Glutathione transferases (GSTs) are a family of Phase II detoxification enzymes that are involved in the development of multi-drug resistance (MDR) phenomena toward chemotherapeutic agents. GST inhibitors are considered candidate compounds able to chemomodulate and reverse MDR. The natural flavonoid myricetin (MYR) has been shown to exhibit a wide range of pharmacological functions, including antitumor activity. In the present work, the interaction of MYR with human glutathione transferase A1–1 (hGSTA1–1) was investigated by kinetics inhibition analysis and molecular modeling studies. The results showed that MYR binds with high affinity to hGSTA1–1 (IC50 2.1 ± 0.2 μΜ). It functions as a non-competitive inhibitor towards the electrophile substrate 1-chloro−2,4-dinitrobenzene (CDNB) and as a competitive inhibitor towards glutathione (GSH). Chemical modification studies with the irreversible inhibitor phenethyl isothiocyanate (PEITC), in combination with in silico molecular docking studies allowed the prediction of the MYR binding site. MYR appears to bind at a distinct location, partially overlapping the GSH binding site (G-site). The results of the present study show that MYR is a potent inhibitor of hGSTA1–1 that can be further exploited towards the development of natural, safe, and effective GST-targeted cancer chemosensitizers. Glutathione transferases (GSTs) are multifunctional enzymes that have evolved from thioredoxins/glutaredoxins family ,2,3,4. TIntense research over the past two decades has established that GSTs are key enzymes that are directly connected to the therapeutic response to chemotherapy. High expression levels of GSTs are connected with an increased resistance of tumors to a variety of anticancer drugs ,5,6,11. Polyphenols are natural compounds with variable structures that are classified into several subclasses including catechins, flavonoids , anthocyanins, chalcones, curcuminoids, and phenolic acids. Flavonoids are being increasingly studied for their diverse biological activities, including anticancer activity, and negligible side effects ,18,19. TThe present work aims to investigate the interaction of myricetin (MYR) with hGSTP1–1. MYR  is a polβ-catenin signaling, and by stimulating ROS-mediated stress, endoplasmic reticulum stress, and DNA damage [MYR is one of the most common flavonoids in fruits, vegetables, herbs, tea, and red wine and displays a range of diverse bioactivities in different cell processes ,22, suchA damage ,22,23,24A damage . MYR hasA damage ,26. SeveA damage  showed tA damage  demonstrConsidering that MYR contributes to chemoresistance and taking into account the key role of hGSTA1–1 in MDR mechanism, the present work was undertaken in order to evaluate whether the activity of hGSTA1–1 can be manipulated by MYR. The results of the present study shine light on the role of MYR as potent inhibitor towards the MDR-involved hGSTA1–1. The outcome of the work provides new insights into the drug design effort towards MDR targets that can facilitate the rational development of GST-targeted chemosensitizers.Reduced glutathione (GSH), 1-chloro−2,4-dinitrobenzene (CDNB), bovine serum albumin (BSA), MYR, and all other buffers, media, and salts were purchased from Sigma–Aldrich .E. coli BL21 (DE3) cells and purified by affinity chromatography on immobilized GSH as described elsewhere [Recombinant hGSTA1–1 was expressed in lsewhere ,32,33. PThe activity of hGSTA1–1 was assessed by measuring the conjugating activity of the 1-chloro−2,4-dinitrobenzene (CDNB)/GSH system as described previously ,32,33. FMeasurement of the dependence of the initial rates of catalytic activity on CDNB concentration was carried out in 0.1 M potassium phosphate, pH 6.5, (37 °C), using 0.02–1 mM CDNB in the presence of 2.5 mM GSH in the presence or in the absence of MYR (0–2 μΜ). Measurement of the dependence of the initial rates of catalytic activity on GSH concentration was achieved using 0.035–2.5 mM GSH and 1.5 mM CDNB, in the presence or in the absence of MYR (0–8 μΜ). Measurements of initial rates were carried out in triplicate. The kinetic data were analyzed by nonlinear regression analysis using the computer program Graph PadPrism 5 .Irreversible inactivation of hGSTA1–1 by PEITC  was perfhttp://www.pymol.org/, accessed on 9 August 2022) [Docking calculations for the MYR and hGSTA1–1 interaction were carried out by AutoDock Vina , with dest 2022)  and UCSFst 2022)  were useThe presented data were obtained from three independent measurements. Graph PadPrism 5  were used for data analysis and evaluation.n-arylimines, and benzophenones and their carbonyl n-analogues were in the range 22–71 μΜ [The interaction of MYR with hGSTA1–1 was studied by employing inhibition studies. The GST substrate system GSH/CDNB was selected for measuring the effect of MYR on enzyme activity. hGSTA1–1 is highly efficient in catalyzing the conjugation of GSH to CDNB, allowing the determination of kinetic constants with high accuracy and reproducibility ,41. Figu22–71 μΜ . However22–71 μΜ . Ethacry22–71 μΜ . Further22–71 μΜ . However22–71 μΜ .n-terminal domain I. The second, “H-site”, is found at the C-terminal domain II, which binds the hydrophobic electrophilic acceptor substrates [Kinetic inhibition studies were carried out aiming to obtain kinetics data for shining light on the type of inhibition as well as a roughly prediction of the location of its binding site on hGSTA1–1. The enzyme possesses three discrete ligand binding sites. The first is the conserved GSH binding site, “G-site”, which is located at the enzyme’s bstrates ,34,36,38bstrates ,51,52. hi = 1.1 ± 0.1 μΜ) (i = 2.1 ± 0.2 μΜ), as the lines of the Lineweaver–Burk plot intersect the velocity axes (The inhibition of hGSTA1–1 by MYR in the form of the Lineweaver–Burk plots is illustrated in  0.1 μΜ) A against 0.1 μΜ) B, suggesity axes C. The deity axes D, suggesTo further validate the inhibition pattern obtained by kinetics inhibition studies chemical modification and inactivation experiments were employed. Phenethyl isothiocyanate (PEITC) A is an iobs)  . The preAs we have already mentioned, PEITC specifically reacts with Cys112 of hGSTA1–1  Figure B. From tPrediction of the binding more of MYR to the hGSTA1–1 was carried out using in silico molecular docking performed by AutoVina . The worThe predicted binding of MYR appears to be in agreement with the results obtained by chemical modification studies, and confirmed the competition observed between PEITC and MYR or S-hexyl-GSH C. FigureThis study expands the role and function of MYR, a widespread flavonoid, and establishes its role as a potent inhibitor towards hGSTA1–1. MYR is able to bind specifically to hGSTA1–1 at a distinct location at the solvent channel partially occupying the entrance of the G-site. The results of the present work suggest that MYR holds the potential for application as adjuvant in combination with drugs known to be substrates of hGSTA1–1 enzyme. The combination of MYR and a drug can affect cancer cells, overexpressing hGSTA1–1, in two discrete ways: firstly by preventing the drug’s metabolism and detoxification through the direct inhibition of hGSTA1–1 catalytic activity, and secondly, MYR can act as an anticancer compound. The outcome of the study can accelerate the development of GST-targeted cancer chemosensitizers. Future work involving in vivo and clinical studies will further evaluate and assess the efficiency, safety, and clinical use of MYR.


We analyzed 208 fish collected in 2016and 2018 at 15 different sites. δ13CFA values of these fish tissues correlated more among each other thanthose of δ2HFA values. Both δ2HFA and δ13CFA valuesshowed tissue-dependent isotopic fractionation, while fish taxa hadonly small effects. The highest site specificity was for δ13CDHA values, while the δ2H isotopicdifference between linoleic acid and alpha-linolenic acid resultedin the highest site specificity. Using linear discrimination analysisof FA isotope values, over 90% of fish could be assigned to theirlocation of origin; however, the accuracy dropped to about 56% whenisotope data from 2016 were used to predict the sites for samplescollected in 2018, suggesting temporal shifts in site specificityof δ2HFA and δ13CFA. However, the predictive power of δ2HFA and δ13CFA over this time intervalwas still higher than site specificity of bulk tissue isotopes fora single time point. In summary, compound-specific isotope analysisof fatty acids may become a highly effective tool for assessing fineand large-scale movement and foraging areas of animals.Methods for identifying origin, movement, and foragingareas ofanimals are essential for understanding ecosystem connectivity, nutrientflows, and other ecological processes. Telemetric methods can providedetailed spatial coverage but are limited to a minimum body size ofspecimen for tagging. In recent years, stable isotopes have been increasinglyused to track animal migration by linking landscape isotope patternsinto movement (isoscapes). However, compared to telemetric methods,the spatial resolution of bulk stable isotopes is low. Here, we examineda novel approach by evaluating the use of compound-specific hydrogenand carbon stable isotopes of fatty acids (δ Compound-specific stable isotopes offatty acids in fishespredict site-specific feeding grounds and track metabolic processesof essential dietary nutrients. Bulk tissue isotope analyses are widely used to investigate larger-scale animal migration in terrestrial,marine, and freshwater ecosystems,19 however, bulk isotope methods rely on the presence of distinctiveisotopic gradients amongst migratory habitats and usually over largerdistances, that is, 100–1000’s of km , and are hence biased to the location of markingeffort, and so do not yield detailed information about finer-scalehabitat use by individuals.13C and δ15Nin amino acids.21 Depending on the metabolicrequirements of individuals, a combination of dietary source acquisitionand metabolic processes may lead to spatially explicit δ valuesof essential and non-essential components that ultimately depend onthe isotopic composition and food web structure of the local environmentin space and time.22 Fatty acids, whoseδ2H and δ13C isotopic compositioncan potentially provide more detailed smaller spatial resolution thanbulk tissue stable isotope methods, have never been tested or exploitedfor animal migration and provenance studies.23Finer-scale spatial resolutionfor isotope provenance assignmentmight be achieved by using compound-specific isotope analysis (CSIA),as shown by using δ13C (bulk tissue) gradients havebeenidentified so far, for example, in correlation with moisture contentfor terrestrial plants,24 as well as apositive increase of 1–2‰ km–1 altitudein terrestrial grazers,25 hummingbirds,26 and terrestrial leaves.28 Temperature and pCO2 are suspected tobe the main driver of increasing δ13C-DIC valuesby altitude, however, soil respiration rate, instream metabolism,and anaerobic respiration cumulatively contribute to significant spatialvariations in δ13C-DIC as well29 and subsequently influence bulk and compound-specific δ13C values of primary producers and consumers.30Several δ2H and/or δ13C of FA in tissues of three fish species  along reaches of a sub-alpine stream catchmentin 2016 and 2018. In addition, bulk FA stable isotope values fromsame fish species were also examined. Bullheads generally have lowmigration range, rarely move beyond several hundred meters, and henceshould exhibit high isotopic site specificity.31 In contrast, salmonids like brown trout can migrate distancesof 100 km or more,32 but in small mountainouscatchments are also often stationary,33 and thus could exhibit high or low isotopic site specificity.The aim of this study was to measure patterns ofFA carbon andhydrogen stable isotope values in fish tissues at small spatial scales(1–5 km) to assess their potential for studying fish migrationand movement ecology and to evaluate the temporal isotope value stability.We evaluated sampling location (site specificity) as variation factorof δ2. The River Ybbs tributaries and mainstem are firstto fifth order streams (Strahler). The maximum distance between fishsampling sites was 35 km between “Weiße Ois” and“Göstling”  were sampled and analyzed from 15 sites of 10 streamsduring base-flow conditions  were collected by electrofishing and euthanized in accordance withthe Austrian Federal Act on the Protection of Animals (http://www.ris.bka.gv.at). Allfish were dissected for samples of dorsal muscle and liver, eyes,and brain for FA analysis. Detailed analysis of fish tissue lipidcomposition for our samples was reported previously.35For δ2. Forfatty acid methyl ester (FAME) formation, lipid samples were incubatedwith a sulfuric acid/methanol mixture (1:100 vol/vol) for 16 h at50 °C, following the addition of KHCO3 and hexane.Samples were then shaken, vortexed, and centrifuged, and the upperorganic layers were collected, pooled, and concentrated under N2.36Lipids of the selected tissues wereextracted as described by Heissenberger et al. (2010). Briefly, freeze-driedtissue samples were homogenized and mixed with chloroform/methanol(2:1 vol/vol) following sonication, and vortexed and centrifuged threetimes to remove non-lipid materials. The solvent-extracted lipidswere evaporated to a final volume of 1.5 mL under N13C and δ2H analyses of FA were conducted followingthe analytical methodology described previously.23 Briefly, a Thermo Trace 1310 GC  was connected via a ConFlo IV (ThermoFisher Scientific)to an isotope ratio mass spectrometer . FAMEs were separated using either a VF-WAXms 60 mcolumn, 0.25 mm ID, film thickness 0.25 μm; ora VF-WAXms 30 m column, 0.32 mm ID, film thickness 1μm  and then for δ13C analysis oxidized to CO2 in a combustion reactor,filled with Ni, Pt, and Cu wires, at a temperature of 1000 °C,or for δ2H analysis reduced to H2 by passingthrough a high thermal conversion reactor kept at 1420 °C. Dueto better isotope-ratio mass spectrometry detection limits for CO2 gas compared to H2, the total number of FA peaksidentified for δ2H was less than for δ13C, which however, did not affect our analysis.All δ13C = −30.53‰, δ2H = −183.9‰,USGS71: δ13C = −10.5‰, δ2H = −4.9‰, and USGS72: δ13C= −1.54‰, δ2H = +348.3‰), whichwere also used to check and correct for instrumental drift and linearity.The δ13C and δ2H values of individualFAME were determined by automated peak integration by defining 0.5mV/s as the start and end point of a FAME peak and by using dynamicbackground removal calculation. All peaks were validated and correctedmanually if necessary. FA δ13C and δ2H values (δIFA) were correctedfor the methyl group addition during methylation according to theformula23IFAME are the δ2H or δ13C values ofthe measured FAME and δIMeOH theδ2H or δ13C values of the methanolused during methylation, and n equals the total numberof H-/C-atoms of the FAME molecule. Values for δ13C are referenced to Vienna PeeDee BelemniteSampleswere reference scale-normalized using three-point calibrationwith certified FAME stable isotope (Me-C20:0) standards  and δ13C (δ13CFAbulk) were calculatedby summing the isotopic value by the mass fraction of all identifiedpeaks of a sample and divided by the sum of their mass fractions.These composite values were used as proxies for “bulk lipid”stable isotope values for comparison with individual FA.Integrated “bulk” FAvalues for both δrstatix, ggplot2, ggpubr, lme4, car, CCA, ccp, candisc, rptR, mass, and Morpho. A Shapiro–Wilks test was used to affirm normaldistribution of the data. Significance of the linear models was evaluatedusing ANOVA using Type II and III sums of squares for models withoutand with significant interaction terms, respectively, thereby controllingfor tissue type, sampling location, and taxa where necessary. Forpost-hoc univariate analysis of compound-specific effects, p-values were adjusted for simultaneous inference by termusing the Holm method.37 Estimated marginalmeans for post-hoc group comparisons were calculated using the emmeanspackage and its pairs function while applying Šidákadjustments. Canonical correlation analysis (CCA) was performed (CCApackage) using the δ13C and δ2Hvalues of FA as covariates. Significance of dimensions was testedusing the ccp package and Wilks’ Lambda F-approximation. Canonical coefficients (ccoef) were calculatedusing the candisc package.Data and graphical analyses were performedin R (version 4.0.2) using the packages 13C and δ2H data wasanalyzed by performing canonical variate analysis using the cvafunction of the Morpho package and linear discriminantanalysis (LDA) provided by the mass package. Overall,site classification accuracy is given before and after (in parenthesis)cross-validation by running 999 bootstrapping analyses. Site-specificrepeatability, describing the relative partitioning of variance intowithin-group and between-group sources of variance,38 was calculated for each isotope and FA compound individually,while controlling for other group variables using the rptR package with 1000 bootstrapping analyses.Site specificityof FA and δ39Unless otherwise mentioned, individual δ values are reportedas the mean ± standard deviation, while site-specific isotopicvalues are provided as estimated marginal means [lower and upper 95%confidence interval].13CFA and δ2HFA), we used fish samples of the larger and comprehensive 2016 data set to ensure robust results.For this data set, 10,138 GC-IRMS FAME peaks were used for δ13C and 9,183 for δ2H analysis . The FA δ13C values differed significantly across sampling sites and tissue types  and to a lesser degree across fish taxa .The δ2HFA values were predominantly influencedby fish taxa  and tissue type  and to a lesser extent bythe sampling site . In a post-hoc analysis ,all FA compounds showed significant  sampling site-specific differences forboth δ13CFA and δ2HFA. The tissue types also explained some of the variation incompound-specific δ13CFA and δ2HFA values, except for the n –6 PUFA ARA and LIN, and additionally for EPA and ETA for δ13CFA. Significant taxa-specific influence of δ13C values was only observed for 16:1 and ALA (p.adj = 0.019), while δ2H had taxa-specific differencesin 14:0, 16:0, 16:1 (p.adj = 0.025), 18:0, LIN (p.adj = 0.020), ALA (p.adj = 0.037), ARA(p.adj = 0.023), 20:3n –3, ETA, DPA, and DHA. Hydrogen isotope differences among taxa werefound mainly between bullheads and salmonids, while the only significantdifference between rainbow and brown trout was observed in δ2HARA . Large hydrogen isotopic differences betweenbullheads and salmonids were observed for δ2H andδ13C values of 20:3n – 3,ETA, and DPA. No significant correlation or patterns between isotopevalues and physiological parameters such as size, weight, or Fulton’s K were identified.For the evaluation of sampling site-specific FA stableisotope values . Correlations among δ13CFA versus δ13CFAbulk were significantly higher compared tothe corresponding δ2H values . The first dimension had a canonical correlationof 0.721  and was primarily explainedby the isotopic variation between the tissue types, particularly thebrain . Separation of brain δ values from othertissues was higher for O. mykiss and S. trutta than for C. gobio (13C16:0 (ccoef= −1.21), δ13CEPA (ccoef = 0.83),δ2H18:1 (ccoef = 0.62), δ2HALA (ccoef = −0.47), δ2H16:0 (ccoef = −0.36), and δ13C18:0 (ccoef = 0.36). Similarly, the second dimension showeda separation of isotope values between eye and other tissues, particularlyin C. gobio. The third dimension  was highly taxa-specificand separated between C. gobio andsalmonids  dueto high canonical coefficients of δ13CALA (1.06), δ13CDHA (−0.78), δ13C14:0 (0.68), δ2HALA (0.66), δ2H18:0 (0.63), and δ2HDHA (−0.49). We thus concluded that FAisotope discrimination among the tissue types was too large to allowfor pooling of the FA isotope data; subsequently, all further analyseswere conducted by tissue type.Paired δC. gobio 3A and waalmonids 3B, but f13C, no individual FA compoundprovided significantly better site repeatability than FABulk [mean: 74% ± 10]. δ13Cn–3 LC-PUFA values had similar site-specificrepeatability as δ13CFABulk, particularlyin the brain and muscle tissue, while the repeatability for n – 6 PUFA was poor across all tissue types. Amongthe non-essential  FA, δ13C16:0 had the best site repeatability, especiallyfor the muscle tissue [81.0% ± 10], which is higher than bulksample repeatability, while δ13C18:1 showedhigh site repeatability in brain [74% ± 10] and eye tissue [71%± 10]. In contrast, several FA showed better site-specific repeatabilityin their δ2H values compared to the δ2H-FABulk [mean: 42% ± 11]. The δ2HALA values performed particularly well among all tissuetypes [mean: 54% ± 12], while site-specific repeatability forδ2HLIN was high for the muscle tissue[59% ± 12]. δ2HDHA showed some site-specificrepeatability only for the liver tissue [52% ± 13], while thoseof 16:0 and 14:0 performed equally or better than FABulk in the brain and eye tissue. The highest repeatability for any ofthe compound-specific δ2H values among all sampleswas achieved when using the difference between δ2HLIN and δ2HALA  revealed several site-specific clusters . In lineardiscrimination analysis, the proportions of variation explained forthe combined first three dimensions were >70% for all tissues  and provided good visual discriminationof the site-specific clusters in a 3D plot (see online Supporting Information).Based on their close Mahalanobisdistance in the δocations 1, the fi2HFA and δ13CFA values in the sedentary bullheads over time(site repeatability), we compared the δ2HFA and δ13CFA data of 2018 with those of2016 from the same sampling sites. Both δ2HFA and δ13CFA values showed significantbetween-year isotopic differences in various tissues and locations. In general, omega-6 PUFA frequentlyhad larger isotopic differences than omega-3 PUFA. Saturated FA showedsmall changes in mean δ13C values. Changes in δ2H18:0 values were accompanied by an inverse proportionalchange in δ2HLIN and δ2HARA.For evaluating the δcaret (R package), a trainingdata set (grandmeans of LDA) of bullhead samples from 2016 was generated to predictthe sites of the bullhead samples from 2018. 34% of samples from 2018were assigned to the correct location from the 15 sampling sites basedon the isotopic composition of FA. This proportion increased to 54%,if accounting for assignments to a spatially close sampling location(< 1 km). In general, samples from tributaries at higher altitudeshad a higher probability of being assigned to the correct locationthan samples from the further downstream sites of this study catchment between sampling sites of thesestreams, fish tissues had unique δ2HFA and δ13CFA values that allowed assignmentof fish origin at a small spatial scale.The δ13CFA values of fish were morenegative at higher altitude sites, and the δ13C valuesof individual FA were highly correlated with each other.The δ13C values with altitude,no such effect was observed for δ2H and the variationamong FA was not explained by a calculated δ2HBulk value. It is possible that different physiological requirementsfor fish at the higher and lower altitude sampling sites altered fishmetabolism, which is already known to change δ2Hvalues of long-chain (>18) PUFA.40 Thus,δ2H values of individual FA, for example, highlyrequired EPA and DHA, likely change because of FA bioconversion processesin case of insufficient dietary supply.30 This is also reflected by the fact that the difference in isotopicvalues δ2HLIN-ALA showed the highestsite specificity for δ2HFA values. Consumersare not able to synthesize LIN or ALA de novo,41 and thus, the isotope values likely reflectthose of the base of the food web, whose structure can also show site-specificdifferences. This suggests that δ2HFA showedless site specificity compared to δ13CFA, although δ2HFA values can reveal fine-scalestructures in small geographical areas where larger-scale δ2H patterns of water are absent.Incontrast to changes in δn – 3 PUFA DHA, EPA, and ALA, as wellas the saturated FA 16:0 and the mono-unsaturated FA 18:1 had highpredictive δ13C values of sampling site specificity.It is likely that these FA are important for maintaining tissue function,are required in high quantities, and are retained in storage and functionalmembrane lipids,42 thereby keeping thedietary isotopic values. While 16:0 and 18:1 are non-essential FAand thus might be also synthesized de novo by consumers,they are also available in high levels from the diet ,and hence, their isotopic values are also more likely to reflect localfood sources. Interestingly, LIN dual-isotope data did not correlatewith the sampling site, in any tissue, despite being an essentialFA. LIN is the precursor of the n – 6 LC-PUFAARA, an important tissue FA regulating inflammatory processes in vertebrates.43 It thus might be used mainly for energy production,as the precursor for elongation to ARA or to regulate n – 3 to n – 6 bioconversion ratesbecause n – 3 and n –6 PUFA use the same bioconverting enzymes.44 All the above involve several biochemical conversion processes thatmight induce significant isotopic fractionation of LIN,thereby blurringsite-specific isotopic information of this FA.The 13CSFA and δ13CMUFA values and lower δ2HSFA and δ2HMUFA values comparedto muscle or liver tissues, which may be due to the unique metabolismof the brain; (1) relying more on carbohydrates than fat for ATP production;45 (2) being separated by the blood–brainbarrier restricting the exchange of molecules between blood and therest of the body, and/or (3) relying on its own FA synthesizing complex,for example, for building very long-chain FA that isolate axons aspart of the myelin sheet.46 Fatty acidsare thereby synthesized, particularly during growth phases of an individual,using ketone bodies as sources, in contrast to acetate, which is used for FA synthesisin the liver.46 These different biochemicalsynthesizing pathways might therefore explain these isotopic differencesin saturated and mono-unsaturated FA between brain and the other tissues.In addition tosampling site-specific FA isotope variation, tissueand species type also differed isotopically. For example, brain FAhad comparatively higher δ2HFA and δ13CFA values as site-specific markers in canonical variationanalysis . Nevertheless, this study provides compound-specific isotopicevidence that the fish origin within this stream network  could be ensured with 54% accuracy across the two years,which was more precise than using only bulk stable isotopes for sitespecificity within a single season. Compound-specific isotope valuesgenerally have higher variation than bulk stable isotope values onlarger isoscapes,48 implying that local food web and communitystructures as well as physiological processes have a higher influenceon stable isotope composition of FA than meteorologic isotopic fractionation.Naturally occurring inter-annual δ2H and δ13C variations at the molecular level might be more sensitiveto small environmental changes, which are largely smoothed out whenanalyzing bulk tissues with longer turnover times , while changes are quickly reflected in high-turnover tissue,such as liver. In this case, tissue FA isotopic differences couldpotentially be used to improve tracking migration of individual fish.This finding might provide a meaningful estimate of the fish origin,especially if considering larger-scale catchments. However, the naturalrange of FA isotopic variation needs to be better defined by locationand over time.The δUnfortunately, there are no movement data forthis retrospectiveassessment, however, we found relatively small isotopic differencesbetween salmonids and bullheads compared to the overall isotopic variations.Therefore, these differences are possibly attributed to differentfeeding behaviors rather than different movement patterns, which remainsto be clarified in future studies.(1)No clear spatio-temporal patternsin the FA isotopic composition can be applied for all cases. Residentfish grown in enclosures, ponds, or with limited capacity for migrationneed to be sampled to assess the isotope variation within the areaof interest before evaluating samples of unknown origin (“known-dietsource approach”). Controlled captive studies using isotopegradients in the sources (water and diet) and different levels ofdietary quality to quantify the mechanisms that drive the isotopicflow and dynamics in FA are needed to understand the strengths andcaveats of these CSIA to better assign the origin of fish in aquaticecosystems.(2)One technicalaspect for the use ofthis isotope tool is how researchers determine the FA-CSIA baselineto distinguish between sites or areas of interest. Whether a residentfish species to discriminate among sites or the use of a lower trophiclevel organism, such as macroinvertebrates or periphyton, dependenton the FA type  and time of sampling.(3)As with bulk tissue isotopemethods,more detailed knowledge is warranted for specific tissue/s FA isotopeturnover times and integration period .(4)The use of multi-isotope molecularapproaches is recommended to improve data and spatial assignment resolutionin other environments. The current study focused on a small sub-alpinestream catchment, and the applicability of this methodological approachto other stream systems or by using anadromous species remains tobe elucidated. Well-known and predictable hydrogen isotope gradientsin water based on latitude or marine versus freshwater studies mayenhance the potential of using FA H-isotopes for this method. Thedual-isotope approach at the FA level makes site discrimination easierwithout increasing time on sample treatment; however, it will requiretwo separate CSIAs.(5)Statistical approach of large datasets: multivariate data analysis including several FA is recommendedfor a priori site specificity, but refined protocols with a widerselection of FA can be investigated. As most of the variation amongthe data originates from the FA compound, CCA can be a useful toolto assess the influence of other factors than location and to decidefor which samples a pooled analysis can or should not be performed.(6)Finally, the additionof CSIA of aminoacids might add another dimension to further increase accuracy andmay be included in future studies.Our findings suggest thatthe FA isotope composition in fish tissuescan help better identify the provenance of fish samples, even at smallgeographical scales. We summarize the following aspects as recommendationsfor future research:13CFA and δ2HFA values providedhigher spatialresolution assignment of fish origin in this stream network than bulkFA isotopic analysis. The isotopic patterns resulted from a combinationof physicochemical processes that had a strong influence on δ13C values, and possible ecophysiological processes with effectsmainly on δ2H values of individual FA. The compound-specificisotopic composition of the samples gained from individuals thus reflectsthe integrated effect of element isoscapes, diet, and metabolism.The latter has the potential to induce significant metabolic fractionation,especially for δ2H values, disrupting the preservationof isotopic values of the diet. In that sense, this method primarilycompares individuals regarding their similarity in life history, amongwhich residency and feeding at a particular location is an importantfactor, without requiring a baseline or knowledge about all potentialfood sources. How topological and ecophysiological properties influencethe isotopic patterns of FA, how isotopic patterns of FA change inresponse to different trophic levels or at larger spatial scales,and if non-migrant fish require to establish local isotopic patternsremain questions for future migration ecology research. This introducedmethod enhances our capacity to trace foraging and migration of fisheswhere other telemetric methods are not applicable, and it can providevital information about habitat degradation of endangered speciesand spread of an invasive species.49 Specificexamples of such application are to determine the impact of verticalbarriers on migration of small fish species .The CSIA of FA of eggs and early life stages recovered at the spawninggrounds can provide important information on foraging grounds of adults50 and lipid-rich tissues with slow turnover  could also help assess the hatchery or wild originof fish stocked to the natural systems.51In conclusion, the combination of δ


Retama raetam (white broom) and the ecologically significant Acacia spp. (acacia thorn trees), two high-calorific and high-burning taxa best suited for such purposes. It was also observed that over the course of the industry, the search for fuel expanded, as evidenced by the later appearance of taxa unsuited for the prevailing regional conditions, hinting at the detrimental toll the industry took on the local ecosystem. Altogether, it is suggested that the lucrative copper industry ended due to limits in the availability of fuel, caused by anthropogenic hastening of desertification and environmental degradation.Economic and industrial progress frequently comes at the expense of environmental sustainability. For the early Iron Age (~ eleventh to ninth centuries BCE) smelters of the ancient copper industry of the Timna Valley, southern Israel, where today the hyper-arid Aravah Desert provides sparse vegetation, woody fuel for metallurgical furnaces constituted the greatest limiting factor for continued operations. This study presents the first investigation into the fuel sources relied upon by this industry during its most intensive period, as reflected by hundreds of charcoal samples collected from two well-stratified and chronologically anchored accumulations of industrial waste. The two sites demonstrate similar results: a heavy reliance on the local vegetation, particularly  Though some of the earliest evidence of extractive metallurgy is attested to here, it was during a roughly half-millennium period within the Late Bronze and Iron Ages (thirteenth-ninth centuries BCE) that the Timna Valley achieved its industrial peak3. Originally attributed solely to the 19th and 20th Dynasties of New Kingdom Egypt  due to the rich corpus of Egyptian finds from a small temple dedicated to Hathor (Site 2004), recent excavations and the results of radiometric and archaeomagnetic analyses6 have revealed that this period of Egyptian hegemony marks only the beginning of the region’s most intensive period of metallurgical activity until the modern era. At the end of the Late Bronze Age, coincident with the collapse of other major and influential polities across the Eastern Mediterranean8—including the Cypriot copper industry (Alashiya) that had been the main supplier of copper throughout the Mediterranean—Egypt, weakened by internal struggles, withdrew from the region9, leaving the lucrative industry in the hands of the local nomadic society . The ensuing period of ca. 300 years of intense production serves as the backdrop for the research presented here. Following Egypt’s departure, operations continued within numerous scattered unwalled sites throughout the Timna Valley, while the copper mines in the Wadi Faynan copper ore district of the northern Aravah also reopened on a massive scale as part of the reorganization of production in the entire region by the local tribes12 , was assumed to have been gathered from the immediate vicinity of the sites. Two charcoal assemblages collected from Slag Mound S at Site 30 and Slag Mound 19 at Site 34 reflect the industrial fuel remains from this critical period of activity in Timna, and are able to elucidate the relationship between the ancient smelters and their desert environment, and how such an industry was, or perhaps was not, sustained. Consequently, this study may be able to explain why the lucrative large-scale metallurgical activities ceased.While the exact reason for the halt in metallurgical activities in the Aravah remains unclear—with the campaign of Hazael and the Arameans into Canaan20. Climatically, the Aravah Valley is the hottest and driest area in the entire Negev/south-western Jordan region and is classified as hyper-arid27. The mean annual rainfall in the Aravah ranges between 30 mm in the south near the Red Sea, to 50 mm in the north close to the Dead Sea  to the end of the Late Bronze Age, a sudden and pronounced dry event—the driest period during the Bronze and Iron Ages—occurred during the thirteenth century BCE and lasted roughly a century and a half. The Iron Age began (ca. 1100 BCE) with a brief moist interval43 during the Iron Age I and IIA followed by a moderate period through the Iron Age IIB-C45. It is assumed that the climate regime during the examined period was similar to that of today45.There are few available paleoclimate records for the northern desert regions of the southern Levant, where most paleoenvironmental studies are derived from locations either within or directly influenced by the Mediterranean climate system. The Timna Valley, part of the Saharo-Arabian Desert Belt, is mostly outside of the influence of this climatic system and over 100 km away from the Mediterranean vegetation zone  are the most characteristic trees of this desert environment, and are a keystone species of the ecosystem, as animal and plant species alike rely on them for food, nutrients, and shade49. Other salt- and heat-tolerant plant species, such as Calligonum comosum (fire bush), Retama raetam (white broom), Zygophyllum dumosum (bushy bean caper), Haloxylon persicum (white saxaul), and Salsola spp.  are found in the alluvial fans, sandy hills, and chalky marls, though these tend to be shrubs and bushes rather than large trees. Within the oases, in areas with fresh or brackish water and saline soils, Phoenix dactylifera  and Juncus arabicus (rushes) grow, while other halophytic species, such as Nitraria retusa (nitre bush) and Suaeda monoica (seepweed) are present in belts around the salt marshes and oases. Trees of Tamarix nilotica and T. aphylla (tamarisks) grow along the channel of the Wadi Aravah33.Currently the Aravah Valley is characterized by sparse Saharo-Arabian halophylic and xerophytic vegetation Fig. . In this2 in area, was first excavated by the Aravah Expedition in 1974 and 197650. The excavators identified three “Layers” of activity at the site: Layers III and II, earlier phases similarly characterized by smaller furnaces and iron-fluxed slag, and Layer I, typified by novel metallurgical technologies—such as larger furnaces and tuyères (blowpipe nozzles) and the use of a manganese flux—not found anywhere else in the Timna Valley50. In 2009, a section of the large, partially excavated slag mound  broken by layers of organic-rich material as well as sandy accumulations, possibly reflective of brief periods of abandonment—represents a significant portion of the occupational history of Site 30  Late Bronze/Egyptian New Kingdom paradigm established by the previous excavators.Site 30, a walled smelting camp ca. 4000 mhttps://www.tau.ac.il/~ebenyose/CTV/), was the extensive surveying and excavations of Site 34, the largest smelting camp in the valley. Located less than 250 m from Site 30, Site 34 is a low sandstone mesa, ca. 3 ha in area, whose steep cliffs provide limited access to the site. Originally termed the “Slaves’ Hill” by the first surveyors and excavators of the valley due to its restrictive access and fortification walls, which gave the impression of a prison camp51, the recent excavations have suggested the opposite: that the workers of the smelting camp were esteemed individuals fed choice cuts of meat and granted access to food and supplies coming from the distant Mediterranean region53, and were adorned in intricate textiles dyed using innovative and complicated chemical procedures requiring exotic plants and mollusks55. One large slag mound—Slag Mound 19—was excavated in 2013  on the basis of the deposition of the slag horizons, which suggest that over the course of the site’s ca. 100 years of activity, the arrangement and positions of the smelting-related activities surrounding the slag mound shifted somewhat, though the activities themselves remained consistent.One major focus of the renewed research on the Timna Valley, commenced in 2012 by the Central Timna Valley Project of Tel Aviv University (Retama raetam), which comprised 45.3% of all examined pieces, or 43.4% and 47.5% of the Site 30 and 34 assemblages, respectively. Acacia trees, (Acacia spp.) constituted the second-largest represented taxon, comprising 28.7% of the combined assemblages or 21% of the Site 30 assemblage and 39.7% of the Site 34 assemblage. These two taxa collectively account for three quarters of all examined samples. In addition, a large array of desert shrubs are reflected in smaller numbers. Amaranthaceae/Chenopodiaceae (goosefoots), a family of annual and perennial shrubs and sub-shrubs well-represented among halophytic and thermophilic flora, comprised the third-largest taxon if considering all examined samples; however, while they comprise 20.7% of the Site 30 assemblage, they are almost totally absent within the Site 34 assemblage (< 1%). Many of the species in this family have similar anatomical structures that make identification to the species or even genus level difficult if not impossible (e.g.56); nonetheless, an attempt was made at differentiation within the Site 30 goosefoot assemblage on a comparative basis with modern references , Mediterranean saltbush , Aellenia lancifolia-type (< 1%), shrubby seablight , eshnan , white saxaul , and anabasis . The wide variety of types witnessed within the goosefoot group suggests that either (as suggested) a variety of different species were used, and/or different parts  of plants of this group were used (e.g.57). Other desert shrubs included tally weed , nitre bush , fire bush , caper bushes , and bushy bean caper . Few taxa that grow in the oases or nearby water sources within the desert were also identified: tamarisks , Sodom’s apple , jujube  and date palm . Few species (each comprising < 1% of the assemblages) not presently encountered in the southern Aravah were identified as well: these include sumac  and the toothbrush tree , two desert elements still present in the Dead Sea region and, in the case of the toothbrush tree, along the Edomite Plateau, while sumac is still present in one stand in the southern Negev Desert. Phoenician juniper (Juniperus phoenicea), a coastal Mediterranean element with extensions into desert regions, was found at Site 30. Pistacia spp., pistacia trees, were also encountered, with three species-types within the Pistacia genus identified according to existing and observed precedents (and following the criteria of58): Atlantic pistacia (Pistacia atlantica) and khinjuk (P. khinjuk), Irano-Turanian relict trees identified at both Site 30 and Site 34, and the terebinth , a Mediterranean element identified at Site 30. Roots of certain taxa—in particular white broom—were also identified, as were unknown types , the latter amounting to 2.8% of the total assemblage, or 3.5% and 1.6% of the Site 30 and Site 34 assemblages. In addition, evidence of fungal hyphae were also noted in some samples  are able to be accounted for or lessened. Charcoals extracted from the slag mound reflect human choices, as opposed to natural accumulations; their association with metallurgical activities (versus domestic ones such as heating or cooking) is clear from the other archaeological signatures of the site and contents of not only the slag mounds but particularly those horizons probed within the slag mounds (e.g. the high levels of crushed slag and fragments of smelting furnaces). While it is possible that other activities are reflected within the assemblages, their contribution is likely minute in comparison to that of the industrial activities . Moreover, while reconstructions of the smelting furnaces are near impossible to reproduce, as they were destroyed following every smelting event in order to extract the copper produced, smelting was a relatively standardized process, at the end of which the remaining charcoal and metallurgical debris were dumped onto the slag mounds, creating, over time, successive layers of smelting events that sealed and secured earlier events for the archaeological record. Since their deposition, the dry environment and isolated nature of the site has likewise aided in lasting preservation. High fragmentation was most prevalent among the goosefoot members, as their secondary phloem creates natural weak points and cleavage planes, but the examination of samples as small as 5 mm was employed in order to compensate in part for this potential bias in results. The two assemblages, observed both in the higher resolution of the individual probes and the lower resolution of the site-wide phases or layers, allow us to interpret the results largely as an “average” of the industrial fuel preferences used over time.Given the context of the present assemblage within well-stratified and chronologically anchored slag mounds located in a marginal, arid and geographically restricted environment, many of the variables that may typically induce interpretative and taphonomic biases within an anthracological study (e.g.63) and the Principle of Least Effort (PLE), according to which resources are gathered from the immediate vicinity of the settlement or site, i.e. what is easiest and hence requiring the least effort to procure67. This means that, unlike certain foods and other goods that had been brought from afar to sustain the mining communities53, fuels were not being subsidized through long-distance trade (despite the significance of the copper trade itself). This further suggests that during the Iron Age, the vegetation of the southern Aravah consisted of a variety of desert and savannoid flora similar to what is witnessed today : acacia trees along with desert shrubs in the steppe zones and wadi beds, desert scrub and shrubs in the dune sands and reg, small halophytes  in the salinas , while closer to oases other varieties of larger trees  and date palms grew. A similar phenomenon was observed in the charcoal studies conducted at the Wadi Faynan copper smelting sites, where it was generally observed that the ancient smelters gathered their fuels from the immediate vicinity of the smelting sites74, though, unlike Timna, Faynan benefits from a greater abundance of water and vegetation due to its location at the base of the Edomite Plateau 80.Most woody taxa represented within the two assemblages largely reflect the vegetation present in the southern Aravah today. This is consistent with Site Catchment Analysis . The hot-burning charcoal of the white broom is even referenced in the Hebrew Bible , while the roots of this bush are also acknowledged (Job 30:4). It is therefore suggested that the ancient smelters were familiar with and understood the effects of the properties of these particular taxa, causing them to be preferential for smelting over the other taxa, which instead would have been used for other activities taking place within the larger mining community likewise requiring woody fuel, such as cooking, heating, lighting, ceramic production, etc. Evidence for such behavioral preferences has been observed since as early as the Mesolithic, such as at the site of Sibudu (77–65 ka YBP) in the KwaZulu-Natal region of South Africa, where the ancient inhabitants seem to have already been aware of the varieties of wood in the area and their different properties (particularly their burning temperatures vis-à-vis their wood densities), and selected their wood accordingly84, though such selective preferences may have begun even as early as the Paleolithic85. This may also explain the change in goosefoot representation within the Site 30 assemblage: while in Layer III, goosefoots make up 20% of the overall assemblage and are well-represented in every probe from this layer, in Layer II, their representation shrinks substantially to less than 2%. While Layer III begins prior to the activities at Site 34, once Site 34 becomes active—a period largely contemporaneous with Layer II of Site 30—the two assemblages more closely resemble each other. It is possible that the higher representation of these “lesser quality” taxa in the Site 30 assemblage is due to the site being much smaller and compacted than the more expansive Site 34, with activities besides smelting taking place in the immediate vicinity of Slag Mound S (as attested to by the ceramic kiln found adjacent to Slag Mound S50), while it is also possible that these taxa were used in the initial charges at the start of the smelting process. However, the near absence of this family in Site 34 (established as a dedicated and protected smelting camp) and their diminished representation in Layer II of Site 30 may reflect a shift in resource management beginning in the late eleventh century, in which the white broom and acacia  were specifically selected for smelting purposes. Meanwhile, the goosefoots may have been earmarked for other activities  or even saved for the grazing flocks that accompanied the metalworking communities.While on the one hand, the PLE suggests that resources are gathered from nearby the site, on the other hand, it also supposes that resources are gathered in direct proportion to their presence in the environment. What is witnessed within the slag mounds, however, is not this case. Instead, two taxa dominate the Site 30 and 34 assemblages—acacia trees and white broom bushes. At Site 34, where Slag Mound 19 reflects roughly a century . Many of the plant taxa in semi-arid and arid environments are tolerant of stresses related to salt, heat, and brief periods of drought, and the continued presence of the toothbrush tree into the Iron Age may be the product of plant resilience in a relatively stable ecological situation, i.e. one without the added pressures of human industrialisation.Of the desert elements identified within the assemblage, of particular note is the toothbrush tree, a Sudanian relict species characteristic of savannahs in the dry tropical regions of Africa34–1) where it makes up only about one third of examined samples, at which point it is subsidized by other taxa as well . In the past, however, juniper was a more prevalent element in the region, and though populations were once found in the Central Negev, where vegetation was denser, the most recent evidence of them in the Negev is dated to ca. 8000 cal. BC88. The disappearance of juniper in the region since the Early Holocene is most likely due to human pressure on the natural environment, further evidence of which can be found at Faynan. During the Early Bronze Age II-III (ca. 2900–2350 BCE), juniper was a major source of copper smelting fuel at Faynan, but is near absent in the Iron Age assemblages (e.g.69). This suggests that the stands presently upon the top of the Plateau may have once extended further down the slopes, and their populations may have been severely reduced by the Early Bronze Age activities coupled with the climate changes that have occurred since the Early Bronze. All three Pistacia-types are found in the vicinity of the Aravah Valley and, like the juniper, may have been better represented in the past, particularly along ravines and upon higher elevations where more water was available. Khinjuk, an Irano-Turanian tree, like the juniper, is totally absent from the Israeli flora, and in antiquity as well as today is found in the Sinai (including at the Iron Age II site of Kuntillet ‘Ajrud in the Eastern Sinai89) and along the Edomite Plateau90. The Atlantic pistacia, an Irano-Turanian element, is still found along the Edomite Plateau as well as throughout the Central Negev Highlands33. It is totally absent in the Aravah Valley and appears only in one stand in the southern Negev ca. 15 km north of Timna. The closest stands of terebinth, a characteristic tree of the Mediterranean zone, are on the higher elevations of the Edomite Plateau, with their furthest southward extensions found ca. 50 km south of Faynan. This is the only location where all four of the aforementioned species  are still found together. In the Iron Age, were any of these species still found in or around the immediate vicinity of the Timna Valley, they were likely already relict species represented by few individuals rather than entire stands or large populations.Irano-Turanian and Mediterranean trees which today are found only in areas of higher elevation and precipitation begin to appear at a similar time in both sites—around the middle of the tenth century and following. Today, Phoenician juniper is totally absent among the Israeli flora; the closest modern stands to Timna can be found in the northern Sinai and along the Edomite Plateau Fig. ;33. In t91. In the case of the Edomite Plateau, the cliffs rise to a precipitous height of ca. 1500 m asl. Though the enhanced elevation allows for the average precipitation to achieve ca. 350 mm per annum in some locations69—thus fostering a much greater array of vegetation—access to these locations is permitted by even fewer ascents than those entering into the Negev. Moreover, it is assumed that charcoal was produced specifically for the sake of metalworking in the vicinity of the smelting sites, rather than being transported as such. Though charcoal production locations are difficult to identify in the archaeological record and have not been found at Timna, charcoal burns hotter and retains its heat longer than fresh wood, and is also better for the chemical process  required for the separation of the metal from the parent mineral92, while ethnographic evidence further points to its preference as a smelting fuel among traditional societies (e.g.95). However, if charcoal is borne over long distances, there is a great risk of the fragile material being crushed into fines  that would stunt airflow within the smelting furnace, rendering it unsuitable for such a volatile dynamic system70. For this reason it is most likely that charcoal was produced nearby the smelting camps, while the wood was culled and transported to the charcoal production centers by groups of people dedicated to the task. For the non-local taxa, a great deal of effort would need to be invested in bringing them from distances of up to 100 km away across steep terrain, likely still as uncarbonized wood.With the exception of a few ascents, the journey into or out of the Aravah Valley from either the Negev Highlands to the west or the Edomite Plateau to the east is treacherous if not impossible. The Negev hills bounding the western margins of the Aravah rise ca. 500 m above the Aravah Valley; to find even a moderate ascent, one must leave the Timna Valley, while most major routes required travelling to the shores of the Red Sea—or at least Yotvata—before heading westward3, therefore would have put a large strain on the local environment. However, unlike the Faynan copper district in the northern Aravah, whose Iron Age industry was fueled largely by fast-growing tamarisks watered by the high water table and perennial streams of the region69, in Timna, vegetation would have been more scarce. Consequently, with no signs of stress in ore qualities from the earliest understood stages of copper production in the Chalcolithic on through the Early Islamic period96, fuel sources, coupled with the scarce availability of water, were a major limiting factor in the ongoing activities in the southern Aravah97. Rough calculations relying on experimental data (e.g. 98) suggest that 10 tons of wood (by weight) were required for the production of one ton of copper97,99:936–938, though estimations as high as 80 tons of wood for one ton of copper have also been suggested98:53,147. It was estimated that 30,000 tons of wood were required for the Iron Age Timna industry99:938, while the scale of wood used in Site 34 alone is estimated to be at least 3300 tons (if a low estimate of only 500 tons of slag are assumed for Site 34) for a period of about 100 years or somewhat less. Considering the ratios of acacia and white broom present within Slag Mound 19, and taking mean values for the biomass estimates of these plants (8.5 kg for white broom and 325 kg for acacia73), this would equate to at least 4100 acacia trees and over 185,000 white broom bushes for this single site.The geographic isolation of the Aravah made fuel procurement for the Timna copper mines a largely local endeavor. The sudden intensification in the production of copper beginning in the middle of the twelfth century BCE and peaking within the eleventh–tenth centuries BCE, as typified by Sites 30 and 34100. Species diversity increases as the number of individual trees increases while, conversely, the opposite is also true102: as the numbers of acacia go down, the number of plant taxa reliant on them will also decrease. This in turn will affect other aspects of the ecosystem, including soil quality and the diversity of faunal species that depend on the acacia and reliant species for forage, shade, and shelter49. Acacia are long-lived trees with infrequent periods of recruitment105, with many issues of decreased recruitment in modern contexts especially driven by anthropogenic actions109. Acacias have also been shown to be susceptible to changes in environmental and ecological conditions, and though in a natural situation the ability for mature specimens to recover following extensive droughts has been demonstrated (e.g.109), the acacia population in Israel has decreased by a third over the last several decades on account of unprecedented exploitation of underground water111.No taxa are better able to reflect the potential impact of such an environmental strain than the acacia trees, a keystone species within the desert ecosystem. Many forms of life, plant and otherwise, are dependent upon these species for their own survival112. Charcoaling is a major source of economic income for these groups, but because the delicate nature shared between the acacia trees and their ecosystems is recognized, the preservation of these trees for future generations is linked with their own preservation. Among the Kushmaan Bedouin clan of the Eastern Desert in Egypt, there is even a belief that ancient forests once grew throughout their desert landscape, but that they were destroyed (interestingly enough) by the overexploitation of acacia trees for the production of charcoal for metallurgical furnaces112: 99. In their own words, “[c]harcoaling is prohibited among us because without the trees, there are no animals, and no Arabs”112: 109. The Handandawa Beja clan of the Red Sea Hills have a similar understanding of the acacia’s role in upholding the life cycle: “[w]hen the last tree is gone, it is the end of the world”79: 41. Nonetheless, in some cases from recent history, the overexploitation of these trees for the sake of producing charcoal has been recorded; Stanley, for example, recounts how in the early nineteenth century, the vegetation in the Sinai had virtually disappeared because of the production of charcoal from the acacias, a situation likely further exacerbated by Muhammad Ali Pasha of Egypt when, in 1823, he compelled certain Bedouin to pay tribute in charcoal for an assault they perpetrated on the Mecca Caravan113: 25.For some modern traditional nomadic societies living in the Middle East and North Africa, such as certain groups among the Bedouin and the Beja, acacias are regarded with reverence and frequently protected and carefully managed3 , the difference in the charcoal assemblages between Faynan and Timna lies in the availability of other fuel sources in the former, in particular fast-regenerating hydrophilic plants such as tamarisk97, while in the southern Aravah there were no alternatives.The special attitude towards acacia witnessed among contemporary autochthonous nomadic tribes of the desert regions is probably reflected in the minimal use of acacia charcoal in early Iron Age Faynan. Though the contemporaneous copper production centers of Faynan and Timna were operated by the same emerging tribal kingdom114. While absolute quantification of roots versus stem and branch wood is difficult, their presence within the assemblages was nonetheless noted, pointing to the high demand for woody fuel in order to sustain the Timna copper industry. Though archaeological parallels for the use of roots are rare, one example can be found from Vallon du Fou in the south of France. Here, the large roots of heather—particularly tree heather (Erica arborea)—a small tree or shrub with very dense wood, were extensively dug up and used to produce charcoal since as early as the European Iron Age (eighth century BCE) through the Middle Ages (thirteenth century CE)115. Given the large amount of biomass provided by the root systems of white broom in particular, as well as the large representation of white broom within the assemblages, it is probable that a sizeable portion of the fuel assemblage in the Iron Age smelting sites of Timna derived from this part of the plant. Moreover, even if roots are left out of the equation, repeated cuttings of plants above the root line, such as in the case of excessive cropping, will severely reduce a plants vitality and ability to produce fresh growth116:171. Pruning for the sake of sustainability, as has been suggested at Faynan74, may have been an option for the relatively fast-growing white broom; however, in the case of acacia, the slow growing nature of this taxon makes this an unlikely scenario. In the case of most members of the Amaranthaceae family, pruning or coppicing would likewise not be an advantageous means of wood sourcing for industrial charcoal production, as many of their branches do not achieve a tremendous volume on their own and the tedium of selecting those branches would produce very little payoff. In this case, roots would be preferred; however, experiments conducted on Haloxylon salicornicum  suggested that uprooted  plants reproduce less than half the amount of fresh biomass compared to surface-cut plants after seven months117, highlighting the unsustainability of such a practice. It is also important to bear in mind that it takes several years for a tree (particularly acacia) to produce branches with dimensions suitable for fuel use, and excessive cropping will severely reduce the tree’s vitality and ability to produce fresh growth116: 171. Of further consideration is the fact that many of the species relied upon by the Iron Age smelters, including acacia and white broom, are also responsible for soil stability, plant recruitment, and halting land degradation118 and are significant sources of food and fodder for wild and shepherded animals119. Moreover, a great degree of water in arid environments is stored in the roots and stems of the desert vegetation, and by removing them, water is likewise removed from this system, further enhancing desertification rates121. It is also worth noting that in the numerous excursions and field surveys conducted by the authors over the past decade, only two small white broom bushes were encountered in the northern part of the Timna Valley, with no presence elsewhere within the area, raising the possibility that the white broom population has never recovered from the Iron Age overexploitation.Also of note in the Timna charcoal assemblage is the selection of roots for fuelwood. Comparison with reference samples collected from the research area suggests that roots were relied upon to some degree. Though stem and root wood bear similarities in their morphologies , variations nonetheless occur to some degree, primarily with regards to the appearance of pith and the size, shape and arrangement of vessels, rays and other cells3. This final period, which lasted into the early ninth century, is also characterized by a radical change in the fuels used: instead of acacia and white broom, the assemblage of the uppermost horizon (Layer I) is dominated by date palms (47.8%) and small bushes (46.9%). Today the nearest date palm stands can be found around the oases of ‘Evrona and Yotvata, both ca. 10–15 km away from the examined sites , a commensurately greater magnitude of wood would have been required to fuel the smelting furnaces. With the evidence provided by the charcoal collected from within two slag mounds excavated at these sites, it can now be understood that the wood for these purposes was sourced locally from within and around the southern Aravah Desert itself. Moreover, it is clear that the ancient smelters during this period were aware of the best woody fuels available to them in this otherwise scarce environment, as made evident by the high representation of white broom and acacia trees, two taxa producing dense and high-burning wood. The reliance on these species in particular, however, as well as the use of roots of these and other taxa, were likely to have had a detrimental effect on the delicate ecosystem of this extreme environment, potentially already exacerbated by the climate crisis that occurred at the end of the Late Bronze Age just prior to the period under study. The region would have quickly approached its carrying capacity with the  arrival of the mining community and their herds, whose sudden and intensive activities would have upset the equilibrium of the ecosystem. The overexploitation of the keystone acacia species, upon which much other plant and animal life depend, would have a ripple effect throughout the entire ecosystem, and together with the uprooting of other desert bushes, would significantly reduce the reproduction and recruitment of the desert flora, limit the available shade and fodder for desert fauna, decrease soil quality, remove much needed water from the system, and ultimately lead to an environmental collapse certainly within the Timna Valley microclimate if not beyond. Evidence for such destruction is implied by the later appearance of taxa (particularly juniper and pistacia trees) unsuited to the extreme conditions of the southern Aravah Desert, suggesting an increasing range of wood collection over time. By the late 10th-early ninth century BCE, a novel and more efficient smelting technology utilizing larger furnaces and requiring even more fuel was in use. In this final stage of the Iron Age industry, when smelting activities were further restricted to Site 30 only, the fuels selected for the furnaces also drastically change; choice fuels all but disappear and small desert bushes and palm trees become the main fuels used. Given that for much of the period examined , the selection of fuels had been based on what was preferred first and available second, the change in the selection suggests that what was most readily available—i.e., small bushes and palms that were likely to have grown in abundance only ca. 10–15 km away in the oases —was significantly limited contra what was available less than two centuries prior. The short-lived nature of this final stage of activity, coupled with the total halt of the lucrative copper industry in the Timna Valley for nearly a thousand years, suggests that the diminishing availability of fuel sources in the southern Aravah was a primary reason for the cessation of this industry that had been operating continuously for about four centuries prior.Near the end of the eleventh century BCE, the copper industry of the Timna Valley would enter its most intensive period of activity in antiquity. At this time, smelting activities, once dispersed throughout the valley, were consolidated within two newly walled sites—Sites 30 and 34—where thousands of tons of slag6, therefore allowing the probes to be correlated both within the established absolute chronologies as well as the relative horizontal stratigraphy for the duration of each slag mound’s use. Probes were made into horizons rich in both charcoal and slag  within the slag mounds and create a relative stratigraphy able to reflect changes within each mound’s respective occupational history. Charcoal samples were collected along with the surrounding sediments and materials, including large amounts of crushed slag . Unfortunately, deterioration of Slag Mound S since its original excavation has resulted in the destruction of the upper portion of the section, preventing sampling from certain contexts. In addition, all of the remaining Layer I material from Slag Mound S , including all of the charcoal found therein, was excavated at the time of probing due to its rapidly deteriorating state. An initial count of 500 examined charcoal samples per slag mound was sought for analysis, though this number was increased slightly to increase the resolution of certain probes and/or Layers/Phases. For Site 30, this number was further increased due to both the greater duration of time reflected within this slag mound as well as the desire to further clarify the various species “-types” encountered (see below). In total, 11 probes from Site 34 (representing 554 examined samples) and eight probes and Layer I from Site 30 (representing 650 examined samples) were used. Charcoal fragments were hand-picked from the probed material. No preference for charcoal of a certain size was employed during collection or analysis so as to lessen the possibility of a bias in the results, though samples were not typically larger than 1–2 cm, while those smaller than 5 mm were avoided as they are frequently too small for a determination.The examined charcoal assemblages derived from two series of dedicated probes made into the western sections of Slag Mound 19 at Site 34 and Slag Mound S at Site 30 in 2014 and 2015. The use of these sections was significant as they both served as the basis for the chronological reconstruction for their respective sites124. Specimens were cut using razor blades and examined along three observational axes—transverse, tangential, and radial—under oblique angled top-lighting using a stereoscopic Carl Zeiss SteREO Discovery.V20 epi-illuminated microscope at magnifications of up to 360 × and a Vega3 TESCAN Scanning Electron Microscope at the Laboratory of Archaeobotany and Ancient Environments, TAU. Characteristic anatomical features, such as vessels and their arrangements, size and arrangement of rays, and abundance and nature of parenchyma were noted and identification at the highest possible systematic level was determined mainly by comparison to the wood and charcoal reference collection of the southern Levant  as well as wood anatomy atlases130.Taxonomic determinations were made on the basis of ligneous cell structure followingAcacia and Pistacia genera, for both of which three “-types” were encountered , as well as the members of the Chenopodiaceae/Amaranthaceae family. Accordingly, besides the information provided in the abovementioned references and modern comparative samples, a directed anatomical investigation of southern Levantine Pistacia species58 was referenced for the identification of this group. For the Chenopodiaceae/Amaranthaceae group, identification followed125 and modern reference samples, with further publications57 being referenced. Acacia species-types were assigned according to125 and modern reference samples.For certain taxa, due primarily to interfamilial or intergeneric similarities and overlaps in characteristic features, determinations were not always able to be definitively assigned at the species level; nonetheless, in these cases, “-types” were assigned in order to ascribe a significant affinity in the anatomical structure towards similarly observed features in modern reference samples and publications. Significantly, this includes the For a complete enumeration of how species were generally assigned within this assemblage, please see the key in the Supplementary Information.Supplementary Information 4.


The common disease hallux valgus results in foot discomfort and dysfunction. Less soft tissue damage and faster wound healing have made minimally invasive surgery (MIS) more popular. However, little research has compared the fixation results of minimally invasive chevron-akin (MICA) osteotomy thus far. In this study, the clinical and radiographic results of MICA with first metatarsal single- or dual-screw fixation are being examined.A total of 107 feet of 103 patients with mild to moderate symptomatic hallux valgus treated MICA from January 2018 to June 2020 were retrospective evaluated, with at least 12-months follow-up. 51 patients underwent single-screw fixation procedures and 52 patients received dual-screw fixation procedures. Patients were assessed preoperatively and at the final follow-up with radiographic measurements  and clinical scores (american orthopaedic foot and ankle society (AOFAS) forefoot score, visual analog scale (VAS) and Manchester-Oxford Foot Questionnaire (MOxFQ) scores). The coughlin satisfaction scores were also obtained.P < 0.001). Regarding clinical outcomes, the AOFAS, VAS and MOxFQ in two categories also significantly improved postoperatively (P < 0.001). There was no obvious difference in the clinical and radiographic outcomes between the two groups . However, the single-screw fixation group showed significantly lower operation time and less number of intraoperative fluoroscopy (P < 0.001). No serious complications were observed in either group. The single-screw fixation technique saves at least $1,086 compared with the dual-screw group.Both groups showed significantly improved HVA, IMA and DMAA at the final follow-up (At the final follow-up, both the single- and dual-screw fixation groups had comparable good to excellent clinical and radiographic outcomes, as well as a similar incidence of complications. Additionally, the single-screw fixation group reduces overall surgical costs, number of intraoperative fluoroscopy and operational time. Hallux valgus is a common forefoot deformity that usually presents as an exostosis of the first metatarsophalangeal joint and an inversion of the first metatarsal, with a prevalence of approximately 23%–35.7% , causingThe minimally chevron-akin (MICA) osteotomy is currently the most commonly used technique for third-generation (3G) hallux valgus MIS. Drs. Redfern and Vernois popularized and improved this procedure in Europe in 2008 . CompareThe aims of this retrospective study were to compare the results of single- and dual-screw MICA for hallux valgus by evaluating the radiographic and clinical outcomes. Furthermore, a cost-effectiveness comparison between the two fixation types was also conducted.This was a retrospective comparative study carried from January 2018 to June 2020, 103 patients (107 feet) with mild to moderate hallux valgus that had been treated with MICA by the senior surgeon were included. The study was approved by the institutional review board of our institution, and written informed consent was obtained from all patients prior to the study.Two case series were reviewed and compared in this study. One series was that of a surgeon who performs MICA using the first metatarsal single-screw fixation technique. The second series was that of using the first metatarsal dual-screw fixation technique. The inclusion criteria were as follows: (1) 18 < age < 65; (2) 15° < Hallux valgus angle (HVA) ≤ 40°; (3) 9° < Intermetatarsal angle (IMA) ≤ 16° ; (4) PatThe patient is placed in a supine position with three-quarters of the lower leg extended at the end of the bed and the foot resting on the mini C-arm . A combination of ultrasound-guided local nerve block combined with laryngeal mask anaesthesia is used and a tourniquet is tied and padded on the thigh.A 5-mm incision was made at the first metatarsal head/neck junction, then a miniature periosteal stripper was used to separate the soft tissue. A low-speed, high-torsional minimally invasive power  was selected to reduce thermal damage to the skin and soft tissues, and a 2*8 mm milling drill was used to perform a V-shaped osteotomy toward the second metatarsal head under image control. After the osteotomy was finished, the first metatarsal head was displaced laterally and assisted reset using a specially designed minimally invasive tool. A 5-mm incision was made at the slightly distal end of the first tarsometatarsal joint, a guidewire was inserted obliquely through this incision under image control. The wire penetrates the medial and lateral cortices of the proximal end of the osteotomy to reach the lateral cortex of the metatarsal head. Then a 4.0 mm fully threaded hollow screw  is inserted under the guidance of the guidewire. In the dual-screw fixation group, the second screw is placed in the same manner and parallel to the distal end of the first screw A postoperative bandage is essential to maintain the position of the hallux and prevent chronic complications such as recurrence. A coil of bandage is placed between the flippers of the first and second toes, and another bandage is placed between the flippers of the 1st and 2nd toes through the ankle joint to hold the hallux in a neutral position for 6 weeks. Two weeks postoperative, the stitches are removed, and walking with walkers or forefoot decompression shoes is permitted. To avoid stiffness of the first metatarsophalangeal joint, full weight bearing is permitted if tolerated, and walking is encouraged. To progressively recover joint motion, light plantarflexion activities can be started. After 1 month, walking in sports shoes is permitted, followed by jogging after 3 months, and intense sports after four to 6 months. Patients are recommended to be reviewed and followed up at 6 weeks, 3 months, 6 months, 12 months and 24 months after surgery.The visual analogue scale (VAS), the american orthopaedic foot and ankle society (AOFAS) forefoot scoring system and Manchester-Oxford Foot Questionnaire (MOxFQ) scores were used preoperatively and postoperatively respectively , 12, witt-tests were performed between normally distributed data. The differences between the two groups of qualitative data such as sex and laterality were tested by chi-square test. Differences were indicated as statistically significant at P < 0.001.The Shapiro-Wilk method was applied to test for normality using SPSS 26.0 statistical software. Measures that conformed to a normal distribution were described as mean ± standard deviation (±s), and P < 0.001). The mean preoperative HVA, IMA and DMAA for the single-screw fixation MICA group were 33.98 ± 6.9°, 10.51 ± 3.0°and 13.99 ± 4.1°, respectively. At final follow-up, this improved to a mean HVA of 10.28 ± 5.11°, IMA of 4.72 ± 2.6° and DMAA of 4.79 ± 3.46° (P < 0.001). The mean preoperative HVA, IMA and DMAA for the dual-screw fixation MICA group were 34.15 ± 8.83°, 10.54 ± 3.0° and 16.16 ± 6.11°, respectively. At final follow-up, this improved to a mean HVA of 10.07 ± 5.18°, IMA of 3.9 ± 2.0° and DMAA of 5.73 ± 4.2° (P < 0.001). Both HVA, IMA and DMAA had comparable ultimate outcomes at the final follow-up when groups were compared . The mean duration of follow-up was 20.98 ± 5.14 months in single-screw fixation group and 21.44 ± 4.10 months in dual-screw fixation group. The comparison of the baseline characteristics of the two groups was statistically insignificant . The proP < 0.001). AOFAS scores of two groups rising from 56.49 ± 10.29 to 90.25 ± 6.15 and 54.94 ± 11.74 to 89.15 ± 8.58, respectively. In the single-screw fixation group, the VAS and MOxFQ score decreased from 5.75 ± 1.63 to 0.62 ± 0.56 and 50.09 ± 13.28 to 12.85 ± 8.28; in the dual-screw fixation group, it decreased from 5.20 ± 1.41 to 0.65 ± 0.55 and 51.94 ± 13.08 to 13.15 ± 11.03. Clinical outcomes between the two groups were not substantially different  and 3 in dual-screw fixation group (5.6%). Hallux valgus recurrence rates (5.7% and 3.7%) were comparable between groups receiving single or dual-screw fixation. Two patients in the single-screw fixation group (3.8%) and 3 patients in the dual-screw fixation group (5.6%) also reported numbness. One additional patient had persistent dysesthesias over the dorsomedial foot in dual-screw fixation group. One foot in the dual-screw fixation group had skin irritation which improved after screw removal. In neither group were any cases of avascular necrosis, nonunion, or dorsal malunion of the distal fragment described.P < 0.001).A cost analysis was also performed comparing the material costs of two different types of surgical fixation. The average cost of a screw was $1,085 and the single-screw fixation technique was found to be more cost-effective. the single-screw fixation group required an average of 15.42 ± 3.00 numer of intraoperative fluoroscopy and took 46.87 ± 5.23 min on average to complete, both of which were significantly less than the average for the dual-screw fixation group .The first metatarsal single-screw fixation group strategy had the lowest proportion of implants, which was a measure of the overall cost of the surgery, compared to dual- screw fication group, it is even more cost-effective than other approaches . MIS is This study has several limitations. To start, since the study only included mild to moderately patients, it is not impossible to draw conclusions about the effectiveness of treating severely patients. Second, our study population involves a small number of subjects. This was caused by a number of factors, e.g., patients who were excluded for missing follow-up appointments or having follow-up periods that were shorter than 2-years were deemed to have incomplete data sets. Third, our overall patient follow-up time is another drawback because this study only looked at short- to medium-term patient outcomes. As a result, we are unable to make any judgments on the long-term effects of this approach. At last, fully threaded hollow screws were used in this study. It is not clear whether other screws, such as double-headed compression screws, can also achieve stability. These restrictions should be considered in future study, and mitigating strategies should ideally be included.In addition to offering outcomes that are equivalent to those of dual-screw MICA for the first metatarsal, the single-screw technique also offers the benefit of lowering overall surgical costs, fluoroscopy number during surgery and operational time. We recommend single-screw MICA as a viable treatment option.


S1 genes of 160 IBV isolates was performed and revealed that QX-type (GI-19), TW-type (GI-7), 4/91-type (GI-13), HN08-type (GI-22),TC07-2-type (GVI-1), and LDT3-type (GI-28) exhibited IBV positive rates of 58.15, 25, 8.12, 1.86, 5.62, and 1.25%. In addition, recombination analyses revealed that the four newly IBV isolates presented different recombination patterns. The CK/CH/JS/YC10-3 isolate likely originated from recombination events between strain YX10 (QX-type) and strain TW2575-98 (TW-type), the pathogenicity of which was assessed, comparing it with strain GZ14 (TW-type) and strain CK/CH/GD/JR07-7 (QX-type). The complete S1 gene data from these isolates indicate that IBV has consistently evolved through genetic recombination or mutation, more likely changing the viral pathogenicity and leading to larger outbreaks in chick populations, in China.Avian coronavirus infectious bronchitis virus (IBV) is a respiratory pathogen of chickens, resulting in severe economic losses in the poultry industry. This study aimed to monitor and isolate the molecular identity of IBV in broiler flocks with respiratory symptoms in eight provinces of China. In total, 910 samples  from broiler flocks showed IBV positive rates of 17.6% (160/910) using PCR assay. Phylogenetic analysis of the complete  Infectiephritis , 3. IBV  GI-28) . QX-type-type GI-, 4/91-ty . QX-typ (GI-28) . The folIn this brief report, we analyzed the molecular characterization of 160 IBV isolates from eight provinces in China in 2020–2021. It provides detailed evidence of the severe prevalence of IBV in China, and they also provide a reminder that more scientific vaccine strategies are needed, including strain selection and cross-protection assessment to provide broad protection against different IBVs.The use of animals in this study was approved by the South China Agricultural University Committee for Animal Experiments [approval ID: SYXK (Guangdong) 2019-0136]. All study procedures and animal care activities were conducted following the national and institutional guidelines for the care and use of laboratory animals.AF093793.1). The reaction conditions were as follows: 50°C for 5 min, 95°C for 30 s, and 45 cycles of 95°C for 5 s and 60°C for 34 s on the Applied Biosystems™ 7500 . The positive judgment value for this assay is between 15 and 35 cycle thresholds (CT) per 2 μL RNA template. The sequences of primers and probes used were as follows: IBV-F (5′-CCTCTAAGGGCTTTTGAG-3′), IBV-R (5′-GTCACTGTCTATTGTATGTC-3′), and IBV-P (5′-FAMCACCACCAGAACCTGTCACCTC BHQ1-3′).Between Sept 2020 and July 2021, several chick flocks, having developed upper respiratory symptoms, including coughing, wheezing, rales, and nasal discharge, were suspected of being infected with IBV. A total of 910 oropharyngeal and cloacal mixed swab samples were collected from the suspected chick flocks from eight provinces in mainland China . The swab specimens were stored in 500 μL of 1 mM phosphate-buffered saline (PBS) containing 200 U/mL of penicillin and 200 μg/mL of streptomycin, and vortexed for 20 min to prepare the virus solution for testing. The total RNA was extracted by using the RaPure Viral RNA/DNA Kit . The viral RNA was measured by quantitative reverse transcription PCR (qRT-PCR) according to the following protocol  were collected and serially diluted 10-fold from 10−1 to 10−3 in PBS, which inoculate into 9-day-old SPF chicken embryonated eggs respectively with 0.2 mL by the allantoic cavity route. Infectious allantoic fluid was harvested from the embryos at 2–3 days post-inoculation and the viral RNA was determined as previously described. Next, specific primer pairs targeting the complete IBV S1 gene were designed for reverse transcription polymerase chain reaction (RT-PCR). The thermal cycling  parameters were as follows: 94°C for 1 min, and 30 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 1.5 min. The complete S1 gene, ~1,640 bp, was amplified using the PrimeScript™ RT-PCR Kit , and its products were separated on agarose gel and purified using a QIAquick Gel Extraction Kit . The purified PCR products were cloned into a pMD™19-T vector  and then sequenced by BGI Genomics Co., Ltd. The sequences of the primers used were as follows: S1-F (5′-AACTGAACAAAAGACCGACT-3′), S1-R (5′-CAAAACCTGCCATAACTAACA-3′)  embryonated eggs were purchased from Guangdong DHN Poultry and Egg Products Co. Ltd. . After the positive swab samples were vortexed shortly and centrifuged at 750 × AACA-3′) .S1 gene sequences, obtained from GenBank, were aligned by the MEGA (version 7.0) ClustalW algorithm and the Test Neighbor-Joining Tree for designing the IBV S1 phylogenetic tree. A total of 1,000 bootstraps were calculated. Finally, the tree was visualized using Interactive Tree Of Life (iTOL) web-based tool (http://itol.embl.de/). All of the reference complete IBV S1 sequences representing all currently recognized types and subtypes used in the present study are listed in the P-values (<10–20), were further investigated by similarity plot and bootscan analyses implemented in Simplot 3.5.1.The sequencing was performed using DNAStar Lasergene software . Additional publicly available complete 6 EID50 per 0.2 mL of GZ14 strains, CK/CH/GD/JR07-7 strains and CK/CH/JS/YC10-3 isolates respectively and used the same volume of sterile allantoic fluid for the control group. Postmortem was carried out 7 dpi, and the kidney and air sac lesions of the chickens were scored as previously described  and housed in the isolators and randomly divided into four groups. The GZ14 strain (TW-type) and strain CK/CH/GD/JR07-7 (QX-type) was isolated and saved in the laboratory. Subsequently, we inoculated the chicks intranasally with 10escribed –14 , p < 0.01 was considered a moderately significant difference (**), and p < 0.001 was considered a highly significant difference (***).The gross lesion scores of kidney and air arc were analyzed using the one-way ANOVA followed by Tukey's multiple comparison tests. A S1 gene revealed that QX-type, TW-type, 4/91-type, HN08-type, TC07-2-type, and LDT3-type exhibited IBV positive rates of 58.15, 25, 8.12, 1.86, 5.62, and 1.25%  and the minor parent strain TW2575-98 (TW-type), was used for the exposure tests.Postmortem, all of the birds exposed to strain GZ14 showed obvious lesions in the kidneys, in which they were mainly preformed nephritis, as well as different degrees of air sac turbidness. In contrast, the birds with strain CK/CH/GD/JR07-7 or the CK/CH/JS/YC10-3 isolate generally showed swollen kidneys and obviously thickened air sacs covered with yellow white caseous exudate. No clinical symptoms were observed in the chickens of the control group . In addiEscherichia coli, Staphylococcus aureus, Mycoplasma gallisepticum (MG), and Mycoplasma synoviae (MS); these pathogens can cause respiratory diseases independently, or together . However, GZ14 strain (TW-type) showed its damage mainly with acute nephritis.S1 gene will urge the corresponding S1 protein to mutate in IBV variants (S1 gene of these isolates definitively proved that IBV is continuously undergoing these processes, and simultaneity its biological characteristics has changed probably. In productive activities, chick flocks in China were received already the IBV H120, 4/91, or QX vaccine in 1-day-old. However, it also happened high positive rate of wild type virus infection. The caveat is that the recombination strain hardly shows weaker virulence than the classical strain, which is a significant challenge for the effectiveness of the vaccine. It is difficult for the commercial vaccine to make a difference in cross-protection against various IBVs.IBV, a single molecule RNA virus, can mutate frequently for complex mechanisms, leading to extensive antigenic variation . Recombivariants . Eventuavariants . In thisIt is particularly notable here that controlling IB in China remains a challenging task due to the co-circulation of numerous IBV genotypes and the increasing isolation of novel strains . TherefoThe original contributions presented in the study are included in the article/The animal study was reviewed and approved by South China Agricultural University.Conceptualization, project administration, and funding acquisition: QX. Methodology: HF and ZWu. Software, writing—original draft preparation, and formal analysis: HF. Validation: ZWu, ZXu, and JL. Investigation: ZWu and ZXu. Resources: ZWu. Data curation: HF and ZXi. Writing—review and editing: ZWu and ZWa. Visualization: XZ. All authors have read and agreed to the published version of the manuscript.ZWu, HF, Zxu, ZWa, and JQ were employed by Wen's Group Academy, Wen's Foodstuffs Group Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


It accepts reduced lipophilic quinones from various dehydrogenases. The enzyme catalyzes the transfer of an electron to cytochrome c from ubiquinol and, in this process, it also transfers protons across the gradient, which is used in the synthesis of ATP by ATP synthases. In plants, the complex cytochrome b6f carries out the equivalent function . ISP is a single-pass transmembrane protein, and the hydrophilic region of ISP contains the iron–sulfur (FES) cluster and is domain-swapped. As commonly observed in many complexes, the core subunits are surrounded by a number of supernumerary subunits in the eukaryotic complex III  and transfers one electron to heme c1 via 2Fe–2S, subsequently reducing cytochrome c. Ubiquinone at the Qi site is reduced by the second electron via heme bL and heme bH generating a semiquinone intermediate. This is accompanied by the translocation of two protons to the intermembrane space (IMS) from the Qo site. The semiquinone intermediate at the Qi site is further reduced by the binding of a second ubiquinol to the Qo site and uptake of protons from the mitochondrial matrix as well as two protons released from the Qo site to IMS. During the enzymatic process, the ISP undergoes a large structural change to occupy multiple positions and the two extreme positions are called b and c to denote the proximity to the cyt b and c1. The necessity for such movement in intramolecular electron transfer has been supported by mutagenesis as well as with inhibitors  and antimycin A (a Qi inhibitor) act as the reference structure, where the hydrophilic domains of ISP from both monomers are locked in one conformation. The consensus map from all the particles of different samples (except the atovaquone bound complex) yielded an overall resolution of ∼2 Å allowing the modelling of water molecules and lipids, and giving an atomistic view of complex III from this species of fungi [Figs. 1(a) and 1(b)In this issue of o site and cyt b. The typical positions occupied by ISP in the enzyme are shown in Fig. 1(c)o site in the b position and to heme c1 in the c position. Some observations include the lack of ISP in the b position in presence of decylubiquinol and antimycin A treated enzyme, more prominent density for quinone at the Qo site in reduced conditions (in the presence of ascorbate), ubiquinone observed in the Qo site and decylubiquinone in the Qi site (quinones in both the sites are not modelled/observed in the same map). Structural analysis shows that the redox state of the enzyme and the substrate/inhibitor bound in the Q sites play a critical role in the interaction of ISP with cyt b and cyt c1. Further, the data also show that two ISP domains in the dimer are independent (at least in the isolated complex from this species), and both symmetric and asymmetric molecules are observed.The cryoEM maps, as expected, revealed poor density for the ISP in many of these conditions, and using focused classification and signal subtraction during image processing, the populations of the enzyme with ISP in different positions in the complex could be separated. The maps of ISP in position b are of higher quality than those when it occupies the intermediate or c position, perhaps due to the interaction with the Qet al., 2021i.e. not just with incubation but with faster mixing of substrate and enzyme followed by freezing (Crofts, 2021The preparation of defined samples and the possibility to analyze the different conformations/populations of the enzyme is one of the major advantages of cryoEM. Although the large movement of ISP has been well documented in earlier studies (Sarewicz