Human T-Lymphotropic Virus type 1 (HTLV-1) is endemic among people of Melanesian descent in Papua New Guinea, Solomon Islands and Vanuatu, and in Indigenous populations from Central Australia. Molecular studies revealed that these Australo-Melanesian strains constitute the highly divergent HTLV-1c subtype. New Caledonia is a French overseas territory located in the Southwest Pacific Ocean. HTLV-1 situation is poorly documented in New Caledonia and the molecular epidemiology of HTLV-1 infection remains unknown.Studying 500 older adults Melanesian natives from New Caledonia, we aim to evaluate the HTLV-1 seroprevalence and to molecularly characterize HTLV-1 proviral strains.Plasma from 262 men and 238 females  were screened for anti-HTLV-1 antibodies by particle agglutination (PA) and indirect immunofluorescence assay (IFA). Serological confirmation was obtained using Western blot assay. DNAs were extracted from peripheral blood buffy coat of HTLV-1 seropositive individuals, and subjected to four series of PCR . Primers were designed from highly common conserved regions of the major HTLV-1 subtypes to characterize the entire HTLV-1 proviral genome.env gene showed the existence of two major clades. The first is composed of strains from Papua New Guinea; the second includes strains from all neighboring archipelagos , and Australia. Interestingly, this second clade itself is divided into two sub-clades: strains from Australia on one hand, and strains from Solomon Islands, Vanuatu and New Caledonia on the other hand.Among 500 samples, 3 were PA and IFA positive. The overall seroprevalence was 0.6%. The DNA sample from 1 New Caledonian woman (NCP201) was found positive by PCR and the complete HTLV-1 proviral genome  was obtained. The full-length HTLV-1 genomic sequence from a native woman from Vanuatu (EM5), obtained in the frame of our previous studies, was also characterized. Both sequences belonged to the HTLV-1c Australo-Melanesian subtype. The NCP201 strain exhibited 0.3% nucleotide divergence with the EM5 strain from Vanuatu. Furthermore, divergence reached 1.1% to 2.9% with the Solomon and Australian sequences respectively. Phylogenetic analyses on a 522-bp-long fragment of the gp21-The HTLV-1 seroprevalence (0.6%) in the studied adult population from New Caledonia appears to be low. This seroprevalence is quite similar to the situation observed in Vanuatu and Solomon Islands. However it is very different to the one encountered in Central Australia. Taken together, these results demonstrated that Australo-Melanesia is endemic for HTLV-1 infection with a high diversity of HTLV-1c strains and a clear geographic clustering according to the island of origin of HTLV-1 infected persons. The human T-lymphotropic virus type 1 (HTLV-1) infects at least 5 to 10 million individuals worldwide. In Australo-Melanesia, a south Pacific region including Papua New Guinea, Solomon Islands, Vanuatu archipelago and Australia, previous studies have shown that HTLV-1 is present in limited remote areas among few ancient Aboriginal populations. The molecular characterization of the HTLV-1 viruses present in such Indigenous individuals indicates that they belong to a specific HTLV-1 genotype called the Australo-Melanesian subtype c. In the present study, we provide evidence that the HTLV-1 endemicity among elderly individuals from New Caledonia is low and quite similar to Vanuatu and Solomon Islands, yet very different to the situation encountered in Central Australia. Furthermore, the newly described full-length HTLV-1 genomic sequences, from two Melanesian natives from New Caledonia and Vanuatu, both belong to the HTLV-1c genotype but are distinct from those of Aboriginal individuals living in neighboring countries. These results suggest that HTLV-1 viral strains were probably introduced among Melanesian native populations during multiple ancient human migration events to these archipelagos. The Human T-lymphotropic virus type 1 (HTLV-1) is a human oncoretrovirus, which causes two major diseases: adult T-cell leukemia/lymphoma and tropical spastic paraparesis/HTLV-1-associated myelopathy. This virus infects at least 5 to 10 million people worldwide –4.In the context of ongoing studies among Aboriginal individuals in Australo-Melanesia –6, we exTherefore, the detection of HTLV-1 molecular variants from southwestern Pacific territories may constitute a powerful epidemiological and phylogenetic tool to reveal the ancient spread of this oncogenic virus, whose presence in this region has not yet been fully elucidated.The aim of the present study was hence to characterize the HTLV-1 strains that infect Melanesian natives from New Caledonia, and to compare them with those previously characterized from Melanesian natives originating from neighboring territories.New Caledonia (NC), a French overseas territory, is a group of islands located in the Southwest Pacific, east of Australia. . Our worThis survey received administrative and ethical clearance in New Caledonia from the “Direction des Affaires Sanitaires et Sociales de Nouvelle-Calédonie” and in metropolitan France from the “Comité consultatif sur le traitement de l’information en matière de recherche dans le domaine de la santé” (N° 08.511) and the “Commission nationale de l’informatique et des libertés “(N° 908425). Information regarding the blood collection and the molecular characterization of HTLV-1 proviral strains was provided to the participants presenting to the blood-taking center.HTLV-1 antibodies in plasma were first detected by a particle agglutination (PA) technique  at the Institut Pasteur (IP) in New Caledonia and then transferred to the IP in Paris for serological confirmatory assays. An indirect immunofluorescence assay (IFA) using the HTLV-1 and HTLV-2 transformed cell lines MT2 and C19 respectively, was performed. Anti-HTLV-1 antibody titers were determined by successive 2-fold dilutions. All positive samples were further tested by Western blot (WB) assay . A sample with reactivity to the two gag proteins (p19 and p24) and both env-coded glycoproteins (the HTLV-1 recombinant gp46-I peptide [MTA-1] and the HTLV-1/HTLV-2 recombinant [rGD21] protein) was considered to be positive for HTLV-1 antibodies.High-molecular weight DNA was extracted from peripheral blood buffy coat using the QIAamp DNA Blood Mini Kit . DNA samples were subjected to a first polymerase chain reaction (PCR) using human beta-globin specific primers, to ensure that DNA was amplifiable . DNA samSeven of the 500 plasma samples studied were tested positive by PA. Among them three were IFA positive with higher immunofluorescence assay titers on MT-2 (HTLV-1) than on C-19 (HTLV-2) cells .These three plasma samples exhibited high PA assay titers  and displayed full reactivity on WB . Taken tAmplification of HTLV-1 proviral genome was tested for the 3 HTLV-1-seropositive women  and a sample from Vanuatu (EM5). Complete HTLV-1 genomic sequences , derived from 4 PCR fragments of the appropriate size, were obtained for a unique sample from New Caledonia  and the sample from Vanuatu . The New Caledonian NCP201 strain exhibited a 0.3% nucleotide divergence with the EM5 strain from Vanuatu. The pairwise comparison of these two new strains with other available complete sequences from Solomon Islands (Mel5) and Australia  revealed an overall nucleotide polymorphism of up to 2.9% (1.1% to 2.9% respectively) .To include in our analyses most of the Central Australian sequences characterized to date , we perfenv gene, we further analyzed this region to better understand the relationships within the HTLV-1c subtype strains. Thus, the nucleotide homology of the NCP201 strain reached [97.1%-97.3%] with the PNG strains . Interestingly, the New Caledonian NCP201 strain exhibited a nucleotide similarity range of [99.6%-100%], [99%-99.4%] and [98.1%-98.5%] with the Vanuatu, Solomon Islands and Australian strains respectively  in the studied adult population appears to be low but is quite similar in Vanuatu and Solomon Islands. Indeed, previous studies showed that HTLV-1 seroprevalence in Vanuatu archipelago reached 0.74% among individuals aged over 60 years old, while among native Solomon islanders over 50 years old, the reported seroprevalence was 2.3% . This reenv gene fragment revealed the existence of a PNG clade. Thus, even among HTLV-1c subtype, a clustering according to the geographic origin of the HTLV-1 infected individuals exists.From a molecular point of view, the NCP201 sequence belongs to the HTLV-1c subtype, together with the Melanesian strains from Papua New Guinea, Solomon Islands and Vanuatu , 13, 14,Though this study is the first demonstration that New Caledonia is endemic for HTLV-1 infection, it has some limitations. The small sample size precluded a detailed epidemiological analysis that could illustrate the geographical dispersal of HTLV-1 infection in the whole New Caledonian archipelago. A further limitation was our inability to molecularly characterize various HTLV-1 strains. This would certainly require HTLV-1-infected individuals exhibiting a higher proviral load. Nevertheless, our findings are consistent with previous studies that have found that HTLV-1 seroprevalence was low among Indigenous Melanesian natives from neighboring archipelagos .Current studies combining both host genetics markers and viral analyses on HTLV-1, but also on Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 (HHV-8), which infects the same aboriginal populations , are ong


Compensation implies the recruitment of additional neuronal resources to prevent the detrimental effect of age-related neuronal decline on cognition. Recently suggested statistical models comprise behavioral performance, brain activation, and measures related to aging- or disease-specific pathological burden to characterize compensation. Higher chronological age as well as the APOE ε4 allele are risk factors for Alzheimer's disease. A more biological approach to characterize aging compared with chronological age is the brain age gap estimation (BrainAGE), taking into account structural brain characteristics. We utilized this estimate in an fMRI experiment together with APOE variant as measures related to pathological burden and aimed at identifying compensatory regions during working memory (WM) processing in a group of 34 healthy older adults. According to published compensation criteria, better performance along with increased brain activation would indicate successful compensation. We examined the moderating effects of BrainAGE on the relationship between task performance and brain activation in prefrontal cortex, as previous studies suggest predominantly frontal compensatory activation. Then we statistically compared them to the effects of chronological age (CA) tested in a previous study. Moreover, we examined the effects of adding APOE variant as a further moderator. Herewith, we strived to uncover neuronal compensation in healthy older adults at risk for neurodegenerative disease. Higher BrainAGE alone was not associated with an increased recruitment in prefrontal cortex. When adding APOE variant as a second moderator, we found an interaction of BrainAGE and APOE variant, such that ε4 carriers recruited right inferior frontal gyrus with higher BrainAGE to maintain WM performance, thus showing a pattern compatible with successful neuronal compensation. Exploratory analyses yielded similar patterns in left inferior and bilateral middle frontal gyrus. These results contrast those from a previous study, where we found no indication of compensation in prefrontal cortex in ε4 carriers with increasing CA. We conclude that BrainAGE together with APOE variant can help to reveal potential neuronal compensation in healthy older adults. Previous results on neuronal compensation in frontal areas corroborate our findings. Compensatory brain regions could be targeted in affected individuals by training or stimulation protocols to maintain cognitive functioning as long as possible. Neuronal compensation as an individual's reaction to cognitive challenge with the aim to maintain cognitive performance has been increasingly investigated over the past years in healthy aging and beginning neurodegenerative disease. Several theoretical frameworks describe compensation as a flexible recruitment of additional neuronal resources when existing networks reach their capacity limits and are not sufficient anymore for successful cognitive performance Cabeza, . TogetheMeasures of task performance and brain activation for compensation models can be derived from task-based fMRI experiments. Approximations of pathological burden specific to healthy aging or a certain neurodegenerative disease can be specified according to the group investigated. In patients with beginning neurodegenerative disease, regional brain volume of an area affected by the disease can be used, e.g., striatal volume in Huntington's disease  were recruited as part of a project investigating neuronal plasticity in aging and early neurodegeneration at the University Medical Center Freiburg. All were right-handed, had normal or corrected-to-normal visual acuity and no history of psychiatric or neurological disease as well as adequate performance in a sensitive cognitive test . Then, the BrainAGE index of all participants was estimated using individuals' segmented T1-weighted images that were derived using the SPM8 package (http://www.fil.ion.ucl.ac.uk/spm) and the VBM8 toolbox (http://www.neuro.uni-jena.de/). We used the affine registered segmentations for gray and white matter that were smoothed with a 4-mm full-width-at-half-maximum smoothing kernel. After smoothing, data were resampled to 8 mm and a data reduction was performed by applying principal component analysis (PCA), utilizing the “MATLAB Toolbox for Dimensionality Reduction” (https://lvdmaaten.github.io/drtoolbox/). PCA was only performed on the training sample and the estimated transformation parameters were subsequently applied to the test sample. The BrainAGE score indicates the deviation (in years) from an individual's CA. A positive BrainAGE score implies an older-appearing brain , e.g., the BrainAGE of +2.5 of an individual with a CA of 79 yields a value of 81.5 years. In the current manuscript, we use “BrainAGE” to label the deviation from CA, and “estimated BrainAGE” to label BrainAGE + CA in years.BrainAGE of each participant was estimated based on the individual T1-weighted anatomical image with a voxel size of 1 mmGenotyping of the sample is described in detail in previous work . In short, functional images were coregistered to respective anatomical scans, which were segmented with the VBM 8 toolbox . First-level analysis was conducted using a general linear model (GLM) approach in native space. Normalization took place before 2nd level analyses, such that images were resampled to a spatial resolution of 1.5 cubic mm and smoothed with a 6 mm full width at half maximum (FWHM) Gaussian kernel. For group multiple regression analyses, contrast images of interest comparing 2-back vs. 0-back conditions were entered in a multiple regression model. Activation peaks were anatomically labeled with the Anatomy Toolbox for SPM  corrected for multiple comparisons, paralleling previous work .All multiple regression analyses were conducted voxel-wise with the in-house developed IFX Toolbox for SPM8 , hence fulfilled prerequisites of multiple regression. CA, BrainAGE as well as task performance in all conditions did not differ between ε4-carriers and non-carriers  as well as BrainAGE and task performance . Of the 34 participants, 14 obtained a positive BrainAGE index pointing to accelerated atrophy; 20 participants obtained a negative BrainAGE index and thus were estimated as younger than their CA . The R2 increase due to the inclusion of the three-way interaction of task performance, BrainAGE, and APOE variant (R2-change) was 0.29, p < 0.001. The effect size Cohen's f2 of 1.61 was large according to the guideline defining f2 ≥ 0.02, f2 ≥ 0.15, and f2 ≥ 0.35 as small, medium and large effects, respectively  to determine if the addition of genetic burden as a moderator would reveal compensatory effects. Indeed, we found a significant three-way interaction in right inferior frontal gyrus pars orbitalis at y Cohen, . Individy Cohen, . The posy Cohen, , while tb = −0.01, standard error (SE) = 0.02, t = −2.12, p = 0.05], which was not the case for CA . The three way interaction of task performance, BrainAGE, and APOE variant proved highly significant as already derived from the analysis with the IFX toolbox . It is possible to report so-called simple effects in multiple regression allowing for interaction effects , and—as already pointed out above—a significant simple effect of BrainAGE , but not a significant effect of accuracy. This underlines that the interaction is driven by APOE allele status and is significant only at positive BrainAGE. An overview of simple and interaction effects in our multiple regression model can be found in Supplement We further investigated the activation peak in rIFG with the PROCESS macro for SPSS. To be able to discern the incremental variability of using BrainAGE instead of CA as a moderator, we implemented CA as an additional nuisance variable. BrainAGE as a predictor significantly contributed to the model [unstandardized regression coefficient p < 0.001). Interestingly, we found a similar interaction in the contralateral homologous area to the above-reported peak in rIFG  as well as in left and right middle frontal gyrus . In all three areas, there was a similar activation pattern compared with the peak in rIFG, with APOE ε4 carriers exhibiting increased recruitment with higher BrainAGE together with better performance, with significance regions beginning around the mean BrainAGE of −0.46 years , lMFG , and rMFG , confirming the incremental benefit of the moderator variables.For exploratory purposes, we tested the same interaction with an uncorrected threshold (p < 0.05) as well as at an uncorrected threshold (p < 0.001), we did not find a moderating effect of CA and APOE on the relationship between performance and prefrontal activation. A voxel-based morphometry (VBM) analysis of the same sample confirmed that there were no differences in gray matter volume between APOE ε4 and non-ε4 carriers (Scheller et al., The same three-way interaction was tested with CA instead of BrainAGE as a moderator in a previous study of the same sample. At an FWE corrected threshold (The aim of the current study was to investigate potential compensatory recruitment in healthy older individuals with a compensation model comprising the components task performance, brain activation and two measures related to pathological burden in aging, BrainAGE, and APOE variant. First, individuals with higher BrainAGE did not require additional neuronal resources to perform a cognitively demanding WM task successfully, hence compensatory brain activation was not detected when BrainAGE alone moderated the relationship between performance and PFC activation. Second, the three-way interaction of performance, namely BrainAGE and APOE variant was examined. Here we found increased activation in bilateral inferior frontal as well as bilateral middle frontal gyrus at higher BrainAGE with better performance, fulfilling one clear-cut criterion of successful compensation (Cabeza and Dennis, When testing the moderating effects of BrainAGE on the relationship between task performance and prefrontal activation, we did not find significant interaction effects and thus no indication of compensation. Fourteen of thirty-four study participants had an estimated higher BrainAGE compared to their CA. Hence, the majority of our sample exhibited younger-appearing brains, which might be the reason why we did not identify compensatory areas when BrainAGE alone was implemented as a moderator. Younger-appearing brains are associated with higher years of education (Steffener et al., After combining BrainAGE with APOE variant as moderator variables to yield a more detailed characterization of pathological burden, we identified increased prefrontal recruitment compatible with successful neuronal compensation. Specifically, the three-way interaction of task performance, BrainAGE, and APOE variant was significant in rIFG and as revealed by exploratory analyses, also in lIFG and bilateral MFG, with large effect sizes. The increased recruitment of these areas along with better task performance confirms one criterion for successful compensation (Cabeza and Dennis, The negative effect in lIFG observed in non-ε4 carriers might be a sign of processing efficiency, i.e., they perform best when using a concise task-related network without additional areas, as their need for compensation might still be small due to lack of genetic burden (Goh and Park, Our compensation-related findings can also be viewed as an elaboration of previous results of the same sample. Prior to the availability of the sample's BrainAGE coefficients, we had already identified bilateral areas on the margin of IFG and insula as compensatory areas in APOE ε4 carriers (Scheller et al., DLPFC is part of the well-established WM network (Owen et al., Why we observe a significant interaction of task performance, BrainAGE, and APOE and not of task performance, CA, and APOE cannot be determined unambiguously, as our sample size was not sufficient for more complex statistical models, i.e., a four-way interaction of task performance, CA, BrainAGE, and APOE variant. A larger sample or a replication sample would be desirable to strengthen our findings. Still, taking brain structure into account when approximating pathological burden helped to obtain a more fine-grained picture of compensatory recruitment. Consequently, we would argue that our ε4 carriers showed compensation in PFC, but that potential compensatory recruitment could only be revealed when taking into account a combination of risk factors, i.e., the most accurate approximation of pathological burden available. Moreover, our cross-sectional design only captures inter-individual variability. To follow individuals' compensation trajectories, i.e., the initialization and further development of compensatory recruitment, longitudinal investigations are needed (Nyberg et al., BrainAGE together with APOE variant has proved a helpful proxy of pathological burden to be implemented in models of neuronal compensation. The suggested combination of structural and functional imaging as well as genetic data translating theoretical frameworks to statistical models of compensation should be transferred to other cognitive domains as well as further samples of healthy older individuals and patients with beginning neurodegenerative disease. As proposed in a review of studies on compensation (Scheller et al., This study was carried out in accordance with the recommendations of the ethics committee of the Albert Ludwigs University Freiburg with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the ethics committee of the Albert Ludwigs University Freiburg.ES, JP, LS, CK, and SK: designed the study; JP, JL, and LS: acquired data; ES, CK, and CG: analyzed data; ES, JW, and SK: interpreted data for the study; JW and ES: created figures; ES and SK: drafted the manuscript and all authors revised it critically for important intellectual content. All authors gave their final approval of the version to be published and agree to be accountable for all aspects of the work.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 impeccable photostability of fluorescent nanodiamonds (FNDs) is an ideal property for use in fluorescence imaging of proteins in living cells. However, such an application requires highly specific labeling of the target proteins with FNDs. Furthermore, the surface of unmodified FNDs tends to adsorb biomolecules nonspecifically, which hinders the reliable targeting of proteins with FNDs. Here, we combined hyperbranched polyglycerol modification of FNDs with the β-lactamase-tag system to develop a strategy for selective imaging of the protein of interest in cells. The combination of these techniques enabled site-specific labeling of Interleukin-18 receptor alpha chain, a membrane receptor, with FNDs, which eventually enabled tracking of the diffusion trajectory of FND-labeled proteins on the membrane surface. Fluorescence imaging of proteins of interest requires highly specific molecular labeling with fluorescent probes, such as cyanine and rhodamine dyes, and fluorescent proteins ,2,3, enaTo observe the fluorescence from a single molecule, total internal reflection fluorescence (TIRF) microscopy has been developed  to monitet al. [In this study, we examined fluorescent nanodiamonds (FNDs) as fluorescent probes for selective labeling of membrane proteins. FNDs fluoresce in the range of 600–800 nm ,17, whicet al. . Howeveret al. ,31. BL-ti.e., BL-tag binds the β-lactam ring in ampicillin, forming a covalent bond [BL-tag is a mutant class A β-lactamase protein that was created to be covalently labeled with molecules with an ampicillin structure; ent bond ,31. In oTo label the BL-tagged proteins with 30 nm-sized FNDs, the FND surface was modified with HPG and the terminal COOH groups were further attached to the ampicillin moieties a. The obex = 488 nm, λem = 507–509 nm) [in situ, in which the labeling specificity was demonstrated for a BL-tagged IL18Rα expressed in HEK293 cells  in a buffer solution in a test tube, in which 20 μL of the BL-EGFP solution (50 mg/mL) was added to 1 mL of Tris-HCl buffer (pH 8.0) containing 2 mg of FND-HPG-Amp. After 2 h of incubation at room temperature, the FNDs were collected by centrifugation and repeatedly washed until the fluorescence signal from the supernatant became negligibly low. The collected FNDs showed the characteristic fluorescence spectrum of EGFP (λ–509 nm) , indicat93 cells .ca. 8 s (2/s (n = 15). Note that the value is given as the mean ± standard error. This value is approximately consistent with that determined by using the TMR probe , and similar to those for transmembrane proteins laterally diffusing on the plasma membrane (0.09~0.59 μm2/s) [We then tested whether the labeling strategy can be utilized in the fluorescence tracking experiments. The FND-HPG-Amp particles were reacted with BL-IL18Rα expressed on the membrane of living HEK293 cells. After extensive washing, fluorescence images of the cells were recorded immediately with oblique illumination fluorescence microscopy before the labeled receptors underwent internalization via endocytosis or cross-reacted with two or more proteins, as most of FNDs were expected to have more than one Amp moiety in our experimental condition. ca. 8 s . Notablyca. 8 s . From th9 μm2/s) .x-y plane. However, with this technique, there is a trade-off between the spatial and temporal resolution. To achieve higher spatial resolution, acquisition of a large number of photons with longer exposure time is generally required, leading to lower temporal resolution. Thus, to attain both high spatial and temporal resolution, stronger laser irradiation is needed; however, this causes rapid photobleaching of the fluorophores. For instance, QDs bleach within 5 s under the 5 ms exposure times to obtain 1 nm scale accuracy [A plausible application of our FND-labeling strategy would be in fluorescence imaging with one nanometer accuracy (FIONA) ,34, whicaccuracy . In the Another promising and attractive application of our strategy is in high sensitivity magnetic sensing in nanospace by employing the optically detected magnetic resonance (ODMR) of NVCs in FNDs. Electron spin resonance of an NVC can be optically measured under ambient conditions with high sensitivity at a single spin level . RecentlAlthough we obtained a reasonable value for the diffusion coefficient of the membrane protein in this study, it is still difficult to completely rule out the possibility that a single FND was connected with two or more proteins, as several ampicillin moieties could be loaded on the surface of a single FND in our preparation condition. To overcome this problem and create a FND-HPG particle labeled with a single ampicillin molecule, monofunctionalization on the nanoparticle surface can be used ,39,40.+ ions for 40 keV at a dosage of 1 × 1013 cm−1 [2SO4:HNO3 (9:1 v/v) at 70 °C for 3 days and then centrifuged for 30 min at 15,000 g, discarding the supernatant. Next, 0.1 M NaOH was added and the suspension was stirred for 2 h at 90 °C. After centrifugation, the nanodiamonds were stirred in 0.1 M HCl for 2 h at 90 °C [In this experiment, 30-nm nanodiamonds  were used. Ion irradiation, which is a common method for creating additional vacancies in nanodiamonds, was applied with He013 cm−1 . The sam013 cm−1 . The sam013 cm−1 . To elimat 90 °C . FinallyFNDs were treated with borane in tetrahydrofuran (1 M) under nitrogen atmosphere with refluxing for 12 h. The reaction was quenched with an aqueous HCl solution and the samples were centrifuged and washed three times with milli-Q water, yielding FND-OH .g for 30 min to collect the hyperbranched polyglycerol-grafted FNDs (FND-HPG). FND-HPG was then centrifuged and washed four times to remove free polyglycerol [FND-OH was dispersed in glycidol , sonicated for 2 h, and stirred for 10 h at 140 °C. The resulting gel was diluted with methanol and centrifuged at 87,000 glycerol .g for 30 min to collect the COOH-terminated hyperbranched polyglycerol-grafted FNDs (FND-HPG-COOH). FND-HPG-COOH was then washed four times to remove excess succinic anhydride and pyridine [FND-HPG was reacted with succinic anhydride  in pyridine under argon atmosphere at 70 °C for 1 h. After the reaction, the samples were centrifuged at 87,000 pyridine .g for 30 min to collect the ampicillin-modified FND-HPG-COOH (FND-HPG-Amp). FND-HPG-Amp was washed four times to remove unreacted materials. Conjugation of Amp to FND-HPG was checked by Fourier transform infrared spectroscopy (FND-HPG-COOH (15.0 mg) was reacted with N-hydroxysuccinimide  and water-soluble carbodiimide  in milli-Q water for 30 min at room temperature to activate the COOH groups. Ampicillin sodium salt (2.6 mg) was then added to the solution and stirred for 3 h. After the reaction, the samples were centrifuged at 87,000 troscopy . After ttroscopy .2. The protein expression of BL-tag fused IL-18Rα for each colonized cells was confirmed with the western blot method.The DNA fragment of BL-tag fused between the signal peptide and the mature coding sequence of IL-18Rα  was cloned into the pcDNA3.1+ plasmid vector . The plasmid vector was transfected to HEK293 cells  using Nucleofector . The cells were cultured in high glucose-containing Dulbecco’s modified Eagle’s medium  supplemented with 10% heat-inactivated fetal bovine serum , and geneticin (400 μg/mL). All cells were incubated at 37 °C in a humidified atmosphere of 5% COSingle FND-labeled membrane proteins were visualized on the apical plasma membrane of HEK293 cells at 37 °C, using a homemade objective lens-type total internal reflection fluorescence (TIRF) microscope with an oblique-mode of illumination, based on a Nikon Ti-E inverted microscope  . It was We established a method that allows for specific labeling of IL18Rα expressed on the plasma membrane with FNDs. HPG surface modification of FNDs dramatically decreased their nonspecific adsorptive nature, with specific conjugation between FNDs and IL18Rα being achieved via the BL-tag system. With this method, we successfully recorded the molecule trajectories of IL18Rα on the membrane. The labeling strategy described in this study can be widely applied to not only various membrane proteins such as receptors and ion channels but also cytosolic proteins. Considering the excellent fluorescence property of FNDs, high spatiotemporal resolution with extended recording time can be expected for various live cell fluorescence imaging techniques, including single-molecule tracking. Recently, NVC-based ODMR has been shown to provide unprecedented information for molecules in nanospace. Our labeling strategy allows for ODMR experiments on FNDs conjugated to specific proteins, which can offer unique information regarding the structure and dynamics of the proteins functioning in subcellular space, expanding new avenues for nanobiology.


This perspective paper focuses on the most recent results suggesting a potential role for UDP-Glucose as a signaling molecule in plants. In animals, UDP-Glucose is well-established as an extracellular signaling molecule that is sensed by G-protein coupled receptors, activating several downstream defense mechanisms. Recent studies have shown that abnormal growth occurred in both vegetative and reproductive tissue of plants with reduced UDP-Glucose levels, and this could be rescued by exogenous UDP-Glucose. In plants with increased biomass accumulation, the genes involved in UDP-Glucose production were up-regulated. However, excessive endogenous accumulation of UDP-Glucose induced programmed cell death (PCD), and this could also be obtained by exogenous UDP-Glucose application. Plants with decreased UDP-glucose were insensitive to pathogen induced PCD. We speculate that UDP-Glucose acts as an extracellular signaling molecule in plants, and that it may be perceived as a damage-associated molecular pattern. Gene expression is regulated by diverse signals and stimuli that are sensed and subsequently transmitted via signaling pathways that ultimately control transcription of genes. For sugar signaling, this starts by sensing the level and nature of a specific sugar. Several mechanisms have been proposed on how these sugars are perceived. Since sugars are readily interconverted in cells, identifying the precise sugar that is sensed is difficult. The mechanisms downstream of sugar sensing is even more complex, and most of the knowledge on sugar signaling in plants were obtained from yeast and mammalian systems.Carbohydrates like sucrose (Suc) and glucose (Glc) have been linked to their role as signaling molecules during plant development and stress conditions. For Glc, the major energy and carbon source in eukaryotic organisms, this is well-established by the identification of HXK which is the first enzyme in Glc catabolism. This enzyme is now characterized as a true Glc sensor with both signaling and catalytic activities . AlthougIn animals, UDP-Glucose (UDP-Glc) acts as extracellular signaling molecule via G-protein-coupled receptors , but litde novo synthesis of numerous genes and proteins occurs, providing evidence of its regulatory role  hexoses, led to significant responses. Increased levels of Suc are known to induce the expression of genes involved in starch biosynthesis such as the ADP-Glc pyrophosphorylase (AGPase) in several species, however, from these reports it is not clear whether this response was specific to Suc . It is aEven though, Suc is the main sugar transported from source to sink tissue in plants, several sugar responses depend on Glc and other signaling sugars directly, or indirectly through energy and metabolite sensors, through the activity of invertases and SuSys . MoreoveFigure 1). In animals, UDP-Glc serves as an extracellular signaling molecule that activates several pathways . UDP-Glc is mainly synthesized from UTP and Glucose-1-phosphate (G1P) through UGPase in source tissues and formed together with Fru via the degradation of Suc by SuSy in sink tissues.Uridine 5′ -diphosphate-glucose (UDP-Glc) serves as the key substrate in the synthesis of both Suc and polysaccharides, and serves as the Glc donor for many glycosylation reactions  mutation (spl29) caused lesion mimics and subsequent leaf senescence  UDP-Glc transporter has been identified to date, although UDP-Glc transporters exist in the endoplasmic reticulum membrane . HoweverSH1 and SUS1) interacting with the anion channel on the mitochondrial membrane in an anoxia-enhanced manner, are believed to be involved in the regulation of solute fluxes in or out of the mitochondria , but showed a low Km (0.21 mM) for UDP-Glc . It will be interesting to investigate whether Arabidopsis UAP2 mutants show similar UDP-Glc accumulation, since UAP2 shows a high affinity for UDP-Glc in vivo  and Arabidopsis UAP2 mutants as compared to WT, to pinpoint the reason for UDP-Glc accumulation.Clearly UAP can catalyze the conversion of a broad range of substrates. The ability of some UAP enzymes to convert UDP-Glc to G1P  remains  in vivo . FurtherUAP1 mutant (Most studies analyzing genes contributing to the UDP-Glc pool do not consider changes in metabolites other than UDP-Glc . This po1 mutant , the ina1 mutant . Althoug1 mutant . Interes1 mutant . TOR is In conclusion, accumulation of UDP-Glc can cause an imbalance in other metabolites that are known to be involved in sugar signaling, instead of being a signaling molecule itself. Both these hypothetical scenarios provide alternative signaling pathways that can arise from increased levels of UDP-Glc.UDP-sugars, or the so called activated sugars, are high energy donor substrates for several biosynthetic reactions in cells. These sugars also play an active role in the glycosylation of proteins in the secretory pathway of the endoplasmic reticulum. In animals, it is well-established that these sugars interact with receptors on the cell surface, and several nucleotide-activated cell surface receptors have been identified with a wide variety of downstream responses . In plan14, shows a high affinity for extracellular UDP-Glc compared to other nucleotides and nucleotide sugars (In animals, the UDP receptor, P2Ye sugars . This ree sugars . The mose sugars . MAP kine sugars . Interese sugars . By manie sugars ? This miIn animals, UDP-Glc receptors seem to play a crucial role in response to stress conditions by activating several downstream cascades serving as protective mechanisms. In plants, however, it is still unclear as to whether UDP-Glc plays any role as a signal molecule, and no receptor has been associated with this nucleotide sugar either. In Arabidopsis, G-protein-receptors have been associated with signal transduction of external sugars and subsequent autophagy , providiFluctuations in sugar levels are clearly associated with metabolic responses, however, the exact mechanisms of how these sugar levels are perceived are not yet fully understood. Glc, Suc, and T6P are the most studied sugars for their role in metabolic signaling in plants, however, several other sugars and intermediates have been proposed as signaling candidates. UDP-Glc has been proposed as potential intracellular mediator of ROS signaling and PCD. Alterations in endogenous levels of UDP-Glc had several downstream responses which could also be simulated with exogenous UDP-Glc. This points to a potential signaling role of UDP-Glc in plants. In animals, UDP-Glc functions as an extracellular signaling molecule perceived by receptors that triggers several downstream kinases. These findings suggest that UDP-Glc can play a similar role in plants to what is found in animals. This opens the door for future work on UDP-Glc as potential signaling molecule and unraveling the mode of perception of this sugar nucleotide in plants.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Mechanistic approaches revealed WIN 55,212-2 to suppress IL-1β-induced TF expression via inhibition of ceramide formation and via decreased phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinases. Further inhibitor experiments demonstrated neutral sphingomyelinase (nSMase) to confer ceramide generation upon IL-1β treatment with the parallel IL-1β-mediated activation of MAPKs occurring via an nSMase-independent pathway. Finally, a receptor-independent inhibition of IL-1β-induced TF protein by WIN 55,212-2 was confirmed in human blood monocytes. Collectively, this data provide a hitherto unknown receptor-independent anticoagulatory action of the cannabinoid WIN 55,212-2.The role of cannabinoids in thrombosis remains controversial. In view of the primary importance of tissue factor (TF) in blood coagulation and its involvement in the pathology of several cardiovascular, inflammatory and neoplastic diseases, a regulation of this initial procoagulant signal seems to be of particular interest. Using human umbilical vein endothelial cells (HUVEC) the present study investigated the impact of the synthetic cannabinoid WIN 55,212-2 on interleukin (IL)-1β-induced TF expression and activity. WIN 55,212-2 caused a time- and concentration-dependent suppression of IL-1β-induced TF protein accompanied by decreases in TF mRNA and activity. Inhibition of TF protein expression by WIN 55,212-2 was mimicked by its cannabinoid receptor-inactive enantiomer WIN 55,212-3 but not by structurally unrelated phyto-, endo- and synthetic cannabinoids. In addition, the inhibitory effect of WIN 55,212-2 was not reversed by antagonists to cannabinoid receptors (CB Tissue factor (TF), formerly known as thromboplastin, is a critical initiator of physiologic and pathophysiologic coagulation. The 47-kDa transmembrane glycoprotein binds factor VIIa to form a complex that cleaves factor IX and factor X, ultimately leading to fibrin formation . TheTF-dependent coagulation has been implicated to promote thrombotic episodes in a variety of clinical disorders, including cardiovascular diseases, septic shock and cancer. Accordingly, TF expression has been identified in all stages of atherosclerotic lesions and significantly higher levels of circulating soluble TF have been found in patients with acute myocardial infarction and unstable angina (for review see ). ClinicTF-dependent signaling has been also implicated in tumor growth, angiogenesis, metastasis and thrombosis in patients with cancer (for review see ). As a m9-tetrahydrocannabinol (THC), the main psychoactive ingredient of marijuana, was previously shown to increase the expression of glycoprotein IIb-IIIa and P-selectin on platelets -1,3-benzene disulfonate (WST-1) by mitochondrial dehydrogenases in metabolically active cells.HUVEC were seeded at a density of 25,000 cells/well in 96-well plates 24 h before stimulation. PBMC were seeded at a density of 100,000 cells/well in 96-well plates followed by a 1-h adhesion period and removal of supernatants. HUVEC or adherent monocytes were incubated with the respective test compounds or vehicles for 8 h in 100 μl of the corresponding medium. Thereafter, cell viability was measured by the colorimetric WST-1 test . This test is based on the cleavage of the tetrazolium salt 4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2P < 0.05.Comparisons between groups were performed with one-way ANOVA plus post hoc Bonferroni (comparison of selected groups within one experiment) or Dunnett test  using GraphPad Prism 5.04 . Results were considered to be statistically significant at values of


Giardia duodenalis and Enterocytozoon bieneusi are widespread pathogens that can infect humans and various animal species. Thus far, there are only a few reports of G. duodenalis and E. bieneusi infections in ruminant wildlife. Thus, the objective of this study was to examine the prevalence of G. duodenalis and E. bieneusi in forest musk deer in Sichuan, China, as well as identifying their genotypes.n = 80) and the Maerkang Breeding Institute (n = 143). Five (2.24%) faecal samples were positive for G. duodenalis; three belonged to assemblage E, and two belonged to assemblage A based on the sequence analysis of the β-giardin (bg) gene. One sample each was found to be positive based on the glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi) gene, respectively. Thirty-eight (17.04%) faecal samples were found to be E. bieneusi-positive based on the internal transcribed spacer (ITS) sequence, and only SC03 genotype was identified, which belonged to the zoonotic group 1 according to the phylogenic analysis. The infection rates were significantly different among the different geographical areas and age groups but had no apparent association with gender or clinical symptoms.In total, we collected 223 faecal samples from musk deer at the Sichuan Institute of Musk Deer Breeding in Dujiangyan (G. duodenalis and E. bieneusi in musk deer. Identification of the zoonotic genotypes indicated a potential public health threat, and our study suggested that the forest musk deer is an important carrier of these parasites.To our knowledge, this was the first molecular characterisation of  Giardia spp. are parasites with a broad host range comprising economic, companion, and wildlife animals, ranging from mammals to amphibians and birds, and humans [Giardia infection [Enterocytozoon bieneusi is another common intestinal parasite that infects the host’s enterocytes, causing gastrointestinal illness such as chronic diarrhoea in animals and humans, particularly in immunosuppressed groups, including organ-transplant recipients, children, the elderly, and patients with cancer, diabetes, or AIDS [d humans , 2. Thesd humans . Accordinfection . Enteroc or AIDS , 6. Inge or AIDS .Giardia duodenalis is the only species of Giardia infecting humans and is comprised of eight assemblages (A-H). Among them, assemblages A and B have a broad host range and zoonotic potential. In particular, subtypes A1, A2, A3, A4, B1, and B4 are closely associated with human infections. In contrast, assemblages C-H have strong host specificity and a narrow host range [E. bieneusi [E. bieneusi has been reported in various economic animals and wildlife, including snakes and birds [E. bieneusi have been identified and divided into eight groups (groups 1–8). Most genotypes in group 1 have zoonotic potential, whereas the other groups have narrow host range and higher host specificity [st range , 9. Apprbieneusi , 11. In nd birds –14. Currcificity .Moschus spp.) is currently considered a class I-protected animal in China. Forest musk deer (Moschus berezovskii) is the largest species of musk deer and mainly found in the Sichuan and Guizhou provinces of China [G. duodenalis or E. bieneusi can result in a significant loss of musk yield. This study aimed to investigate the presence of these parasites in musk deer, which may pose a threat to the health of both forest musk deer and humans.As an endangered species, musk deer . DNA samples were stored in 200 μl of the kit Solution Buffer at -20 °C until use.Faecal samples were washed with distilled water and centrifuged at 3000× G. duodenalis and E. bieneusi were identified using nested PCR amplification of the β-giardin (bg) gene and internal transcribed spacer (ITS) sequence, respectively. bg-positive specimens were subjected to further amplification of the glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi) genes, whereas ITS-positive specimens were subjected to amplification of three microsatellites  and one minisatellite (MS4). The primers and amplification conditions were as previously described [Taq PCR Master Mix , 8.5 μl deionized water (Tiangen), 2 μl DNA, and 1 μl each of upstream and downstream primers. Positive and negative controls were included in each test. Secondary PCR products were subjected to 1% agarose gel electrophoresis.escribed –21 . Assemblages and subtypes were determined by the alignment of the nucleotide sequences with known reference sequences for the G. duodenalis and E. bieneusi sequences was utilised to assess genetic clustering of the genotypes. A total of 1000 replicates were used for the bootstrap analysis.Neighbor-joining phylogenetic analysis of the aligned bg, gdh, and tpi genes of G. duodenalis, and ITS, MS1, MS3, MS4, and MS7 of E. bieneusi isolated from forest musk deer in this study were deposited in the GenBank database under accession numbers MF497406–MF497412 and MF942581–MF942596, respectively.All nucleotide sequences of the G. duodenalis and E. bieneusi are emerging zoonotic pathogens. To our knowledge, this study is the first to report the presence of G. duodenalis and E. bieneusi in musk deer, with an infection rate of 2.24% (5/223) and 17.04% (38/223), respectively. G. duodenalis infection rate in the Dujiangyan breeding centre (3.75%) was slightly higher than that in the Maerkang breeding centre (1.40%), whereas E. bieneusi infection rate was much lower in Dujiangyan than in Maerkang  , which may be caused by incomplete development of the immune system of young animals compared with adult animals. The proportion of infected females and males was similar. Several infected animals had obvious diarrhoea , which may be due to the individual’s low resistance to infection. There was no apparent age- or gender-associated difference for the infections in this study, in agreement with the findings of Zhang et al. [G. duodenalis was obtained only from young forest musk deer in the Maerkang breeding location, whereas assemblage E was obtained from adult forest musk deer in the Sichuan Institute of Musk Deer Breeding in Dujiangyan  accounted for more than half of the g et al. . Here, aan Table .Table 3GG. duodenalis in musk deer has not been reported, there are few reports of these parasites infecting other species in the families Cervidae and Bovidae, in the same suborder as the forest musk deer. G. duodenalis identified in these animals was mainly assemblage A, and in several studies, the rate of infection in these species was higher than that in forest musk deer in our study. For example, Lalle et al. [G. duodenalis was 11.5% in fallow deer (Dama dama) which was also higher in fawns than in older deer, and the genotype was assemblage A. García-Presedo et al. [Capreolus capreolus) samples were positive for G. duodenalis, and the genotype was AII. In Norway, 12.3% of moose , 1.7% of red deer (Cervus elaphus), 15.5% of roe deer, and 7.1% of reindeer (Rangifer tarandus) were found to be infected with G. duodenalis [Odocoileus virginianus) was found positive for G. duodenalis, and assemblage A was identified [G. duodenalis found in red deer and roe deer was AIII and zoonotic AI, respectively. Also, sheep faecal specimens from China were found to be positive for G. duodenalis assemblage A genotype [G. duodenalis assemblage A was previously identified in humans, forest musk deer can play a role in transmitting G. duodenalis to humans.Although the distribution of e et al.  reportedo et al.  reportedodenalis . In the entified . Solarczentified  reportedgenotype . Given tE. bieneusi analysis, ITS sequencing showed that all E. bieneusi isolates from Maerkang and Dujiangyan were characterised as SC03 (n = 38), which had been previously found in sika deer (Cervus nippon) at zoological gardens in China [E. bieneusi isolated in this study belonged to group 1 (subgroup 1d)  of faecal samples from Père David’s deer (Elaphurus davidianus) in China were E. bieneusi-positive [E. bieneusi, including 28 sika deer and one red deer [E. bieneusi in golden takins (Budorcas taxicolor bedfordi) in China, and Shi et al. [E. bieneusi in 28.8% (176/611) of goats and 42.8% (177/414) of sheep, with 42 genotypes identified. Twenty-three (7.0%) yaks in China were E. bieneusi-positive; three genotypes  from group 2 that were previously reported in humans and two group 1 genotypes were identified [E. bieneusi genotypes were identified in 26 (32.5%) white-tailed deer in the United States [E. bieneusi genotype we identified in forest musk deer, and most E. bieneusi genotypes reported in the Cervidae and Bovidae can infect both humans and animals. However, E. bieneusi isolated from forest musk deer appeared to be from a single genotype, in contrast to those found in other deer species, yaks, and goats.From the 38 ITS-positive specimens, there were nine, five, one, and three isolates successfully sequenced at MS1, MS3, MS4, and MS7 loci, respectively. Analysis of sequence polymorphisms and single nucleotide polymorphisms (SNPs) at MS3 locus revealed two distinct types (type I and II) (Table positive . Anotherred deer . Zhao eti et al.  found E..8% 176/6 of goatsG. duodenalis and E. bieneusi is well described, their method of transmission is still not clear. Investigations on their epidemiology, detection methods, and diagnosis are required to provide experimental bases for ensuring the health and safety of both animals and humans.Although the genetic heterogeneity of G. duodenalis and E. bieneusi in forest musk deer in China. Furthermore, to our knowledge, this is the first report of G. duodenalis and E. bieneusi infections in musk deer and thus demonstrating that the host range of these parasites is wider than previously reported. Zoonotic genotypes identified in this study showed the transmission potential of G. duodenalis and E. bieneusi from forest musk deer to humans or other animals. Currently, there is no known effective vaccine or drug to treat infection with these parasites. Hence, measures should be taken to prevent humans and animals from being infected by these parasites.This study demonstrated the prevalence of


However, measuring ROS  content in vivo is now possible using the MitoB probe. Here, we extend and refine the MitoB method to make it suitable for ecological studies of oxidative stress using the brown trout Salmo trutta as model. The MitoB method allows an evaluation of H2O2 levels in living organisms over a timescale from hours to days. The method is flexible with regard to the duration of exposure and initial concentration of the MitoB probe, and there is no transfer of the MitoB probe between fish. H2O2 levels were consistent across subsamples of the same liver but differed between muscle subsamples and between tissues of the same animal. The MitoB method provides a convenient method for measuring ROS levels in living animals over a significant period of time. Given its wide range of possible applications, it opens the opportunity to study the role of ROS in mediating life history trade-offs in ecological settings.In recent years evolutionary ecologists have become increasingly interested in the effects of reactive oxygen species (ROS) on the life-histories of animals. ROS levels have mostly been inferred indirectly due to the limitations of estimating ROS from  Over the last two decades evolutionary ecologists have shown a growing interest in the role of oxidative stress in shaping life-histories12367891011in vitro assays of the rate of mitochondrial ROS production1516in vitro results to the in vivo situationin vitro assays require considerable expertise and laboratory facilities and must be conducted on fresh samples , so virtually precluding assays of ROS in wild animals.The vast majority of the ROS present in organisms are generated by the mitochondriaet al.in vitro assays. This method uses a newly-developed ratiometric probe, called MitoB, to measure levels of one major ROS, hydrogen peroxide (H2O2), within living animals2O2 into MitoP  after controlling for effects of the mass of liver used for the extraction of Mito compounds . These results suggest that the MitoP/MitoB ratio in trout is unlikely to be affected by differential rates of disappearance of the two molecules, at least over the 72 h period covered by this trial. Moreover, the probe appears to accumulate rapidly within the tissues, as indicated by the large amount of MitoB present in the liver within 3 hours of injection , with two aliquots from each tissue being flash-frozen for separate extraction of compounds. White muscle was taken dorsally to the lateral line (to avoid contamination with red fibres) and just behind the caudal fin; one aliquot was collected from one side of the fish while the other aliquot was collected from the other side. Intra-class correlation coefficients (ICC) were used to test for the consistency of the quantification of the compounds between the two measures from the same tissue extract. The consistency was calculated using a two-way mixed effect ICC with an absolute agreement definition of concordance. This type of ICC was employed for all ICC used in our study as it assesses not only the correlation but also the exact similitude between measurements.In order to evaluate whether the Hr = 0.785, P = 0.001), but not for the white muscle . White muscle showed a significantly higher average MitoP/MitoB ratio than the liver within individuals . However, there was no correlation between the individual MitoP/MitoB ratio in the liver with that in the muscle . Importantly, such differences in the MitoP/MitoB ratio within the muscle aliquots of an individual cannot be triggered by variation in the extraction and quantification of the compounds. Variation among samples in the efficiency of the extraction step would have an equal effect on both MitoB and MitoP, so would only change their absolute concentrations but not the MitoP/MitoB ratio. In a separate experiment, we showed that the quantification of each of the compounds and their isotopic spikes was very consistent, so that the very high repeatability in the calculated MitoP/MitoB ratio from duplicated quantification of individual extracts  is unlikely to be a source of variation in the estimation of the MitoP/MitoB ratio  but all fish received the same absolute dose of MitoB, the initial concentration of MitoB in the body (nmol MitoB g−1 fish) varied four–fold among individuals  or MitoB content . However, the duration of exposure to the MitoB probe  had a small but significant negative effect on final MitoB content . Finally, final MitoB content (i.e. about 28 h after injection) was slightly higher in smaller fish, i.e. those that started with a higher concentration of MitoB . These results suggested that it might be prudent to take initial whole-body MitoB concentration and exact duration of exposure into account when calculating MitoB content of the sampled tissue (whereas in our study MitoP levels did not require correction). In order to correct the MitoP/MitoB ratio for these effects, the MitoB content could be first standardized for the mean values of the exposure duration and the initial concentration of MitoB in the body. However, such corrections were found to have relatively little effect on calculated MitoP/MitoB ratios, since the ratios calculated from uncorrected and corrected MitoB content were very similar  and 24-fold (range: 74.82 to 1782.02 pmol) respectively . The size of the liver samples used for the extractions  had no effect on the measured MitoP . However, our results clearly show that the method can be used even if animals are not recaptured until several days after injection, so making it feasible to use the assay in the field. Note that this time course is likely to be temperature-dependent: MitoB was found to be barely detectable 12 h after injection in Drosophila living at 25 °CWe have developed a step-by-step procedure (Steps 1–3) that is required in order to apply the MitoB method to a new animal model, with an additional step that is necessary before using the MitoB method on aquatic organisms (Step 4). In juvenile brown trout kept at 12 °C, we found that MitoB had already accumulated in the tissues 3 h post-injection, but levels of MitoP were in some cases too low to be accurately quantified. Larger tissue samples or a more sensitive HPLC-MS may overcome this detection limit and allow more reliable measurement of MitoP/MitoB ratios soon after MitoB injection. More importantly, MitoB was still present at high levels in the fish 72 h after injection, so allowing measurements of HAfter the selection of an appropriate experimental timescale (about 24 h in our case), the effects of (i) the dose of MitoB injected, and (ii) the exact duration of MitoB exposure need to be evaluated. We chose to inject a standardized dose of MitoB (constant volume and amount) instead of a body mass-normalized dose (constant amount per unit of body mass) since this represents the situation faced by many ecologists where measuring the body weight of mobile animals and adjusting the dose accordingly is not always convenient (e.g. when working in the field). In addition, the duration of MitoB exposure will depend on the recapture time of the animal, which can be variable when sampling free-living organisms. Our results show that the MitoB content at the end of the exposure was influenced by exposure duration and concentration of MitoB at injection; while these had a minimal effect on the variation in MitoP/MitoB ratio found in our data, this may not be the case in other contexts and so both should be quantified and their importance evaluated. Although the MitoB method necessities the capture, injection, release and then recapture of the animal, this is a very commonly used protocol for a number of well-established assays that assess physiological processes in an ecological context , since the sample can be frozen and stored at −80 °C before analysis19232O2 in the mitochondria. For instance, H2O2 levels as measured using MitoB increase with age in flies but do not explain variation in lifespan and fecundity192O2 also functions as a signalling molecule controlling essential cellular processes422O2 is involved in developmental control and immune responses332O2 needs attention: when, where and by how much do H2O2 levels constrain the animal? ROS have been suggested to act as universal life-history constraints1In summary, this method provides the means to move towards direct assessment of ROS levels in living animals from the size of a fly upwards, across a range of ecological contexts (i.e. field as well as laboratory) and over timescales from hours to days, all of which are relevant criteria for ecologists and evolutionary biologistsThis study adheres to the animal welfare standards of the U.K. Home Office. All animal experiments were conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals. The study was covered by Home Office Project Licence No. 60/4292 and approved by the University of Glasgow Animal Welfare and Ethical Review Board.15-MitoB (d15MitoB) and deuterium15-MitoP (d15MitoP) were synthesized at the University of Glasgow according to Cairns, et al.2O), with a final ethanol content of 1% (v/v). A 504 μM MitoP solution was produced in the same way. Aliquots of MitoB, MitoP and 0.9% saline solutions were stored at −70 °C and thawed at the time of the experiment. Standard solutions for the calibration curve and for the deuterium spikes were made by serially diluting the compounds in 100% ethanol; these were also kept at −70 °C.The probe compounds MitoB, MitoP, and their deuterium spikes deuteriumSalmo trutta at the University of Glasgow in 2014 and 2015. For each experiment, trout were initially held in a common tank under standard conditions of temperature (12 °C), photoperiod (12 L: 12 D), and ad libitum food . At the start of each experiment, animals were transferred to individual tanks, unless specified, containing 2 L of aerated water, allowed to acclimate for 24 h before being injected with the probe, and maintained in these conditions throughout the period of probe exposure.All the experiments were conducted on juvenile brown trout 2O) in the case of controls. Insulin syringes have a very fine needle, but to avoid loss of any injected solution, a finger was kept on the punctured skin which was slowly massaged for a few seconds. The injected fish were put back in their well-aerated tanks which allowed a complete recovery according to Kennedy, et al.Fish were lightly anaesthetised (50 mg benzocaine per litre water) and then injected with MitoB into their intraperitoneal (IP) cavity using an insulin syringe . The injection was anterior to the base of the pelvic fins. Control animals were included in order to obtain a blank control value for the subsequent quantification analysis. Each fish was injected with the same volume (100 μl) of fluid, which was a standard dose of 504 μM MitoB (50 nmol/fish) or a saline solution (0.9% (weight in g/volume in mL) NaCl/Het al.et al.et al.et al.d15MitoP, MitoB and d15MitoB) was analysed by Xcalibur software  per sample were corrected using these individual coefficients. The HPLC-MS response for “blank” samples, i.e. those which have been exposed to saline solution with no MitoB, must be measured for each new animal model or tissue. In our study the AA of MitoP and MitoB for the control fish injected with saline solution were below the detection level, and so were not considered for calculation. For all other fish we calculated the ratio pmolMitoP/pmolMitoB , hereafter referred to as the “MitoP/MitoB ratio”; higher values indicate a higher content of H2O2 in the mitochondria of the living animalPeaks from the standards were used to produce calibration curves relating AA for each of the four compounds to pmol of MitoP, How to cite this article: Salin, K. et al. Using the MitoB method to assess levels of reactive oxygen species in ecological studies of oxidative stress. Sci. Rep.7, 41228; doi: 10.1038/srep41228 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Gender and sex diverse individuals—transgender individuals and those with disorders of sex development (DSD)—both face medical treatments that may impair biological fertility potential. Young DSD patients also often have abnormal gonadal development. Fertility preservation for these populations has historically been poorly understood and rarely addressed. Future fertility should be discussed with gender and sex diverse individuals, particularly given recent advances in fertility preservation technologies and evolving views of fertility potential. Key ethical issues include parental proxy decision-making and uncertainty regarding prepubertal fertility preservation technologies. Many opportunities exist for advancing fertility-related care and research for transgender and DSD patients. Although many distinctions exist between transgender individuals and individuals with DSD, an important area of commonality lies in the area of fertility—the ability to have a biological child.The term “gender and sex diversity” refers to (1) transgender individuals for whom gender identity is incongruent with birth-assigned sex and (2) individuals with differences (disorders) of sex development (DSD), also known as intersex, for whom development of chromosomal, gonadal, or anatomic sex is atypical.Many medical and surgical therapies available to transgender adolescents/young adults (AYA), and children and AYA with DSD, can affect future fertility. Individuals with DSD frequently also have subfertility related to their medical condition. Gender and sex diverse individuals often choose to build a family through options such as adoption or surrogacy. For some, however, it may also be important to have the option of a biological child. Fertility preservation technologies  and societal attitudes toward “nontraditional” family structures have expanded significantly in recent years such that having a biological child is now possible for many transgender adults and adults with DSD.The purpose of this perspectives article is to provide an introduction and overview of preservation of biological fertility potential for gender and sex diverse individuals. We also propose next steps for advancing this new clinical and research focus.2 Transgender individuals and their healthcare teams have now begun to recognize that options for preserving reproductive potential exist. A majority of transgender adults believe that fertility preservation should be discussed with and offered to them.4 The Endocrine Society Guidelines recommend counseling for transgender individuals regarding fertility before initiating gender-affirming hormonal therapy.5 Knowledge of the effects of medical treatments on fertility, familiarity with available fertility preservation options, and an appreciation for potential ethical issues are all necessary to effectively counsel patients.Loss of biological fertility was once thought to be a given consequence of gender transition.6 However, many transgender individuals initiate gender-affirming hormone therapy concurrently with pubertal suppression,7 and thus, germ cells never fully mature. This highlights the need for discussion of fertility options before initiating GnRH-a treatment.Pubertal suppression with gonadotropin releasing agonists (GnRH-a) not only prevents development of potentially distressing secondary sex characteristics but also suspends germ cell maturation. Puberty appears to progress normally after discontinuation.9 Testosterone use by transgender men causes ovarian stromal hyperplasia11 and follicular atresia.10 Gonadal effects of gender-affirming hormones are thought to be at least partially reversible. For example, pregnancy has been reported in transgender men who have previously used testosterone.13 Thresholds have not been established for the amount and duration of exogenous testosterone or estrogen exposure necessary to have a permanent negative effect on fertility.Gender-affirming hormones produce impairments in gonadal histology that can cause infertility. Estrogen use by transgender women results in impaired spermatogenesis and an absence of Leydig cells in the testis.For patients who elect surgical transition, gonadectomy will render them permanently sterile. Thus, this is the last opportunity for fertility preservation for patients who choose gender reassignment surgery that includes gonadal removal.14 OTC is a potential area of future investigation for peripubertal affirmed males, as it does not require hormonal stimulation. Similarly, testicular cryopreservation may offer fertility potential for peripubertal affirmed females. Prepubertal tissue cryopreservation protocols for oncology patients could be expanded to transgender individuals in the future. Although the technology is still developing, fertility experts expect that precursor egg and sperm cells will soon be able to be matured and used with assisted reproductive technologies to produce a biological child.15–17Ovarian tissue cryopreservation (OTC), an experimental procedure in which prepubertal ovarian tissue is harvested and preserved, is offered to women with cancer.A primary ethical concern regarding fertility preservation for transgender AYA is that parents must make decisions about fertility preservation if it is to occur before initiation of pubertal suppression and/or gender-affirming hormone therapy. Although this is done with patient assent, children are often not intellectually or emotionally mature enough to predict their future desires or understand the implications of their present decisions. This results in transgenerational fertility decision-making, which may not align with future patient desires; disagreement between the patient and parent regarding fertility preservation may also arise. In addition, there may be patient distress associated with procurement processes, given that the gamete type is not congruent with gender identity. Furthermore, peripubertal tissue cryopreservation is experimental. It is unknown if and when assisted reproductive technologies will be available to mature germ cells. Thus, patients are potentially incurring surgical risk and storing gonadal tissue without the guarantee of future fertility.As for transgender patients, infertility or sterility has often been assumed for many DSD patients. The approach to DSD care has shifted in the last few decades from the assumption that individuals were psychosexually neutral and early sex reassignment surgery was preferred, without concern for future fertility, to one in which it is acknowledged that the formation of gender identity is poorly understood and that irreversible surgeries should be avoided or performed only with great caution and counseling. In current practice, the future fertility potential is considered and discussed, but there remain little evidence and infrequent counseling about fertility preservation.The term DSD refers to a diverse group of conditions that result in atypical sex development. This includes sex chromosome abnormalities, including Turner and Klinefelter syndrome, disorders of gonadal development, including complete and partial gonadal dysgenesis, and disorders of androgen synthesis or action—complete and partial androgen insensitivity , congenital adrenal hyperplasia, and 5-alpha reductase deficiency. These diverse DSD conditions differ in etiology and fertility potential. DSD patients are at risk for potential infertility or sterility due to the following: (1) abnormal gonadal development, (2) gonadectomy for malignancy risk, (3) abnormal hormonal function, and (4) discordance between gonadal type and gender identity. These often overlapping etiologies are outlined in 18–21Currently, the treatment for infertility in DSD is limited. General options for pre and postpubertal fertility preservation are the same as for transgender patients . HoweverEthical concerns related to proxy decision-making, patient maturity, and the experimental nature of prepubertal cryopreservation mirror those that exist for families of transgender individuals. Unique to DSD, some families are now opting to postpone gonadectomy for fertility potential purposes, with an unclear trade-off in terms of gonadal malignancy risk. Researchers are still working to define the fertility potential of many DSD conditions, so counseling regarding the balance between surgical risk, risk of malignancy, and possibility of future fertility is occurring in context of great clinical uncertainty. Finally, many DSDs are a result of genetic mutations, which could be passed to the offspring. Thus, genetic counseling is crucial, and preimplantation genetic diagnosis will play an important role in fertility preservation for DSD patients.Transgender and DSD patients are largely disparate, but the medical expertise necessary to care for both overlaps, and the potential for infertility is shared. Both groups have previously been told infertility or sterility was an eventuality. Advancing scientific techniques and broadening perspectives can change this assumption. Fertility preservation for individuals with gender and sex diversity is a new frontier that requires research and collaboration. Priorities include delineating the fertility desires of patients and families, gaining a comprehensive understanding of treatment effects on fertility potential, further defining the gonadal potential of the diversity of DSD conditions, and expanding possibilities for reproductive tissue preservation and maturation. We must establish research protocols, develop clinical tools to guide fertility-related care, and broaden the medical community's knowledge of current and future fertility preservation possibilities.


High serum uric acid (UA) level has been assumed to be a risk factor for left ventricular (LV) dysfunction; however, the precise relationship between these conditions has not been fully examined because many confounding factors are associated with UA level. We herein examined the precise relationship by proposing structural equation models. The study population consisted of 1432 cases with ischemic heart disease who underwent cardiac catheterization. Multiple regression analyses and covariance structure analyses were performed to elucidate the cause-and-effect relationship between UA level and LV ejection fraction (LVEF). A path model exploring the factors contributing to LVEF showed that high UA was a significant cause of reduced LVEF (P = 0.004), independent of other significant factors. The degree of atherosclerosis, as estimated by the number of diseased coronary vessels, was significantly affected by high UA (P = 0.005); and the number of diseased coronary vessels subsequently led to reduced LVEF (P < 0.001). Another path model exploring the factors contributing to UA level showed that LVEF was a significant cause of high UA (P = 0.001), while other risk factors were also independent contributing factors. This study clearly demonstrated that there was a close link between high UA and LV dysfunction, which was represented by possible cause-and-effect relationship. According to epidemiological research and meta-analyses, the serum UA level predicts the progression of chronic kidney disease and the development of stroke5. Furthermore, elevated UA levels are associated with the presence of hypertension, diabetes, and metabolic syndrome8, while the relationship between ischemic heart disease (IHD) and serum UA level remains controversial. Another recent meta-analysis studying the relationship between serum UA and IHD showed that a high UA level was not likely to be a main determinant of IHD and that it may not significantly contribute to the prediction of IHD in the general population9.Patients with high uric acid (UA) levels are predisposed to gout as a major clinical complication. With the increased awareness of the hazardousness of high UA levels, discussion has also recently resurfaced in the cardiovascular field10. Nonetheless, few studies have shown a precise relationship between high UA levels and cardiac function, as measured in detail by echocardiography, cardiac catheterization, and other methods. Thus, the direct relationship between cardiac systolic dysfunction and high UA levels remains unclear and an investigation should be performed in which the effects of high UA levels on the process of coronary atherosclerosis are considered.In contrast, in patients with heart failure (rather than IHD), there has been increasing evidence to indicate that high UA levels predict an increase in morbidity and mortalityWhen taken together, although it has almost been accepted that high UA levels are associated with most cardiovascular diseases, including heart failure, many more studies are required to confirm the positioning of high UA level as a risk factor for the respective cardiovascular disorders. A statistical analysis would be helpful; however, there is a degree of intractableness associated with performing a precise analysis because serum UA level is likely to be associated with many other risk factors, including -but not limited to- male gender, obesity, dyslipidemia. All of these risk factors may –both individually and collectively- affect the progression of cardiovascular diseases. It therefore seems quite difficult to confirm the risk that a high UA level itself poses in relation to cardiovascular diseases. Advanced statistical methods will be needed in order to elucidate the precise risk of high UA levels.11.In a statistical analysis, confounders are extraneous variables that affect the variables being studied—when confounders are present, the results do not reflect the actual relationships between the studied variables. Confounding variables are often defined as variables that are correlated—either positively or negatively—with both the dependent variable and the independent variable. There are several ways to eliminate confounding bias, for example, by adjustment of the independent variables. Still, a confounding bias can be difficult to control if multiple potential confounding variables are present or if the study population is of insufficient size. A covariance structure analysis plays an important role in understanding how the relationships between observed variables may be generated in many areas using hypothesized latent variables. A covariance structure analysis is useful for exploratory and explanatory factor analyses. This analysis can be performed based on the confounding bias. Another merit of using a covariance structure analysis is that the analysis can package Bayesian networks to infer cause-and-effect relationships. However, the factors that are included in these analyses should be carefully selected, and the path model based on covariance structure analysis should be proposed based on consistent concepts and the clear direction of the study. In a tangible way, we successfully proposed a path model based on a covariance structure analysis in order to explain a complex phenomenon involving possible causalityIn the present study, we examined the effects of high UA levels on cardiac function and the cause-and-effect relationship between them using cardiovascular disease patients, all of whom underwent cardiac catheterization in our institution. We performed a step-by-step statistical analysis to identify the risk of high UA per se for cardiac dysfunction while taking into account the influence of high UA levels on IHD—as evaluated by organic stenosis in the coronary arteries and the effect of IHD on the cardiac function.The clinical characteristics of the 1432 cases are shown in Table A multiple regression analysis was performed to examine the effects of risk factors on the LVEF  was proposed by positioning the serum UA level in parallel with the other risk factors of age, gender, BMI, TG, smoking, AOsys, eGFR, and HbA1c  are shown in Table The theoretical path model (B) was proposed by positioning LVEF in parallel with the risk factors of age, gender, BMI, TG, AOsys, smoking, eGFR, and HbA1c, all of which possibly affect the serum UA level Fig. . The serThe precise results of path models (B) and (C) are shown in Tables 2; however, the precise positioning of UA in these conditions remains unclear and controversial behind the high impact of metabolic syndrome. One of the difficulties in research the impact of UA is probably attributable to the intractableness of the statistical approach because the serum UA levels are tightly associated with numerous other risk factors. In this study, we used a covariance structure analysis together with single and multiple regression analyses. A covariance structure analysis can eliminate confounding bias and clarify possible cause-and-effect relationships. As a result, we found that a high UA level was causally related to LV dysfunction and vice versa, suggesting that there is a possible cause-and-effect linkage between these factors.It has recently been reported that a high UA level is a significant risk factor for many cardiovascular diseases, including heart failureThe current study showed that high UA levels were thought to be causally reduced LVEF. In this study, we took account of the severity of IHD because IHD is affected by numerous risk factors, and subsequently affects LV dysfunction. The path model (A) was proposed in order to simultaneously estimate the effects of risk factors for both IHD and LVEF in a single equation model. This analysis indicated that high UA levels were causally linked to the severity of IHD and then that IHD affected LVEF. Importantly, it was clarified that high UA levels reduced LVEF independently of the severity of IHD.13 and various metabolic disorders, such as glucose intolerance, elevated blood pressure, dyslipidemia, and atherosclerotic cardiovascular diseases, which are conceptualized as metabolic syndrome18. However, smoking fell short of being significantly associated with hyperuricemia, in contrast to the findings in previous reports20.We found that an older age, male gender, obesity, hypertriglyceridemia, hypertension, renal dysfunction, and diabetic condition were major factors associated with high UA levels in Path model B. The current results are mostly in agreement with those of previous reports; hyperuricemia is closely associated with visceral fat accumulationIn this study, 85.5% of the patients were male and 14.7% were female. Generally, the serum UA level is elevated during puberty in men and after menopause in women. In addition, the UA clearance in the kidney is low in men. Therefore, there is a gender difference in the serum UA level, which might have influenced the results of the current study. However, given the small number of female patients in the study, we are unable to make a conclusive decision at present, and analyses concerning gender differences in serum UA levels should be performed in the future.22 are likely involved. Furthermore, elevated UA levels reflect upregulated xanthine oxidase (XO) activity, which induces oxidative stress24. These findings suggest that a high UA level is a major risk factor for the development of cardiovascular diseases. On the other hand, this study also indicated that LV dysfunction causally induced high UA levels. The molecular mechanisms are also unclear at present; however, the proposed mechanisms are as follows: adenosine triphosphate (ATP), which is synthesized in the mitochondria is transferred to myofibril by phosphocreatine (PCr) through the Cr kinase energy shuttle, and is used by the contractile mechanism to form adenosine diphosphate (ADP), which is resynthesized by oxidative phosphorylation in the mitochondria27. In states of ATP depletion and reduced Cr kinase shuttle, such as heart failure28, the metabolic turnover of the purine metabolism is increased, and the breakdown paths below ADP are activated, eventually reaching the breakdown endpoint of UA by the activation of XO29. At present, it is unclear whether this biological reaction occurs in the cardiomyocytes. Future research should be focused on the precise energetic metabolic changes that occur in connection with high UA levels in an in vitro analysis.Although the molecular mechanisms underlying the harmful effects of high UA levels have not been fully examined, the activation of redox-dependent effects, extracellular regulated kinase (ERK), proinflammatory pathway, and endothelin-1vice versa , drifting into a vicious cycle. The current study strongly supports the previous reports that show the significance of high UA levels as a harmful effect in heart failure31. Overall, the present findings indicate that UA is not only an active player but also an indirect marker of the pathogenesis of IHD. XO inhibitors could theoretically reverse the harmful reactions of XO and those of high UA35. Inhibition of XO can be expected to decrease the formation of cytosolic ROS, which ought to be increased in heart failure. XO inhibitors would thus in turn lessen the inhibition imposed by ROS on the cytosolic CK, which simulates the formation of ATP from PCr, thereby providing energy for contraction in HF. In fact, a recent study reported that XO inhibitors accelerated Cr kinase energy shuttle29. In addition, XO inhibitors theoretically block the purine metabolism, namely, the paths of ATP breakdown. Taken together, XO inhibitors may have the potential to produce and conserve ATP in the failing heart. In this context, it would be interesting to examine the effect of XO inhibitors on LVEF independently of serum UA. Unfortunately, the number of patients taking XO inhibitors in the present study was not sufficient for a statistical analysis, and details regarding the medications being taken by patients were missing. However, the findings of such an analysis along with those of a covariance structure analysis would thus support the need to conduct a prospective tracking study of XO inhibitors in patients with heart failure.When taken together, this study suggests that high UA levels lead to the development of heart failure; and In the present study, the mean UA level was 6.1 ± 1.4 mg/dL, and UA-lowering agents were administered to 17.2% of cases. In other words, the UA level in most subjects was considered to be within the normal range, and the number of the patients with hyperuricemia were small in this study. Given these clinical characteristics, the present results may indicate a harmful effect of not-very-high levels of serum UA on the cardiac function, although we have no way of determining the ideal levels of serum UA.37. However, this study did not include precise data about AF. This important analysis should also be performed in the future. Finally, it is quite natural for serum levels of UA to change substantially over time and due to the effects of various drugs, although diuretics are typically stopped before catheterization in our facility. Food intake, alcohol consumption and physical stress also contribute to rapid changes in the serum UA. In contrast, the progress of atherosclerosis and the development of heart failure is relatively slow in general. However, we used the serum UA level obtained at the point of cardiac catheterization in this study. Although the study population was relatively large, this represents another study limitation.The present study is associated with some limitations. First, we used the TG level to represent dyslipidemia because we preliminarily performed an association study of UA with TG and low-density lipoprotein (LDL) and found a substantial link between TG and UA rather than LDL and UA (data not shown). In the future, we should perform an association study to determine the associations between UA and precise lipid profiles, including LDL, HDL, TG, and oxidized-LDL using a covariance structure analysis and attempt to clarify the combinations that are truly hazardous with regard to the progression of coronary atherosclerosis and heart failure. Second, the mean age of the subjects was 66.5 years old, which seems relatively young among subjects with IHD. This population characteristic is believed to be due to the exclusion of elderly or critically ill patients who did not meet the indications for cardiac catheterization. There may therefore be a selection bias among our study patients, and further studies in a more general population are warranted. It will also be interesting to investigate the pathological conditions of IHD in greater detail, such as by dividing patients into groups of acute coronary syndrome (excluded in the current study) and stable effort angina. Furthermore, patients with non-IHD were excluded from this study; a study of non-IHD patients should be performed in another series. Third, atrial fibrillation is likely to have an important association with high UA levelsvice versa, indicating that there is a direct cause-and-effect linkage between these factors. Stratified analyses, for example, by gender difference and the degree of obesity, are needed to further explore the condition in which this linkage becomes more evident.In summary, while elevated UA levels are often associated with established cardiovascular risk factors, the question as to whether UA is an independent risk factor for IHD and/or heart failure remains controversial. This is probably attributable to the intractableness of statistical approaches because the serum UA levels are tightly associated with many other risk factors. In the present study, we examined the relative importance of the associations between UA levels and cardiac function, with consideration for the severity of IHD using a covariance structure analysis. After eliminating multiple confounding biases, we found that a high UA level is thought to be causally related to LV dysfunction and The study population consisted of 1432 cases with IHD who were consecutively admitted to our institutions from 2012 to 2016. All of the patients underwent cardiac catheterization for an evaluation of IHD. We excluded the patients who underwent hemodialysis because their cardiac function was significantly altered by artificial volume control. Emergency cases (i.e. acute coronary syndrome) were also excluded, as their hemodynamics (including LVEF) and miscellaneous biomarkers are highly variable and do not reflect the values under stable conditions. The ethics committee of The Jikei University School of Medicine approved the study protocol (24–355[7121]), and we complied with the routine ethical regulations of our institution. This was a retrospective study, and informed consent could not be obtained from each patient. Instead of obtaining informed consent from each patient, we posted a notice about the study design and contact information at a public location in our institution.38. The number of diseased vessels was counted as the number of three major coronary arteries  with organic lesions that were indicated for treatment by revascularization and/or standard medical therapy. Diagonal and high lateral branches were included in LAD and LCx, respectively, if they had a substantial myocardial perfusion area and were indicated for treatment. In the present study, we divided the patients into four groups based on the number of diseased vessels with significant organic stenosis . Patients with a left main trunk lesion were included in the 2-vessel group. The number of diseased vessels was counted at the time of cardiac catheterization in this study, and lesions already treated by revascularization were not included. Patients with coronary spasm  were placed into the 0-vessel group if there was no organic stenosis after nitroglycerin administration. Some of the patients had comorbid cardiovascular diseases, such as valvular disease, arrhythmia, cardiomyopathy, and other conditions. Hypertension, Diabetes mellitus (DM) and dyslipidemia were defined as described previously39. The eGFR was calculated according to the Modification of Diet in Renal Disease (MDRD) Study equation shown below40, with coefficients modified for Japanese patients41.IHD was diagnosed based on symptoms, electrocardiography (ECG), blood sampling, and the coronary artery morphology. Organic lesions producing ≥75% luminal stenosis of the coronary arteries on coronary angiography were defined based on the modified American Heart Association (AHA) coronary tree segment classification2, according to the guidelines of the Japanese Society of Nephrology.Renal dysfunction was defined by an eGFR of <60 mL/min/1.73 m42.We collected blood samples and hemodynamic data during cardiac catheterization. The serum biochemical analyses were performed in a central laboratory of our hospital during the study period. In this statistical analysis, we included serum levels of Cr, UA, and TG, AOsys, and HbA1c level. We included smoking habit . LVEF was measured at the time of left ventriculographyContinuous variables are expressed as the mean ± standard deviation (SD) or medians. The correlation between two factors was investigated by a single regression analysis and expressed as Spearman’s correlation coefficient. A multiple regression analysis was performed to compare multiple values. The above-mentioned statistical analyses were performed using the SPSS Statistics software program . P values of <0.05 were considered to indicate statistical significance.11. In brief, the possible causality model defined some hierarchical regression models between clinical factors and LVEF or serum UA level. For every regression, the total variance in dependent variable is theorized to be caused by either independent variables that are included in the model or by extraneous variables (e). The indirect effect was determined by multiplying the path coefficients of the intervening variables. The structural equation models that were obtained were tested and confirmed; P values of < 0.05 were considered to indicate statistical significance.A path analysis based on a covariance structure analysis was used to investigate the relationship between clinical factors in this study population and to survey the probable causal effects on LVEF or serum UA level. The path analysis was performed using the IBM SPSS AMOS software program . We have previously described how to write a path modelSupplementary Information