De novo mutations are implicated in a variety of genetic diseases and arise primarily in the male germline. We investigated whether male germ cells have unique mechanisms for spontaneous or chemically-induced mutation relative to somatic cells using the MutaMouse model. We recovered lacZ transgenes from sperm 42 days after a 28-day exposure to benzo(a)pyrene  to assess mutations arising in dividing spermatogonia. BaP caused a 3.4-fold increase in lacZ mutant frequency over controls which increased to 4.1-fold after clonal correction. We then used next generation sequencing to compare the spontaneous and BaP-induced mutation spectra in sperm and bone marrow. The spontaneous spectrum in sperm had significantly more G:C to A:T transitions and fewer mutations at A:T basepairs than bone marrow. BaP predominantly induced G:C to T:A transversions in both cell types, and both were enriched for mutations at CpG dinucleotides. However, BaP induced significantly more deletions in sperm, but more G:C to A:T transitions and G:C to C:G transversions in bone marrow. Differences in error-prone translesion DNA synthesis polymerases may underlie the observed spectrum differences between sperm and bone marrow. These findings suggest that mutations in sperm can arise via mechanisms that are unique to male germ cells. However, short term increases in mutation rates can lead to unwanted increases in the incidence of genetic disease and cancer. Recent studies using next generation sequencing confirm that the male germline is the primary source of Elevated mutagenicity in the male germline is believed to be due to the continuous replication of spermatogonial stem cells36in vivo mutagenicity of chemicals121314cII transgene due to technical restrictions. However, when coupled with next generation sequencing (NGS) technologies, the TGR assay can be used to generate a high-resolution mutation spectrum of the larger lacZ transgene, providing coverage of a much greater number of mutants16lacZ analysis allows the detection of subtle differences in mutation spectra that may otherwise be missed when analyzing a smaller sample of mutants.The transgenic rodent (TGR) mutation assay is an internationally supported method for testing the a)pyrene (BaP) is a ubiquitous environmental pollutant and Group 1 human carcinogen12131918a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE). BPDE adducts occur predominantly at the N2 position of guanosine, resulting in an increased frequency of G:C to T:A transversions22231213192025Benzo. The lacZ mutant frequency (MF) in the BaP group (MF = 13.1 × 10−5) was 3.4-fold higher (p < 0.001) than in controls (MF = 3.9 × 10−5), as shown in The animals used in the present study are a subset from a previously reported dose-response study on the induction of lacZ mutant plaques and 200 titre plaques (assumed wildtype) derived from spermatozoa from the cauda epididymis in five control (578 mutant plaques) and six BaP-treated animals (1013 mutant plaques). Sequence data are available on the NCBI Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra) under the BioProject accession number PRJNA342798. Sequencing generated 80, 327, 390 reads with an average read-length of 129 bp resulting in a total of 9.2 × 109 high quality base calls (≥Q20). For the purpose of this study, sequencing depth is defined as the average number of base calls at any nucleotide position along lacZ. Alignment to the reference sequence resulted in an average sequencing depth of approximately 2700-fold (range = 1370-fold to 5124-fold) per identified copy of lacZ (i.e. each base in each mutant copy of lacZ that we identified was sequenced on average approximately 2700 times). A similar study found that a sequencing depth of 35-fold was sufficient for highly accurate mutation identificationlacZ MFs were then corrected for the varying degrees of clonality  with respect to the control group when compared using a Monte Carlo generated X2 distribution. The proportional spectra of mutation types for each treatment group are shown in The type, position, and frequency of independent mutations in the control and BaP groups are shown graphically along the To analyze the influence of the sequence context on mutation formation, we compared the proportion of mutations that occurred at G:C or A:T base-pairs in each treatment group, and checked whether the nucleotide position of each mutation occurred within or adjacent to a nucleotide homopolymer repeat or at areas with one or more CpG dinucleotides . These rlacZ gene were mutated in more than one animal , BaP-induced mutation spectra in sperm and bone marrow were corrected for differences in background mutation spectrum and for differences in the BaP-induced mutation frequency  from germ cells1315Using NGS we sequenced ~5-fold more mutants than the combined number of mutants sequenced from all previous studies that have recovered transgenes  of the spontaneous G:C transition mutations occurred at CpGs, which is not significantly different from chance . This is not consistent with cytosine deamination as the primary mechanism for spontaneous G:C transition mutations, given that all of the CpGs of the lacZ transgene are known to be heavily methylated in all tissues, including testes31lacI and cII transgenes of Big Blue rats and miceWe found that spontaneous mutations occurred almost entirely at G:C basepairs 96.2%, , which ilacZ gene are in a dipyrimidine context. These results suggest that the occurrence of dipyrimidines may be an important factor in the observed spontaneous mutation spectrum, especially for spontaneous G:C to A:T transitions.Other proposed mechanisms for the G:C to A:T spontaneous mutation bias include DNA damage from ROS29Spontaneous G:C to T:A transversions are likely associated with damage from ROS produced by aerobic metabolism3637The spectrum of spontaneous mutations in sperm was comparable to that observed in bone marrow . As in set al.cII genes from spermatozoa of BaP-treated mice. BaP also induces mutations via several other minor mechanisms. BPDE forms adducts at N6 of adenosine and N4 of cytosine, but at a lower frequency than with guanosine. BaP metabolism also results in the production of ROS species that can form various types of DNA damage, including 8-oxo-dGBaP’s mutagenic mode of action in germ cells is assumed to be similar to somatic tissues. BaP is metabolized by cytochrome P450s and epoxide hydrolase to form the predominant mutagenic metabolite BPDE, which forms DNA adducts on N2 of guanosine causing G:C to T:A transversions during replication394042The BaP-induced mutation spectrum in germ cells and bone marrow shared many characteristics . The pre45464550In conclusion, analysis of high-resolution mutational spectra by NGS showed that there is overlap in BaP’s mutagenic mode of action between germ cells and bone marrow, although some notable differences were observed. Differential distribution of BaP metabolites between tissuesAll protocols involving animal use were approved by the Health Canada Ottawa Animal Care Committee and animal manipulations were carried out in accordance with the guidelines and regulations of the Canadian Council on Animal Care. Experiments were conducted following the recommendations in OECD guideline TG488lacZ MF was determined in genomic DNA as previously describedE. Coli  were infected with the recovered viral vectors and grown on agar containing 0.3% phenyl-β-D-galactopyranoside  to detect mutants, or on agar without P-Gal to determine the total number of plaque-forming units (pfu). A minimum of 125,000 total pfu were scored for each animal. MF was calculated as the number of mutant plaques divided by the total pfu count. Statistical differences in MF between treatment groups were determined by logistic regression using the glm function in RGenomic DNA was isolated from spermatozoa from the cauda epididymis by digestion with proteinase K and β-mercaptoethanol, followed by phenol/chloroform extraction, and ethanol precipitation as described previouslylacZ gene. To control for PCR errors, each sample was amplified and sequenced in duplicate. The PCR products were purified using QIAquick PCR purification kit (Qiagen). Libraries were prepared from each purified PCR sample using Ion Xpress Plus kits and an AB Library Builder (Ion Xpress Plus Library Protocol v. 1.00) following the manufacturer’s directions with the following exceptions: we used a custom shearing time of 10 minutes and replaced the Agencourt AMPure XP Reagent with SPRIselect Reagent . The ends of each library were ligated with P1 adapters and a unique barcode A adapter from the Xpress barcode adapter kit. Fragments between ~175–225 bp were selected from each library using an E-Gel Safe Imager, purified using the Invitrogen PureLink Quick Gel Extraction kit, and amplified using the Library Amplification Primer Mix (Ion Plus Fragment Library Adapters kit) and High Fidelity Platinum PCR SuperMix. Barcoded libraries were pooled together to a final total concentration of 8 pM. Pooled libraries were amplified by emulsion PCR using Ion Sphere particles on an Ion OneTouch2 system. Particles containing PCR products were enriched using an Ion OneTouch ES. Enriched particles were loaded onto an Ion P1 chip (version 1) and sequenced.Mutant plaques recovered from spermatozoa from the cauda epididymis were collected for sequencing. Sequencing was performed using an Ion Proton sequencer  as described previouslylacZ sequence (GenBank ID: J01636.1) using Bowtie2Bioinformatic analysis of sequence data was conducted as described previouslyX2 distributionOnly the independent mutations were used to determine the mutation spectra for each treatment group. Significant differences in mutation spectra, when considering both nucleotide position and mutation type, were determined using a Monte Carlo generated How to cite this article: O’Brien, J. M. et al. Next generation sequencing of benzo(a)pyrene-induced lacZ mutants identifies a germ cell-specific mutation spectrum. Sci. Rep. 6, 36743; doi: 10.1038/srep36743 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Zebrafish spontaneously regenerate the retina after injury. Although the gene expression profile has been extensively studied in this species during regeneration, this does not reflect protein function. To further understand the regenerative process in the zebrafish, we compared the proteomic profile of the retina during injury and upon regeneration. Using two-dimensional difference gel electrophoresis (2D-DIGE) and label-free quantitative proteomics (quadrupole time of flight LC-MS/MS), we analysed the retina of adult longfin wildtype zebrafish at 0, 3 and 18 days after Ouabain injection. Gene ontology analysis indicates reduced metabolic processing, and increase in fibrin clot formation, with significant upregulation of fibrinogen gamma polypeptide, apolipoproteins A-Ib and A-II, galectin-1, and vitellogenin-6 during degeneration when compared to normal retina. In addition, cytoskeleton and membrane transport proteins were considerably altered during regeneration, with the highest fold upregulation observed for tubulin beta 2 A, histone H2B and brain type fatty acid binding protein. Key proteins identified in this study may play an important role in the regeneration of the zebrafish retina and investigations on the potential regulation of these proteins may lead to the design of protocols to promote endogenous regeneration of the mammalian retina following retinal degenerative disease. Fish and amphibians have the ability to completely regenerate a functional retina after injury and this capability continues throughout adult life134in vitro1112in vivo. Currently, most of our understanding of the retinal regenerative process in the zebrafish is based upon gene expression of isolated retina914The zebrafish has been increasingly used as a model to study complex tissue regeneration of the retina. Several models of retinal insult in this species including surgical removal, light damage and neurotoxin damage result in complete recovery of the functional retina78The aim of the current study was to identify protein expression changes in the zebrafish retina that may regulate the regenerative processes after injury in this species. We compared the protein expression profile of normal zebrafish retina with that of retina damaged by intravitreal injection of ouabain undergoing degeneration and during the process of regeneration. We conducted 2D Difference Gel Electrophoresis (2D-DIGE) and label free proteomic analysis on these samples. We have shown that metabolic processing is negatively regulated, and fibrin clot formation was upregulated during retinal degeneration in the zebrafish. In addition, apolipoproteins A-Ib and A-II and the carbohydrate-binding protein extracellular matrix factor galectin-1 were highly upregulated in the degenerated zebrafish retina as compared to normal retina. Similarly, in the regenerating retina we observed activation of cytoskeletal and extracellular matrix proteins during regeneration. More specifically, cytoskeletal proteins such as tubulin beta 2A are highly upregulated in the regenerating zebrafish retina as compared to control. The validation of galectin-1 expression was supported by RT-PCR and western blot.Factors identified are likely to be important for the regenerative process in the zebrafish retina and further investigations on the roles of these molecules in the mammalian retina may aid to design regenerative approaches to induce endogenous repair of the human retina.Examination of the zebrafish retina after injection of 200 μM ouabain showed evident morphological changes occurring after toxin-induced damage. Bright field images were taken from the same site of the peripheral retina of treated and control animals at various time intervals after injection . The thiAnalysis of the proteomic profile of zebrafish retina by label free mass spectrometry identified the presence of 2042 proteins in the control retina, 1514 proteins in the degenerated retina and 1445 proteins in the regenerating retina. Further examination of the proteins detected in the zebrafish retinae identified several proteins that were differentially expressed between control, degenerated and regenerating retina. Using the normalised abundancies, proteins considered as differentially expressed were either ≥2-fold upregulated or ≤0.5-fold downregulated between the different time points. In the degenerated retina, 192 proteins were found to be differentially expressed as compared to the control retina, with 21 being ≥2-fold upregulated and 171 being ≤0.5-fold downregulated . Fewer cIndividual protein analysis showed that only one protein, annexin A2A, was ≥2-fold upregulated in both the degenerated and regenerating zebrafish retina as compared to control. In the degenerated retina the glycoprotein vitellogenin 6 exhibited the highest fold upregulation when compared to control retina (32.12-fold increase) . This waApproximately 135 spots were detected in the 2D-DIGE gel containing samples of control, degenerated and regenerating zebrafish retinae . CompariProteins that were differentially expressed between degenerated and control zebrafish retina by 2D-DIGE and label free proteomics were subject to gene ontology (GO) enrichment and pathway analysis to identify key functional areas of interest. Differentially expressed proteins between regenerating retina (18 dpi) and control retina obtained from the label free mass spectrometry were also subject to gene ontology and pathway enrichment. Out of the 55 proteins in this dataset, 16 could not be mapped by the enrichment. It was shown that proteins involved in the structural constituent of the cytoskeleton were the most significantly enriched within this group. These proteins included tubulin alpha 1B chain and an uncharacterised tubulin (Q6PE34), which were ≤0.5-fold downregulated and tubulin beta 2B chain, tubulin beta 5, and vimentin, which were ≥2-fold upregulated in the regenerating retina as compared to control retina. Proteins involved in GTPase activity and transporter activity were also significantly enriched . InteresTo validate our MS proteomic results, we conducted PCR and western blot analyses to examine the mRNA and protein expression. We selected three of the highly upregulated proteins to which antibodies were readily available for the zebrafish, including adenylate kinase isoenzyme1, tubulin beta 2 and galectin 1. Analysis of mRNA for galectin 1 correlated with proteomic results, and showed a significant increase in expression in the degenerated retina (3 dpi) as compared to normal retina (P = 0.0461). In contrast, mRNA levels of tubulin beta 2 did not correlate to the proteomics results and showed a significant decrease in the degenerated retina as compared to control (P = 0.0270) that returned to normal levels in the regenerating retina. A significant reduction in mRNA expression of adenylate kinase isoenzyme was observed in the degenerated retina as compared to control, and regenerating retina (P < 0.0020) . SimilarThis study has applied a proteomics approach to investigate molecular events occurring during degeneration and regeneration in the zebrafish retina. We observed that upon degeneration proteins involved in fibrin deposition and cytoskeletal proteins were highly upregulated, whereas energy metabolism in the retina is mostly negatively regulated. Similarly in the regenerating retina, cytoskeletal proteins such as tubulin beta 2 were highly upregulated and proteins involved in transporter activity were downregulated. Validations of galectin1 by western blot and RT-PCR correlated with results. However mRNA and protein expression of tubulin beta 2 and adenylate kinase largely conflicted with proteomic findings. The differences for beta tubulin 2 expression can be accounted for as fragments of this particular protein was identified across seven fractions and then averaged, as generated by Progenesis software. In comparison, galectin1 and adenylate kinase was identified in only one out of the ten fractions analysed. It is also well documented that mRNA and protein expression levels may differ in the same systemZebrafish retinae are small in size (1 mm × 1 mm), hence samples used in this study were pooled to obtain a single representative sample. Creating a sample pool can also reduce the sample bias and biological variance, leading to more precise data estimatesAnalyses of zebrafish retina by 2D-DIGE and label-free mass spectrometry were comparable, supporting the validity of the results. Data presented by 2D-DIGE analysis showed that apolipoprotein A-1b (ApoA1b), fibrinogen beta polypeptide, and heat shock protein 90-alpha2 were ≥2-fold upregulated in the degenerating retina as compared to the control retina. These results supported the findings from the label free mass spectrometry analysis, which also showed upregulation of apolipoproteins ApoA2, ApoA1b and ApoC in the degenerated retina. Previous observations have also shown increased apolipoprotein expression, including ApoA1 and ApoE, after injury in zebrafish tissues, that are capable of regeneration including the eye and fin20Gene ontology and pathway enrichment analyses of zebrafish proteins can be challenging due to the incomplete proteome database for this species. This may lead to partial or incorrect annotation of the dataset, and results should therefore be carefully analysed. Despite these limitations gene ontology analysis showed that proteins involved in energy metabolism were highly represented. However, in both the degenerated (3 dpi) and regenerating (18 dpi) retina the majority of these proteins were negatively regulated in comparison to control retina. Metabolic regulation in the retina is tightly controlled and is one of the most energy demanding tissues, with photo-transduction, neurotransmitter signalling, oxygen consumption and glucose metabolism all playing a significant role in energy consumption25in vitro investigations of vitellogenins have shown hemagglutination activity, as well as inhibition of bacterial growthVitellogenin 6, a glycoprotein involved in the response to activation of oestrogen receptors, showed the highest fold upregulation in the degenerated retina as compared to control retina. Although vitellogenin has been mainly described in the embryo and early developmental stages, expression has also been observed in adult tissues such as skin, brain and eye, however the function in these tissues is largely unknown29Many changes were observed in the regulation of cytoskeleton proteins between the control, degenerated and regenerating retina. Specifically, striking changes in the expression of several tubulins were observed between the control, degenerated and regenerating retina, with tubulin beta 2A showing the greatest fold increase in the regenerating retina as compared to control. Various tubulins are involved in retina remodelling and previous studies have shown changes in α-1 tubulin levels in Müller glia from the regenerating zebrafish retina37Neural retina remodelling may also occur through extracellular matrix regulation. Galectin 1, a beta galactoside-binding protein, was observed to be 2-fold upregulated in the degenerated zebrafish retina as compared to controls. This protein is expressed in the cytoplasm and can be translocated into the nucleus, or secreted into the extracellular space, where it can bind to cell surface. Galectin-1 plays a role in cell adhesion, transmembrane signalling and cell-cell interactions as well as inflammatory responsesIn conclusion, comparison of protein expression profile between the degenerated and regenerating zebrafish retina has highlighted key differences in protein expression. The data presented suggest that retinal regeneration in this species is likely to involve complex cellular processes involving proliferation, remodelling and metabolic changes, which might influence one another throughout the process. The study has highlighted proteins that may be important to examine for their function in order to create a permissible environment for endogenous regeneration of the mammalian retina.et al., in which an injection of 200 μM was given to yield a final concentration of 10 μM in the vitreousLongfin wildtype Zebrafish, 7–9 months were obtained from UCL biological services Fish facility and maintained according to local and Home Office regulations for the care and use of laboratory animals and the UK (Scientific Procedures) Act at the UCL Institute of Ophthalmology. UCL approved all procedures for experimental protocols, which were conducted in accordance with the approved guidelines at UCL Institute of Ophthalmology. Zebrafish were anaesthetised using 1 mg/ml Tricane in aquarium water for 1 min. Injections were performed by making an incision into the temporal side of the zebrafish eye using a double edge sapphire blade , where the needle was inserted and Ouabain  was injected intravitreally. The needle set up consisted of a 10 μl Gastight Hamilton syringe  with a retinal pigment epithelium (RPE) kit . A total of 0.2–0.4 μl of 200 μM Ouabain in injectable saline solution was injected into each eye to produce a final intravitreal concentration of 10 μM. The protocol used was based upon the original paper by Sherpa Zebrafish eyes were fixed for 24 hrs in 4% paraformaldehyde (PFA) at 4 °C. They were dehydrated through a graded ethanol series and then embedded in JB-4 plastic resin as described previouslyRetinal samples were washed in phosphate buffered saline (PBS) prior to homogenisation in the presence of 50 nM Ammonium Bicarbonate + 2%ASB-14  pH8.2, followed by sonication. Protein content was estimated using the Bicinchoninic Acid protein assay kit . Samples were pooled to obtain three separate conditions, consisting of control, degenerated and regenerating zebrafish retina to a total of 80 μg. Samples were run on a 1D gel  as previously describedhttp://www.pantherdb.org/), and gene ontology classification by WebGestalt .Data was processed using ProteinLynx Global Server (PLGS) 2.4 software  and peptides were matched using downloaded Uniprot reference proteome for Danio rerio. The protein sequence for the internal standard, yeast enolase1 (Accession: P00924) was also added to the database. Identified peptide data was imported into Progensis LC-MS (non-linear dynamics) for comparative analysis. Peptides were normalised to housekeeping protein beta actin (E9QD59). Further bioinformatics analysis of the results were conducted on web-based platforms which included the Panther Classification System , given an n number of 1 for each condition. Therefore the high cut-off values of ≥2-fold upregulated or ≤0.5-fold downregulated were chosen to obtain higher confidence in analysing the data from a pooled cohort.http://proteomecentral.proteomexchange.org) via the PRIDE partner repositoryThe mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium database .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Interestingly, two components exhibiting characteristic spectral and distribution profiles of proteoglycans were found, and relative component/tissue maps of these components (labelled PG1 and PG2) showed distinct distributions in non-degenerate versus mildly degenerate goat samples. MCR-ALS analysis of human IVD sections resulted in comparable spectral profiles to those observed in the goat samples, highlighting the inter species transferability of the presented methodology. Multivariate FTIR image analysis of a set of 43 goat IVD sections allowed the extraction of semi-quantitative information from component/tissue gradients taken across the IVD width of collagen type I, collagen type II, PG1 and PG2. Regional component/tissue parameters were calculated and significant correlations were found between histological grades of degeneration and PG parameters ; glycosaminoglycan (GAG) content and PGs ; and MRI T2* measurements and PGs . Additionally, component/tissue parameters for collagen type I and II showed significant correlations with total collagen content . In conclusion, the presented findings illustrate, that the described multivariate FTIR imaging approach affords the necessary chemical specificity to be considered an important tool in the study of IVD degeneration in goat and human IVDs.Traditionally tissue samples are analysed using protein or enzyme specific stains on serial sections to build up a picture of the distribution of components contained within them. In this study we investigated the potential of multivariate curve resolution-alternating least squares (MCR-ALS) to deconvolute 2nd derivative spectra of Fourier transform infrared (FTIR) microscopic images measured in transflectance mode of goat and human paraffin embedded intervertebral disc (IVD) tissue sections, to see if this methodology can provide analogous information to that provided by immunohistochemical stains and bioassays but from a single section. MCR-ALS analysis of non-degenerate and enzymatically  This together with technological developments in this field,18 which now allow a typical experiment to be completed and analysed within hours, make this technique a viable tool for rapid disease screening and diagnosis in pathologies where tissue biopsies are collected as part of routine diagnosis.18 FTIR imaging has been used for the analysis of biological samples such as cells, bone and cartilaginous tissue in its native, repaired and regenerated state.25 Different components in the extracellular matrix of connective tissue e.g. collagens and proteoglycans, show discriminatory spectroscopic characteristics and information about multiple species can be derived from the measurement of a single section without the need for lengthy immunohistochemical staining from multiple successive sections.25Fourier transform infrared (FTIR) spectroscopy provides information about the chemical species in a sample based on the frequency of the vibrations of its covalent bonds.26 The analysis of FTIR spectroscopic imaging data can be further hindered by spectral artefacts related to sample preparation28 and collection mode.30 This is particularly true of spectra collected in transflectance mode, which has some advantages over transmission mode, for example the use of low cost substrates, ease of sample preparation and a great potential for automation, which has been reported to be affected by spectral distortions due to reflective and optical phenomena such as Mie scattering and electric field standing wave effects.38 For FTIR spectroscopy to become a diagnostically useful tool the challenge lies in the development of analytical strategies which enable the development of robust methods with high chemical specificity. Current methodologies utilise 2nd derivative infrared spectra and multivariate analysis methods such as principal component analysis (PCA), partial least squares (PLS) and cluster analysis to enhance the data interpretation of the FTIR imaging data of cartilaginous tissue.43 Only recently the potential of multivariate curve resolution-alternating least squares (MCR-ALS) methods for the analysis of biological samples was highlighted.44 Like PCA, MCR methods are able to describe data matrices of multi-component measurements, without any prior knowledge of the nature and composition of the mixtures. The only assumption is a bilinear structure of the data matrices. In spectroscopic data, a bilinear structure arises through obedience of the Beer–Lambert law which implies a linear relationship between the absorbance and concentration of a component and the additivity of these absorbencies in a mixture.46 However, while PCA analysis of infrared images results in abstract factors, MCR analysis transforms abstract results into noise filtered, physically and/or chemically meaningful spectra and distribution maps of single components through the application of constraints related to the physicochemical or mathematical properties of the mixture components.48 Iterative MCR methods such as MCR-ALS have been shown to work especially well for the resolution of vibrational spectroscopic images of biological samples.53 Furthermore, MCR-ALS results can then be used as input information into segmentation analysis for histopathological classification.44However, despite its great potential FTIR imaging of biological samples is not without drawbacks; significant overlap of infrared spectral profiles of different tissue components  complica55 In this work, we examine the potential of MCR-ALS analysis to extract spatially resolved biochemical information of non-degenerated and enzymatically in vivo degenerated goat IVDs from 2nd derivative FTIR microscopic imaging data collected in transflectance mode. Additionally, the transferability of the described multivariate imaging approach from goat to human IVD sections is investigated. As a second objective, the derived methodology is applied to a second set of goat IVDs discs (n = 43) obtained from an in vivo goat study investigating the effect of two different growth factors (BMP2 and BMP2/7) in combination with a fibrin hyaluronic acid hydrogel on the regeneration of the intervertebral disc. Extracted multivariate spectroscopic parameters are correlated with parameters derived from histological grading, biochemical and MRI T2* measurements.To date, there have been limited studies of IVDs using FTIR spectroscopy and multivariate data analysis.–1 using a Nexus FTIR spectrometer  fitted with a diamond Golden Gate ATR accessory . ATR-FTIR spectra were vector normalised  and second derivatives of the spectra were generated using the Savitzky–Golay transformation .Collagen type II , collagen type I (collagen from bovine achilles tendon), chondroitin sulphate A sodium salt  and elastin (elastin from bovine neck ligament) were purchased from Sigma Aldrich . Attenuated total reflectance (ATR) FTIR spectra of reference material were collected at a spectral resolution of 4 cmet al.56 In short, eight skeletally mature female Dutch milk goats were obtained from a local farmer. During a surgical procedure three out of six lumbar IVDs in each goat were injected with chondroitinase ABC (CABC), an enzyme which cleaves proteoglycans and more specifically glycosaminoglycans (GAGs), which thus induces the onset of degeneration.60 The research protocol was approved by the Scientific Board and the Animal Ethics Committee of VUmc. To confirm that mild degeneration had occurred, radiographic and MRI analyses were conducted. After biomechanical and viscoelastic testing the samples were stored at –20 °C until further analysis. After thawing, IVDs were cut from the endplates with a surgical knife, formalin fixed  and paraffin embedded . Consecutive 4 μm sections of each sample were mounted on glass slides  for histological and immunohistochemical staining, and on custom made reflective 316 stainless steel slides for FTIR imaging analysis.Non-degenerated (control) and enzyme induced mildly degenerated goat IVDs were provided by the VU University Medical Centre, Amsterdam. The samples originate from a larger set of samples used to study the biomechanical and NP viscoelastic properties of non-degenerate and mildly degenerated goat IVDs. A detailed description of the experimental set-up is outlined in Detiger Sections were deparaffinised, rehydrated and subjected to H&E, Alcian blue (pH 2.5) and Masson Trichrome  staining using standard protocols. Additionally, immunohistochemistry was used to visualise the distribution of collagen type I and collagen type II. For the immunohistochemical staining paraffin sections were deparaffinised, rehydrated and endogenous peroxidase blocked using hydrogen peroxide. After washing in distilled water, sections for collagen type II staining were treated with a hyaluronidase-protease enzyme antigen retrieval solution for 1 h at 37 °C . Following washing, non-specific binding sites were blocked at room temperature for 45 minutes in 25% w/v rabbit serum  in 1% w/v BSA/TBS . Sections were incubated overnight at 4 °C with type II collagen monoclonal primary antibody . Additional sections were treated with mouse IgG1 isotype controls (NCG01)  and used as negative controls. To determine collagen type I content within a section, a heat/citric acid antigen retrieval method ) was used and non-specific binding sites were blocked at room temperature for 45 minutes with 25% w/v donkey serum in 1% w/v BSA/TBS . Sections were incubated overnight at 4 °C with rabbit polyclonal to collagen type I  and rabbit polyclonal IgG isotype controls  were used for negative controls. On the following day all sections were incubated with secondary antibodies for 30 min at room temperature  and collagen type I: 1 : 200 dilution of Donkey anti-rabbit IgG H&L (Biotin) ). Following washes in TBS sections were incubated in ABC solution  for 30 minutes at room temperature. Following TBS washes, antibody staining was disclosed by incubation with 3,3′-diaminobenzidine tetrahydrochloride solution  for 20 min at room temperature and counterstained with Mayers Haematoxylin . Sections were dehydrated and mounted in Pertex .–1. Infrared data matrices of the control and injected samples were collated resulting in a 184 × 228 pixels data matrix. Data was pre-processed by performing a second derivative transformation on the spectra . Pixels within the imaging data set where spectra indicated only the presence of paraffin or substrate were identified and masked using spectral statistics . The data sets were analysed using a MCR-ALS algorithm described in detail by Wang et al.61 The basic procedures of the MCR-ALS algorithm comprised the transformation of abstract factors deduced from non-linear iterative partial least squares (NIPALS) decomposition into chemical and concentration information by applying a modified iterative alternating least-square optimisation.61 MCR-ALS was carried out using the MCR-ALSv1.6 software with the following settings; decomposition method: NIPALS; maximum number of iterations: 500; soft constraints: MALS-2D . Two spectral regions 950–1300 and 950–1600 cm–1 were analysed using a range of four to six components in the MCR-ALS model. No spectral correction or exclusion of the spectral region ∼1400–1500 cm–1, which is dominated by absorbance peaks of the embedding medium paraffin, was carried out. Both paraffin and potential spectral distortions are considered as additional signal contributions during MCR-MLS resolution.62Mid-infrared microscopic images were collected using an Agilent 680-IR FTIR spectrometer coupled to an Agilent 620-IR FTIR imaging microscope. The microscope was fitted with a liquid nitrogen cooled 64 × 64 mercury–cadmium–telluride focal plane array detector (FPA) and an automated sampling stage. FTIR mosaic images  covering an area of approximately 8.05 × 19.95 mm of the tissue sections were collected in transflectance mode at a spectral resolution of 4 cmTwo human cadaveric discs were obtained within 72 h of death from the Leeds tissue bank . Samples were obtained from a 45 year old male L4/5 disc and 74 year old female L1/2 disc.et.al.63 Discs were graded as: male L4/5 disc: grade 2 (non-degenerate); female L1/2 disc: grade 8 (degenerate).A wedge of IVD tissue encompassing AF and NP was formalin fixed (10% v/v) and paraffin embedded. Consecutive 4 μm sections of each disc were mounted on glass slides  for histological analysis and on custom made reflective 316 stainless steel slides for FTIR imaging analysis. Sections were histologically graded between 0 and 12 based on the presence of cell clusters, fissures, loss of demarcation and haematoxophilia (indicating reduced proteoglycan content) with each component scored out of 3. A score of 0 to 3 indicates a histologically non-degenerate IVD and a grade of ≥4 indicates evidence of degeneration, as described previously by Le Maitre FTIR mosaic images of human sections were collected covering an area of approximately 9.8 × 21.7 mm  and the same data analysis as described for the goat samples was applied.in vivo goat study investigating the effect of two different growth factors (BMP2 and BMP2/7) in combination with a fibrin hyaluronic acid hydrogel on the regeneration of the intervertebral disc as reported in Peeters et al.64 The research protocol was approved by both a Scientific Board as well as the Animal Ethics Committee of the VU University Medical Center. Seven skeletally mature female Dutch milk goats were used for this study. In the first surgical procedure, mild intervertebral disc degeneration was induced by injecting ±200 μl chondroitinase ABC (CABC) 0.25 Units per ml in the lumbar IVDs using a 29 G needle of 1 cm length. Another disc (T13-L1) was left as a non-degenerate control.60 During the second surgery, twelve weeks later, IVDs were injected with hydrogel in combination with either the BMP-2 or BMP-2/7 growth factor and one level was injected with hydrogel only. Detailed discussions about the safety and efficacy of the nanobiopolymeric fibrin–hyaluronic acid (FBG–HA) conjugated hydrogels, alone or in combination with BMP-2 or BMP2/7 growth factors are reported in Peeters et al.64 In short it was found, that although mild degeneration was induced after injection with CABC, which was indicated by a significant disc height loss of injected IVDs, no significant differences in histological grades, MRI T2* mapping, GAG and total collagen content were found between the control and treated IVDs. However, it was found that MRI T2* mapping showed strong and significant correlations with biochemistry and histology in the NP.A second set of 43 IVDs were obtained from an 57 Each IVD was divided into 5 different regions of interest (ROI) covering the IVD from anterior to posterior where ROI 1 and 3 covered 27.5% of the total disc diameter whereas ROI 2,4 and 5 each covered 15% . This way ROI 1, 3 and 5 represent the anterior annulus fibrosus (aAF), NP and posterior annulus fibrosus (pAF) respectively. ROI 2 and 4 represent the transition zones between AF and NP and were not used for analysis. Mean T2* relaxation times were calculated for each ROI fitting the signal intensities of the five echo times by a linear-log least-squares method using Microsoft Excel (Microsoft Office 2010).MRI scans were acquired from all lumbar spines within 2–3 hours after autopsy using a 1.5 T MR scanner . Sagittal scans were performed using a T2-weighted turbo spin echo sequence, followed by a multi-echo gradient echo sequence for T2* mapping .et al.59 Differences in scoring were resolved by consensus, resulting in a final histological scoring ranging between 0 (non-degenerate) and 6 (degenerate).After obtaining MRI scans, IVDs including endplate were dissected from the spine using an oscillating band saw. Mid-sagittal slices of 4 mm were cut, fixed in 4% formalin, decalcified in Kristense fluid and subsequently embedded in paraffin. Consecutive 3 and 4 μm sections of each sample were mounted on glass slides for histological staining, and on custom made reflective 316 stainless steel slides for FTIR imaging analysis respectively. Sections were stained with Alcian blue (Alcian Blue-Periodic Acid Schiff). Stained sections were scored for disc degeneration by two independent researchers using a histological grading scale adapted for goats as described by Hoogendoorn FTIR mosaic images  of the tissue sections were collected. For the correlation of spectroscopic parameters with results from biochemical, histological and magnetic resonance imaging only areas of interest (AoI: 10 × 208 pixels) across each section  were coll-cysteine, 0.01 M EDTA and 0.33% papain (w/v) , the pH of the papain solution was titrated to 6.6 using 1 M NaOH. Samples were digested overnight at 65 °C in a continuously shaking warm water bath. GAG content was measured using a colorimetric 1,9 dimethyl-methylene blue assay according to the manufacturer's protocol . Hydroxyproline (HYP) content, representing total collagen content of the tissue, was quantified using a dimethylamino-benzaldehyde assay adapted from Paul et al.65 Both GAG and HYP were normalized by tissue dry weight (μg (mg DW)–1).Tissue samples for biochemical analysis were obtained from consecutive 4 mm slices used for histological, immunohistochemical and FTIR imaging analyses. Tissue samples were harvested from the anterior AF, NP and posterior AF analogous to ROI 1, 3 and 5 respectively, as used for MRI T2* mapping. The samples were freeze dried (speed vac) and digested in 1.5 ml papain digestion solution containing 0.1 M sodium acetate, 0.01 M P values ≤ 0.05 were considered statistically significant and scatter diagrams were plotted using the GraphPad Prism® software (Version 6.05).Non-parametric linear regression analysis was performed between extracted MCR-ALS parameters and histological grades, biochemical parameters and MRI T2* measurements (StatsDirect 3.0126). –1) of a non-degenerated IVD section was generated, and the posterior AF , anterior AF  and NP (red) regions of an IVD were highlighted (–1) to zero (–1). While paraffin peaks at 1473 and 1464 cm–1 have similar intensities across the sample regions; the amide I, amide II and amide III peaks show varying intensities. The band intensities of amide I, II and III in the aAF spectrum are approximately double the band intensities exhibited in the NP spectrum. The amide I peak of the aAF spectrum shows a peak shift to a higher wavenumber and asymmetry. The convoluted peak associated with polysaccharide species (900–1100 cm–1) shows a comparatively higher intensity in relation to the amide bands in the pAF and NP spectra, indicating a higher concentration of PGs in the pAF and NP regions. Performing a second derivative of FTIR spectra can enhance the discrimination of broad overlapping bands,36 and more detailed differences between the aAF, pAF and NP regions, especially in the polysaccharide region are revealed. Average second derivative spectra are shown in –1. The second derivative amide II bands in the pAF and aAF spectra show a subtle shift to higher wavenumbers compared to the NP spectrum. The amide I band of the aAF spectrum shows a different peak shape and a shift to higher wavenumbers. Paraffin embedded goat IVD sections measured in transflectance mode show region dependent changes in spectral intensity leading to a saturation of the signal in some sample areas. Based on these findings the amide I spectral region traditionally used as a biomarker for collagens25 was excluded from further analysis and MCR-ALS deconvolution was carried out in the spectral region 950–1600 cm–1.Sagittal sections of paraffin embedded goat IVDs were measured and an average intensity map  Intermolecular interactions between matrix components can lead to peak shifts in the spectral profiles.17 (III) Iterative MCR-ALS deconvolution leads to a set of feasible solution and the final results depend on the initial estimates and chosen optimisation processes.47 In this work, the MCR-ALS deconvolution is carried out without any input of a priori information such as the spectral or distribution characteristics of an IVD. Initial estimates are based on abstract factors derived from eigenanalysis. The four most commonly used mathematical methods to perform eigenanalysis are singular value decomposition (SVD), NIPALS, the power method and the Jacobi method.46 Although these techniques use different approaches to extract abstract factors the results should be the same.46 Initial investigations of loadings and scores derived from SVD and NIPALS deconvolution of the spectral regions 950–1600 cm–1 and 950–1300 cm–1 modelled using 5 factors, however showed that results can vary (S3 and S4). Nevertheless, it was found that after iterative partial least squares optimisation both sets of abstract factors lead to almost identical MCR-ALS solutions. Further inspections of the extracted MCR-ALS spectral profiles showed deviations from the reference spectra, including increased/decreased relative peak heights. One possible explanation for the observed differences could be that the used combination of abstract factors as initial estimates together with the modified iterative alternating least-square optimisation61 leads to spectral profiles, which emphasise the biggest spectral change in the data set rather than pure spectral profiles. For example, this could explain the observed differences between the extracted spectral profiles for PGs and the reference spectrum of chondroitin sulphate  would be expected.43 However the extracted spectral profiles accentuate the peak at 1064 cm–1 previously found to show the biggest relative changes in the investigated wavenumber region before and after enzymatic removal of chondroitin sulphate.43 (IV) In spectroscopic data matrices linear or near-linear relationships within a set of variables, also referred to as co-linearity, can affect the deconvolution power and stability of the MCR-ALS model. Examples of incomplete deconvolution can be seen in the spectral profiles of collagen type I and II, which show contributions of a strong peak at ∼1470 cm–1 characteristic of paraffin  reference spectra were measured with ATR-FTIR, in comparison IVD sections were measured using FTIR transflectance microscopy meaning that the relative peak heights and peak positionssulphate . Both naparaffin . Althougparaffin . (V) FinAs discussed in the previous section, because of differences between the extracted spectral profiles and collected reference spectra, the biochemical identity of each MCR-ALS factor was established based on a visual assessment, firstly on the correlation between the reference spectra and extracted spectral profiles , and sec–1) are proposed as a measure of overall extracellular matrix or tissue structure and match H&E stained tissue sections. Like the H&E stained sections, tissue distribution maps highlight the overall structure of an IVD  shows a decrease in intensity from the NP towards the AF typical for collagen type II  and Factor 2 (950–1300 cm–1) show a decrease in intensity from the NP region towards the AF region and are in good agreement with the Alcian blue stain  shows a high intensity in the AF regions and matches well with the red areas of the Masson Trichrome stained sections. The calculated spectral profile of this factor resembles the reference spectrum of elastin  and PG2: Factor 2 (950–1300 cm–1)), which show characteristic spectral and distribution maps of PGs. Information about PGs and more specifically aggrecan and GAG content in the NP, is of particular interest as it can be directly related to disc degeneration.67 PG/amide I ratios have previously been used as a measure of relative PG content in cartilage.21 In this work PG distributions are normalised with second derivative peak integration maps of the amide III spectral region (1186–1297 cm–1) (further referred to as tissue map) and relative PG distribution maps reveal distinct PG distributions in non-degenerate versus degenerate samples  to PG2 . A peak shift of the C–O–S peak has previously been observed by Rieppo et al.,43 where the authors noted a peak shift to lower wavenumbers in samples treated with the CABC enzyme, which facilitates the breakdown of GAG chains. Based on these findings PG1 would represent the “original” PG/GAG population which is decreased in degenerated samples, while PG2 could represent an altered PG/GAG population which is increased in degenerated discs. As the goat degeneration model is mimicking the onset of degeneration, much of the long term remodelling has not yet taken place and the altered PG/GAG molecules are localised in the NP.Interestingly, two factors were observed (PG1: Factor 5 (950–1300 cm samples . While t–1) show a lower presence in the degenerated disc. Additionally, a localisation of PG1 within cells in the posterior AF region can be seen (–1) shows the highest intensity in the posterior AF region of the degenerated disc . Interestingly, PG2 shows a higher correlation with GAG content in the NP, while PG1 shows a higher correlation with GAG content in the pAF.MCR-ALS extracted parameters for PG1 and PG2 were correlated with MRI T2*, GAG concentration and histological grades in the aAF, NP and pAF IVD areas. Both MCR-ALS parameters for PGs show significant correlation with the histological grades, MRI T2* and GAG content in the NP  and betwSignificant correlation between collagen type I and type II and GAG content, total collagen content and MRI T2* was observed only for the NP region .No significant correlations were found between the MCR-ALS extracted parameters and GAG or total collagen content for the aAF region. One possible explanation might be that volumes taken for the biochemical analysis are relatively small (∼10% IVD width) and cannot be taken from exactly the same part of the IVDs used for sectioning. This is particularly relevant in the pAF, as this region is relatively large 1–27% IVD width and in comparison to the NP structurally more organised . This difference in sample size and position used for the different techniques may reduce the correlation between the investigated parameters. Furthermore, in this study MCR-ALS parameters for collagen type I and type II were correlated with total collagen and further studies using more specific biochemical assays are recommended.p < 0.001), GAG content (p < 0.01) and MRI T2* measurements (p < 0.01). The FTIR derived parameters for collagen type I and II show significant correlations with the total collagen content .In this study, we have examined the potential of MCR-ALS analysis to extract spatially resolved biochemical information of non-degenerated and degenerated goat IVDs from 2nd derivative FTIR microscopic imaging data collected in transflectance mode. The presented results show that MCR-ALS allows the resolution of 2nd derivative FTIR microscopic imaging spectra of paraffin embedded IVD sections and important biochemical matrix components can be detected and their distribution visualised. Furthermore, matrix components specifically related to healthy and degenerate states were found. An analytical strategy was presented for the extraction of semi-quantitative biochemical information from IVD sections. Multivariate FTIR image analysis reveals the relative distribution gradients for collagen type I, collagen type II and two PG components. MCR-ALS parameters extracted from the NP region of the IVDs of both PG components show significant correlations with histology (–1 data set, however distinct factors for collagen type I and II can still be resolved, and good matches between the extracted spectral profiles and distribution maps with immunohistochemical stains were found.Exploratory MCR-ALS results highlight the ability of multivariate curve resolution techniques to deconvolute the overall signal into single component contributions, but also the importance of understanding the molecular information contained in spectral profiles extracted using MCR-ALS analysis. Collinearity was observed in the 950–1600 cmvia directed immunohistochemical analysis.While more molecular specific comparative experiments are recommended to further investigate the chemical nature of the extracted PG parameters and to validate correlations between the extracted collagen type I and type II parameters with collagen type I and II content, the presented results show the potential to study IVD degeneration and enable the characterisation of extracellular matrix components and their distribution in a single slice of IVD tissue, therefore allowing extensive investigations to be performed on limited tissue samples. Additionally, the described multivariate FTIR imaging method was shown to be easily transferable to human sections. The facile application across species provides a major advantage of traditional techniques such as immunohistochemistry where selective antibodies for each species are required, which involve extensive optimisation. In addition the ability of FTIR imaging to screen for variants of the extracellular matrix molecules enables identification of novel components, modifications or degradation products which is not possible


To treat a cluster of tuberculosis (TB) transmission cases in a long-term care facility for cognitively impaired adults located in Puerto Rico (facility A), the Puerto Rico TB Control Program used a novel video directly observed therapy (VDOT) application. In 2016, active TB disease was diagnosed in 11 residents and latent TB infection (LTBI) was diagnosed in six residents of facility A. Asynchronous VDOT was used to monitor treatment for these 17 residents. One of the patients with active TB disease had received a diagnosis of LTBI during an investigation at facility A during 2011–2012.During 2010–2012, seven residents of facility A received a diagnosis of active TB disease; four of these diagnoses were culture-confirmed, with isolates that had the same rare genotype . Six other residents with diagnosed LTBI were prescribed 4R treatment. Because of staffing shortages, Puerto Rico Department of Health (PRDH) TB field personnel were not available to administer daily directly observed therapy (DOT) at facility A and facility A did not have the personnel needed to provide daily patient transport to the PRDH clinic.,† was implemented to ensure proper treatment for TB and LTBI patients. Use of this asynchronous system avoided audio/visual interruption related to poor connectivity, which can be problematic in standard live VDOT applications  to facilitate adoption of these practices.As of July 12, 2017, all 11 patients with active TB disease and all six with LTBI had completed treatment with recommended ≥80% compliance   (6). Act


While the dose did decrease inflammation and protein oxidation in dystrophic muscles, there was no improvement in muscle strength (in contrast with benefits observed with the lower dose) and growth of the young mice was significantly restricted. We present novel data that a high taurine dose increases the cysteine content of both mdx liver and plasma, a possible result of down regulation of the taurine synthesis pathway in the liver . These data caution that a high dose of taurine can have adverse effects and may be less efficacious than lower taurine doses. Therefore, monitoring of taurine dosage needs to be considered in future pre-clinical trials, in anticipation of using taurine as a clinical therapy for growing DMD boys (and other conditions).Duchenne Muscular Dystrophy (DMD) is a fatal muscle wasting disease manifested in young boys, for which there is no current cure. We have shown that the amino acid taurine is safe and effective at preventing dystropathology in the mdx mouse model for DMD. This study aimed to establish if treating growing mdx mice with a higher dose of taurine was more effective at improving strength and reducing inflammation and oxidative stress. Mice were treated with a dose of taurine estimated to be 16 g/kg/day, in drinking water from 1–6 weeks of age, after which  Duchenne Muscular Dystrophy (DMD) is a lethal, X-chromosome linked muscle disease affecting about 1 in 3500–6000 boys worldwide (Reviewed in ). DMD isin vivo and ex vivo muscle strength, increased fatigue resistance and decreased other indices of mdx pathology such as inflammation and oxidative stress. Recently we showed that an oral dose of approximately 4 g/kg/day  had benefits on many indices of dystropathology (with improved in vivo and ex vivo muscle strength and decreased inflammation and oxidative stress) in mdx mice . Grip strength measurements were performed on three separate occasions during the final week of treatment to accustom mice, however only data from the final testing session was used for analysis.The grip strength of all mice was measured using a Chatillon Digital Force Gauge (DFE-002) and a triangle metal bar, as per the TREAT-NMD recommended standard protocol “Use of grip strength meter to assess limb strength of mdx mice– M.2.2_001 “Mice (at 6 weeks of age) were anaesthetized via an intraperitoneal (IP) injection of sodium pentobarbitone (40 mg/kg body weight). Anaesthetized mice were placed on a heated plate at 37°C to maintain core body temperature. Following dissection of the EDL muscle , blood was removed via cardiac puncture and other tissues, including the quadriceps muscle and liver, were removed and snap frozen for further analysis.Ex vivo muscle function testing was performed as per [in vitro muscle test system  filled with mammalian ringer solution (containing (in mM) NaCl (121); KCl (5.4); MgSO4.7H2O (1.2); NaHCO3 (25); HEPES (5); glucose (11.5) and CaCl2 (2.5)), bubbled with Carbogen and maintained at 25°C. The muscle was stimulated with 0.2 ms square wave pulses  via parallel electrodes situated on both sides of the suspended muscle.d as per . In brieo) by manually adjusting the lever arm and recording the twitch force response. The length at which the maximum twitch force was recorded was taken as Lo. All subsequent measurements of contractile function were performed at Lo. The optimal fibre length (Lf) in the EDL was calculated from a pre-defined fibre-length to muscle-length ratio of 0.44 [At the start of each experiment the muscle was set to the optimal length (L of 0.44 . The for2). CSA was estimated by dividing the wet muscle mass (g) by Lf and the density of mammalian skeletal muscle (1.056 mg / mm3).Force recordings were normalized relative to muscle cross sectional area (CSA) and presented as specific force  as previously described . In brieg for 10 min. The protein concentrations of supernatants were quantitated using the DC protein assay (Bio-Rad). Samples were resolved on 4–15% SDS-PAGE TGX Stain-Free gels (Bio-Rad) and transferred onto nitrocellulose membrane using a Trans Turbo Blot system (Bio-Rad), after imaging for total protein using the Stain-Free imaging program on the ChemiDoc MP Imaging System (Bio-Rad). Immuno-blotting was performed with rabbit polyclonal antibodies to neutrophil elastase , cysteine dioxygenase type 1  and cysteine sulfinate decarboxylase  all dissolved 1:1000 in 5% bovine serum albumin (BSA). HRP-conjugated goat anti-rabbit secondary antibodies were from Thermo Fisher Scientific. Chemiluminescence signal was captured using the ChemiDoc MP Imaging System (Bio-Rad). Resultant images were quantified using ImageJ software [Frozen livers and quadriceps muscles were crushed using a mortar and pestle under liquid nitrogen and homogenized in 1 ml ice-cold 1% NP40, 1 mM EDTA in phosphate buffered saline (PBS), supplemented with complete EDTA free protease inhibitor tablets and PhosSTOP phosphatase inhibitor tablets (Roche), and centrifuged at 10,000 software . A commosoftware .+ and 25 mM hydroxylamine hydrochloride at pH 5.9 was added. To both tubes, a mixture containing 40 mM cysteine and 0.50 mM bathocuproinedisulfonic acid was added. 20% sulfosalicylic acid was immediately added to the blank tubes, and all samples were incubated for 5 minutes. 20% sulfosalicylic acid was added to the rest of the tubes, and samples were incubated on ice for 15 minutes before centrifugation to remove the precipitated protein. Supernatants were removed and stored at -80°C before analysis. Analytes were separated using HPLC with fluorescent detection, with pre-column derivitisation with OPA and 2ME as per analysis of taurine and cysteine as above. Activity of cysteine dioxygenase was determined to be the amount of cysteine sulfinate formed per minute, by subtracting the cysteine sulfinate content of the sample with cysteine substrate added minus the cysteine sulfinate content of the sample without cysteine substrate, divided by 5 (samples were incubated for 5 minutes).Cysteine dioxygenase activity was measured by quantification of cysteine sulfinate from substrate cysteine, as per , with soData were analysed using GraphPad Prism software. One-way ANOVA tests with post-hoc (LMS) comparisons were used to identify significant differences between experimental groups. Two-way repeated measures ANOVAs were used to analyse data for the force-frequency protocols. Statistical significance was accepted at p<0.05. Phenotype and force data are presented as mean ± SEM, all data points are presented ± SEM for all other measures.Body, muscle and liver weights were measured at 6 weeks of age to identify potential detrimental effects of treatment with taurine in young growing mdx mice. While body weights were similar for C57 and untreated mdx mice, taurine treatment reduced mdx body weight by 22% . Liver win vivo measure of mouse limb strength. Mdx mice had weaker (15%) normalised (to body weight) grip strength  in the drinking water to mdx mice is highly effective at increasing levels of taurine in the liver and plasma; however, this high dose taurine causes a  build-up of cysteine.Immunoblotting quantification of protein levels of enzymes related to cysteine metabolism showed that cysteine dioxygenase protein was 140% higher in livers of untreated mdx mice compared with C57 , and tauThese data show that treating juvenile mdx mice with a high dose of taurine likely causes a down-regulation of taurine synthesis in mdx liver, leading to a build-up of cysteine in mdx liver and plasma.The key novel observation in this study is that delivery of high dose taurine in drinking water, estimated to be about 16 g/kg/day, caused growth deficits in young mdx mice. We propose that the cause of the growth deficit is an indirect consequence of an increase in cysteine levels resulting from an inhibition of the taurine synthesis pathway (which disposes of excess cysteine) in mdx mice. Nevertheless, consistent with previous work, there was good evidence that taurine decreased inflammation and protein oxidation in dystrophic tissue.We and others have shown that taurine treatment of mdx mice (with doses ranging from 1 to 16 g/kg/day) decreases dystropathology, with an improvement in muscle strength and a decrease in myofibre necrosis, inflammation and oxidative stress –21. Likein vivo and ex vivo force. However this dose did not affect in vivo and ex vivo muscle strength when normalised to body weight (grip strength) or to cross sectional area calculated from muscle weight (specific force). This indicates that taurine was not detrimental to the inherent contractile properties of muscle(s). Instead, the loss of force can be attributed to smaller body and muscle weights in taurine treated young mdx mice, relative to age-matched untreated mdx mice.The high dose taurine in the present study resulted in a decrease in total The decrease in body weight and muscle size was also associated with a decrease in tibia length of taurine treated mdx mice, indicating that the high dose taurine caused growth restriction in these young mdx mice. Whilst studies have shown that dietary taurine restriction can lead to growth restriction , 35, eviOur data indicate that a high dose of taurine may not be an effective or safe treatment for very young growing mdx mice. In contrast, De Luca and colleagues did not observe any deleterious effects of taurine when treating mdx mice aged 4 weeks (until 8 weeks) with an equivalently high dose of taurine . Their dose was also highly effective at improving strength of exercised mdx muscles, and was more beneficial than treatment with prednisolone, the current gold standard treatment of DMD boys . By treaWe propose that the detrimental effects of taurine on growth are an indirect effect caused by increased cysteine levels in the liver and plasma. Excess cysteine is considered toxic (reviewed in ) and excCysteine is catabolised by the enzyme cysteine dioxygenase to cysteine sulfinate, which is decarboxylated by the enzyme cysteine sulfinate decarboxylase to form hypotaurine, which is then oxidised to taurine . We founOur observation that taurine exerts a strong feedback effect on this pathway appears to be novel. Although a high protein diet has also been shown to affect activity and levels of these enzymes, this response was suggested to be wholly dependent on the sulfur containing amino acids cysteine and methionine within the protein diet –46. One We and others have provided strong data from pre-clinical studies to support the use of taurine as a highly promising and inexpensive therapeutic nutritional intervention for DMD. The issue of growth restriction with high taurine dosage in very young dystrophic animals adds a cautionary note. We propose that this concern can be addressed by carefully monitoring levels of metabolites of taurine and cysteine in the blood and urine of growing animal models and DMD boys. As a consequence, further research is required to identify the dosing range of taurine that maximises the protective benefits of taurine while minimising the likelihood of growth restriction, before considering clinical translation to young DMD boys.S1 TableSymbol (*) denotes significant differences (p<0.05) between male and female mice in that treatment group.(DOCX)Click here for additional data file.


However, the Desulfobulbaceae Operational Taxonomic Units (OTUs) from the 16S sequencing did not cluster closely with other putative cable bacteria sequences suggesting that the taxonomic delineation of cable bacteria is far from complete.Multicellular, filamentous, sulfur-oxidizing bacteria, known as cable bacteria, were discovered attached to fibers of a carbon brush electrode serving as an anode of a benthic microbial fuel cell (BMFC). The BMFC had been operated in a temperate estuarine environment for over a year before collecting anode samples for scanning electron microscopy and phylogenetic analyses. Individual filaments were attached by single terminus cells with networks of pilus-like nano-filaments radiating out from these cells, across the anode fiber surface, and between adjacent attachment locations. Current harvesting by the BMFC poised the anode at potentials of ~170–250 mV vs. SHE, and these surface potentials appear to have allowed the cable bacteria to use the anode as an electron acceptor in a completely anaerobic environment. A combination of catalyzed reporter deposition fluorescent  Significant quantities of the dissolved sulfide react chemically with iron (oxydr)oxides to form FeS, S°, and FeS2, while a fraction is typically reoxidized after transport by diffusion or bioturbation to overlying oxic zones oxides and S° often coincide with a redox potential gradient between oxic and anoxic conditions Berner, . In coasDesulfobulbaceae mediate intra-species cell-to-cell electron transport over mm to cm-scale distances that coincide with suboxic zones  and 16S rRNA gene sequence analysis confirmed the phylogeny as Desulfobulbaceae, and the BMFC performance history indicates that these bacteria were using the anode as an electron acceptor after being deprived of access to oxygen for many months. In the attached mode, the cable bacteria co-occur with nano-filament networks and other electrogenic bacteria, especially those belonging to the Desulfuromonadaceae family. The evidence for these assertions and their possible implications for understanding the mechanisms for long-range electron transfer through cable bacteria are the focal points of this research article. We also re-evaluate representations of the microbial metabolic networks that may catalyze electron transfer to marine BMFCs.As serendipity has often led to discoveries of unknown microbial processes and associations, we were excited to observe by scanning electron microscopy (SEM) cable bacteria filaments firmly attached by their terminus cells to carbon fibers of a benthic microbial fuel cell (BMFC) anode after it had harvested electricity in the marine environment for over 1 year. Subsequent catalyzed reporter deposition fluorescent 2, open at the bottom for insertion into the seafloor) to isolate an anode from dissolved oxygen, while the cathode was exposed to oxic seawater in a turbulent environment  intertwined mid-length in #11 titanium stem wire . Individual carbon fibers are cylindrical providing a high surface area for bioelectrochemical reactions. The anode brush was coiled on top of the upper PVC divider in the chamber. The cathode was supported above the chamber as described below.The BMFC operation was controlled by a power management platform (PMP) Reimers,  with cus−1 (based on near bottom SonTek Argonaut Acoustic Doppler Velocimeter measurements made adjacent to the BMFC during deployment days 91–135). The BMFC operated continuously for a total of 412 days until recovered using a shore-side crane to pull it from the seafloor and out of the water. Logged data included sensor readings (dissolved oxygen and temperature), and energy harvesting parameters that included hourly records of the cell voltage of the BMFC , energy out of the BMFC, and the voltages of the rechargeable Li-ion batteries. BMFC performance was assessed further by conducting a polarization  the PMP did relax energy harvesting, allowing the BMFC to approach open circuit. This condition occurred periodically 80–300 days after initial deployment when large energy demands were not being placed on the BMFC.where Voperating declined and current was drawn continuously (except for 6 min of open circuit at the start of each daily polarization cycle). Current densities were calculated as the measured current normalized to the footprint area inside the chamber (0.259 m2).Over the last ~110 days of the experiment, more frequent, and then by the last 75 days more prolonged, modem transmissions were conducted purposely to increase the energy drawn from the Li-ion cells through the PMP and from the BMFC. Consequently, during the last 75 days the average VTwo hours after hoisting the BMFC from its deployment location, the anode chamber was opened to expose the anode and internal sediment Figures . Clippin3 sample was transferred to a beaker, and whole sediment pH measured at 15°C with a glass combination electrode. The pH electrode was calibrated using National Bureau of Standards (NBS) buffer standards before insertion into the sediment. Additional samples of internal sediment were transferred to 50 cm3 centrifuge tubes and centrifuged at 3,800 rpm for 10 min at 15°C. Separated pore waters were then vacuum filtered through 1.2 μm glass fiber filters (GF/C), and the filtrate frozen for later nutrient analyses by autoanalyzer. The solids in each centrifuge tube were also frozen. Subsamples of the sediment solids were later dried, ground and analyzed for organic C and N contents after carbonates were removed by concentrated HCl fumigation. These analyses were made using a NC2500 ThermoQuest Elemental Analyzer with acetanilide as the calibration standard  using a Desulfobulbaceae-specific oligonucleotide probe  modified with horseradish peroxidase  for 30 mins at room temperature (~25°C). The hybridization process was performed in standard hybridization buffer at 46°C with 45% formamide for 7 h. Catalyzed reporter deposition was performed by using Alexa Fluro 488 tyramides reagent . 4′,6-diamidino-2-phenylindole (DAPI) was applied as a counter stain in some treatments. Hybridization samples were visualized using confocal laser scanning microscopy .To verify that the multi-cellular filaments observed as attached to carbon fibers by SEM were cable bacteria, anode carbon fiber samples that had been frozen were examined by catalyzed reporter deposition-fluorescence Desulfuromonadales-specific oligonucleotide probe  modified with horseradish peroxidase. To perform the multicolor CARD-FISH, samples were treated with 0.15% H2O2 after the first catalyzed reporter deposition to inactivate the horseradish peroxidase from the first hybridization. The inactivated samples were then hybridized with the DRM 432 probe in standard hybridization buffer at 46°C with 40% formamide for 4 h and sequentially stained by a secondary catalyzed reporter deposition using Alexa Fluro 555 tyramides reagent.To investigate the potential synergistic relationship on carbon fibers, multicolor CARD-FISH was performed on a few samples by using an additional Three types of controls were performed and visualized: (1) sterile carbon fibers from an unused carbon brush, (2) anode carbon fibers from the deployed BMFC that went through all procedures except the oligonucleotide probe was omitted during the hybridization step, and (3) anode fibers from the deployed BMFC that did not receive any of the hybridization and catalyzed reporter deposition processing Figure . It is nTotal DNA was extracted from three anode carbon fiber samples, from a sample of sediment from inside the anode chamber, and from sediment collected by a diver next to the BMFC at the study site. DNA extraction, amplification, and sequencing  was performed by the Research and Testing Laboratory . DNA was extracted using a MoBio PowerSoil DNA Extraction Kit with a bead beating step, and bacterial and archaeal 16S rRNA genes were amplified by PCR with primers 357wF (5′-CCTACGGGNGGCWGCAG-3′) and 785R (5′-GACTACHVGGGTATCTAATCC-3′).vegan package for R  as described previously  initially, but contained only 3.0 ± 1.9 wt. % organic C and 0.29 ± 0.16 wt. % N (n = 4) at the time of recovery, indicating significant organic matter remineralization. The black color of the sediment and black deposits on the anode chamber wall  helped to confirm concentrations of these known electrogenic bacteria on the carbon fibers surrounding points of cable bacteria attachment . In contrast, Desulfobulbaceae contributed <2% to the observed community dissimilarity, and were slightly higher in relative abundance in sediments outside the BMFC than in anode-associated communities. Communities inside the BMFC were also depleted in members of the class Gammaproteobacteria compared to sediments outside the BMFC. Microbial community diversity .Total microbial community composition differed significantly between anode-associated communities within the BMFC and sediments from inside or outside the BMFC ,  relative abundance estimates of cable bacteria are especially sensitive to DNA-extraction methodologies.The contributions of cable bacteria to microbial communities associated with electrodes harvesting electricity from aquatic sediments or waste water may have been inadvertently missed in past studies because their taxonomy has only been described recently and their outer cell structures may hamper cell lysis and DNA extraction  to sulfate with the electrode as electron acceptor or else to recycle S° by a disproportionation reaction. Many of the members of the microbial community within the anodic chamber of a marine BMFC appear likely to take advantage of multiple-step oxidations from sulfide to S0, 42Sulfurovum and Dethiosulfovibrio  what is the full spectrum of electron donors utilizable by cable bacteria and where along a filament may electrons be harvested from external donors; (2) what is the mechanism of cell-to-cell electron conduction from end-to-end of a cable; (3) what causes the filaments to form bundles; (4) how do cable bacteria interact with other bacteria capable of extracellular electron transfer; (5) how do they attach to a solid electron acceptor; (6) do they produce conductive pili or form connections to pili produced by other microrganisms; (7) how genetically diverse are cable bacteria; (8) are all cable bacteria facultative anaerobes; and (9) will the enrichment of cable bacteria in microbial communities on a BMFC anode enhance the device's electrical energy recovery.CR: Primary author of the paper, designed field experiment and follow-up analyses, recognized the discovery. CL: Performed CARD-FISH and wrote paper sections on this method and results. MG: Did phylogenetic analysis, prepared figures, and contributed to writing. PS: Maintained field experiment and collected samples. MW: Developed the power management system that maintained the microbial fuel cell and collected data on its performance.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer KN and handling Editor declared their shared affiliation.


We tested whether in vivo neuroinflammation relates to the distinctive distributions of pathology in Alzheimer disease (AD) and progressive supranuclear palsy (PSP).11C]PK11195 PET scanning, which was used as an in vivo index of neuroinflammation.Sixteen patients with symptomatic AD (including amnestic mild cognitive impairment with amyloid-positive PET scan), 16 patients with PSP–Richardson syndrome, and 13 age-, sex-, and education-matched healthy controls were included in this case-control study. Participants underwent [11C]PK11195 binding in the medial temporal lobe and occipital, temporal, and parietal cortices was increased in patients with AD, relative both to patients with PSP and to controls. Compared to controls, patients with PSP showed elevated [11C]PK11195 binding in the thalamus, putamen, and pallidum. [11C]PK11195 binding in the cuneus/precuneus correlated with episodic memory impairment in AD, while [11C]PK11195 binding in the pallidum, midbrain, and pons correlated with disease severity in PSP.[Together, our results suggest that neuroinflammation has an important pathogenic role in the 2 very different human neurodegenerative disorders of AD and PSP. The increase and distribution of microglial activation suggest that immunotherapeutic strategies may be useful in slowing the progression of both diseases. Despite the importance of neuroinflammation, there is still insufficient information regarding the extent and regional distribution of microglial activation in patients with neurodegenerative conditions, and their association with clinical markers of disease severity or their relationship to systemic inflammatory markers.11C]PK11195 is a well-established PET marker of in vivo microglial activation,6–14 although microglial activation represents only part of the complex cascade of events in neuroinflammation.15 Here, we assessed the magnitude and patterns of [11C]PK11195 binding in 2 very different neurodegenerative entities, AD and PSP, characterized by distinct anatomical distributions of pathology. The value of this comparison does not lie in the differential diagnosis between these clinically diverse entities but rather in establishing the distribution of neuroinflammation in 2 distinct tauopathies. We tested whether [11C]PK11195 binding relates to the distinctive distributions of pathology in typical amnestic AD and PSP–Richardson syndrome, and whether [11C]PK11195 binding relates to different measures of clinical severity in AD and PSP.-PET scan) (MCI+). Thirteen age-, sex-, and education-matched healthy controls with no history of major psychiatric or neurologic illnesses, head injury, or any other significant medical comorbidity were also recruited. All participants were older than 50 years, had sufficient proficiency in English for cognitive testing, did not have any acute infectious or symptomatic systemic inflammatory disorder , and had no contraindications to MRI. Patients were identified from the specialist clinics at the Cambridge University Hospitals NHS Trust and the Dementias and Neurodegenerative Diseases Research Network, while healthy controls were recruited via the Dementias and Neurodegenerative Diseases Research Network, which is part of the National Institute for Health Research (NIHR) Clinical Research Network (nihr.ac.uk/nihr-in-your-area/dementias-and-neurodegeneration/).The current study was conducted within the context of the NIMROD (Neuroimaging of Inflammation in Memory and Related Other Disorders) Study.All participants had mental capacity, and we obtained informed written consent from patients  and patients' designated informants (for providing informant information) in accordance with the Declaration of Helsinki. The study was approved by the local ethics committee.21 Demographic measures and neuropsychological tests  as well as a blood sample to assess the levels of 3 basic peripheral markers of inflammation  were also obtained from all participants. The CRP, ESR, and white blood cell biomarkers were included on the basis that peripheral inflammation may facilitate the development of neuroinflammation and neurodegeneration,22–24 and such peripheral markers might augment the monitoring of immunotherapeutic trials if related to central inflammation.Participants' assessment included clinical indices of disease severity, such as Rey Auditory Verbal Learning Test (RAVLT) in AD/MCI+ patients and Progressive Supranuclear Palsy Rating Scale (PSPRS) in patients with PSP.medical.siemens.com) using a magnetization-prepared rapid-acquisition gradient echo T1-weighted sequence. The T1-weighted sequence  was used to facilitate tissue class segmentation  and to allow nonrigid registration of standard space regions of interest (ROIs) to subject MRI space . Each T1 image was nonrigidly registered to the ICBM2009a template brain using ANTS (picsl.upenn.edu/ANTS/), and the inverse transform was applied to the modified Hammers atlas (resliced from MNI152 to ICBM2009a space) to bring the ROIs to subject MRI space.All participants underwent MRI on a 3T Siemens Magnetom Tim Trio or Verio scanner , with [11C]PiB having a specific activity >150 GBq/μmol at the end of synthesis, while [11C]PK11195 specific activity was around 85 GBq/μmol at the end of synthesis. PET scanning was performed with a GE Advance PET scanner  and a GE Discovery 690 PET/CT, with attenuation correction provided by a 15-minute 68Ge/68Ga transmission scan and a low-dose CT scan, respectively. The emission protocols were 550 MBq [11C]PiB injection followed by imaging from 40 to 70 minutes post injection, and 75 minutes of dynamic imaging (55 frames) starting concurrently with a 500-MBq [11C]PK11195 injection. Each emission frame was reconstructed using the PROMIS 3-dimensional filtered back projection algorithm into a 128 × 128 matrix 30-cm transaxial field of view, with a transaxial Hann filter cutoff at the Nyquist frequency.25 Corrections were applied for randoms, dead time, normalization, scatter, attenuation, and sensitivity. Each emission image series was aligned using SPM8 to reduce the effect of patient motion during data acquisition (fil.ion.ucl.ac.uk).All participants underwent [11C]PiB, we used reference tissue ROI defined by ≥90% on the SPM8 gray matter probability map (smoothed to PET resolution) in the superior cerebellar cortex.26 For [11C]PK11195, supervised cluster analysis was used to determine the reference tissue time-activity curve.27 All ROI data were corrected for CSF contamination through division with the mean ROI probability  of gray + white matter, using SPM8 probability maps smoothed to PET resolution. To test whether correction for CSF affected the main results, we repeated all the [11C]PK11195 ROI PET analyses using data not corrected for CSF contamination .The mean aligned PET image  was rigidly registered to the T1-weighted MRI. For [11C]PiB data were quantified using standardized uptake value ratio by dividing the mean CSF-corrected radioactivity concentration in each Hammers atlas ROI by the corresponding mean CSF-corrected radioactivity concentration in the reference tissue ROI. For [11C]PK11195, nondisplaceable binding potential (BPND), a measure of specific binding, was determined for each ROI using a basis function implementation of the simplified reference tissue model, both with and without CSF contamination correction.28 [11C]PK11195 BPND maps were also generated using this basis function simplified reference tissue model approach. [11C]PiB data were treated as dichotomous measures  and considered positive if the average standardized uptake value ratio across the cortical ROIs was >1.5.29[11C]PK11195 binding across groups , individual ROI BPND values for [11C]PK11195 were used in a repeated-measures general linear model to test for the main effect of ROI, main effect of group, and group × ROI interaction. Age and sex were included as covariates of no interest. For the AD/MCI+ and PSP groups, we also tested Pearson correlations between regional [11C]PK11195 BPND and disease severity using the RAVLT scores for AD/MCI+ patients and the PSPRS for patients with PSP. Finally, we tested for associations between neuroinflammation and peripheral markers of inflammation using Pearson correlations.To compare PK11195 BPND values that were not corrected for CSF partial volume effects yielded similar results . We then tested whether regional [11C]PK11195 BPND related to disease severity in each clinical group. In the AD/MCI+ group, there was a significant negative correlation between the RAVLT scores  and [11C]PK11195 BPND in the precuneus  and interleukin 1β and transforming growth factor β expression showed a disease-specific topological relationship with the pathologic hallmarks of AD and PSP.3 More specifically, the previous postmortem study3 found that patients with AD had significantly higher microglia density and interleukin 1β expression in the parietal cortices compared to patients with PSP and controls, while the microglia density and cytokine expression was greater in the substantia nigra of patients with PSP relative to patients with AD and controls.3 The expression of transforming growth factor β was also increased in frontal and parietal cortices in patients with AD relative to patients with PSP or controls.3 Together with our findings, these data suggest that microglia activation and cytokine expression coexist with the pathogenic processes underlying AD and PSP and could contribute to the process of ongoing neurodegeneration.3 If so, this would warrant further investigation of immune-therapeutic strategies to modulate neuroinflammation in AD and PSP, although evidence from earlier anti-inflammatory trials in AD remains controversial,37 and no such clinical trials have been conducted in PSP.Our PET data are also in keeping with previous postmortem findings,11C]PK11195 binding correlates with disease severity in both AD/MCI+ and PSP; more specifically, with severity of episodic memory impairment as assessed via the RAVLT in AD/MCI+, and with PSP severity as measured via the PSPRS in PSP. Again, these effects were not global correlations but adhered to the functional anatomy of cognitive and motor symptoms in AD and PSP .Our data also confirmed the hypothesis that PK11195 PET could provide helpful information to stratify patients in future clinical trials or to track the effects of treatments targeting neuroinflammation in neurodegenerative disorders such as AD and PSP. However, to fully meet its potential toward these directions, additional properties are necessary to show for this biomarker of neuroinflammation. Specifically, although recent longitudinal studies in AD have demonstrated that changes in [11C]PK11195 binding may be associated with disease progression,38 such a correlation has not been established in PSP. Neuroinflammation might be stable in symptomatic stages of PSP, as suggested by a pilot study of 2 patients with PSP.10 Furthermore, increased levels of serum neurofilament light protein have been found in both AD and PSP, and correlated with disease severity39–41; hence, future studies may test whether markers of neuroinflammation are associated with neurofilament light protein levels in the serum. Perhaps more importantly, it remains to be determined whether the putative effects of anti-inflammatory therapies can reduce the elevated [11C]PK11195 binding in AD and PSP and, consequently, could help slow the progression of these disorders. This would also enable mediation analysis to test the causality between immune-reactivity and disease progression in dementia and related disorders. Furthermore, we suggest that multitracer PET studies will be useful to formally assess how neuroinflammation relates to other important molecular aspects in dementia and related disorders including, for example, studying how neuroinflammation is associated with amyloid load in AD14 as well as with tau burden in AD and PSP. A cross-sectional and single-tracer study like the present one is not able to address such interesting and open questions, although it represents the necessary first step toward achieving this goal.Overall, the use of [11C]PK11195 BPND PET methods should also be considered. In particular, our main regional PET analyses used partial volume correction for CSF, which controlled for differences in CSF signal contamination within each region and across the different diagnostic groups . Although this approach is important to reduce the potential influence of brain volume loss seen in AD/MCI+ and PSP, this MRI-guided method is subject to error because of imperfect registration of PET and MRIs, together with errors in segmentation and point spread function modeling. However, we note that using uncorrected PET data yielded similar results in terms of the main effect of ROI, main effect of group, and group × ROI interaction, which provides substantiation of the CSF-corrected results. The supervised cluster method for estimating [11C]PK11195 BPND could also have introduced an underestimation bias, as the reference tissue may have still included specific binding of the radioligand. In any case, this may have only reduced the effect sizes without altering the risk of reporting false-positive results.Technical considerations regarding the [11C]PK11195 and do not inevitably generalize to second-generation translocator protein (TSPO) ligands  or alternative tracers of neuroinflammation over and above those that bind to TSPO .42–44 Further studies should assess the utility of such novel markers for in vivo imaging of neuroinflammation, bearing in mind that the binding of second-generation TSPO tracers like PBR28 can be affected by genetic variations .45We also highlight that our data are specific to [11C]PK11195 is a sensitive PET ligand for in vivo studies of neuroinflammation in clinical populations with AD and its prodromal stage of amnestic MCI, as well as in a non-AD tauopathy, PSP–Richardson syndrome. The brain regions that showed increased [11C]PK11195 binding were those predicted from the well-established pattern of regional cortical and subcortical neurodegeneration in each disease. Our data support the further use of [11C]PK11195 PET to study microglia activation in neurodegenerative disorders and in clinical trials that aim to modulate neuroinflammation in neurodegenerative disease.In conclusion, we have provided clear evidence that [


Saccharomyces cerevisiae , [URE3], [SWI+], and [RNQ+]  [PSI+] called [PSI+]Sc37 and a strong variant of [RNQ+] called [RNQ+]STR; even when experiments are conducted in a single strain initially maintaining both prions, either prion can be selected at the expense of the other depending upon which construct of Sis1 is expressed  –20—but tvariants . TogethePSI+] and [RNQ+] [SWI+] revealed that it additionally requires the function of the C-terminal peptide-binding domains of Ydj1, the most abundant J protein in the yeast cytosol [SWI+] propagon fragmentation by virtue of direct binding to [SWI+] aggregates via these domains  , an inve cytosol . Recent  cytosol , 25–28.  domains , 29.URE3] revealed an essential role for the J protein Swa2, the yeast ortholog of the mammalian protein auxilin, normally responsible for initiating the disassembly of the clathrin lattice on clathrin-coated vesicles following endocytosis [URE3], indicating that Swa2’s mechanism of action in prion propagation is independent of its role in mediating clathrin dynamics [URE3] propagation, forming a novel multichaperone complex to increase prion fragmentation efficiency  variants present distinct surfaces for chaperone binding  and [SWI+], are vastly dissimilar from those of Rnq1 and Sup35, which form [RNQ+] and [PSI+] [PSI+] and polyQ aggregates  , 29, 36.d [PSI+] –40. Likegregates –43, and URE3] and [SWI+] exhibit low propagon numbers and relatively large intracellular aggregates, indicating that it may be difficult to productively fragment aggregates of these prions to produce propagons relative to other prions. [URE3] and [SWI+] may then require additional chaperone complexes to increase sites of fiber fragmentation for continued propagation, explaining their secondary J-protein requirements, although some sensitivities to subtle changes in chaperone function may simply be a consequence of reduced propagon numbers alone. However, these hypotheses are based on a limited data set, so further examination of additional prion–chaperone interactions is necessary to resolve them.These observations led us to speculate that perhaps both the variations in heritable prion propagons per cell and sensitivities to ectopic chaperone expression found among yeast prions may be primarily dictated by the amino acid composition of the prion protein . For exaPSI+], [RNQ+], or both when expressed in place of Sis1 in yeast   . Considein yeast , the posBecause J proteins often act as targeting factors for Hsp70s, they may constitute the first chaperone response to the presence of amyloid. Due to the general requirement for Sis1 by all studied yeast prions , the fac