EGR1 is implicated in the etiology of neuropsychiatric disorders, and is linked to gene pathways associated with social behavior. Despite extensive knowledge of EGR1 gene regulation at the molecular level, it remains unclear how EGR1 deficits might affect the social component of these disorders. Here, we examined the social behavior of zebrafish with a mutation in the homologous gene egr1. Mutant fish exhibited reduced social approach and orienting, whereas other sensorimotor behaviors were unaffected. On a molecular level, expression of the dopaminergic biosynthetic enzyme, tyrosine hydroxylase (TH), was strongly decreased in TH-positive neurons of the anterior parvocellular preoptic nucleus. These neurons are connected with basal forebrain (BF) neurons associated with social behavior. Chemogenetic ablation of around 30% of TH-positive neurons in this preoptic region reduced social attraction to a similar extent as the egr1 mutation. These results demonstrate the requirement of Egr1 and dopamine signaling during social interactions, and identify novel circuitry underlying this behavior.Finding the link between behaviors and their regulatory molecular pathways is a major obstacle in treating neuropsychiatric disorders. The immediate early gene (IEG)  Egr1 is an immediate early gene (IEG) linked to neuropsychiatric disorders, particularly those with social components. However, the role it plays in control of social behavior remains elusive. We found that zebrafish with a mutation in the egr1 gene present deficits in social approach and orienting behavior. Furthermore, egr1 mutants have reduced tyrosine hydroxylase (TH) expression, particularly in dopaminergic neurons of the preoptic area (POA) synaptically connected to neurons already identified within a social circuit. Our study provides evidence of a role for egr1 in establishing social behavior, and adds to our understanding of the underlying circuitry.Intraspecies interaction, or social behavior, is a widespread phenomenon among animals. It is considered to be beneficial for reasons of predator evasion, reproduction and cooperative foraging . Brain rEGR1. In fact, this gene has been evaluated as a biomarker for certain neuropsychiatric disorders  isorders . EGR1 hadisorder . In addider ASD; . EGR1 caof genes Egr1 in social behaviors. Social defeat stress, a common depression model, reduces Egr1 expression in the prefrontal cortex  as a response to recognition of social opportunity and ascension to dominance  project, contain at least one Egr1 binding motif in their promoter region  neurons. Chemogenetic ablation of about one third of the identified PPa neurons resulted in social behavior deficits, similar to the reduction observed in egr1 mutants. Our results suggest that egr1 is required for expression of th in a BF population of dopaminergic neurons necessary for normal social interactions, thereby expanding our understanding of the precise circuitry for social behavior.Development of robust social behavior was recently described in the zebrafish  and a reon assay . We findsa64egr1, y321Et  [Ety321], y405Et  [Ety405], and c264Tg(14xuas:nfsB-mCherry). The sa64egr1line, which was generated by ENU mutagenesis during the Sanger Zebrafish Mutation Project (ZMP), was outcrossed to AB/Tübingen for 7 generations before the analysis described. Genotyping was performed for the sa64egr1 allele using a Competitive Allele-Specific PCR SNP genotyping system  according to manufacturer’s protocols after behavioral assays and before processing for Western blotting and immunolabeling. Animals with unknown genotypes were excluded from experiments and analysis.All zebrafish embryos, larvae, and adults were raised and maintained at 28.5°C according to standard protocols with a 14/10 h light/dark cycle . Lines usa64egr1 heterozygotes (hets) were placed in paired, isolated tanks (50 mm in length × 20 mm in width × 20 mm in depth) separated by an opaque divider and allowed to habituate for 5 min, then the divider was removed and the animals allowed to interact for an additional 5 min. Both the presocial and social stimulus periods were recorded to determine the baseline exploratory and locomotor behavior. Recordings were obtained from below at 10 fps using a Mightex SME-B050-U camera and illuminated by an overhead white LED panel . For numbers of animals and their genotypes see Socially-motivated place preference and visually-evoked orienting behavior of 14 d postfertilization (dpf) zebrafish were measured using a split dyad assay . Roughlysa64egr1 hets were placed individually in watch glasses filled with facility water. The watch glasses were located in a shallow aquarium on translucent sheets, with a cold mirror and IR LEDs below. A projector, located to the side, projected images onto the translucent sheets by way of the cold mirror. A digital camera with an IR filter was located above the watch glasses. Projector and camera were controlled by bespoke software in Bonsai. Dots of varying sizes were projected intermittently, moving in a synthetic knot shape for a 5-min period. Fish were maintained for 2–3 h in the watch glasses. At each frame, animal and stimulus parameters were streamed to a text file for offline analysis. Fish were tested in facility water at room temperature (25°C). For numbers of animals and their genotypes see To explore social engagement, we tested social approach using a virtual social assay that is based on detection of biological motion . The stic264Tg(14xuas:nfsB-mCherry)] and [Ety405] fish were incubated in the dark for two nights in static facility water with 2.5 μm nifurpirinol , a nitroaromatic antibiotic that is converted into a toxic metabolite by the enzyme nitroreductase. Water flow was restored and larvae were fed during the day. As a control, sibling larvae only expressing GFP, and not the nitroreductase transgene, under the influence of the y405Et  enhancer trap, were analyzed in parallel. Water flow was restored for a minimum of 1 h before biological motion social assays. A total of 60 animals were tested from two experiments with 28 animals expressing GFP only and 32 animals expressing both GFP and nitroreductase.Fourteen days postfertilization larvae from a cross between  was recently identified (th1 and th2 genes in larvae and adults (A synaptic connection between TH-positive neurons and cholinergic BF neurons (cBFNs) defined by the enhancer trap line entified . Chemogeentified . Images entified E, includentified . TH-posientified , so we wd adults .sa64egr1mutants relative to wt siblings at 14 dpf , analogous to results from the Western blot experiments  line expresses in neurons in the PPa, and the neuropeptide galanin has been implicated in various mammalian social behaviors y405], in which the construct integrated in the robo2 gene, also drives expression in the PPa. In contrast to the galanin driver, ∼30% of neurons labeled by the y405Et  driver overlapped with TH-positive dPPaNs . Biological motion-based social behavior was significantly reduced in larvae with ablated dPPaNs compared with control animals at dot sizes 2.0, 4.0, 8.0, and 16.0  Egr1 is implicated in regulating social behavior, and (2) dPPaNs represent a novel population of neurons implicated in driving social behavior. We discuss these findings further below.Our findings using egr1 mutants appears to be relatively specific to social behavior, as it does not affect vision or general motility. This specificity may lie in the egr1 expression pattern. From the first day of development, egr1 is specifically expressed in the ventral telencephalon and diencephalon (egr1 expression, dPPaNs presumably synthesize and release less dopamine, reducing critical input to cBFNs and a putative social network in the BF, resulting in aberrant social behavior. Our finding is reminiscent of the loss of another IEG, FosB, in mammals. Most behaviors are largely the same in FosB knock-out mice, suggesting no ongoing deficit in neuronal function across most of the brain. However, nurturing of neonates is almost absent in knock-out dams, which was explained by deficient signaling in the POA (First, the behavioral deficit in cephalon . This excephalon . Interescephalon . Therefo the POA . This maegr1 mutation is consistent with the identification of EGR1 as a susceptibility gene for neuropsychiatric disorders including schizophrenia and depression (EGR1 is a hub for other genes involved in synaptic transmission that are affected in ASD (EGR1 mutations are severe or lethal, and that only other mutations or environmental influences that modulate EGR1 activity or expression levels result in neuropsychiatric symptoms. This postulated exclusive mode of EGR1 modulation via other effectors may be unique to humans, as the gene has been implicated in a human-only network of genes aberrant in ASD cases (Indeed, the effect we describe in zebrafish induced by the pression . Our resd in ASD . Althougd in ASD , it is iSD cases .egr1 mutants implicates a population of neurons, dPPaNs, and their connections in driving social behavior. The PPa region lies within the POA, an integral part of the conserved vertebrate social behavior network (y321 arbors suggests that the dPPaNs identified here are presynaptic to cBFNs (Second, our study of the molecular mechanisms underlying the social phenotype of  network . Consist network . Our resto cBFNs . It is yto cBFNs , implicaFinally, based on anatomic marker studies of the developing zebrafish brain, the TH-positive PPaNs are most probably dopaminergic . The put


Ammotragus lervia Pallas 1777) is an ungulate species, native to the mountain ranges of North Africa. In the second half of the twentieth century, it was successfully introduced in some European countries, mainly for hunting purposes, i.e. in Croatia, the Czech Republic, Italy, and Spain. We used neutral genetic markers, the mitochondrial DNA control region sequence and microsatellite loci, to characterize and compare genetic diversity and spatial pattern of genetic structure on different timeframes among all European aoudad populations. Four distinct control region haplotypes found in European aoudad populations indicate that the aoudad has been introduced in Europe from multiple genetic sources, with the population in the Sierra Espuña as the only population in which more than one haplotype was detected. The number of detected microsatellite alleles within all populations (< 3.61) and mean proportion of shared alleles within all analysed populations (< 0.55) indicates relatively low genetic variability, as expected for new populations funded by a small number of individuals. In STRUCTURE results with K = 2–4, Croatian and Czech populations cluster in the same genetic cluster, indicating joined origin. Among three populations from Spain, Almeria population shows as genetically distinct from others in results, while other Spanish populations diverge at K = 4. Maintenance of genetic diversity should be included in the management of populations to sustain their viability, specially for small Czech population with high proportion of shared alleles (0.85) and Croatian population that had the smallest estimated effective population size (Ne = 5.4).The aoudad ( Newly created populations are often established with relatively few individuals of unknown genetic background and therefore may be particularly susceptible to loss of genetic variation due to inbreeding and genetic drift4. Within such recently established populations the genetic diversity might decrease, whereas population differentiation might increase over time5. Reduced gene flow and the low level of genetic diversity can also enhance the rate of inbreeding, which is considered to reduce a populations’ adaptability to a changing environment. Homozygous recombination of deleterious recessive alleles become more likely, which may lead to inbreeding depression6.During the nineteenth and twentieth century, large number of non-native animal species have been widely introduced in Europe. One of the most important drivers for such introductions was hunting, with introductions aiming to create or improve hunting opportunities, especially when native game species had become scarce7. Consequently, both distribution and genetics of ungulate populations across Europe have been profoundly influenced by such introductions8. Among ungulates introduced into Europe, the aoudad  is one of the species that was successfully established in the wild beyond its natural range8, with established populations in Croatia, Czech Republic, Italy, and Spain9.Among all introduced groups of mammal species in Europe, ungulates stand out from the others with 73.5%, since this is one of the most important game groups in all European countries10. As a result of mostly poaching and habitat loss, population numbers of aoudad in its native range are declining12, classifying this species as Vulnerable in the IUCN Red List13. The aoudad is a generalist herbivore and extremely plastic in its utilization of available food resources14. It is a polygynous species with high reproductive rate. Large and strong individuals have high fitness and reproductive success15, increasing their potential to colonise different localities whenever conditions are appropriate11.The aoudad, or Barbary sheep, is native to the mountain ranges of North Africa. Small groups scattered irregularly on large territories from Mauritania in the west to the Red Sea in the east show a typical pattern of the distribution of the species in the wild16. The population in the Czech Republic was established following the escape of several individuals from the Plzeň Zoo in 1976, and it consisted of a few dozen animals17, while today this population is believed to be extinct . In Italy, one small population of about 20 individuals is known to be present at the Beigua Regional Park in the province of Savona (Liguria) since 200715. Previously, another population was present in the province of Varese (Lombardy), where a group of six individuals  escaped from a private enclosure in 1993 and established a small breeding population that reached the maximum size of about 20 individuals. This population has been eradicated by the personnel of the Province of Varese accordingly to a plan approved by the Italian National Institute for Environmental Protection and Research (ISPRA) since 2005 . In Sierra Espuña, Spain, 36 aoudads, from zoos in Germany and Morocco, were intentionally introduced by the regional administrations in 1970. This founding population reproduced with great success and naturally dispersed very rapidly from their release area to nearby montane enclaves. Its current population, estimated at around 2000 individuals, is still expanding19. In 1972 sixteen animals from Sierra Espuña were released into La Palma island, Canary Islands, and established successfully since then18.In Croatia, during 2002 a number of aoudads of unknown origin were illegally released in the southern Dinaric region (Mosor Mountain). Current data obtained by GPS tracking of aoudad movements showed that the population is limited to the Mosor Mountain range and it is highly unlikely that it will expand its range beyond that area20. There is no information on environmental impacts for the other non-native aoudad populations15.So far, there is no evidence of the negative impact of the aoudad on host ecosystems in mainland Spain, except for La Palma island, where they have critically affected the survival and diversity of native, endemic flora and caused high levels of soil erosion5, as adaptation in such species occurs mainly through selection on pre-existing genetic variation21. Since scientific literature about the genetic structure of European aoudad populations is scarce, it is thus important to study genetic diversity and structure of those populations to assess their sustainability. Unfortunately, no comprehensive genetic analyzes have yet been carried out on original aoudad populations from their native range, except for a few studies of specific populations . In addition, insight into the genetic structure and origin of European aoudad populations might contribute to the ex-situ conservation of the species that is threatened in its native range.When the introduction of species into new areas is human-mediated, the new populations are often founded by only a few individuals that are completely isolated from the source populations. In the case of the European aoudad populations, sudden and substantial reduction in effective population size during first introductions and lack of gene flow into the established populations are assumed to have led to the loss of genetic variation through genetic drift. Severe reductions in genetic diversity may limit viability and adaptive potential of introduced populations under environmental change23. We aimed to understand how current levels of genetic diversity and structuring vary among European aoudad populations that differ with regard to the time and source of introduction.Here we report the first attempt to characterize the genetic diversity and population genetic structure of all non-native European aoudad populations. We used neutral genetic markers, mitochondrial DNA (mtDNA) control region sequence and microsatellite loci, to characterize and compare genetic diversity and spatial pattern of genetic structure on different timeframes among study populations. While variability of maternally inherited and more conserved mtDNA control region reflect maternal lineages present in the founder individuals, microsatellites, due to their codominant nature and high variability can reflect more recent events that shaped current genetic structureBased on samples collected from all known locations in Europe, the objectives of this study were to characterize the patterns of neutral genetic structure of recently established non native populations of aoudad and gain insights into the number of maternal lineages of these populations.24. In the full sample, four mtDNA haplotypes . None of the samples collected in Italy were successfully sequenced, due to the low quality of DNA. Low quality of the DNA isolated from faecal samples is known to dramatically reduce the genotyping success of such samplesHaplotype Amle01 was the most frequent (70%) and the only one found in Croatian, Czech and La Palma samples. Samples from Almeria were monomorphic for Amle02 haplotype, therefore haplotype and nucleotide diversity was zero in these populations. Samples from Sierra Espuña comprised of three haplotypes  with haplotype diversity of 0.69 and nucleotide diversity of 0.06. Observed four haplotypes were defined by 59 variable sites of which 11 were parsimony informative. The least number of mutation (12) was present between haplotypes Amle02 and Amle03, while the haplotype Amle04 stood out as remarkably divergent from all others, with the maximum of 55 variable sites separating Amle04 and Amle02  to 9 (INRA040), with a mean number of alleles per locus of 5.07 (Supplementary Table f estimator ranged between − 0.029 (Almeria) to 0.070 (Sierra Espuña) with a mean positive value of 0.302 ± 0.057.Linkage disequilibrium was observed for: 14 pairs of loci in Croatia, four pairs in Sierra Espuña, and for one pair of loci in La Palma and Almeria. After applying FDR corrections, linkage disequilibrium was significant only for MM12/SR-CSRP12 and TGLA073/SR-CSRP12 in Croatia. The multilocus value of the Estimated effective population size was the smallest in Croatian population reflecting the small number of founding individuals, and the largest in Sierra Espuña.Mean proportion of shared alleles between all individuals within each population was: 0.56 in Sierra Espuña, 0.57 in La Palma, 0.66 in Croatia, 0.75 in Almeria and 0.85 in the Czech population.ST value was 0.296 (95% CI = 0.249–0.347). The lowest pairwise FST value was observed between populations from Czech and Sierra Espuña (FST = 0.109), while the highest value was found between Croatian and Almeria populations (FST = 0.459) .Global FAmong STRUCTURE runs, the highest ΔK value was observed for K = 3, followed by that for K = 4  between individuals from this population and the largest estimated effective population size of 36.25. There is no available data on subspecies/lineages of aoudads used in introductions to Sierra Espuña (or in preceding introductions to Frankfurt and Ain Sebad Zoos), nor published mtDNA sequences from original populations that would allow for identification of subspecies/lineages present in this population, but variability of detected haplotypes might reflect introductions from multiple distinct lineages. This is especially valid for haplotype Amle04, which differs by at least 46 mutations from other haplotypes detected in this population, indicating its origin from evolutionary distinct units. Reported numbers of mutations between haplotypes from same subspecies of ungulates are usually lower. For example, in the study of chamois (Rupicapra sp.)26, native European mountain ungulate, D-loop haplotypes from the same subspecies were up to 15 mutations apart. In its native range in North Africa, six aoudad subspecies have been described, based on their distribution and morphological differences in coat color and horns27. Derouiche et al.22 aimed to further evaluate the validity of the subspecies and determine the geographical limits of the valid ones using mitochondrial cytochrome b gene. Based on the haplotypes obtained, their results indicated a deep Mediterranean-Saharan genetic break in the species, suggesting the presence of two highly distinct evolutionary lineages.Sierra Espuña population is known to have originated from individuals from the Frankfurt Zoo in Germany and the Ain Sebad Zoo in Casablanca, Moroccoar was less than 3.61 in all analysed populations) and mean proportions of shared alleles within all populations (> 0.55) which confirm their poor genetic diversity. The theoretical expectations for the loss of allelic variation after bottlenecks are described by Nei et al.28 who showed that critical factors determining this loss are the size of founding population and the growth rate of a newly established population. They indicated that the average number of alleles per locus will be more sensitive to founding population size than heterozygosity. This is illustrated in most of sampled aoudad populations, in which allelic richness is substantially low , where it was attributed to isolation and poor management practices. Largest number of detected alleles and the highest observed heterozygosity in Sierra Espuña population is another indication of the highest genetic variability in this population, supporting the theory of multiple origins.All analyzed aoudad populations in Europe were only recently introduced, with relatively small number of founding individuals, so it is expected that their genetic diversity is low in comparison to the viable free mating populations of native ungulates. This is confirmed by the number of detected alleles within all populations  back in 1975; so that it presumably belongs to the Saharan aoudad subspecies, while the other ones analyzed in our study might be of admixed origin, based on the fact that it is a population formed by individuals coming from the Casablanca Zoo in Morocco , and from the Frankfurt Zoo, of unknown origin. In addition, Almeria population had the second highest mean proportion of shared alleles (0.75), lower only than the small Czech population.30. Implications of detected low genetic diversity on management of these populations can be discussed only after management goals are clearly defined. Commonly accepted practice is to increase genetic variability of introduced species by introducing more individuals with different genetic background. The effects of such practices were reviewed by Dlugosch and Parker31 who quantitatively summarized the genetic diversity data available at the time for 80 introduced or invasive species of animals, plants and fungi. Their review shows that multiple introductions had only small positive effects on diversity on average, disapproving the argument that multiple introductions have been critical in providing genetic rescue from severe and deleterious founder effects in most cases. According to this review, increases in genetic diversity caused by multiple introductions and/or gene flow occur after many decades, during which time the range expansion of the founder population is successful. Later introductions of genetically distinct individuals can increase genetic diversity through admixture, and the strength of this effect will depend on the reproductive rate of the individuals from different “waves” of introductions.Management of European aoudad populations varies between countries and the views change with new insights of their coexistence with native species and ecosystemsA. lervia individuals from Croatia , Italy  and Spain . These populations represent the overall distribution range of free-ranging aoudad populations in Europe. Also, we obtained samples from a captive population from the Czech Republic  and those of the Saharan aoudad, A. l. sahariensis, in Almeria, Spain; 36°49′48″N 2°24′29″W.From 2016 to 2018, we collected 92 samples of 17. Since the aoudad population in Italy is small and we were not able to collect any tissue samples, we collected seven fresh faeces samples. In Spain 47 tissue samples were collected from three locations: Sierra Espuña n = 17, La Palma n = 15, during regular hunting, and from captivity in Almeria n = 15. Geographic locations of the sampled populations are presented in Fig. In Croatia, 34 tissue samples were collected during regular hunting. In the Czech Republic, four blood samples were collected from live aoudad because they are protected and not huntedSampling was done according to the Ethical and Welfare Standards presented in the , Regulation on the Protection of Animals Used for Scientific Purposes , with the approval of the Bioethical Committee for the Protection and Welfare of Animals of the University of Zagreb Faculty of Agriculture (UR.BR. 251-71-29-02/19-21-1). All the research was done in accordance with arrive guidelines.32. For the fluorescent labeling of PCR fragments, we used the M13-tailed primer method33. Singleplex PCRs were performed in a final 8 µL reaction volumes consisting of 1 × GoTaq G2 Hot Start Colorless Master Mix (Promega), 0.2 µM of each reverse and M13 primer, 0.05 µM of forward primer and 1 µL of template DNA. Information about PCR hybridization temperatures of all loci and thermocycler programme is given in Beja-Pereira et al.32. PCR products were analyzed on an ABI 3730 Genetic Analyser with GeneScan 350 ROX internal size standard (Applied Biosystems) by Macrogen (South Korea). GeneMapper v3.2. (Applied Biosystems) was used for scoring alleles.Total genomic DNA from tissue samples was extracted using the Promega Wizard Genomic Purification Kit following the manufacturer’s instructions. From faeces samples, DNA extraction was done using a liquid handling robot (Hamilton Starlet) and optimization by Applied Biosystems MagMAX DNA extraction Kit. All individuals were genotyped using fifteen polymorphic microsatellite loci: BM143, BM302, BM415, BM1443, BM1818, ETH225, ILSTS030Q, INRA005, INRA040, MAF70, MB25, MM12, SR-CSRP12, SR-CSRP24, TGLA07334, following the PCR protocol given in Mereu et al.34. The PCR products were sequenced in forward direction using an ABI 3730 automated DNA sequencer (Applied Biosystems). Electropherograms were visually inspected using Applied Biosystems SEQSCAPE software and the sequences were trimmed to 767 bp which could be unequivocally called.Mitochondrial control region was amplified using tRNAPro and tRNAPhe primers35. The presence of tandem repeats in the 5′ part of the control region led to ambiguity in the position of the gaps and for this reason we excluded the ambiguous region from final sequences used in further analyses. A final alignment was obtained by merging the two trimmed fragments at positions 15,448–15,568 and 15,730–16,214 with respect to the GenBank sequence (accession number FJ207522) and consisted of 606 nucleotides. DnaSP v.6 was used to estimate number of haplotypes, haplotype (Hd) and nucleotide (π) diversity36. The evolutionary relationships between the haplotypes were analyzed by a median-joining (MJ) network37 which was constructed using PopART38. The parameter ε was set to zero (default) to obtain a sparse spanning network.The resulting sequences were aligned with two previously published sequences of the aoudad complete mitogenome (GenBank accession numbers FJ207522 and EF466060) using the ClustalW algorithm implemented in MEGA X39 was used to check amplified microsatellite genotypes for large allele dropout, scoring errors due to stuttering and the presence of null alleles. Null allele frequencies were then estimated for each locus and population using the expectation maximization algorithm40 implemented in FreeNA41. Polymorphism Information Content (PIC) was calculated for each microsatellite marker per population using CERVUS v.3.0.742. GENETIX v.4.0543 was used to estimate the mean number of alleles, the observed heterozygosity (Ho), expected hererozygosity (He) and f estimator of FIS44 per locus and population. Linkage disequilibrium was tested among all pairs of loci in each population using a permutation procedure (n = 1000) using GENETIX v.4.05. Departures from Hardy–Weinberg equilibrium (HWE) were tested for each population using the score tests for heterozygote deficiency implemented in GENEPOP v.4.7.245. Allelic richness [Nar(g)] and private allelic richness [Npar(g)] were estimated using a rarefaction procedure implemented in HP-RARE46, where g represents the minimum number of alleles observed at a locus in one of the populations . The minimum number of genes in analysed populations was 28 , so this was used as a basis for rarefraction.For all population based analyses we used only genotypes from populations with more than 10 samples . MICRO-CHECKER 2.2.347, as implemented in NEESTIMATOR 2.0148. As suggested by Waples and Do49, we excluded alleles with frequencies below 0.02 in order to avoid bias caused by rare alleles.We estimated effective population size (Ne) for each population using the linkage disequilibrium methodhttps://www.microsoft.com/en-ca/microsoft-365/excel).Finally, to estimate the genetic similarity of individuals within populations, we estimated the mean proportion of shared alleles between all individuals within each population using custom script in Microsoft Excel 2016 , with K ranging from 1 to 7, were carried out with 106 iterations after a burn-in period of 105 iterations. We considered no admixture model and uncorrelated allele frequencies. The most likely number of clusters (K) was estimated by estimation of the ΔK statistic that reflects the rate of change of log-likelihood values between sets of runs with successive K values, using STRUCTURE HARVESTER51. To account for label switching between results of different runs with same K, results from 10 runs with selected K were combined using CLUMPP v.1.1.252. Results were displayed graphically using DISTRUCT v.1.153 and modified with Microsoft PowerPoint 2016 .We estimated overall and pairwise genetic differentiation with the Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.


COI sequencing with two different primer sets reached an amplification success rate of 87.3 and 97.2%. The investigation revealed four major frauds (5.8%) and four minor ones (5.8%). Results highlighted a decrease in incorrect labeling in Italy from 32% to 11.6% over the last decade, although a recurrent involvement of “endangered” species sensu IUCN was still observed.The problem of fish traceability in processed products is still an important issue in food safety. Major attention is nowadays dedicated to consumer health and prevention of possible frauds regulated by national and international laws. For this reason, a technical approach is fundamental in revealing mislabeling at different levels. In particular, the use of genetic markers has been standardized and DNA barcoding is considered the gold-standard strategy to examine and prevent species substitution. Considering the richness of available DNA databases, it is nowadays possible to rapidly reach a reliable taxonomy at the species level. Among different approaches, an innovative method based on DNA mini barcoding has recently been proposed at an international level. Starting from this evidence, we herein illustrate an investigation dealing with the evolution of this topic in Italy over the last decade. The molecular analysis of 71 commercial fish samples based on mini- Pangasius, Salmo and Tilapia whose aquaculture exploitation makes them easy substitutes for wild species [The consumption of seafood products has increased all over the world during the last 50 years as demonstrated by data issued by the United Nations Food and Agriculture Organization (FAO) ,2 that e species .Precautionary measures are, therefore, necessary, particularly for products that are not visually recognizable at sight and are indistinguishable on a morphological basis after processing. Deliberate mislabeling and replacement of high-value species with cheaper ones is an Economically Motivated Adulteration (EMA) and is considered as fraud . In a reAlthough the European Union labeling law (EU Regulation No. 1379/2013) .COI) gene is the favorite sequence to act as a ‘‘barcode” to identify and delineate the animal lifeform [COI barcode [In recent years, the gold-standard strategy to examine and prevent species fraud has been DNA barcoding, a fast and cost-effective method for correct classification at a species level . The orilifeform . Althoug barcode ,12. Sinc barcode , and the barcode , the mos barcode ,16,17.COI mini-barcoding is, therefore, useful to assess the correct taxonomy in processed products. This is particularly the case with commercial fish, for which the correct preservation under stable refrigerated conditions is a major issue during fishing, transportation and distribution. In addition, fish processing for preservation is the major cause of DNA degradation. Consequently, molecular analyses to reveal species substitution may face DNA degradation limitations and, therefore, become biased by technical questions. The possibility of analyzing short DNA fragments such as COI mini-barcoding seems to solve this issue. In the field of ichthyology, the application of mini-barcoding is nowadays possible thanks to the availability of specific databases accounting for millions of COI sequences. To give a general idea, BOLD database reports cover more than 24,000 barcoded species among Actinopterygii and Elasmobranchii. On the other hand, it is noteworthy to observe that data redundancy may generate confusion or technical biases due to contradictory attribution within a genus level.The methodology refers to the analysis of short DNA fragments and it can be applied in different fields of systematics (from museum collection research to forensic applications). COI and CytB barcoding, besides the use of additional markers [COI mini-barcoding as an innovative methodology to substitute for classic barcoding. The research was focused on sampling and analyzing the same taxa purchased in the same department stores to assess the evolution of cryptic species mislabeling after a decade, also considering the application of new European regulations [COI mini-barcoding and the evolution of the mislabeling issue are herein discussed, also considering some critical ecological, taxonomic and commercial aspects that have been arisen by experts based on their judgments.Starting from past experience of the application of  markers , for the markers , a new iulations . The suiFresh and frozen commercial fish products were acquired in 10 different department stores located in the Emilia Romagna region during 2020 and 2021. The different department stores belonged to the major brands fully distributed over the entire country, with their own national fish provider. In this way, although executed locally, the research had national coverage.A small sample of the edible tissue of approximately 50 mg was collected and fixed in absolute ethanol under refrigerated conditions. Samples were stored at −20 °C to be processed within one week of purchasing. A total number of 71 specimens belonging to 27 putative seawater species were analyzed. The samples dataset was prepared, listing the declared names and areas of fishing. The entire dataset is reported in ® Genomic DNA Purification Kit . Purified DNA was evaluated by means of 1% agarose gel electrophoresis. No samples were discharged due to bad DNA quality although some of them were considered borderline (as outlined in the Results section).Genomic DNA was extracted and purified from about 10 mg of ethanol-preserved samples using the WizardCOI gene was amplified through polymerase chain reaction (PCR) amplification using a Bio-Rad T100 Thermal Cycler. The cycling conditions were 95 °C for 10 min, followed by 34 cycles of 95 °C for 45 s, 57 °C for 45 s, 72 °C for 45 s and a final step at 72 °C for 10 min.Two different sets of primers were tested. The first pair of universal primers, Fish_miniFW: ATCACAAAGACATTGGCACCCT and Fish_miniRV: AATGAAGGGGGGAGGAGTCAGAA, specifically proposed by Sultana et al.  for fishThe second set of primers was tested following the protocol suggested by Shokralla et al. . Amplico2+ 1.5 mM, dNTPs 0.2 mM and 10 pmol of each primer—was used.The chemical conditions for both approaches were the following: a reaction volume of 20 µL—containing 1 U of GoTaq Polymerase , MgCOI sequencing of both amplified regions was performed by the MACROGEN Europe service . Twenty percent of the samples were locally reanalyzed using a CEQ8000 DNA Analysis System  based on capillary electrophoresis. The analytical conditions are reported in detail in Filonzi et al. [Amplicons were separated by 2.5% agarose gel electrophoresis and purified using the Qiagen MinElute PCR Purification Kit. The quality of the purified sample (1 μL) was visualized in 1.5% agarose gel that yielded clear bands. DNA quantity was evaluated using the Qubit dsDNA HS (High Sensitivity) Assay Kit  with a Qubit 3.0 Fluorometer . i et al. .The obtained sequences were manually corrected using MEGA 7.0  and compData concerning 71 analyzed samples are reported in The sequencing results and their comparison with available genomic databases allowed the recognition and identification of various fish species present in the analyzed commercial fish products. The obtained identity scores ranged between 96.5 and 100% in BLAST and 97.1 and 100% in BOLD. Mismatches between BLAST and BOLD were appropriately corrected by integrating either result. In fact, according to the 98% threshold value for species identification , three sKatsowomus pelamis (MB19 and MB25) turned out to be either Thunnus thynnus (GenBank) or Thunnus maccoyii (BOLD). Similarly, Chelidonichtys lucerna (BAR38) was found to be Merluccius paradoxus (GenBank and BOLD), and Lamna nasus (MB44) was Ixurus oxyrinchus (GenBank and BOLD). Concerning minor frauds, the four labeled species displayed lower identity percentages in BLAST and BOLD within the same genus: MB07 (respectively 97.1% and 99.1%), MB20 (96.4% and 97.5%), MB55 (97.1% for both databases), MB68 (97.5% and 97.1%).Among the 69 samples successfully attributed, a total number of eight species substitutions were detected (11.6%). In particular, four major frauds  related to mislabeling at the genus level (5.8%) and four minor  ones concerning different species within the same genus were detected (5.8%). The single names and substitutions are fully described in Discrepancies between GenBank and BOLD were also evaluated and were evident in eight samples  (11.6%).Interestingly, among the eight species substitutions, application of the IUCN index  revealedCOI fragments proved to be a useful methodology to define the correct taxonomy of commercial fish products. Among previously proposed primer sets, a choice was made based on the most reliable ones [The mini-barcoding approach based on analysis of short ble ones ,17, and ble ones , which wble ones , and thuCOI, the need for a continuous update of databases is important to reach a wide coverage of different populations over a large geographic scale.Although new markers are emerging for charismatic fish species and add technological improvements to this topic ,25,26, tKatsuwonus pelamis. BLAST and BOLD analyses returned two different Bluefin tuna species (Thunnus thynnus and T. maccoyii), therefore, revealing a mislabeled sample. Expert judgment was important in assigning the sample to Southern Bluefin Tuna rather than to Atlantic Bluefin Tuna according to both the identity score and the area of origin, the former species being a Pacific taxon consistent with the declared fishing zone.In this research, the intervention of expert judgment was, therefore, necessary to evaluate genetic results that had to be integrated with sampling information useful to determine species-specific ecological characteristics . To better clarify this concept, that was the case of sample MB19, which was labeled as Pacific Chelidonichtys lucerna and Merluccius paradoxus have completely different meat colors and morphologies, even after processing, in their fillet appearance. Fillets of both species were also close to each other in the exposition counter. The product was classified anyway as a major fraud since the final delivery still belongs to the entire traceability process.Similarly, sample MB38 was not properly considered a fraud but rather hypothesized as an involuntary substitution that happened during delivery at the moment of purchasing. In fact, From a general point of view, the research evidenced 5.8% major frauds and 5.8% minor ones. Major and minor frauds were considered in relation to the taxonomic level of erroneous labeling. A final percentage of 11.6% of species substitutions were, therefore, discovered. It must be remarked that data are greatly variable on a world scale. As a matter of comparison, mislabeling was detected in 9.3% of seafood products in Germany , 24% in Besides the generalized assessment of multiple specimens, the experimental design considered a long time comparison starting from the previous work by Filonzi et al.  see Fig. Our preNonetheless, another major issue is the illegal sacrifice and trade of endangered species widely protected by an international fishing ban. Fraudulent substitutions seem to continue, particularly in sharks and tunas, despite apparently increased control since the first published papers focusing on the problem ,33. Intesensu IUCN whose involvement in mislabeling is recurrent and suggests still existing inappropriate control processes at different levels.Mini-barcoding is a valuable tool to assess seafood species substitution, particularly in the case of processed products, displaying a high success rate. Despite DNA degradation, which may limit its diagnostic reliability, the majority of samples were correctly classified using BOLD and BLAST databases when supported by expert evaluation to solve cryptic nomenclature cases or apparent database redundancies. Although a general decreasing trend in fish species substitutions over the last decade was observed, consistent with similar trends in other European countries, a continuous update of datasets is important to reach a wide coverage of different species and populations. In particular, special attention should be focused on critically endangered species


Introduction: Maternal tobacco smoking during pregnancy is a global health problem leading to an increased risk for fetal and neonatal morbidities. So far, there are no data of the potential impact of maternal smoking during pregnancy on the most vulnerable period after birth – the immediate postnatal transition. The aim of the present study was therefore, to compare cerebral oxygenation during immediate postnatal transition in term neonates with and without prenatal tobacco exposure.Methods: Included in this post-hoc analysis were healthy term neonates, with measurements of cerebral oxygenation (INVOS 5100C) during the first 15 min after birth, and for whom information on maternal smoking behavior during pregnancy was available. Neonates with prenatal tobacco exposure (smoking group) were matched 1:1 according to gestational age (±1 week), birth weight (±100 grams) and hematocrit (±5 %) to neonates without (non-smoking group). Cerebral regional tissue oxygen saturation (crSO2), cerebral fractional tissue oxygen extraction (cFTOE), arterial oxygen saturation (SpO2) and heart rate (HR) within the first 15 min after birth were compared between the two groups.Results: Twelve neonates in the smoking group with a median (IQR) gestational age of 39.1 (38.8–39.3) weeks and a birth weight of 3,155  grams were compared to 12 neonates in the non-smoking group with 39.1 (38.7–39.2) weeks and 3,134  grams. In the smoking group, crSO2 was significantly lower and cFTOE significantly higher until min 5 after birth. HR was significantly higher in the smoking group in min 3 after birth. Beyond this period, there were no significant differences between the two groups.Conclusion: Cerebral oxygenation within the first 5 min after birth was compromised in neonates with prenatal tobacco exposure. This observation suggests a higher risk for cerebral hypoxia immediately after birth due to fetal tobacco exposure. Tobacco smoking during pregnancy is a global health problem with a prevalence of 1.7%, whereby the highest prevalence of 8.1% is seen in Europe. Regrettably, 30.6% of European women, who smoke daily before pregnancy, continue smoking during pregnancy , which enables continuous and non-invasive monitoring of cerebral regional tissue oxygen saturation (crSO2) and cerebral fractional tissue oxygen extraction (cFTOE) by measuring oxygenated (HbO2) and deoxygenated hemoglobin (Hb) –15. WhenPrenatal tobacco exposure influences cerebral oxygenation during the first 2 days after birth in preterm neonates, who show lower crSO2 and higher cFTOE compared to neonates without prenatal tobacco exposure . A prospHowever, so far no study has investigated the potential influence of maternal smoking during pregnancy on neonates during the most vulnerable period after birth—the immediate postnatal transition.The aim of the present study was to evaluate the potential influence of prenatal tobacco exposure on cerebral oxygenation in clinically stable term neonates during the immediate neonatal transition after birth. Therefore, cerebral oxygenation of neonates from mothers who had smoked during pregnancy were compared to those from mothers who had not smoked during pregnancy. Based on existing studies, we hypothesized that cerebral oxygenation in neonates from mothers who had smoked during pregnancy would be compromised.post-hoc analysis of secondary outcome parameters of a prospective observational study  or intubation], to eliminate any influence on cerebral oxygenation.The neonates were stratified into two groups according to maternal smoking during pregnancy: neonates with maternal smoking were assigned to the “smoking group,” and neonates from mothers who had not smoked during pregnancy to the “non-smoking group.” Neonates in the smoking group were matched for gestational age ± 1 week and birth weight ± 100 g to healthy term neonates in the non-smoking group. In addition, groups were matched for hematocrit ± 5% to rule out a potential influence of hemoglobin on cerebral oxygenation.Information on maternal smoking behavior during pregnancy including number of daily smoked cigarettes, maternal characteristics, antepartum medical history, and neonatal demographic data including gestational age, birth weight, sex, umbilical artery pH, Apgar scores and results of the first, routinely performed blood gas analysis were documented.The neonates were delivered by planed Cesarean section, without labor and cord clamping was routinely delayed for 30 s after birth. Immediately after the cord was clamped the neonates were placed under an overhead heater in supine position dried and wrapped in warm towels.2 and arterial oxygen saturation (SpO2) using the following formula: cFTOE = (SpO2-crSO2)/SpO2  and a neonatal transducer. cFTOE was calculated out of crSOO2)/SpO2 . Accordihan SpO2 .2 and heart rate (HR) were measured with pulse oximetry, applied on the neonates' right hand or wrist, using the IntelliVue MP30 monitor .SpO2 SpO2 and HR were stored every second.All variables were recorded continuously during the first 15 min after birth and were stored in a multichannel system  for subsequent analyses. Values of crSOUsing a pneumatic cuff on the neonates' right upper arm, non-invasive blood pressure (MABP) was measured once in minute 15 after birth. The IntelliVue MP50 monitor  was used for this purpose. Rectal body temperature was also measured once in minute 15 after birth.2) and partial carbon dioxide pressure (pCO2) were measured by blood gas analysis from a capillary sample within 25 min after birth. Samples were obtained by a heel-stick only at the discretion of the attending neonatologist. The analysis was performed according to local standard operating procedures with a blood gas analyzer .Hematocrit, hemoglobin and fetal hemoglobin (HbF) values as well as pH, bicarbonate, base excess, partial oxygen pressure  are shown. A p-value of p < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS 26.0 .Data are presented as mean and standard deviation (SD) or median and interquartile range (IQR) for continuous data and absolute and relative frequency for categorical data, respectively. Baseline differences between groups were analyzed using Eligible were 158 term neonates, who were included in the prospective observational NIRS study. Eighteen neonates were stratified to the smoking group and 140 to the non-smoking group. Two neonates in the smoking group and 25 neonates in the non-smoking group were excluded due to the need for medical support, including supplementary oxygen and/or respiratory support. None of the neonates in the smoking group and three neonates in the non-smoking group were excluded as no NIRS measurements were available within the first 15 min after birth. Three neonates in the smoking group and 29 neonates in the non-smoking group had no blood gas analysis performed within the first 25 min after birth and were therefore excluded. Furthermore, one neonate in the smoking group and 71 neonates in the non-smoking group were excluded, as no matching according to gestational age, birth weight and hematocrit was possible. Finally, 12 neonates in the smoking group and 12 neonates in the non-smoking group fulfilled inclusion and matching criteria .p = 0.743] and parturition . Mothers in the smoking group had a body-mass index of 31.8 ± 8.4 and in the non-smoking group 28.3 ± 6.0; p = 0.387. In both groups, there were no severe preexisting maternal morbidities. Except for medication treating hypothyroidism , the mothers had no additional medications. The mothers in the smoking group reported to smoke median (IQR) 8  years in the smoking group and 31.0 26.6–34.2) years in the non-smoking group; .2 years  (IQR) 8  cigarettn = 7), breech position (n = 3), and maternal request (n = 2) in the smoking group and previous Cesarean section (n = 6), breech position (n = 3), maternal request (n = 2), and preeclampsia (n = 1) in the non-smoking group. Anesthesia during Cesarean section was in all cases epidural anesthesia, except of one mother in the smoking group, who received general anesthesia.Indications for the Cesarean sections were previous Cesarean section  and cFTOE was significantly higher until min 5 after birth  in the smoking group compared to the non-smoking group. SpO2 and HR during the first 15 min after birth are demonstrated in The courses of crSO2 and cFTOE during the first 15 min after birth are demonstrated for the two groups in To the best of our knowledge, this study is the first investigating a potential influence of maternal smoking during pregnancy on cerebral oxygenation during the immediate neonatal transition after birth in healthy term neonates. We found that cerebral oxygenation within the first 5 min after birth was lower in infants whose mothers had smoked during pregnancy compared to those of the non-smoking group. These results support our hypothesis that maternal smoking during pregnancy is associated with compromised cerebral oxygenation immediately after birth.In neonates from mothers who had smoked during pregnancy, polyglobulia  and highin utero. Nicotine moderates the release of catecholamines from adrenal glands and nerve cells, leading to vasoconstriction in placental and umbilical vessels (Cerebral oxygenation immediately after birth in those neonates from mothers who had smoked during pregnancy may display the fetal conditions  vessels , 41. Tob vessels . This mi vessels . HoweverHR was significantly higher in min 3 and a trend toward higher values in min 4 after birth in the smoking group was observed in the present study. This is in accordance with reports of increased maternal and fetal HR due to tobacco exposure , 43. An An increase in cerebral blood flow velocity between 20 and 42 h after birth has already been described in newborn infants from mothers who had smoked during pregnancy , 44. ThiWe have some limitations to acknowledge. First, the number of cigarettes smoked during pregnancy may be underreported, since they were self-reported by the mothers using a questionnaire; moreover, some mothers in the non-smoking group could have neglected smoking during pregnancy. We did not perform measurements of nicotine levels in hair or urine, which would have informed us on the actual tobacco consumption. Second, background factors that might be associated with smoking were not collected/documented and may have had an impact on the reported results. Third, the number of included neonates in this study is small, therefore interpretation of these results has to be performed with caution. However, the main reason for the small sample size was the strict matching criteria to obtain comparable groups and even in this small cohort the results were not only significantly different, but also differences between groups were of clinical relevance. Matching according to birth weight was performed to rule out any potential influence of fetal growth restriction due to smoking on cerebral oxygenation. Therefore, the matching allowed us to achieve homogeneity in birth weight between the two groups.Immediately after birth, cerebral oxygenation was compromised with lower crSO2 and higher cFTOE values in stable term neonates delivered by Cesarean section from mothers who had smoked during pregnancy, compared to neonates without prenatal tobacco exposure. We assume that in the included stable term neonates, with an inconspicuous postnatal transition period, the observed lower cerebral oxygenation in the smoking group might be of questionable clinical relevance. However, our results suggest that neonates with prenatal tobacco exposure are at a higher risk of cerebral hypoxia immediately after birth. This could be even more pronounced in compromised term neonates and/or preterm neonates potentially causing cerebral injury. This observation should be implemented in the education about the harmful effects of tobacco smoking especially for women of childbearing age and those being pregnant.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by Regional Committee on Biomedical Research Ethics, Medical University of Graz. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.CW and GP conceived the research idea and finalized the methods. CW, GP, and AA analyzed the data and CW wrote the first draft. CW, MB, BS, LM, EP, NH, BU, and GP contributed to data collection, interpretation of the results, and drafting and finalizing the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


Thoracic trauma has decisive influence on the outcome of multiply-injured patients and is often associated with clavicle fractures. The affected patients are prone to lung dysfunction and multiple organ failure. A multi-center, retrospective analysis of patient records documented in the TraumaRegister DGU was performed to assess the influence of surgical stabilization of clavicle fractures in patients with thoracic trauma. A total of 3,209 patients were included in the analysis. In 1362 patients (42%) the clavicle fracture was treated operatively after 7.1 ± 5.3 days. Surgically treated patients had a significant reduction in lung failure , multiple organ failure , intubation time  and length of hospital stay  compared to non-operative treatment. Moreover, surgical fixation of the clavicle within five days following hospital admission significantly reduced the rates of lung failure , multiple organ failure  and length of hospital stay . Based on our results, multiply-injured patients with thoracic trauma and concomitant clavicle fracture may benefit significantly from surgical stabilization of a clavicle fracture, especially when surgery is performed within the first five days after hospital admission. Early detection of life-threatening injuries and administration of optimal emergency care  are essential to achieve the best possible outcome4. However, late or inadequate treatment of thoracic trauma increases the risk of developing severe complications such as respiratory failure, ARDS, long-term morbidity, and higher mortality rates7.More than 50% of all multiply-injured patients with an injury severity score ≥ 16 suffer from thoracic trauma8. In multiply-injured patients sustaining a clavicle fracture concomitant thoracic injuries occur in 77%9. Therefore, the clavicle can be considered the gatekeeper of the thorax11.A reliable indicator for underlying thoracic injuries is a clavicle fracture which is easily diagnosed during the primary survey in early trauma patient care14. Since clavicular injuries represent a severe loss of integrity of the chest and upper quarter of the body, disorders in breathing mechanics can occur15. This might be attributed to either direct functional deficiency or as a consequence of associated pain15, especially since the breathing mechanism does not only consist of the chest wall itself and the diaphragm. An intact and stable clavicle, as the origin of the accessory inspiratory muscles, is a crucial prerequisite for effective respiration and oxygenation17.Despite advances in ventilatory management like lung-protective, non-invasive ventilation protocols, patients with thoracic trauma often require prolonged ventilatory support and protracted ICU and hospital staysTherefore, the purpose of this study was to investigate the influence of surgical stabilization of clavicular fractures in multiply-injured patients with thoracic trauma on lung failure, multiple organ failure, sepsis rate, intubation time, length of intensive care unit stay and length of hospital stay. We hypothesized that osteosynthesis of the clavicle is associated with reduced complication rates and has an influence on ICU and hospital stay.The initial database contained 145,518 patients. After applying inclusion and exclusion criteria a total of 3,209 multiply-injured patients with thoracic trauma and concomitant clavicle fracture remained for analysis , had a mean age of 49.0 ± 19.3 years and a mean ISS of 22.9 ± 8.8. In 1362 patients (42.0%) the clavicular fracture was treated operatively after 7.1 ± 5.3 days. 19.1% had an AISThe majority of patients sustained their injuries during road traffic accidents, mainly riding motorcycles 26.9%) and cars (24.2%). Figure 6.9% and Most patients (83.2%) were treated at a supra-regional level I trauma center, 15.1% at a regional level II center and 1.7% at a local level III trauma center.In level I and II centers patients received non-operative treatment in the majority of cases , whereas in level III trauma centers operative therapy was chosen more frequently (55.6%), as shown in Fig. Overall, non-operative therapy was preferred especially in patients under 20 and over 60 years of age Fig. .Figure 419.3% of finally analyzed patients suffered from subsequent lung failure, 19.7% from multiple organ failure and 6.4% sustained sepsis. For all included patients, mean time spent on ICU was 9.4 ± 11.7 days, mean intubation time 7.4 ± 11.4 days and mean hospital stay 22.4 ± 18.0 days.Patients with operative treated clavicle fractures also seemed to have lower rates of lung, multiple organ failure and sepsis and spent less time intubated in ICU as well as in hospital Table .Table 2OWhether these differences were statistically significant and attributable to operative treatment we sought to examine with the following logistic regression analysis.In the regression analysis surgically treated patients showed significantly reduced rates of lung failure  and multiple organ failure .While no significant effect was found on sepsis rates and time on ICU, operatively treated patients had significant less intubation time  and less total days in hospital  compared to non-operatively treated patients Table .Table 3ITo assess a possible effect of early operative intervention within the first 5 days after hospital admission within the subgroup of 620 surgically treated patients, we performed an additional logistic regression analysis.When considering the operatively treated patients only, early surgical fixation of the clavicle within the first five days led to significant reduction of lung failure  multiple organ failure  and total days spent in hospital  (Table 4–0.88) mThe most important finding of our present retrospective study evaluating 3209 multiply-injured patients is that operative stabilization of concomitant clavicle fractures in patients with thoracic trauma has decisive positive effects on clinically relevant outcome parameters.18. Affection of the clavicle is the third most common fracture in multiply-injured patients (10.4%), only surpassed by femoral (16.5%) and tibial (12.6%) fractures19. Regarding all multiply-injured patients with bony injuries to the chest wall, an additional clavicular fracture is present in even 18.8% of the cases20. Thus, clavicle fractures are common injuries in patients suffering from thoracic trauma.In addition to the serious, life-threatening thoracic organ injuries, the bony chest wall is at great risk in multiply-injured patients11 or a British collective from Leeds21. In these populations patients were also predominantly male and had a mean age of 39.1 and 47.5 years, respectively. While our reported mean age of 49.0 years is slightly higher, their reported ISS was higher with 28.7 and 29.2, compared to our mean ISS of 22.921.Basic characteristics of our study cohort correspond to other studies describing the epidemiology of clavicle fractures in thoracic trauma, like the Dutch National Trauma Database23.Similar to previously published data on thoracic trauma, motorcycle crashes could be identified as the most frequent injury mechanism for developing a severe thoracic trauma with a clavicle fractureOne concern in most studies dealing with multiply-injured patients suffering from thoracic trauma is the heterogeneous patient collective regarding the leading injury and the immanent implication on the intensive care course . By deliberately excluding more than moderate brain injuries in our patient collective, we sought to limit this risk of bias.25. In line with this recommendation, our patients were treated mainly at level 1 and level 2 trauma centers (98.3%).It has been suggested that patients sustaining serious trauma to the chest and suffering from three or more rib fractures should be transferred to a high-volume level 1 trauma center® described an even higher rate of 52.4% operative treatments26, which might be due to more generous inclusion criteria, as all patients with an ISS ≥ 16 were included.In 42.0% of cases the clavicular fracture was treated operatively. A study with older data reviewing the years 2002–2013 in the TraumaRegister DGU3. Yet, our findings are comparable with previously published data on patients with thoracic trauma .Our reported patients suffering from thoracic trauma and clavicle fracture showed a prolonged period of ICU stay (mean 9.4 days) and intubation time (mean 7.4 days) compared to published multiply-injured patients without thoracic trauma 28. However, to the best of our knowledge, the implication of surgical management of clavicle fractures in thoracic trauma on clinical outcome parameters had yet to be evaluated. This is therefore the first study addressing the impact of operative treatment of the clavicle fracture in patients with relevant thoracic trauma on outcome parameters in a large collective. There are various theoretical reasons supporting surgical stabilization in this subgroup.In expert opinions and small case series, several authors recommend surgical stabilization of clavicular fractures in thoracic trauma, particularly in the case of displaced fractures and associated serial rib fractures or flail chest injuries30.In patients with high-energy mechanism of injury clavicle fractures are prone to fracture displacement, also when initial imaging shows nondisplacement10.Since the clavicle acts as a stabilizer of the upper quadrant of the chest fracture displacement is causing pain, especially in patients with concomitant ipsilateral rib fractures. The skeleton of the shoulder and chest loses stability and can lead to a loss of function of the shoulder and a pronounced deformation of the chest wall17.In particular, the breathing mechanism is affected. A fractured and instable clavicle as the origin of the accessory inspiratory muscles  can cause an ineffective respiration and oxygenation32. Furthermore, it is well known that 80% of patients with multiple organ failure start with lung failure21, and that severe thoracic trauma is an independent risk factor for developing multiple organ failure34.In combination with a severe thoracic trauma it can thereby further the development of multiple organ dysfunction and pulmonary failure35, The operative treatment of flail chest injuries shows a clear advantage with regard to the clinical course and outcomes in polytrauma35. According to the metaanalysis of Leinicke et al. patients with early stabilization have less intubation time and both the length of stay in the intensive care unit and the total length of stay in the hospital could be significantly reduced by surgical therapy13.To support the thesis that surgical fixation of fractures to the thoracic wall (or origins of inspiratory muscles) can support patient recovery in thoracic trauma several studies of rib fracture fixation have been published. Concerning flail chest injuries there is valid data supporting operative care of the injury to stabilize the thoracic aperture. Flail chest occurs when three or more adjacent ribs are fractured in at least two places, creating a chest wall segment that moves paradoxically from the chest wallIn our present study we are the first to demonstrate similar results concerning surgical stabilization of clavicle fractures.Thorax ≥ 3). Therefore, to analyze the effect of operative treatment of the clavicle fracture for all thoracic trauma patients the independent variable AISThorax was included into to the regression model. We also included other possible confounders  as independent variables into the regression model, and therefore sought to control for confounding.80.9% of our patients suffered from at least serious thoracic trauma  in our study and, as a result, our findings cannot be readily transferred to severely injured patients sustaining additional major trauma to this body region. We are not able to comment on the performed surgical procedures  and treatment protocols, since this information is not available in the TR-DGUAdditionally, we are unable to comment on the fracture morphology, displacement and fractured part of the clavicle, since this information is not available in the TR-DGU® database, either. Therefore, we were unable to include this information in our statistical regression model.38. For small bone fractures no recommendations or studies exist on when to surgically stabilize these injuries in multiply injured patients. For long bone and spinal fractures research exists and some favor stabilization within 72 h after hospital admission39. Since long bone and spinal fractures are more relevant to patient management on the ICU and are preferably stabilized before small bone fractures, we concluded to examine the time for early clavicular stabilization until 5 days after hospital admission. Although, as mentioned above, this timing is chosen in lack of scientific evidence we based our decision on reasonable clinical experience.There is an ongoing discussion on the management of multiply-injured patients and timing of osteosynthesisBesides the above stated limitations, to the best of our knowledge, we are the first study group to present comprehensive data on clavicle surgery and timing in multiply- injured patients suffering from concomitant thoracic trauma.Based on our data, surgical stabilization of clavicle fractures in multiply-injured patients with thoracic trauma may be beneficial and significantly reduces lung and multiple organ failure rates as well as time of invasive ventilation and length of hospital stay. The effect of reduced lung und multiple organ failure rates, as well as shorter hospital stay, was significantly associated with the clavicle stabilization being performed within 5 days of hospital admission. Further studies regarding patient-tailored surgery are warranted to elucidate which multiply-injured patients profit from clavicular stabilization.The TraumaRegister DGU® (TR-DGU) of the German Trauma Society (DGU) was founded in 1993. The aim of this multicenter database is the anonymized and standardized documentation of severely injured patients.Data are collected prospectively in four consecutive time periods from the accident site until hospital discharge: (A) pre-hospital phase, (B) emergency room and initial surgery, (C) ICU, and (D) discharge. The documentation includes detailed information on demographics, injury patterns, comorbidities, pre- and in-hospital management, course in the ICU, relevant laboratory findings including each individual’s data on transfusions, and outcome.The inclusion criterion is admission to the hospital via the emergency room with subsequent ICU care or reaching the hospital with vital signs and dying before admission to the ICU.The infrastructure for documentation, data management, and data analysis is provided by the Academy for Trauma Surgery (AUC), a company affiliated with the German Trauma Society.Scientific leadership is provided by the Committee on Emergency Medicine, Intensive Care and Trauma Management (Section NIS) of the German Trauma Society. The participating hospitals submit their anonymized data into a central database via a web-based application. The quality of the scientific data analysis is monitored by peer review procedure established by Section NIS.The participating hospitals are primarily located in Germany (90%), but a rising number of hospitals in other countries are contributing data as well . Approximately 35,000 cases from more than 700 hospitals have been entered into the database per year.Participation in TR-DGU is voluntary. For hospitals associated with TraumaNetzwerk DGU®, however, the entry of at least a basic data set is obligatory for reasons of quality assurance. Hospitals interested in trauma research must enter a standard data collection form that contains more comprehensive information  on the patient course compared with the basic data set.Patients documented between 2009 and 2015 in the TR-DGU® were analyzed for eligibility in this investigation. Patient selection based on the following criteria: (1) standard documentation (including surgery and organ failure assessment) from German trauma centers, (2) diagnosed clavicle fracture with documentation of treatment modality (3) maximum AIS severity ≥ 3 and (4) requirement of initial intensive care.Head 4–6, were excluded since serious head trauma may pose an indication for intubation and prolonged mechanical ventilation itself41. Also, patients admitted more than 24 h after trauma to a referring hospital and patients transferred into another institution within 2 days after hospital admission were excluded to prevent confounding. Finally, patients who died within 24 h were excluded from analysis since the surgical treatment of their clavicle fracture was deemed unlikely  according to the clavicle fracture treatment. The operative therapy group comprises all stabilization methods performed .We performed an additional subgroup analysis of patients undergoing surgical clavicular stabilization regarding the timing of surgery to evaluate the impact of early operation. Therefore, we defined the time frame for early clavicular surgery to be performed within 5 days after trauma.42, and the injury severity score (ISS)43 was calculated as described. The ISS is calculated from the three worst affected body regions as the sum of squares of the respective AIS severity levels44.Injuries were graded according to the 2008 version of the Abbreviated Injury Scale (AIS)45. Each organ system was graded to evaluate the severity of organ dysfunction or failure. Patients with organ failure entered in the TR-DGU database had to have met the SOFA score criteria for organ failure (3 or 4 points per organ) for at least 2 days.Lung failure was assessed by the sepsis-related organ failure assessment (SOFA) score. The SOFA score describes organ function in the respiratory, cardiovascular, renal, hematologic, hepatic, and central nervous systems46. Sepsis was defined as a systemic response to infection (proven presence of microorganisms). Data on respiratory failure, MOF, and sepsis were only available from patients documented with the standard data collection form.Multiple organ failure (MOF) and sepsis were assessed according to published guidelinesLength of intensive care unit stay was defined as days spent in the intensive care unit and length of mechanical ventilation was defined as the number of days spent in the ICU with endotracheal intubation or tracheostomy and mandatory mechanical ventilation (e.g. excluding non-invasive ventilation). Length of hospital stay was defined as the time spent in the hospital.47 was calculated for outcome adjustment. The model consists of the following predictors: worst and second-worst injury (AIS severity level), head injury, age, sex, mechanism, pupil reactivity and size, pre-injury health status, blood pressure, acidosis (base deficit), coagulation, hemoglobin, and cardiopulmonary resuscitation47.The Revised Injury Severity Classification II Score (RISC II)Demographic and clinical characteristics comparing two treatment groups were evaluated using descriptive statistics. Continuous variables are presented as mean with standard deviation (SD), while categorical variables are presented as number of cases with percentages. The respective statistics refer to patients with valid data sets only. Data of lung failure, MOF and sepsis are not part of the basic data set. Therefore, the total number of patients or characteristics may vary.Head, AISThorax, AISAbdomen, AISExtremities), hemodynamic parameters (shock defined as RRsyst ≤ 90 mmHg on admission) and therapeutic interventions .Statistical testing for the effect of operative treatment on the outcome was performed using multivariate logistic or linear regression analysis. In this analysis, the dependent variables were lung failure, MOF, sepsis, length of ICU stay, length of mechanical ventilation and length of hospital stay, respectively. The independent variables consisted of patient characteristics , injury severity .The present study is in line with the publication guidelines of the TR-DGU® and registered as TR-DGU® project ID 2017-006. The study was approved by the University of Freiburg Ethics Committee (EK 167/20) and informed consent was waived. All research was performed in accordance with relevant guidelines/regulations.


Late patterns had higher levels  compared to active and early patterns , and giant capillaries (p = 0.01) showed higher levels of LTs. Levels of pCysLT were higher in patients with RP at examination vs. patients without RP; saCysLT and LTB4 were higher in SSc group with vs. without capillary loss. LTs could be involved in the pathophysiology of vascular abnormalities. Further research is required to determine if blocking LTs could be a therapeutic target for SSc vascular manifestations.The role of leukotrienes (LTs) in the pathogenesis of systemic sclerosis (SSc) needs clarification. We analyzed the association of salivary (sa) and plasma (p) levels (pg/mL) of cysteinyl-leukotrienes (CysLT) and LTB4 with SSc vascular manifestations and nailfold capillaroscopy (NFC) in a cross-sectional study. Patients and healthy controls were evaluated for vascular manifestations and NFC. LTs were compared between groups as follows: SSc with or SSc without vascular features and controls, and by NFC parameters. Twenty SSc patients and 16 volunteers were recruited; Raynaud’s phenomenon (RP) history (SSc: saCysLT 99.4 ± 21.8 vs. controls: 23.05 ± 23.7,  Vasculaeceptors ,16 in fieceptors  or blockeceptors  or theireceptors , and LTAeceptors , were reeceptors ,17,20. Oeceptors .Regarding the vascular activities of LTs, they have a potent vasoconstrictor effect in many vascular beds  includin1 receptor and endothelial cell proliferation through the CysLT2 receptor [In addition, CysLT are produced during vascular injury, and they modify vascular function with a wide array of effects such as constriction of microvasculature, promotion of post-capillary venules permeability, reduction of coronary flow and pathological retinal angiogenesis . CysLT ireceptor . All theCross-sectional studies have shown high levels of LTB4 and LTE4 in bronchoalveolar lavage fluid of patients with scleroderma lung disease ,29; highEthical Considerations. The cross-sectional study was conducted according to the guidelines of the Helsinki Declaration and local regulations, and we followed appropriate ethical research design and the relevant standards to ensure the protection of human subjects. The protocol was approved by the Research Commission and Ethics Committee of Instituto Nacional de Cardiología Ignacio Chávez (IRB: 12-776). Informed consent was obtained from all subjects involved in the study.Patients and controls. Patients and healthy volunteers fulfilled all inclusion criteria including age greater than or equal to 18 years and agreement to participate under informed consent; neither healthy volunteers nor patients that had history of bronchial asthma, rhinitis, allergic diseases or other systemic diseases were enrolled. Patients were recruited from the Rheumatology Department at the Instituto Nacional de Cardiología Ignacio Chávez and all patients fulfilled the 2013 American College of Rheumatology/European League Against Rheumatism criteria for the classification of SSc , patientClinical assessment. A complete medical history was taken for all individuals. Demographic information and vascular manifestations found at recruitment were collected. Vascular features included Raynaud’s phenomenon (RP), digital ulcers (DU) and fingertip scars both since the onset of the disease  and RP, DU and fingertip scars found at the time of physical examination. Clinical photographs were also taken A,B.Nailfold capillaroscopy (NFC). NFC was performed in all patients on the fourth finger of their right hand at room temperature with the patient in a sitting position. We chose to evaluate the same finger in all subjects to reduce the variability that could be found when comparing different fingers. The fourth finger was chosen for assessment because it has been previously reported that it is in this finger where the presence of enlarged capillaries and hemorrhages is most frequent in patients with SSc. Additionally, it has a greater transparency of the skin compared to other fingers . This saPrimary endpoint. The primary endpoint was to detect any differences in LT levels between the groups classified by vascular features and capillaroscopy findings. Other endpoints. Levels of LTs between patients and healthy volunteers were compared, and LT levels by clinical groups were also analyzed. In addition, we looked for correlation between capillaroscopy parameters and LT levels.Procedures. All samples  were collected between 7:00 and 9:00 after fasting on the same day of clinical assessment. Peripheral venous blood was collected in sodium heparin BD Vacutainers. Non stimulated saliva was collected from most subjects between 7:00 and 9:00 on the same day before clinical evaluation. Only patients with insufficient saliva chewed paraffin gum to take a stimulated saliva sample. Patients and controls were asked to continuously spit out all their saliva for 5 min in plastic cones connected to 15 mL polypropylene centrifuge tubes (corning) according to a previously published method .g for 15 min at 4 °C and stored at −80 °C until processing. The LTs in all samples were identified and quantified by analytical services of Cayman Chemical in Ann Harbor, Michigan, USA. Levels of LTB4 and CysLT in plasma and saliva were determined with Cysteinyl Leukotriene Express EIA and Leukotriene B4 Express EIA kits in triplicate. The Cayman kit protocols were followed by the analytical services. For CysLT and LTB4 assays for saliva and plasma, samples were purified using the Purification Protocol for each type of kit to avoid contaminants, which can interfere in the assay.Enzyme immunoassay (EIA). Peripheral blood and saliva samples were collected, centrifuged at 2600× p < 0.05 was recognized as statistically significant. We used the Holm–Bonferroni Sequential Correction procedure to protect against Type I errors for correlation analyses. Correlations were evaluated by the Spearman rank test. Data analysis was performed using JMP software, version 13.0  and an excel calculator version 1.2 (2013) to calculate the Holm–Bonferroni sequential correction [Statistical analysis. Demographic, clinical, and laboratory characteristics of SSc patients were summarized as mean ± standard error of the mean or number (%). Associations between participant groups and LTs and clinical findings were analyzed by ANCOVA, with patient age as a covariate. The following procedure was used to create the models. First, an ANCOVA model in which the participant groups were the explanatory variable, the subject’s age the covariate, and the product of these two as the interaction term was constructed for each of SSc-related variables as the respective outcomes; second, ANCOVA model assumption sufficiency was tested through the goodness of fit of studentized residuals to normal distribution. When this assumption was satisfied, the ANCOVA model was subjected to the variable selection procedure by the backward stepwise method. In the case it was not, the Box-Cox transformation was applied to the outcome variable. After these steps, if the effect of the explanatory variable, subject groups, was statistically significant, the differences among groups were determined by Tukey’s honestly significant difference test (Tukey’s HSD). For all statistical tests, rrection .Twenty patients (12 with limited and 8 with diffuse SSc) and 16 healthy volunteers were recruited, and the baseline characteristics of subjects, clinical features and vascular manifestations are shown in n = 16), F = 6.33, (n = 17). SSc patients with RP at the time of examination had higher levels of salivary CysLT  compared to controls , but not compared to patients without RP at examination , F = 5.31 (n = 15), compared to SSc without RP at examination , F = 5.31. With plasma, all samples were analyzed.LTs and vascular abnormalities. Patients with RP history showed higher levels of salivary CysLT (99.4 ± 21.8 pg/mL) compared to controls  = 5.31 B. A simin = 11) levels were significantly higher in the SSc group with capillary loss compared to SSc without capillary loss  and controls , F = 7.0  = 7.0 A. A simi3) = 7.0 B.n = 7) had the highest salivary CysLT (177.8 ± 32 pg/mL) levels compared with groups with SSc without loss (47.96 ± 27.93 pg/mL) and controls which had salivary CysLT of 23.05 ± 19.75 pg/mL, F = 7.97 (n = 9) had the highest levels of salivary LTB4 compared to ectasia SSc group and controls, F = 8.98; giant capillaries vs. ectasia capillaries (p = 0.001) and giant vs. controls (p = 0.01). However, in this case the ANCOVA model was also significant for age (p = 0.03). SSc patients with dilated capillaries showed higher levels of salivary LTB4 were compared to controls (p = 0.04). Scleroderma patterns. Patients with the “late” pattern (n = 6) had higher salivary CysLT  capillaroscopy  and compared to “active” pattern . In addition, LTB4 levels were higher in patients with the “late” pattern than in patients with an “early” pattern  = 7.97 C. A simi) = 7.97 D. CapillLevels of LT between the SSc and control groups. Higher CysLT concentrations in saliva and plasma were found in SSc patients (data not shown), but no statistical significance was found.Leukotrienes and clinical groups. Patients were classified according to skin involvement in diffuse (dcSSc) and limited (lcSSc) subsets, and the level of leukotrienes was compared between the subsets and controls, but no significant differences were found.p = 0.001), no other correlations between LTs were found. Correlations between leukotrienes and NFC. Levels of salivary CysLT were negatively correlated with the number of capillaries .Correlations between leukotrienes and demographic variables. Correlations between age and body mass index was not found (data no shown). Correlations between LTs. Salivary levels of LTB4 and CysLT were positively correlated (ρ of Spearman 0.51, Considering the relationship between LTs and vascular function, we found significantly higher concentrations of LTs in SSc patients that had vascular features. We found higher levels of salivary and plasma CysLT in patients with RP and higher salivary levels of both LTB4 and CysLT were associated with loss of capillaries and “late” scleroderma pattern. In addition, higher levels of salivary LTB4 were associated with giant capillaries and dilated capillaries. Such features are caused by tissue hypoxia, one of the pathogenic mechanisms of SSc. This suggests that they could be related with LTs synthesis as these findings are consistent with the reported biological activity of leukotrienes ,15,38,39To our knowledge, few studies have focused on a potential association between RP and LTs. Lau and colleagues  investigPrevious studies suggest a link between LTs and atherosclerosis ,57,58,59Previous research supports LTs synthesis by cells of the vascular system ,65,66 anThe use of saliva to test levels of LTs has several advantages, the main one being the lack of harm to the patient . The higWhether the LTs found in saliva and plasma participate in cell recruitment or result from the production by cells recruited by other cytokines is still an open question, but current knowledge from multiple research groups suggests that LTs could somehow be involved in the complex network of mediators participating in SSc pathogenesis ,30,31,41As far as we know, only Liang et al.  have resAlthough a modest size and cross-sectional design of the study are potential limitations, a follow-up study is planned to assess whether the findings hold over time and are associated with disease progression. We are aware of other limitations of the present study. An important limitation is that some patients were under immunosuppressive treatment, so we included the treatment as a covariable and did not find differences between levels of LTs in the groups analyzed. In addition, it is important to mention that many of the patients evolve despite of current SSc treatments. Therefore, we postulate that even though some patients have immunosuppressants, which can affect in some degree the levels of inflammatory mediators, if LTs have a role in pathogenesis in SSc, on the one hand their levels would be unaffected or could be only partially affected since they are not specific antagonist blockers of LTs and on the other hand it may have a complex interaction with other mediators of the disease, so this could explain why the disease in many cases evolves despite of the current immunosuppressive treatment.The value of the present study lies in two main issues. Firstly, it provides the observational data for a generation of new hypotheses regarding the pathophysiology of the vascular and cutaneous manifestations of this autoimmune disease, with possible implications in its treatment. Secondly, we believe the present study shows the importance of stratifying patients according to their clinical profile in order to identify differences in the study variables. In this study, we found similar levels of LTs between asymptomatic patients and controls, the same was observed for NFC parameters. Those who had normal or few abnormalities had similar levels to the controls. However, symptomatic patients or with important abnormalities in NFC showed higher levels of LTs pointing to a possible role in their pathogenesis.CysLT and LTB4 levels, mainly in saliva samples, were associated with RP and capillaroscopic manifestations of SSc. Saliva proved to be a useful source of LTs to study their involvement in SSc.The results are further confirmation of the potential involvement of LTs as mediators in SSc and encourage translational research on the role of LTs to find out their relevance in this complex pathogenesis, focusing on the processes of vascular damage and fibrosis in SSc.Including LTs in the context of SSc is very important, not only from the point of view of pathogenesis, but also to consider possible combined treatment strategies to improve survival and life quality for patients.