SCN1A encodes the voltage-gated Na+ channel Nav1.1, and FHM caused by mutations of SCN1A is named FHM3. For 7 of the 12 known FHM3-causing SCNA1 mutations functional consequences have been investigated, and even if gain of function effect seems to be a predominant phenotype, for several mutations conflicting results have been obtained and the available data do not reveal a univocal FHM3 pathomechanism.Familial hemiplegic migraine (FHM) is a group of genetic migraine, associated with hemiparesis and aura. Three causative different genes have been identified, all of which are involved in membrane ion transport. Among these, To obtain a more complete picture, here, we characterized by patch clamp approach the remaining 5 mutations  in heterologous expression systems.+ current blocker, on the above mentioned mutants as well as on previously characterized ones . GS967 inhibited persistent currents of all SCNA1 FMH3-related mutants and dramatically slowed the recovery from fast inactivation of WT and mutants, consistent with the hypothesis that GS967 specifically binds to and thereby stabilizes the fast inactivated state.With the exception of I1498M, all mutants exhibited the same current density as WT and exhibited a shift of the steady state inactivation to more positive voltages, an accelerated recovery from inactivation, and an increase of the persistent current, revealing that most FHM3 mutations induce a gain of function. We also determined the effect of GS967, a late Na+ and/or exhaustion of neuronal energy resources. In silico application of GS967 largely reduced net ionic currents in neurons without impairing excitability.Simulation of neuronal firing showed that enhanced persistent currents cause an increase of ionic fluxes during action potential repolarization and consequent accumulation of K+ current blockers appear a promising specific pharmacological treatment of FHM3.In conclusion, late Na Several functionally studied mutations exhibit defects of the fast inactivation process such as a rightward shift of the steady-state inactivation, accelerated recovery from inactivation, and enhanced persistent currents [Familial hemiplegic migraine (FHM) is a severe form of hereditary autosomal dominant migraine with aura caused by mutations in three different genes encoding neuronal or glial ion transporting membrane proteins. FHM1 is caused by mutations in the gene encoding the presynaptic neuronal Caepilepsy . The preionality . Comparacurrents –14. Thiscurrents , 8, 11. currents , 13, 14.currents . Based o+ channel gene SCN4A lead to muscle hyperexcitability and myotonia [SCN5A gene to Long QT (LQTS) and Brugada syndrome (BS) [+ current blockers”, like ranolazine, have been proposed as specific correctors of mutation-induced disruption of inactivation [Gain of function mutations with impaired fast inactivation in the muscle Namyotonia  and in tome (BS) . For LQTtivation . Hence, + current blocker, has been shown to exert a potent antiarrhythmic effect on rabbit myocytes and on canine or porcine models, targeting Nav1.5 heart channels [+ currents in cardiac myocytes and neurons in the different mouse models.Also GS697, a late Nachannels –20. In achannels –23. The SCNA1 mutations in heterologous expression systems, confirming for four of them a defective inactivation process. One mutation (I1498M) exhibited dramatically reduced functional expression. We then tested the effect of the late Na+ current blocker GS967 [+ concentration and an important energetic burden on firing neurons. Is noteworthy that simulating a reduction of persistent current similar to that experimentally obtained by the application of GS697 ameliorated these effects without compromising excitability.In the present study, in order to further test the generality of the assumption that FHM3 is associated with a gain of function, we examined the effects of five so far uncharacterized er GS967  on theseAll salts were of highest available grade and were obtained from Sigma-Aldrich . GS967 was obtained from MedChemtronica AB The European branch of MCE (Sweden). Stock solution aliquots at 2 mM in DMSO were kept at − 20 °C and diluted in the experimental solution at the day of the experiment.SCN1A we employed our previously characterized optimized SCN1A expression plasmid which encodes the shorter SCN1A splice variant and in which codons are changed to render the cDNA more similar to the cardiac SCN5A isoform, and which is much easier to handle than traditional SCNA1 expression plasmids [SCNA1 construct [Xenopus oocytes the original pCDNA3-based constructs were transferred into the pFrog vector [Xenopus oocytes we co-expressed SCN1A with the human β1 subunit which is known to reduce slow components of inactivation [For heterologous expression of WT and mutant plasmids . Expressonstruct . All newonstruct  and the g vector  using Hitivation . The β1 tivation . In additivation .2, and 95% humidity. For transfection we used the Effectene Kit (Qiagen), co-transfecting 500 ng SCN1A plasmid and 50 ng of a vector expressing GFP. Currents were recorded 24-48 h after transfection.HEK-293 cells were grown and transfected as described earlier . BrieflyXenopus oocytes, cRNA was transcribed by the mMessage mMachine T7 kit  after linearization with Mlu I (SCNA1) or NheI (β1). cRNA (5 ng of SCN1A + 5 ng of β1) was injected in defolliculated oocytes, which were incubated at 18 °C in the maintaining solution containing (in mM): 90 NaCl, 2 KCl, 1 MgCl2, 1 CaCl2, and 10 Hepes (pH 7.5).For expression in 2, 1 MgCl2, 10 Hepes, pH 7.4. Internal solution contained 40 CsCl, 10 NaCl, 80 CsF, 11 EGTA, 10 Hepes, 1 CaCl2, pH 7.3. Cell capacitance was between 10 and 25 pF. We controlled that series resistance, RS, was not larger than 4 MΩ and often it was around 2 MΩ, for which a maximum accepted current level would be 2.5 nA to keep the voltage error smaller than 5 mV. The current levels shown in Fig. Currents in HEK cells were recorded using the whole-cell configuration of the patch clamp technique  using anXenopus oocytes were measured using the two-electrode voltage clamp technique (TEVC) using an npi-electronics amplifier  as described previously [2, 2 MgCl2, pH 7.3.Currents in eviously . To reduhttp://users.ge.ibf.cnr.it/pusch/programs-mik.htm). Between the application of pulse protocols, the membrane was kept at a holding potential of − 90 mV (See Supplementary Methods).All measurements were performed using the custom acquisition program GePulse , m the activation variable, h the fast inactivation variable and s the slow inactivation variable. Activation (m) and slow inactivation (s) variables were parameterized as in [h, is usually modelled as a two state processA (“activated”) denotes the non-inactivated state, I the inactivated state, βh the inactivation rate constant and αh the rate constant of recovery from inactivation. The standard inactivation rate constants αh and βh were calculated as in [Gating of the sodium channel was modelled as described earlier , 11, 30 ed as in , 11. Theed as in , 11.In order to implement the assumption that GS967 binds exclusively to the inactivated state, we modelled inactivation as a three state process:IGS represents the inactivated channel with a molecule of GS967 bound. Binding of GS967, and unbinding rate constants were assumed to have the values: λ = 10 μM− 1 s− 1, μ = 1 s− 1.Here, Ppersist induced by a mutation, the inactivation rate constants (βh and αh) were modified to Further, in order to impose a certain “percentage of persistent current” in the absence of GS967, h∞, is given byIn the standard model, steady state inactivation, Ppersist we defined the modified steady state inactivation, To introduce Thus the fully extended scheme of fast inactivation including persistent currents and GS967 binding is given byhttp://users.ge.ibf.cnr.it/pusch/programs-mik.htm). These protocols included the standard IV pulses, and the protocol of recovery from fast inactivation. Holding voltage in these simulations was − 90 mV as in the experiments. In the program MarkovEditor, the kinetic equations are solved by calculation of eigenvectors and eigenvalues of the system of linear differential equations employing code from the Alglib C++ library .Voltage step responses to typical voltage protocols Fig. b-e were + conductance, the neuronal model harbored a K+ conductance and a leak conductance with the same characteristics as described in [K = -85 mV. Simulations were performed for current injections between 10 and 350 pA (in 10 pA steps) and the membrane potential, the K+ current and the Na+ current were followed for 400 ms.The model was then used to predict the effect of GS967 in a simplified single compartment neuronal model as in , 11 and ribed in , 11. TheLocalization of FHM3 mutations in the Na+channel structure+ channel protein structures. In Fig. + channel [Since FHM3 mutations affect critical biophysical functional properties of the voltage-gated sodium channel it is insightful to map their location onto the recently obtained Na channel . Interes channel . All thr channel  at the b channel  are mutaSCN1A expression plasmid: Q1489H and F1499 L were found in families in which FHM was associated with elicited repetitive daily blindness [+ currents of practically the same size as WT, I1498M induced only small currents, rendering a detailed functional analysis practically impossible ; I1498Mlindness ; M1500 Vlindness . We firsble Fig. i. Thus, + current blockers have been proposed as possible treatment options for cardiac arrhythmia causing mutations of SCN5A that led to similar gain of function effects as seen here for SCN1A [+ current blocker GS967 on the above investigated four mutations  as well as on three previously characterized ones [Xenopus oocytes that allowed more stable recordings upon application of the drug compared to HEK cells. In addition, we exploited the fact that mutants L1649Q and L1670 W expressed rather large currents in oocytes, whereas these are difficult to express in HEK293 cells [Xenopus oocytes (18 °C) compared to HEK cells (30-37 °C). Even though the absolute values of most gating parameters are slightly different in oocytes compared to HEK cells, the effects induced by the mutations are well reproduced in this system  corresponds to F1760 in g [SCN5A , in agre+ channel [Overall, the kinetic effects of GS967 are well explained by a preferential binding to the inactivated state of the channel, and indeed the recovery from inactivation is dramatically slowed by the drug. The effects found here are qualitatively very similar to those described for the cardiac Na channel . These a channel . HoweverOur results support the hypothesis that FHM3 is caused by hyperactivity of the Nav1.1 channel due to a defect in the inactivation process and that GS967 may have beneficial effects inhibiting Nav1.1 persistent currents, which are greatly increased in FHM3 mutations. A preclinical testing of GS967 could be a valid approach to explore specific pharmacological treatment of FHM3. Genetic hemiplegic migraines are rare in themselves, but share many molecular mechanisms with the more common forms of migraine with or without aura. Thus, any compound that turns out to be a remedy for FHM3 could also have a therapeutic effect on common forms of migraine that affect a very large number of patients.Additional file 1. Supplementary Methods and Tables


FABP4) on milk production traits in Greek Sfakiasheep. Polymerase chain reaction – single-stranded conformationalpolymorphism (PCR-SSCP) analysis was used to genotype a total of 374 Sfakiaewes for two regions of FABP4 located around exon 2–intron 2 (Region 1) andexon 3–intron 3 (Region 2). Each month, for a period of 6 months, milksamples were collected from the ewes to measure total milk yield, fatcontent, protein content, lactose content, non-fat solid content, pH, andsomatic cell count (SCC). A general linear model was used to test theassociation between the variation observed in FABP4 and milk production traits.Four gene variants (A1–A4) were found in Region 1 and two variants(C1–C2) were found in Region 2. In the first region, the FABP4 genotypesignificantly affected (A2 variant was significantly associated (A4 was significantly associated withdecreased milk yield (FABP4 genotype had an effect (C2 variant wasassociated (FABP4 and milkproduction traits in Greek Sfakia sheep. Nevertheless, further analyses inindependent sheep populations of increased size will strengthen thesefindings.The aim of this study was to estimate the effect of variation in the fattyacid binding protein 4 gene ( The Sfakia breed is one of the most populardual-purpose breeds of Greek sheep and its milk is used for consumption andfor processing into cheese and yogurt. Sheep milk has advantages over cowmilk because it contains higher levels of protein, lower levels of cholesterol,and large amounts of vitamins A and E, which act as antioxidants . With sheep milk, producing 1 kg of cheese takesapproximately 4 kg of milk, compared to 8 to 10 kg forcows .Increasing the productivity of milking sheep is very important if thepurpose is to increase the income of Greek farmers. Accordingly, it isimportant to understand the genetic basis of milk production traits. Oneapproach, which has gained in popularity, is to search for the genetic basisof the important traits and select superior breeding stock usingmarker-assisted selection. The use of marker-assisted selection can speed upthe identification of genetically superior animals by increasing theaccuracy in which genetic merit can be ascertained, and by decreasinggeneration interval . To achieve this outcome, you firstneed to identify the major genes that affect any valued traits .The various components of milk originate from blood plasma substrates andthey are synthesized in the epithelial cells of the mammary gland . Milk lipids are synthesized from fatty acids that bind to specificproteins called fatty acid binding proteins (FABPs). The FABPs are a smallfamily of cytoplasmic proteins. They are thought to affect various cellularprocesses, in-particular lipid metabolism. They do this by transferring thefatty acids, heme, retinoids, and different vitamins across the cytoplasmicmembrane to the sites of FABP gene family (FABP1–FABP9) have been described.Fatty acid binding protein 4 (FABP4), also known as adipocyte FABP (A-FABP),is a protein found in abundance in the mammary gland, and also in matureadipocytes and adipose tissue . The gene for FABP4 (FABP4) isexpressed during lactation  and the main function ofthis protein is thought to be in lipid metabolism, where it binds bothlong-chain fatty acids and retinoic acid and delivers them to receptors inthe nucleus of adipocytes .To date, nine members of the FABP4-deficient mice suggest that thermogenesis and whole-bodyenergy expenditure are decreased after feeding on a high-fat diet, indicating the importance of FABP4 in the maintenance of normal lipid metabolism. Furthermore, FABP4 was found to increase thermogenesis bypromoting the conversion of thyroid hormone from its inactive form (T4) to active form (T3) in brown adipocytes, the levels ofFABP4 were found to increase in the bloodstream, and in both brown and whiteadipose tissues, in response to thermogenic stimuli . In thisrespect, it has been shown that the performance of lactating animals isnot only limited by the intrinsic properties of mammary glands but also bycompetition from heat production including thermogenesis in brown adiposetissue . Taken together, these suggest that the performanceof lactating ewes could be affected by FABP4.Investigations of FABP4 affects milkproduction traits in sheep have not been undertaken but Yan et al. (2012)analyzed two regions of the gene  using polymerase chain reaction – single-stranded conformationalpolymorphism (PCR-SSCP) and DNA sequencing. They detected fivedifferent SSCP patterns derived from three nucleotide substitutions and onedeletion in Region 1 and four different SSCP patterns derived from fournucleotide substitutions in Region 2.The FABP4 gene has been identified in sheep, cattle, chickens, and humans.Across these species it has a conserved structure with four exons beingseparated by three introns. Studies on whether variation in FABP4 isconsidered to be a candidate gene for milk production traits in sheep. Theobjective of the present study was therefore to look for genetic variationin two separate regions of ovine FABP4 gene and, if found, test its associationwith milk production traits in Greek Sfakia ewes.It is assumed that the lipid metabolism, thermogenesis, and whole-body energyexpenditure affects most biological functions in the cells of varioustissues and organs, including the mammary gland; therefore, 22.1Twenty  and by flow cell cytometry forsomatic cell count (SCC) using the Fossomatic™ . Fat, protein, lactose, and non-fat solid contents inthe milk were expressed in grams per 100 mL and the logarithmic value of somaticcell count in milk was recorded.All ewes were milked twice daily and the daily milk yield was measured usinggraduated measuring cylinders. Milk samples for analysis were collected oncea month for a period of 6 months. These samples were analyzed at the StateMilk Quality Laboratory (ELOGAK) in Rethymno. First, the samples were heatedto 25 2.3FABP4 gene (Region 1and Region 2) that are located on exon 2–intron 2 and exon 3–intron 3.Blood samples were collected from 374 Sfakia ewes onto FTA cards (Flinders Technology Associates);genomic DNA was purified for PCR analysis using a two-step procedure that isdescribed by Zhou et al. (2006). Two pairs of specific primers (Table 1) were usedto study the genetic variation in two regions of the Taq DNApolymerase . The thermal profile consisted of a 2 min denaturation at 94 The PCR reaction was carried out in a total reaction volume of 20 2.4FABP4 were used as templates for Sanger sequencing of DNA at the LincolnUniversity DNA Sequencing Facility. For each heterozygous SSCP pattern, twosheep were sequenced using the method of Gong et al. (2011). Briefly, one of theunique bands of the heterozygous pattern was cut out of the gel, washed with300 The amplicons from two sheep that produced each homozygous SSCP pattern for2.5FABP4 genotype, lactation number,lambing period, and management system. Sheep with FABP4 genotypes with frequenciesless than 4 % were omitted from the statistical analysis to reduce thechances of obtaining false positive results during the multiple comparisons.Data were analyzed using the general linear model (GLM) procedure of SASsoftware  to estimate the effect of FAPB4 genotype . These had frequencies of17.1 %, 31.6 %, 14.4 %, 6.3 %, 15.9 %, 8.8 %, 3.8 %, 0.7 %,0.7 %, and 0.5 %, respectively. They were comprised of four uniquevariant sequences  with frequencies of43.3 %, 38.0 %, 12.8 %, and 5.9 %, respectively.Four different SSCP banding patterns were observed for amplicons derivedfrom the exon 2–intron 2 region of FABP4 in the Sfakia sheep were73.0 % and 27.0 % for variants that were named C1 and C2, respectively. Threegenotypes were observed: C1C1 (frequency 53.0 %), C1C2 (40.0 %), and C2C2 (7.0 %) .The variant frequencies for the second region of 3.2The nongenetic factors  significantly  and listed in GenBank with accession numbersJX290313-JX290316. It is evident from Tables 3 and 4 that there aresignificant associations for the variation observed in Region 1 of FAPB4 withmilk yield, non-fat milk solid content, fat content, lactose content, pH, andSCC. While the nucleotide substitutions that were found in this region arelocated in intron 2, and do not change the amino acid sequence, it ispossible that these substitutions might still influence FABP4 expression. Forexample they might affect the splicing of mRNA or be linked to variationelsewhere in a regulatory region , or the 5This is the first report describing the effect of FABP4 and carcass traits, meat quality traits, and fatty acid composition inadipocytes . Limited information is availableregarding the effect of FABP4 on milk production traits in livestock. In Jerseycattle, Kulig et al. (2010) studied variation in two regions of the bovine FABP4 gene(from nucleotides 5433 to 6106 and from nucleotides 7417 to 7868), for theaccession number AAFC01136716, and found no associations between the FABP4 polymorphismand daily milk yield, percentage fat content, or percentage protein content.For the same regions of FABP4 in Polish Holstein-Friesian cows, Kulig et al. (2013)revealed a significant effect for the single-nucleotide polymorphism , un-saturated fatty acids(UFA), monounsaturated fatty acids (MUFA), polyunsaturated fatty acids(PUFA), and the SFA/UFA ratio. Zhou et al. (2015) identified three PCR-SSCPpatterns derived from five SNPs: two of these SNPs were detected in exon 3and the other SNPs were detected in intron 3; these five SNPs defined threehaplotype sequences . It is notable that the C2variant, which is associated with increased protein content, decreased SCC andlower pH, and carries the nucleotide substitution c.317A/G that would lead tothe putative amino acid substitution of lysine with arginine at residue 106.Arginine is one of the most multifunctional amino acids in animal cells. Itturns into polyamines, which play a key role in the synthesis of protein inmammary epithelial cells . Previous studies revealed theimportance of arginine in increasing milk protein content. For example,Moreira et al. (2018) described how dietary supplementation with high levels ofarginine increased milk production and milk protein in pigs. Clark et al. (1975)found that arginine uptake by the mammary gland associated with milk yieldin rabbits and cows; however, O'Quinn et al. (2002) revealed that the rate ofarginine catabolism in the lactating sows was greater than that for thepregnant or non-lactating sows. Furthermore, Tian et al. (2017) stated thatarginine deficiencies had negative effects on milk yield and milk proteinyield. The investigators attributed the effect of arginine to its crucialrole in regulating protein turnover in mammary epithelial cells byactivating the mechanistic target of the rapamycin (mTOR) cell signalingpathway .Comparison of the exon 3–intron 3 sequences in this study with the sequencesdescribed by Yan et al. (2012) revealed that the C2 variant might beattributable to the substitution of lysine with arginine in FABP4. Manystudies have described a positive effect for arginine on reducing the SCC inmilk produced from mammals. For example, Chacher et al. (2013) suggested thatpromotion of the synthesis and metabolism of endogenous L-Arginine, throughsupplementation with feed additives that elevate blood plasma argininelevels, enhanced immunity, reduced SCC, and reduced the incidence ofmastitis in sows. McCoard et al. (2013) found that administration of L-arginine toRomney ewes improved mammary gland health and decreased SCC in the milkproduced. Woloszyn (2007) revealed that the cows with high SCC had asignificantly lower concentration of arginine in their blood plasma.Alternatively, studies  have described apositive correlation between the number of somatic cells in milk and its pH.Given that the ewes that carry the C2 variant also produced milk with lowerpH, the effect of C2 on SCC may either be mediated via pH or argininelevels.The lower SCC for milk produced from ewes which carry the FABP4 variation on milk production traits might also be explainedby other phenomena. Besides its direct effects on milk production traits,FABP4 has been shown to have an effect on residual feed intake and feedefficiency , subcutaneous fat depth and fatty acidcomposition of carcass , intramuscular fat ,marbling score and back-fat thickness , and insulinresistance . Many investigations haveshown associations between milk production traits and these traits.Casper (2008) stated that the lower residual feed intake was, the higherfeed efficiency was, leading to increased milk production in dairy cows.Marston et al. (1998) speculated that increased milk production was positivelyassociated with greater marbling in beef cows. A strong relationship wasfound between the development of back-fat thickness and milk protein contentin dairy cows . A positive correlationof milk yield and milk fat content, with increased subcutaneous fat depth, indairy cows was revealed by Nogalski et al. (2012). Elevated levels of insulin inplasma were also found to be associated with decreases in milk productionand milk lactose content and increases in milk fat and protein contents inHolstein dairy cows and goats .The effects of 5FABP4 has an effect on milkproduction traits. However, further investigations have to be undertaken ona larger population of Sfakia sheep, or other breeds of sheep, to confirmthese findings before they can be recommended to breeding programs toimprove milk production traits in sheep.It could be concluded that the variation in ovine


SSM-MFC with PVA and CPD as binders were producing on average the same level of power (1226 ± 90 μW), which was 24% less than the SSM-MFC having PTFE-based cathodes. When balancing the power by the cost and environmental impact, results clearly show that PVA was the best alternative. Power wise, the SAC cathodes were shown being the less performing (≈1070 μW). Nonetheless, the lower power of SAC was balanced by its inexpensiveness. Overall results indicate that (i) PTFE is yet the best binder to employ, and (ii) SAC and PVA-based cathodes are promising alternatives that would benefit from further improvements.The recently developed self-stratifying membraneless microbial fuel cell (SSM-MFC) has been shown as a promising concept for urine treatment. The first prototypes employed cathodes made of activated carbon (AC) and polytetrafluoroethylene (PTFE) mixture. Here, we explored the possibility to substitute PTFE with either polyvinyl-alcohol (PVA) or PlastiDip  as binder for AC-based cathode in SSM-MFC. Sintered activated carbon (SAC) was also tested due to its ease of manufacturing and the fact that no stainless steel collector is needed. Results indicate that the SSM-MFC having PTFE cathodes were the most powerful measuring 1617 μW (11 W·m •Activated carbon based cathodes were investigated in SSM-MFCs.•PTFE showed to have the higher power production.•Binder concentration did not affect directly the power production.•PVA-based cathodes and sintered activated carbon were the most cost effective. The utilisation of biocatalysts (i.e. microorganisms) for the direct production of electricity from organics was demonstrated firstly in 1911 ,3, identAs a single MFC is intrinsically a low power source, a plurality of MFC must be assembled in stacks to produce exploitable power levels ,25. In p2)2CO) is primarily hydrolysed by urease enzyme into ammonia (NH3) and carbamate (NH2COOH), which is subsequently degraded to ammonia and carbon dioxide [Urine is a very complex biofluid that may contain up to 2651–3079 different metabolites  each wit dioxide . When trThe cathodic reactions involved in the functioning of SSM-MFCs are yet to be determined. The cathodic reactions could involve the reduction of oxygen and/or oxidised nitrogen, in the form of, for example, nitrate or nitrite. However, urine typically contains low amounts of nitrate and nitrite and they would be the product of the autotrophic ammonium oxidation processes that involve oxygen, as represented by Eqs. − reduction mechanism that occurs, as represented by Eq. Under these conditions, the autotrophic microorganisms involved in the aerobic oxidation of ammonium would be competing for oxygen. This agrees with most microbial fuel cell systems employing activated carbon based cathodes, in which it is the oxygen 2e3− could be reduced into N2 on an anaerobic cathode (denitrification), whilst a simultaneous nitrification was occurring on a separated aerobic cathode [Studies have demonstrated that NO cathode . In the Nonetheless, the concept of SSM-MFC is as a cost-effective system worth developing further. Up to now, this research concept was developed by our group  as a pow22.1−2) (−2). The PVA-5 (i.e. PVA 5%wt) mix comprised 47.2 g activated carbon powder and 50 mL water-based 5% PVA (w/v) stock solution final . Once applied onto the stainless-steel mesh, the paste was dried overnight. Once dried, the PVA cathodes were placed in an oven at 180 °C for 1 h. The paste applied on the stainless-steel mesh for the CPD-5 (i.e. CPD 5%) cathodes consisted of 19 g activated carbon powder mixed to 2.5 g of PlastiDip and 20 mL of solvent (petroleum ether 80°–110°) – 2.5 g of fresh PlastiDip has a dry weight of ≈1 g. The CPD paste was rapidly applied and then put 1 h in the fume hood for the solvent to evaporate (both the PlastiDip solvent and the added petroleum ether). The CPD cathodes final weight was of 19.48 ± 0.77 g, which correspond to an AC/CPD loading of 248 ± 14 mg cm−2. Although the loadings between the cathodes are rather different, the objective here was not to compare the mixture from a quantitative perspective (i.e. at similar loading), but from an implementation perspective. In fact, the cathodes needed to have those specific dimensions  to fit a specific design adopted during the experimentation. Hence, the difference in loadings is the result of the production procedures.All the tested materials were using AC as catalyst . The mai−2) . This caThe cathodes materials  were tes2.22 carbon fibre veil  that was folded down to a projected surface area of 50 cm2 (50 mm × 100 mm). Stainless steel 316 M3 bolts were maintaining the electrodes in position and maintaining a good physical contact with them, whilst protruding 20 mm from the side. These bolts, which serve as current collectors, were also used to connect the monitoring cables and the resistive loads using crocodile clips. All the electrodes were submerged in the same electrolyte, which in the present study was undiluted human urine. In every case, 10 mm of the cathode heights were emerging above the urine level  [Human urine (pH between 6.4 and 7.4) was collected and pooled daily, from anonymous and healthy individuals with no known previous medical conditions. As the urine was kept in a tank (max. 24 h), it had already gone through partial hydrolysis by naturally occurring microflora. Hence, the pH and solution conductivity of the urine feeding the MFCs had values varying between 8.3 and 8.9 and 24–28 mS·cms (≈50%) . The SSM2.3– in Amperes (A) – was calculated according to the Ohm's law, I = V/R, where V is the measured voltage in Volts (V) and R is the known value of the resistor. The power output P in Watts (W) was calculated as P = I × V.Voltage outputs were monitored over time using an Agilent LXI 34972A data acquisition/switch unit . Data were recorded every 3 min. The polarisation scan was performed on mature modules . The polarisation sweeps were performed using an automated resistorstat  with res3Due to its high porosity, AC is not amongst the most conductive carbon materials. Usually carbon black (CB) is added to the mixture to increase the electrical conductivity of the cathode material and therefore the overall performance ,44. HoweResults show that under 500 Ω and 300 Ω, the sintered activated carbon cathodes (SAC) were producing similar level of power as the PTFE (≈1150 μW) and slightly more than the PVA-10 cathodes ≈850 μW; a. ConverWei et al. have shown that binder loading does not affect significantly the cathodes performance . It was PVA-5 cathodes were able to produce more power than the CPD-5, but these performance levels were not stable over time. As illustrated by the results, the power of the SSM-MFC using PVA-5 cathodes drastically decreased a. This dComparing all the power curves indicates that the PTFE cathodes were the best performing cathodes amongst the tested ones a. SSM-MF−1) is much cheaper than 60% PTFE dispersion (≈6 $ kg−1). Even though CPD cathodes were on par with the PVA ones in terms of absolute power, from a cost perspective, CPD were less efficient as cathode material (−1). Regarding the power produced per amount of material used, the SAC cathodes were the least efficient (−1). A previous study investigating PTFE alternatives has shown, with a different MFC design (i.e. ceramic membrane), that chitosan was a good alternative [The power output was also normalised by the mixture loading that lead to new considerations related to the performance of the tested cathodes binder. From the cost perspective, PVA is more efficient than the PTFE since more power is produced per amount of loaded mixture . Of partfficient b, but alernative . The MFCernative . CompareAlthough PVA binder would be a good alternative to PTFE, the instability of PVA cathodes over time, as observed with the PVA-5 cathodes a, indica4The sintered activated‑carbon cathodes present high performance to cost ratio as they are self-supported and could be easily mass manufactured. However, the tested material would need a composition change to increase its robustness. Such improvement should focus on utilizing smaller particle size to have a more homogenous repartition of the HMW-PE binder at the scale of the used cathodes (3 mm thick). Between CPD and PVA binder, results clearly demonstrated that PVA based cathodes were the only one that would be a good alternative to PTFE-based cathodes. The low cost and biocompatibility of PVA further support that conclusion. Unfortunately, their stability over time was low and their production protocol would need to be further improved. For the whole duration of the experiments, and amongst all the tested cathodes, SSM-MFCs mounted with PTFE cathodes were the more powerful. Hence, results demonstrated that at present and with regards to the specific SSM-MFC design and power production, PTFE are still the most suited binder for activated carbon based cathodes.


The role of ONOO− being a double-edged sword in biological systems drives the development of effective detection tools. In this work, a boronate-based polymeric fluorescent probe PB-PVA was synthesized and the probe performance was evaluated. The probe exhibits ratiometric sensing of ONOO− in a range of 0–6 µM. There is good linear relationship between the probe fluorescence intensity ratio and ONOO− concentration. The probe also displays moderate selectivity towards ONOO− over other ROS. Moreover, it is water-soluble and possesses good biocompatibility which aids the imaging of ONOO− in living cells. These properties could make the probe a promising tool in in vitro study related to ONOO−.Peroxynitrite (ONOO There is growing evidence on its role in the physiological and pathophysiological pathway in living systems +; found, 397.1718.Compound −, H2O2, NO·, NO2−, NO3−, and cysteine obtained from commercial sources were diluted or dissolved in water. ONOO- was prepared by mixing pre-cooled 0.6 M NaNO2, 0.6 M HCl, and 0.7 M H2O2 into 3 M NaOH at 0 °C. Manganese dioxide was then added to the ONOO− solution to eliminate the residual H2O2 and was removed by simple filtration. The ONOO− solution was stored at −80 °C. The concentration of peroxynitrite was estimated by its extinction coefficient of 1670 M−1 cm−1 at 302 nm before use [ClOfore use ,20.− were measured at room temperature. The probe was measured 3 min after the addition of ONOO−. The absorption spectra were scanned from 300 to 800 nm while the emission spectra were scanned from 510 to 800 nm. The ratio between the emission intensities of two peaks from the probe when exposed to different concentrations of ONOO− was calculated in order to investigate the relationship between the ratio and the concentration of ONOO− exposed. The probe was also tested for its selectivity towards ONOO− by investigating the fluorescence intensity ratios when exposed to various ROS. The time course of the probe response to ONOO− was measured by tracking the fluorescence intensity at 600 nm with 10 s interval.The absorption and emission spectra of the probe at different concentrations of ONOO2 in a humidified environment. MTT assay was performed to evaluate the probe cytotoxicity with the method mentioned [−, the cells were incubated with the probe (5 μM) for at least 2 h. DMSO (1% v/v) was added to culture medium to increase the solubility of probe. The cells were washed with PBS and further incubated with SIN-1 dissolved in fresh culture medium (1 mM) for 1 h. The treated HeLa cells were observed with a Carl Zeiss LSM710 confocal microscope.HeLa cells were obtained from the Faculty of Health Sciences, University of Macau. The HeLa cells were cultured in RPMI-1640 medium supplemented with 10% of fetal bovine serum (FBS) and 1% of penicillin-streptomycin solution at 37 °C and 5% COentioned . For con− and imaging of ONOO− in living cells. This cell-permeable probe displays good ratiometric detection of ONOO− at low concentration with good selectivity and fast response. These properties could allow the probe to be utilized in ONOO− related in vitro studies as a promising measuring instrument in the future.In conclusion, we have developed a polymeric fluorescent probe PB-PVA for ratiometric measurement of ONOO


We observe a non-uniform spatial distribution and temporal activity of ion channels in mouse brain neurons.Neurons communicate through electrochemical signaling within a complex network. These signals are composed of changes in membrane potentials and are traditionally measured with the aid of (toxic) fluorescent labels or invasive electrical probes. Here, we demonstrate an improvement in label-free second harmonic neuroimaging sensitivity by ~3 orders of magnitude using a wide-field medium repetition rate illumination. We perform a side-by-side patch-clamp and second harmonic imaging comparison to demonstrate the theoretically predicted linear correlation between whole neuron membrane potential changes and the square root of the second harmonic intensity. We assign the ion induced changes to the second harmonic intensity to changes in the orientation of membrane interfacial water, which is used to image spatiotemporal changes in the membrane potential and K Non-invasive spatiotemporal probing of electric potentials in living neurons without chemical or genetic modification provides a major advancement to neuroscience. Here, the authors demonstrate the use of membrane water as a probe for neuronal membrane potentials and ionic flux. The movement of these ions through specific ion channels leads to an adjustment of the membrane potential.Neuronal signaling occurs through rapid changes in the membrane potential that originate from a redistribution of ionic charges across the plasma membrane. In mammalian neurons, Na2. In neurons the membrane potential is further regulated by ion channels, whose functions were discovered in experiments by Hodgkin, Huxley and Katz4. By varying the extracellular concentration of K+ ions around the axons of a squid, while electrically recording the change in resting membrane potential Hodgkin, Huxley and Katz demonstrated the essence of the membrane resting potential regulating mechanism. Ion channels present in the membrane regulate the imbalance in ionic strength and thus change the membrane potential1. This behavior is modeled by the Goldman, Hodgkin and Katz (GHK) equation that relates the membrane potential (ΔΦ0) to the concentration of ionic species  and the permeability (P) of ion channels:A membrane potential arises as a consequence of an imbalance in the ionic strength between two aqueous solutions that are separated by an impermeable membrane. This static difference in the membrane potential is described by the Nernst equation+ ions1 and changing it from 5 to 50 mM results in a less negative membrane-resting potential. Such a reduction in membrane potential magnitude is called depolarization. Using for Eq. 1, ΔΦ0 changes from −75.4 mV ([K+]out = 5 mM) to −33.3 mV when [K+]out is changed to 50 mM.For neurons, the potential gradient is mainly controlled by K8: electrophysiological and imaging. The electrophysiological approach, with invasive patch-clamp techniques7, is used to record whole-cell changes in membrane potential4. Patch-clamp measurements rarely exceed the time scale of several minutes and are limited to the soma . Recent schemes using chronic implants can reduce but not eliminate these effects9.Mapping neuronal electrical activity in cells in vitro and in real time is a challenging task that follows two main approaches11, optogenetic approaches12, and interferometric microscopy14. These techniques rely on measuring physiological changes in the cell that are indirectly correlated to neuronal activity, such as changes in refractive index16, stress-induced birefringence17 or changes in the one or two-photon fluorescence emission from Ca2+-responsive labels19. Recent work with stimulated Raman spectroscopic imaging has shown promising results in relating spectral C–H stretch bands to membrane potential changes in erythrocite ghost cells, still with an empirical connection between the Raman shift amplitudes and the membrane potential20. Second harmonic generation (SHG) is an elastic two-photon scattering process in which two photons with frequency ω are transformed by a non-centrosymmetric or anisotropic material into one photon with twice the frequency (2ω). Non-centrosymmetric crystals, interfaces21, or a broken centrosymmetric orientational distribution of (dipolar) fluorescent molecules by an electric field can generate second harmonic (SH) photons22. SH imaging is used to image non-isotropic polar structures such as collagen24 and muscle myosin26. Additionally, fluorescent voltage sensitive dyes have been used together with resonantly excited SHG to image membrane potentials30. In these studies the optical response was correlated to the reorientation of the chromophore in the membrane29. Membrane potential sensitive chromophores can significantly alter the electric property of the membrane and modify the normal physiological behavior of the cell, can be instable and toxic31 thereby severely limiting the applicability of the methods32.The main imaging techniques involve calcium imaging23. Recently, we demonstrated high throughput wide-field second harmonic imaging of glass/water interfaces33 for which we improved the throughput of a SH microscope to a factor of ~5000, enabling the measurement of interfacial water that is oriented by the presence of surface charges inside a borosilicate micro-capillary. We used the interfacial water response to construct maps of the electric surface potential by relating the interfacial electrostatic field (EDC) and corresponding surface potential (Φ0) via36:f3 is an interference term that takes the value 1 for a transmission experiment37. Given the low photo toxicity of the high throughput microscope38, it is possible to use the same approach, namely the reorientation of water in the electric double layer of a membrane to map membrane potentials in living neurons optically and label-free.As SHG is an inherently weak optical process, label-free imaging of neurological membrane potentials has not yet been achieved; an improvement in detection efficiency of several orders of magnitude was foreseen to be needed3 by detecting the membrane potential of primary cortical neurons during a K+-induced depolarization. Using the nonlinear optical response of interfacial water and nonlinear optical theory we convert the SH images into membrane potential and K+ ion flux maps. While the average temporal response agrees with the theoretically expected membrane potential changes from the GHK equation and the electrophysiological recordings obtained with the same protocol, the images show clear spatiotemporal fluctuations across the neuron. Within the framework of the GHK description, we observe a non-uniform density distribution and temporal activity of permeant ion channels. Being able to non-invasively probe membrane potentials and ion flux in active neurons, opens up avenues to understand signaling mechanisms, either on the single neuron level or in a larger network.Here, we demonstrate the possibility of label-free imaging of the electrical neuronal activity of primary cortical neurons in vitro employing the membrane water as a probe for membrane potentials. We first characterize the SH throughput for neuroimaging, showing that label-free SH images of neurons can be recorded on 600 ms timescale. We then revisit the experiments of Hodgkin, Huxley and Katz+-induced swelling/shrinking using phase contrast imaging39  from cultured neurons. Figure + ions from 5 mM  in the extracellular buffer solution to 50 mM. We then record the voltage difference induced by this K+ depolarization in two ways: by means of an electrophysiological patch clamp and with SH imaging. We subsequently verify the connection between the two predicted by Eq.  changes from −65 to −34 mV. According to Eq.  it is necessary to take them into account as well as the presence of a lipid membrane (mem). Equation  is a constant that converts counts to response units (m4 V−2) and includes I, the intensity profile of the incoming beams. s1 and s2, and ΔΦ is the membrane potential difference. To determine ΔΦ0 from the reorientation of water by the interfacial electrostatic field, we need to determine solutions for C, and 37 and liposome solutions have shown that −22 m2/V2, and −24 m2/V. To determine C(x.y), we use the GHK equation and two known values of ΔΦ0, assuming that at steady state the membrane potential distribution across the neuron is constant. At steady state conditions, when the extracellular concentration of K+ is constant at [K+]1 = 5 mM  and at [K+]2 = 50 mM . Comparing the average intensity over 10 frames (Δt) and subtracting the two resulting time-averaged SH intensities to eliminate the contribution of every X(2) value, we can determine C via:Φ0,1, C. The resulting spatially varying range of values for −22 < −22 m2/V, is in agreement with expectations, as they are an order of magnitude larger than an oriented interfacial monolayer37.Having recorded the dependence of electrophysiological membrane potential and SH intensity during Kdate Eq.  to refleΦ are determined by substituting the previously calculated C, Finally, the spatio-temporal fluctuations of the calculated membrane potential Δinto Eq. .+ from 5 mM to 50 mM  the spatially averaged SH intensity changes, dropping by 25%. When the flow is reversed back to 5 mM K+ the SH intensity recovers, although not fully due to the short waiting time, and then drops back down when the extracellular K+ concentration is switched back to 50 mM. The computed space-averaged membrane potential value ΔΦ0 is shown on the right axis and follows the trend of the SH intensity. The average value agrees well with the electrophysiological measurements of Fig. 43. During the depolarization, the membrane potential reaches more positive values in the somas, while during recovery the neurites display more positive membrane potential values. Hence, different parts of the cells (soma or neurites) are active at different points of the depolarization cycle. We can display these changes more clearly by considering that the membrane potential change is determined mainly by the K+ activity1. Assuming that K+ ions are the only potential determining ions, the ion flux (ΔQ/Δt) can be computed44 using the relationship between the difference in charge (ΔQ) of two frames (separated by Δt), the membrane capacitance (Cm), and the membrane potential difference (ΔΦ0), ΔQ = CmΔΦ0. The capacitance of the membrane has a typical value45 of 1 µF/µm2. Knowing ΔΦ0 from our SH measurements, we can construct ion flux maps. Figure + ion efflux for a few 600 ms intervals during the continuous depolarization cycle from t1 = 85 to 86.2 s and during the recovery period from t2 = 147 to 148.2 s. Supplementary Movie + ionic efflux.Figure + concentration initiated a change in the resting membrane potential of the neuron. Electrophysiological recordings confirmed this well-known response. The temporal square-rooted response of the SH intensity that originates from the oriented water consistently followed the same trend confirming expectations from nonlinear optical theory. We then analyzed the recorded images and constructed maps of the membrane potential as well as the K+ ion flux. We observed temporal variations and spatial variations in the membrane potential and K+ ion flux. These changes are due to a non-uniform distribution of ion channels within the neurons. Different parts of the cells were active at different parts of the depolarization cycle. Thus, within the framework of the GHK description, there is not only a non-uniform distribution and density of permeant ion channels, but also a different temporal activity. We should note here, though, that we used the GHK equation as an approximation, which undoubtedly has simplified the real complexity of a living system. Several aspects of electrical pulse propagation have been reported to suggest a higher complexity involving a coupling between electrical and mechanical responses51 and a more complicated energy dissipation52 than what would be expected from simple Ohmic heating. Future experiments, using multiple modalities, such as SH imaging of water in combination with techniques such as iSCAT53, digital holography54, and stimulated Raman scattering20 will potentially allow one to understand the coupling between electrical and mechanical activity related to neuro-signaling.Summarizing, we propose the nonlinear optical response of membrane water as a mechanism for probing membrane potentials and ion fluxes label-free. We have used the endogenous response of interfacial water to image the sub-cellular and time dependent response of living cortical mammalian neurons to a potassium-enriched solution. The enrichment in KBeing able to non-invasively probe membrane potentials and ion fluxes, opens up avenues for understanding the signaling mechanism inside single neurons or within a larger network. For the present work our method is limited to the mapping of membrane resting potentials and processes occurring on time scales of ~0.1–1 s. While the present 600 ms recordings are still longer than the duration of action potentials, future improvements to the optical layout and noise reduction may bring the recording time down to the 1 ms time scale. The possibility of simultaneous multisite membrane potential or ion flux recordings allows one to resolve the local neuronal network activity; a prerequisite for comprehensive studies of the relationships between spatiotemporal activity patterns, neuronal network development, and information processing. Measurements on tissues are also possible, and endoscopic human clinical applications may become within reach one day, given the straightforward relationship between membrane potential and the probed water, the absence of toxic changes, or the need for genetic modifications.2 per coverslip. Cultured neurons were maintained at 37°C in a humidified atmosphere of 95% air/5% CO2 and were used for experiments after 14 days in vitro.Primary cultures of cortical neurons were prepared from E17 OF1 mice embryos. The average plated density of cells was 15,000 cells/cm2HPO4, 2 KH2PO4, 3 KCl, pH 7.3 ± 0.1, with an osmolarity of 290 ± 10 mOsm.Single whole-cell recordings (current clamp) were performed using a EPC10 amplifier  controlled by PatchMaster software. Patch pipettes with tip resistance of 5–7 MΩ were pulled and fire polished prior to the measurements. Intracellular solution  contained (mM): 140 NaCl, 10 Na68 fs laser pulses and described in detail in ref. 33. The pulse energy delivered to the sample is 36 µJ and shaped into a circular illumination spot size with a FWHM of 150 µm in the sample plane, reaching a fluence of 2.15 mJ/cm2 and peak intensity of 12.79 GW/cm2. The laser beam illuminates a spatial light modulator  under normal incidence. Used as a reflective diffraction grating, the SLM generates a gray scale hologram image of a rotatable slit pattern. The pattern is filtered in the Fourier plane of a lens placed after the SLM, such that only the 1st and −1st orders are retained. These beams are then imaged onto the sample plane using an Olympus LUMPLFLN ×60 1 NA water immersion objective such that it is illuminated with two wide-field beams with a tunable opening angle that can be set by varying the grating spacing period on the SLM. The polarization of the two incoming beams is controlled by a zero-order half-wave plate . The generated SH light was collected in the forward direction using a high NA Olympus, LUMFLN 60 × 1.1NA objective and analyzed with an analyzer placed in the collection path after a 515 nm band pass filter . The endogenous 2PEF response was recorded using a 50 nm-broad bandpass filter (BP550/25) filter. Imaging was performed with a gated detection on an electron-multiplying intensified charge-couple device . The microscope also integrates a path for phase contrast imaging with white light. The SH K+-induced depolarization experiments were repeated 7 times on different cultures under the same experimental conditions.The imaging system is composed of a Yb:KGW amplified laser (Pharos Light Conversion) with 200 kHz, 1036 nm, 12·2H2O, 2 mM MgCl2·6H2O, 5 mM glucose and 10 mM Hepes (denoted as HEPES solution) was used at a rate of 1 mL/mn associated with a suction speed of 1.2 mL/mn. After 2 minutes the HEPES solution was switched to a solution enriched in K+ with 93 mM NaCl, 50 mM KCl, (denoted as K+ solution). The solution was switched back (syringe pump Harvard Apparatus PHD 2000) to HEPES after 2 min and the wash out lasted for 5 min.Coverslips with plated neurons were inserted in a Quick Change Imaging Chamber from Warner Instruments (Series 40 RC-41LP). This chamber provides a maximum liquid content of 3 mL, a laminar flow across the chamber and an open bath. A constant flow of extracellular solution with 140 mM NaCl, 5 mM KCl, 3 mM CaClISH,max, the maximum SH intensity and ISH,min the minimum SH intensity found in the image. Denoising was done by subtracting an offset, applying a Fast Fourier Transform filter along the temporal dimension, and applying thresholding and a median filter in the spatial dimension for each frame (kernel = 3).The raw imaging data were collected from LightField (Princeton Instruments), stored as a TIF stack and exported to Matlab (R2016b). The specific set of data mentioned in the paper is composed of a stack of 500 frames with a temporal acquisition of 0.6 s per frame. The Michaelson contrast of Fig. Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Reporting Summary


Measuring total nitrogen, nitrate, and nitrite is critical for compliance with water safety standards. Previous methods for measuring total nitrogen were hazardous, time consuming, and expensive. Here we report a method for measuring total nitrogen in water and soil using alkaline persulfate digestion combined with a Nitrate Reductase assay. In this method the alkaline persulfate reaction oxidizes all nitrogen present in the sample to nitrate, Nitrate Reductase then is used to catalyze the reduction of nitrate to nitrite in the presence of NADH. The nitrite is then treated with Griess reagents to produce a pink color. The absorbance of this color is measured at 540 nm using a spectrophotometer and when compared to a standard curve of nitrate, treated with both the reduction and colorizing steps, can be used to determine the total nitrogen content of measured samples. This method customizes the measurement of total nitrogen by combining alkaline persulfate digestion with a Nitrate Reductase assay using enzyme based green chemistry.•Customization of total nitrogen analysis by combining alkaline persulfate digestion, driving all nitrogen to nitrate, with a colorimetric nitrate reductase assay•Nitrate reductase catalyzes all nitrate, produced by alkaline persulfate digestion and present in the original sample, to nitrite•Nitrite is measured by the addition of sulfanilamide and N-(1-napthyl)ethylenediamine dihydrochloride, resulting in a pink color Specification TableThe alkaline persulfate digestion is used to completely oxidize nitrogenous compounds to nitrate for the determination of total nitrogen . The quaMost persulfate digestion reactions initially start alkaline , althougSamples containing chloride  can be problematic, since chloride oxidizes to form chlorate ions . ChloratA persulfate to hydroxide ratio of 1:2 must be present in the digesting solution, this will maintain a highly alkaline pH. The concentration of the digesting solution is not important but maintaining the ratio is required. The decomposition of persulfate is dependent on thermal degradation and not pH so nitrogen oxidation can occur in the presence of chloride without chlorate interference downstream.The alkaline persulfate reaction oxidizes all organic nitrogen and ammonia in the sample to nitrate. This is a well known and accepted step in the determination of total nitrogen . The nexg for 10 min then collect supernatant into a 10 mL volumetric flask, leaving pellet behind. Bring volumes to 10.0 mL with distilled water, concentrations are now 0.1 g/mL. Dilute samples appropriately , using a volumetric flask, for digestion reaction. After 1.0 mL of digestion solution is added, the concentration will become 0.083 g/mL which is important for calculating PPM N/g forage sample. For water samples take 5 mL of sample, the working range for the standard nitrate reductase assay is 0.5–10 Nitrogen mg/L, and mix with 1 mL of digestion solution. Dilute water samples with HPLC grade water to bring into the range of the standard assay.Collect forage samples and dry in an oven at approximately 60 °C for at least 24 h, or until the mass no longer changes. Dried forage samples are weighed to 1.0 g and placed in a 25 mL Erlenmeyer flask  along with 20 mL distilled water. Boil samples for a total of 20 min ensuring no samples boil dry. Recover extracts using a garlic press to remove as much extract as possible and store in labeled 15 mL Falcon™ tube . Centrifuge tubes at 5k x 2S2O8; Sigma, Catalog #S6172) in 50 mL 0.5 M sodium hydroxide , prepared fresh day of use. All water used was HPLC grade , and all reagents used were ACS Reagent grade or better. For total nitrogen samples, 5 mL sample of diluted extract, 5 mL of water sample, or 5 mL of nitrate standard was added to a Hach test tubes , along with 1.0 mL alkaline digesting solution and mixed by vortexing. The volume of sample is not critical but the sample to digesting solution ratio of 5:1 must be maintained. For determining amount of nitrate in sample simply add 1.0 mL HPLC grade water in place of the alkaline digesting solution. Tubes are loosely capped then autoclaved at 121 °C, 15 psi, for 60 min. Allow tubes to cool to room temperature, assay for total nitrogen the day of digestion. The Instant Pot® serves as a cheap alternative to the autoclave. If using the Instant Pot® add 1 L of deionized water (not HPLC grade) in pot, place tubes in autoclave safe rack in pot. Attach lid and ensure steam release valve and handle are in closed position. Plug in pot and turn on pressure cook setting to high pressure, turn mode indicator to “More” and set timer for 1 h. Be sure to turn off the keep warm setting. After the run allow the cooker to cool down, wait until float valve has dropped. The entire cycle takes between 1.5 and 2 h. Samples were then quantitatively measured using Nitrate Reductase and the colorimetric Griess reagent [The alkaline digesting solution was prepared by dissolving 1.31 g sodium persulfate , in 200 mL HPLC water. Standards were then diluted to 10 ppm using a volumetric flask, prepared daily, and then diluted again to produce a standard range of 0.05–5.00 ppm for 7 data points. The standards are digested with alkaline persulfate or left undigested. Both sets of samples (digested and undigested) are tested with the nitrate reductase assay to determine the standard curve and show that the alkaline persulfate digestion does not impede nitrate recovery and measurement . The sta4Cl; Sigma-Aldrich, Catalog #09718) in 200 mL HPLC water. To test for chlorate formation based on pH, two different methods were used. One experiment used the acidic persulfate method where the pH started highly acidic and remained highly acidic throughout the digestion. The other experiment used the low alkaline persulfate digestion where the pH starts highly basic and remains highly basic. The acidic persulfate digested ammonium chloride clearly shows inhibition with increased concentrations. Subsequently, the low alkaline persulfate method shows adequate nitrate-N recovery and the slope is agreeable with the nitrate standards  and vortex then allow tubes to sit for 10 min. The contents of the reaction tubes are transferred to a polystyrene (PS) cuvette  and the absorbance is read at 540 nm ± 20 nm in colorimeter or spectrophotometer for the samples and Nitrate Standards. To ensure accurate results, read absorbance between 10 and 30 min after color reagents are added. The absorbance at 540 nm for digested sample is compared to a digested standard curve prepared with certified KNO3 standard 1000 ppm diluted in distilled water to the range of 0.5–5.0 ppm. Using the linear regression equation of the standard curve, the total nitrogen content of each forage sample or water sample is calculated and recorded. If the results are outside the range of the nitrate standards simply dilute the digested sample appropriately and re-run the nitrate reductase assay.Add 50 μL of digested or undigested sample to a labeled glass nitrate-free test tube . Add 900 μL assay buffer (0.1 M potassium phosphate containing 1 mM ethylenediaminetetraacetic acid (EDTA), pH 7.5) and 50 μL 2 mM nicotinamide adenine dinucleotide (NADH) to the sample tubes and vortex. Add 40 μL (0.5 U/mL) recombinant yeast NAD(P)H nitrate reductase  , vortex, and let tubes sit for one hour at room temperature. Add 500 μL color reagent #1 (1 % Sulfanilamide in 3 N HCl) and vortex. Add 500 μL color reagent #2 (0.02 % Total nitrogen determination by alkaline persulfate digestion has several advantages over the traditional Kjeldahl method, these advantages have been described in previous literature ,3 and we


Bioenergy is central for the future energy mix to mitigate climate change impacts; however, its intricate link with the water cycle calls for an evaluation of the carbon–water nexus in biomass production. The great challenge is to optimize trade-offs between carbon harvest and water use by choosing cultivars that combine low water use with high productivity.Regional scenarios were simulated over a range of willow genotype × environment interactions for the major UK soil × climate variations with the process-based model LUCASS. Soil available water capacity (SAWC) ranged from 51 to 251 mm and weather represented the north-west , north-east , south-west  and south-east  of the UK. Scenario simulations were evaluated for small/open narrow-leaf (NL) versus large/closed broad-leaf (BL) willow canopy phenotypes using baseline (1965–89) and warmer recent (1990–2014) weather data.−1, especially in the north. The modern NL cultivar ‘Resolution’ had the smallest and most efficient water use. On marginal soils (SAWC <100 mm), yields remained below an economic threshold of 9 t ha−1 more frequently under baseline than recent climate. In the drought-prone south-east, ‘Endurance’ yielded less than ‘Resolution’, which consumed on average 17 mm year−1 less water. Assuming a planting area of 10 000 ha, in droughty years between 1.3 and 4.5 × 106 m3 of water could be saved, with a small yield penalty, for ‘Resolution’.The low productivity under baseline climate in the north could be compensated by choosing BL cultivars (e.g. ‘Endurance’). Recent warmer climate increased average productivity by 0.5–2.5 t haWith an increase in air temperature and occasional water scarcities expected with climate change, high-yielding NL cultivars should be the preferred choice for sustainable use of marginal lands and reduced competition with agricultural food crops. Salix spp.), grown in short-rotation coppice (SRC), are an important source of biomass, bring multiple environmental benefits and offer a potential additional source of income for farmers  demand and high precipitation , of whicSalix viminalis compared with broad-leaved (BL) species  becomes an important trait for sustainable yield −1 H2O; . EventuaThe overall aim of this study was to find the optimal G × E combination for growing SRC willow to resource the bioeconomy with a low water footprint. Specific objectives were: (1) to determine the cultivar with the highest sustainable productivity, including the least variability under varying environmental constraints (soil × meteorology); and (2) to optimize the water footprint, by selecting phenotypes for a high WUE and low water use, depending on their canopy size and leaf typology. Particular focus was given to productivity on marginal soils and areas of low water availability. Our work combines agronomic productivity with the environmental water footprint challenge and evaluates how management can modify these at the regional and sub-regional scales.Salix species) model  cultivars: two BL (leaf width 20–27 mm), closed-canopy cultivars, ‘Endurance’ (S. rehderiana × S. dasyclados) and ‘Terra Nova’ [ × S. miyabeana]; and two NL (leaf width 14–19 mm), open-canopy cultivars, ‘Resolution’  and ‘Tora’ [S. schwerinii × ]. It was also validated for final harvest after a 3-year rotation at Rothamsted Research and Long Ashton (south-west England) for ‘Endurance’, ‘Resolution’ and ‘Tora’. Key parameters  approach  as descrA field trial was established in a randomized complete block design at Rothamsted Research in 2009 for the four SRC willow cultivars described above .The data show a clear separation of the regions and a general warming trend . The lowSeventeen soil types with different textures, bulk densities and depths were selected to have a variation of soil available water capacity (SAWC) ranging from 51 to 251 mm . Soil paScenario simulations were run for the four willow cultivars for both climatic periods and all 17 soils and eight sites for 2-year and 3-year rotations, generating yields and hydrological variables for twelve 2-year and eight 3-year coppicing cycles for each period. For each genotype × site climate × soil, we obtained specific annual values for stem dry biomass (harvestable yield) and various other indicators, such as the minimal water stress coefficient, ET and crop yield-related WUE  tests (P < 0.05) were performed using the statistical software R with the stats package (Three-way ANOVA (genotype × region × period) and  package  on annuaThe hydrological model performance was evaluated with the root mean squared error (RMSE) and the Nash–Sutcliffe modelling efficiency index (EF):where where 3 m−3 for ‘Tora’ and 0.044 m3 m−3 for ‘Resolution’ during the total validation period  with an increase in LAI. The extraction was greater for the BL (‘Endurance’ and ‘Terra Nova’) than for the NL (‘Resolution’ and ‘Tora’) cultivars . Moreove2010–16, .The results showed regional and temporal differences, which are driven by global radiation, air temperature, water availability and drought controlled by a combination of precipitation and SAWC. Because these regional and temporal patterns were similar between 2- and 3-year growth cycles, only 2-year averages will be discussed except where specified.P < 0.05). In the south, only yields for the NL cultivar ‘Tora’ were significantly lower, while the NL cultivar ‘Resolution’ was overall as productive as the highest-yielding cultivar, ‘Endurance’  north than in the south, and in the (drier) east than in the west. Overall, the different phenotypes showed a consistent ranking of productivity (‘Endurance’ > ‘Terra Nova’ > ‘Resolution’ > ‘Tora’), which was found to be significant in the north . The shift to greater yield distributions was more accentuated in the north  were clearly superior to small (NL) canopies according our model. In the warmer south all cultivars produced within a similar range . HoweverUnder baseline weather, modelled water use (ET) ranged under water limitation from 150 to 440 mm in the SW and from 175 to 375 mm in the NW/NE, with some extremely low annual ET rates of <150 mm for the BL cultivar ‘Endurance’ . Under i−1 H2O (P < 0.05).Overall, simulated WUE ranged from <2 to >5.2 g DM kg−1 H2O . WUE sho−1 H2O . In termY) difference [ΔY = Y(BL) – Y(NL)] was superior (i.e. ΔY < 0) for the NL cultivar ‘Resolution’, in terms of both frequency and magnitude. A significant yield advantage (ΔY < −1 t ha−1) was likely to be twice as frequent and doubled on average (−0.37 versus −0.15 t ha−1) under the warmer recent weather scenario. In addition, ‘Resolution’ saved water, as its average simulated ET was 24 mm lower in drought-prone years.As shown above , the low−2 when compared with ‘Endurance’. On average, growing ‘Resolution’ on marginal soils could virtually save water annually at 16.5 L m−2, which would correspond to 1 650 000 m3 of water, assuming willow plantations of 10 000 ha.Except for two out of 46 years, ‘Resolution’ could have given an annual water saving of between 5 and 55 L mWe addressed the question of whether, beyond selection of cultivar × location, other management options might reduce the risk of low yields on marginal soils. The climate change scenario analysis for SRC willow plantations presented here is based on the combination of a validated G × E optimization model (kws) regulates photosynthesis and growth in the model, which enables the simulation of regional-scale climatic scenarios (kws < 0.5) was more likely for ‘Endurance’ (BL) than for ‘Resolution’ (NL), reflecting greater water use and stress for the cultivar with BL traits forming a large canopy. For light interception, Salix genotypes. However, these authors found a significant genotype × water treatment (drought) interaction for plant biomass, height and leaf angle, which confirmed a potential relevance for breeding. Various greenhouse experiments confirmed variable responses to different forms of water stress, expressing large genetic variability for drought tolerance and WUE, found earlier within the Salix genus , which exceeded grass and wheat by 58 and 93 mm, respectively (−1) due to changes in canopy size, together with greater specific impact when allocated to droughty soils and years (24 mm year−1), are likely to be of substantial benefit. The potential of reducing the water footprint became clearer when compared with the range of 23–68 mm in groundwater recharge in dry years  in terms of production and water use. These high-yielding cultivars can be regionally selected for carbon capture and water use as follows.(1) For (up to 1 °C) warmer and more drought-prone climates and regions (e.g. SE England) NL phenotypes with high photosynthetic capacity (e.g. ‘Resolution’) are advantageous due to high yield with low variation and reduced water consumption (delaying drought stress).(2) For areas with more available water and lower evaporative demands, not affected by occasional drought (e.g. NW UK) BL cultivars with greater light interception and light use efficiency, such as ‘Endurance’, are superior to NL cultivars, such as ‘Resolution’, in terms of yield.−1) and water use. High-productivity NL cultivars can save >20 mm of water in droughty years, which is a considerable resource with expanding plantations and changing climate.(3) On marginal land with high drought frequency and severity the use of NL cultivars like ‘Resolution’ is superior in terms of yield  to have an annual yield higher than an economical threshold of 9 t ha−1 for four regions in the UK and four willow cultivars estimated for two sites per region and 17 soils per sites for the baseline and recent scenarios. Figure S1: weather station locations and partitioning of the climatic areas in the UK. Figure S2: Simulated and observed SWC between 0.1 m and rooting depth, and simulated ET for the willow cultivars ‘Endurance’ and ‘Tora’ at Rothamsted Research in 2011. Figure S3: average modelled annual yields and associated coefficients of variation of four willow cultivars for a 2-year growth cycle management between 1966 and 1988 (started in 1965) for two weather stations in the NW, NE and SW of the UK regarding the soil water capacities of 17 soils. Figure S4: cumulative simulated ET of SRC willow cultivars ‘Endurance’ and ‘Resolution’ and a 2-year growth cycle management in the NE and SW of the UK (two weather stations and 17 soils per region) between 1966 and 1988 and between 1991 and 2013. Figure S5: modelled WUE distribution comparison between ‘Endurance’ and ‘Resolution’ for baseline and recent scenarios in the NE and SW of the UK (two weather stations and 17 soils per region). Figure S6: Frequencies of the annual minimum water-stress coefficient values simulated in the NW and SE for the baseline and recent scenarios for the willow cultivars ‘Endurance’ and ‘Resolution’.Supplementary data are available online at mcz006_suppl_Supplementary_MaterialClick here for additional data file.


By employing ferromagnetic resonance driven spin pumping and the inverse spin Hall effect measurement, we find a dramatic change in the spin pumping signal (decrease by > 80%) and charge-spin conversion efficiency (increase by five times) upon insulator to metal transition. The abrupt change in the structural and electrical properties of this material therefore provides useful insights on the spin related physics in a strongly correlated material undergoing a phase transition.The charge-to-spin conversion efficiency is a crucial parameter in determining the performance of many useful spintronic materials. Usually, this conversion efficiency is predetermined by the intrinsic nature of solid-state materials, which cannot be easily modified without invoking chemical or structural changes in the underlying system. Here we report on successful modulation of charge-spin conversion efficiency via the metal-insulator transition in a quintessential strongly correlated electron compound vanadium dioxide (VO The interconversion of spin and charge is fundamental to the operation of spintronic devices. Here the authors demonstrate spin-to-charge conversion in the correlated material vanadium dioxide, and show that the efficiency changes dramatically across the metal-insulator transition. Ferromagnetic resonance driven spin pumping is one of the most versatile and powerful tools to study the generation and detection of pure spin currents. Since its inception, people have utilized this technique to characterize the spin-orbit interaction induced charge—spin conversions in various materials and heterostructures, including paramagnetic, ferromagnetic and antiferromagnetic metals, semiconductors, and superconductors9. Recently, using materials with magnetic phase transitions as spin sinks, it has been demonstrated that the spin pumping efficiency undergoes a dramatic change across the transition point. This change was attributed to the change in magnetic ordering that modulates the spin mixing conductance at the ferromagnet/spin sink interface14. To date, most experiments related to spin-charge conversions are performed in materials with fixed structural and electrical properties. Having the additional ability to modulate the spin-charge conversion by varying these properties can lead to new functionalities for spintronic materials.The functionality of spintronic devices is determined by three key processes: spin injection, spin manipulation, and spin detection. Since the pioneering spin injection experiment of Johnson and Silsbee in 1987, the focus has been to inject pure spin currents without any transfer of charge current in hopes of realizing low energy cost, high efficiency electronics2) is a transition metal oxide with a unique property of a dramatic structural phase transition under external stimuli near room temperature. When the temperature rises above ~340 K, VO2 transitions from a monoclinic insulating phase into a rutile metallic phase accompanied with large and abrupt change in the electrical, optical, thermal, and magnetic properties17. Nowadays extensive research effort is geared at utilizing these properties for applications such as Mott transition field effect transistors, coupled relaxation oscillators for non-Boolean computing, etc21. Exploring the spin-charge conversion phenomena in this material can therefore open up potential applications as an active spintronic material. Moreover, this material provides a viable platform to study the fundamental spin related physics under phase transitions. The resistivity of VO2 varies by several orders of magnitude over a narrow temperature range across transition allowing an assessment of spin-charge conversion efficiency variation over a wide resistivity range. In addition, the structural change in VO2 (Peierls transition) is accompanied with an electron correlation transition (Mott transition), allowing us to probe the charge-spin conversion in a strongly correlated system16.Vanadium dioxide  across the insulator-metal transition of VO2, we started with YIG films grown on gadolinium gallium garnet (GGG) substrate with ultralow magnetic damping. Two different types of YIG films deposited with liquid phase epitaxy (~3 um thick) and radio frequency (RF) sputtering (100 nm thick) were employed in our experiment. Single phase, polycrystalline VO2 thin films (15–70 nm) were deposited on the YIG/GGG stack subsequently with RF sputtering (see methods). Figure 2/YIG samples, we measure the resistivity, ρ, of the samples as a function of temperature T. Figure ρ(T) result from a typical sample where a sharp decrease in resistivity (101–10−2 Ω.cm) is observed at the transition temperature (Tt) of ~60 °C. The hysteresis in the ρ vs T curves from the subsequent heating and cooling scans is typical for the metal-insulator transition of VO2, reflecting the martensitic nature of the phase transition22. Magnetic measurements on YIG films were performed using SQUID magnetometer and were found to be unaltered pre- and post-deposition of VO2. Figure Ms) of a 3 μm YIG film as a function of temperature after the deposition and annealing of VO2. The Ms value of ~140 emu/cc at 300 K is in good agreement with the values reported for single crystal YIG; furthermore, Ms changes by less than 30% across the temperature range of our experiment.To study the spin-charge conversion properties of VO2/YIG bilayer film facing down (see methods). At resonance, the spin current Js from the magnetic resonance of YIG layer is injected along the y-axis into VO2 and is converted into a transverse spin pumping voltage (VSP) therein. Figure 2(68 nm)/YIG(3 μm) bilayer at θH = 90° and 270° (in-plane fields) at T = 20 °C. As is expected, the inverse spin Hall effect (ISHE) voltage (VSP) changes sign upon the reversal of external magnetic field, Hext, due to the flipping of the injected spin orientation. Note that as YIG is an insulating ferromagnet there is no contribution from anisotropic magnetoresistance (AMR) and anomalous Hall effect (AHE) and the measured voltages are exclusively from the ISHE in VO2 layer. We also performed control experiments and ruled out the existence of other thermal contributions /YIG(3 μm) sample between T = 20 °C and 100 °C at a microwave frequency of 8 GHz. The peak values of VSP at different temperatures are summarized in Fig. VSP variation with the resistivity change, we can clearly see that the dramatic decrease in VSP coincides with the transition of VO2 from insulator to metal. In the transition region, VSP lies between the extreme values of the metallic and insulating phases, reflecting the mixed state nature of this multi-domain regime. Besides the VO2/YIG(3 μm) sample, a similar transition behavior in spin pumping signal was also observed in samples with thinner YIG film /YIG(100 nm) sample), indicating that the observed phenomena does not rely on specific YIG film qualities. In addition to the change in the magnitude of VSP, we also observe a shift in the peak position of spin pumping signal as temperature varies, which can be explained by the reduction of Ms at higher temperatures. According to Kittel’s formula 23, for a fixed driving frequency, a decrease in Ms necessitates a higher external field to satisfy the resonance condition. The resonance field Hres as a function of temperature is plotted in Fig. Ms(T) data from Fig. 2 films with different thicknesses /YIG μm sampl2 has been shown in literature, where the higher switching speed (nanoseconds with voltage gating and sub-picoseconds with laser irradiation) and lower power consumption can help in realizing practical devices that are easier to modulate30. Recently, gate tunable spin-charge conversions in two-dimensional electron gas and ultrathin platinum have also been shown32. The discovery of a metal-insulator transition induced change of conversion efficiency in our experiment provides alternative mechanisms and material candidates for realizing this important goal.The large change in the spin pumping voltage with temperature variation provides the first evidence of phase-dependent spin-charge conversions in this material, where the sample temperature is used as a tuning knob. We note that recently electric-field, irradiation or strain mediated phase transitions in VO2) under continuous spin generations from spin pumping35. Assuming that the thickness of the spin sink layer is much larger than its spin diffusion length, the measured inverse spin Hall voltage is given by33,e is the electron charge, σS, tS and λSD are the conductivity, thickness and spin diffusion length of spin sink, geff↑↓ is the effective spin mixing conductance, L is the sample length, f is the microwave frequency, and θmis the magnetization precession cone angle. At resonance, ΔH is the linewidth and hrf is the microwave magnetic field33. Using the calibrated rf magnetic field in our experiment and the FMR linewidth, we determine that θm~0.008o. P is a correction factor accounting for the ellipticity of the magnetization precession, which is calculated to be P ≈ 1.09 ± 0.04 using the relationship given in ref. 36.The variable spin pumping signal across the metal-insulator transition requires a deeper understanding of the underlying mechanism that causes the aforementioned observations. In general, the spin pumping voltage depends on several fundamental material parameters such as resistivity, spin mixing conductance, spin Hall angle, etc. For example, an increase in the spin mixing conductance will enhance the injected spin current and raise the detected signal, while a decrease of resistivity across the transition can directly lower down the spin pumping voltage. In order to disentangle these factors and understand the observed variation, we start with the spin transport model in spin sink layer /YIG(100 nm) sample at different temperatures are summarized in Fig. 2/YIG sample exhibits a sudden increase when the temperature rises above Tt, indicating an enhancement in spin pumping across the metal-insulator transition. In our experiment, the effective spin mixing conductance changes from (4.0 ± 0.20) × 1017m−2 to (8.5 ± 0.20) × 1017m−2 as the temperature increases. The increase in geff↑↓for higher temperature is consistent with the picture of a more transparent interface in the metallic regime due to the larger density of states of conduction electrons. Here, we recognize that the standard drift-diffusion model may not accurately describe spin transport in a high resistivity material such as VO2, where the electron hopping time can be significantly longer than the spin decay time. However, if the same spin-orbit coupling mechanism dominates the spin relaxation leading to Gilbert damping enhancement and the spin pumping voltage VISHE, they can still be correlated through a single phenomenological parameter geff↑↓ , which are comparable to the reported values40. In contrast to the abrupt change across the MIT transition for VO2, the spin Hall angle exhibits no noticeable change over the temperature range for Pt  change in the spin-charge conversion within the same material brings forth opportunities for engineering spintronic materials with variable functionalities. Even larger variations in the conversion efficiency are expected in optimized VO2 films with higher quality and sharper phase transitions.To conclude, we have experimentally demonstrated phase-dependent spin-charge conversions in VO2 bilayer films. YIG films with thicknesses in the range of 2.5–3 μm were grown on gadolinium gallium garnet (GGG) substrate using liquid phase epitaxy. Thinner YIG samples with thicknesses ranging from 55 to 100 nm were grown using rf-sputtering from a YIG target. The samples were subsequently annealed in a furnace for two hours at 850 °C in 100% O2.VO2 thin films (15–70 nm) were sputtered on the YIG/GGG stack using radio frequency sputtering from a VO2 target. The advantage of using a VO2 target, as compared to reactive sputtering, is the stoichiometric stability of the deposited film which is determined mainly by the V–O stoichiometry in the sputtering target. The substrate (YIG/GGG stack) was kept at 300 °C during deposition. The films were sputtered at a working pressure of 5 mTorr (Ar gas) and subsequently annealed by rapid thermal treatment at 450 °C for 2 min in an O2 (4%) + N2 (96%) mixture.The samples used in this study are yttrium iron garnet (YIG)/ VO2 (4 × 4 mm2) films  in sample plane (xz). The ISHE voltage is measured across the VO2 layer along the x-axis. For temperature dependent measurements, we mounted the CPW onto a ceramic heater. The sample temperature was monitored with a thermocouple attached to the film surface and carefully controlled with a PID temperature controller. At each setpoint the temperature was maintained within ±0.5 °C. We observed no significant variation in the transmission coefficient of the microwave signal through the CPW over the experimental temperature range of 20–120 °C, indicating negligible changes in the driving power of spin pumping. We excited the FMR by sweeping the external magnetic field while fixing the excitation microwave frequency (between 3 and 10 GHz), and measured the ISHE voltage across the sample with a DC voltmeter.The schematic of the setup used for spin pumping measurement is illustrated in Fig. Supplementary Information