C. elegans as a model system to investigate the effects of GGA. First, we show that GGA-mediated activation of the HSR is conserved in worms. Then, we show that GGA can ameliorate beta-amyloid toxicity in both muscle and neuronal worm Alzheimer’s disease models. Finally, we find that exposure to GGA is sufficient to extend the lifespan of wild-type worms. Significantly, the beneficial effects of GGA on both beta-amyloid toxicity and lifespan are dependent on HSR activation. Taken together, this research supports further development of GGA as a therapeutic for Alzheimer’s disease, provides evidence that HSR activation is a relevant therapeutic mechanism, and indicates that the beneficial effects of GGA are not limited to disease.Activation of a cytoprotective cellular pathway known as the heat shock response (HSR) is a promising strategy for the treatment of Alzheimer’s disease and other neurodegenerative diseases. Geranylgeranylacetone (GGA) is a commonly used anti-ulcer drug in Japan that has been shown to activate the HSR. Here, we establish  Translation of biomedical science discoveries into disease-modifying therapeutics is a slow and laborious process that requires enormous financial resources. The estimated cost of developing a new pharmaceutical is between 1 and 3 billion dollars . One anaRepurposing of current therapeutics is a strategy that can substantially reduce development costs . Drug reAlzheimer’s disease (AD) is a devastating neurodegenerative disease with an urgent and desperate need for therapeutic development . Two of The heat shock response (HSR) is a cytoprotective, cellular stress response that responds to protein misfolding induced by temperature and other stresses . The HSRGenetic activation of HSR genes has been shown to have beneficial effects in disease models for Alzheimer’s disease and other neurodegenerative diseases. For example, overexpression of the HSP70 heat shock gene suppresses toxicity in a mouse AD model . FurtherGeranylgeranylacetone (GGA), also known as Teprenone, is a drug that has had widespread use in Japan as an anti-ulcer agent since the 1980s . While tAdministration of GGA was found to have dramatic effects in a mouse Alzheimer’s disease model. GGA treated APP23 mice exhibited decreased levels of beta-amyloid peptide, beta-amyloid aggregates, and synaptic loss . ImportaCaenorhabditis elegans as a model to study the mechanism of GGA and determine if the beneficial effects of GGA against beta-amyloid toxicity requires HSR activation.The promising results with GGA in the mouse Alzheimer’s disease model and its established clinical use in humans makes GGA an ideal candidate for drug repurposing. Continued development of GGA would benefit from a model system that is experimentally feasible and genetically tractable. Therefore, we establish unc-54/human Aβ peptide 3-42 minigene) + rol-6(su1006)] (snb-1:Aβ 1-42:3′ UTR (long) + mtl-2:GFP]I (unc-54 (vector) + (pCL26) mtl-2::GFP] (hsf-1(sy441) (daf-16(mu86) (Nematodes were maintained using standard laboratory techniques on NGM plates seeded with OP50 bacteria at 20°C unless otherwise indicated . Worms wsu1006)] ; 3) CL23-2:GFP]I ; 4) CL21-2::GFP] ; 5) PS351(sy441) ; 6) CF1016(mu86) .hsp-16.2 forward (ACTTTACCACTATTTCCGTCCAGC), hsp-16.2 reverse (CCTTGAACCGCTTCTTTCTTTG), hsp-16.11 forward (GGCTCAGATGGAACGTCAA), hsp-16.11 reverse (GCTTGAACTGCGAGACATTG), F44E5.5 forward (CAACTGCTGGTGATACCCATCTC), F44E5.5 reverse (CTTGAAAGTGTTCTCTTGGCACG), hsp-70 forward (GTACTACGTACTCATGTGTCGGTATTTATC), hsp-70 reverse (ACGGGCTTTCCTTGTTTTCC), cdc-42 forward (TTTGCTTCTCCGTGGTTGCTCC), and cdc-42 reverse (TCCGTTGACACTGGTTTCTGCTTG).GGA was obtained from Santa Cruz Biotechnology. Diacetyl was obtained from TCI America. RNAi constructs were obtained from the Ahringer RNAi library and used for feeding RNAi as previously described . The folcdc-42. All qPCR experiments were performed with biological triplicates and p-values were calculated using the Student’s t-test.N2 worms were grown at 20°C on NGM control or 10 μM GGA plates starting at the L1 larval stage until day one of adulthood. RNA was isolated using Trizol Reagent (Thermo Fisher) as previously described with the addition of a freeze-thaw in liquid nitrogen and use of a Direct-zol RNA kit (Zymo Research) . RNA washsf-1 RNAi containing bacteria starting at the L1 larval stage. Worms were scored for paralysis starting on day 1 of adulthood. Individuals were considered paralyzed if the body of the worm did not move when gently prodded. Worms were transferred by picking onto new plates as needed to separate adults from eggs and larva. Data was analyzed using OASIS 2 (Transgenic CL 2006 (muscle beta-amyloid) worms were grown at 20°C on NGM control or 10 μM GGA plates seeded with empty vector RNAi (L4440) or  OASIS 2 .Transgenic CL2122 (control) and CL2355  worms were grown at 16°C on NGM control or 10 μM GGA plates starting at the L1 larval stage until day one of adulthood. Then, worms were shifted to 25°C for 24 h to induce neuronal beta-amyloid expression. Day 2 adults were conditioned by transferring to bacteria-free NGM plates with 6 μL of 0.04% diacetyl added to the plate lid . Controlhsf-1 mutant), and CF1038 (daf-16 mutant) worms were grown at 20°C on NGM control or 10 μM GGA plates starting at the L1 or L4 larval stages. Viability was scored starting on day 1 of adulthood. Individuals were considered dead if they did not have a motile response to gentle prodding and lacked pharyngeal pumping. Worms were transferred by picking onto new control or GGA plates as needed to separate adults from eggs and larva. Data was analyzed using OASIS 2  in cultured human cells and in mice  to ameliorate beta-amyloid toxicity in mice motivated further investigation of this drug using the genetically tractable model nematode  in mice . Therefo RT-qPCR . GGA alsPreviously, GGA was shown to have beneficial effects on a mouse model for Alzheimer’s disease (AD) containing expression of the human beta-amyloid peptide . TherefoHaving established that the effects of GGA on HSR activation and beta-amyloid-induced paralysis were conserved in worms, we next tested whether the effects on beta-amyloid were dependent on the HSR. The HSR was inhibited by RNAi knockdown of the HSF1 transcription factor that is required for the response. Consistent with previous work, we found that inhibition of HSF1 alone enhanced the toxicity in this disease model, with median paralysis occurring around day 6 of adulthood. In contrast to its effects in wild-type worms, GGA did not have a significant effect on beta-amyloid toxicity when HSF-1 was inhibited. These results indicate that activation of the HSR is required for the beneficial effects of GGA.verses 0.66 conditioned).Having shown that GGA can reverse the toxicity from beta-amyloid expression in muscle tissue, we next tested the effects of GGA in neurons, a more relevant tissue type for AD. In a second worm AD model, neuronal expression of human beta-amyloid has been shown to cause neuronal toxicity and results in a defect in associative learning . In the verses 0.47 conditioned). Significantly, exposure to GGA restored associative learning in worms with neuronal beta-amyloid (0.68 naïve verses 0.39 conditioned). Taken together, these experiments demonstrate that the beneficial effects of incubation with GGA in worms extend to neurons.We tested the ability of GGA to reverse the associative learning defect in worms expressing neuronal beta-amyloid. GGA did not affect chemotaxis nor associative learning in wild-type worms    . Together, these data indicate that GGA can extend lifespan in worms via activation of the HSR and that its beneficial effects are not limited to neurodegenerative disease models.Having established the effects of GGA on disease models, we next tested whether its beneficial effects could extend to longevity. A wide range of protein aggregates accumulate during the normal process of aging and disruption of protein folding homeostasis, or proteostasis, is considered a hallmark of aging . Overexpue 0.03) ; Table 1e 0.002) . We testue 0.54) ; Table 1C. elegans as a model system for investigation of the anti-ulcer drug geranylgeranylacetone (GGA). First, conservation of the effects of GGA in worms was shown with respect to both HSR activation and amelioration of beta-amyloid toxicity. Then, genetic manipulation of the system was used to demonstrate that activation of the HSR is required for GGA’s beneficial effects. Finally, it was discovered that GGA can also extend lifespan in wild-type worms. Taken together, this research not only supports further development of GGA as a novel Alzheimer’s disease (AD) therapeutic, but also provides a foundation that can be used for new insights into HSR regulation and lifespan.This manuscript establishes C. elegans HSR field. This activation is not surprising given the high degree of conservation in the HSF1 transcription factor that mediates this response. However, worms are resistant to the effects of many small molecules due to the barrier function of their cuticle. The ability to activate the HSR independent of temperature increases and associated protein misfolding stress will enable new experimental approaches to explore HSR regulatory mechanisms. Furthermore, as HSR-inducible promoters have been one of the mainstays for ectopic expression in C. elegans, this tool may also be useful for other fields of biology that use worms as a model organism including developmental biology and neuroscience.Activation of the HSR by GGA in worms establishes a new tool in the Amelioration of the detrimental effects of beta-amyloid expression in worm muscle and neuronal AD models by GGA is consistent with previous results showing that GGA has beneficial effects in a mouse AD model. However, the establishment of its effects in worms facilitated an explicit test of its dependence on the HSR. The powerful genetic tools and other advantages of this model system should set the stage for further exploration of the effects of GGA on beta-amyloid toxicity. Moreover, the use of worms can facilitate investigation of GGA in other neurodegenerative disease models such as Parkinson’s disease, ALS, and Huntington’s disease.Excitingly, we discovered that GGA can extend the lifespan of wild-type worms. This lifespan extension is also dependent on HSF1 but not dependent on insulin-like signaling, one of the major cellular pathways that influences longevity. These results are consistent with disruption of proteostasis as a hallmark of aging, further supporting that interventions targeting this pathway can have beneficial effects. To our knowledge, our results for the first time provide experimental evidence that a small molecule HSR activator can positively affect longevity in worms. Importantly, GGA is already approved for human use to treat ulcers, indicating that it is both bioavailable and does not have substantial off-target toxicity. Therefore, these findings provide strong support for future work on GGA and other HSR inducers.


Phosphorus magnetic resonance spectroscopy is the only in vivo technique that can assess the biochemistry associated with high-energy phosphate and membrane phospholipid metabolism across different cortical, subcortical and white matter areas. In this cross-sectional study, whole-brain, multi-voxel phosphorus magnetic resonance spectroscopy was acquired at 3 T on 14 patients with Pelizaeus–Merzbacher disease with protein, proteolipid protein 1 duplications and 23 healthy controls . Anabolic and catabolic levels of membrane phospholipids , as well as phosphocreatine, inorganic orthophosphate and adenosine triphosphate levels relative to the total phosphorus magnetic resonance spectroscopy signal from 12 different cortical and subcortical areas were compared between the two groups. Independent of brain area, phosphocholine, glycerophosphoethanolamine and inorganic orthophosphate levels were significantly lower  and phosphocreatine levels were significantly higher (P < 0.0001) in Pelizaeus–Merzbacher disease patients compared with controls. Additionally, there was a significant group-by-brain area interaction for phosphocreatine with post-hoc analyses demonstrating significantly higher phosphocreatine levels in patients with Pelizaeus–Merzbacher disease compared with controls across multiple brain areas . Phosphoethanolamine, glycerophosphoethanolamine and adenosine triphosphate levels were not significantly different between groups. For the first-time, widespread alterations in phosphorus magnetic resonance spectroscopy metabolite levels of Pelizaeus–Merzbacher disease patients are being reported. Specifically, increased high-energy phosphate storage levels of phosphocreatine concomitant with decreased inorganic orthophosphate across multiple areas suggest a widespread reduction in the high-energy phosphate utilization in Pelizaeus–Merzbacher disease, and the membrane phospholipid metabolite deficits suggest a widespread degradation in the neuropil content/maintenance of patients with Pelizaeus–Merzbacher disease which includes axons, dendrites and astrocytes within cortex and the myelin microstructure and oligodendrocytes within white matter. These results provide greater insight into the neuropathology of Pelizaeus–Merzbacher disease both in terms of energy expenditure and membrane phospholipid metabolites. Future longitudinal studies are warranted to investigate the utility of phosphorus magnetic resonance spectroscopy as surrogate biomarkers in monitoring treatment intervention for Pelizaeus–Merzbacher disease.Pelizaeus–Merzbacher disease is an X-linked recessive leucodystrophy of the central nervous system caused by mutations affecting the major myelin protein, proteolipid protein 1. The extent of the altered  in vivo neurochemistry in Pelizaeus–Merzbacher disease, an X-linked recessive leucodystrophy, is poorly understood. In this study, Laukka et al. report widespread biochemical alterations in patients with Pelizaeus–Merzbacher disease compared with controls utilizing phosphorous magnetic resonance spectroscopy. Results implicate energy expenditure and membrane phospholipid metabolism in Pelizaeus–Merzbacher disease.The extent of the altered   Overexpression of PLP1 causes diffuse demyelination, axonal loss and infiltration of myelin with both macrophages and microglia, but does not alter normal myelin development.2 The cellular and molecular mechanisms causing demyelination in patients with PLP1 duplications are not well understood but may include decreased energy utilization.4Pelizaeus–Merzbacher disease is an X-linked leucodystrophy caused by mutations in the gene encoding of the major CNS myelin structural protein, proteolipid protein 1 (PLP1). The most common cause of Pelizaeus–Merzbacher disease is through the duplication of the X-chromosome that encodes the PLP1 gene, which accounts for 50 to 75% of cases.1- and T2-weighted MRI studies have demonstrated distinct differences in the signal intensity of white matter (WM) areas in patients with Pelizaeus–Merzbacher disease6 with a variety of PLP1 mutations, suggesting hypomyelination, dysmyelination and demyelination due to alteration in both oligodendrocyte function and PLP1 function and expression.7 Structural MRI studies have also demonstrated volume reductions in frontal WM, arcuate fibres and the internal capsule,8 as well as diffuse atrophy throughout the brain of patients with Pelizaeus–Merzbacher disease9 although with similar proportion of grey matter (GM) to WM compared with healthy individuals.8 Lastly, diffusion tensor imaging studies have also implicated primarily WM areas in patients with Pelizaeus–Merzbacher disease with some alterations in GM areas.10 In all, the evidence provides the extent of the known areas of brain pathology in patients with Pelizaeus–Merzbacher disease but provide little insight into the underlying cellular and molecular pathophysiology of the effects of PLP1 duplications.Structural Tet al.3 and Appikatla et al.4 have demonstrated in transgenic mice that overexpress PLP1, that PLP1 can be inserted into the mitochondrial membrane as well as into myelin. Transgenic mice with increased PLP1 gene dosage have markedly decreased concentrations of brain adenosine triphosphate (ATP) and altered expression of cytochrome oxidase, Complex IV of the mitochondrial respiratory chain. In addition, tissue culture cells transfected with wild-type PLP1 acidify their culture media, also suggesting that mitochondrial function has been altered. Together, this suggests that mitochondrial dysfunction impacting energy utilization may play a role in the pathogenesis of Pelizaeus–Merzbacher disease.Hüttemann 11–14 Moreover, both PC and PE act as both anabolic and catabolic products of sphingomyelin and hence, reflect the metabolism of oligodendrocytes and myelin.15 Stained sections from the brain of a patient with Pelizaeus–Merzbacher disease with PLP1 duplication in Phosphorus magnetic resonance spectroscopy (³¹P MRS) is the only non-invasive method that can assess the neurobiological basis related to high-energy phosphate  and membrane phospholipid (MPL) metabolites . The latter reflects the molecular biochemistry and cellular density of axons, dendrites, astrocytes (neuropil).in vivo ³¹P MRS was applied to investigate regional differences in high-energy phosphate metabolites and in MPL precursor and breakdown products between patients with Pelizaeus–Merzbacher disease with PLP1 gene duplications and healthy individuals. We hypothesized widespread differences in the ³¹P MRS biochemistry between both groups that would implicate the high-energy phosphate utilization and the cellular density of both WM and GM in the pathology of Pelizaeus–Merzbacher disease.To advance our understanding of the cellular and molecular biochemistry of this disease, http://pmdfoundation.org/?page_id=343). The 23 healthy individuals matched in age and sex  were recruited between 2010 and 2014 from Detroit and the Detroit metropolitan area. The upper and lower age range was selected to restrict the sample to adolescents and young adults and hence, minimize any age effects.The 14 patients with Pelizaeus–Merzbacher disease and PLP1 duplications  were recruited between 2012 and 2014 with the help of the Pelizaeus–Merzbacher Disease Foundation , elliptical k-space sampling, with ¹H-decoupling and acquisition time 23 min. A set of T1-weighted MRI images was also collected during the ³¹P MRS session as well as with a single-tuned 32-channel ¹H volume head coil, which proceeded the ³¹P MRS. The T1-weighted MRI images  collected with the dual-tuned head coil was used to co-register the subject space of the ³¹P MRS to the high-quality T1-weighted MRI images  collected with the single-tuned ¹H volume head coil.The structural MRI and the ³¹P MRS data collection were conducted on a 3 T Siemens Verio whole-body system  using a using a dual-tuned ³¹P/¹H transmit/receive volume head coil . A three-plane MRI localizer was first collected followed by a set of sagittal and axial scout MRI images using the 2D fast spin-echo sequence, which were used to prescribe the ³¹P MRS measurement. The ³¹P MRS acquisition sequence included a single slab-selective excitation radiofrequency pulse followed by phase encoding pulses to spatially encode in all three directions. The slab in the axial plane was placed parallel to the AC-PC line and utilized to limit the localization within the brain. The experimental parameters were: 3D FID_CSI sequence (Siemens system) modified with a pre-acquisition delay of 1.43 ms, FOV = 340 × 340 × 170 mm16 using in-house software  and FSL17 and FreeSurfer19 tools as well as the Marquardt20 non-linear time-domain MRS fitting programme . Twelve different right and left voxel locations of interest were predefined anatomically on the MNI template brain and co-registered to the subject space by co-registering the T1-weighted images of the subject to the MNI template brain (12 degrees of freedom) and re-mapping the voxels to the subject space via the inverse transformation.16 The coordinates of each voxel location in subject space were then used to shift mathematically the 3D multi-voxel grid by applying a phase shift in the k-space to ensure the ³¹P MRS voxel was centred at each location and then the ³¹P signal at each location was extracted for quantification. This highly innovative procedure ensured that the voxel placements were consistently and systematically placed in the specified anatomical locations between subjects and has been demonstrated to be highly accurate and reliable.16 The voxel locations of interest included the right and left anterior and posterior WM (aWM and pWM), dorsolateral prefrontal cortex, inferior and superior parietal lobe (iPL and sPL), superior temporal gyrus (STG), anterior/body of the hippocampus (HIP), occipital cortex, striatum (STR) thalamus (THA) and the medial anterior and posterior cingulate (ACC and PCC). The post-processing included extracting and quantifying the ³¹P MRS signal from the different voxel locations. The procedure from start to finish was 100% automated (i.e. fully independent of operator input)3; an estimate that also accounted for the reduced k-space sampling during the acquisition. In the time domain of the ³¹P MRS dimension, a 5 Hz Gaussian line broadening was applied prior to quantification of the data. In quantifying the PE, PC, Pi, GPE, GPC, MPL, PCr, dinucleotides and ATP (two doublets and one triplet) resonances, the ³¹P MRS signal was modelled with 21 Gaussian damped sinusoids in the time domain using the appropriate a priori knowledge.21 PE, PC, GPE and GPC were modelled as singlets due to the applied ¹H-decoupling and the chemical shift of 2.23 p.p.m. for the MPL is consistent with prior reports of identifying a signal from less mobile phosphodiester moieties that have a relatively intermediate correlation time and are part of small MPL structures when the pre-acquisition delay time is relatively short .22–27 ³¹P MRS spectra of poor spectral quality with spectral linewidth >30 Hz for PCr, or >50 Hz for PE, PC, GPC or GPE were excluded from the statistical analysis. In total, 35 out of 814 spectra (or 4% of the data) were excluded. The outcome metabolite measurements were expressed as mole% relative to the total ³¹P MRS signal, which have been demonstrated to provide equivalent results compared with expressing values as an absolute concentration.28 An example of a modelled spectrum from a key region of interest is shown in In the k-space domain of the ³¹P MRS data, a 75% Hamming window was applied, which resulted in estimated effective voxel size of approximately 15 cmP-values of ≤0.0042 were considered significant for the post-hoc analyses in the second model reflecting the Bonferroni correction.Two regression analyses based on the generalized estimating equation methodology  were used, which included: (i) subject group (Pelizaeus–Merzbacher disease versus HC), age, region (12 voxel regions) and side (right and left) as the main effect terms to access the main group effect independent of region effects; and (ii) subject group, age, region, side and the group-by-region interaction as the main terms to access any group-by-region interactions. The dependent variables included PE, PC, GPE, GPC, PCr, Pi and ATP expressed as a mole% relative to the total ³¹P MRS signal from the 12 areas of interest, the right and left measurements across regions were treated as repeated measures in each analysis, and The ¹H-decoupling was effective in reducing the spectral broadening and subsequently allowed the delineation and quantification of the individual phosphomonoesters (PE and PC) and phosphodiesters (GPE and GPC), as demonstrated in 2χ = 9.17, P = 0.0025), MPL breakdown products, GPC  and Pi  compared with healthy participants  compared with healthy participants  as well as oligodendrocytes and myelin. During periods of neuropil development or myelination of WM tracts, in vivo human and ex vivo rat brain ³¹P MRS studies have consistently shown high MPL precursor levels of PE and PC reflecting the high demand of active MPL synthesis and metabolism of myelin.30 At the time and site of neuritic sprouting in an hippocampal lesion rat model, elevated MPL precursor levels, PC, concomitant with elevated MPL levels, phosphatidylcholine, have also been reported.31 Additionally, MPL are also constantly being turned over reflecting the ongoing reorganization/plasticity and maintenance of synaptic connections. Therefore, the observed deficits of MPL precursor levels and breakdown products, which were specific to PC and GPC, respectively, in patients with Pelizaeus–Merzbacher disease compared with healthy individuals suggests significant and widespread decrease both in axon density in cortex and in myelin in WM. This is consistent with evidence of decreased number of oligodendrocytes and related known processes of demyelination and axonal injury in patients with Pelizaeus–Merzbacher disease.32 In addition, there might also be metabolic abnormalities in oligodendrocyte function and myelin maintenance.Although the spatial resolution of post-hoc analyses for PCr (P < 0.0042). Past ³¹P MRS studies have demonstrated the opposite of decreased PCr and increased Pi during processes reflecting increased high-energy utilization.33 In this study, increased PCr levels and decreased Pi levels concomitant with non-significant differences in ATP levels between groups suggest decreased energy utilization in the brains of patients with Pelizaeus–Merzbacher disease, possibly a result of decreased cytosolic CK activity34 as suggested by Hüttemann et al.3 and Appikatla et al.4.4 The lack of group differences in ATP in our study is probably a result of strong buffering of ATP pools. Interestingly, Zhang and co-workers35 also found increased levels of PCr in the brains of mouse models of Huntington disease, as well as the brain from an individual with this disease. In addition, they also found decreased levels of CK activity, both the brain-specific BB isoform and the mitochondrial isoform, suggesting that the decrease in CK activity may lead the accumulation of PCr that cannot utilize its high-energy phosphate. Taken together, our data from ³¹P MRS analysis of individuals with Pelizaeus–Merzbacher disease are consistent with the notion that a deficit in high-energy metabolism is a part of the pathophysiology of this disease. These data may serve as a way to identify potential surrogate biomarkers to follow treatment of patients in the future.All cells native to the CNS, including neurons and glia, express creatine kinase (CK), the enzyme that catalyses the equilibrium among, PCr, adenosine diphosphate (ADP), creatine and ATP. Through CK equilibrium, PCr preserves a high-energy ratio of ATP/ADP by maintaining low ADP concentration. PCr is a high-energy compound that is used by the cell to maintain adequate ATP stores. PCr is synthesized in mitochondria by mitochondrial creatine phosphokinase, transported to the cytoplasm, and then used to regenerate ATP from ADP by the cytosolic creatine phosphokinase. 36–38 In contrast, there are in vivo ¹H MRS studies40 in patients with PLP1 duplications demonstrating increased N-acetyl-aspartate, PCr plus creatine (PCr + Cr) and myo-Inositol in WM but not in GM.40 ¹H MRS, however, lacks the biological specificity with respect to the interpretation of these results. For example, both PCr and Cr spectral peaks are indistinguishable on a ¹H MRS spectrum, and PCr and Cr are both reactants in the CK reaction and therefore, insensitive to reflect CK utilization. Likewise, GPC and PC are also indistinguishable and lack the specificity of implicating either the synthesis or degradation of MPL. Additionally, several studies in Pelizaeus–Merzbacher disease have reported ¹H MRS results expressing outcome measures as a ratio of metabolite levels, which further complicate the interpretation of results.41–43 Although these data are informative, ours is the first patient study using 31P MRS to quantify information on ATP and measure alterations in the bioenergetics in Pelizaeus–Merzbacher disease in a patient population with a uniform mutational aetiology.The number of pathologic studies focused on examining autopsy tissue from human patients with PLP1 duplications is limited.28There are limitations to this study including the following: (i) small sample size; (ii) age range of sample spanning from adolescents to young adulthood; (iii) lack of clinical data on symptom severity on the patients with Pelizaeus–Merzbacher disease for correlation analyses; (iv) relatively large voxel sizes leading to potential partial volume effects (i.e. heterogeneity in the portion of tissue types within voxels); (v) the process of shifting the CSI grid to ensure voxels were centred in the predefined anatomical locations only had 3 degrees of freedom , which may lead to subtle inaccuracies in the localization of the anatomical locations across participants; and (vi) expressing the biochemical levels as a mole% relative to the total ³¹P MRS signal and not as absolute concentration levels.in vivo ³¹P MRS is not only useful to characterizing the biochemical differences in vivo associated with Pelizaeus–Merzbacher disease but could also be used as a tool for tracking treatment progress. Several new treatments are being targeted at restoring function to the genetically abnormal oligodendrocytes or using stem cells to transplant healthy oligodendrocytes into patients with Pelizaeus–Merzbacher disease.7 ³¹P MRS would be able to localize differences in the regional biochemistry of different areas of the brain after treatment was started. The increasing myelination, and concomitant increased synthesis of MPLs would alter the levels of MPL precursor and breakdown products; differences that would be observable with ³¹P MRS. This shows that in vivo ³¹P MRS has a role in characterizing the biochemical differences in Pelizaeus–Merzbacher disease; however, future studies are warranted to investigate whether ³¹P MRS could be used to monitor responses to treatment.The ability to observe widespread biochemical differences in the brains of patients with Pelizaeus–Merzbacher disease and PLP1 duplications demonstrates that fcac202_Supplementary_DataClick here for additional data file.


Tc at moderate pressures. Here, we report a discovery of giant enhancement of Tc in CeH9 obtained via random substitution of half Ce by La, leading to equal-atomic H9 alloy stabilized by maximum configurational entropy, containing the LaH9 unit that is unstable in pure compound form. The synthesized H9 alloy exhibits Tc of 148–178 K in the pressure range of 97–172 GPa, representing up to 80% enhancement of Tc compared to pure CeH9 and showcasing the highest Tc at sub-megabar pressure among the known superhydrides. This work demonstrates substitutional alloying as a highly effective enabling tool for substantially enhancing Tc via atypical compositional modulation inside suitably selected host crystal. This optimal substitutional alloying approach opens a promising avenue for synthesis of high-entropy multinary superhydrides that may exhibit further increased Tc at even lower pressures.A sharp focus of current research on superconducting superhydrides is to raise their critical temperature  9 with superconducting temperature (Tc) of 57 K at pressures below 1 megabar. Here, the authors show that Tc can be increased to 148 K in the substitutional alloy H9, while maintaining a pressure below 1 megabar.Superconductivity was recently discovered in the clathrate hydride CeH The long-sought overarching goal is to find materials with sufficiently high superconducting critical temperatures (Tcs) to facilitate experimental exploration and practical implementation, ultimately allowing ambient-environment applications. Over the years, the search for room-temperature superconductors has been among the most luring and challenging topics in condensed matter physics; notable progress includes materials with Tc in liquid-hydrogen and liquid-nitrogen temperature ranges achieved at ambient pressure3, making it more easily manageable to utilize superconductors in advanced devices. More recently, superconductivity with Tc approaching or even exceeding the room-temperature has been predicted in a class of hydrogen-rich compounds, termed superhydrides, under extreme compression. Theoretical prediction followed by experimental synthesis led to the discovery of superconductors with Tc of 203 K in covalent SH36 and 250–260 K in ionic LaH1010, obtained at very high pressures between 150–200 GPa. A Tc of 288 K was reportedly achieved at extremely high pressures around 270 GPa in a C-S-H system with unknown composition and structure11, but it has caused intense debate14 and still awaits confirmation.Ever since Heike Kamerlingh Onnes made the astonishing discovery615, extensive ensuing studies have established a large family of clathrate superhydrides, including CaH617, YH618, YH920, CeH923, CeH1021, and LaH1010, which comprise an array of distinct hydrogen cages anchored at the center by a variety of metal atoms. The diverse structural and compositional forms of these binary compounds offer a platform for exploring material parameters to optimize the energetic and superconducting states. Systematic studies indicate that atomic radius, electronegativity, and valence electron of metal atom play important roles in tuning the most concerned properties of superhydrides, i.e., superconductivity and stability24. Among these ionic superhydrides, LaH10 faces an inescapable problem of its harsh synthesis pressure (>150 GPa), despite its very high Tc10; whereas CeH9 or CeH10 with relatively low Tc of 95–115 K can be synthesized around megabar pressure21. Improving the superconducting properties of these superhydrides with moderate synthesis and stability pressures presents pressing challenges in this active research field.Meanwhile, inspired by the prediction of high-temperature superconductive CaH25. The potential of this approach is demonstrated in a theoretical work26 showing that Li doping introduces an extra electron into the molecular-like hydrogen in MgH16 to generate atomic-like hydrogen, and the formed ternary Li2MgH16 has a Tc of 473 K at 250 GPa. In addition, theoretical studies have also designed a series of ternary superhydrides27, such as XYH830, Ca-Y-H31, and Ca-Mg-H32 that are expected to possess desirable high-Tc features or prospects. A sticking point, however, is to find ternary superhydrides that host high-temperature superconductivity at moderate, near or below megabar, pressures.Binary superhydrides are highly restricted in their configurational space with limited structural and compositional variations. To expand the material horizon, related research recently shifted focus to ternary systems with much higher degrees of freedom, which offer a larger number and richer variety of structural prototypes for superconducting superhydride screening34. This offers a viable avenue to use suitably selected binary compounds as the base template to construct ternary alloy superhydrides that share the same crystal structure. It has been shown that LaH6 and YH10 units, which are experimentally unreachable in the binary systems, do appear in La-Y alloy hydrides at pressures of 170–196 GPa35, although superconductivity is not improved. It is of great interest to explore the cubic La-H36 and hexagonal close packed (hcp) Ce-H23 systems to find a desirable ternary platform for experimental realization of high-temperature superconductivity in materials that can be stabilized at relatively low pressures.Rare-earth (RE) metals have similar electronegativities, electronic configurations, and atomic radii, so their disordered solid solution alloys are easy to form9 with essentially equal metal-atom occupancy was obtained using the starting materials of equiatomic La-Ce alloy and ammonia borane (NH3BH3) under the synthesis conditions of about 110 GPa and 2100 K. The synthesized ternary alloy was maintained to at least 90 GPa during decompression. Compared with binary CeH9, the Tc is dramatically increased by up to 80 K in ternary H9, which contains an unprecedented LaH9 unit that is unstable in pure compound form but stabilized in the base binary CeH9 structural framework. This atypical compositional structure unit, together with the reduction of possible Abrikosov-Gor’kov suppression-like effect37 of Ce by replacing with La, have a critical impact on the enhancement of Tc of the ternary H9 alloy. The present findings demonstrate that substitutional alloying is effective in tuning and improving high-temperature superconductivity in superhydrides by stabilizing new and unusual structural and compositional configurations that are conducive to greatly enhanced Tc, and this approach may be extended to constructing additional ternary and higher multinary alloys to usher in more breakthrough discoveries in the quest of finding optimal clathrate superhydrides through a thoughtful choice of substitutional alloying metal-element combinations.In this work, we experimentally investigated the crystal structure, superconductivity, and stability pressure range of a La-Ce alloy superhydride. Substitutional H3BH3 as the pressure transmitting medium and hydrogen source. A total of seven samples were compressed to 110–125 GPa at room-temperature and then heated to 2100–2300 K with pulsed radiation from an yttrium-aluminum-garnet laser  with NHhcp (P63/mmc) lattice with cell parameters of a = 3.762(6) Å and c = 5.675(5) Å  experiments in synchrotron radiation sources. The representative XRD pattern of the product in Cell-4 at 110 GPa is shown in Fig. hcp P6/mmc lattVlattice) can be used to estimate the hydrogen concentration of the superhydride. While the volumes  of La, Ce, and H atoms are not known, they have to be estimated from their elemental phases. Specifically, at 110 GPa, phase structures of distorted-fcc for La38, of bct for Ce39, and of hcp for H240 are adopted where VLa, VCe, and VH are calculated to be ~16.04, ~14.59, and ~2.30 Å3/atom, respectively. As a result, the hydrogen content (n) of the synthesized La-Ce alloy hydrides was determined as ~8.5 using the formula of n = (Vlattice – VLa – VCe)/VH/2, which has a small deviation with the ideal value of 9. Numerical error in this calculation is inevitable, and it comes from the direct use of elemental phases for the evaluation of volumes. It is expected that once a compound is formed the volume of constituted elements will somewhat change compared with their elemental phases. Furthermore, the experimental equation of state (EOS) during decompression is highly consistent with the simulated EOS H9 H9 with a tiny amount of impurity (~3.8 At%) being as I4/mmm-H4 H4 would not hold such high superconductivity at Tc = 148–178 K observed in this study. For a good comparison, its isostructural phase in a lighter hydride of YH4 shows much lower superconductivity at 76–88 K43. It is expected that the heavier hydride of H4 should possess even lower Tc values than that in YH4. Therefore, the measured superconductivity is associated with major H9 rather than minor H4. With decreasing pressure, the superconducting transition tends to disappear at about 90 GPa H4 Fig. . In our 20. However, due to the Pauli paramagnetic effect of electron spin polarization and the diamagnetic effect of orbital motion, an applied external field can disrupt the Cooper pairs, thereby reducing the value of Tc. As shown in Fig. μ0H = 9 T, the application of the magnetic field reduces Tc by about 12 K (14 K). The extrapolated values of the upper critical field μ0Hc2(T) and the coherence lengths towards T = 0 K are 56 T (52 T) and 24 Å (25 Å) and 76 T (70 T) and 21 Å (22 Å), respectively, by Ginzburg-Landau (GL)44 and Werthamer-Helfand-Hohenberg (WHH)45 model fits. The obtained μ0Hc2 data and coherence lengths following the 10% criterion of the metallic state resistance are similar to those derived from the use of 90% resistance. The magnitude of the short coherence lengths and high upper critical fields indicate that ternary H9 is a typical type-II superconductor. We note that the similar superconductivity of ternary La-Ce-H system was observed by two different groups47 during the preparation of our manuscript.Owing to the rather small size of the samples, it is still a challenging task to measure the weak signals of the magnetic flux expulsion  effect under high pressures9 phase with H29 cage was predicted8 to be a high-temperature superconductor in 2017, and this structural prototype has since been experimentally verified by a series of RE metal superhydrides51. Of particular interest among these compounds is CeH9 that was synthesized at easily achievable megabar pressure and exhibited an experimental Tc of ~95 K21. Compared to the higher-Tc LaH1036, the low Tc of CeH9 stems from its low logarithmic average phonon frequency, which may be related to the 4f electron in Ce at high pressure24. Furthermore, no superconductivity or only low-temperature superconductivity was observed in other similar RE metal superhydrides, where strongly correlated interactions of 4f electrons dramatically suppress the superconductivity52. It has been reported that the superconductivity of LaH10 was dramatically suppressed by doped Nd53 (each atomic percent of Nd causes a decrease in Tc of 10–11 K), which can be reasonably understood by the well-known impure superconductor theory proposed by Abrikosov and Gor’kov37. If half of the weakly magnetic Ce in CeH9 is replaced by non-magnetic La, Abrikosov-Gor’kov suppression-like effect of Ce should be significantly weakened, resulting in the giant lift of Tc in H9 over that in CeH9. This analysis points to rational improvement of superconductivity in CeH9 via substitution of Ce by La that hosts no 4f electron but otherwise shares most chemical characters.The clathrate REHTc varies slightly in different experiments, which may originate from subtle differences in molar ratios of the La-Ce alloy polyhydride samples or different degrees of anisotropic stress that leads to variable deformation of the lattice in different experiments54. Moreover, the observed c/a ratio (1.511) of the synthesized hcp H9 at 110 GPa is smaller than the ideal value of 1.633. This indicates that the structure is not isotropic, which may play somewhat role in regulating superconductivity.In addition, the pressure dependence of hcp LaH9 is calculated to be thermodynamically unstable8 and unlikely to be synthesized among binary system at the corresponding pressure of ~100 GPa, the LaH9 structural unit appears in synthesized ternary La-Ce alloy superhydride via La-substitution modification of the native CeH9 units. This result can be elucidated by the Hume-Rothery rule55, because the ratio of La/Ce atomic radii (~1.032 at 110 GPa)39 is well within the rule’s stated 15% range, and La and Ce have similar electronegativity values33, thus satisfying the conditions for the formation of a disordered solid solution compound. Moreover, laser heating helps to overcome the kinetic reaction barrier and stabilize substitutional H9 via the contribution of the configurational entropy to the total free energy, overcoming the contribution of mixing enthalpy and the energy increment of LaH9 relative to LaH10 at a sufficiently high temperature34. In particular, the configurational entropy reaches the maximum in an equal-atomic distribution and plays a significant role in stabilizing ternary H9. This effect should be more pronounced in higher multinary superhydrides, such as equiatomic medium- or high-entropy alloy superhydrides. Further theoretical examination and experimental verification are needed.Although Tc enhancement in equal-atomic substitutional ternary alloy H9 that exhibits high Tc values of 148–178 K in the pressure range of 97–172 GPa examined in this work. These Tc values are significantly higher, by up to 80 K, compared to the results found in binary CeH9, which represents a giant, 80% enhancement. This ternary alloy superhydride integrates the native CeH9 structural units and the atypical LaH9 units that are unstable in pure compound form but stabilized by optimal configurational entropy under the laser-heated DAC compression synthesis conditions. These results show that substitutional alloying is an effective and promising approach to tune and improve superconducting superhydrides, leading to the long-sought scenario of high-temperature superconductivity at relatively lower pressures. The present findings also provide insights for expanding the scope of ongoing studies in search of room-temperature superconductors among diverse compounds via construction of multinary alloys via suitable metal-atom substitution.In conclusion, we discovered a giant La-Ce alloy  was prepared by levitation melting technology, where mixed elemental La and Ce with purity of 99.7% were heated to completely melt in an Ar atmosphere at a pressure of 60 KPa for 1–2 min and then were annealed for about 40 min. To ensure the uniformity of the composition, the specimen ingot needed to be turned over and the above process was repeated more than twice. The 1:1 molar ratio and homogeneity of La-Ce alloy were further confirmed by our own experiments using inductively coupled plasma atomic emission spectroscopy (Thermo Fisher iCAP PRO) and scanning electron microscope  equipped for the energy dispersive X-ray spectroscopy.3BH3  in diamond anvil cells (DACs). The diamond anvils with culets of 60–100 µm in diameter were beveled at 8.5° to about 250 µm in diameter. A composite gasket consisting of a rhenium outer annulus and an aluminum oxide (Al2O3) epoxy mixture insert was employed to contain the sample while isolating the electrical leads in the electrical measurements. La-Ce alloy foil with a thickness of 2–3 µm was sandwiched between NH3BH3. Sample preparation and loading were done in an inert Ar atmosphere with residual O2 and H2O contents of <0.01 ppm to guarantee that the sample was properly isolated from the surrounding air atmosphere. The samples were compressed to target pressures at room-temperature. The pressure values in cells were determined from room-temperature first-order diamond Raman edge calibrated by Akahama57. One-side laser-heating experiments were performed using a pulsed YAG laser (1064 nm) with spots of ~10 μm in diameter. The temperature was determined using the emission spectrum of the black body radiation within Planck’s radiation law.We synthesized the superhydrides via a reaction of La-Ce alloy and NHλ = 0.6199 Å) of Shanghai Synchrotron Radiation Facility and BL10XU (λ = 0.4131 Å) of at the SPring-8 facility58 with focused monochromatic X-ray beams (5 × 12 and 3 × 2 µm2). A Mar165 CCD detector and an imaging plate detector  were used to collect the angle-dispersive XRD data. The sample to detector distance and other geometric parameters were calibrated using a CeO2 standard. The software package Dioptas was used to integrate powder diffraction rings and convert the 2-dimensional data to 1-dimensional profiles59. The full profile analysis of the diffraction patterns and the Rietveld refinements were done using GSAS and EXPGUI packages60.X-ray diffraction (XRD) patterns were obtained at BL15U1 . The data was taken upon warming, as it yielded a more accurate temperature reading. Symmetric Cu-Be alloy DACs were used for electronic transport measurements under external magnetic fields up to 9 T.The resistance was all measured via the four-probe van der Pauw method where four Au electrodes were placed on the NHSupplementary Information


Groundwater was sampled in the period from April 2018 to December 2019 at 10 different sites by pumping the wells, and directly in the gravel pit in Šijanec. Representative solid samples of plants, soil, manure, and synthetic fertilizers were collected from arable land in two field campaigns (July and October 2019). The presented dataset can be used as a baseline for identification of nitrate sources in groundwater and possible nitrate attenuation processes. The data is related to the research article “Tracking the nitrogen cycle in a vulnerable alluvial system using a multi proxy approach: case study Varaždin alluvial aquifer, Croatia.” The Varaždin aquifer is the only source of drinking water for inhabitants of the Varaždin County. In the last decades, groundwater contamination with nitrate has become an increasing problem. Therefore, there is a need to define the origin of nitrate as the first step in groundwater remediation. The data in this article consist of δ Specifications Table•Presented data provide a unique insight into nitrogen dynamics, enable identification of nitrate sources in groundwater and indicate possible processes that could attenuate nitrate concentrations in groundwater.•Data can be useful for researchers dealing with the nitrogen cycle, especially hydrogeologists and geochemists working on groundwater contamination related problems. Nitrogen transformation processes are mediated by microorganisms, so the data could be interesting to microbiologists dealing with bacteria linked to the nitrogen cycle. As groundwater is an important source of drinking water, researchers involved in water quality management may also be interested in data, e.g. to formulate management strategies and specific measures to reduce nitrate contamination in groundwater.•15N values in solid matter present a valuable data of various nitrogen sources from which nitrate in groundwater originates, i.e. δ15N fingerprint. The contribution of each nitrate source can be quantified using mixing models.The dataset can be used for development of groundwater model, primarily for defining the origin of nitrate in groundwater and exploring the possibility for simulation of the retardation processes. Characteristic δ1The presented data article is related to an original research article “Tracking the nitrogen cycle in a vulnerable alluvial system using a multi proxy approach: case study Varaždin alluvial aquifer, Croatia” published in Science of The Total Environment. The data was an important factor in establishing the source of groundwater contamination with nitrates, on the basis of which further groundwater protection measures can be taken.218O and δ15N in nitrate in groundwater and δ15N in solid matter. The location of sampling sites in the Varaždin area are depicted in The data presented include stable isotope ratios of δ3-content, δ18O and δ15N in nitrate in 60 groundwater samples distributed in 10 sampling sites. Solid matter data contains sampling date and δ15N values in plants, soil, manure, and synthetic fertilizers.The dataset in XLSX format includes two tables: (i) Groundwater and (ii) Solid matter and is deposited in Mendeley Data online repository 33.115N and δ18O in nitrate was determined. A successful isotope measurement requires 1 to 2 mg of NO3-N. The samples for nitrate content analyses were transported in a portable refrigerator and measured by ion chromatography (Dionex ICS 6000) in the Hydrochemical Laboratory of the Croatian Geological Survey immediately upon returning from the field. Samples for isotope analyses of δ15N and δ18O in nitrate in groundwater were filtered through 0.2 µm membrane filters into the HDPE 200/500/1000 mL bottles with a tight-fitting cap, transported in a portable refrigerator and frozen upon arrival at the laboratory. The frozen groundwater samples were transported to the National Environmental Isotope Facility of the British Geological Survey (UK), where isotope analyses of δ15N and δ18O in nitrate were performed.Groundwater samples (n = 60) were collected in the field from 10 sampling locations. Preliminary sampling campaign was carried out in April 2018, followed by nine monthly sampling campaigns from April 2019 to December 2019. The groundwater was sampled by pumping from nine observation wells, except sampling site in Šijanec where sample was taken by installed water pump and by submerging the bottle below the water level in the gravel pit. Technically, the gravel pit is surface water that was formed by the excavation of aquifer material, but is presented here as groundwater because it is a groundwater discharge zone, and the water level in the pit is actually at groundwater level. At least three volumes of groundwater from each observation well were pumped out before sampling to provide a representative sample from the aquifer. Samples for nitrate content analyses were filtered through 0.45 µm membrane filters into a 50 mL HDPE plastic bottles with a tight-fitting cap, on the basis of which the volume required for isotopic analyzes of δ3 salt according to the methods described in Chang et al. (1999) and Silva et al. (2000) 2O. The solution was filtered through a 0.2 µm polycarbonate filter to remove the AgCl, and 4 ml of 1 M BaCl2 was added and left overnight to precipitate non-nitrate oxygen-bearing anions. The solution was then passed through cation resin column to remove excess barium, re-neutralized with 1 g of Ag2O, and filtered before freezing overnight. The frozen sample was freeze-dried to obtain solid AgNO3, then re-dissolved in 1 mL of MilliQ water, after which the samples were centrifuged and ready for mass spectrometry. An appropriate volume of AgNO3 solution was pipetted into a silver capsule, freeze-dryed and wrapped for measurements of oxygen isotopes (δ18O-NO3). The analysis was performed on an TC pyrolysis elemental analyser (EA) coupled to a Thermo Fisher Delta XL Isotope Ratio Mass Spectrometer (IRMS). Before analysis, the EA-IRMS was checked for stability through a series of reference gas (CO) injections and tuned to achieve optimum sensitivity. Silver nitrate samples were introduced to the EA via a zero blank autosampler first flushed by lab grade He (99.9%). The sample was pyrolyzed at 1450 °C in the presence of glassy carbon and graphite, silver residuals are captured in a graphite crucible for disposal. The sample gas then passed onto the IRMS for isotope analysis with 0% sample dilution on CO. Samples for nitrogen isotope analysis (δ15N-NO3) were prepared by pipetting an appropriate volume of AgNO3 solution to a silver capsule, freeze-drying, adding 0.5 mg of sucrose, closing and wrapping in a tin capsule. The analysis was performed on a Flash elemental analyser (EA) coupled to a Thermo Fisher Delta XL IRMS. Before analysis, the EA-IRMS is checked for stability through a series of reference gas (N2) injections and tuned to achieve optimum sensitivity. Silver nitrate samples were introduced to the EA via zero blank autosampler first flushed by lab grade He (99.9%). The sample was combusted at 1050 °C in the presence of oxygen before passing over copper wires to reduce any residual oxygen, silver and tin residuals are captured in a glass crucible for disposal. The sample gas then passed onto the IRMS for isotope analysis with 0% sample dilution on N. Raw isotope values were produced on the Thermo software by comparing the peak area of the sample with laboratory working tank gas. Whilst this "working gas" was injected directly into the IRMS, all external reference materials were weighed into caps and pyrolyzed/combusted during the same run as the samples: IAEA-N1, IAEA-N2, and IAEA-NO3 for δ 15N-NO3 and USGS-32, USGS-34, USGS-35, and IAEA-NO3 for δ 18O-NO3. To achieve accurate measurements, raw isotope values were corrected offline for blank, drift and linearity issues before correction using a two, or three-point calibration with IAEA-NO3 as a check standard. All stable isotope results are reported using conventional delta (δ) notation in permil (‰) with respect to international standards: VSMOW (Vienna Standard Mean Ocean Water) for oxygen, and AIR (atmospheric N2) for nitrogen. The analytical precision of measurements is <1.5‰ for δ18O-NO3, and <0.3‰ for δ15N-NO3, based on within run replication of reference materials.Dissolved nitrate in groundwater samples was converted to AgNO3.2δ15N measurements in solids were performed.Solid matter samples (n = 17) were collected in two field sampling campaigns (July and October 2019). Samples were taken directly from arable land to represent the typical soil, plants, manure and synthetic fertilizers of the Varaždin area. Solid samples were collected into plastic bags and transported to the Geochemical Laboratory of the Croatian Geological Survey, where they were frozen, freeze-dryed, and finely ground. The prepared samples were transported to Department of Environmental Sciences of the Jožef Stefan Institute, where 15N). The prepared samples were measured using the Elementar PyroCube preparation module coupled to an Isoprime 100 IRMS. The accuracy of the measurements was controlled with reference materials IAEA-N1 (ammonium sulphate) with value of +0.4‰, and IAEA-310 (A1) with value of 47.2‰.An appropriate amount of each sample was added into tin capsules  to determine the isotopic composition of nitrogen (δThe authors declare that the hereby presented data and data article fully comply with the Journal's policy in terms of authors’ duties, data integrity, and experimental requirements.Igor Karlović: Investigation, Conceptualization, Methodology, Writing – original draft. Tamara Marković: Investigation, Data curation, Supervision, Writing – review & editing. Andrew Smith: Methodology, Writing – review & editing. Tjaša Kanduč: Methodology, Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.


Global variance-based sensitivity analysis using Sobol’ indices revealed oxygen concentration in off-gas and dissolved oxygen as the most important input quantities to determine α-factors accurately. Measurement uncertainty of other instruments was negligible. These findings are transferable to in situ off-gas hoods because the methods are similar. Random measurement error of α-factors was estimated with uncertainty analysis and comparison measurements to a relative standard deviation of about ± 2.8% for our ex situ pilot setup. Diffuser fouling, biofilm growth, or sensor drift caused systematic errors avoidable by maintenance. Additional mixing of bubble column due to sludge inflow into ex situ tanks led to a systematic overestimation of α-factors at lower airflow rates. Hence, the ex situ off-gas method is not suitable to determine α-factors for the design of aeration systems but offers unique possibilities for research of oxygen transfer dynamics and development of aeration equipment because ex situ columns can be operated independently from a full-scale activated sludge tank.Performance of aeration systems in wastewater treatment plants (WWTP) under process conditions can be monitored with off-gas tests. The ex situ off-gas method transfers activated sludge from an adjacent aeration tank into aerated columns to determine oxygen transfer parameters . This method is an alternative to in situ off-gas testing with hoods at the tank surface; however, its application and measurement uncertainty have not been examined yet. We outline our experience from long-term off-gas testing with two pilot-scale test reactors (8.3 m Aeration is an energy-intensive process in activated sludge (AS) biological wastewater treatment. Measurement of oxygen transfer parameters in activated sludge tanks is essential for design and operation of aeration systems. Clean water testing is an established method to determine oxygen transfer performance of diffusers (ASCE 2–06 2O) in a conventional activated sludge (CAS) WWTP and estimated volumetric mass transfer coefficient of nitrous oxide based on the mass transfer coefficient for oxygen. Baeten et al.  in an aerobic granular sludge WWTP. So far, off-gas analyses have been used in numerous studies, but application of gas analyzers is not part of the typical instrumentation on WWTPs yet.Off-gas testing methods have been used for numerous applications in design and operation of aeration systems as well as research of gas transfer in activated sludge, as the following examples show. In several studies, off-gas tests were used to examine impacts on oxygen transfer by activated sludge characteristics or WWTP operation and process layout  or fouling related (F) causes. In contrast, ex situ columns allow to mitigate fouling by regular cleaning of diffusers and therefore distinguish the α-factor from the fouling factor. In addition, clean water testing is mandatory to determine the α-factor and easier to perform in ex situ columns than in a full-scale AS tank. Overall, an ex situ column allows to change certain properties of the aeration system and operation without interfering with the WWTP operation. Applications for research purposes could include varying tank geometry , changing diffusers to find suitable types for a certain application, examining the effect of maintenance methods  on reinstating pressure loss of diffusers, or performing spiking experiments to change wastewater characteristics and study the resulting effect on oxygen transfer. While off-gas hoods can only be placed in aerated zones, the ex situ method allows to transfer sludge from non-aerated zones into the column and determine oxygen transfer parameters. This also allows to determine oxygen transfer parameters for activated sludge in tanks without submerged aeration systems or covered tanks where hoses for sludge transfer can be installed unlike off-gas hoods. This represents a unique advantage of the ex situ off-gas method to research potential emissions from non-aerated zones in the future.Placing multiple off-gas hoods to cover an activated sludge tank is generally less expensive than using ex situ columns to reach the same coverage. Determination of an overall α-factor of the process design is especially relevant in plug-flow reactors, tapered aeration or tanks with varying oxygen concentrations in different tank areas , oxygen transfer efficiency (OTE), or the α-factor. These measurement models define a functional relationship between several input quantities recorded by sensors or instruments and an output quantity, e.g., the α-factor as a measurand. Currently, guidelines are missing a stochastic component that considers measurement uncertainty of instruments recording input quantities. Instead, guidelines propose a measurement uncertainty to be expected for results if the applied method was conducted according to standard. For the use of in situ off-gas hoods DWA-M 209  estimateNone of the studies examined the ex situ column off-gas method. In addition, it remains unclear which measured input quantity is most important when determining oxygen transfer parameters or the α-factor. Another detail that is often accepted without further revision is the use of correction terms for standardization. For off-gas methods β and θ correction factors are applied to consider the effect of salts on the effective oxygen saturation and temperature on the oxygen transfer, respectively. These empirically determined correction terms are also estimates of quantities which are known imperfectly and could vary between applications of off-gas tests  we determine the most influential input quantities for determination of α-factors according to ASCE 18–18 with a sensitivity analysis, (2) we estimate the method’s random and systematic measurement error, and (3) we discuss causes of these errors and other constraints of the ex situ column off-gas method.Pilot-scale test reactors were used to determine oxygen transfer parameters applying an off-gas method described in Appendix D.1.4.4 of ASCE/EWRI 18–18 . The met3. The tank height was chosen to resemble typical water depths of AS tanks and therefore bubble rise conditions in the column. Columns were equipped with fine-bubble disc diffusers with a diffuser density of 13.5% . Oil-free rotary vane vacuum blowers  were controlled by frequency converters to set airflow rates  between 0.5 and 2.5 Nm3∙m−3∙h−1. Airflow rate was standardized  and measured with thermal mass flowmeters  in the inflow only. As with in situ off-gas hoods, this assumes that inert gas constituents such as nitrogen are conservative within the reactor and therefore net transfer of these gases is negligible. Pressure in air pipes  was measured after blowers and before diffuser distribution frame to determine pipe pressure loss and diffuser pressure loss. Sludge transfer pumps  pumped AS from a nearby AS tank through DN 100 hoses into the columns at the height of the disc diffusers. Depending on the examined AS tank zone a hose length of up to 100 m was installed and the maximum transfer time of the AS to the test columns was 90 s. Sludge flow was measured with electromagnetic flowmeters  and transfer pumps controlled by frequency converters to set a constant hydraulic retention time (HRT) of 15 min as recommended by ASCE/EWRI 18–18. Effluent sludge was directed in free flow through a DN 150 hose from an overflow edge back into the nearby AS tank downstream of sludge intake.Tank dimensions were 1.2 m × 1.2 m × 5.8 m (L × W × H) with a volume of 8.3 m−1 total dissolved solids). Off-gas concentrations of oxygen (paramagnetic sensor) and carbon dioxide (NDIR) were recorded with a gas analyzer  that received dry off-gas free of particles from a gas conditioning unit . Off-gas was collected from the sealed column hood. To quickly monitor changing process conditions, a low hood height of 0.2 m above water surface was implemented on top of the columns. Depending on airflow rate setting the mean gas sample residence time in the hood was between 2 and 4 min, which included off-gas transport from hood to gas analyzer. Foaming could complicate off-gas collection in low hoods. Thus, the pilot plant was equipped with a U-shaped off-gas pipe that withheld foam from off-gas collection.Determining oxygen transfer parameters of AS in the columns required further sensors and instruments for measurement. Atmospheric pressure , atmospheric temperature , and electrical conductivity in AS  were measured for standardization of oxygen transfer parameters , which is a commonly used design criterion to maintain solids in suspension  in a single ideal CSTR can be expressed as a cumulative distribution function asA suitable interval for data compression must be short enough to record changes in WWTP operation or wastewater composition that could affect oxygen transfer and long enough to produce distinguishable datapoints for further analysis. Depending on the response time of equipped sensors in a pilot plant, determining α-factors in intervals of a few minutes is possible. A typical measurement period for off-gas testing is 30 min to 2 h ASCE . From thBased on this ideal relationship, in our pilot plant, 63% of activated sludge transferred into the test column was exchanged within the HRT of 15 min. Accordingly, after 30, 45, and 60 min, 86%, 95%, and 98% of sludge were replaced. As a result, a 1-h interval is a suitable interval for data compression for an ex situ reactor operated at an HRT of 15 min to determine α-factors.To maintain steady-state conditions within a selected interval of data compression, some parameters  should remain constant to determine oxygen transfer parameters Boyle . TherefoIn our setup, data was recorded in 30-s intervals by online sensors and compressed as 1-h averages. This results in high resolution data that can detect variations within the diurnal cycle of WWTP operation. It also prevents autocorrelation of measured values and converts the collected time series data to resemble a cross-sectional dataset. From our experience, the required constant conditions as described above were met within a 1-h interval unless airflow rate or DO setpoint were changed manually or according to a schedule within an interval.Determination of the α-factor and other oxygen transfer parameters is based on the well-established equation for actual oxygen transfer rate under process conditions (AOTR) which represents the transfer of oxygen without any standardization in activated sludge  is calculated from the oxygen transfer efficiency (OTE) at a certain airflow rate. ASCE 18–18 describes how OTE is calculated from a mass balance of inlet and outlet oxygen and carbon dioxide concentrations measured with an off-gas analyzer. It also defines dimensionless standardization parameters to calculate standard oxygen transfer rate (SOTR) where τ is the oxygen saturation ratio at operating temperature and at 20 °C, Ω is the oxygen saturation pressure correction factor for 101.325 kPa, β is the ratio of oxygen saturation in process water and clean water, and θ is the temperature correction coefficient for water temperatures of 20 °C. Although ASCE 18–18 provides a general description of the ex situ column method, we provide further explanations based on practical experience below and added all equations to determine the α-factor in Appendix 1 of this paper.In off-gas measurements, AOTR  produced similar results but at lower accuracy. These were used in process conditions as long-term testing did not require a fast response time and their lower maintenance allowed more reliable operation in activated sludge. Furthermore, off-gas measurements were performed at a steady sludge inflow, while non-steady-state clean water tests were not. Consequently, differences of bubble rise and gas holdup in the columns could have occurred between test methods as discussed later. A steady-state clean water test is neither described in technical guidelines nor practically feasible at the setup’s scale.Clean water (cw) testing is required to determine the denominator of the α-factor which is based on the linear relationship between airflow rate and SOTR in clean water. We used different probes in clean water and process water because clean water tests required faster dissolved oxygen (DO) probes than off-gas measurements at high airflow rates. Electrochemical DO probes  with a fast response time C*20 which would result in the activated sludge at zero respiration rate. Therefore, it was estimated by a mid-depth model also considering influence of temperature and pressure   and carbon dioxide concentrations , water temperature (Tw), dissolved oxygen (DO and C(t)), electrical conductivity (EC) of the AS, atmospheric pressure (patm), and the airflow rate (qair). In the underlying model to determine α-factors, some input quantities are correlated , e.g., higher CO2,e values generally correlate with lower O2,e values. The model is non-additive because input quantities interact with each other. This means that changing two inputs has a different effect on the output than the sum of their individual effects which must be considered in sensitivity analysis  is to identify the effect of changes of input quantities on the model output  Turányi . ExamineApplying the methodology of sensitivity and uncertainty analysis, we examined the following aspects of α-factor determination with the ex situ off-gas method:An elementary “one factor at a time” (OAT) analysis only considers the relationship between the output and the variation of one individual input quantity around one baseline case where all other input quantities are kept at their nominal values Saltelli . This loWe applied the same method as in Method 1 and varied the correction factors θ and β as well as the conversion factor for TDS/EC according to their ranges found in literature.n = 10,700). A detailed overview of a priori instrument measurement uncertainties and their distributions which are specific to our pilot setup is listed in Appendix 2 of this paper and technical information of each instrument is also provided by manufacturers online. Most measurement uncertainties were chosen according to technical information by the manufacturer. However, because optical sensors for measurement of dissolved oxygen were operated in activated sludge the uncertainty of ± 1% of reading stated by manufacturer was considerably lower than our own measurements. Therefore, we assumed an uncertainty of ± 0.1 mg·L−1 (uniform distribution) ± 5% of reading  as described in Appendix 2. In total, 42 million theoretical α-factors were determined based on the instrument measurement uncertainty that represented the expected uncertainty of the α-factors defined as “true” values. Finally, theoretical α-factors were compared with the measured “true” α-factors in our dataset.We performed an uncertainty analysis to estimate the measurement uncertainty to expect when determining α-factors with our ex situ off-gas columns. The measurement uncertainty of the α-factor was affected by the measurement uncertainty of all instruments involved to measure input quantities. A common approach is to use a derivative based method for error propagation to determine a combined standard uncertainty  and illustrate the importance of all input quantities’ measurement uncertainty when performing off-gas tests with the ex situ method. The general concept is described in Saltelli et al.  and firsStatistics and visualization were done using R 3.6.3 . The resultant first-order and total effects Sobol’ sensitivity indices for all input quantities are sorted in descending importance from left to right in Fig. O2,e) has the highest first-order Sobol’ index followed by dissolved oxygen (DO). These Sobol’ indices show the influence of each input quantities’ measurement uncertainty on the variance of the output . Compared with the previous OAT sensitivity analysis . On average, sum of first-order and total effects Sobol’ indices were close to 1, which also confirms that interactions between input quantities were negligible. Some first-order Sobol’ indices were negative for the less influential input quantities. This was not caused by correlated input quantities because sampling was random. Negative first-order Sobol’ indices can occur when the sample size is insufficient .Next, we estimated the measurement uncertainty of our setup by considering measurement error from comparison measurements. Our dataset included periods where both pilot reactors were operated at the same airflow rate and hydraulic retention time while transferring AS from the same aeration zone. In total, 1400 pairs of simultaneously determined α-factors collected at 1-h intervals provided a direct comparison to estimate the pilot setup’s measurement uncertainty. This direct comparison of two identically equipped and operated ex situ off-gas columns was a suitable method to estimate the method’s measurement uncertainty because no activated sludge with a known α-factor can be used for calibration. The results are shown in Fig. Figure µ) and standard deviation (σ) can be derived from:The measurement error equals random measurement error plus systematic measurement error. As discussed in the next section, systematic measurement errors could not be excluded or corrected as the offset shifted between measurement periods. Figure Nμobs = μ1 − μ2 = −0.0044 ≈ 0 and standard deviation σobs = 0.026Normal distribution of the observed (measured) differences of α-factors as shown in Figure 3 with mean NNormal distribution of the individual setups determining α-factor #1 and #2.σ1 and σ2) were assumed identical they can be calculated as follows:Because standard deviations for the individual setups  within that period (maintenance excluded). Diagrams on top present the distribution of relative difference with a rug marking individual datapoints while diagrams on the bottom show the direct comparison of each pair of α-factors. As in Fig. Comparative measurements were performed in five distinct periods of more than 10 days that are separated in Fig. The comparison shows that the distribution of relative differences and their average varied between measurement periods. Relative differences were not always normally distributed. This suggests that the ex situ off-gas measurement was subject to systematic measurement errors that changed between or within measurement periods. The systematic measurement error as a relative difference was within a range lower than ± 1.5% in our setup. It is worth mentioning that this cannot identify systematic measurement errors occurring at the same time and evenly in both setups. Therefore, a systematic measurement error could be higher than the reported relative difference of ± 1.5%.Systematic measurement error could be caused, among other reasons, by fouling of diffusers, biofilm growth, sensor drift, or imperfect clean water testing and therefore reduced by proper maintenance of the setup and extensive clean water testing. Based on our data, systematic measurement error could not be quantitatively attributed to potential causes as discussed below. Consequently, an unknown systematic measurement error cannot be corrected when estimating the measurement uncertainty. The comparison in Fig. Fouling, scaling, and aging of diffusers affects the oxygen transfer performance of an aeration system. Odize et al.  found thPreventing excessive biofilm growth within the reactors is critical. Sessile biomass in the ex situ columns increases overall oxygen respiration and therefore alters DO concentrations and oxygen driving force in the columns when compared to suspended biomass in the AS tank. Consequently, ex situ columns could fail to accurately measure oxygen transfer conditions in an AS tank because of this systematic error. Reactor tank walls were cleaned regularly concurrently with diffusers to prevent this effect. Overall, no significant biofilm production was observed during testing. However, the impact of biofilm growth remains an unquantifiable source of error.Sensor drift of off-gas analyzer or DO sensors could result in a systematic measurement error. A small drift of oxygen concentrations in the off-gas would have a disproportionate effect on the α-factor as shown by the sensitivity analysis. Regular calibration depending on the gas analyzer’s requirement is advisable. Outliers in collected data could be identified a posteriori by large drifts marked in a calibration protocol. Moreover, biofilm growth on DO sensors submerged in AS affected their accuracy and required regular cleaning. A duplicate or triplicate measurement is advisable as it allows to identify outliers of single defective sensors a posteriori. Once these outliers were detected and removed from our dataset, no correlation with the relative difference of the α-factor was apparent. From our experience, other sensors and instruments involved in the measurement were less error-prone. Details on implementations in our setup are stated in section the “qair) were similar for both reactors, but deviations were more probable at extreme airflow rates. Extensive clean water testing beyond the usual range of set airflow rates is advisable. Nonetheless, at airflow rates below 0.5 Nm3·m−3·h−1 accuracy of the airflow meter was insufficient in our setup. Once α-factors determined at low airflow rates were excluded, no correlation with the relative deviation between pilot reactors was apparent.Clean water testing results are the denominator of the α-factor. Results of linear regression equations  relies on accurate measurement of power consumption of blowers. Blowers equipped in a pilot-scale ex situ setup cannot accurately represent the power consumption of aeration in a full-scale AS tank. In contrast, in situ off-gas measurements with off-gas hoods use the blowers of the AS tank and should therefore be preferred to determine SAE.We determined the most important input quantities of the ex situ off-gas method with a “one factor at a time” (OAT) sensitivity analysis and a global variance-based sensitivity analysis using Sobol' indices. The analysis was based on measurement uncertainties of required instruments and revealed that oxygen concentration in off-gas was the most important input quantity to determine oxygen transfer parameters . It was followed by dissolved oxygen concentration because its measurement in activated sludge could be unreliable. The uncertainties of all other input quantities were negligible.Method 3. In any case, repeating measurements is advisable to produce more accurate results and reporting a measurement uncertainty of the method is beneficial to interpret results. Nonetheless, systematic measurement errors can be present and caused, e.g., by fouling of diffusers, biofilm growth, sensor drift, or imperfect clean water testing. In our experience, systematic measurement errors of about ± 1.5% of α-factor can be caused by these issues which can rarely be identified a posteriori and only reduced by proper maintenance of the setup.We performed an uncertainty analysis for a dataset of long-term measurements based on the measurement uncertainties of instruments in our pilot setup and estimated measurement uncertainty of the α-factor as a relative standard deviation of about ± 3.7%. A direct comparison of α-factors from parallel operation of ex situ pilot reactors under the same conditions transferring AS from the same aeration zone resulted in a similar relative standard deviation of about ± 2.8%. This value represents the measurement uncertainty of a single value recorded with the ex situ off-gas method. The theoretically determined relative standard deviation of ± 3.7% and the relative standard deviation of ± 2.8% determined from practice in our pilot setup are lower than a measurement uncertainty of ± 5 to 10% estimated in literature before. Thus, a more accurate off-gas measurement seems possible. We recommend estimating the measurement uncertainty of α-factors theoretically for the installed instruments when planning an ex situ pilot setup as shown in The α-factor is standardized with correction factors to consider the influence of temperature and total dissolved solids on oxygen transfer according to standard guidelines. OAT sensitivity analysis revealed that impact of correction factors on the α-factor was lower than measurement uncertainty of the most important input quantities (oxygen concentration in off-gas and activated sludge). However, temperature correction factor θ became increasingly important when off-gas testing was conducted in activated sludge at water temperatures deviating from 20 °C. Because θ was empirically estimated as 1.024, an unknown systematic measurement error could result when comparing oxygen transfer results from tests at significantly different temperatures. The influence of salts on the effective saturation concentration as represented by the β-factor was estimated with a formula that has negligible effect on α-factor. Nonetheless, for off-gas tests in AS treating industrial wastewater with high salt contents the β-factor should be validated by additional tests to avoid a systematic measurement error.In general, the findings for the ex situ off-gas method are transferable to in situ off-gas hoods because the same instruments are used to determine the α-factor. We outlined systematic influences that differentiate the methods from each other, such as changes of oxygen balance in inflow or higher turbulence in ex situ columns due to sludge transfer. We conclude that the ex situ method is not suitable to determine α-factors to design aeration systems because a systematic overestimation of α-factor at low airflow rates is probable. In contrast, off-gas hoods are suitable to monitor oxygen transfer in activated sludge tanks, e.g., for compliance testing, because resulting α-factors represent in situ conditions. In addition, full coverage of tanks is less expensive and operation easier to maintain than with ex situ reactors. However, the possibility to operate ex situ reactors independently from AS tanks offers unique possibilities for research of oxygen transfer dynamics in AS and development of aeration equipment. It could see a future application in the parallel measurement of oxygen transfer and greenhouse gas emissions (such as nitrous oxide) in aerated and non-aerated zones.Below, we summarize our findings about the application of ex situ column off-gas testing and its measurement uncertainty to determine α-factors in activated sludge tanks.


GAP43–CaM interaction plays a key role in intracellular Ca2+ homeostasis and, consequently, in skeletal muscle activity. The control of intracellular Ca2+ signaling is also an important functional requisite in cardiac physiology. The aim of this study is to define the impact of GAP43 on cardiac tissue at macroscopic and cellular levels, using GAP43 knockout (GAP43−/−) newborn C57/BL6 mice. Hearts from newborn GAP43−/− mice were heavier than hearts from wild-type (WT) ones. In these GAP43−/− hearts, histological section analyses revealed a thicker ventricular wall and interventricular septum with a reduced ventricular chamber area. In addition, increased collagen deposits between fibers and increased expression levels of myosin were observed in hearts from GAP43−/− mice. Cardiac tropism and rhythm are controlled by multiple intrinsic and extrinsic factors, including cellular events such those linked to intracellular Ca2+ dynamics, in which GAP43 plays a role. Our data revealed that, in the absence of GAP43, there were cardiac morphological alterations and signs of hypertrophy, suggesting that GAP43 could play a role in the functional processes of the whole cardiac muscle. This paves the way for further studies investigating GAP43 involvement in signaling dynamics at the cellular level.Growth-associated protein 43 (GAP43) is found in skeletal muscle, localized near the calcium release units. In interaction with calmodulin (CaM), it indirectly modulates the activity of dihydropyridine and ryanodine Ca Growth-associated protein 43 (GAP43) is a highly conserved protein expressed in vertebrate species , and extends towards the C-terminal domain , a protein that binds CaData obtained in our laboratory, on the expression levels and intracellular localization of GAP43, showed that the protein expression follows a specific evolution related to the differentiation process from myoblasts to myotubes. Indeed, our experiments revealed that GAP43 was mainly localized within the nucleus in undifferentiated cells with diffuse patches within the cytoplasm, while in myotubes the protein localized at the cytoplasmic level, forming streaks transversal to the muscle fiber  muscle of the hind legs of C57BL/6 mice, it was possible to clarify in detail the position of GAP43 in relation to functional skeletal muscle fiber proteins, such as sarcomeric α-actinin and components of the calcium release units (CRUs). We demonstrated that GAP43 was positioned between CRUs and mitochondria, suggesting that GAP43 may play a key role in Ca−/− mice have been shown to generate less force, and exhibit reduced body weight. Ultrastructural analyses of the diaphragm muscle (early maturing) and EDL (late maturing) from GAP43−/− mice showed a delay in the degree of triads’ maturation as well as a reduced number of triad-junction complexes, despite the formation of the neuromuscular junctions being normal. These data highlight a possible role of GAP43 in those processes that accompany the development and functional maturation of skeletal muscle. Interestingly, Rahmati and Taherabad found lower levels of GAP43 in atrophied gastrocnemius muscle in diabetic animals, suggesting that the level of GAP43 could be a critical factor for skeletal muscle mass and size (Rahmati and Taherabadi Adult GAP432+ homeostasis was investigated in myotubes deriving from differentiated satellite cells from wild-type (WT) and GAP43−/− mice. Of note is the observation that GAP43−/− myotubes showed profound alterations with Ca2+ release. Indeed, in GAP43−/− samples, there was an increase in the number of myotubes with spontaneous oscillations having a higher frequency and amplitude than WT myotubes. Electrophysiological analyses showed that these alterations are due to increased L-type Ca2+ currents linked to an alteration of the CaM-operated control. It has been hypothesized that, due to its vicinity to the CRUs, GAP43, by interacting with CaM, provides a “functional microdomain” that locates the CaM near the CRUs. Thus, GAP43 indirectly modulates the activity of dihydropyridine receptor (DHPR) and ryanodine receptor (RyR) Ca2+ channels, thus influencing the dynamics of intracellular Ca2+ and the functional activity of myotubes . GAP43−/− newborns show decreased survival compared with GAP43+/− and WT mice, which have a normal lifespan. In fact, about 45–60% of GAP43−/− mice die within the first 48 h. About 45% die after 2 or 3 weeks after birth, at the time of weaning. Only a very small percentage of the knockout offspring survive past 3 weeks (about 5–10%) , and reagents and standards from Merck Life Science .+/−) mice were kindly provided by Dr Karina F. Meiri   and the Italian Government law on the protection of animals for experimental procedures in research laboratory (92/116). The experimentation and housing of the animals were conducted at the Center for Advanced Studies and Technology (CAST) with the consent of the “Organismo preposto al benessere animale” (OpBA) of the “G. d’Annunzio” University of Chieti-Pescara and with authorization no. 4739.n.ias of the Italian Ministry of Health [principal investigator (PI) S.G.].C57BL/6 GAP43 heterozygous , according to the manufacturer instructions. The DNA samples were amplified by PCR using the following primers: p1 (5′-GGCTCATAAGGC TGCAACCAAAAT-3′), p2 (5′-CCATCTCCCTCC TTCTTCTCCACA-3′), p3 (5′-CCGGCCGCTTGG GTGGAGAG-3′), and p4 (5′-TCGGCAGGAGCA AGGTGAGATGAC-3′).The offspring of the GAP43Newborn mice were weighed and sacrificed according to the authorized procedure. Hearts were removed after performing midsection thoracotomy, rinsed in cooled phosphate buffer, and blotted dry; then the heart’s weight was measured. The cardiac mass index was calculated as ratio of heart weight to body weight. The values obtained were used to calculate the mean ± standard error of the mean (SEM).−/− mice were sacrificed as described above, and after two washings in phosphate-buffered saline (PBS), isolated hearts were fixed in a solution of 10% formalin for 24 h. Fixed hearts were washed in PBS, appropriately oriented in the biocassettes, and treated using the EG-1160 embedding center and ASP 300 tissue processor . Paraffin-embedded hearts were sectioned in slices of 6 μm at intervals of 150 μm from the cardiac base to the apex using RM2125 RT microtome (Leica Microsystems).WT and GAP43Sections from the medial region of each paraffin-embedded heart were stained with H&E using an automated TST 44 slide stainer  and mounted on glass slides. After H&E staining, cellular nuclei appeared stained blue, whereas the cytoplasm and extracellular matrix showed varying degrees of pink staining. The stained samples were observed using a straight transmitted light microscope Leica DMRD equipped with a 5×/0.8 numerical aperture (NA) objective (Leica Microsystems). Images were acquired using a DFC550 camera (Leica Microsystems) and analyzed with the software of Leica LAS  to perform quantitative measurements. For each section, the wall thickness of the left ventricle was measured at three different points . The values obtained were used to calculate the mean ± SEM.Masson’s trichrome staining was used to distinguish collagen from cardiac muscle tissue. Paraffin-embedded heart sections, obtained as reported above, were incubated for 10 min with iron hematoxylin solution, washed in distilled water, and processed with picric acid alcoholic solution for 1 min. Then, samples were washed in distilled water and treated with Ponceau acid fuchsin for 1 min. After washes, samples were fixed with phosphomolybdic acid 1% for 5 min, stained with aniline blue for 1 min, and dehydrated in ethanol and xylene. The collagen appeared stained blue, nuclei black, myocardium red, and erythrocytes yellow. The stained samples were observed using a straight transmitted light microscope Leica DMRD . The images were acquired using a Leica camera  and analyzed with the software of MetaMorph 6.1 . The area of collagen and the area of cardiac muscle are expressed as percentage ratio to total area, and data are reported as mean ± SEM.Papillary muscles were dissected from hearts of adult (3 months) WT mice and fixed with 4% paraformaldehyde for 10 min at room temperature. After three washes in PBS, the samples were permeabilized with a 0.2% Triton X-100 solution for 10 min, incubated in blocking buffer (PBS containing 10% goat serum) for 1 h at room temperature, then incubated overnight at 4 °C with primary antibodies anti-GAP43  and mouse monoclonal anti-α-actinin 2 . The primary antibody anti-GAP43  was validated by western blot using muscle GAP43 knockout extracts as negative control and brain tissue homogenates from WT mice as positive control  diluted 1:100 in PBS for 30 min at room temperature. After washing with PBS, sections were treated with antifade mounting medium . The samples were observed using a Zeiss LSM800 URGB confocal microscope equipped with an upright AxioObserver Z1 microscope and a 40×/1.3 NA oil immersion objective and ZEN Blue 2.1 software (Zeiss). For each section, at least 40 cardiac fibers in three different fields were acquired. Acquired images were analyzed with ImageJ software . Fiber area was calculated by tracing the WGA-TRITC fluorescence signal. Data are reported as mean ± SEM.g, for 10 min at 4 °C), the protein concentrations in the supernatants were determined . Samples (20 μg) were resuspended in Laemmli buffer and separated by SDS-PAGE on 8%, (w/v) homogeneous slab gels, and then electroblotted onto nitrocellulose membranes . Equal loading of the protein samples and transfer efficiency were monitored using Red Ponceau S (Merck Life Science) staining of each membrane, and Coomassie blue staining  of the gels. The membranes were blocked with a TBS-Tween 0.1% solution with milk 5%, and then incubated with the following primary antibodies: HPA-GAP43 rabbit polyclonal anti-GAP43 ; mouse monoclonal antibody anti-β-myosin heavy chain (MYH) ; mouse monoclonal antibody anti-MYH7 . The membranes were then incubated with horseradish-peroxidase-conjugated sheep anti-IgG, with the relevant proteins detected using chemiluminescence kits  and an image acquisition system . A mouse monoclonal anti-GAPDH antibody  was used as the loading control.Protein extracts for western blotting were isolated from brain and heart of the mice. The tissues were homogenized with a Kontes glass potter  in ice-cold RIPA lysis buffer . After centrifugation . p-Values < 0.05 were considered statistically significant.Data are expressed as the mean ± standard error of the mean (SEM) and compared using Student’s Heart muscle homogenates were obtained from WT C57BL/6 mice at 24 h after birth and in adult mice (3 months), and used to define the expression levels of the GAP43 in the heart. Heart protein samples and homogenates from WT mice brain (representing a positive control for GAP43 immunodetection) were separated by electrophoresis in SDS-PAGE, and subjected to immunoblotting. In the samples obtained from the heart tissue (at 24 h or at 3 months of age), there was a specific band at the molecular weight characteristic of GAP43 (~ 43KDa) as well as in the brain sample used as positive control see Fig. a. ComparTo analyze the localization of the GAP43 in the heart muscle, we used confocal microscopy and dissociated fibers derived from heart papillary muscles obtained from WT C57BL/6 adult mice. The localization of GAP43 was compared with the labeling of the α-actinin whose localization is known at the level of the Z line. The immunofluorescence signal for GAP43 revealed a dotted pattern that drew transverse lines Fig. b. The me−/− mice appeared very similar to the WT littermates, but their body weights were slightly, but significantly, lower than those of WT     WT and GAP43−/− mice was further evaluated by quantifying the expression levels of myosin protein and, in particular, the expression levels of total myosin heavy chain (MYH) and its ventricle-specific isoforms (MYH7). Western blot analyses revealed that MYH and MYH7 were both significantly overexpressed in GAP43−/− hearts compared with WT ones , while that of ventricular myosin isoform MHY7 increased by about 1.4-fold (WT: 0.8685 ± 0.1122 N = 3 versus GAP43−/−: 2.635 ± 0.2461 N = 3) , which are the major components of the thick filaments in the sarcomere. These proteins show distinct developmental expression profiles and distributions in the heart. In fact, the MYH7 is the predominant isoform expressed in the ventricular chambers, while MYH6 is preferentially expressed in the atrial chambers . A similar pattern was found in skeletal muscle, where the levels of GAP43 expression were more than doubled in neonatal mice compared with those found in adult (4 months) or elderly (1 year) mice. The decrease in protein expression over time appeared to be related to the maturation of the muscle excitation–contraction structures; thus, due to its peculiar localization near the CRUs, it was proposed that GAP43 is involved in muscle maturation  and produces concentric hypertrophy. In sharp contrast, volume overload is common in aortic and mitral regurgitation, and occurs during diastole (preload) and yields eccentric hypertrophy . These also orchestrate cardiac hypertrophy under mechanical overload , c-Jun NH2-terminal kinase (JNK), and p38 MAPK (He et al. 2+ oscillations in myotubes. We proposed that the location of GAP43 close to the CRUs in skeletal muscle generates a “functional microdomain” able to anchor CaM near to functional sites like the CRUs (Benowitz and Routtenberg GAP43 interacts with CaM in not only neurons but also skeletal muscle. In our previous work, we hypothesized that, by interacting with CaM, GAP43 indirectly modulates the activity of DHPR and RyR channels, and the spontaneous Ca−/− mice do not survive beyond weaning (Maier et al. As reported in other studies, GAP43 expression was not required for embryonic viability, although a high percentage of GAP43These results pave the way for further studies investigating GAP43 involvement in signaling dynamics at the cellular level.