholesterol, triglyceride, and glucose concentration in control and obese RAG mice. Figure C: Representative flow cytometry demonstrating enrichment of DCs after magnetic bead selection. Figure D: Mean number of migrating DCs in lean wild-type and RAG mice 36 hours after FITC skin painting. Figure E: Representative low power  and higher power  photomicrographs of back punch tissue sections from control and obese mice stained for Podoplanin (brown). Boxes in right panel represent area magnified in high power views. Figure F: Mean number of Podoplanin+ lymphatic vessels/area in back punch biopsies of control and obese mice.(ZIP)Click here for additional data file.


Our results demonstrated that caprylic triglyceride alleviates ALS-type motor impairment through restoration of energy metabolism in SOD1-G93A ALS mice, especially during the overt stage of the disease. These data indicate the feasibility of using caprylic acid as an easily administered treatment with a high impact on the quality of life of ALS patients.Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder of motor neurons causing progressive muscle weakness, paralysis, and finally death. ALS patients suffer from asthenia and their progressive weakness negatively impacts quality of life, limiting their daily activities. They have impaired energy balance linked to lower activity of mitochondrial electron transport chain enzymes in ALS spinal cord, suggesting that improving mitochondrial function may present a therapeutic approach for ALS. When fed a ketogenic diet, the G93A ALS mouse shows a significant increase in serum ketones as well as a significantly slower progression of weakness and lower mortality rate. In this study, we treated SOD1-G93A mice with caprylic triglyceride, a medium chain triglyceride that is metabolized into ketone bodies and can serve as an alternate energy substrate for neuronal metabolism. Treatment with caprylic triglyceride attenuated progression of weakness and protected spinal cord motor neuron loss in SOD1-G93A transgenic animals, significantly improving their performance even though there was no significant benefit regarding the survival of the ALS transgenic animals. We found that caprylic triglyceride significantly promoted the mitochondrial oxygen consumption rate  Amyotrophic Lateral Sclerosis (ALS) is a relentlessly progressive neurodegenerative disease causing weakness in skeletal muscles of the limbs, respiration and swallowing. It is invariably fatal and is caused by the death of neurons responsible for voluntary muscle control in the spinal cord and brain in vitro and in vivo. In recent clinical trials, the mitochondrial modulator dexpramipexole Mitochondrial abnormalities in ALS can be seen in the spinal motor neurons of ALS patients Ketones (or ketonic bodies), consisting essentially of D-β-3 hydroxybutyrate (DBH) and acetoacetate are derived from fat catabolism in liver mitochondria and are inversely proportional to the levels of glucose in the blood Medium chain triglyceride diets were developed as a more palatable modification of the ketogenic diet In this study, we treated SOD1-G93A transgenic mice with caprylic triglyceride to test whether this medium chain triglyceride may alter the progression of the motor impairment through modulation of mitochondrial energy metabolism.All procedures were approved by the Mount Sinai Institutional Animal Care and Use Committee (IACUC).in vitro and in vivo studies. At 50 days of age, SOD1-G93A animals or their wild type littermates were placed on solid diets containing 10% (w/w) caprylic triglyceride  or a control isocaloric diet . Mice had access to food and water ad libitum. Body weight, food intake and motor function assessment were monitored weekly. The survival study endpoint was defined as meeting any one of the following conditions: no spontaneous breathing or movement for 60 seconds with no response to pain; the animal was unable to roll over to the normal position within 10 s following a push over; or complete hind limb paralysis.Male SOD1-G93A mutant transgenic mice (stock #002297) were obtained from the Jackson Laboratory . Only males were used because of the background and gender effects on survival in this mouse model of ALS Mice were tested on an accelerating rotarod  as previously described. In brief, mice are placed onto a grooved cylinder (facing away from the experimenter) rotating at a predetermined speed that incrementally increases to a maximal rotation at 300 s; the time maintained on the rod by each mouse (latency) is then recorded (300 s max). A diminishing latency indicates declining performance and at values of 0 s is suggestive of severe muscular weakness and impaired coordination. Mice were tested weekly, beginning at 60 days of age, until they could no longer perform the test. Before testing, mice underwent a 1 week training period wherein they were introduced to the apparatus and handled by the experimenter daily. Testing was conducted during the last 4 h of the day portion of the light cycle in an environment with minimal stimuli such as noise, movement, or changes in light or temperature.Mice were fasted overnight in clean cages with free access to water in new clean bottles. The next morning each mouse was weighed, and a baseline fasted blood glucose measurement was taken by applying tail blood to a Contour Blood Glucose Monitoring System (Bayer). Each mouse was injected intraperitoneally with a filter-sterilized solution of 20% (w/v) D-glucose, with the size of the bolus determined by animal weight (2 mg glucose/g body weight). Blood glucose measurements were taken as described above for each animal at 15, 30, 60 and 120 min. The data were plotted as blood glucose concentration (mg/dL) over time (minutes).Blood ketone level was obtained at pre-symptomatic stage (9 weeks) and post-symptomatic stage (17 weeks) by applying tail blood to a Precision Xtra® Blood Ketone Monitoring System (Abbott Laboratories) following the manufacturer’s instructions. Plasma corticosterone level was determined using Corticosterone ELISA (Enzo Life Sciences). Free glycerol and total triglyceride levels in the plasma were measured using Serum Triglyceride Determination Kit (Sigma Aldrich).Control and caprylic triglyceride treated mice were sacrificed at 110 days of age, when the motor deficits were apparent. The mice were anesthetized and perfused with phosphate buffered saline, followed by 4% para-formaldehyde. The lumbar spinal cord was dissected out and post-fixed in 4% para-formaldehyde. Spinal cords were embedded in paraffin and sectioned using a microtome to give 15 µm serial sections spaced 230 µm apart. The sections were stained with cresyl violet (Sigma Aldrich) using the following procedure: deparaffinization in Histo-Clear II (3×10 min), re-hydration in graded ethanol series 100% ethanol (5 min), 90% ethanol (3 min), 70% ethanol (3 min), 50% ethanol (3 min), rinse in tap water and then distilled water, staining in 0.1% Cresyl Violet (w/v) in 0.3% (v/v) glacial acetic acid (30 min at 50°C), rinse in distilled water, differentiation in series 50% ethanol (3 s), 70% acetic acid in ethanol (3 s), 95% ethanol (3 s), 100% ethanol (3 s), clearing in xylene (2 times), air drying (5 min) and mounting in Paramount media. Mosaic image acquisition covering the entire area of each section was performed under the 40×oil immersion objective of an Axioplan 2IE microscope with a motorized stage , using a Zeiss AxioCam MRc digital camera. The images from each section were stitched together using the Axiovision software. Neurons in the ventral horn were counted and were identified as large (>25 µm) cells having a pale nucleus with a distinct nucleolus and dark Nissl bodies in the cytoplasm SOD1 G93A mice on control and caprylic triglyceride diet were sacrificed by carbon dioxide inhalation at 110 days of age (post-symptomatic stage). The spinal cords were dissected out and immediately processed for isolation of mitochondria. Mitochondria were prepared by differential centrifugation and mitochondrial respiratory function was measured using the Seahorse XF24 Extracellular Flux Analyzer  as previously published Caprylic triglyceride did not result in any change in body weight  or motorImportant behavioral and physiological characteristics of SOD1-G93A transgenic mice include impaired motor performance, weight loss and reduced survival. SOD1-G93A mice fed caprylic acid showed no significant difference in weight as compared to those on control diet . There wWe also monitored the survival of the experimental animals . SOD1-G9Impaired glucose tolerance has been reported in ALS patients Caprylic triglyceride is metabolized into ketone bodies in the liver. To determine whether caprylic triglyceride treatment could result in increased concentrations of ketone in the SOD1-G93A animals, we measured the blood ketone level in mice fed caprylic triglyceride or control isocaloric diet. The caprylic triglyceride fed mice showed about a 2.5 fold increase in the blood concentration of circulating ketones compared to animals on control diet  at post-symptomatic stage . We alsoTo determine whether caprylic triglyceride can protect against the motor neuron loss that accompanies the clinical symptoms of ALS, we counted the number of motor neurons in the lumbar spinal cord in mice on the caprylic triglyceride diet compared to those on control isocaloric diet at the post-symptomatic stage (day 110) . There wTo evaluate the effect of caprylic triglyceride in the mitochondrial bioenergetic profile, we isolated the spinal cord mitochondria of WT and SOD1-G93A animals fed 10% caprylic triglyceride or control isocaloric diet at post-symptomatic stage (day 110) and measured oxygen consumption rate using the Seahorse XF24 extracellular flux analyzer. WT mice on control and caprylic triglyceride diets had comparable OCRs . We founOur study demonstrated that SOD1-G93A transgenic animals fed caprylic triglyceride showed significant improvement in the clinical signs of ALS and in motor neuron survival in the spinal cord. The improvement of motor performance in caprylic triglyceride-treated animals  was accoIt has been reported that ALS patients with elevated triglyceride and cholesterol serum levels have a prolonged survival Caprylic triglyceride can be metabolized into ketone bodies, and serve as an alternate energy substrate for neuronal metabolism. Our results showed increased basal and maximal mitochondrial oxygen consumption rates in the spinal cord together with elevated ketone levels in the blood of caprylic triglyceride-treated ALS transgenic animals, suggesting that the additional ketones provided by caprylic triglyceride might be used as an alternate substrate for energy metabolism in ALS animals. The increased maximal respiration  may reprIt has been reported that caprylic triglyceride, when used as a medical food, attenuates cognitive dysfunction in Alzheimer’s disease


The ability of invasive species to adapt to novel conditions depends on population size and environmental mismatch, but also on genetic variation. Away from their native range, invasive species confronted with novel selective pressures may display different levels of neutral versus functional genetic variation. However, the majority of invasion studies have only examined genetic variation at neutral markers, which may reveal little about how invaders adapt to novel environments. Salmonids are good model systems to examine adaptation to novel pressures because they have been translocated all over the world and represent major threats to freshwater biodiversity in the Southern Hemisphere, where they have become invasive. We examined patterns of genetic differentiation at seven putatively neutral (microsatellites) loci and one immune-related major histocompatibility complex (MHC class II-β) locus among introduced rainbow trout living in captivity (farmed) or under natural conditions  in Chilean Patagonia. A significant positive association was found between differentiation at neutral and functional markers, highlighting the role of neutral evolutionary forces in shaping genetic variation at immune-related genes in salmonids. However, functional (MHC) genetic diversity (but not microsatellite diversity) decreased with time spent in the wild since introduction, suggesting that there was selection against alleles associated with captive rearing of donor populations that do not provide an advantage in the wild. Thus, although high genetic diversity may initially enhance fitness in translocated populations, it does not necessarily reflect invasion success, as adaptation to novel conditions may result in rapid loss of functional MHC diversity. Human-mediated global change is causing the local extinction of some species but also the spread of others, resulting in novel species interactions and changes in ecosystem functioning. However, we are still far from understanding why some introduced species become invasive or whether successful invaders are also the most capable of transforming ecosystems  is one of the most widely translocated invasive fishes in the world, having been introduced to 90 different countries and being now present in all continents except Antarctica  and at a functional, immune-related locus  among translocated rainbow in Chilean Patagonia. Along with sexual selection, pathogen-driven natural selection is involved in maintaining MHC diversity in vertebrates (Piertney and Oliver n = 85), (b) recent escapees (n = 33), and (c) hybrids between escapees and naturalized trout (n = 92) as described in Consuegra et al.  fish caught by single-pass electrofishing at 10 different rivers  and 94 farmed fish collected at four nearby fish farms in the Los Lagos Region of Chilean Patagonia during 2007–2009 , 1× buffer, and 2 mmol/L MgCl2. Thermal conditions consisted of 5 min initial denaturation cycle (94°C) followed by 35 cycles of 1 min at 94°C, 1 min at 57°C, 1 min at 72°C, and a final extension cycle of 10 min at 72°C. Amplified fragments were directly sequenced using the same PCR primers and resolved in a 3130 automated sequencer . The resulting sequences were aligned using BioEdit 7.0.5.3  fragment of the exon 2 of the MHC class II-β gene, including part of the peptide-binding region (PBR), was amplified by polymerase chain reaction (PCR) using the forward primer B1RA  was calculated for the 14 study populations (10 free living and four captive) using the individual rarefaction method in PAST , as well as departures from Hardy–Weinberg equilibrium, were assessed using Arlequin 3.5. We employed analysis of molecular variance  to explore how MHC variation compared between free-living and captive populations. The relationship between population diversity at MHC and neutral markers was investigated by examining the magnitude and significance of the Pearson correlation coefficient at AR and Ho and the extent of genetic admixture was calculated using Pielou's J' evenness index  at microsatellite and MHC markers grouping the trout according to the time spent from introduction . AR  was estimated in PAST using an adjusted sample size of 30 individuals/group. Differences in allelic diversity between pairs of groups were determined by resampling 1000 times with replacement a population with the same size as the smallest of the pair  to compare patterns of change in genetic diversity (AR and Ho) between both types of markers in relation to time spent as free living in the wild. To determine which MHC class II-β alleles were contributing most to group separation we used the SIMPER permutation approach . AR and observed heterozygosity (Ho) were not statistically different between captive and free-living trout . In addition, MHC AR was positively correlated with the extent of population admixture (J') in all cases , suggesting that populations with higher neutral diversity and multiple genetic origins were also more diverse at the MHC class II locus.A total of 37 MHC class II-β alleles were identified, 12 of which represent novel sequences  based on MHC class II-β was 0.093 among all samples (P < 0.001). Differentiation was higher among trout living in captivity than among free-living populations , with pairwise FST values ranging from 0.004 to 0.215 (Genetic differentiation (to 0.215 . AMOVA aP = 0.030) and between naturalized and farmed trout (P = 0.001) but not between any of the other pairs (P > 0.9). MHC AR was lower in farmed fish than in escapees and then decreased with increasing time as free living . In contrast, no significant differences in AR  or heterozygosity  were detected among the four trout groups for neutral microsatellite loci, suggesting that diversity at these markers could be less affected by the time spent in the wild. ANCOVA analyses comparing the patterns of variation with time spent as free living between both markers revealed that the slopes between both markers were significantly different for AR (F = 168.5 P = 0.006) but not for Ho . All pairwise comparisons at microsatellite loci revealed significant genetic differentiation between the four groups of fish  and both the FEL and REL methods identified several sites to be under selection. FEL identified three positively selected and two negatively selected sites, whereas REL found 18 positively selected (three of them coincident with those identified by FEL) and two negatively selected sites. Of these, codon 47 was also identified as a potential recombination breakpoint.The Human-mediated species translocations are widely recognized as one of the main threats to global biodiversity  FST, can be problematic and must be interpreted with caution in invasive populations given that the assumption of equilibrium between mutation, drift, and gene flow may not be applicable , similar to those reported for natural populations at the species' native range  and population admixture suggests that populations founded from multiple origins not only display higher levels of neutral genetic diversity  among invasive rainbow trout with increasing time spent in the wild. We suggest that despite the potential effects of bottlenecks and genetic drift associated with invasion and the massive number of escapees and continuous introgression of domesticated trout (Consuegra et al.


Reduced left ventricular function in patients with severe symptomatic valvular aortic stenosis is associated with impaired clinical outcome in patients undergoing surgical aortic valve replacement (SAVR). Transcatheter Aortic Valve Implantation (TAVI) has been shown non-inferior to SAVR in high-risk patients with respect to mortality and may result in faster left ventricular recovery.We investigated clinical outcomes of high-risk patients with severe aortic stenosis undergoing medical treatment (n = 71) or TAVI (n = 256) stratified by left ventricular ejection fraction (LVEF) in a prospective single center registry.Twenty-five patients (35%) among the medical cohort were found to have an LVEF≤30% (mean 26.7±4.1%) and 37 patients (14%) among the TAVI patients (mean 25.2±4.4%). Estimated peri-interventional risk as assessed by logistic EuroSCORE was significantly higher in patients with severely impaired LVEF as compared to patients with LVEF>30% . In patients undergoing TAVI, there was no significant difference in the combined endpoint of death, myocardial infarction, major stroke, life-threatening bleeding, major access-site complications, valvular re-intervention, or renal failure at 30 days between the two groups . After TAVI, patients with LVEF≤30% experienced a rapid improvement in LVEF  associated with improved NYHA functional class at 30 days (decrease ≥1 NYHA class in 95%). During long-term follow-up no difference in survival was observed in patients undergoing TAVI irrespective of baseline LVEF (p = 0.29), whereas there was a significantly higher mortality in medically treated patients with severely reduced LVEF (log rank p = 0.001).TAVI in patients with severely reduced left ventricular function may be performed safely and is associated with rapid recovery of systolic left ventricular function and heart failure symptoms. Reduced left ventricular ejection fraction (LVEF) among patients with severe aortic stenosis importantly impacts prognosis in patients treated conservatively, and increases peri-operative risk in patients undergoing surgical aortic valve replacement (SAVR) 2; (2) age ≥80 years in the presence of a logistic EuroSCORE >15%. Patients <80 years of age were eligible if at least one of the following comorbid conditions was present: previous cardiac surgery, chronic obstructive pulmonary disease (forced expiratory volume during one second <1.0), severe pulmonary hypertension (≥60 mmHg), porcelain aorta, history of radiation therapy to the mediastinum, or frailty (BMI <18 kg/m2). Patients with severe aortic regurgitation due to degenerated aortic valve prosthesis were excluded.High-risk patients with symptomatic, severe aortic stenosis deemed at increased surgical risk have been consecutively included in a prospective single center registry initiated in July 2007. Inclusion criteria involved (1) symptomatic, severe aortic stenosis with an echocardiographic mean gradient >40 mmHg or a calculated aortic valve area <1 cmwww.kek-bern.ch). All subjects gave informed written consent.The study complies with the Declaration of Helsinki and was approved by the Ethics Committee of the University of Bern, Switzerland  and transesophageal echocardiography (TEE), and CT angiography of the chest, abdomen and pelvis an interdisciplinary team of interventional cardiologists and cardiac surgeons reviewed the cases and formed a consensus on treatment allocation to medical treatment (1), SAVR (2) or TAVI (3) based on risk assessment, anatomical considerations and patient preference Latter was performed through a tranfemoral, transapical, or transsubclavian approach according to anatomical characteristics using either the Medtronic CoreValve Revalving system or the Edwards Sapien valve as previously described Patients treated medically were included into this registry at the time of the in-hospital evaluation for a potential intervention, whereas the date of the intervention was considered the time of inclusion among patients undergoing TAVI. Follow-up was performed regularly at 1, 6, and 12 months during a clinic visit or by means of a standardized telephone interview. Furthermore, all patients were contacted within two months of data freezing for the purpose of the present analysis. Hospital records and municipal civil registries were consulted to ascertain vital status. Medical records, discharge letters, and documentations of hospitalizations were systematically collected and all suspected events were adjudicated by an unblinded clinical event committee consisting of cardiac surgeons and interventional cardiologists.th percentile upper reference limit (URL), or a peak value exceeding 5x the 99th percentile URL with new pathological Q waves in at least two contiguous leads) within 72 hours after the index procedure. Major stroke encompassed a rapid onset of focal or global neurological deficit of ≥24 hours duration necessitating therapeutic intervention, or documentation of a new intracranial defect using MRI or CT-scan. The modified RIFLE  classification was used for the definition of kidney injury which was based upon changes in serum creatinine up to 72 hours after the procedure. Stage 1 was determined as an increase of serum creatinine to 150–200% (or an increase of ≥26.4 µmol/l), stage 2 required an increase of the baseline creatinine to 200–300%, and stage 3 was considered in case of an increase in creatinine of ≥300% with an acute increase of at least 44 µmol/l.Assessment of left ventricular ejection fraction at baseline was based on measurements from TTE using planimetry. All endpoint definitions were in accordance with the criteria suggested by the Valve Academic Research Consortium All analyses were performed using SPSS Statistics Version 17.0. Continuous variables are presented as mean ± standard deviation (SD) and were compared by means of a two-sided students T-test. Categorical data are expressed as frequency (percentages), and were compared using the chi-square and Fishers exact tests. Survival was estimated using the Kaplan Meier method. We performed an univariate analysis and included in addition to age and gender all variables with a p<0.1 into a cox multivariate regression model to adjust for potential confounders. and Among 452 patients with severe aortic stenosis at increased surgical risk, 10 patients died before treatment allocation, and 107 patients underwent SAVR leaving 335 patients allocated to medical treatment or TAVI as basis of the present study. After exclusion of two patients with cross-over from medical treatment to SAVR, one patient with cross-over from TAVI to SAVR, and five patients with cross-over from medical treatment to TAVI, 71 patients treated medically and 256 patients treated by TAVI remained for the purpose of the present analysis. The patient population was divided into four groups according to treatment strategy  and left ventricular ejection fraction (LVEF≤30% versus LVEF>30%). Severely impaired LVEF (≤30%) was observed in 25 (35%) patients treated medically (M30-), whereas 46 (65%) patients treated medically had normal or only moderately reduced LVEF (M30+). A total of 37 (14%) TAVI patients had severely diminished LVEF (T30-), whereas 219 (86%) TAVI patients showed normal or moderately reduced LVEF (T30+) Figure 1Mean LVEF amounted to 56.2±9.7% (M30+) and 26.7±4.1% (M30-) in patients assigned to a medical strategy, and to 55.6±10.0% (T30+) and 25.2±4.4% (T30-) in patients undergoing TAVI. While mean gradients were lower in patients with LVEF≤30% among TAVI patients , the numerical difference in the medical cohort fell short of statistical significance . Among T30- the prevalence of patients with a mean gradient ≤30 mmHg amounted to 45.9%. Furthermore, LVEF ≤30% in patients undergoing TAVI went along with a higher rate of severe pulmonary hypertension . In contrast, patients with LVEF≤30% in the medical cohort had a higher prevalence of triple vessel coronary artery disease  Table 2. and Peri-procedural characteristics and short-term clinical outcome of patients undergoing TAVI are summarized in Exercise intolerance as assessed by NYHA functional class was documented at 30 days and is illustrated in Crude and adjusted long-term survival of patients undergoing TAVI and medical treatment as a function of LVEF is shown in Echocardiographic follow-up was available in 93% of patients at discharge and in 79% at a mean follow-up of 144±130 days. No echocardiographic follow-up was performed in patients allocated to medical treatment. Patients of the T30- group experienced recovery of LVEF from baseline to discharge  that continued to increase during mid-term follow-up (41±13%) Figure 5Severely diminished left ventricular function among patients with severe aortic stenosis treated medically has an important impact on clinical outcome with dismal prognosis.TAVI in patients with severely impaired left ventricular function may be performed safely and may not be associated with an increased peri-procedural risk.Patients with severely diminished LVEF undergoing TAVI demonstrate a rapid improvement in LV function and may have a similar prognosis as compared to patients with normal or moderately reduced LVEF.Among patients with severely diminished LVEF, TAVI disproportionally improves NYHA functional class as compared with patients with normal or moderately reduced LVEF.The observational data of the present single center experience demonstrate the favorable impact of TAVI on recovery of LVEF and long-term clinical outcome as compared with medical treatment. Moreover, the risk of peri-procedural complications in patients with severely impaired LVEF appears to be comparable to patients with normal or mildly reduced LVEF.There are several limitations to be considered. First, the observational, non-randomized nature of the present analysis is susceptible to a selection bias attributable to the primary allocation of the selected treatment strategy. The decision whether to perform TAVI or medical treatment in patients with severely impaired LVEF was driven by the decision of the interdisciplinary heart team, by the feasibility based on anatomic and technical features, and by the final decision of the patient. Second, the threshold values for categorization of the overall patient cohort according to LVEF, although arbitrary, were based on previous clinical studies of patients undergoing SAVR indicating an association of impaired LVEF with adverse clinical outcome Placement of AoRtic TraNscathetER Valve) trial Our data highlight the dismal prognosis of patients with severe, symptomatic aortic stenosis undergoing medical treatment. The overall estimated rate of mortality after one year amounted to 55% and is in line with the results of the medical group of the PARTNER B cohort  and were found to have a considerably higher mortality rate as compared to medically treated patients with LVEF>30%.Among patients with severely impaired LVEF symptom status as assessed by NYHA functional class improved disproportionally along with a rapid recovery in systolic left ventricular function. These findings suggest that patients with severely reduced LVEF may substantially benefit from TAVI.TAVI in patients with severely reduced left ventricular function may be performed safely and is associated with rapid recovery of systolic left ventricular function and heart failure symptoms.