Brugia pahangi as another zoonotic lymphatic filarial nematode. In this report, a genomic study was conducted to understand this species at molecular level.Efforts to completely eradicate lymphatic filariasis from human population may be challenged by the emergence of B. pahangi-harbouring cat, the Aedes togoi mosquitoes were maintained to harvest infective third stage larvae, which were then injected into male Mongolian gerbils. Subsequently, adult B. pahangi were obtained from the infected gerbil for genomic DNA extraction. Sequencing and subsequently, construction of genomic libraries were performed. This was followed by genomic analyses and gene annotation analysis. By using archived protein sequences of B. malayi and a few other nematodes, clustering of gene orthologs and phylogenetics were conducted.After blood meal on a B. pahangi shared high similarity to that B. malayi genome, particularly genes annotated to fundamental processes. Nevertheless, 166 genes were considered to be unique to B. pahangi, which may be responsible for the distinct properties of B. pahangi as compared to other filarial nematodes. In addition, 803 genes were deduced to be derived from Wolbachia, an endosymbiont bacterium, with 44 of these genes intercalate into the nematode genome.A total of 9687 coding genes were predicted. The genome of B. pahangi draft genome contributes to genomic archive. Albeit with high similarity to B. malayi genome, the B. pahangi-unique genes found in this study may serve as new focus to study differences in virulence, vector selection and host adaptability among different Brugia spp.The reporting of The online version of this article (doi:10.1186/s13071-015-1064-2) contains supplementary material, which is available to authorized users. Wuchereria bancrofti, Brugia malayi, and Brugia timori) [Mansonia, Anopheles, Culex, and Aedes. Lymphatic filariasis causes enlargement of body parts and skin exfoliation, which can result in severe disability, disfigurement, and social stigma [W. bancrofti [Brugia spp. have been found to routinely cause filariasis in various Asian countries including India, Malaysia, Indonesia, the Philippines, and China [. W. bancrofti, B. malayi , and B. timori exclusively infect humans, whereas the nocturnal subperiodic strain of B. malayi  is zoonotic and transmitted to humans (via mosquitoes) from cats, dogs, and wild carnivores [Brugia spp. commonly infect humans, recent evidence has also indicated that Brugia pahangi, a filarial nematode that is naturally found in cats, can cause clinical infection in humans, with clinical presentations that are consistent with lymphatic filariasis [B. pahangi infection may be more prevalent in humans than previously thought. Nevertheless, it is known that the physiology and vector competence of B. pahangi and B. malayi differ from each other [B. pahangi and compared it with those of other nematodes in order to deduce biological similarities and differences among these nematodes.Lymphatic filariasis, which is also known as elephantiasis tropica, affects more than 120 million people worldwide [l stigma . The mosnd China . W. bancrnivores , 5. Althlariasis . This such other , 8. CleaAedes togoi mosquitoes were fed on a cat naturally infected with B. pahangi. The infective third stage larvale (L3) of B. pahangi were recovered from the infected mosquitoes. Subsequently, 100 L3 larvae suspended in 0.5 ml RPMI were injected into each of the recruited male Mongolian gerbils (Meriones unguiculatus) via intra peritoneal and subcutaneous routes of administration. Sluggish and inactive larvae were not used. Three months later, thick blood smears of the gerbils were made and stained with Giemsa for microfilariae detection [Female etection . At 95 detection . The aduB. pahangi using DNeasy Blood and Tissue Kit . The specific identity of the worm was verified by PCR amplification using primer pairs specific for the cytochrome oxidase I (COXI) gene of B. pahangi  and DNA sequencing (Reference GenBank accession no. AJ271611) [Qubit fluorometer dsDNA HS Kit, Invitrogen); DNA integrity was verified by agarose gel electrophoresis and using a Bioanalyzer .Female worms were collected and washed 3 times with sterile physiological saline before genomic DNA extraction. High molecular weight genomic DNA was isolated from a single female worm of J271611) . The DNAMus muculus and Wolbachia of B. malayi (accession number AE017321) were identified by comparison to the ENSEMBL (release 72) and NCBI (build 37) databases respectively. To do this, all libraries were aligned to the Mus musculus genome using SOAPaligner v.2.21 [Wolbachia genomes using BLAST v.2.2.26 [Paired-end (PE) genomic libraries  and Jumping (J) genomic libraries  were constructed , 14. To r v.2.21 , and reav.2.2.26 .B. pahangi and B. malayi and other genomes were detected using the program LASTZ [Pre-processed genomic PE-libraries and J-libraries were assembled and scaffolded using the program SOAPdenovo with k-mer of 29 . During am LASTZ . A pairwde novo approaches. The homology approach used standard databases containing known repetitive sequences (Repbase), and predicted TEs at both the DNA and protein levels. For the former, RepeatMasker was applied [de novo approach employed LTR_FINDER [B. pahangi, and to categorize them  were identified using the software Tandem Repeat Finder (4.04) with the following settings: Match = 2, Mismatching penalty = 7, Delta = 7, PM = 80, PI = 10, Minscore = 50, MaxPeriod = 2000. TEs were identified using homology and  applied , 22. TheR_FINDER  and REPER_FINDER  and REPER_FINDER  to identde novo- and homology-based predictions. For homolog predictions, we used protein sets of B. pahangi, B. malayi, Caenorhabditis briggsae, Caenorhabditis elegans, and Pristionchus pacificus from ENSEMBL [-5. After that, we selected the most homologous protein for each genomic locus showing multiple matches. Subsequently, we removed regions with < 25 % homology of the query protein. We extended 500 bp at both ends of the alignment regions and predicted gene structures using the program GeneWise2.2.0 [de novo predictions, we used the programs AUGUSTUS [B. pahangi genes has revealed key data that can further our understanding of differences among different filarial species. A number of genes and pathways of interest were selected for case study analysis. In this regard, we used BlastP against GO [To predict gene structures in the draft genome assembly, we performed both  ENSEMBL , and mapise2.2.0 . For de AUGUSTUS , SNAP [2AUGUSTUS  and GLIMAUGUSTUS . Genes wAUGUSTUS . FunctioAUGUSTUS  and TrEMAUGUSTUS  databaseAUGUSTUS  against AUGUSTUS , PRINTS AUGUSTUS , PROSITEAUGUSTUS , ProDom AUGUSTUS  and SMARAUGUSTUS . Gene OnAUGUSTUS , 40. EacAUGUSTUS . Functioainst GO  and KEGGainst GO . The gen-7), and con-joined fragment alignments for every gene were used using the program Solar (Skiena) when necessary. We assigned a connection (edge) between 2 nodes (genes) if more than 1/3 of the region aligned to both genes. H score (0 to 100) was used to weight similarity (edge) between the nodes. For two genes G1 and G2, the H score was defined as score (G1G2)/max (score (G1G1), score (G2G2), the score used here was the BLAST raw score. Extracting gene families using clustering by Hcluster_sg [All protein sequences were compared against database containing protein dataset of all species covered in this study  based on Kimura model was applied [Wolbachia genes.We constructed a phylogenetic tree of  applied . SimilarThis Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession JRWH00000000 . The version described in this paper is version JRWH01000000.B. pahangi genome at 168-fold coverage  chromosomal genome sequences   appears to have orthologs (BLASTp cut-off: 10-5) in B. malayi, as compared to that in C. elegans  and C. briggsae . Overall, 6795 genes were found to be orthologous among all 4 species under comparison. Another 62 predicted B. pahangi genes were found to be shared among B. malayi and C. briggsae, and 1624 B. pahangi genes were shared exclusively with B. malayi. A total of 569 genes were predicted to be unique to B. pahangi  coded for known proteins involved in various cellular pathways, and another 853 genes  encoded for proteins of mostly unknown functions (i.e. orphans). These KEGG-mapped genes were further divided into 6 different function classes, namely cellular pathways , environmental information processes , genetic information processing , metabolism  and organismal systems .A total of 7801 out of the 9687 predicted Brugia genome, comparative analyses of the functional annotation in genes shared between B. pahangi and other nematodes were performed. KEGG database was used to examine the 6795 genes shared among B. pahangi, B. malayi, C. elegens and C. briggsae. Of these, 5140 (~75.64 %) could be mapped to biological pathways. Of these mapped genes, 5131 (~99.82 %) were coded for known proteins whereas 9 genes (~0.18 %) encoded for orphan proteins. The encoded known proteins encompassed a number of functional classes, namely genetic information processing , metabolism , environmental information processing , cellular processes , and organismal systems . Interestingly, comparison between B. pahangi and B. malayi revealed that the shared gene set was closely associated with phosphorylation activity. Most of the Brugia spp. conserved genes are coded for fundamental and common biological processes such as oxidation-reduction transports, DNA replication machinery and transcription regulation. As expected, comparisons of B. pahangi genome with those of Caenorhabditis spp. reflected larger differences. For instance, GTPase-mediated signal transduction machinery could be found in both species of Caenorhabditis spp. studied. However, this signal transduction machinery was not available in B. pahangi genome. On the other hand, protein histidine kinase activity was encoded by B. pahangi genes, but not available in C. briggsae and C. elegans.To find out the shared and conserved elements encoded in the B. pahangi  prediction of B. pahangi can be due to larger amount of repetitive regions in B. pahangi genome. Overall, from the whole genome sequencing, the relative amount of AT nucleotides was higher than GC nucleotides (AT bias). Interestingly, it was reported previously that the amount of coding genes in a genome is correlated to the relative amount of GC content [B. pahangi. Nevertheless, without additional measures such as RNA sequencing, the actual causes of such discrepancy in gene numbers between these closely related Brugia spp. cannot be answered by this study. Meanwhile, whole genome conserved synteny analysis was conducted to identify and characterize the functionally related stretches of clustered genes between different species. Large segments exhibiting high similarity between homologous sequences from several species indicate conserved and important biological features. Therefore, the strength of evolutionary constraints and the importance of functional elements can be quantitatively measured by determining the local evolution rates in multiple alignments. The genome similarity shared between B. pahangi and B. malayi in many aspects of fundamental cellular activities may be one of the factors that enable survival and growth of B. pahangi in humans apart from animals as its natural host.The high detection rate of core essential genes from our . malayi . Besidesematodes –49. As eted Fig. . The gen content . This maB. pahangi genome comprise only a small portion of the whole draft genome (~5.87 %). Nevertheless, the functions encoded by these unique genes may be responsible for the unique properties of B. pahangi that make it a distinct species. We identified 9 genes that mapped to metabolic pathways: glycan biosynthesis and metabolism (Brugia.Pahangi_GLEAN_10002876); cofactor and vitamin synthesis ; amino acid metabolism (Brugia.Pahangi_GLEAN_10004798); secondary metabolite processing (Brugia.Pahangi_GLEAN_10000930); carbohydrate metabolism ; enzyme biosynthesis (Brugia.Pahangi_GLEAN_10004319); and a yet-unclassified pathway (Brugia.Pahangi_GLEAN_10004951). In addition, 4 genes were linked to other metabolic pathways as well .Genes unique to B. pahangi is mapped to N-glycan biosynthesis and metabolism, which is critical for survival of eukaryotic organisms and can confer unique characteristics to distinct species. There was evidence of 2 Wolbachia genes  predicted to participate in terpenoid backbone biosynthesis, which contributes to N-glycan biosynthesis and degradation, suggesting successful endosymbiotic relationship that exists between B. pahangi and Wolbachia.N-glycans are essential for proper protein folding in eukaryotic cells. Without the correct folding, proteins are subjected to proteolysis, which represents a quality control measure that is vital for ensuring functional efficiency of newly produced proteins. Furthermore, N-glycans are involved in cell–cell communication and protein–protein interactions. Therefore, it is not surprising that one of the unique genes in B. pahangi unique genes  mapped to porphyrin and cobalamin (vitamin B12) metabolism. Cobalamin is involved in fundamental cellular metabolic activities, whereas porphyrin is the key ingredient for heme biosynthesis. Heme is a crucial component for several important classes of proteins , which are involved in a variety of critical biological processes, including oxidative metabolism and electron transport [B. pahangi and Dirofilaria immitis [B. pahangi and D. immitis are unable to synthesize ecdysteroids from cholesterol [B. pahangi unique genes  are inadequate to complete the whole interactions involved in heme synthesis, we identified 4 non-integrated Wolbachia genes that are mapped to the same heme biosynthesis pathway . This finding suggests that the 4 genes of bacterial origin may complement heme biosynthesis in B. pahangi. In addition, the metabolism of some amino acids  is associated with porphyrin and cobalamin synthesis. Interestingly, we also identified two non-integrated Wolbachia genes that mapped to glycine and threonine metabolism  as well as another non-integrated Wolbachia gene (Brugia.Pahangi_GLEAN_10010462) mapped to alanine, aspartate and glutamate metabolism in the B. pahangi genome assembly. This finding might suggest that B. pahangi achieves enhanced metabolic efficiency through successful endosymbiotic relationship with its Wolbachia endosymbiont.Two ransport . Heme haransport , 53. Morransport , which a immitis , 56. Altlesterol , 58, ecdlesterol . ClearlyB. pahangi through a pathway involving one of its unique genes (Brugia.Pahangi_GLEAN_10000930). Interestingly, this B. pahangi gene also mapped to pathways participating in environmental information processing via two-component system (TCS) signal transduction, which is commonly found in bacteria [Wolbachia gene (Brugia.Pahangi_GLEAN_10010525) that is involved in TCS signalling. With TCS signalling pathway, the parasite could sense, respond, and react to chemicals encountered in its surroundings, which may help the parasite to identify its vectors and hosts. Apart from TCS system, protein kinases also play key roles in cell signaling pathways. The biosynthesis of protein kinases is mapped to one of the unique B. pahangi genes (Brugia.Pahangi_GLEAN_10004319). This gene may code for synthesis and regulation of distinct protein kinases that are involved in specific cellular signaling pathways in B. pahangi, thereby conferring differential host/vector adaptability and virulence as compared to other filarial nematodes.The ability to process and metabolize secondary metabolites can confer survival advantages to parasites. In this regard, various drug molecules, such as beta-lactams , can be metabolized by bacteria . Indeed,B. pahangi that mapped to metabolism of cysteine and methionine (Brugia.Pahangi_GLEAN_10004798). Notably, this gene also mapped to another non-metabolic function involving a DNA repair and replication regulatory pathway. Indeed, it has been demonstrated that a high level of intracellular cysteine and homocysteine (metabolite of methionine) can affect the efficiency of the DNA repair machinery [We also identified another unique gene in achinery , 61. TheB. pahangi genes were found to be involved in genetic information processing. A total of 10 unique genes were mapped to this group, revolving around 3 functions: transcription and transcription factors ; folding, sorting and degradation ; and DNA replication/repair . Eight of these genes were mapped to functions and pathways specific to gene regulation and processing, whereas the remaining 2 genes were involved in cross- KEGG class functions and pathways. Besides, 4 B. pahangi-specific genes  are mapped to ubiquitin-related regulation. Ubiquitin is a post-translational regulatory protein that is exclusive to eukaryotic organisms. Following ubiquitination, proteins can be targeted for degradation through ubiquitin-activated proteolysis. Genes annotated to transcription factors and ubiquitin-related regulations may play roles in epigenetic regulation of B. pahangi as well. Indeed, epigenetic regulation via microRNAs (miRNAs) has been reported in filarial nematodes [A number of unique ematodes .B. pahangi genes. These include homologous recombination, non-homologous joining, DNA replication, DNA repair, DNA recombination, and chromosome repair machinery.Moreover, an additional gene was found to map to a eukaryotic-exclusive sorting and folding function involving SNARE interactions during vesicular transport (Brugia.Pahangi_GLEAN_10005156). This endoplasmic reticulum-dependent function enables synthesized proteins to be exocytosed. Finally, several DNA replication and repair functions were mapped to unique B. pahangi genes . These findings indicate that B. pahangi may employ unique strategies for cell–cell communication and cellular regulation.We identified 5 genes that mapped to environmental information processing pathways . These genes were mapped to signal transduction pathways and linked to other KEGG pathway classes as well, including metabolism, genetic information processing, cellular processes, human disease pathways, and organismal systems. Such diverse functional mapping highlights the importance of signal transduction in the regulation of vital cellular activities. Indeed, several specific signal transduction pathways were mapped to these unique According to the KEGG classification, genes mapping to “cellular processes” participate in cell growth/death, movement, communication, catabolism, and transport. A total of 8 unique genes were found to be mapped to this category. Of these, 2 were mapped to endocytosis , while 5 genes were linked to cell communication via tight junctions  or focal adhesions . Both tight junctions and focal adhesions are responsible for cell anchorage. In addition, tight junctions play vital role in barrier protection and regulate signal transduction . SimilarB. pahangi genes that coded for organismal system pathways were also linked to other functional groups, including pathways associated with human diseases, cellular processes, and environmental information processing . The mapped function pathways include the insulin and gonadotropin-releasing hormone (GnRH) signaling, as well as chemokine signaling pathways. Notably, chemokine signaling pathways are important for cellular growth, differentiation, migration, and survival, as well as regulation of reactive oxygen species (ROS) production.Organismal system pathways include all cascade reactions that are associated with the immune responses, endocrine system, nervous system, circulatory system, and other organ system pathways. All 4 unique B. pahangi and pathogenesis of B. pahangi infection may revolve around the dysregulation of host MAPK signaling cascade, be it the definitive host, vector or reservoir. Indeed, filarial worms are known for its manipulation of host’s immune system towards their benefit of survival [Three unique genes were mapped to human disease pathway . All of them coded for multiple functions spanning different pathway categories. One of these (Brugia.Pahangi_GLEAN_10009860) was mapped to pathways of genetic information processing, environmental information processing, cellular processes, and organismal systems. The other 2 were linked to functions of cellular processes and organismal systems. Of particular interest, one gene (Brugia.Pahangi_GLEAN_10009860) was linked to pathogenesis pathway related to leishmaniasis, another parasitic infection. This pathway revolves around dysregulation of MAPK cascade. The interference of MAPK cascade will affect various downstream cellular events such as DNA proliferation, gene expression and regulation, cell survival and differentiation, protein synthesis and apoptosis. Indeed, dysregulation of MAPK cascade is one of the key players in endometrial cancer, prion disease and leishmaniasis pathogeneses. For endometrial cancer, this leads to uncontrolled cell growth and proliferation . In priosurvival . While tsurvival , 70.B. pahangi unique gene was the machinery of epithelial cell invasion. This machinery is derived from bacteria. Although the gene coded for this function (Brugia.Pahangi_GLEAN_10001231) is not one of those Wolbachia-derived genes, we believe that B. pahangi has acquired this gene from Wolbachia since the beginning of their endosymbiotic relationship. The ability of invading epithelial cell is critical for the parasite developmental stages within the mosquito vector and migration from circulation to the lymph nodes in the mammalian hosts. There are two models for the epithelial invasion machinery [B. pahangi unique gene may also be responsible for the ability of B. pahangi to survive in Armigeres subalbatus, which is different from B. malayi [Another pathogenesis-related function mapped to achinery , 72. Bot. malayi .Wolbachia was sequenced concomitantly. In fact, the presence of Wolbachia in B. pahangi worm used for genome sequencing is the major limitation of our study, where the endosymbiont bacteria should be removed prior to sequencing. Nevertheless, many studies have shown that killing of Wolbachia resulted in growth retardation or death of the nematode [B. pahangi specimens and separate the Wolbachia genome from the filarial nematode genome after sequencing by referring to the draft genome of Wolbachia-B. malayi (WBm) [Wolbachia origin to have a better understanding on the genetic interactions between B. pahangi and its endosymbiont Wolbachia. Here, we have selected a few interesting Wolbachia-B. pahangi (WBp) genes to discuss.In this project, the genome of endosymbiont nematode , 74. Couyi (WBm) . ThereafB. pahangi may be benefit from metabolic processes of at least 11 amino acids, owing to unique WBp genes . These amino acids included lysine, histidine, tyrosine, glycine, serine, threonine, alanine, aspartate, glutamate, arginine, and proline. In addition, one gene was annotated to metabolism of selenocompounds (Brugia.Pahangi_GLEAN_10010573). Interestingly, there were as many as 4 genes (50 % of the genes involved in amino acid biosynthesis) participating in lysine biosynthesis. Lysine is known to be an important ingredient for collagen and elastin cross-linkage [B. pahangi and the endosymbiont Wolbachia. This is indeed similar to situations in B. malayi [Based on analysis using KEGG database, -linkage , 76. Nev. malayi , 52.Wolbachia gene products also displayed multiple closely related functions. For instance, the gene linked to histidine and tyrosine metabolism (Brugia.Pahangi_GLEAN_10010481) was also responsible for biodegradation of xenobiotics and polycyclic aromatic hydrocarbons. Indeed, histidine is the precursor of carnosine, an antioxidant capable of neutralizing ROS derived from xenobiotics and polycyclic aromatic hydrocarbons [WBp genes . Riboflavin (vitamin B2) is crucial for the synthesis of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN). These factors function as important coenzymes in diverse oxidation–reduction reactions during intermediate metabolism.Specific ocarbons . Likewisocarbons . Interesocarbons . RiboflaWBp genes may also contribute significantly to the complexity of the environmental information and cellular processing machinery of B. pahangi, particularly in signal transduction. For example, a gene linked to G protein-coupled receptors (GPCRs) (Brugia.Pahangi_GLEAN_10005033) was identified. GPCRs are trans-membrane receptors that regulate cellular activity by triggering intracellular signaling cascades in response to extracellular signals, biomolecules, and environmental changes [B. pahangi unique genes were found to code for products involved in phosphatidylinositol signaling . Since GPCRs are involved in the regulation of various cellular activities (e.g. homeostasis and immunity), it may contribute to the specific parasite–vector relationship between B. pahangi and Ar. subalbatus, as well as the broader mammalian host range as compared to the nocturnal periodic B. malayi. This signal transduction machinery may enable B. pahangi to interact with its surroundings, including the immune cells of mosquitoes. Sensing environmental cues allows the parasite to respond by evading or impairing the mosquito’s immune system, thereby facilitating parasite survival within the mosquito vector. Similar signal transduction events may permit B. pahangi to cause asymptomatic infections in mammalian hosts, including humans.In addition, unique  changes , 81. TheB. pahangi genome and WBp genome that are annotated to functions that may play critical role in determining vector specificity and host adaptability of B. pahangi. Nevertheless, it would be difficult to draw a solid conclusion on this subject based on draft genome comparison. Besides, there are 2 strains of B. malayi, the nocturnal periodic type that infects only humans, and the nocturnal subperiodic type that is zoonotic in nature. Genomic comparison and analysis on this subject would be more powerful if genomic data of the nocturnal subperiodic B. malayi and its endosymbiont Wolbachia are available. Nevertheless, our findings assist in narrowing the focus for further studies to seek answers for this matter.As mentioned earlier, we found a number of unique genes, both from Wolbachia origin were integrated into the B. pahangi genome, suggestive of lateral gene transfer. Indeed, the integration of Wolbachia genes into their host genome has been reported previously [B. malayi as well, where many of the integrated genes are pseudogenized and cannot be expressed by the nematode [B. pahangi.In this project, we found that a number of genes of eviously , 83. Sucnematode . NeverthB. pahangi shares high similarity with that of B. malayi. Nevertheless, there are distinct features in B. pahangi genome that may confer unique physiological and pathological differences, as well as features that may influence its interaction with vectors and mammalian hosts. The unique genes found in B. pahangi genome may serve as reference to study biological and virulence differences among different filarial nematodes in future. Moreover, study of the B. pahangi genome has illuminated the genetic interaction between B. pahangi and its endosymbiont Wolbachia. Indeed, it appears that Wolbachia-derived genes contribute and complement the content and uniqueness of the B. pahangi genome. Such interaction between B. pahangi and its endosymbiont bacteria at genetic level may serve as additional reference to better understandings on cellular biology of other filarial nematodes.As expected, the genome of


Marshosaurus, Megalosauridae cf. Torvosaurus and probably Ceratosauria. The formerly reported presence of Dromaeosauridae in the Late Jurassic of northern Germany could not be confirmed. Some teeth of this study resemble specimens described as pertaining to Carcharodontosauria (morphotype A) and Abelisauridae (morphotype K). This interpretation is however, not supported by discriminant function analysis and cladistic analysis. Two smaller morphotypes (N and Q) differ only in some probably size-related characteristics from larger morphotypes (B and C) and could well represent juveniles of adult specimens. The similarity of the northern German theropods with groups from contemporaneous localities suggests faunal exchange via land-connections in the Late Jurassic between Germany, Portugal and North America.Remains of theropod dinosaurs are very rare in Northern Germany because the area was repeatedly submerged by a shallow epicontinental sea during the Mesozoic. Here, 80 Late Jurassic theropod teeth are described of which the majority were collected over decades from marine carbonates in nowadays abandoned and backfilled quarries of the 19th century. Eighteen different morphotypes (A—R) could be distinguished and 3D models based on micro-CT scans of the best examples of all morphotypes are included as supplements. The teeth were identified with the assistance of discriminant function analysis and cladistic analysis based on updated datamatrices. The results show that a large variety of theropod groups were present in the Late Jurassic of northern Germany. Identified specimens comprise basal Tyrannosauroidea, as well as Allosauroidea, Megalosauroidea cf.  Isolated teeth probably represent the most abundant theropod dinosaur body fossils. Even sedimentary deposits where terrestrial vertebrate fossils are very rare, such as the marine Late Jurassic carbonates in Northern Germany, yield isolated teeth. Unfortunately, theropod teeth are comparatively simple structures, so that a taxonomic assignment is often difficult, especially when more diagnostic material is absent. In such cases, statistical analysis of isolated theropod teeth can often facilitate assignment to a higher taxonomic level , 2, someStatistical identification of isolated theropod teeth has a history of 25 years. Farlow et al.  were theHere, 80 theropod teeth (mainly 19th century collections) from the Late Jurassic of northern Germany are examined; most of them are previously undescribed specimens. They are compared with updated published databases using cladistic analysis and discriminant function analysis (DFA).Saurocephalus monasterii”. Struckmann [Megalosaurus monasterii”. Von Koenen et al. [Megalosaurus from the Gigas-Beds  from the Langenberg Quarry near Goslar (Harz Mountains) were described by van der Lubbe et al. [Among the hitherto described material, the oldest published specimen was portrayed by Graf Georg zu Münster in 1846 [ruckmann , not Winruckmann , as erroruckmann , noticedn et al.  mentioneian, see ) of Holzian, see  also lise et al. .Only limited stratigraphic information is available for most specimens because many were collected from historic sites, which have long been abandoned and in part backfilled. One of the first reference of all localitities described below was reported by Roemer in 1836  in his mAll sites  are locaThe three historic localities in Hannover  yielded 52 of the 80 teeth . These lSerpula sp. They are overlain by various thin-bedded marly layers with occasional stromatolite bioherms [Serpula sp., the ooliths are generally free of invertebrates [One theropod tooth NLMH101379) was collected in Thüste and its stratigraphic level was labeled as “Gigas-Beds”. The limestones (used as building stone) of the still active quarry are now assigned to the “Münder Mergel”- Formation (Tithonian) , 36. Thebioherms . Beside tebrates , 36. Ver9 was colMegalosaurus in Oxfordian sediments of the now abandoned quarry southwest of Marienhagen, but provided no description of the material. The exposed sediments of the active quarry comprise in the lower section thick-banked, partly oolithic and dolomitic limestones that grade into marly layers alternating with thin-bedded partly fossil-rich limestone layers at top of the sequence [The age of the marine carbonates from the Marienhagen locality spans from Oxfordian  to Early Kimmeridgian , 39. Twosequence . The fossequence . VertebrLate Jurassic and Early Cretaceous sediments can be found in the area around the asphalt-rich limestones near Holzen/Ith, which were quarried (now abandoned) for more than 120 years . The KimMegalosaurus and that they derive from the Gigas-Beds. The exposed Gigas-Beds in the Holzen area are now interpreted as Late Kimmeridgian age [Five theropod teeth  from Holzen are labeled “Oxfordium?” and one (MB.R.2800) as “Purbeck/Serpulit”. Von Koenen et al.  mentionegian age .th century at the “Kahleberg” (nowadays: Kahlberg) [Megalosaurus monasterii were found [Megalosaurus monasterii derive from the marly, oolithic Lepidotus-Beds, which were dated with invertebrates as Late Kimmeridgian [Megalosaurus monasterii, Late Kimmeridgian”), these remains probably represent morphotype K (GZG.V.010.334) of this study.Several quarries were active in the 19ahlberg) , 41. There found . The remeridgian . FollowiEuropasaurus and several theropod teeth described herein) were found in bed 83, not in bed 93 as erroneously stated in recent publications [The Langenberg Quarry is a classic and well-studied outcrop exposing excellent sections of shallow marine strata –44. It iications –47. Thisications , 28.Europasaurus holgeri has been found [Theriosuchus [Thalassemys and Plesiochelys [The Langenberg Quarry is the only locality where exquisitely three-dimensionally preserved material of the dwarfed sauropod dinosaur en found –47, whicen found . This inen found , teeth aiosuchus , 51 and iosuchus . Diverseiochelys . In addiiochelys –56, and iochelys , 58.Europasaurus, the general distribution of vertebrate remains in bed 83 is scarce. Skeletal remains were accumulated in certain areas, probably lenses or channels. The bone-bearing sections of bed 83 are usually 30–50 cm thick and also contain a large number of well-rounded micritic intraclasts in all bone-rich areas.Throughout the sequence, changes in water depth and brackish influences are apparent due to sediment composition and invertebrate faunal content, but there is no evidence of subaerial exposure , 44. DesDFMMh/FV: Dinosaurier-Freilichtmuseum Münchehagen/Verein zur Förderung der Niedersächsischen Paläontologie (e.V.), Germany; NLMH: Niedersächsisches Landesmuseum Hannover, Germany; GZG: Geowissenschaftliches Zentrum der Universität Göttingen, Museum, Germany; MB: Museum für Naturkunde, Berlin, Germany; GSUB: Geowissenschaftliche Sammlung der Universität Bremen, Germany; RPM: Roemer- Pelizaeus Museum Hildesheim, Germany; ML: Museu da Lourinhã, Lourinhã, PortugalAL: apical length (in mm); CA: crown angle, using the formula of the law of cosine [CAA: crown apical angle; CBL: crown base length (in mm); CBR: crown base ratio (CBW/CBL); CBW: crown base width (in mm); CDA: crown distal angle; CH: crown height (in mm); CHR: crown height ratio (CH/CBL); DA: distal apical-crown serration count per 5 mm; DC: distal mid-crown serration count per 5 mm; DB: distal base-crown serration count per 5 mm; MA: mesial apical-crown serration count per 5 mm; MC: mesial mid-crown serration count per 5 mm; MB: mesial base-crown serration count per 5 mm; DSDI: denticle size difference index, MC divided by DCf cosine ; CAA: crth century, with the exception of the material from the Langenberg locality , recurvature of the crown and outline of the basal cross-section in morphotype E, F and G; relatively straight distal margin, strongly twisted mesial carinae that terminates at the cervix in morphotype K. The morphotypes were coded  was performed with the free statistics software R  using thThe graphics  were creLiliensternus, Ceratosaurus, Masiakasaurus, Majungasaurus, Duriavenator, Baryonyx, Suchomimus, Allosaurus, Acrocanthosaurus, Carcharodontosaurus, Daspletosaurus, Tyrannosaurus, Deinonychus, Dromaeosaurus, Velociraptor, Troodon, Zanabazar, and Marshosaurus, with recalculated ratio variables CBR, CHR and CA, as during an early stage of this study some incongruences in the dataset were observed. Measurements provided by Rauhut et al. [Proceratosaurus were also included, with missing data (CBW and AL) measured on photographs that were scaled for CBL and CH. The dataset of this study was completed with taxa Hendrickx et al. [Coelophysis, Berberosaurus, Genyodectes, Erectopus, Piatnitzkysaurus, Afrovenator, Dubreuillosaurus, Torvosaurus, an “unpublished megaraptoran”  in these taxa  are taken into account , Neovenry teeth ) of Ceraese taxa . As cutory teeth , 61, so ount see . Applyin results  in a poo results  were traZanabazar. The variables used for the LDA comprise log(x+1) transformed CBW, CH, AL, CHR, MC and DC. The LDA was executed with prior probability set that the groups were treated as equal under the “options” menu; the other settings were retained with default options. To obtain the classification, posterior probability and statistics the corresponding boxes were ticked under the “Export” and “Show” menu. The values returned by the LDA indicate the probability (in percent) that a tooth is correctly assigned to a certain group. In teeth where the posterior probabilities are rather weak, the second rank classification is also indicated. For tooth crowns where MC is not preserved, log(x+1) transformed CBW, CH, AL, CHR, CBR and DC were used. The reclassification rates are 86.87% for the dataset with MC and DC and 82.09% when only DC was taken into account. The variable selection of the two LDA represents the best possible solution for the used dataset regarding reclassification rate. The differences in the reclassification rates indicate that variable MC is crucial in discrimination between theropod teeth based on morphometric data.For the DFA the variables were log transformed to obtain a better normal distribution  using thDubreillosaurus (MNHN 1998–13) a CA of 70.48°. Measuring CA of this tooth  and one from the Gigas-Beds of Thüste (NLMH101379).The specimens of morphotype A are nearly complete, except for NLMH101379, where only the tip is preserved. With a CH of 56.5–77mm, they represent the largest teeth in our study. The crowns are moderately recurved with a CA around 63–64°. The basal cross section is lanceolate with a CBR of 0.5–0.59. The distal carinae run along the midline in distal view, only in GZG.V.010.389 it is displaced labially, and terminates well below the cervix. The non-twisted mesial carinae are centrally positioned on the crowns in mesial view and extend also down to the cervix, except for GZG.V.010.333 where it terminates slightly above. The enamel surface is well preserved and has a braided texture. The denticles are very coarse, DC and MC varies from 6–7 denticles per 5 mm with a DSDI of 0.85 to 1.17. The apical mesial denticles are quadrangular in lateral view and slightly inclined apically. Distal denticles are rather horizontal rectangular, and also slightly inclined apically at midcrown. In GZG.V.010.333 and GZG.V.010.389, this feature varies as some denticles are more perpendicular to the mesial margin. Well-developed interdenticular sulci run diagonally towards the base at midcrown of the distal carinae and become shorter at the basalmost denticles. Except for GZG.V.010.333, the basal denticles are devoid of this feature. The interdenticular sulci along the mesial carinae are less pronounced than that of the distal. They are not preserved due to wear in GZG.V.010.389 and absent at the apex in MB.R.2800. Large, numerous, transversal undulations run mesio-distally on the labial and lingual surface of the crown (to a lesser extent in GZG.V.010.389). The marginal undulations are short, shallow, and mesio-distally oriented. They are present on the mesial side of the teeth, in GZG.V.010.333 also on the distal.Megalosaurus teeth mentioned by von Koenen et al [Torvosaurus, agreeing in part with DFA results . Morphotype A bears very coarse denticles on the mesial and distal carinae . A similar denticle count is seen only in larger teeth of Tyrannosaurus, Torvosaurus and Carcharodontosaurus [Following the collection labels of the specimens, the stratigraphic range of morphotype A spans from the? Oxfordian to the lower Berriasian. No exact localities and stratigraphic positions are recorded for the material. GZG.V.010.333, GZG.V.010.389 and MB.R.2800 were found at Holzen and could represent the en et al . These ben et al . The claTorvosaurus teeth [Torvosaurus tooth from Portugal  ingens from the Late Jurassic (late Tithonian) of Tanzania with similar denticle count and serrated mesial carinae that reach the cervix. Rauhut [Veterupristisaurus milneri, the oldest member of this clade (Kimmeridgian to earliest Tithonian). By comparison, the earliest European occurrences of Carcharodontosauria  the mes  are Neo England  and Conc England . Morphot England . PossiblDFMMh/FV1205, DFMMh/FV1206, GZG.V.010.381, NLMH101375b, cf. RPM.NKP.14356 Morphotype B comprises two teeth from the Langenberg Quarry near Goslar ; two teeth from Marienhagen  and one tooth (NLMH101375b) from Ahlem in Hannover (Kimmeridgian).Morphotype B comprises rather poorly preserved teeth: two casts ; one poorly preserved tooth with a missing tip (GZG.V.010.381); a lower two thirds of a tooth with partial root (NLMH101375b); and a complete tooth (RPM.NKP.14356) where only the labial side is visible as the lingual side is still enclosed in matrix. Morphotype B shows some similarities with morphotype A, such as the distal carinae that terminate well below the cervix; comparatively coarse denticulation; lanceolate outline of basal cross-section; existence of marginal undulations; and well developed interdenticular sulci. However, some differences exist that allow us to separate both morphotypes: the mesial carinae terminate well above the cervix; apical mesial denticles are rather vertical rectangular and perpendicular-oriented to the mesial margin. The mesial denticles are also more chisel-shaped, with narrower interdenticular space and a MC of 7 to 9 denticles. The midcrown denticles (DC 8 to 10) of the distal carinae are perpendicular to distal margin. CH ranges from 41.3–57 and CBR from 0.48–0.53.Torvosaurus and Megalosaurus. The DFA assigns (Afrovenator and Duriavenator. NLMH101375b is classified as Majungasaurus (39.91%), or possibly Duriavenator (26.16%), but as this tooth is very incomplete, the results should be viewed with caution. Comparison of morphotype B with the megalosaurid teeth described by Hendrickx et al. [Torvosaurus [Torvosaurus sp. This interpretation appears probable given the presence of tooth and bone material described as Torvosaurus from the Late Jurassic of Portugal [The cladistic analysis recovers  morphoty assigns  four teex et al.  and Bensx et al.  shows thvosaurus , 63 are Portugal , demonstGZG.V.010.320, GZG.V.010.329, GZG.V.010.344, NLMH101376a, NLMH101377 Morphotype C comprises four teeth from Ahlem, Lindener Berg and Tönniesberg in Hannover (Kimmeridgian). GZG.V.010.329 was collected in Holzen (Late Kimmeridgian).The crowns of morphotype C are almost complete, except for NLMH101376a, NLMH101377, and GZG.V.010.320 were the tips are missing. Mesial denticles are only preserved in GZG.V.010.320 and are quadrangular in lateral view. The distal denticles are horizontal rectangular; their orientation varies from perpendicular to slightly inclined apically. The denticulation is comparatively fine, e.g. in GZG.V.010.329 (CH 30) MC counts 16.25 and DC 16.5. The centered, non-twisted mesial carinae extend down to the cervix. The distal carinae are labially displaced, in GZG.V.010.320 also sigmoid-shaped in distal view and terminate at the cervix. With a CHR of 1.54–1.71 the crowns are rather stout. The basal cross-section is eight-shaped, in GZG.V.010.320 bean-shaped and there is always a shallow depression present on the labial side of the crown. The CBR of 0.41–0.47 denotes morphotype C as labio-lingually compressed. More or less well developed interdenticular sulci are present at the denticles of the distal carinae, as well as pronounced transversal undulations running mesio-distally across the crown.Liliensternus, Berberosaurus, Raptorex and Neovenator. This differs considerable from the results of the cladistic analysis and shows the size dependent classification of the DFA. Only NLMH101377 is classified (63.12%) as Berberosaurus. However, when the second rank is taken into account, GZG.V.010.329 (22.74%) and NLMH101376a (23.9%) are also regarded as Berberosaurus. Morphotype C shows also some similarities with tooth crowns described for the Chinese Late Jurassic theropod Sinraptor, e.g. the eight or bean-shaped basal cross-section, distal carinae that terminate at the cervix. Sinraptor belongs to the family Metriacanthosauridae [Metriacanthosaurus from the Oxfordian of England, however, as no teeth were reported [Ceratosaurus are reported from the Late Jurassic of Europe [incerta sedis.Morphotype C is classified  with a ssauridae , which ireported , 96 for reported  and concf Europe . We regaGZG.V.010.325, GZG.V.010.332, NLMH16480 Morphotype D consists of three teeth; two from Ahlem and Lindener Berg in Hannover (Kimmeridgian) and one from the “?Oxfordian” (probably Kimmeridgian ) of HolzThe teeth of morphotype D are similar to morphotype C . The CH Ceratosaurus are that in morphotype D the mesial carinae terminate well above the cervix [Berberosaurus, but with a poor probability (48.22% and 36.69%). The smallest crown is, however, assigned to Alioramus (42.81%). Similar to morphotype C, morphotype D is considered as Ceratosauria incerta sedis.Given the similarities to morphotype C  it is noe cervix  and the GSUBV4023, GZG.V.010.374, GZG.V.010.377, GZG.V.010.378, GZG.V.010.379, GZG.V.010.380, NLMH101378c, NLMH101380e, NLMH106235c, RPM.NKP.14359 All ten teeth of morphohotype E were collected at the Lindener Berg and Tönniesberg in Hannover (Kimmeridgian).The crowns of morphotype E are with a CH of 8.2–15 rather small and stout (CHR 1.26–1.50). Most obvious character is that the mesial denticles are smaller than the distals; the DSDI ranges from 1.25–2. The distal margin of the teeth varies from relatively straight, so that the apex does not extend beyond the crown base, to recurved (CA 49.36°–62°) in lateral view. The basal cross-section is lanceolate to slightly elliptical (CBR 0.53–0.7); only in GZG.V.010.379 there is lingually a shallow basal depression present. The mesial carinae terminate around midcrown and in NLMH101378c and GZG.V.010.380 they are twisted lingually. Profile and position of the distal carina varies; in some crowns it is offset from the midline and slightly sigmoid-shaped in distal view. Where preserved, the mesial denticles (22.5–35) are vertical rectangular and the apicals are perpendicular-oriented to the mesial margin. The distal denticles (DC 15–20) are horizontal rectangular, mainly perpendicular-oriented and possess an asymmetrically convex distal margin in lateral view. Few weak transversal undulations are present, as well as interdenticular sulci, but only at midcrown of the distal carinae. The enamel surface has a faint braided-oriented texture [Piatnitzkysaurus and Tyrannosauroidea. When the very similar morphotypes F—G are included, or Piatnitzkysaurus was excluded from the analysis, morphotype E is recovered as Tyrannosauroidea. The DFA assigns (Liliensternus (48.83–94.68%); only GZG.V.010.378 and NLMH101380e are classified as Deinonychus (56.86–74.63%) and NLMH106235c as Dubreuillosaurus (32.12%). This is not surprising as Liliensternus and Deinonychus have comparable crown sizes and serration densities, whereas the teeth of basal Tyrannosauroidea  have, by similar crown sizes, either finer or coarser serrations. The results of the cladistic analysis coincide with rare skeletal material described in the fossil record of Europe. This material comprises the basal Tyrannosauroidea Avityrannis [Juratyrant [The cladistic analysis classifies  morphoty assigns  most teetyrannis  from theratyrant , 101 froGZG.V.010.326, GZG.V.010.328, GZG.V.010.335, GZG.V.010.345, GZG.V.010.370, GZG.V.010.371, NLMH101378b, NLMH101380a, NLMH101380b, NLMH106235d, NLMH16416a All eleven teeth of morphotype F were collected at the Lindener Berg and Tönniesberg in Hannover (Kimmeridgian).Similar to morphotype E, morphotype F has mesial denticles larger than the distals (DSDI 1.21–2) and show comparable CBR (0.52–0.70). Where preserved, both morphotypes have similar denticle morphology as well as MC and DC  range. The surface structure is also braided-oriented, with interdenticular sulci present only at midcrown of the distal carinae. The transversal undulations are sparsely distributed. Most obvious differences are that morphotype F is larger (CH 14.7–21.8) and more elongate (CHR 1.54–2.04). There is always a shallow depression present on the labial side and in some crowns also lingually, giving the basal cross-section a bean or eight shape. Some crowns possess a shallow depression that runs along the labial side of the distal carinae. GZG.V.010.335 is the only one tooth of this study where the root is preserved. In lateral view, the root-margins are sub-parallel and not thicker than the base of the crown. There are numerous transversal undulations visible whereas in the tooth crown only few are present. The cross-section at midroot is eight-shaped and the lingual fossa for the erupting tooth is deeper than the labial depression.Deinonychus, Piatnitzkysaurus, Raptorex and Proceratosaurus with an as well variable classification probability (40.99–75.76%). As these results are rather indecisively, a separate DFA and principal component analysis (PCA) for morphotypes E—G  and Dromaeosauridae . The variables used comprise the same as for the main analysis and the DFA was executed with default options (priors =“observed”). This analysis classifies morphotype F as “Tyrannosauroidea” and the classification probabilities are relatively robust (71.88–99.01%). Dromaeosaurid teeth are very similar to crowns of basal Tyrannosauroidea [The cladistic analysis places  morphotyypes E—G  was run ty 40.99–.76%. As auroidea , 103, whty 40.99–.76%. As auroidea . Older rty 40.99–.76%. As ty 40.99–.76%. As auroidea  show simGZG.V.010.318, GZG.V.010.321, GZG.V.010.322, GZG.V.010.323 GZG.V.010.331, GZG.V.010.372, NLMH101376, NLMH101376d, NLMH101376e, NLMH101378a, RPM.NKP.14358 Ten teeth were collected at the Lindener Berg and Tönniesberg in Hannover (Kimmeridgian); GZG.V.010.331 was found in Holzen (Late Kimmeridgian).The teeth of morphotype G are very similar to morphotype F so that the transition is sometimes indistinct. They are of similar size (CH 10.5–26) and possess a comparable elongation of the crowns (CHR 1.62–1.96), as well as distal denticles that are larger than the mesials (DSDI 1.33–1.57). They share a similar CBR (0.54–0.75) but differ from morphotype F in the absence of a labial depression resulting in a lanceolate to ovoid basal cross-section. In some crowns, however, there is also a very shallow lingual depression present so that the basal cross-section is rather bean-shaped. Contrary to morphotype F, the distal carinae are always sigmoid-shaped in distal view and in some crowns they bear interdenticular sulci not only at midcrown but also near the cervix. The distal denticles are quadrangular to slightly horizontal rectangular-shaped and the external margin is asymmetrically convex in lateral view.Piatnitzkysaurus and Tyrannosauroidea. When Piatnitzkysaurus is removed or morphotypes E and F are included then morphotype G is classified as Tyrannosauroidea. The DFA assigns  classification probability. Given the similarities of morphotype G to F it is possible that these teeth belong to one taxon. As in morphotype F, some crowns of morphotype G resemble teeth described from the Kimmeridgian of Portugal [Like morphotype E, the cladistic analysis classifies  these te assigns  morphotyPortugal . For detGSUBV4022, GZG.V.010.369, GZG.V.010.373 All three teeth were collected at the Lindener Berg and Tönniesberg in Hannover (Late Kimmeridgian).The most obvious character of morphotype H is a near circular basal cross-section (CBR 0.79–0.82). In lateral view the distal margin is rather straight so that the apex does not extend beyond the base. Morphotype H comprises rather small teeth (CH 10–17.2) and the CHR ranges from 1.59–2.06. Mesial denticles are not preserved but apparently present as in GZG.V.010.373 faint imprints could be seen where the enamel is broken off. If this interpretation is correct, GZG.V.010.373 has a high DSDI of 1.66. The denticulated distal carinae terminate at the cervix, except for GZG.V.010.373 where it terminates slightly below. The distal denticles resemble those of morphotype E—G and have also a similar serration density (DC 15).Deinonychus (53.73%) and on the second rank to Proceratosaurus (28.71%). The second DFA, with CBR instead of MC, recovers . Morphotype H resembles the premaxillary teeth of Dubreillosaurus in some details, however, differences exists as well, e.g. the presence of interdenticular sulci and asymmetrically convex distal margin of the distal denticles in lateral view. Morphotype H is regarded here as probable mesialmost tooth and could not be classified beyond Theropoda indet.Morphotype H was initially coded as lateral teeth, as it shares many similarities with morphotype G . The only differences are that GZG.V.010.373 has a more circular cross-section (CBR 0.81); GZG.V.010.372 is more elliptical to lanceolate (CBR 0.7) and the distal carina terminates slightly below the cervix. Both crowns are not well-preserved, with, e.g. the mesial denticles missing. The results  of the crecovers  all teetGZG.V.010.319, GZG.V.010.327, NLMH101375a, NLMH101376c, NLMH16416c, cf. RPM.NKP.14357 All six teeth were found in Ahlem, Lindener Berg and Tönniesberg .The tooth crowns (CH 25.5–39) of morphotype I are of a moderate to elongated appearance (CHR 1.97–2.47). They are slightly recurved (CA 55.04°–65.7°) and moderate labio-lingually compressed (CBR of 0.49–0.55) with a lanceolate basal cross-section. The lingual surface is relatively flat, the enamel texture finely braided and well-developed interdenticular sulci are present even at the mesial carinae. The distal carinae extend to the cervix. The profiles of the distal carinae are sigmoid-shaped, in NLMH101376c more straight, and labially displaced in distal view. The non-twisted mesial carinae terminate at midcrown. The midcrown distal denticles are horizontal rectangular-shaped and perpendicular oriented to the distal margin in lateral view, whereas the mesials are rather quadrangular. The interdenticular space of the midcrown distal denticles is broad. With a DSDI of 1.07–1.15 the distals are slightly larger than the mesial denticles. GZG.V.010.327 represents the most complete crown of morphotype I, whereas in RPM.NKP.14357 the lingual side is still enclosed in matrix. In NLMH101376c there is a part of the lingual base of the crown and the mesial carina missing.Sinraptor, a theropod of the family Metriacanthosauridae [Metriacanthosaurus [Sinraptor, several differences exist, such as the outline of the basal cross-section and the rather straight and non-twisted distal carinae in distal view. The Late Jurassic taxon Ceratosaurus from North America, also reported from Europe [Ceratosaurus crowns [Neovenator and GZG.V.010.327 (49.49%), NLMH101376c (with CBR instead of MC 17.12%) and RPM.NKP.14357 (39.77%) as Erectopus. The DFA results support therefore the assignment of the cladistic analysis of morphotype I to Allosauroidea, if Erectopus remains within the Allosauroidea [Saurocephalus Monasterii” [Megalosaurus [The cladistic analysis places  morphotysauridae  from thehosaurus , 96 fromm Europe , possessm Europe , 105, alm Europe . Howeverauroidea . Followiauroidea , one tooasterii” , which wlosaurus .GZG.V.010.330, GZG.V.010.392, NLMH106235b Morphotype J comprises two teeth from the Lindener Berg and Tönniesberg of Hannover (Kimmeridgian) and one tooth from Holzen, labeled as “?Oxfordian” .The specimens representing morphotype J are similar to the smaller teeth of morphotype I regarding the morphometric measurements CH (29.8–30.5), CBR (0.54–0.58), CHR (1.85–2.14) and DSDI (1–1.2). They differ from morphotype I in possessing a slightly narrower interdenticular space, absence of interdenticular sulci at the lower part of the distal carinae which terminate well below the cervix, and slightly larger mesial denticles at midcrown that become coarser towards the apex.Neovenator (41.27 and 46.97%) and NLMH106235b to Piatnitzkysaurus (50%), however, with a rather poor posterior probability. The cladistic analysis places . Madsen [Marshosaurus are rather incomplete, hampering further comparisons. Morphotype J is treated here as pertaining to a member of the clade Megalosauroidea. The referral to Marshosaurus is only tentatively and requires further description of its dentition.The DFA classifies  two teets places  morphotyhosaurus , a membehosaurus : A slend. Madsen  publisheGZG.V.010.334 One complete tooth collected at the Kahlberg near Echte (Late Kimmeridgian)The crown high measures 37.3. With a CHR of 1.96 and a CBR of 0.6 it is of a stout appearance. The denticle counts are 11 for DC and 12 for MC (DSDI 1.09). The basal cross-section is lanceolate and the distal margin only weakly recurved in lateral view. The mesial denticles are worn. The denticulated mesial carina runs at the first third of the apex straight and twists then strongly lingually near the base in mesial view. The distal carina is labially displaced and slightly sigmoid-shaped in distal view. The somewhat worn distal denticles are inclined apically and horizontal rectangular in lateral view and become progressively larger from the base to the apex. The interdenticular space of the distal denticles is rather wide and bears well-developed, basally curving interdenticular sulci at the entire carina. Contrary to most other morphotypes, the enamel surface is smooth to fine irregularly textured.Megalosaurus (26.98%) and on the second rank to Genyodectes (21.36%). GZG.V.010.334 shows many similarities with teeth (ML327 and ML966) described by Hendrickx and Mateus [Allosaurus (43.43%). Hendrickx and Mateus [Allosaurus [Allosaurus they are smaller apically than at midcrown. Despite of the noticeable similarity of morphotype K with abelisaurid teeth, we consider it plausible to regard GZG.V.010.334 as Allosaurus sp. until more complete material is found.The cladistic analysis classifies  morphotyodectes 2.36%. GZGodectes 2.36%. GZGd Mateus  and Lated Mateus . Howeverd Mateus . Morphotodectes 2.36%. GZGlosaurus , namely NLMH16416b, NLMH106235a Both teeth were found at the Tönniesberg (Late Kimmeridgian) in Hannover.Morphotype L comprises a tooth fragment (NLMH16416b) and a tooth crown (NLMH106235a) with the apical half of its distal part missing. Because NLMH16416b is very fragmentary, only NLMH106235a is described. With a CH of 40.7 it is one of the larger tooth of this study. NLMH106235a is elongated (CHR 2.39) with a subcircular to elliptical basal cross-section (CBR 0.72). The non-twisted mesial carina terminates around midcrown. The relatively coarse mesial denticles (MC 8) are worn. The preserved part of the distal carina is centrally positioned in distal view and terminates at the cervix. With a DC of 9 the distal denticles are smaller (DSDI 0.88) than the mesials and of quadrangular to somewhat subrectangular shape in lateral view. Interdenticular sulci are present and the interdenticular space is rather wide.Allosaurus. This is not a surprising result as Allosaurus teeth are comparable regarding morphometric measurements, but they differ from morphotype L in possessing often twisted and displaced mesial and distal carinae, distal carinae terminating well below the cervix and a more irregular surface texture [Torvosaurus and Dubreillosaurus are either larger or smaller than NLMH106235a. Confirmed by the results of the cladistic analysis morphotype L is treated as mesialmost tooth of a megalosaurid theropod that shows great overlap in characteristics with a tooth (ML962) described from Late Jurassic of Portugal [Torvosaurus. It is possible that the teeth of morphotype L were the mesialmost of morphotype B and therefore pertain to one taxon.With a subcircular to elliptical basal cross-section NLMH106235a resembles in most details crowns described as megalosaurid mesialmost teeth . The claGZG.V.010.399 GZG.V.010.399 was collected at the Lindener Berg in Hannover (Kimmeridgian).With a CH of 7 mm morphotype M is a relatively small tooth. Most obvious character of GZG.V.010.399 is a series of longitudinal grooves and ridges (flutes ) at the Proceratosaurus teeth. This result is supported by the DFA  are restricted to the crown base [Paronychodon  possesses teeth that are often devoid of denticles [Paronychodon-like teeth from the Lower Cretaceous of Spain [Paronychodon from the Late Cretaceous of Spain [Euronychodon [Paronychodon [Velociraptor these structures are rather rounded ridges [Ceratosaurus premaxillary teeth they are only present on one side [Ostafrikasaurus [Ostafrikasaurus in possessing considerably finer denticles; distinct size difference between the mesial and distal denticles (DSDI 1.67); smooth surface structure and strongly curved, displaced carinae. The assignment of GZG.V.010.399 by the DFA and cladistic analysis to basal Tyrannosauroidea would be supported by skeletal material of the contemporary taxa Aviatyrannis [Juratyrant [Aviatyrannis [GZG.V.010.399 possesses a unique combination of features: fine lingual and coarser fluted labial side, restricted to the upper two thirds of the crown; mesial denticles considerable smaller than the distals and a smooth surface structure. For the cladistic analysis morphotype M was coded as mesialmost. When the basal cross-section is coded as subcircular this results  in a str the DFA  which asown base . Fluted enticles  and whenenticles ; Paronycof Spain , 114 andof Spain , 116, thof Spain  and Portnychodon , transfed ridges ; in Ceraone side ; morphotkasaurus  in that ratyrant . The absDFMMh/FV382, NLMH105652 Morphotype N comprises two teeth from the Langenberg quarry near Goslar . DFMMh/FV382 was like morphotype O—R included in the study of van der Lubbe et al. .Morphotype N comprises two small teeth (CH 4.9 and 10.2) that are rather stout (CHR 1.63 and 1.62) in lateral view. They have a lanceolate basal cross-section (CBR 0.50 and 0.52). The enamel surface texture of DFMMh/FV382 is obscured by hardener but seems to be braided-oriented near the apex. Morphotype N has faint, numerous (only few in NLMH105652) transversal undulations, as well as faint interdenticular sulci at the distal and mesial denticles (in DFMMh/FV382 obscured by hardener). The mesial carina terminates at midcrown and twists slightly lingually in mesial view in DFMMh/FV382, whereas in NLMH105652 it is centrally positioned on the crown. The vertical rectangular mesial denticles (MC 27.5 and 22.5) are perpendicular oriented to the mesial margin with a parabolic to flattened external margin. The distal carinae are slightly sigmoid-shaped and located at the midline in distal view. The rounded distal denticles (DC 30 and 25) are more quadrangular-shaped and perpendicular oriented to the distal margin.Dromaeosaurus) [Liliensternus (56.46%) and NLMH105652 as Velociraptor (82.19%). The cladistic analysis recovers  , 61. Nonrecovers  morphotyurus sp.  where, iDFMMh/FV530 Morphotype O was collected at Langenberg/Goslar .With a CH of 7.1 mm, DFMMh/FV530 is not a large tooth. It is very stout (CHR 1.2) and strongly recurved (CA 39.32°). The CBR (0.47) denotes this tooth as somewhat flattened and the basal cross-section is rather indistinctly lanceolate to eight-shaped, but the depressions, if any, on both sides are very inconspicuous. The enamel texture is obscured by hardener and seems to be braided-oriented at the apical part. Transversal undulations are like in DFMMh/FV382 numerous but faint and interdenticular sulci are present at the midcrown distal denticles. The non-twisted mesial carina terminates at midcrown where the crown becomes more recurved. There is a flat surface adjacent to the lingual side of the mesial carina. The apically inclined denticles (MC 30) are quadrangular-shaped with an asymmetrically convex external margin. The distal carina is centrally-positioned and only slightly bowed in distal view. The orientations of the midcrown distal denticles (DC 27.5) are indistinct because of the strong recurvature of the crown. They are horizontal rectangular-shaped and their external margin is slightly obscured by wear, but seems to be more asymmetrically in lateral view.Sciurumimus [Proceratosaurus [Liliensternus (84.79%) and on the second rank to Proceratosaurus (13.7%). For the cladistic analysis morphotype O was coded with an eight-shaped basal cross-section despite the fact that this character is not obvious in this tooth. TNT recovers  of Langenberg/Goslar.DFMMh/FV658 is a relatively complete tooth, with only the enamel at the apex and small pieces of the mesial and distal carina missing. The tooth has an irregular to smooth enamel texture. Faint interdenticular sulci are present at midcrown and basal distal denticles. The transversal undulations are sparsely distributed but well visible in lateral view. With a CH of 21.4 it is the largest crown collected at Bed 83 from the Langenberg/Goslar quarry. The CBR is 0.58 and the CHR (2.43) denotes morphotype P as slender and elongated. The basal cross-section is lanceolate. The mesial carina starts straight at the apex and twists then lingually at midcrown in mesial view. Apical denticles are not preserved on the mesial carina; the midcrowns (MC 25) are quadrangular-shaped and perpendicular-oriented to the mesial margin. The distal carina is centrally positioned and shows a sigmoid-shaped curvature in distal view. The midcrown denticles (DC 25) are more horizontal rectangular than quadrangular-shaped and the orientation is variable with some perpendicular-oriented and others slightly inclined apically.Proceratosaurus. Proceratosaurus teeth [Eocarcharia and therefore within Allosauroidea. This classification would be supported by skeletal material of the contemporaneous european genera Metriacanthosaurus [Allosaurus from Portugal [Metriacanthosaurus no teeth were reported [Allosaurus material only the posteriormost tooth of the maxilla is preserved, but not described [Allosaurus crowns of North America described by Hendrickx and Mateus [The result of the DFA  is puzzlus teeth  are smalhosaurus , 96 fromPortugal . For Metreported , 96 and escribed , preventd Mateus  only in Velociraptor, Itemirus, Adasaurus, Tsaagan and Linheraptor [Velociraptor and Tsaagan were included in the cladistic analysis presented here .Morphotype Q has a CH of 10.8, is more laterally compressed (CBR 0.48) and the basal cross-section is eight-shaped. The surface texture is braided-oriented and the presence of faint interdenticular sulci is slightly obscured by hardener, as well as the rather inconspicuously transversal undulations. The non-twisted mesial carina terminates slightly above the cervix. The mesial denticles (MC 25) are worn and exhibit a rounded cross-section in mesial view. At two thirds of the crown they are rather quadrangular-shaped in lateral view. The distal carina is straight and somewhat displaced in distal view. The denticles (DC 22.5) are horizontal rectangular-shaped and perpendicular-oriented to slightly inclined apically in lateral view.Liliensternus (94.73%). The cladistic analysis is more ambiguous and places (incerta sedis).The results of the DFA  allocated places  DFMMh/FVDFMMh/FV790.5, NLMH105654 Morphotype R comprises two teeth from Bed 83 (Kimmeridgian) of Langenberg/Goslar.Morphotype R represents the smallest (CH 2.8–3) teeth in our study. NLMH105654 was recently collected by screen-washing for mammal teeth . As it iProceratosaurus mesialmost teeth (92.85%). However Proceratosaurus mesialmost teeth exhibit basal striations [Juravenator [Compsognathus [Sciurumimus [The DFA assigns  morphotyriations , 61, 75,avenator –125, Comognathus  and Sciuurumimus  from theurumimus  but diffDFMMh/FV.CL340L, DFMMh/FV.CL340S, DFMMh/FV707.1, DFMMh/FV1194, DFMMh/FV1202, DFMMh/FV1203, DFMMh/FV1204, NLMH105653 .DFMMh/FV.CL340L is a very incomplete tooth crown (CH ~30) from the Kimmeridgian of the Langenberg Quarry. The apical one third of the mesial carina and most of the distal, except for some denticles near the cervix, are worn down. However, the surface texture of both sides is well preserved and shows a braided-oriented structure with well-developed interdenticular sulci and transversal undulations. At the basal mesial carina there are also short marginal undulations present. The few preserved distal denticles are perpendicular oriented to the distal margin and have a rounded to parabolic external margin in lateral view. With a DSDI of 1 the mesial denticles are as large as the distals. The mesial carina is centrally positioned in mesial view and the distal slightly displaced labially in distal view. The basal cross-section is lanceolate with a CBR of 0.55.Ceratosaurus premaxillary (48.44%) and on the second rank to Duriavenator (33.54%). The second rank classification coincides with the cladistic analysis that recovers (The DFA classifies  DFMMh/FVrecovers  this tooDFMMh/FV.CL340S was collected in the Langenberg/Goslar Quarry (Kimmeridgian). The tooth is well preserved, except for the worn mesial denticles and apical part of the crown. There is a deep, tongue-shaped depression that terminates 3.5 mm above the cervix resulting probably from tooth contact with the opposite jaw and as the mesial and distal carinae are centrally positioned on their margins it is uncertain on which side it is located. DFMMh/FV.CL340S is relatively small (CH ~15) and has a stout appearance (CHR 1.39) in lateral view. The distal carina terminates well below the cervix. DFMMh/FV.CL340S shows many similarities with DFMMh/FV.CL340L, e.g. nearly identical CBR (0.56); the braided-oriented surface texture; well-developed transversal undulations; position of the mesial carinae on the crown and perpendicular oriented, rounded distal denticles.Dubreillosaurus (78.07%). The cladistic analysis supports this assignment (The DFA classifies  DFMMh/FVsignment  and showDFMMh/FV707.1 was collected in the Langenberg/Goslar quarry (Kimmeridgian) and also included in the study of van der Lubbe et al. . It lackBased on the limited morphometric data, DFMMh/FV707.1 was only included in the cladistic analysis that recovers  it as NeEuropasaurus from the Langenberg Quarry  and found during preparation. The lingual side is still enclosed in matrix and the visible part is highly coated with glue and hardener. The mesial and distal denticles are worn, except for some denticles located near the lower third of the distal carina, so only limited data could be recovered from DFMMh/FV1194. It is a small (CH 7.5), elongated (CHR 2.5) and recurved tooth crown. The distal denticles (DC 50) are as large as the mesials (MC 50). The distal carina has a sigmoid shape in distal view and the non-twisted mesial is centrally positioned on the mesial margin. The preserved basal distal denticles are quadrangular-shaped and have a symmetrical, parabolic external margin in lateral view.This tooth was associated with bone material of the sauropod Juravenator [Compsognathus [Sciurumimus [Eodromaeus. Given the limited data available for this tooth it could not be classified beyond Theropoda indet.A DFA was not conducted for DFMMh/FV1194 because of the missing CBW. The theropods avenator –125, Comognathus  and Sciuurumimus  from theEuropasaurus bone material. It is partly enclosed by bone, however given the fragmentary state of this specimen it must remain unclear if this bone fragment is part of the jaw in which the tooth was embedded. Most obvious feature of DFMMh/FV1202 is that the mesial margin bears a non-serrated ridge that twists lingually near the base. However, at midcrown of the carina there are three bumps visible so it remains unclear if this feature is a result of imperfect preservation. The distal carina bears well-developed, quadrangular-shaped denticles, with a symmetrical, parabolic distal margin in lateral view. The cervix is not visible as the base of the tooth is broken and the distal carina still enclosed in matrix. The distal margin of DFMMh/FV1202 is straight in lateral view. The surface texture is braided-oriented and there are no transversal undulations and interdenticular sulci visible.With a CH of 3.5 mm, DFMMh/FV1202 is the second smallest tooth of this study and was found, like DFMMh/FV1194, during preparation of The rather unserrated state of the mesial carina coincides with teeth described for the southern German theropods Juravenator –125, ComDFMMh/FV1203 was collected from Bed 56  of the LProceratosaurus premaxillary (68.43%) and on the second rank as Proceratosaurus lateral (17.3%). The cladistic analysis recovers  tooth is more complete than the previously described specimens; only the apex and small pieces near the base missing. It was also collected in the Langenberg Quarry. The mesial denticles are heavily worn and the non-twisted mesial carina terminates at midcrown. The mesial denticles (MC 27.5) are slightly smaller than the distals (DC 25). The distal denticles are horizontal rectangular-shaped, with a parabolic to rounded distal margin and perpendicular-oriented in lateral view. The distal carina is rather straight and offset from the midline in distal view. The surface structure is braided-oriented and there are only few transversal undulations visible. The interdenticular space is rather narrow and there are also faint interdenticular sulci present on the lingual side.Liliensternus (50.18%) and on the second rank similar to Proceratosaurus (24.07%). The cladistic analysis recovers . With the strongly twisted carina and elliptical to subcircular basal cross-section NLMH105653 was coded as mesialmost [Juravenator [Compsognathus [Sciurumimus [Compsognathus [The DFA classifies  NLMH1056sialmost  for the avenator –125, Comognathus  and Sciuurumimus . With thognathus , howeverFor the DFA we confined the number of variables and from the known ratio variables  only CHR was included instead its dependent CBL as it results in a higher reclassification rate. Keeping CBL together with CHR does not improve the results in this study. A higher number of included variables do not mean better resolution of the taxa. This is shown when a DFA on our dataset was run with all known variables  includeda priori assigned to different morphotypes because of their characteristics and this view is supported by the reclassification table of a DFA , comparable CBR, interdenticular sulci present only at midcrown of the distal carinae and a similar surface structure. Because of this resemblance it could be assumed that these morphotypes represent the same taxon. The differences in morphology could well represent tooth position and/or ontogenetic change, with the small teeth of morphotype E probably located in more distal position of the maxilla and dentary.Allosaurus [Torvosaurus as apex predator. The similar size range of the northern German theropod fauna suggests that at least during part of the Late Jurassic (Late Oxfordian -Tithonian) large land masses existed that supported a comparable theropod fauna.Jurassic and Cretaceous theropod guilds are often dominated by a diversity of large-bodied theropods from several clades , 132. ThTorvosaurus, Ceratosaurus and Allosaurus. This is further supported by the occurrence of basal Tyrannosauroidea remains in the Morrison Formation, Portugal and England [The ecological sympatry of the theropod fauna from the Morrison Formation of North America and Portugal is well established , 115 wit England , 101. Th England  and seemThe similarities of morphotype A with teeth tentatively assigned to carcharodontosaurid remains of the Late Jurassic of Tanzania  and morpVeterupristisaurus from the Late Jurassic of Tendaguru suggests that there could have been also faunal exchange between Northern Germany and the Southern Hemisphere. Further description of the Tendaguru material is required to support this assumption. With a potentially Late Kimmeridgian age it was roughly contemporaneous to Veterupristisaurus the oldest member of this clade [Megalosaurus(?) ingens [Veterupristisaurus is only tentatively [The resemblance of morphotype A with teeth referred to the carcharodontosaurid is clade . However) ingens , 90 to Vtatively  and coulSciurumimus, Compsognathus and Juravenator are small and could easily be overlooked when embedded in matrix. The theropod fauna from Northern Germany shares many similarities with specimens described from Portugal  and the North American Morrison Formation (morphotype J). This could imply that at least some faunal exchange via temporary land connections between these localities existed in the Late Jurassic.This study reveals ecologic sympatry of several major clades of Theropoda in the Late Jurassic of Northern Germany. Basal Tyrannosauroidea, Allosauroidea, Megalosauridae and probably Ceratosauria can be established with a high probability. The presence of Dromaeosauridae  could noS1 AppendixMorphometric measurements, character codings of the morphotypes and results of the DFA and classification for 80 theropod teeth from Northern Germany. Character list adopted of Hendrickx and Mateus , 61.(XLS)Click here for additional data file.S2 AppendixModified datamatrix used for the cladistic analysis that is based on the supermatrix of Hendricks and Mateus .(TNT)Click here for additional data file.S3 AppendixCompilation of changed character codings for the data- and supermatrix of Hendrickx and Mateus .(DOC)Click here for additional data file.S4 AppendixResults cladistic analysis of the morphotypes following protocol of Hendrickx and Mateus , 61.(DOC)Click here for additional data file.S5 AppendixModified datamatrix of Hendricks and Mateus  with den(TNT)Click here for additional data file.S6 AppendixDataset with morphometric measurements for the DFA. This dataset comprises measurements of theropod teeth provided by Smith and Lamanna , Hendric(XLS)Click here for additional data file.S7 AppendixReduced morphometric dataset for the comparison of overlapping taxa of Smith and Lamanna  and Hend(XLS)Click here for additional data file.S8 AppendixDFA and PCA results for morphotype E–G. Dataset that contains only taxa with a DSDI of >1.2.(XLS)Click here for additional data file.S9 AppendixDFA results for morphotype K with isolated teeth (ML927 and ML966) described by Hendrickx and Mateus  included(XLS)Click here for additional data file.S10 AppendixDFA and PCA results of the morphotypes.(XLS)Click here for additional data file.S11 AppendixMB.R.2800 of morphotype A in 3D-View.(ZIP)Click here for additional data file.S12 AppendixGZG.V.010.381 of morphotype B in 3D-View.(ZIP)Click here for additional data file.S13 AppendixNLMH101376a of morphotype C in 3D-View.(ZIP)Click here for additional data file.S14 AppendixGZG.V.010.325 of morphotype D in 3D-View.(ZIP)Click here for additional data file.S15 AppendixGZG.V.010.379 of morphotype E in 3D-View.(ZIP)Click here for additional data file.S16 AppendixNLMH101380a of morphotype F in 3D-View.(ZIP)Click here for additional data file.S17 AppendixNLMH101378a of morphotype G in 3D-View.(ZIP)Click here for additional data file.S18 AppendixGZG.V.010.373 of morphotype H in 3D-View.(ZIP)Click here for additional data file.S19 AppendixGZG.V.010.327 of morphotype I in 3D-View.(ZIP)Click here for additional data file.S20 AppendixNLMH106235b of morphotype J in 3D-View.(ZIP)Click here for additional data file.S21 AppendixGZG.V.010.334 in 3D-View.(ZIP)Click here for additional data file.S22 AppendixNLMH106235a of morphotype L in 3D-View.(ZIP)Click here for additional data file.S23 AppendixGZG.V.010.399 in 3D-View.(ZIP)Click here for additional data file.S24 AppendixDMMhFV382 in 3D-View.(ZIP)Click here for additional data file.S25 AppendixDMMhFV530 in 3D-View.(ZIP)Click here for additional data file.S26 AppendixDMMhFV658 in 3D-View.(ZIP)Click here for additional data file.S27 AppendixDMMhFV383 in 3D-View.(ZIP)Click here for additional data file.S28 AppendixDMMhFV709.1 of morphotype R in 3D-View.(ZIP)Click here for additional data file.


Artemisia asiatica extract and eupatilin in kidney epithelial (LLC-PK1) cells. Although cisplatin is effective against several cancers, its use is limited due to severe nephrotoxicity. Eupatilin is a flavonoid compound isolated from the Artemisia plant and possesses antioxidant as well as potent anticancer properties. In the LLC-PK1 cellular model, the decline in cell viability induced by oxidative stress, such as that induced by cisplatin, was significantly and dose-dependently inhibited by the A. asiatica extract and eupatilin. The increased protein expressions of phosphorylated JNK and p38 by cisplatin in cells were markedly reduced after A. asiatica extract or eupatilin cotreatment. The elevated expression of cleaved caspase-3 was significantly reduced by A. asiatica extract and eupatilin, and the elevated percentage of apoptotic cells after cisplatin treatment in LLC-PK1 cells was markedly decreased by cotreatment with A. asiatica extract or eupatilin. Taken together, these results suggest that A. asiatica extract and eupatilin could cure or prevent cisplatin-induced renal toxicity without any adverse effect; thus, it can be used in combination with cisplatin to prevent nephrotoxicity.The present study investigated the renoprotective effect of an  Cisplatin is a potent chemotherapeutic agent for the treatment of multiple human malignancies , 2. It a Silybum marianum was found to attenuate nephrotoxicity induced by gentamicin in dogs , where A0 is the absorbance of the control (without extract) and A1 is the absorbance in the presence of the extract.The radical scavenging activity ofd method . The finμg/lane) were separated by electrophoresis in a precast 4–15% Mini-PROTEAN TGX gel  blotted onto PVDF transfer membranes as reported previously [Proteins  . Propidip values of less than 0.05 were considered statistically significant.Statistical significance was determined through analysis of variance (ANOVA).  A. asiatica and eupatilin were tested using DPPH, a stable free radical. DPPH decolorizes in the presence of antioxidants. The scavenging ability of A. asiatica and eupatilin was represented by a line diagram and compared with vitamin C ( A. asiatica. As shown in p = 0.0004) after 25 μM cisplatin treatment. However, pretreatment with the A. asiatica extract and eupatilin markedly restored cell viability to 80 and 82%, respectively, in a dose-dependent manner  and eupatilin (10 and 50 μg/mL) treatment. As shown in  A. asiatica and eupatilin treatments. In addition, the elevated protein expression of cleaved caspase-3 was also markedly reduced by A. asiatica and eupatilin treatments. ERK protein expression in the LLC-PK1 cells was slightly decreased by cisplatin treatment and increased by A. asiatica and eupatilin treatment; however, the differences were not of significant effect. A. asiatica extract and eupatilin on apoptosis in LLC-PK1 cells. As shown in  A. asiatica extract (p = 0.008) and more significantly eupatilin (p = 0.003). The elevated percentage of apoptotic cells after cisplatin treatment was markedly decreased after cotreatment with the A. asiatica extract and eupatilin (upatilin . A. asiatica extract and eupatilin against cisplatin-induced nephrotoxicity was tested using LLC-PK1 cells, which are the most vulnerable renal tubular cells to oxidative stress [ A. asiatica. In addition, pretreatment with the A. asiatica extract and eupatilin markedly ameliorated reduced LLC-PK1 cell viability by cisplatin in a dose-dependent manner.The protective effect of thee stress , 28. It e stress –31. Our  A. asiatica extract or eupatilin cotreatment. To further investigate the ability of A. asiatica and eupatilin to prevent apoptosis, we measured the expression levels of cleaved caspase-3, known as an index of apoptosis, in the kidney. As shown in the results, A. asiatica and eupatilin significantly reduced the expression of cleaved caspase-3. These results suggest that cisplatin-induced increases in the ROS level activate MAPKs in LLC-PK1 cells, whereas the A. asiatica extract and eupatilin inhibit the activation of p38 and JNK.It has been reported that ROS act as a second messenger initiating signal transduction cascades, including the MAPKs signaling pathway , 33. The A. asiatica extract and eupatilin.Cisplatin-induced renal cell damage is dependent on apoptosis induced by DNA damage , 37. Apo A. asiatica extract can ameliorate nephrotoxicity in LLC-PK1 cells. Eupatilin serves as one of the major components by blocking the MAPKs-caspase-3 signaling cascade.In conclusion, our results suggest that the