We found that ERα binding sites overlap with a subset of PR binding sites. To investigate further, we re-analysed raw data to remove any biases introduced by the use of distinct tools in the original publications. We identified 22,152 PR and 18,560 ERα binding sites  with 4,358 overlapping regions among the two datasets. BiSA statistical analysis revealed a non-significant overall overlap correlation between the two factors, suggesting that ERα and PR are not partner factors and do not require each other for binding to occur. However, Monte Carlo simulation by Binary Interval Search (BITS), Relevant Distance, Absolute Distance, Jaccard and Projection tests by Genometricorr revealed a statistically significant spatial correlation of binding regions on chromosome between the two factors. Motif analysis revealed that the shared binding regions were enriched with binding motifs for ERα, PR and a number of other transcription and pioneer factors. Some of these factors are known to co-locate with ERα and PR binding. Therefore spatially close proximity of ERα binding sites with PR binding sites suggests that ERα and PR, in general function independently at the molecular level, but that their activities converge on a specific subset of transcriptional targets.Chromatin factors interact with each other in a cell and sequence-specific manner in order to regulate transcription and a wealth of publically available datasets exists describing the genomic locations of these interactions. Our recently published BiSA  database contains transcription factor binding locations and epigenetic modifications collected from published studies and provides tools to analyse stored and imported data. Using BiSA we investigated the overlapping cis-regulatory role of estrogen receptor alpha (ER We selected the E2-treated dataset for our study. Datasets from both studies were of 36 base pair lengths on the Illumina platform. The PR data were generated using an Illumina Genome Analyzer IIx while ERα libraries were sequenced on Illumina HiSeq 2000. The data used in this study have been derived from peer-reviewed publications, suggesting that they are of an acceptable quality, in addition we also ensured standard quality control checks prior to our re-analysis of the raw data. The two studies used different genome assemblies and different tools to align the reads and to call the peaks. Therefore, to remove any biases we re-analysed the raw ERα and PR data. We mapped the raw data to the GRCh37/hg19 assembly using Bowtie version 2  were removed . The OCV ranges from 0 to 1, the closer the value to 1 the stronger the significance of overlap of two datasets. The OCV represents the fraction of regions in the query dataset with a p-value less than a specified threshold. In BiSA, we have set the threshold p-value to 0.05 and used a number of domains such as whole genome and promoter proximal regions for this analysis.Overlapping features were studied in BiSA . BiSA isWe also investigated the spatial correlation of regions of whole datasets being closer to each other by Binary Interval Search (BITS)  and Genoα-PR common cis-regulatory regions using GREAT (Genomic Regions Enrichment of Annotations Tool)  . GREAT iα ChIP-seq data from T-47D breast cancer cells revealed 22,152 PR and 18,560 ERα binding regions with FDR <5%. HOMER motif analysis on the top ranked 1,000 regions by peak score revealed the strong presence of a PRE motif (59.40%) and ERE motif (48.80%)  peaks and known conserved PR and ERα recognition sequences confirmed the success of the alignment and peak-calling process.Analysis of PR and ER(48.80%)  and 2. T(48.80%) . In addi(48.80%) . Interes factors  . The prα  and PR  binding regions were visualised by drawing a histogram and box plot  were greater than 1 kb, whereas most ERα binding regions (∼95%) were less than 1 kb. The longer PR regions may be due to longer input DNA fragment lengths in the original samples  overlap with 3,870 unique PR binding regions. This revealed that some long PR binding regions spanned more than one ERα binding region and the reverse was also true for large ERα binding regions. In total, we found 4,358 sections that were common to the two datasets. The Venn diagram in α and PR binding region is shown in α binding region (red dotted lines) is completely contained within the 813 bp PR binding region (blue dotted lines) and the two regions share the peak centre location  of ERlocation .α and PR binding was statistically significant, statistical analysis was performed in BiSA, BITS and Genometricorr. In BiSA, using a whole genome domain and selecting the ERα cistrome as query and PR as reference revealed an overlap correlation value of 0.33. The value decreased to 0.26 when PR was selected as query and ERα as reference. This showed that, although a considerable proportion of ERα binding regions are also bound by PR, the two receptors do not cooperate for binding at all sites. To determine whether the significance of ERα-PR binding overlap was greater in functionally relevant genomic regions, we compared the level of binding overlap over a range of genomic domains from promoter proximal (within 500 b of a TSS) to more distal regions (α region length (mean = 601) and performed the OCV test again. This did not change the OCV (0.33) for the whole genome dataset, and there was negligible change in OCV observed for other domains   . We repeα-PR were searched for known motifs. Known motif analysis in these common sections revealed a strong presence of ERE, forkhead protein and PRE motifs. In p-value. A PRE motif was found in 41.88%  of the total 4,358 regions, which was much higher than the number of ERE motifs detected 14.3% (623) of the sequences. However, this may reflect the higher stringency of the position specific scoring matrix used to identify ERE motif occurrence than the matrix used to find PRE motifs since the p-value for ERE motif detection (1e–291) was much stronger than the p-value for PRE motif occurrence in the dataset (1e–179). The presence of FOXA1 motifs in these regions confirms that the factor facilitates the binding of ERα and PR on these regions as previously reported  are not associated with any gene and 3,687 (∼85%) regions are associated with 2 genes  where 137 regions were found regulating 24 genes associated with this pathway. The FOXA1 transcription factor network and IL6-dedicated signalling events were also significant terms . Mouse phenotype analysis revealed two breast cancer related ontologies  as the most significant terms. There were 32 regions associated with 5 genes linked to abnormal mammary gland epithelium physiology and 189 regions associated with 52 genes linked to mammary gland development. The We used GREAT (Genomic Regions Enrichment of Annotations Tool)  to inter 2 genes . Most of 2 genes  confirms 2 genes . Gene on 2 genes . Epithel 2 genes , revealeα and PR binding in T-47D cells, utilizing the published binding regions, revealed an overlap of ∼27% of ERα binding regions with the published PR cistrome (data not shown). This suggested an interesting functional relationship between the receptors, which justified further study. To perform a more rigorous exploration of their overlapping binding patterns, we reanalysed the raw ERα and PR ChIP-seq data using a standardized pipeline. This illustrates the great value of BiSA as an easy to implement first pass tool to investigate potential functional relationships in transcription factor binding and epigenomic datasets.The BiSA database provides a good starting point for studying overlapping binding by a range of transcription factors from a comprehensive collection of published studies . The datp-values calculated by the IntervalStat tool and reflects the overall correlation of two binding site datasets. IntervalStat calculates a p-value for each query region against the closest reference region within the given domain. It is designed to identify factors that target the same genomic locations. As described in examples in our previous study  represents a statistical summary value of the set of us study  the OCV us study . Similarus study . Therefoα and PR binding regions were highly statistically significant binding sites for both receptors, as determined by a strong p-value and low FDR value in MACS, suggesting that these are biologically valid binding regions for these receptors and that their overlap reflects converging function on a subset of gene targets.A consistent overlap was found both proximal and distal to gene promoters . It is aα binding regions in the MCF-7 cell line  to observe the impact of converging ERα and PR regulation in comparison to individual stimulation.The ERt cancer . PR itseα in a subset of binding sites in breast cancer cells. Our analysis demonstrated the utility of our previously published software BiSA  suggesting that the two factors are not obligate cofactors. However, spatial correlation testing using Monte Carlo simulation by BITS, Relevant Distance, Absolute Distance, Jaccard and Projection tests by Genometricorr revealed a statistically significant correlation between the two factors. In addition, the discovery that ERα, FOXA1, PR, AP-2 and TEAD4 binding motifs are significantly enriched in regions that are bound by both ERα and PR suggests that their overlap is biologically relevant.In summary, we have evidence for a biologically relevant interplay between PR and ERare BiSA , which h10.7717/peerj.654/supp-1File S1Click here for additional data file.


Arabidopsis thaliana to correlate differences in PSII organization with the role of SOQ1, a recently discovered thylakoid protein that prevents formation of a slowly reversible NPQ state. We developed a statistical image analysis suite to discriminate disordered from crystalline particles and classify crystalline arrays according to their unit cell properties. Through detailed analysis of the local organization of PSII supercomplexes in ordered and disordered phases, we found evidence that interactions among light-harvesting antenna complexes are weakened in the absence of SOQ1, inducing protein rearrangements that favor larger separations between PSII complexes in the majority (disordered) phase and reshaping the PSII crystallization landscape. The features we observe are distinct from known protein rearrangements associated with NPQ, providing further support for a role of SOQ1 in a novel NPQ pathway. The particle clustering and unit cell methodology developed here is generalizable to multiple types of microscopy and will enable unbiased analysis and comparison of large data sets.Photoautotrophic organisms efficiently regulate absorption of light energy to sustain photochemistry while promoting photoprotection. Photoprotection is achieved in part by triggering a series of dissipative processes termed non-photochemical quenching (NPQ), which depend on the re-organization of photosystem (PS) II supercomplexes in thylakoid membranes. Using atomic force microscopy, we characterized the structural attributes of grana thylakoids from  Arabidopsis thylakoids, the major type of supercomplex found is identified as C2S2M2, and consists of two reaction center core (C) complexes, two strongly (S) bound LHCII trimers, and two additional moderately (M) bound LHCII trimers; supercomplexes lacking one or both M-trimers are also observed  Plants are exposed to fluctuations in light quantity and quality, and therefore need to balance productive photochemistry and dissipative photoprotection. This goal is achieved by dynamic regulation of the structure and organization of pigment-proteins throughout the thylakoid membrane. In particular, photosystem II (PSII) and its closely associated light-harvesting complex II (LHCII) form supercomplexes within the grana that undergo reversible molecular modifications and large-scale rearrangements 2S2, C2S2M, or C2S2M2 particle with a different placement and orientation Electron and atomic force microscopies have been valuable tools for characterizing the global morphology and the distribution of photosynthetic complexes within the thylakoid membrane. In particular, AFM scanning is gentle enough to preserve the membrane integrity, and the resolution is high enough to comfortably assign PSII supercomplexes and characterize their spatial distribution 2S2M2 unit cells Because many grana membranes appear to be at crystal-fluid phase coexistence in vivo, separating particles with crystalline local environments  from those with disordered local environments (''disordered particles'') is a common step in the analysis of nano-resolution micrographs, even when the crystalline unit cell has not been previously characterized. Although qualitative distinctions have been drawn between sets of Csuppressor of quenching 1 (soq1) grana membranes from Arabidopsis. SOQ1 is a recently discovered thylakoid protein that functions in a novel NPQ pathway soq1 membranes may give us insights into the mechanism of this new quenching pathway.To demonstrate the usefulness of our PSII unit cell analysis methodology, we applied it to a high-resolution AFM data set of wild-type (WT) and soq1 grana membranes collected before and after exposure to excess light were imaged by high-resolution AFM, and it was found that our analysis is robust enough to be consistent with previous reports of total crystallinity in WT, while powerful enough to quantitatively discriminate between coexisting PSII crystals. Based on detailed analysis of nearest-neighbor organizational motifs within the crystalline and disordered phases of PSII complexes, we determined the first PSII nanoscale structural signatures of soq1 mutant membranes and compared them with those known for WT. Our findings suggest that protein-protein interactions are altered in the absence of SOQ1, leading to changes in typical PSII separations in the fluid phase and in typical PSII unit cells in the crystalline phase. These structural properties are likely to affect the reorganization dynamics of PSII and LHCII during illumination, and thus the photoprotective responses.WT and soq1 mutant grown in control light (175 µmol photons m−2 s−1) or exposed to photoinhibitory high light .PSII-enriched (grana) membranes were prepared from Arabidopsis leaves of the WT and AFM showed a heterogeneous mixture of grana membrane patches, as shown in Each grana disc was densely packed with particles, with approximate diameters of 15–25 nm and protrusions above the lipid bilayer of 2–4 nm. High-resolution images revealed internal structure within the particles  coexist a and b of the two Bravais lattice vectors, and the angle ϑ between them. Feature sets for local positional order that are prevalent in the literature, including in the fields of shape-matching To determine the local unit cell around each PSII particle, our algorithm fits a Bravais lattice to the coordinates of other particles in the central particle's nearest-neighbor shell . Bravais unit cells are convenient for our analysis because they are intuitive and lower-dimensional than these feature sets, being characterized simply by the lengths k that produces the most meaningful clustering; additional model selection criteria are necessary if k is not known a priori. We used Bayesian Information Criterion (BIC) to select the best value of k for many bootstrap-resampled data sets k = 6 was most frequently selected as the best number of clusters. Yet overall, k = 6 was selected in only 27% of the bootstrap resamplings, while k = 5, 7, and 10 were each selected in 15–20% of resamplings. While this model selection process guided us to present results with k = 6 in this work, it also suggests that one should use caution when drawing conclusions about the “ground truth” number and identity of classes of PSII unit cells in this data set. For all three features, the mean µ and standard deviation σ of each Gaussian mixture component of the non-resampled model fell within the bootstrapped 95% confidence interval  clustering method. A GMM is a probabilistic model that fits several multivariate Gaussian distributions to the data, typically using the expectation-maximization algorithm interval .2S2M2 data set of Kouřil et al. (2013), allowing us to assign it as being composed of C2S2M2 particles. Classes (b) and (c) had slightly smaller average areas than class (a); although both are large enough to accommodate a C2S2M2 supercomplex, we cannot rule out the possibility that these classes are composed of slightly smaller supercomplexes . We assigned class (d) as C2S2 because it contained the C2S2 unit cells 2S2 unit cells b value, which manifests as a larger spacing between rows and the largest average area, and was almost entirely composed of C2S2M2 unit cells like those in Kouřil et al.’s “normal light” and “low light” conditions 2S2M2 unit cells from Ref. 9, as well as some outliers with unusually acute angles (θ<65°) that may be artifacts of the unit cell determination algorithm.soq1 mutant membranes, we collected a larger data set of lower-resolution AFM micrographs (500 nm×500 nm). Each PSII particle in these images was assigned to its maximum-likelihood crystal class based on our GMM, or assigned as disordered if its local unit cell was absent or an outlier. To evaluate the effect of inhibitory light on the WT and Examination of the classification results for representative images  revealedsoq1 membranes: the average net crystallinity decreased from 9.2% to 3.5% in WT membranes, but only from 8.1% to 5.3% in soq1 membranes  by approximately 2-fold. The fraction of particles in class (b) was 0.017–0.020 in all conditions except low-light-acclimated soq1, where the fraction was more than 2.5 fold higher. Photoinhibitory treatment had opposite effects on the fraction of particles in class (c) in WT and soq1 membranes: the fraction decreased in the former and increased in the latter.The frequencies of different crystal types were also found to depend on the light treatment and the Surprisingly, some crystalline domains appeared to alternate rows of wide and tall particles with short and narrow ones . To invesoq1 membranes was very pronounced  for particles in micrographs with no crystalline particles . The NNDAlthough diverse types of PSII supercomplex crystalline arrays have been detected in both intact and partially solubilized thylakoid membranes from several species for decades, our understanding of the biological implications of such crystalline organization is still developing. Several laboratories have used a variety of high-resolution microscopy techniques to correlate the PSII propensity to form ordered arrays with photon starvation soq1 mutant Arabidopsis thaliana grana membranes treated with control or excess light conditions to test the method's robustness and potentially gain further insights on the structural organization of this mutant.With our crystal classification methodology , we have developed an objective tool for structural comparisons between membranes exposed to diverse environmental conditions and with different genetic backgrounds. We applied this methodology to detect differences in crystalline structural features between WT and Arabidopsis chloroplasts, and the reported fraction of crystalline membranes agrees with those from solubilized membranes, including our results In control light conditions, our results indicate that, regardless of the genetic background, a small fraction of particles are organized into crystalline arrays  . Under csoq1 membranes. Our results agree with observations reported for fully high-light-acclimated membranes, in which the fraction of crystal-containing micrographs was severely decreased We found that even relatively short exposure (90 min) to high light triggers crystal dissolution in both WT and 2S2M2 particle at the center of each unit cell and determining particle orientations that do not result in steric clashes. Our models for the dominant classes (a), (b), and (c) are depicted in 2S2M.Based on the nanoscale AFM data, we propose internal structures for each PSII-LHCII crystal class by placing a COur crystal classification revealed that photoinhibitory conditions have specific effects on individual types of crystals, and that those effects are different in the absence of SOQ1. The molecular basis by which the recently discovered SOQ1 protein induces high-light NPQs independent of pH gradient, zeaxanthin accumulation, or LHCII phosphorylation are unknown soq1 control-light membranes, indicates that it may be stabilized by different protein-protein interactions and that a cooperative transition would be necessary to convert between crystal (b) and the other crystals. Slight changes to the relative organization of chlorophyll transition dipoles can have dramatic effects on fluorescence and energy transfer efficiency and/or pathway The modeled molecular arrays suggest an intriguing hypothesis for the structural modulations that occur in the absence of SOQ1. Crystals (a) and (c) may be able to locally interconvert via slight rotations or translations while maintaining similar antenna contacts. On the other hand, the rotational restriction and distinct minor antenna contacts of crystal (b), which dominates in In addition to particle locations, AFM micrographs contain additional important information about particle size and shape, which we studied by segmentation analysis . SurprisThis result is unexpected based on previous EM studies of PSII crystalline arrays, which (to the best of our knowledge) have only observed crystals composed of homogenous PSII supercomplex unit cells. This discrepancy can be explained if the two populations of particles that we detect have differently-sized protrusions above the membrane but indistinguishable projected electron densities, or if multiple populations of electron densities have been present in EM micrographs but tend to be averaged together during data analysis. We cannot rule out membrane distortions during deposition or drying due to surface defects, inhomogeneous electrostatic interactions, or membrane rippling.Our finding is also intriguing from the perspective of the thermodynamics of crystallization: a perfect crystal contains homogeneous unit cells by definition, and is destabilized by defects that disrupt steric or attractive energetic contacts between particles. On the other hand, inhomogeneities that have a negligible effect on inter-particle interactions will have a negligible effect on crystal formation and stability. For instance, we speculate that mixtures of PSII supercomplexes with identical antenna organization but different extrinsic protein subunits could form co-crystals like those we observe. Such mixtures could occur if variation in extrinsic proteins exists in vivo, as has been suggested soq1 mutation also affects the local structure within the fluid phase of the grana membrane. In control light conditions, disordered PSII complexes in soq1 grana are significantly more densely packed than those in WT grana  at 21.5 °C for 8 weeks. The growth of plants and preparation of grana membranes from WT and soq1 were done at the same time in order to exclude differences that might arise from growth conditions and/or sample preparation. For high-light treated samples, several plants were exposed to 1,200 µmol photons m−2 s−1 for 90 min before isolating thylakoids. Grana membranes were isolated as described n-dodecyl-α maltoside (α-DM), 15 mM NaCl, 5 mM MgCl2 was added and incubated with gentle rocking for 20 min at 4 °C in the dark. The detergent solubilization was carefully adjusted to insure high concentration of isolated but intact grana discs; α-DM for 20 min was used rather than Triton X-100, as this treatment yielded larger and more homogeneous membranes (as shown in Wild-type and soq-1 mutant low-light-acclimated (soq1) and photoinhibited (soq1-PI) membranes. Grana aliquots were deposited on freshly cleaved mica , and incubated at room temperature for 1–3 hours. Mica was rinsed with water ten times and dried under N2 gas flow. AFM measurements were performed with a Multimode AFM Nanoscope V (Bruker Co.). The samples were imaged in tapping mode; the silicon cantilevers (Nanosensors) were excited at their resonance frequency (280–350 kHz) with free amplitudes of 2–15 nm. The image amplitude (set point As) and free amplitude (A0) ratio (As/A0) was kept at ∼0.8. All samples were imaged at room temperature in air, at a relative humidity of 30%. More than eight different grana patches were scanned per each type of membrane. Bi-layered patches were fully mapped at scans of 500 nm×500 nm. Higher resolution of 150 nm×150 nm images were also recorded.Four different types of samples were imaged: WT low-light-acclimated (WT) and photoinhibited (WT-PI) grana membranes; and Raw AFM images were flattened and leveled using Gwyddion 2.3 i at position ri, a local unit cell ⊝i =  was extracted by the following algorithm.For particle iA of neighboring particles j around particle i, iA = [rj | rmin<ijr<rmax], where the cutoffs rmin = 14 nm and rmax = 33 nm are chosen to select the first PSII coordination shell based on typical PSII g(r) data. Let in be the number of particles in iA.1. Determine the set iB centered at ri spanned by the real-space lattice vectors  that minimizes the penalty function π(i)Br = 7 nm. COBYLA π(i) subject to the constraints rmin<|ui|<rmax and rmin<|vi|<rmax.2. Find the Bravais lattice in<4 or π(i)>πmin = 0.2, then ⊝i does not exist. Otherwise, ⊝i exists; continue.3. If rk in iB in polar coodinates, rk = uknui+vknvi = . For any pair of distinct points rk, rl in iB, define the unit cell ⊝kl =  by kla = kr, klb = lr, and klϑ = kϑ–lϑ ∈ ]. Then we select ⊝i viakla<klb+er and ϑmin<klϑ<ϑmax. The angular parameters ϑmin = 50°, ϑmax = 120°, and ϑ* = 75° were chosen to favor unit cells in the range of the unit cells in er = 1 nm is a small error term on the scale of the pixel size that allows the angular constraints to be satisfied consistently when kla≈klb. The cutoff parameters were generously chosen to favor a small fraction of false positives , which could be screened out later during classification.4. We can write any point ia, ib, iϑ) for each of 101 unit cells from published PSII crystalline arrays k of components in the Gaussian mixture, 1000 bootstrap-resampled data sets were generated and the best value of k in the range 1–10 was selected for each data set via BIC . Second, a spatial k-nearest neighbor majority-rule filter, where k is the number of particles within a radius rmax of particle i, was used on the categorical output of the classifier to reduce the noise.For classification, a two-step pipeline was used. First, a probabilistic classifier was constructed from the We have implemented the algorithms for unit cell identification, clustering, model selection, and classification in Picolo. Picolo is a Python package for analyzing local spatial order in sets of 2d coordinates, which we have made freely available on GitHub Figure S1AFM micrographs of grana thylakoid solubilized under different detergent conditions.(DOCX)Click here for additional data file.Figure S2Model selection metrics for the Gaussian mixture model.(DOCX)Click here for additional data file.Figure S3Histograms of net particle crystallinity by AFM image.(DOCX)Click here for additional data file.Figure S4Structural models of the six types of crystal arrays clustered by Picolo analysis package.(DOCX)Click here for additional data file.Table S1Gaussian mixture model parameters, with bootstrapped 95% confidence intervals.(DOCX)Click here for additional data file.Table S2Comparison of grana complexes height and area parameter distributions from representative patches with different type of packing.(DOCX)Click here for additional data file.Table S3Comparison of nearest neighbor distribution's fitting parameters.(DOCX)Click here for additional data file.


Although a moderate decrease in SPAK phosphorylation and a marked increase in WNK1 expression were evident in the kidneys of WNK4−/− mice, the amount of phosphorylated and total NCC decreased to almost undetectable levels, indicating that WNK4 is the major WNK positively regulating NCC, and that WNK1 cannot compensate for WNK4 deficiency in the DCT. Insulin- and low-potassium diet-induced NCC phosphorylation were abolished in WNK4−/− mice, establishing that both signals to NCC were mediated by WNK4. As shown previously, a high-salt diet decreases phosphorylated and total NCC in WNK4+/+ mice via AngII (angiotensin II) and aldosterone suppression. This was not ameliorated by WNK4 knock out, excluding the negative regulation of WNK4 on NCC postulated to be active in the absence of AngII stimulation. Thus, WNK4 is the major positive regulator of NCC in the kidneys.By analysing the pathogenesis of a hereditary hypertensive disease, PHAII , we previously discovered that WNK (with-no-lysine kinase)–OSR1/SPAK  cascade regulates NCC (Na–Cl co-transporter) in the DCT  of the kidney. However, the role of WNK4 in the regulation of NCC remains controversial. To address this, we generated and analysed WNK4 The analyses of WNK4 (with-no-lysine kinase 4) knockout mice help to end a long-standing controversy about the role of WNK4 on NCC (Na–Cl co-transporter) regulations in the kidney. WNK4 is a strong positive regulator of NCC. WNK (with-no-lysine kinase) 1 and WNK4 were identified as the genes responsible for PHAII , high- [4% NaCl (w/w)] or low- [0.01% (w/w)] sodium diets for 7 days. As shown in −/− mice was significantly lower than that of WNK4+/+ mice during the resting period , the DCT were dilated, cell height was decreased and staining for NCC was reduced in the WNK4−/− mice. The WNK1 antibody used does not usually detect significant WNK1 expression in the kidneys of WNK4+/+ mice. However, increased WNK1 expression, co-localized with NCC expression, was evident in WNK4−/− mice, suggesting that a compensatory increase in WNK1 occurred in the DCT, but that this was insufficient to maintain NCC activity. This compensation might be caused by the increased transcription of the WNK1 gene and/or by the reduced degradation of WNK1 protein. WNK3 protein expression was not detected by immunoblotting in the kidneys of either WNK4+/+ or WNK4−/− mice (results not shown), confirming that WNK3 is not a major WNK in the kidneys in vivo  for 7 days. A low-potassium diet-induced increase in total and phosphorylated NCC observed in WNK4ium diet A. We hypium diet B.−/− mice by administering insulin intraperitoneally at a dose of 5 U/kg, and killed the mice 60 min after injection. As shown in −/− mice, indicating that the effect of insulin signalling on SPAK and NCC is exclusively mediated by WNK4.We then performed an acute insulin infusion experiment in WNK4in vivo [+/+ and WNK4−/− mice  responsible for PHAII [in vivo evidence validating the negative effect of wild-type WNK4 on NCC suggested by in vitro studies [There is now general consensus that the phosphorylation status of NCC reflects its activity . NCC phoactivity , and alsactivity ,35. We hactivity ,11. We bactivity . In thisin vitro . Furtheror PHAII ,15 suppoor PHAII . Before or PHAII  reported studies . However studies  should b studies .−/− mice exhibited an almost complete absence of total and phosphorylated NCC on immunoblots, consistent with the data of Castañeda-Bueno et al. [in vivo. As shown by our results, NCC was never up-regulated or activated by the deletion of WNK4 even in the absence of AngII stimulation. We are not denying the inhibitory effect of wild-type WNK4 on NCC observed in Xenopus oocytes [in vivo, overwhelmed by the strong positive regulation of NCC by WNK4 via its phosphorylation. The importance of phosphorylation to the regulation of NCC is confirmed by the absence of PHAII phenotypes in NCC TG mice [In this study, WNK4o et al. . Althoug oocytes ,6 and cu oocytes . However TG mice . An incr−/− mice and those used by Castañeda-Bueno et al. [−/− mice did not exhibit hypokalaemia. We observed a slight but significant decrease in total and phosphorylated SPAK in our WNK4−/− mice, and an increase in total OSR1, but those used by Castañeda-Bueno et al. [−/− mice is not necessarily unexpected. It is also true that data on serum potassium in mice vary significantly depending on how blood samples are collected . As for the differences in SPAK and OSR1, these may originate from the antibodies used in the two studies.We observed some minor phenotypic differences between our WNK4o et al. . WNK4 kno et al.  exhibiteo et al.  did not o et al. , suggest−/− mice, further establishing the minor involvement of WNK3 in NCC regulation in the kidneys. However, this increase in WNK1 in the DCT may be insufficient to compensate for WNK4 deletion, as NCC phosphorylation was significantly decreased in WNK4−/− mice. Although insufficient to compensate for the absence of WNK4, this finding suggests that WNK1 has the same role as WNK4 in the DCT. As no controversy surrounds the positive influence of WNK1 on NCC, this data also supports the positive role of WNK4 in NCC regulation. An increase in WNK1 expression was also evident in WNK4−/− mice fed a low-potassium diet (−/− mice fed a low-potassium diet occurs in nephron segments other than the DCT.We also obtained data on WNK-related molecules not described in the study by Castañeda-Bueno et al. . We demoium diet B. Increa−/− mice. Previously, ROMK was shown to be inhibited by wild-type WNK4, and strongly inhibited by PHAII-causing mutant WNK4 [WNK4D561A/+ PHAII model mice crossed with Osr1 and Spak knock-in mice, we showed that the pathogenesis of hypokalaemia is also the result of NCC activation by WNK–OSR1/SPAK signalling, but is not caused by a direct effect of mutant WNK4 on ROMK. However, the effect of wild-type WNK4 on ROMK has not been clarified in vivo. We observed a clear increase in ROMK in the immunoblots of the total kidney samples from WNK4−/− mice exhibiting normal serum potassium levels. Although further detailed analyses focusing on intracellular localization are necessary, our data suggest that wild-type WNK4 behaves as a negative regulator of ROMK in the kidneys, consistent with in vitro studies [For the first time, we have presented the status of ROMK in WNK4ant WNK4 . Through studies .−/− mice under various conditions clearly show that WNK4 is a strong positive regulator, and never a negative regulator, of NCC in the mouse kidney in vivo.In conclusion, data obtained from our WNK4


The primary reservoir of LASV is the multimammate rat , which is found throughout sub-Saharan Africa. LASV outbreaks among humans occur only in West Africa in 2 noncontiguous areas: 1 in Guinea, Liberia, and Sierra Leone; and 1 in Nigeria. Rare cases and evidence of exposure of humans have been documented in neighboring countries   dry bushland in northern Côte d’Ivoire, around Korhogo . Traps wM. natalensis captured in Gbalôhô, near Korhogo . This site is much farther north in Côte d’Ivoire than previously examined sites (Tissue samples were collected from all animals according to standard protocols. Total nucleic acids were extracted from lung samples and tested for the presence of LASV RNA by using a real-time PCR system amplifying a 400-bp fragment of the large genomic segment . LASV RNo online  Figure. o online  Figure. Further studies will be needed to investigate the geographic distribution of LASV in Côte d’Ivoire and the frequency of human infections. The current lack of diagnosed cases in the area may be caused by underreporting. Sensitization campaigns are needed to increase awareness of the risk for LASV infection among the local population and to improve detection of cases by health workers.Detailed methods and results, Lassa virus in multimammate rats, Côte d’Ivoire, 2013.


The results of the Bayesian mixing model (SIAR) however showed little difference in the diets of riverine (0 m), riparian (10 m) and upland (25 m) spiders. We found that aquatic insects made up ∼50% of the diet for web-spinning spiders collected at 0 m, 10 m, and 25 m from the stream. This study highlights the importance of aquatic insects as a food source for web-spinning spiders despite the taxonomic differences in assemblages at different distances from the stream.Web-spinning spiders that inhabit stream channels are considered specialists of aquatic ecosystems and are major consumers of emerging aquatic insects, while other spider taxa are more commonly found in riparian forests and as a result may consume more terrestrial insects. To determine if there was a difference in spider taxa abundance between riverine web-spinning spider assemblages within the stream channel and the assemblages 10 m into the riparian forest, we compared abundances for all web-spinning spiders along a headwater stream in El Yunque National Forest in northeast Puerto Rico. By using a nonmetric dimensional scaling (NMDS) abundance analysis we were able to see a clear separation of the two spider assemblages. The second objective of the study was to determine if aquatic insects contributed more to the diet of the spider assemblages closest to the stream channel and therefore stable isotope analyses of  Riparian zones have been identified as areas of high importance for maintaining biodiversity in aquatic and terrestrial habitats along with being an important interface for the exchange of resources, resulting in an ecosystem with unique environmental dynamics . The impTetragnatha (Tetragnathidae) and Wendilgarda (Theridiosomatidae)   . The dismatidae) . The genarctica) . Juvenileshwater . Wendilg insects . Along w insects .δ15N and δ13C into the tissue of the consumer. A third advantage is that naturally occurring stable isotopes have been shown to be effective at identifying the contribution of different prey items in the diets of consumers through the use of mixing models  . Anotherg models . This fig models .This study had two main objectives. The first was to determine if there were differences in the composition of taxa in the assemblages of web-spinning spiders that were found over the stream channel compared to those 10 m from the stream. Because some spiders are specialists of aquatic ecosystems we predicted that these taxa would be in far greater abundance over the stream channel. The second objective was to determine if there were differences in the diets of these two assemblages using stable isotopic analyses. Most emerging aquatic insects remain very close to the stream channel and their abundance can drop exponentially only a few meters into the riparian area , so we pDacryodes excels Vahl) forest which is the dominant tree species in the Luquillo Mountains up to 600 m in elevation (Sloanea berteriana Choisy) and palms (Prestoea montana Graham and Nicolson).The macroinvertebrate community of Quebrada Prieta is diverse and is composed of a variety of aquatic insects with the most abundant being trichopterans and ephemeropterans . During A 100 m reach of Quebrada Prieta was selected and then divided into four 25 m subsections. Field work for this portion of the project was conducted from April to August 2012, with at least one week between sampling dates to minimize the possibility of impacting the study area. A 3 m × 3 m riverine quadrat was selected within the stream channel in the first 25 m section of stream, measuring 3 m from the stream edge into the stream channel. Each quadrat was selected to contain a random mixture of available substrates, such as boulders, vegetation and deadwood which may affect web-spinning spider distribution. All web-spinning spiders within the quadrat up to 2.5 m in height were hand collected and preserved in 70% ethanol for later identification. This process was then repeated for a riparian site 10 m laterally from the stream edge into the riparian forest from the riverine sampling site. The same sampling procedure was then repeated for the next 25 m section of the stream. On the following sampling date we would sample the remaining two 25 m sections of the stream not sampled during the previous visit.Tetragnatha and Chrysometa) are more active at night and rarely build webs during the day. Nocturnal sampling on average was conducted from 19:00–23:00, while diurnal sampling on average was from 10:00–14:00. Sampling was only conducted during favorable weather conditions, because spider webs are many times easily destroyed by wind and rain  analysis along with a post-hoc Analysis of Similarity (ANOSIM) were used to determine if there were differences in taxa composition of the two web-spinning spider assemblages. A secondary post-hoc analysis, Similarity Percentages (SIMPER), was used to determine which particular taxa of spiders were causing a difference in the composition of the two assemblages. All of these analyses were conducted with the statistical program PAST .To verify that the spider assemblages and their prey had stable isotope signatures that fell within realistic ranges of the basal C sources we sampled the three principal energy sources for aquatic and terrestrial arthropods. The three C sources sampled were stream leaf litter, periphyton and terrestrial vegetation. Stream leaf litter was collected at random throughout the 100 m stream transect and was gently rinsed to remove any macroinvertebrates. Periphyton was also sampled randomly by collecting rocks from the stream, gently rinsing them to remove any macroinvertebrates, and then scrubbing them with a small wire brush. The resultant slurry was then collected into glass vials to be dried later. For terrestrial vegetation samples, green leaves were collected at random from C3 plants within the riparian forest.Possible insect prey of the spider assemblages were collected for isotope analysis using two methods. Flying insects were collected using a passive sampling method with three Malaise traps that were placed within the stream channel for approximately four hours during the diurnal and nocturnal spider sampling. Traps were placed within three different sections of the stream reach  during each sampling period. This was done in order to have a representation of the available prey of web-spinning spiders flying along the stream channel. Aquatic insect larvae were collected using hand nets throughout the 100 m stream reach. Sampling was conducted in pools, riffles, and cascades to ensure that all major microhabitats were sampled. The larval stages of Ephemeroptera, Trichoptera and Chironomidae were used for isotopic analysis because they no longer feed as adults and thus their isotopic signature is fixed during the aquatic larval stage.δ15N and δ13C stable isotope signals for the different spider assemblages, individuals were collected from a riverine transect within the stream channel, from a riparian transect 10 m parallel from the stream edge and from an upland transect 25 m parallel from the stream edge. In each transect, web-spinning spiders were collected from the four most abundant families: Tetragnathidae, Theridiosomatidae, Pholcidae and Uloboridae. Spiders were collected and maintained live in small containers for a day, to allow for the digestion of prey that may have been recently consumed to reduce the influence of the isotopic signal from what they were consuming.To compare the Wendilgarda clara Keyserling 1886, identified to species). Composite taxa samples of a minimum of four individuals for spiders 1 ± 0.05 mg of animal tissue and 5 ± 0.05 mg of plant tissue was measured for the natural abundances of 15N and 13C using ratio mass spectrometry at the Miami Stable Isotope Ecology Lab at the University of Miami in Florida. Natural abundances of stable isotopes for δ13C and δ15N were calculated as: Rsample = 13C:12C or 15N:14N ratio in the sample and Rstandard = 13C/12C ratio in Pee Dee Belemnite for δ13C and Rstandard = 15N/14N ratio in the atmosphere for δ15N (Specimens were frozen (−20 °C) for a minimum of 24 h, then placed in a drying oven for a minimum of 48 h (70 °C) and finally ground to a fine powder for isotopic analysis. Insects were identified to family (except for Lepidoptera identified to order) and spiders were identified to genus , filterers (n = 2), predators (n = 3), scrapers (n = 2) and shredders (n = 2) (n = 3) or predacious (n = 2). Terrestrial dipterans were only identified to order, and due to their varied feeding behaviors they were not placed into a particular feeding group. The isotopic values for the five aquatic functional groups were then combined into a single aquatic insect group and the values for the three terrestrial insect groups  were combined as well. Spider taxa were identified to genus and were grouped as either having been collected in riverine (n = 7), riparian (n = 5) or upland (n = 5) transects. Mean averages of δ13C and δ15N for each group  were used in subsequent biplots and dietary analyses.The stable isotopes  (n = 2) . Terrestδ13C: 0.08 ± SD 1.90 and δ15N: 2.75 ± SD 2.20) were adopted from the work by Dietary analyses were conducted utilizing Bayesian mixing models in the SIAR package version 4.2 with Stable Isotope Analysis in R (SIAR)  for R veTheridiosoma sp. and Wendilgarda clara (Keyserling)   were collected in varying abundances from riverine and riparian quadrats . The leaerling)  . The secragnatha . Ulobories genus . Pholcidus genus . There wp < 0.002, R = 0.722)  . An addiδ13C in terrestrial vegetation, periphyton and stream leaf litter. Terrestrial vegetation (−34.90‰) was more depleted in δ13C than aquatic periphyton (−32.40‰) and stream leaf litter (−25.50‰)  and periphyton (−0.80‰) while stream leaf litter had the highest δ15N value (0.80‰). Despite these differences in δ13C, there was no clear separation between aquatic and terrestrial C signatures.Stable isotope analyses of the basal C sources showed a difference of −25.50‰) . δ15N vaδ13C and δ15N values for individual insect groups varied among taxa. The family Helicopsychidae (Trichoptera) was the most depleted in δ13C (−34.88‰), while the family Lampyridae (Coleoptera) was the most enriched in δ13C (−25.30‰) , while Lampyridae, a terrestrial predator, was not only the most enriched in δ13C but was also the most enriched in δ15N (6.31‰) , followed by collector-gatherers (−26.63‰), aquatic predators (−27.26 ± SD0.68‰), terrestrial herbivores (−28.05 ± SD0.91‰), shredders (−28.55‰), filterers (−28.59 ± SD1.17‰) and scrapers (−31.52 ± SD4.75‰). Terrestrial predators (5.07 ± SD1.75‰) were the most enriched in δ15N, followed by aquatic predators (4.35 ± SD1.20‰), filterers (3.16 ± SD0.76‰), collector-gatherers (2.63‰), scrapers (2.27 ± SD0.45‰), shredders (0.78‰) and terrestrial herbivores (0.69 ± SD1.76‰). When insect taxa were grouped together and analyzed as either terrestrial (n = 5) or aquatic (n = 10), no significant difference was found in δ13C and δ15N values between the two groups  were overall more enriched than aquatic insects (−28.66 ± SD2.69‰). Terrestrial insects (3.17 ± SD2.60‰) were also more enriched in δ15N although they also showed greater variation than aquatic insects (3.04 ± SD1.47‰).−25.30‰) . The fam (6.31‰) . There wo groups . Althougδ13C and δ15N values than was seen in the insect taxa. The genus Miagrammopes (Uloboridae) along the upland transect was the most depleted in δ13C (−33.30‰), while Theridiosoma] (Theridiosomatidae) also from the upland transect was the most enriched in δ13C (−26.50‰) (Chrysometa (Tetragnathidae) from the riverine transect had the highest δ15N value (5.19‰), while upland Miagrammopes (Uloboridae) had the lowest δ15N value (0.10‰) , followed by riparian spiders (−27.62 ± SD0.52‰) and finally upland spiders (−28.30 ± SD2.82‰) , followed by riparian spiders (3.76 ± SD0.89‰) and finally upland spiders (2.92 ± SD1.93‰)  . The gen (0.10‰) . There wSD2.82‰) . SimilarSD1.93‰) . The gre spiders .Bayesian mixing models determined that the proportion of aquatic insects in the diets of the three spider groups was relatively similar. However, the proportion of aquatic insects in the diets of the spiders increased slightly in the groups further away from the stream channel . The anaTetragnatha and Wendilgarda), within the stream corridor compared to 10 m into the riparian forest. We then proposed that because the majority of aquatic insects congregate within only a few meters of the stream, that the riverine spider assemblage in the stream corridor would be consuming more aquatic insects than riparian and upland spiders. We found that there was indeed a significant difference between the riverine and riparian assemblages and that around 48% of the dissimilarity between the assemblages was attributed to the abundance of Wendilgarda, a specialist of aquatic habitats. The results did not entirely support our second hypothesis. The analyses of stable isotopes showed no clear separation between the δ13C signature for aquatic and terrestrial prey due to the fact that the aquatic food web was driven by leaf litter inputs from the terrestrial vegetation that resulted in similar δ13C ranges for both terrestrial and aquatic primary consumers. As a result, the biplot of δ13C and δ15N showed significant overlapping of the three spider groups along with the aquatic and terrestrial insects. This resulted in the inability to visually separate the consumer groups or their prey. The Bayesian SIAR dietary analysis showed that the upland group of spiders relied the most heavily on aquatic insects (50–53%) although only slightly more so than riverine (45–47%) and riparian (47–49%) spiders  depending on several factors such as plant taxa, water velocity, and canopy cover , prey items  and web spinning spiders   were all the two . The difpy cover . Basal cδ13C signal, −28.05 ± 0.91‰ and −26.06 ± 1.08‰ respectively. The enriched δ13C signal in the predators is most likely associated with bioaccumulation more so than a change in C sources. Of all the insect groups, terrestrial herbivores and predators had the lowest and highest δ15N values, respectively, similar to what was reported in a study done by Isotopic values of insect taxa were all found to be within the range of basal C sources; however there was no clear separation in the isotopic signals between terrestrial and aquatic insects . Terrestδ13C signature for the aquatic insect groups, as mentioned earlier, was not statistically different from the terrestrial insects and most of the functional feeding groups had overlapping values with terrestrial herbivores emphasizing the importance of leaf litter inputs in the aquatic food web. Overall, aquatic insects were more depleted in δ13C and δ15N than terrestrial insects, similarly to what has been found in other studies . This variation is most likely the result of the two taxa that were collected for this functional group. Helicopsychidae were severely depleted in δ13C due to them being obligate scrapers, feeding on C sources depleted in δ13C such as periphyton and possibly other more depleted C sources that were not sampled in this study . Leptophlebiidae are considered to be more generalists and at times may feed as collector-gatherers, despite the families overall classification as scrapers (δ13C: −24.25 ± 0.72‰ and δ15N: 2.51 ± 0.20‰)  and shredders (0.78‰) having respectively the highest and lowest δ15N signatures. Collector-gatherers, scrapers and filterers were found to be intermediary with relatively little variation in their δ15N values (1.96–3.69‰).The  studies . Scraperscrapers . Similar± 0.20‰) . The smaδ13C and δ15N signatures with upland spiders being the most depleted in both instances. A study conducted in Japan similarly found only minimal changes in the δ13C and δ15N values between riparian and upland web spinning spiders of the same taxa (The three spider groups showed only slight differences in their ame taxa . Howeverame taxa .Tetragantha, which have been found to be specialists in trapping emerging aquatic insects. Along our site there were extremely few Tetragnatha and were therefore not included in our isotopic analyses. Our results were more similar to those reported for other tropical (The Bayesian analysis in SIAR found that upland spiders relied the most upon aquatic insects although there were only slight differences among the three spider groups. Although the vast majority of aquatic insect biomass is concentrated within the first few meters or so from the stream edge, some taxa are known to disperse laterally up to hundreds of meters from the stream . For an tropical  and sub-tropical  sites. IOur study highlights the importance of riparian ecotones as areas that contain a unique biodiversity of web-spinning spider taxa that are specialists in aquatic habitats and are rarely found even after only a few meters from the water’s edge. Dietary analyses revealed that aquatic insects comprised ∼50% of the diet in riverine, riparian and upland spiders with only a slightly greater dependence on aquatic insects in the upland spider group. We found that isotopic signals between terrestrial and aquatic insects were not exclusively distinct and this can impact the effectiveness of isotopic mixing models, which has been shown to be a problem for other studies along forested headwater streams. Despite the overlapping of isotopic signals, the results of the dietary analysis were similar to other studies conducted along tropical streams. Our study provides further evidence for the importance of aquatic subsidies for terrestrial consumers even within upland areas from the stream.The environment provided by the stream channel and that of the riparian forest clearly created two unique web-spinning spider assemblages, in which specialized taxa of aquatic ecosystems were shown to be the major difference between the two study areas. However, differences between these two habitats were potentially the result of structure and microenvironment, rather than prey resources.10.7717/peerj.1324/supp-1Supplemental Information 1Click here for additional data file.


Synphilin-1 is a cytoplasmic protein that has been shown to be involved in the control of energy balance. Previously, we reported on the generation of a human synphilin-1 transgenic mouse model (SP1), in which overexpression of human synphilin-1 resulted in hyperphagia and obesity. Here, behavioral measures in SP1 mice were compared with those of their age-matched controls (NTg) at two time points: when there was not yet a group body weight difference (“pre-obese”) and when SP1 mice were heavier (“obese”). At both time points, meal pattern analyses revealed that SP1 mice displayed higher daily chow intake than non-transgenic control mice. Furthermore, there was an increase in meal size in SP1 mice compared with NTg control mice at the obese stage. In contrast, there was no meal number change between SP1 and NTg control mice. In a brief-access taste procedure, both “pre-obese” and “obese“ SP1 mice displayed concentration-dependent licking across a sucrose concentration range similar to their NTg controls. However, at the pre-obese stage, SP1 mice initiated significantly more trials to sucrose across the testing sessions and licked more vigorously at the highest concentration presented, than the NTg counterparts. These group differences in responsiveness to sucrose were no longer apparent in obese SP1 mice. These results suggest that at the pre-obese stage, the increased trials to sucrose in the SP1 mice reflects increased appetitive behavior to sucrose that may be indicative of the behavioral changes that may contribute to hyperphagia and development of obesity in SP1 mice. These studies provide new insight into synphilin-1 contributions to energy homeostasis. Drosophila and mouse models have revealed that overexpression of human synphilin-1 results in increases in food intake, body weight and fat deposition, resembling key features of human obesity Synphilin-1 (919 aa) is a cellular protein predominantly expressed in the cytosol Hyperphagia is a core feature of many obesity models and alterations in multiple signaling pathways can contribute to hyperphagia In the present studies, we tested separate cohorts of 6–8-week–old (“pre-obese) and 4-month-old (“obese”) SP1 male mice and age-matched NTg controls to assess 1) how meal pattern parameters change to reflect the increased food intake in SP1 mice, 2) whether synphilin-1 expression alters appetitive behaviors or unconditioned licking responses to sucrose that differed from those of NTg control mice, and 3) whether there are developmental feeding behavior changes related to hyperphagia and obesity. The results from these behavioral assessments would provide an increased understanding of how synphilin-1-expression results in hyperphagia and obesity.The SP1 mice that expressed human synphilin-1 in neurons under the mouse prion protein promoter were generated as described previously The brains of Synphilin-1 and NTg control mice were homogenated as described previously ad libitum access to powdered chow  and water. Food intake was monitored continuously over 23-h daily test sessions as previously described ad libitum in a food jar placed on a scale in the feeding compartment of the cage. The animals had access to the food jar via an opening in the wall of the cage. A water bottle was mounted on an adjacent wall of the cage. Water was available ad libitum.Mice for the “pre-obese” comparisons (n = 9–10/group) were single-housed in DietMax System food intake monitoring cages   with Testing began after 7 days of habituation to the experimental environment and maintenance on chow diet. Meal pattern measures to powdered chow were taken for three consecutive days. For the 4–6 weeks old mice, intake of the powdered high fat diet  was measured for the next 3 consecutive days A feeding bout was operationally defined as requiring ≥0.02 g food. An interval of ≥10 min without food intake defined the termination of a meal. This bout criteria on average accounted for ∼90% of the feeding data in the current study. One mouse produced excessive spillage of the standard chow thus data from that animal were excluded for data analysis.For the “obese” comparisons (n = 6–7/group), mice were single-housed under similar test conditions but in test cages  (19 cm×19 cm) equipped with a pellet dispenser that delivers 20-mg chow pellets  as previously described This behavior assessment was performed as previously described with slight modification Training and testing was performed in a lickometer  during the light cycle as described previously Animals were subjected to a ∼23 h water-restriction schedule for the four days of behavioral training during which animals were allowed free access water only during the daily sessions. On days 1 and 2, animals were allowed access a stationary spout of water for 30-min sessions. On days 3 and 4, animals were allowed access to seven spouts of water in 5-s trials across 25-min sessions. After the end of session 4, animals were allowed free access water in their home cages. The next week, animals were subjected to the tests with water and six concentrations of sucrose across three 25-min daily sessions under a partial water and food restriction condition with one repletion day interspersed between testing days.(x-c)b), where x = log10 stimulus concentration, a = asymptotic lick response, b = slope and c = log10 concentration at the inflection point.For each sucrose concentration, the mean number of licks was determined by all trials across the three test sessions. A Licks Relative to Water Value was calculated by subtracting the mean number of licks to water from the mean number of licks at each concentration as described previously The cohorts of both pre-obese (6–10 weeks of age) and obese (4 months of age) SP1 transgenic mice were generated as described previously At the pre-obese time point, there was no significant group difference in body weights between SP1 and NTg mice. However, SP1 mice consumed significantly more standard chow than did controls mice {When animals were switched to a high-fat diet, a statistical difference in daily intake between the SP-1 and NTg mice was no longer apparent {In the older “obese” cohort, SP1 mice were significantly heavier than NTg controls and SP1 mice consumed significantly more chow than age-matched controls {Increased intake and larger meal sizes may be driven by elevated orosensory stimulation. Thus, to assess orosensory responsivity, unconditioned licking responses to a concentration array of sucrose was measured in SP1 and NTg controls in a brief-access taste test. The brief-access taste procedure involves presenting a range of taste solution concentrations in short (5-s) trials across one session and thus minimizes the effect of postingestive cues. The procedure also allows for some segregation of the appetitive and consummatory components of ingestive behavior. The mouse’s approach behavior to the spout and initiating licking can be considered appetitive behavior. The licking response within a 5-s trial follows contact with the taste stimulus and can be considered consummatory behavior.At the pre-obese stage, a two-way ANOVA comparing body weights between the two groups across brief assess test days revealed no main effect of group {F = 2.403, p = 0.143}, a main effect of day {F = 214.831, p<0.001} and a significant interaction {F = 4.242, p<0.001}. The SP1 group was significantly lighter on two testing days but this group difference did not reach Bonferonni correction . The groAt the obese stage (4 months of age), SP1 mice were significantly heavier than their NTg controls as confirmed by a main effect of group {F = 26.586, p<0.001}, a main effect of day {F = 88.759, p<0.001} and a significant interaction{ = 7.568, p<0.001}. To a stationary spout of water, total number of licks across a 30-min session was significantly lower in the SP1 group compared to the NTg controls {t(14) = −2.303, p = 0.037; In this study, we assessed various aspects of feeding behavior to further characterize the hyperphagia in SP1 mice. Meal pattern assessment revealed increases of meal size but no change of meal numbers in SP1 mice compared with non-transgenic controls. SP1 mice at pre-obese stage initiated significantly more trials to sucrose across the testing sessions and licked more vigorously to the highest concentration presented, compared to NTg controls. These findings indicate that the hyperphagia in SP1 mice is at least partly due to an increased appetitive behavioral component that may be indicative of enhanced food reward-related mechanisms.Hyperphagia can be expressed as increases in meal size without significant changes in meal number in some obesity models When the diet was switched from standard chow to a 45% high-fat diet, both SP1 mice and NTg controls decreased meal number and increased meal size. Although SP1 mice showed significantly higher intake to standard chow compared to the NTg group, the group difference was no longer apparent when the mice were maintained on the high-fat diet. In the SP1 mice, total caloric intake was already relatively high on chow diet, thus it is plausible that in these mice, although high-fat diet elicits an increase in meal size, there is a ceiling effect preventing further increase in total daily calories.Increased orosensory stimulation contributes greatly to human obesity and other obesity animal models At the older time point (obese stage), SP1 mice displayed significantly fewer licks to a stationary spout of water during a 30-min session when tested under a water-restricted condition. This was not observed at the “pre-obese” time point but only at the “obese” time point when the SP1 mice were significantly heavier. The two groups did not significantly differ in interlick-interval values, a measure of local lick rate In summary, synphilin-1 overexpression induced hyperphagia appears to present as increases in meal size but not meal number. SP1 mice displayed an increased appetitive behavioral response to sucrose at pre-obese stage but no change with the addition of a high-fat diet. These findings indicate that synphilin-1-mediated hyperphagia is associated with meal size contribution factors, such as appetitive response to sucrose. These studies provide a novel insight into synphilin-1 regulating food intake behavior and energy homeostasis.


Here we describe light fluence in the spectral domain and introduce eigenspectra multispectral optoacoustic tomography (eMSOT) to account for wavelength-dependent light attenuation, and estimate blood sO2 within deep tissue. We validate eMSOT in simulations, phantoms and animal measurements and spatially resolve sO2 in muscle and tumours, validating our measurements with histology data. eMSOT shows substantial sO2 accuracy enhancement over previous optoacoustic methods, potentially serving as a valuable tool for imaging tissue pathophysiology.Light propagating in tissue attains a spectrum that varies with location due to wavelength-dependent fluence attenuation, an effect that causes spectral corruption. Spectral corruption has limited the quantification accuracy of optical and optoacoustic spectroscopic methods, and impeded the goal of imaging blood oxygen saturation (sO Spectral corruption impedes imaging of blood oxygen saturation. Here Tzoumas et al. describe light fluence in the spectral domain and introduce eigenspectra Multispectral Optoacoustic Tomography to account for wavelength-dependent light attenuation and estimate blood oxygen saturation within deep tissue. Positron emission tomography or single-photon emission computed tomography assess cell hypoxia through the administration of radioactive tracers2, suffer from low-spatial resolution and are unable to provide longitudinal or dynamic-imaging capabilities. Electron paramagnetic resonance imaging2, but is not widely used and offers limited spatial and temporal resolution. Imaging methods using tracers may be further limited by restricted tracer bio-distribution, in particular to hypoxic areas. Tracer-free modalities have also been researched, in particular blood-oxygen-level dependent MRIThe assessment of tissue oxygenation is crucial for understanding tissue physiology and characterizing a multitude of conditions, including cardiovascular disease, diabetes, cancer hypoxia2) is a vital tissue physiology measurement and can provide an alternative way to infer pO2 and hypoxia. Arterial sO2 is widely assessed by the pulse oximeter, but this technology cannot be applied to measurements other than arterial blood. Optical microscopy methods like phosphorescence quenching microscopy2 in tissue, the values reported by diffuse optical methods are often hard to interpret.Measurement of blood oxygen saturation levels , a common simplification that can introduce substantial errors in deep tissue.Multispectral optoacoustic tomography (MSOT) detects the spectra of oxygenated and deoxygenated haemoglobin in high resolution deep within tissues, since signal detection and image reconstruction are not significantly affected by photon scattering102 in deep tissue. We demonstrate the superior performance of the method with >2,000 simulations, phantom measurements and in vivo controlled experiments. Then, using eMSOT, we image oxygen gradients in the skeletal muscle in vivo, previously only accessible through invasive methods. Furthermore, we show the application of eMSOT in quantifying blood oxygenation gradients in tumours during tumour growth or O2 challenge, and relate label-free non-invasive eMSOT readings to tumour hypoxia; demonstrating the ability to measure quantitatively the perfusion hypoxia level in tumours, as confirmed with invasive histological gold standards.In this work, we found that the spectral patterns of light fluence expected within the tissue can be modelled as an affine function of a few reference base spectra, independently of the specific distribution of tissue optical properties or the depth of the observation. We show how this principle can be employed to solve the spectral corruption problem without knowledge of the tissue optical properties, and significantly increase the accuracy of spectral optoacoustic methods. The proposed method, termed eigenspectra Multispectral Optoacoustic Tomography (eMSOT), can provide quantitative estimation of blood sOA new concept of treating light fluence in diffusive media/tissues is introduced, based on the observation that the light fluence spectrum at different locations in tissue is the result of a cumulative light absorption operation by tissue chromophores, such as haemoglobin. We, therefore, hypothesized that there exists a small number of base spectra that can be combined to predict any fluence spectrum present in tissue; therefore, avoiding the unattainable task of knowing the distribution of tissue optical properties at high resolution. To prove this hypothesis, we first applied principal component analysis (PCA) on 1,470 light fluence spectral patterns, which were computed by simulating light propagation in tissues at 21 different (uniform) oxygenation states of haemoglobin and 70 different discrete depths (Methods). PCA analysis yielded four significant base spectra, that is, a mean light fluence spectrum  and threΦM) and the three Eigenspectra (Φi(λ), i=1..3) multiplied by appropriate scalars m1, m2 and m3, termed Eigenfluence parameters. To validate this hypothesis we computed the light fluence in >500 simulated tissue structures of different and non-uniform optical properties and haemoglobin oxygenation values  and a fitting residual value. The residual value represents the error of the Eigenspectra model in matching the simulated data and typically assumed values below 1%  is mainly associated with the modifications of the light fluence spectrum due to depth and the average optical properties of the surrounding tissue, while the first Eigenspectrum Φ1(λ) is also associated with the ‘spectral shape' of light fluence that relates to the average oxygenation of the surrounding tissue. Localized measurements of light fluence spectra obtained in vivo and post mortem corroborated these observations , Φ2(λ), Φ3(λ), as a method that formulates the blood sO2 estimation problem as a nonlinear spectral unmixing problem (see Methods), that is:Following these observations, we propose eigenspectra MSOT (eMSOT), based on three eigenspectra P is the multispectral optoacoustic image intensity obtained at a position r and wavelength λ, ɛHb(λ) are the wavelength-dependent molar extinction coefficients of oxygenated and deoxygenated haemoglobin, c′Hb(r) are the relative concentrations of oxygenated and deoxygenated haemoglobin , and Φ′=ΦM(λ)+m1(r)Φ1(λ)+m2(r)Φ2(λ)+m3(r)Φ3(λ). c′Hb(r), m1(r), m2(r), m3(r) and is solved as a constrained optimization problem  for each pixel using these parameters. c′Hb(r), and sO2 is computed (see Methods).where 2 map and visually showcases the differences between the eMSOT sO2 image (middle), the sO2 image obtained using linear unmixing (left), and the original sO2 simulated image (right). eMSOT offered significantly lower sO2 estimation error with depth, compared with the linear fitting method  applied on >2,000 randomly created maps of different optical properties, simulating different tissue physiological states . Figure g method . A substdata set . In partunmixing . A thoro2 estimation derived by eMSOT, as compared with conventional linear unmixing (n=4) were performed in vivo, under gas anaesthesia, by rectally inserting capillary tubes containing blood at 100 and 0% sO2 levels (Methods). The mice were imaged in the lower abdominal area under 100% O2 and 20% O2 breathing conditions (2 maps obtained with linear unmixing (middle column) and with eMSOT (right column). The spectral fitting of linear unmixing (left) and eMSOT (right) corresponding to a pixel in the area of the capillary tube (yellow arrows in a) are presented in 2 values. In the controlled in vivo experiments, the mean linear unmixing error ranged from 16 to 35% while eMSOT offered a mean sO2 error ranging from 1 to 4%, indicating an order of magnitude improved accuracy ; three of the mice were then killed with an overdose of CO2, the latter binding to haemoglobin and deoxygenating blood.Blood oxygenation and oxygen exchange in the microcirculation have been traditionally studied through invasive, single-point polarography or microscopy measurements in vessels and capillaries of the skeletal musclein vivo  states.eMSOT applied in the hindlimb muscle area  captured prominent vascular structures likely corresponding to femoral vessels or their branches  with imp vessels . Averagereathing .2172 in smaller arterioles202 under normal breathing conditions, reaching up to 70% at 100% O2 breathing212 with a large standard deviation2 in the skeletal muscle, while the values measured at ROI-2, which anatomically presents no prominent vasculature, relate more to capillary sO2 measurements.The low blood saturation values in tissue (35–50%) cannot be explained by considering arterial and venous blood saturation. However, previous studies based on direct microscopy measurements in vessels and capillaries through polarography, haemoglobin spectrophotometry and phosphorescence quenching microscopy have revealed similar oxygenation gradient in the skeletal muscle2 values below a threshold which varied from 50 to 25% sO2) over the total tumour area also increased during tumour progression 2 imaging. We hereby followed an alternative approach that describes the spectral features of light fluence as a combination of spectral base functions. Using this principle, we formulated the sO2 quantification problem as a nonlinear spectral unmixing problem that does not require knowledge of tissue optical properties or the inversion of a light transport operator. Effectively, eMSOT converts sO2 imaging from a problem that is spatially dependent on light propagation and optical properties, as common in traditional optical methods, to a problem solved in the spectral domain. Therefore, sO2 can be directly quantified without estimating tissue optical properties.Spectral corruption has so far limited the potential of optical and optoacoustic methods to offer accurate, quantitative assessment of sOeMSOT requires theoretically at least five excitation wavelengths for resolving spectral domain parameters and the relative oxygenated and deoxygenated haemoglobin concentrations. We hereby utilized 21 wavelengths for ensuring high accuracy. The recent evolution of video-rate MSOT imaging systems, based on fast tuning optical parametric oscillator lasersin vivo (muscle and tumour) in high resolution, showing good correlation with the expected physiological state or the histologically observed spatial distribution of perfusion and hypoxia. eMSOT measures blood oxygenation. We hypothesized that a correlation exists to tissue oxygenation and hypoxia measurements by assuming a wide presence of haemoglobin in tissues. We demonstrated congruence  were imaged in vivo using MSOT after the tumours reached a suitable size. All imaging procedures were performed under anaesthesia using 1.8% isoflurane. In the O2 challenge experiment, the mouse was initially breathing 100% O2 and in the following medical air (20% O2). During the O2 Challenge, the mice were stabilized for a period of 10 min under each breathing condition before MSOT acquisition. For controlled mouse measurements (n=4), MSOT acquisition was performed on mice under gas anaesthesia and breathing 100% O2 or 20% O2 by rectally inserting a capillary tube containing pig blood at 100 or 0% sO2 oxygenation levels. Mice were killed during MSOT imaging with an overdose of CO2 or after MSOT acquisition by a ketamine/xylazine overdose. In the following the mice were stored at −80 °C for further analysis.All procedures involving animal experiments were approved by the Government of Upper Bavaria. For the preparation of orthotopic 4T1 tumour models, 8-week-old, adult female, athymic, Nude-Foxn1 mice  were orthotopically inoculated in the mammary pad with cell suspensions (0.5 million 4T1 cells (CRL-2539)). Animals . The cells were authenticated by the Americal type culture collection (ATCC) by several analysis tests: post-freeze viability, morphology, mycoplasma contamination, post-freeze cell growth, interspecies determination; bacteria and fungal contamination. Additional mycoplasma contamination tests were also performed. For the animal studies no randomization, blinding or statistical methods were performed.Optoacoustic imaging was performed using a real-time whole-body mouse imaging scanner, MSOT In Vision 256-TF . The system utilizes a cylindrically focused 256-element transducer array at 5 MHz central frequency covering an angle of 270 degrees. The system acquires cross-sectional (transverse) images through the animal. The animals are placed onto a thin-clear polyethylene membrane. The membrane separates the animals from a water bath, which is maintained at 34 °C and is used for acoustic coupling and maintaining animal temperature while imaging. Image acquisition speed is at 10 Hz29P obtained over excitation wavelength λ were calibrated to correct for the intensity of laser power per pulse, and for the absorption of water surrounding the tissue. With HbO2 and Hb being the main tissue absorbers in the near-infrared, multispectral optoacoustic images can be related to the concentrations of oxy- and deoxy-haemoglobin through All optoacoustic images Φ is the space and wavelength-dependent optical fluence, C(r) is a spatially varying scaling factor corresponding to the effects of the system's spatial sensitivity field and the Grüneisen coefficient, ɛHb(λ) are the wavelength-dependent molar extinction coefficients of oxygenated and deoxygenated haemoglobin, while cHb(r) the associated concentrations at a position r. Φ(r) is a vector corresponding to the light fluence spectrum at position r, and ||Φ(r)||2 is its norm across all excitation wavelengths at a position r. We define Φ′=Φ/||Φ(r)||2, which corresponds to the normalized wavelength dependence of light fluence at a specific position .In C(r) and ||Φ(r)||2 do not affect the estimation of blood sO2, which is calculated as a ratio once the relative concentrations of HbO2 and Hb are known. We define C′(r)c′Hb(r)=C′(r)cHb(r), respectively, where C′(r)=C(r)||Φ(r)||2 is a common, space-only dependent scaling factor. Using this notation, c′Hb(r), sO2 can be computed as in:The space-only dependent factors c′Hb(r), accounting for, or estimating the wavelength dependence of the light fluence Φ′ is further required.For the accurate quantitative extraction of the relative concentrations The Eigenspectra model. eMSOT is based on the observation that the spectral patterns of light fluence present in tissue can be modelled as an affine function of only a few base spectra, independently of tissue depth and the specific distribution of optical properties of the tissue imaged. This hypothesis stems from the notion that the spectrum of light fluence is the result of the cumulative light absorption by haemoglobin; thus the spectrum of light fluence will always be related to the spectra of haemoglobin in a complex nonlinear manner. This complex relation can be linearized using a data-driven approach, that is, through the application of PCA on a selected set of light fluence spectra.ΦΜ(λ) and a linear combination of a number of light fluence Eigenspectra Φi(λ). This model was derived by applying PCA on a training data set comprised of a set of light-fluence spectral patterns. Briefly, a training data set was formed through the creation of multispectral light fluence simulations using the 1D analytical solution of the diffusion equation for infinite media. A set of light-fluence spectral patterns Φz,ox(λ) were computed for high physiological tissue optical properties , tissue depths ranging from z=0 to z=1 cm with a step size of 0.143 mm  and for 21 different uniform background blood sO2 levels . The so computed set of light fluence spectra Φz,ox(λ) was normalized =Φz,ox(λ)/||Φz,ox||2) and used in the following as training data in the context of PCA in order to create an affine, 3-dimensional model consisting of a mean fluence spectrum ΦΜ(λ) and three Eigenspectra Φi(λ). PCA was used for offering a minimum square error property in capturing the spectral variability of the simulated light fluence spectra, in a linear manner. Three components were selected for providing a relatively simple model while also offering a small model error with respect to the training data set  at any arbitrary tissue position r can thus be modelled as a superposition of the mean fluence spectrum and three fluence Eigenspectra multiplied by appropriate scalar parameters m1(r), m2(r) and m3(r), (hereby referred to as Eigenfluence parameters) as per The wavelength dependence of the light fluence was herein modelled as a superposition of a mean fluence spectrum data set . The wavin vivo and ex vivo light fluence measurements, obtained from controlled experiments  by minimizing f, m2(r), m3(r), c′Hb(r)), for brevity noted f(r), defined according to m1…3(r) and the relative blood concentrations c′Hb(r)) can be obtained using a nonlinear optimization algorithm and at least five excitation wavelengths. The relative blood concentrations c′Hb(r) are proportional to the actual ones (cHb(r)) with regard to a common scaling factor. However, as stated before, this fact does not affect the computation of sO2.A solution for the five unknowns  and L radial lines , mi, …, mi, mi, …, mi, …,mi]T correspond to the values of the Eigenfluence parameter i  over all such points, the new inverse problem is defined as the minimization of cost function fgrid defined in Assuming a polar grid of ines see  with a tm1 and m3. The matrix W describes a connectivity graph defined on the grid of points assumed , which are constant for all grid points and correspond to the maximum and minimum values of m2 on the training data set  and m3, which are computed per grid point in a preprocessing step as described in In  assumed  and its data set . and , w2 quantificationFluence correction and sO. The minimization of cost function fgrid  for each Eigenfluence parameter i and each grid point rp,l. The Eigenfluence parameters in the convex hull of the grid are in the following estimated by means of cubic interpolation. We note that due to the nature of diffuse light propagation, the Eigenfluence parameters are expected to vary smoothly in tissue and thus their interpolation is not expected to introduce large errors in the result =ΦM(λ)+m1(r)Φ1(λ)+m2(r)Φ2(λ)+m3(r)Φ3(λ), where Φi(λ) is the ith fluence Eigenspectrum. Finally, a spectrally corrected eMSOT image is obtained after dividing the original image P with the normalized wavelength-dependent light fluence Φ′ at each position r and wavelength λ, i.e., PeMSOT=P/ Φ′. The relative concentrations of HbO2 and Hb (c′HbeMSOT(r)) are computed for each pixel of PeMSOT image independently through non-negative constrained least squares fitting with the spectra of oxygenated and deoxygenated haemoglobin. Thus, the eMSOT blood sO2 maps retain the original resolution of the MSOT imaging system.on fgrid  under thsult see . The wavWe note that both the Eigenspectra model and the inversion scheme were hereby optimized for the application of small-animal imaging. The Eigenspectra model was trained for a maximum depth of 1 cm and the inversion scheme was designed with respect to the same tissue depth and optical properties within the physiological range .Φ=Φ(r) and by assuming haemoglobin as the major absorber in tissue, optoacoustic spectra can be modelled as a linear combination of the spectra of oxy- and deoxy-haemoglobin. The term linear unmixing refers hereby to the computation of the relative concentrations of HbO2 and Hb (cHblu(r)) and subsequently blood sO2, through non-negative constrained least squares fitting of the original image P with the spectra of Hb and HbO2.Under the simplifying assumption that the light fluence attains a flat spectrum irrespective of the tissue position 2O4S2) (deoxygenation process)For validating the accuracy of eMSOT in quantifying blood oxygenation in deep tissue, we prepared tissue mimicking phantoms, containing blood at known oxygenations levels. Specifically, for simulating tissue background, 2-cm-diameter cylindrical solid phantoms were created by using 1.5% Agarose Type I, Sigma-Aldrich (solidifying in <37°), 2% intralipid and 3–5% freshly extracted pig blood diluted in NaCl. Different blood oxygenation levels were achieved by diluting oxygen in whole blood (oxygenation process) or by mixing the blood with different amounts of sodium dithionite (Nan=4) were killed and examined for tumour and tissue anatomy. Mice were embedded in an optimal cutting temperature compound  and frozen at −80 °C. In the following, the mice were sliced at an orientation similar to the one of MSOT imaging and colour photographs were recorded. The cryoslicing imaging system is based on a cryotome , fitted with CCD-based detection camera. During this process, 10 μm slices throughout the whole tumour volume were collected for further histological analysis.After MSOT acquisition, a subset of the mice bearing 4T1 mammarian tumours  . Then, they were rinsed with distilled water and incubated 30 s with Haemotoxylin acide by Meyer  to stain the cell nuclei. The slides were then rinsed in tap water again before incubation for 1 s in Eosin G  to stain cellular cytoplasm. After rinsing in distilled water, the slides were dehydrated in 70%, 94% and 100% ethanol and incubated for 5 min in Xylene  before being cover slipped with Rotimount  cover media. Representative slides were observed using Zeiss Axio Imager M2 microscope with AxioCam 105 colour, and pictures were then processed using a motorized stitching Zen Imaging Software .n=4) was examined for functional characteristics of the tumours by Pimonidazole histological staining. The hypoxia marker Pimonidazole  was injected intraperitonially at 100 mg/kg body weight in a volume of 0.1 ml saline ≈1.5 h before tumour excision, and the perfusion marker Hoechst 33342  was administered intravenous at 15 mg/kg body weight in a volume of 0.1 ml saline 1 min before the tumour-bearing mice were killed. The tumours were excised immediately after the animals were killed. The orientation of the tumours with respect to the mouse body was retained. Eight micrometers cryosections were sliced throughout the tumour. The cryosections were fixed in cold (4 °C) acetone, air dried and rehydrated in PBS before staining. Pimonidazole was stained with the FITC-labelled anti-Pimonidazole antibody  diluted 1:50 in primary antibody diluent  by incubating for 1 h at 37 °C in the dark.A subset of the tumour-bearing mice .Supplementary Figures 1-8, Supplementary Tables 1 & 2, Supplementary Notes 1-6 and Supplementary References.