LRRFIP1, RCAN1 and RHOBTB1) and IF  have an important role to play in muscle cell degradation, development and motility. In general, transcripts were found for both traits with biological process GO terms that were involved in pathways related to protein ubiquitination, muscle differentiation, lipids and hormonal systems. Our results reinforce the biological importance of these known processes but also reveal new insights into the complexity of the whole cell muscle mRNA of Nelore cattle.Transcript data obtained by RNA-Seq were used to identify differentially expressed alternatively spliced genes in ribeye muscle tissue between Nelore cattle that differed in their ribeye area (REA) or intramuscular fat content (IF). A total of 166 alternatively spliced transcripts from 125 genes were significantly differentially expressed in ribeye muscle between the highest and lowest REA groups (p ≤ 0.05). For animals selected on their IF content, 269 alternatively spliced transcripts from 219 genes were differentially expressed in ribeye muscle between the highest and lowest IF animals. Cassette exons and alternative 3′ splice sites were the most frequently found alternatively spliced transcripts for REA and IF content. For both traits, some differentially expressed alternatively spliced transcripts belonged to myosin and myotilin gene families. The hub transcripts were identified for REA ( RNA sequencing (RNA-Seq), is an accurate method for measuring gene expression and can measure all genes in the transcriptome at the same time. This technology could be used to screen the expression of functional candidate genes and identifying essential molecular processes creating a diversity in pathways leading to different tissue phenotypes. One of the usages of RNA-Seq is the identification of alternative splicing events4.The alternative processing of messenger RNAs (mRNAs) produces several transcripts and protein isoforms that have similar or quite different roles in mammalian tissues5 which may be classified into: cassette, mutually exclusive and coordinate cassette exons; alternative 5′ and 3′ splice sites; intron retention; or alternative first and last exon events7. The most common alternative splicing events in mammals are cassette exon and 3′ or 5′ alternative splice sites. Intron retention is a rare alternative splicing event in mammals8.The spliceosomal mechanism may produce several isoforms by interpreting the limits between exon and intron in many different ways. For the most part, these distinct interpretations, fall into subgroups of alternative splicing events5. Several studies have reported the frequencies of different types of alternative splicing events in different species10. In bovine, alternative splicing events have been associated with variation in mastitis susceptibility2, embryonic development11 and muscle development12. Currently, knowledge concerning the molecular mechanisms regulated by alternative splicing events, especially in the context of individual differences in carcass and meat traits in bovine, is limited.Whole transcriptomics analysis through splicing-sensitive identified from RNA-Seq or microarrays dataset, provides the capacity to gain an accurate and deep understanding of the contribution of distinct alternative splicing mechanisms to the developmental specific gene expression programs or establishment of tissue13. Identifying and selecting animals with REA genetic advantages could improve carcass quality, thereby increasing the production of high commercial value meat cut. One of the meat quality traits is the intramuscular fat (IF) content, it is representing the sum of triglycerides and phospholipids; it is, the amount of fat stored between inside muscle cells or muscle fibers. The extent of IF content of the ribeye muscle is a primary determinant of meat palatability and consumer satisfaction with the eating experience. Muscle lipid traits, which determine meat flavor and contribute to beef color, can influence the juiciness and tenderness of the meat14.All along the growth process, the animal's weight and body size increase, and the proportion of tissue, mainly the muscle tissue proportion, changes. Ribeye muscle area (REA) in conjunction with carcass weight provides an indication of carcass muscularity and meat yieldREA development and IF content are polygenic traits controlled by multiple genes directly (or not) related in muscle and fat biological. However, some genes and biological process that have yet to be elucidated, particularly alternatively spliced genes that are cell-tissue specific. It could play an essential role in overall biological systems. Most of the Brazilian herd is a composite of Zebu breeds, notably among them, the Nelore breed that has been shown to be efficient in terms of rusticity and adaptability to the Brazilian pasture conditions, in addition to presenting excellent maternal ability. The principal bovine breed in Brazil is Nelore cattle, because of its strategic importance to Brazil's GDP (Gross Domestic Product) as the one of the world’s biggest exporter of beef. Nelore cattle became more commercially competitive, then cattle ranchers and researchers have been looking for novel strategies to increase and improvement production, as example, the improvement of different carcass and meat traits.Zebu animals, as Nelore breed, show a higher frequency of genes/alleles that are unfavorable to carcass and beef quality traits, such as REA development and IF content, when compared to Taurine breeds. Hence, understanding the functional and biological process from spliced genes that may control REA development and IF content in Nelore cattle is a compelling issue in meat science because this knowledge is not yet clear. Wherefore, we used RNA-Seq approach to find candidate genes that produced alternatively spliced transcripts that were differentially expressed in the Nelore’s ribeye muscle that differed for REA and IF content.Table 15 reports, on overage, 65% and 22% of reads were mapped within the exonic and intronic regions of the genes, respectively. Another 13% of reads were mapped to intergenic regions. Mean of number of reads with splice junctions was approximately 16.8 million reads. Splicing junction analysis showed, approximately, 24% novel, 66% known and 10% partly known splicing junctions  which are specific for RNA-Seq data were summarized in Supplementary Figures There were 166 alternatively spliced transcripts produced from 125 genes that were differentially expressed between the high ribeye area (HREA) and low ribeye muscle area (LREA) groups , myotilin (MYOT) and myomesin (MYOM2) have an important role in the stability of thin filaments along muscle contraction. The transcript isoforms produced by these gene families differ by cassette exon, intron retention, alternative 3′ and 5′ splice sites events  produced DAS transcripts with alternative 3′ splice sites. Solute carrier gene family play roles as transporters16. Among DAS transcripts, we found BOLA-DOA . In cattle, BOLA is a major histocompatibility complex class II gene that exhibits very little sequence variation, especially at the protein level17.Some DAS transcripts belong to important gene families, such as myosin  terms for the DAS genes identified in the pairwise comparison is summarized in Supplementary Table CAMK2B)—represented by a novel cassette exon transcript isoform—and Adenylate Cyclase 2 (ADCY2)—represented by novel alternative 3′ splice site isoform—were present in the pathways mentioned above and also in six other pathways ; oxytocin signaling (bta04921) and inflammatory mediator regulation of TRP channels (bta04750). Calcium/Calmodulin Dependent Protein Kinase II Beta  are isolated, that is, at that moment they are not being co-expressed with the set of genes. From co-expression network the hub DAS genes were predicted and then, the top three hub DAS genes were chosen to define the subnetwork18  involving 65 genes (nodes) for REA, density = 0.030 and clustering coefficient = 0.130 for REA Fig. A. Network18 Fig. B. The suLRRFIP1), Regulator of Calcineurin 1 (RCAN1) and Rho Related BTB Domain Containing 1 (RHOBTB1). The high interconnection between hub genes and nodes within network, have been shown to be biologically significant19. The hub DAS genes identified, could have putative functions as regulators on genes that play a role in development and growth of muscle cells. In general, LRRFIP1, RCAN1 and RHOBTB1 have functions in the proliferation of smooth muscle cells20, and interact with the calcineurin A21 and actin filament systems22, respectively.We predicted three top hub DAS genes: LRR Binding FLII Interacting Protein 1  alternatively spliced transcripts from 219 genes were differentially expressed between highest intramuscular fat (HIF) and lowest intramuscular fat (LIF) animals  and myotilin (MYOT) gene families were among the DAS transcripts. These genes, produced transcripts that differed by alternative first exons, alternative 3′ splice sites, alternative 5′ splice sites and retained introns. In addition to these, members of the myozenin (MYOZ1 and MYOZ3) family were found to produce DAS transcripts: alternative first exons, alternative 3′ and 5′ splice sites. These genes play an important role in the modulation of calcineurin signaling. Another DAS transcripts belong ubiquitin family (UBAC2 and UBE3C). These genes produced transcripts with cassette exons, alternative 3′ splice sites and alternative 5′ splice sites. The ubiquitin–proteasome system plays an important role in muscle loss23.Members of myosin (STK11) and Eukaryotic Translation Initiation Factor 4B (EIF4B) produce transcript isoforms with alternative 3′ splice sites and were present in these pathways. All of the GO terms and pathways mentioned above play important roles in muscle and lipid metabolism.Most biological terms found for animals selected to differ for IF content were associated with growth, regulation and proteolysis of muscle cells , Heat Shock Protein Family E member 1 (HSPE1) and Mitogen-Activated Protein Kinase Kinase 6 (MAP2K6). TRIP12, HSPE1 and MAP2K6 genes are involved in ubiquitin mediated proteolysis24, facilitates the correct folding of imported proteins25, the regulation of the mitogen-activated protein kinase pathway26, respectively. The subnetwork genes  involving 101 genes (nodes), density = 0.018 and a clustering coefficient = 0.152 . The exons are included or excluded from the mature RNA leading to extended or shortened transcripts variants. Bland et al.27 studied mice myogenic differentiation and found 95 alternative splicing events. Most of the splicing events found for Bland et al.27 was cassette exons (86%).Cassette exon and alternative 3′ splice site are the most common alternative splicing events in mammalian pre-mRNAs28. In addition, a functional enrichment analysis was performed for the gene sets found for each trait. In general, for both traits, the results indicated that hub DAS genes had functions in protein ubiquitination, muscle differentiation and regulation of lipolysis in adipocytes.The co-expression analyses were performed with gene sets that contained the isoforms that were differentially expressed between the highest and lowest REA and IF content groups. Through co-expression analyses hub DAS genes for each trait were predicted that may be the primary regulators of the other genes with differentially expressed transcript isoforms found in this study. Hub genes are expected to play significant functions in organism’sCAMK2B  was present in nine pathways . This gene was differentially expressed between animals phenotypically divergent for IF content and by a known isoform with an alternative 3′ splice site, regulates cell polarity and functions as a tumor suppressor31. STK11 is involved in TORC1 signaling (GO:1904262); mTOR signaling (bta04150) and PI3K-Akt signaling (bta04151) , myotilin (MYOT), myomesin (MYOM2), solute carrier  and BOLA (BOLA-DOA) genes families. Myosin, myotilin and myomesin proteins have a significant role in the stability of thin filaments during muscle contraction and are the main components of myofilaments33. SLC gene family play roles as transporters and have been associated with REA34 in Nelore cattle. BOLA-DOA belongs to the major histocompatibility complex and has effects in the immune system. Members of the BOLA gene family have previously been associated with tenderness35 in Nelore cattle and growth traits in Limousin, Simmental Charolais, Holstein, Angus and Hereford, beef cattle37.Several differentially expressed alternatively spliced transcripts were produced by myosin  with alternatively spliced transcripts were differentially expressed between Nelore bulls selected to differ for REA. The differences in transcript splicing event usage for LRRFIP1, RCAN1 and RHOBTB1 found here may be due to the tuning of the spliceosome machinery. The spliceosome is a complex snRNA. The introns are removed from pre-mRNAs through two consecutive phosphoryl transfer reactions. The spliceosome is assembled de novo on a imature mRNA for each splicing event and a complex series of assembly steps result to the active conformation.Three hub genes  was predicted. The alternative 3′ splice sites usage in RHOBTB1 was more abundant in the LREA animals, which could result in the likely morphological changes that occur in the muscle structure of these animals. RCAN1 has previously been associated with REA in a Wagyu × Angus F1 population22. RCAN1 produces a protein that inhibits the transcriptional responses of the calcineurin-dependent mediated by the binding to the catalytic domain of calcineurin A.The protein encoded by 38. RCAN1 has a tissue-specific expression pattern and produces transcripts that differ by known alternative first exons in mice39, humans40 and cattle41. In this study, RCAN1 transcripts that differ by known alternative first exons were more abundant in the LREA animals, which could inhibit the calcineurin-dependent transcriptional responses, causing muscle loss in this animal group.It is known that calcineurin A signaling is important for the normal function of cardiac and skeletal muscle. In skeletal muscle, calcineurin A is involved in different processes, as dystrophic muscle damage, fiber type specification and myotube differentiation42. For example, we found the known and important pathways: focal adhesion (bta04510), cAMP (adenylyl cyclase) signaling (bta04024) and ECM-receptor (extracellular matrix-receptor) interaction (bta04512). The cAMP pathway contributes to an increase in myofiber size and metabolic phenotype over the long term43. It also participates in the differentiation of cell muscle precursors44. The cellular events described above, are necessary for the efficient regeneration of skeletal muscle43.The gene set enrichment analysis based on transcript isoforms that were differentially expressed in animals differing for REA revealed GO terms and pathways related to muscle growth and muscle protein ubiquitination  is continuous and ensures the skeletal muscle quality and functional integrity. In addition, the hormones are essential regulators of this remodeling process.Pathways related to hormonal systems were also found, among them: oxytocin (bta04921) and glucagon signaling (bta04922). 48. Among these, the ubiquitin–proteasome system is very conserved across vertebrates, and works as a degrader of the main proteins of the contractile skeletal muscle, that is, it has an important role in muscle loss23.In contrast to muscle growth, muscle loss is an undermining consequence of a range of pathology and metabolic conditions, for example, an increase in protein degradation. We found processes related to the proteolytic system , some of which were involved in muscle degradationADCY2) gene which also is annotated with several other GO terms and is a member of other pathways including those related to the neural and hormonal systems. There are complex interactions between the skeletal muscle and the immune systems that regulates muscle regeneration49. ADCY2 is a membrane-associated enzyme that catalyzes the formation of cyclic adenosine monophosphate50, a secondary messenger. An alternative 3′ splice site event was found for this gene which is a member of the cAMP signaling pathway (bta04024). ADCY2 may be related in different biological processes, and has previously been associated with meat tenderness in Yanbian cattle51.Genes encoding differentially expressed alternatively spliced transcripts associated with REA were also related to immune function, such as the Adenylate Cyclase 2 , myotilin (MYOT), myozenin (MYOZ1 and MYOZ3) and ubiquitin (UBAC2 and UBE3C) families. Skeletal muscle fiber metabolism is a factor that may affect intramuscular fat deposition. There is a positive correlation between the extent of intramuscular fat and oxidative muscle fiber percentage52. When the rate of protein deposition decreases (possibly due to ubitiquation processes) lipid deposition becomes the major component of weight gain and the energy request to fat tissues increases53.We found differentially expressed alternatively spliced transcripts belonging to members of myosin  found in HSP family genes were associated with thermal tolerance traits in Chinese Holstein56 and Angus cattle57. Heat shock proteins provide subsidies for cells to identify and promote the refolding of damaged proteins or target them to appropriate proteolytic systems, thereby helping to eliminate proteins whose damage is not suitable for recovery. As long as the animal is under stress, HSP levels will be high. This high expression increases the synthesis and maturation of new proteins that will replace those affected by stress. The increase of HSP in damaged cells not only contributes to protein repair, but also plays an important role in maintaining viability because it inhibits apoptosis58. Therefore, the HSPE1 transcript found in this study could be aiding in the repair of proteins in HIF group.This study predicted a putative novel MAP2K6 produces three known transcripts one off them differ by known alternative first exons, which were found in this study and more abundant in the LIF animals. Single nucleotide polymorphisms in MAP2K6 have previously been associated with marbling score, back fat thickness and carcass weight in Hanwoo cattle59. MAP2K6 is a member of the dual specificity protein kinase family and plays a role in the regulation of the mitogen-activated protein kinase pathway26. Mitogen-activated protein kinase (MAPK or MAP kinase) is a protein kinase that is specific to the serine and threonine aminoacids. Mapk protein is involved in directing the response of cells to a variety of stimulus, such as osmotic stress, mitogens, proinflammatory cytokines and heat shock. They regulate cell functions including apoptosis, gene expression, proliferation, mitosis, differentiation, and cell survival. Therefore, the MAP2K6 transcript, as well as the HSPE1 transcript, found this study, could be aiding in the repair of proteins in LIF animals.62. Intramuscular fat accumulates both within (intramyocellular) and externally (extramyocellular) of muscle fibers62. The intramyocellular lipids are stored as spherical droplets in muscle cells and are reportedly associated with aerobic metabolism, whereas the extramyocellular lipids are located in long fatty septa of laminar shape, along muscle fiber bundles. Intramyocellular lipids play an important role in muscle metabolism62.The enrichment analysis suggested that the most relevant function of the genes with significant isoform expression shifts for IF was related to the growth, regulation and proteolysis of muscle cells  that control the expression of genes encoding proteins involved in lipid and cholesterol homeostasis65. The PI3K-AKT pathway is an intracellular signaling pathway essential in regulating the cell cycle. PI3K activation phosphorylates and activates AKT. AKT can have a number of downstream effects such as activating mTOR66.The PI3K-Akt (bta04151), mTOR signaling (bta04150) and negative regulation of TORC1 signaling (GO:1904262) pathways were overrepresented in this study. 67, Wagyu and Angus68, Wagyu and Holstein69 and Nelore70, suggesting that some of these genes regulate lipid composition and deposition in intramuscular fat70. These findings and our results enable a better understanding of the mechanisms underlying the gene transcription associated with IF content in Nelore cattle.We found candidate genes that encode differentially expressed alternatively spliced transcripts associated with IF content in Nelore cattle. Previous studies have identified differentially expressed genes in Simmental-Luxi crossRNA-Seq was used to identify candidate genes that encode alternatively spliced transcripts that were associated with REA and IF content in Nelore bulls. The results suggest that REA and IF content may be influenced by sets of genes that encode alternatively spliced transcripts (both known and novel transcripts). For both traits, we found genes with biological process GO terms that are involved in pathways related to protein ubiquitination, muscle differentiation, regulation of lipolysis in adipocytes, and hormonal systems. These results provide a foundation for further research into the specific functions of candidate genes encoding alternatively spliced transcripts that are differentially expressed in animals differing for REA and IF content.Brachiaria sp. and Panicum sp. forages, and had free access to mineral salt and were finished in a feedlot for approximately 90 days. The diet was based on whole-plant silage and mix of sorghum grain, soybean meal or sunflower seeds were used as concentrate, with a concentrate/roughage ratio from 50/50 to 70/30. The animals were slaughtered at an average age of 24 months in a commercial slaughterhouse, in accordance with Brazilian Federal Inspection Service procedures.The intact Nelore bulls used in this study (N = 80) were from the Capivara Farm, which participates in the Nelore Qualitas Breeding Program. The animals from a single contemporary group (that remained together from birth until slaughter) were reared on longissimus thoracis muscle were collected, between the 12th and 13th ribs of the left half of each carcass, at two time points: immediately following slaughter for the RNA-Seq analysis  and again at 24 h post-slaughter for the evaluation of REA and determination of IF content. REA was measured by the grid method in units of centimeters squared71. The Bligh and Dyer72 method was used to determine IF content. From the 80 animals, groups of animals were selected for RNA-Seq analysis of ribeye muscle tissue: (1) REA, the 15 animals with the highest and 15 with the lowest REA (HREA and LREA); and (2) IF, 15 animals with the highest and 15 with the lowest IF content (HIF and LIF). A Student's t-test was applied to determine if there were statistically significant differences between the selected groups  according to the manufacturer's protocol. Differently bar-coded libraries were pooled to enable multiplexed sequencing and generated, on average, approximately 25 M read-pairs per sample. RNA-Seq was performed using a HiSeq 2500 System (Illumina®) that generated 100 bp paired-end reads.High-quality total RNA (approximately 500 ng) extracted from the samples was processed using a TruSeq RNA Sample Preparation Kit73. Trimmed reads data were obtained using Trimmomatic (v.0.36)74, with following parameters: PE ILLUMINACLIP: TruSeq3-PE.fa:2:40:15 LEADING:20 TRAILING:20 CROP:100 SLIDINGWINDOW:4:20 AVGQUAL:20 MINLEN:50. All downstream analyses were performed on the trimmed data with high quality reads. HISAT2 (v.2.0.5)75 was used to align the paired-end trimmed data to the bovine reference genome (UMD3.1.1 Bos taurus) and chrY (Btau 4.6.1), both deposited in National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/). The parameters used were: − p 12, − dta, − known-splicesite-infile, − x, − 1, − 2, − S. The annotation was merged both, UMC3.1.1 and chrY (Btau 4.6.1). Table 15 was used to estimates metrics and bias, including reads genomic origin, junction analysis, transcript coverage and 5′–3′ bias computation.The quality of RNA-Seq reads  was checked using FastQC v.0.11.4) software1.4 softw3 was used to identify and classify exon-centric alternative splicing events  based on splice junction reads predicted using the Cufflinks2 (v.2.1.1) suite of tools 76.JuncBASE (v.0.9) software 3.For each RNA-Seq sample, the spliced alignment is given as input, in BAM format. Splice junctions passing an entropy score threshold were combined with exon coordinates from transcript reference annotations and optional novel transcripts to identify alternative splicing events. The inclusion and exclusion isoforms of each alternative splicing event are quantified using the RNA-Seq read alignments and Percentage Spliced Index (PSI) values are calculated. Differential splicing analysis is performed from isoform abundances or PSI values. For more details, the following eight steps were usedStep 1 Building annotation database: the annotation database was used to identify all exons in the transcriptome and it was derived from a GTF file. We created two different databases. The first, was GTF file that combined Cufflinks2 and Cuffmerge2 identified transcripts with those that are annotated in a reference set, and it was used to identify all internal exons (not the first or last exon in a transcript). The second database was derived from just a reference annotation. This second database was used to define alternative first and last exons.Step 2 Determination of junction entropy cutoff: for all subset of samples, was built an entropy plot of annotated and novel junctions. Cutoff of 1.1 was used to reduce the number of potential false positive novel junctions.Step 3 BAM files processing: first, the junctions were filtered (based on entropy score obtained in step 2). From this step, for each sample, it was obtained a BED file which contained all junctions and a TXT file, which contained all the non-junction reads from the BAM file.Step 4 Identification of all junctions: this step created one BED file that combined junctions from all samples.Step 5 Building of exon–intron junction count files: this step created a counts file of the number of reads aligning to every exon–intron junction.Step 6 Building of pseudo/“all junction” sample: The pseudo sample was only used to define the set of all junctions and was not used for quantification. The pseudo sample was linked to the BED files created in step 4. An arbitrary sample had given in order to create other pseudo files that were not used.Step 7 Alternative splicing events identification and quantification of the events from each sample: this step identified, classified, and quantified alternative splicing events. The result of this step was a table with details of each alternative splicing event identified in the set of samples examined. The details of each splicing event include coordinates, and raw and length-normalized read counts to specific junctions and regions involved in the alternative splicing event. Also, we built tables of raw and length-normalized read counts of exclusion and inclusion isoforms.Step 8 Differential splicing analysis: this last step identified differentially spliced events between two specified groups (Highest and Lowest) of animals (REA and IF). We obtained a set of uniform transcripts for every sample in the previous steps, and then, the Percentage Spliced Index (PSI) was estimated as the ratio of the number of reads including exons to the sum of the numbers of reads including or excluding exons3. The PSI value was calculated and Fisher's exact test was used to test for differences in alternative splicing events between the high and low REA and IF groups (p < 0.05). Only alternatively spliced transcripts that were supported by at least ten reads and that had PSI differences (ΔPSI) between the respective high and low groups of greater than 5% were considered significant.77. Fisher’s exact test was used and p values were adjusted for multiple testing using the Benjamini–Hochberg method78. DAVID Pathway was used to map the gene enriched pathways using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database79.The enrichment and pathway analyses of genes set DAS transcripts associated with REA or IF content were performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID v.6.8)80. For co-expression network analyses, the ExpressionCorrelation plugin81 was used to identify consensus networks of gene sets in which differentially expressed alternatively spliced transcripts were associated with REA or IF content. Similarity matrices were computed using the Pearson correlations among PSI values. A histogram tool was used for the screening criteria at node score cut-offs > 0.75 and <  − 0.75 and employed to identify statistical significance of the pairwise correlations. We established a co-expression network between the significantly co-expressed DAS genes. The cytoHubba plugin82 was used to explore co-expression network nodes (hub genes). The maximal clique centrality (MCC) was used for analyses, this method has a better performance on the precision of predicting essential genes from the co-expression network78 and to generate a subnetwork83. The top three hub genes were chosen to define gene co-expression subnetwork and then, it was used to analyze the functional enrichment of biological processes.All analyses were performed with plugins or applications from Cytoscape (v.3.7)All experimental procedures were approved by Ethics Committee of the School of Agricultural and Veterinarian Sciences, São Paulo State University (UNESP), Jaboticabal, SP, Brazil (protocol number 18.340/16). The animals were provided by Qualitas Nelore breeding program company and they were slaughtered in commercial slaughterhouses. These slaughterhouses have animal welfare departments staffed by professionals trained by WAG  to ensure that the animals are killed humanely using a captive bolt pistol for stunning.Supplementary Figures.Supplementary Tables.


Microraptor, troodontids and avialans). Here, we studied the microscopic features of paravian non-avian theropod and avialan teeth using high-spatial-resolution synchrotron transmission X-ray microscopy and scanning electron microscopy.Tooth morphology within theropod dinosaurs has been extensively investigated and shows high disparity throughout the Cretaceous. Changes or diversification in feeding ecology, i.e., adoption of an herbivorous diet , is proposed as a major driver of tooth evolution in Paraves (e.g., Microraptor specimen, implying that shifts in diet in avialans from that of closely related dinosaurs may correlate with a shift in feeding ecology during the transition from non-avian dinosaurs to birds.We show that avialan teeth are characterized by the presence of simple enamel structures and a lack of porous mantle dentin between the enamel and orthodentin. Reduced internal structures of teeth took place independently in Early Cretaceous birds and a Different lines of evidence all suggest a large reduction in biting force affecting the evolution of teeth in the dinosaur-bird transition. Changes in teeth microstructure and associated dietary shift may have contributed to the early evolutionary success of stemward birds in the shadow of other non-avian theropods. Tooth reduction is one of the most conspicuous modifications characterizing the dinosaur-bird transition . MorpholAlthough reductions in tooth number and distribution occurred in many stem avialan lineages toward the origin of extant birds, a great number of Mesozoic taxa retained dentition, including most enantiornithines, non-ornithothoracine avialans, and ornithuromorphs . AnalysiAnchiornis huxleyi skeleton, suggests that they were opportunistic feeders [Microraptor gui reportedly preyed on mammals, birds, and even fish based on gut contents [Evidence of direct dietary preferences by paravian theropods, including lizard and fish pellets that are associated with a complete  feeders . Microracontents . An herbcontents . For Mescontents , 11, teecontents , 3.Microraptor species and other Early Cretaceous birds. The lack of mantle dentin with interglobular porous spaces (IGS) between the enamel and orthodentin [The adaptive changes in internal tooth structure related to a shift in feeding ecology during this critical transition are not fully understood, despite a few studies focusing on non-avian dinosaurs that are distantly related to avialans , 12. Herhodentin , 14 in aSapeornis chaoyangensis and Jeholornis prima , measured from the lateral side of the base of tooth crown  significantly exceeds other basal avialans sampled here. Proportionally greater enamel thickness in Enantiornithes is a feature that distinguishes them from other ornithuromorph species, in addition to the recognizable gross osteological features of other skeletal elements [The presence of enamel in all five bird species in this study was confirmed using SEM and TXM imaging of thin sections prepared from the tooth samples Figs. , 2. Enamima Fig. e–h, all ima Fig. . The meas dispar , 15. Theelements .Fig. 1EJeholornis was largely reduced, with only a few tiny dentary teeth and/or maxillary teeth present in known specimens [Jeholornis have a rounded crown with a cylinder-like root, and the tooth crown is nearly straight  of several taxa , 16. Forsurfaces . The crysurfaces . The prehyosaurs . The lachyosaurs , 31. In hyosaurs , even enhyosaurs .Given that most non-avian theropods and other saurischian dinosaurs possess interglobular porous mantle dentin, the loss of this particular tissue is a key feature in bird teeth , 33. A pIn addition to tooth number reduction, the loss of interglobular porous mantle dentin that occurred in the non-avian dinosaur-to-bird transition is associated with changes in feeding ecology and the functional role of teeth during food acquisition. Interestingly, the lack of interglobular porous mantle dentin found in avialan species has also been observed in a newly discovered Microraptorian tooth, which lacks serrations Fig. d. ConverMicroraptor and Anchiornis), and this structure is absent from all avialans. Reductions of enamel thickness and the structure of schmelzmuster in ornithurine and other early bird teeth may be further associated with the appearance of the gastric mill used for grinding. Changing to an herbivorous diet could relax the selection pressure to maintain the tooth shape and denticles used for a carnivorous diet, giving rise to increased raw teeth variations. For these reasons, we infer that collecting and/or holding food items rather than processing is the major function of such bird teeth.New data have also revealed diversity in bird teeth regarding both gross and microscopic structures. For example, tooth denticles were proposed as an indicator of a puncture-and-pull function in carnivorous theropods . ReducedSmall-bodied theropods diversified quickly during the Late Jurassic period. In the face of intensified competition from carnivorous theropods and pterosaurs, changes of feeding ecology to a more herbivorous diet could be one of the major selective factors ruling on the evolution of Cretaceous stem birds, as exemplified by tooth changes in both exterior morphology and internal microstructures. In addition to feeding adaptation, a recently hypothesized developmental process  may also have constrained tooth growth . Fast emMicroraptor feeding on enantiornithines suggests that biting remained vital for foraging behavior in some Microraptorine taxa. Moreover, we identified a new Microraptorian specimen that resembled avialan species, which featured unserrated teeth across both the upper and lower jaws, and more importantly also the similar internal ultramicrostructures. Increased cranial kinesis and reduced robustness occurred concurrent with modifications in tooth structure in Paraves [We propose that a large reduction in biting force due to an innovative dietary shift acted as a major factor affecting the evolution of teeth in the dinosaur-bird transition. This was characterized by the loss of interglobular porous mantle dentin and simplification of the enamel structure. Direct evidence from one specimen of  Paraves . These lWe sampled five representatives of avialan teeth Table  to invesLongipteryx (IVPP V21702) dentary tooth, one isolated Troodontid tooth specimen (PWM 5400400036) and one Anchiornis dentary tooth (STM 0–69), as well as half of an isolated tooth from one Microraptorine (1) specimen (STM 5–48) and a complete dentary tooth from another Microraptorine (2) specimen (STM 5–151). Other avian sample numbers are as follows: Ornithuromorph sp. (IVPP V14606), Jeholornis prima (IVPP V13886) and Sapeornis chanyangensis (IVPP V13759).Our sampling covers major avialan lineages, including stem ornithuromorphs, enantiornithine, basal avialans, and also the paravian dinosaurs (Table 11 photons s− 1 (0.1% bw)− 1 in the energy range 5–20 keV. A double crystal monochromator exploiting a pair of Ge (111) crystals selected x-rays with 8–11 keV of energy. A Fresnel zone-plate was used as an objective lens to magnify x-ray images. Conjugated with a 20× downstream scintillator-based optical magnification, the microscope provided a total magnification of 880×. The field of view for each image was 15 × 15 μm2, however, a millimeter scale field of view can also be provided by stitching images of the specimens obtained from sequential positions.In preparing thin sections for observation with transmission X-ray microscopy (TXM), we sliced the epoxy-embedded fossil teeth to a thickness of 50–100 μm using a Leica SP1600 Saw Microtome, and sequentially hand-polished the sliced teeth to a thickness of 20–30 μm using silicon carbide 120, 240, 500, 800, 1200, 2500, and 4000 polishing paper to minimize the scratches on the specimen surfaces. No other complicated preparation processes were needed for observation. The original internal structure inside the tooth section was not destroyed during the preparation process. The TXM at beamline BL01B1 of the Taiwan Light Source provided two-dimensional radiography and three-dimensional tomography with an approximately 60-nm spatial resolution. A superconducting wavelength shifter source of BL01B1 beamline provided a photon flux of 4 × 10Polished sections of bird teeth were etched with 0.1 mol/L phosphoric acid for about 90 s, treated in an ultrasonic bath, and air-dried. Air-dried specimens were examined and acquired on a Zeiss Gemini SEM 500 at the Instrument Analysis Center of Xi’an Jiaotong University and the Chinese Academy of Geological Sciences (FEI Quanta 450 FEG) in Beijing. Backscattered-Electron (BSE) Imaging were taken at voltages of 1–20 kV.Additional file 1: Figure S1 The rostrum of indet. Enantiornitine, IVPP V 16041 . White arrow indicates the dentary taken from this small-sized enantiornithine specimen. The conical-shaped tooth is small and only slightly curved caudally. The cervix of the tooth is rather wide with a narrow crown. Figure S2 The rostrum of a referred specimen of Longipteryx chaoyangensis (IVPP V 21702). White arrow indicates the dentary tooth fell off from the rostrum. The tooth is relatively large and strongly curved caudally with a wide cervix (see the inserted image showing the 3D surface rendering from micro-CT scan of the tooth taken). Figure S3 Skull of a referred specimen of Sapeornis chaoyangensis (IVPP V13759). The isolated maxillae tooth was sampled (White arrow pointed). The tooth is rather large and columnar in shape. No significant expansion is present on the tooth cervix. The tip of tooth become slightly pointed. Figure S4 Close up image of the tooth sampled from a referred specimen of Jeholornis prima (IVPP V 13886). Cross-section of the tooth is very rounded from the root to the crown and only gradual decrease of diameter is visible in the 3D surface rendering model of the micro-CT scan (right inset). Figure S5 The tooth sampled from a new ornithuromorph specimen (IVPP V 14606). The tooth shape is distinct with regards to its expanded columnar shaped root with a constriction of the crown (see the inserted image of the 3D surface rendering model from the micro-CT scan). Figure S6 Skull of a new Microraptorine specimen 2 (STM 5–151). The isolated tooth with a sickle-like shape was sampled for sectioning. Figure S7 Skull of a new Microraptorine specimen 1 (STM 5–48). One isolated half dentary tooth was sampled from this specimen. Figure S8. Disarticulated skull of a new specimen of Anchiornis huxleyi (STM 0–69). White arrow indicates the dentary tooth sampled. Figure S9 SEM imaging to show the enamel structure and measurements taken from each tooth.


There is a high incidence of calcified coronary artery disease in patients with severe valvular aortic stenosis (AS). With transcutaneous aortic valve replacement (TAVR) as one of the promising options for severe AS in high and intermediate surgical risk patients; we will encounter more and more patients who will require both complex percutaneous coronary intervention (PCI) with rotablation (RA) and TAVR. The timing of PCI in patients undergoing TAVR; however remains indecisive. Due to the complexity of procedures and the risks involved, very few cases of concomitant TAVR and coronary RA have been reported so far. Seventy-five years old high surgical risk female had severe AS with calcified left main (LM) distal and ostial left anterior descending (LAD) artery lesion. Successful PCI with RA to LM-LAD lesion was done followed by uneventful transfemoral TAVR in the same setting.This is probably one of the very few cases reported where PCI to LM with RA and TAVR was done successfully in the same setting. Since the calcified lesion was focal and left ventricular ejection fraction of the patient was normal, we went ahead with PCI without prior balloon dilatation of aortic valve (BAV) which was a deviation from the prior reported cases, where BAV was performed prior to complex PCI to improve the cardiac output. We herein discuss our case and thoughts about concomitant complex PCI and TAVR. With more experience in transcutaneous aortic valve replacement (TAVR) and positive results of SURTAVI and PARTNER II, a large number of patients will now be candidates for concomitant percutaneous coronary intervention (PCI) and TAVR.In carefully selected patients, complex PCI requiring rotablation can be done in the same setting as TAVR.Till more data are available, the timing of PCI in patients planned for TAVR needs to be individualized.,Patients with severe valvular aortic stenosis (AS) often have concomitant calcified coronary artery disease (CAD).2. Coronary evaluation revealed calcified significant distal LM and ostial left anterior descending (LAD) and borderline right coronary artery (RCA) lesions. The left ventricular systolic ejection fraction (LVEF) was 60%. With a Society of Thoracic Surgery (STS) risk score of 16 and EUROSCORE risk of in-hospital mortality of 8.25%, she was considered high risk for surgical management. A detailed computed tomography angiogram  was performed for the evaluation of a possible TAVR. It showed valve morphology and peripheral vasculature suitable for transfemoral TAVR  and thus, she was offered an option of PCI to LM and LAD with RA and a transfemoral TAVR. As the patient was planned for both PCI and TAVR, she was loaded with Aspirin 300 mg and Clopidogrel 300 mg a day prior to the procedure.Seventy-five years old frail, insulin-dependent diabetic, hypertensive female with chronic kidney disease Stage III was symptomatic with New York Heart Association (NYHA) Class III dyspnoea. She had tricuspid aortic valve, severe AS with maximum, and mean gradient of 110 and 65 mmHg and an effective orifice area of 0.6 cmTM (Asahi Intecc) guidewire, it was exchanged with RA wire over a microcatheter. A 1.5 mm burr was used for RA of LM to LAD  . Following RA, the lesion was predilated with 3.0 mm non-compliant (NC) balloon and stented with a 3.5 mm × 24 mm SYNERGYTM everolimus-eluting stent (Boston Scientific). Proximal optimization of stent in LM was performed with a 4.5 mm NC balloon. There was good expansion at distal LM and ostial LAD without any significant carina/plaque shift towards left circumflex . Total fluoroscopy time was 8 min, and contrast used was only 30 mL for PCI. As the patient tolerated the procedure well with minimal contrast, we decided to go ahead with transfemoral TAVR using the right groyne as the access. Two suture based Perclose ProGlide™ devices (Abbot Vascular) were pre-deployed at 10 and 2'o clock position in the right femoral artery and a 14 Fr expandable PythonTM (Meril Life Sciences) sheath was inserted under fluoroscopic guidance upto the right iliac artery. Native aortic valve was dilated with a 16 mm MammothTM balloon (Meril Life Sciences) over the AmplatzTM super-stiff wire (Boston Scientific) followed by deployment of 21 mm balloon-expandable MyvalTM (Meril Life Sciences) . Transoesophageal echocardiography showed perfect positioning and function of the valve with no paravalvular regurgitation. Right groin access was successfully closed with two pre-deployed ProGlides™. The procedure was uneventful, and the patient was discharged on Day 3 after confirmation of no new renal dysfunction or access site vascular complications. She was continued on dual antiplatelets (Aspirin and Clopidogrel) for another 6 months without any oral anticoagulants. At a follow-up of 6 months, she is doing well and is in NYHA Class I.We obtained a 7 French (Fr) access in the left femoral artery for the PCI. A 7 Fr extra backup left guide catheter was used to engage LM ostium. After crossing the lesion with Sion Blue,,,,Sixty percent of patients undergoing surgical AVR (SAVR) and 40–75% of those being taken for TAVR have an associated significant CAD.,,The timing of PCI in relation to TAVR is a matter of debate and the controversy is still not resolved. A staged procedure reduces the procedural time, contrast used, and risk of haemodynamic instability,,,,Very few cases have been reported where a complex PCI to LM with RA and TAVR has been performed in the same settingTo conclude in carefully selected patients: complex PCI and TAVR may be combined in a single procedure and whether PCI to be performed first or BAV, should depend on the type of coronary lesion, patient’s LVEF, and local expertise and experience.Dr Parag Barwad MD, DM, Associate Professor of Cardiology, PGIMER (Corresponding author) had special interest in structural heart disease, peripheral artery disease, and arrhythmias. European Heart Journal - Case Reports online.ytaa196_Supplementary_DataClick here for additional data file.


As a notable part of lotic systems are temporary, and small lentic systems can substantially contribute to GHG emissions, dry-wet cycles in these ecosystems can play a major role on N2O emissions.Sediments frequently exposed to dry-wet cycles are potential biogeochemical hotspots for greenhouse gas (GHG) emissions during dry, wet and transitional phases. While the effects of drying and rewetting on carbon fluxes have been studied extensively in terrestrial and aquatic systems, less is known about the effects of dry-wet cycles on N2O emissions and related biogeochemical processes in sediments of lentic and lotic ecosystems.This study compiles literature focusing on the effects of drying, rewetting, flooding, and water level fluctuations on N2O pulses were observed following sediment drying and rewetting events. Moreover, exposed sediments during dry phases can be active spots for N2O emissions. The general mechanisms behind N2O emissions during dry-wet cycles are comparable to those of soils and are mainly related to physical mechanisms and enhanced microbial processing in lotic and lentic systems. Physical processes driving N2O emissions are mainly regulated by water fluctuations in the sediment. The period of enhanced microbial activity is driven by increased nutrient availability. Higher processing rates and N2O fluxes have been mainly observed when nitrification and denitrification are coupled, under conditions largely determined by O2 availability.N2O emissions in sediments from a wide array of aquatic habitats. Peak fluxes appear to be of short duration, however, their relevance for global emission estimates as well as N2O emissions from dry inland waters has not been quantified. Future research should address the temporal development during drying-rewetting phases in more detail, capturing rapid flux changes at early stages, and further explore the functional impacts of the frequency and intensity of dry-wet cycles.The studies evidence the driving role of dry-wet cycles leading to temporarily high N Climate change is expected to alter the frequency and intensity of extreme events. Climate change scenarios predict an increase in both prolonged drought periods and heavy rainfall events, intensifying seasonal runoff and flood risk even in areas where precipitation is expected to decline . This wiTemporary rivers and streams, which are characterized by a periodic cease in water flow, constitute over 50% of the length of the global river network . FurtherTemporary systems have been less studied than perennial systems, not least because of their complexity. 2O) emissions has been extensively studied in soils , effects of rewetting by rainfall events  and effects of flooding and water level fluctuations  on N2O emissions. The effects of rewetting were separated in rainfall events, and flooding and water level fluctuations to make a clearer distinction between these rewetting events, as they differ in their magnitude and frequency. Each subsection describes how the physical mechanisms and N processes are affected during each phase, followed by the effects on N2O emissions. ‘Effects of dry-wet cycles on microbial community composition and function’ briefly addresses the effects of dry-wet cycles on microbial community and function. Finally, concluding remarks and research gaps are highlighted. Exploring the relevance of N2O emissions from sediments prone to dry-wet cycles and considering emissions from temporary systems and water drawdown areas is important to improve global estimates of N2O fluxes from inland waters.This synthesis compiles literature focusing on Ns so far . The aim2O emissions from sediments was performed using the literature database search engines Web of Science and Google Scholar, with a combination of search terms: dry* wet* OR dry* rewet* OR dry* flood* OR drought AND “nitrous oxide” OR ”N2O” AND sediment*. The asterisk (*) enables key word variations. The reference lists within the selected papers was also examined. The initial screening of titles and abstracts excluded papers considering N2O emissions from soils exclusively, and from sediments other than from freshwater ecosystems. The following studies were excluded from this synthesis: soils (including wetland soils and peatlands), saline or brackish water environments, aquaculture ponds, and rice/paddy field studies. The studies include both field and laboratory observations. Rewetting of dry sediments include events caused by rainfall , flooding and water table fluctuations. Drying events include water drainage from the sediment and sediment desiccation. N2O emission peaks were considered as a direct response to sediment drying and rewetting events. Last database search: 28 of September 2020.A search for relevant published studies focusing on dry-wet cycling and N2O emissions /redox and moisture content (MC) conditions, within oxic/anoxic microsites in sediments  performed by heterotrophic bacteria (denitrifiers). Denitrifying microorganisms also include ammonia-oxidizing chemolithotrophic bacteria, which reduce NO2O aerobically, archaea, fungi and other eukaryotes ), including water content, NODenitrification, a facultative anaerobic process, is the reduction of nitrate (NOkaryotes . Part of release . Denitrification . Apart fument}3− .3) or ammonium  and chemodenitrification (abiotic). The main factors influencing hydroxylamine oxidation are aerobic conditions and NH3 availability, whilst chemodenitrification is limited by NO3 to N2 gas. It is supported by different O2 conditions, having both oxidation and reduction steps. The first steps are oxidative (ammonia is oxidized to NO2O and N2). Factors influencing N2O production from nitrifier denitrification include O2 conditions, NHNitrification and nitrifier denitrification occur under different environmental conditions and both oxidize ammonia. Nitrification is the oxidation of ammonia  and intensity . As a notable fraction of watercourses is temporary , and eveoduction .2O production by creating oxic-anoxic conditions along the sediment strata. Biogeochemical processes such as nitrification and denitrification are therefore affected by dry-wet cycles, potentially impacting N2O production and emission. Microbial activity is strongly coupled with water-filled pore space (WFPS), a measure of moisture’s influence on biogeochemical processes which normalizes the effect of texture  cycling in temporary systems, namely microbial community structure and functioning, and organic matter (OM) quality and availability . At the  texture . Maximumof %WFPS . Differe2O pulse during sediment desiccation. The increase in gas diffusivity  and the lack of physical trapping, related to water content and oxygen fluctuations, determine the magnitude and temporal development of N2O outgassing from the sediments 2O pulse 2O production are also made available from the release of intracellular solutes from microbial cell lysis, and from mineralization of OM, including previously protected organic matter from aggregate disruption (shrinking), due to sediment desiccation  ratios, since denitrifiers prefer to transfer electrons to NO2O.In general, studies in both lotic and lentic systems suggest that initial sediment desiccation stimulates N processing rates such as N mineralization and nitrification. A MC/WPFS threshold exists after water drainage and/or the onset of sediment drying when denitrification is also enhanced and coupled to nitrification . This wahis time . NO\docudecrease . During y period ; Table 1y period , the dury period , with co2O fluxes have been observed in different freshwater systems during initial sediment desiccation  occurred during an initial drying period of high N processing activity . After an initial lag phase, most likely limited by NO2O flux peaked. During an intermediate state between flooding and sediment drying, a patchy distribution of oxic and anoxic microsites was observed. This high spatial heterogeneity in redox conditions increases the area of the oxic-anoxic boundary where coupled nitrification-denitrification can occur, explaining the high denitrification activity detected during this period. The denitrification rates during desiccation exceeded those during re-flooding. A previous study in the same system detected higher N2O and denitrification rates in exposed sediments, and with greater distance to the waterline  and denitrification enzyme activities remained very high in sediment after 1 month of drought, and communities were able to maintain their enzymatic pool even under severe environmental conditions. Short-term variations in sediment water content strongly affected microbial functioning, but not the metabolic structure of the community.Similar observations were made in a seasonal pond ; Table 12O production potentials were measured along one transect, from flooded sediments to drier sediments at the water margin 2, is alsmissions .Nitrification and denitrification response to rewetting following dry periods of varying duration have yielded contrasting results. In an experimental stream, nitrification and denitrification rates recovered to levels equal or greater than those in the wet controls within 1 day of being rewetted in sediments dried less than 7 days, but failed to fully recover in sediments dried more than 7 days ; Table 1g events . Previoug events .2O emissions were significantly higher after a rainfall pulse following an extensive period of dry conditions  (2O fluxes was further modulated by the duration of the preceding drying period (6 and 9 weeks). Intense rainfall (21 mm) caused a decrease in N2O fluxes, while following modest rainfall (4 mm) emissions increased immediately after the water pulse 24 h later. The decrease of the average fluxes was most likely related to physical trapping preventing gas exchange and displacement during the monitoring period (24 h). Comparing N2O pulses immediately after drainage and rewetting, the former pulse was of smaller magnitude  when considering the rewetting pulse after the longer preceding drying period under moderate rainfall  . The effrainfall ; Table 1ns e.g., .2O emission by denitrification remained high (60%) after rewetting and similar to the one measured during the dry period, suggesting a lack of recovery of the physiological ability of the denitrifying community to reduce N2O, and an impact of drought on the community structure. Potential nitrification rates also slightly increased. Preceding dry conditions also affect sediment microbial activity. Sediments subject to frequent dry-wet cycles displayed a stronger stimulation of potential microbial activities (denitrification enzyme activity) and higher potential for N2O emissions after rewetting compared to sediments undergoing infrequent drying phases. For instance, the potential denitrification rates after rewetting were highest at sites with longer dry periods, namely at the pond edges. The potential denitrification rates triggered by the rewetting pulse were comparable to the maximum rates measured during drying . These include gravel bars and the parafluvial zone in lotic systems (rivers and streams) and the littoral zones of lentic systems , in which potentially large areas of sediments are exposed to the atmosphere during water level fluctuations, and are potential sources of Nrocesses  and 3. Rundation . Intensiundation .2O efflux were found in frequently flooded river gravel bars, including co-occurrence of high-rates of both, nitrification and denitrification . Furthermore, 2O emissions. Similarly, High N turnover rates and locally high Nfication ; Table 12O emissions were measured along a hydrological gradient covering rarely-flooded and frequently flooded sediments, as well as elevated non-flooded soils in the floodplain , decreasing the N2:N2O ratio. At lower water table, N2O emissions were most likely the by-product of nitrification, as seen by the high concentration of NO2O diffusion to the atmosphere was unrestricted, increasing N2O fluxes. Similarly, N2O emissions were detected in exposed sediments during the low water period, but were never detected in flooded lake sediments, as nitrification and NO2 conditions are favorable for OM mineralization, increasing NH2O was presumed to derive from coupled nitrification-denitrification.The short-term effects of flood pulses on Nnditions ; Table 1stricted ; Table 12O sources, where drying and wetting cycles are a significant factor controlling denitrification and N2O emissions. Denitrification and N2O emission rates were found to be higher in the exposed regions and increased with the degree of sediment dryness, whereas in the permanently flooded littoral zone rates were near zero , which were depleted in NH2O emissions.Kettle holes have likewise been identified as significant sources of N2O ; Table 1fication ; Table 1fication . These r2O emissions. Repeated dry–wet cycles may result in a permanent change of bacterial community composition and function , as well as after water drainage and/or during sediment desiccation. Moreover and as recently emphasized for CO2 emissions, studies suggest that exposed sediments during dry phases can be active spots for N2O emissions. Fluxes from exposed sediments are in the range of those observed from terrestrial soils. Nevertheless, the highest N2O emission peaks have been measured for the latter so far , and hot moments may have been overlooked. Addressing this gap may be of significance when modeling ecosystem responses to dry-wet cycles, as the direction of the fluctuation may influence N2O predictions. For instance (in soils), it has been observed that the intensities of N2O emission peaks can be higher during the drying phase compared to the wetting (near-saturation) phase, and correlated with the amount of water drained, although with shorter peak duration  shape the network interactions in microbial communities, as well as the functional potentials of the active microbial associations . Most studies assume the controlling N2O production pathway depending on the predominant redox conditions, and more efforts should be made to determine the relative contribution of each production pathway to N2O emissions during each phase (wet to dry or dry to wet). Nevertheless, these studies tend to oversimplify ecosystem interactions, namely between surface and subsurface sediments, potentially excluding significant N2O input from deeper sediments (2O in freshwater sediments under dry-wet stress.The importance of the dry-wet cycling legacy is also recognized, as it shapes the microbial community composition and function. Future research efforts tackling Nediments . Despite


The Managing Cancer and Living Meaningfully (CALM) therapy for patients with advanced cancer was tested against a supportive psycho-oncological counseling intervention (SPI) in a randomized controlled trial (RCT). We investigated whether CALM was delivered as intended (therapists’ adherence); whether CALM therapists with less experience in psycho-oncological care show higher adherence scores; and whether potential overlapping treatment elements between CALM and SPI can be identified (treatment differentiation).Two trained and blinded raters assessed on 19 items four subscales of the Treatment Integrity Scale covering treatment domains of CALM . A random sample of 150 audio recordings  were rated on a three-point Likert scale with 1 = “adherent to some extent,” 2 = “adherent to a sufficient extent,” 3 = “very adherent.”applied = 62; 83%) and allowed expression of sadness and anxiety about the progression of disease . The exploration of CALM treatment element SC_1 was most frequently implemented in a satisfactory or excellent manner , whereas the treatment element SMP_4: Therapist promotes acknowledgment that some life goals may no longer be achievable  was not implemented in a satisfactory manner. In terms of treatment differentiation, no treatment elements could be identified which were applied significantly more often by CALM therapists than by SPI therapists.All 19 treatment elements were applied, but in various frequencies. CALM therapists most frequently explored symptoms and/or relationship to health care providers (SC_1: n_Results verify the application of CALM treatment domains. However, CALM therapists’ adherence scores indicated manual deviations. Furthermore, raters were not able to significantly distinguish CALM from SPI, implying that overlapping treatment elements were delivered to patients. In order to draw internally valid conclusions about treatment effects in clinical trials, it is important to monitor treatment fidelity . The TreSymptom Management and Communication with Health Care Providers, (2) Changes in Self and Relationship with Others, (3) Spiritual Well-being and the Sense of Meaning and Purpose, and (4) Preparing for the Future, Sustaining Hope and Facing Mortality  were different from one another regarding the application of treatment elements (treatment differentiation).The objectives of our study are (1) to verify the application of CALM manual interventions within CALM therapy sessions, (2) to quantify the extent to which CALM interventions were delivered as intended by CALM therapists, (3) to test whether CALM therapists’ experience is associated with the frequency of applied treatment elements, and (4) to check whether blinded raters can distinguish the two treatment conditions (CALM vs. SPI).clinicaltrials.gov NCT02051660), which was conducted between August 2013 and January 2017  stage III or IV], ≥18 years, fluent in German, and who reported ≥ 9 on the depression module of the Patient Health Questionnaire PHQ-9,  and/or ≥Cancer and Living Meaningfully therapy consists of four therapeutic key domains which are supposed to be addressed during treatment :(1)SC: Symptom Management and Communication with Health Care Providers: The first domain focuses on the cooperation and improvement of communication with health care providers and offers help regarding medical decision-making to ensure best care and control of symptoms.(2)CSR: Changes in Self and Relations with Close Others: The second domain focuses on patients’ adjustment of self-esteem and identity due to cancer-related changes and takes into account relationships with close others, especially sustaining and providing required care and support.(3)SMP: Spirituality, Sense of Meaning, and Purpose: The third domain focuses on understanding the individual meaning of suffering and dying, and includes an evaluation of priorities and goals facing an advanced disease.(4)FHM: Preparing for the Future, Sustaining Hope and Facing Mortality: The last domain deals with the acknowledgment of anticipatory fears, balancing life and death, planning advanced treatment, and preparing for the process of dying.The CALM therapy manual  was tranSupportive Psycho-Oncological Counseling Intervention patients received a non-manualized SPI, which is the usual care for patients reporting heightened mental distress in both study centers. Like CALM therapy, patients in the SPI group received three to eight individual therapy sessions lasting 50 min each over a period of 6 months. The SPI integrative approach helps patients to deal with distressing emotions, to reinforce pre-existing resources, and to promote adaptive coping with cancer . It contCancer and Living Meaningfully therapists received the CALM manual and were trained in a 2-day CALM workshop held by CALM-experienced psycho-oncologists who were previously trained by Gary Rodin and colleagues (AM-T and FS-K). Therapists received supervision with a CALM-experienced supervisor every 4 weeks. Therapists did not receive feedback about their individual scoring on the Treatment Integrity Scale during supervision. However, every case was discussed with focus on high treatment integrity. SPI therapists received the same amount of supervision as CALM therapists with non-CALM-trained supervisors and were blinded to the content of the CALM manual.Therapists were licensed as clinical psychotherapists. For the RCT, we aimed to balance sociodemographic characteristics  and therapists’ experience in psycho-oncological care between groups by building pairs of comparable therapists and allocating one in each pair randomly to CALM and to SPI, respectively. For the present study, we randomly selected therapy sessions of 10 CALM therapists and eight SPI therapists in order to rate therapists’ adherence and treatment differentiation.Therapeutic process subscales: (1) Therapeutic Relationship; (2) Modulating Affect; (3) Shifting Frame; (4) Interpretations; Therapeutic content subscales: (5) Symptom Management and Communication with Health Care Providers; (6) Changes in Self and Relations with Close Others; (7) Spirituality, Sense of Meaning, and Purpose; (8) Preparing for the Future, Sustaining Hope and Facing Mortality. For the present study, we rated the four Therapeutic content subscales with the following treatment elements on 19 items:Rodin and a group of CALM experts developed a Treatment Integrity Scale adapted from (1)SC_1: Therapist explores symptoms and/or relationship to health care providers.(2)SC_2: Therapist encourages better understanding of disease symptoms.(3)SC_3: Therapist promotes patient’s consideration of treatment options.(4)SC_4: Therapist encourages patient’s active involvement in medical care and supports communication with health care providers for better symptom control and medical decision-making.(5)CSR_1: Therapist explores patient’s feelings about his or her life story.(6)CSR_2: Therapist validates patient’s sense of worth in light of his or her accomplishments in the domains of family, work, and community.(7)CSR_3: Therapist acknowledges disappointments or regrets that the patient has experienced in those domains.(8)CSR_4: Therapist explores the relational changes imposed by disease.(9)CSR_5: Therapist explores fears and anxieties about dependency and loss of autonomy.(10)CSR_6: Therapist encourages appropriate communication .(11)SMP_1: Therapist explores the patient’s spiritual beliefs and/or sense of meaning and purpose in life.(12)SMP_2: Therapist explores personal meaning of suffering and dying.(13)SMP_3: Therapist evaluates priorities and goals in the face of advanced disease.(14)SMP_4: Therapist promotes acknowledgment that some life goals may no longer be achievable.(15)FHM_1: Therapist explores patient’s attitudes toward the future.(16)FHM_2: Therapist allows expression of sadness and anxiety about the progression of disease.(17)FHM_3: Therapist explores feelings about death and dying.(18)FHM_4: Therapist promotes discussion of advanced care planning.(19)FHM_5: Therapist helps to sustain realistic hope and engagement in life while acknowledging mortality.Depending on the patient’s individual needs, CALM therapists were free to choose treatment elements. Accordingly, not all 19 items were applied in every therapy session. Thus, raters first had to indicate whether treatment elements were “applied” or “not applied.” After an item was coded as “applied,” the extent of therapists’ adherence had to be estimated on a three-point Likert scale with “1 = adherent to some extent” for an acceptable, but not satisfactory implementation, “2 = adherent to a sufficient extent” for a satisfactory implementation, “3 = very adherent” for an excellent implementation of a CALM therapy technique as described in the CALM manual.All therapy sessions throughout the CALM RCT were recorded. We randomly selected therapy sessions from 50 patients , in order to measure treatment integrity in about 25% of the total CALM RCT patient sample which is slightly above the average amount of adherence ratings in previous psycho-oncological intervention studies . For eacAll audio recordings were independently rated by two psychologists who both had a master’s degree in clinical psychology. They were not involved in the CALM RCT and were not familiar with any of the CALM or SPI therapists. Both raters were trained in a 2-day CALM workshop .Both raters were blinded to the objective 4 (treatment differentiation) of the present study: They were instructed to rate the audio recordings “derived from CALM treatment group”; we did not inform raters that half of the audio recordings contained therapy sessions from SPI condition.]. If an item had Cohen’s κ ≥ 0.40, ICCs ≥ 0.75 represented good, ICCs between 0.50 and 0.75 moderate, and ICC < 0.50 poor interrater reliability  for each item. To quantify the extent to which CALM interventions were delivered as intended by CALM therapists (objective 2), we summed up frequencies of score 2 “adherent to a sufficient extent” and score 3 “very adherent” ratings (n_sufficiently or very adherent).In order to verify the application of the CALM manual interventions within CALM therapy sessions (objective 1), we counted the frequencies of “applied” (n_2 tests with a significance level of 0.003 corrected for multiple testing according to Bonferroni (objective 3).We examined whether frequencies of applied CALM techniques differed with regard to the level of their experience using χ2 tests with a significance level of p = 0.003 corrected for multiple testing according to Bonferroni (objective 4).To check if the two treatment conditions (CALM vs. SPI) can be significantly distinguished by blinded raters, treatment differentiation was tested using χ®, Version 26.Data analyses were conducted using IBM SPSSp = 0.18), age (p = 0.72), UICC tumor stage (p = 0.68), or tumor localization (p = 0.21).Patients of the present study (n = 50) were 70% female with a mean age of 57.4 years (SD = 11.7). UICC III tumor stage was diagnosed in 14%, UICC IV in 86% of the patients with various tumor entities: gastrointestinal (37%), gynecological (20%), breast (12%), lung (10%), urogenital (8%), head or neck (4%), and other (8%). The present random patient sample did not differ statistically significant from the total CALM RCT patient sample with regard to sex (p = 0.11). The range of CALM therapists’ experience in psycho-oncological care was 8–30 years  within the group of CALM therapists and 5–30 years  within the group of SPI therapists.Therapists were mostly female, with 70% in the CALM group and 75% in the SPI group. CALM and SPI therapists did not differ statistically significant in their mean age , moderate for 10 items , and poor for four items  and FHM_2: Therapist allows expression of sadness and anxiety about the progression of disease .All 19 treatment elements were applied, but in various frequencies . Three isufficient or very adherent = 34; 45%), lowest for SMP_4: Therapist promotes acknowledgment that some life goals may no longer be achievable (n_sufficient or very adherent = 0).Four out of 19 CALM treatment elements  were administered to a sufficient or very adherent extent in at least 30% of CALM therapy sessions. The highest percentage of “adherent to a sufficient extent” or “very adherent” ratings was obtained for item SC_1: Therapist explores symptoms and/or relationship to health care providers .We classified CALM therapists according to their experience in psycho-oncological care by calculating 50% percentiles  contained overlapping treatment elements.Results verified the application of CALM manual interventions. All 19 CALM treatment elements were addressed within CALM therapy sessions, yet to different extents. Furthermore, none of the CALM treatment elements seemed inapplicable. We also found that not all of the 19 CALM treatment elements were administered in every CALM therapy session. This selection of treatment elements is prescribed in the manual; or more specifically, CALM therapists are instructed to choose in each therapy session the suitable treatment element depending on the patient’s needs.Overall, results of CALM therapists’ adherence indicate deviations from the CALM manual. In contrast, previous CALM trials obtained satisfactory to excellent treatment integrity ratings referring to CALM therapy , indicatThe frequencies of applied CALM treatment elements were moderate as well. One reason might be that raters judged only three therapy sessions instead of the total number of therapy sessions per patient. Audio recordings included the first and the last therapy session which represent both a special stage in the course of therapy. The first session is mainly characterized by attempts to build a therapeutic relationship, whereas the last CALM therapy session addresses the termination of treatment while providing still an open-door policy to terminally ill patients and expressing that the therapist has not “given up” the patient. Thus, it is unlikely that CALM therapists applied in the first and last session as much content-related CALM treatment elements as in the middle sessions which in turn might have led to an underestimation of frequency scores. It has been shown in psychotherapy research that 5–10 therapy sessions per patient are needed to achieve sufficient generalizability for ratings of therapists’ adherence and competence . HoweverChanges in Self and Relations with Close Others domain), whereas treatment elements dealing with patients’ spiritual well-being and the sense of meaning and purpose  were less commonly applied in our study. The therapeutic goals of the Spirituality, Sense of Meaning, and Purpose domain are to aid in evaluation of priorities and goals in the face of advanced disease and to facilitate a rather active approach to the dying experience  of the CALM RCT did not differ significantly from each other with regard to the frequency of the applied treatment elements. This low level of treatment differentiation implies that on average raters could not distinguish CALM from SPI therapy and that SPI and CALM therapists delivered similar treatment elements. CALM therapy draws upon several manualized effective psychotherapeutic interventions that have been developed to address distress in patients with advanced cancer. It is based on empirical research results, clinical observations, and theoretical concepts of supportive-expressive, existential, and psychodynamic therapy as well as stress- and problem-solving trainings. Therefore, it is reasonable that SPI therapists who worked in psycho-oncological care centers for several years deliver overlapping treatment elements to CALM therapy.A shortcoming of this study is that calculations of internal consistency were not possible, since most items were not applied in each session. In addition, another limitation of the present study includes the insufficient interrater reliability. Therefore, the interpretation of the data should be done under consideration of the restriction that raters had difficulties using the rating instrument in a concordant manner. We attenuated this problem by using a consensus version of data after discussing deviations with both raters, which was a time-consuming process and related to higher costs. The treatment integrity rating instrument should only be used in future CALM trials, if an evaluation of psychometric properties was carried out and a subsequent adaption of items followed.Furthermore, we cannot exclude possible selection and confounding biases due to our approach and the relatively small sample size. We also cannot answer the question of whether patient characteristics  might have an influence on therapists’ adherence and thereby on the implementation of the CALM intervention since we did not follow a stratified approach.Finally, our study may contribute to a more transparent evaluation of treatment integrity in future psycho-oncological intervention research. In future research, the correlation between therapists’ adherence and treatment outcome could be addressed using CALM RCT audio recordings. The question arises whether adherent and competently conducted CALM therapies also lead to improved treatment outcome.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethik-Kommission an der Medizinischen Fakultät der Universität Leipzig; Ethik-Kommission der Ärztekammer Hamburg. The patients/participants provided their written informed consent to participate in this study.SK, LQ, and AM-T contributed to conception and design. AM-T and MH provided administrative support. AM-T, FS-K, and GR contributed to provision of study materials and patients. SK, LQ, KS, and RP contributed to collection and assembly of data. SK and RP contributed to data analysis and interpretation. All authors wrote the manuscript and gave final approval of the manuscript.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.


The postoperative seizure freedom represents an important secondary outcome measure in glioblastoma surgery. Recently, supra-total glioblastoma resection in terms of anterior temporal lobectomy (ATL) has gained growing attention with regard to superior long-term disease control for temporal-located glioblastoma compared to conventional gross-total resections (GTR). However, the impact of ATL on seizure outcome in these patients is unknown. We therefore analyzed ATL and GTR as differing extents of resection in regard of postoperative seizure control in patients with temporal glioblastoma and preoperative symptomatic seizures.Between 2012 and 2018, 33 patients with preoperative seizures underwent GTR or ATL for temporal glioblastoma at the authors’ institution. Seizure outcome was assessed postoperatively and 6 months after tumor resection according to the International League Against Epilepsy (ILAE) classification and stratified into favorable (ILAE class 1) versus unfavorable (ILAE class 2–6).Overall, 23 out of 33 patients (70%) with preoperative seizures achieved favorable seizure outcome following resection of temporal located glioblastoma. For the ATL group, postoperative seizure freedom was present in 13 out of 13 patients (100%). In comparison, respective rates for the GTR group were 10 out of 20 patients (50%) .ATL in terms of a supra-total resection strategy was associated with superior favorable seizure outcome following temporal glioblastoma resection compared to GTR. Regarding above mentioned survival benefit following ATL compared to GTR, ATL as an aggressive supra-total resection regime might constitute the surgical modality of choice for temporal-located glioblastoma. Seizures are the most common symptoms in patients with malignant brain tumors and often constitute the first clinical manifestation of an intracranial lesion. Tumor location and histopathological classification are decisive for the frequency of seizures. Supratentorial tumors, especially those with superficial and/or temporal localization, are more epileptogenic. However, the incidence of seizures inversely correlate with the degree of malignancy with seizures occurring more frequently in low-grade tumors.Nevertheless, as neurosurgical, radiological, chemotherapeutic and supportive therapies for glioblastoma have been improved over the last decades –5 and ovPrevious study results indicated that a larger extent of resection might improve the survival of glioblastoma patients –8. In thHere, we present for the first time data on the influence of this aggressive onco-surgical therapy approach on the outcome of seizures after surgical treatment of temporally located glioblastomas.Between 2012 and 2018, all patients with temporal glioblastoma and preoperative seizures aged 18 years or older were included in further analysis. All patients who underwent only biopsy were excluded. After approval by the institutional ethics committee, the medical records were retrospectively reviewed and relevant clinical information were collected and entered into a computerized database . These information included patient age, gender, Karnofsky Performance Score (KPS), tumor size and extent, seizure status and semiology  according to the International League Against Epilepsy (ILAE), molecular histopathological parameters and postoperative seizure outcome.Using the KPS, patients were categorized according to their neurological functional status both preoperatively and in the further postoperative course. KPS ≥ 70 was defined as a favourable outcome during postoperative follow-up immediately after surgery and 3 and 12 months postoperatively.The institutional interdisciplinary central nervous system (CNS) tumor board made the decision on treatment modality on a case-by-case basis at the initial presentation of the patient and during postoperative follow-up. Within the framework of the weekly CNS tumor board, a consensus treatment algorithm is determined for each patient by specialists in neuro-oncology from the fields of neurology, neurosurgery, neuroradiology, neuropathology and radio-oncology. The specification of neurosurgical procedures is primarily based on the individual assessment of the neurosurgeons participating in the CNS tumor board meeting. The decision whether the patient was assigned to ATL or conventional tumor resection was primarily based upon the prevalence of the responsible treating neurosurgeon. Given by the fact, that authors’ institution have a long tradition of epilepsy surgery, those neurosurgeons with epilepsy surgery skills performed more likely ATL. All leading neurosurgeons involved in the surgical treatment of neuro-oncological patients fulfilled the requirements for Neuro-Oncology Centers certificated by the German Cancer Society. Patients with temporal glioblastoma were only selected in this series if the temporal tumor manifestation was within the margin of 4–5 cm in the dominant and 5–6 cm in the non-dominant hemisphere from the temporal pole. Patients with additional infiltration of the mesial temporal lobe structures  in preoperative gadolinium enhanced MR-imaging were also included in the current series. As known from reports on patients with low-grade glioma, the resection of mesial temporal lobe structures is more likely to achieve a better post-operative seizure control . Thus, pFor further analysis the patients were divided into two groups: (1) patients undergoing gross-total resection (GTR) of the temporal contrast-enhancing tumor, and (2) those patients who underwent temporal tumor removal by means of additional anterior temporal lobectomy (ATL). ATL was performed with the inclusion of the contrast-enhanced tumor portion as well as unaffected brain parenchyma as indicated by the preoperatively performed magnetic resonance imaging (MRI). Furthermore, in selected cases resection of the temporal glioblastoma with additional amygdalohippocampectomy was performed. In these cases, however, an oncosurgical rather than an epilepsy-surgical therapeutic approach was pursued, which was derived from the accompanying tumor infiltration of the temporo-mesial structures. All surgical procedures were performed using intraoperative neuronavigation, 5-aminolevulinic acid (5-ALA) fluorescence, and if indicated intraoperative neurophysiological monitoring with motor evoked and somatosensory evoked potentials. When performing ATL, the temporal lobe was removed until the margin of maximum 4.5 cm from temporal pole in dominant and 6.5 cm in non-dominant hemisphere . The resThe extent of resection was evaluated according to the early post-operative MRI within 72 h after surgery. The GTR was defined as complete resection of gadolinium enhancing tissue. In addition, the result of the neuroradiological report of the post-operative MRI examination was taken into account. For this purpose, two independent neuroradiologists, without any knowledge of the clinical course, assessed the neuroimaging. The required information was available for all included patients, extracted from these reports, and categorized according to the above definition. Only patients with GTR were included in the further analysis.Peri- and postoperative complications were defined as any unfavorable event that occurred within 30 days following initial temporal glioblastoma resection .After histopathological confirmation of glioblastoma, the methylation status of the MGMT promoter was analyzed by pyrosequencing and combined bisulfite restriction analysis.After surgical removal of the tumor, all patients received adjuvant treatment consisting of radiotherapy, chemotherapy or combined radiochemotherapy, as recommended by the institutional interdisciplinary tumor board.According to ILAE, tumor-associated epilepsy was defined as (1) at least two unprovoked seizures occurring at intervals of > 24 h or (2) one unprovoked seizure with an increased probability of further seizures similar to the general risk after two unprovoked seizures. Therefore, glioblastoma patients with both 1 and > 1 symptomatic seizure were included for further analysis. According to the semiology, the seizures were distinguished into focal aware seizures, focal impaired awareness seizures and focal to bilateral tonic–clonic seizures.Full seizure control as defined by ILAE Class 1 was determined after 6 months of follow-up instead of the recommended 12 months in the investigated glioblastoma patients and was considered favorable. An unfavorable outcome was defined as ILAE classes 2–6, i.e. pure auras, rare to no improvement and worsening of seizure frequency and/or seizure disabling quality. Detailed information on the ILAE classification system can be found in Fig. Data analysis was performed using the SPSS  computer software package. An unpaired t-test was used for parametric statistics. Categorical variables were analyzed in contingency tables using the exact Fisher test. Results with p values < 0.05 were considered statistically significant.Between 2012 and 2018, a total of 33 patients with temporally located glioblastoma and preoperative seizures were treated at the authors’ institution. The patients ‘ mean age was 59 ± 14 years. The median KPS score at presentation was 90 (range 60–100). Anterior temporal lobectomy (ATL) was performed in 13 patients (39%), whereas the temporal GTR was performed in 20 patients (61%). The median overall survival (OS) was 22 months (95% CI 11.3–32.7). Further details on patient and tumor characteristics are given in Table Surgery-related unfavorable events were present in 4 out of 33 patients (12%). Thereby, postoperative hemorrhage was found in 2 patients (6%) and postoperative meningitis and wound dehiscence in 1 patient, respectively (3%). Among the patients with postoperative hemorrhage, revision surgery was required in one case. Analysis of peri- and postoperative complications did not reveal any profound new language deficit.As shown in Table Overall, favorable postoperative seizure outcome in terms of ILAE class 1 was achieved in 23 patients (70%) with temporal glioblastoma and preoperative TRE. There was no statistically significant difference between seizure free patients and patients with continuous seizures according to the preoperative seizure semiology. The analysis of the correlation between seizure control and overall survival (OS) revealed a median OS of 23 months IQR 13–33.5) in seizure free patients (ILAE 1) vs. 26 months (IQR 3–33.5) in patients with postoperatively persistent seizures (p = 0.34). From the total of 33 patients, adjuvant treatment according to the Stupp protocol was performed in 26 patients (80%), 4 patients (12%) received adjuvant treatment according to the protocol of the CeTeG-trial [–33.5 in A total of 17 patients (52%) suffering from temporally located glioblastoma and preoperative TRE had additional temporo-mesial tumorous infiltration. In detail, 3 of the 17 patients exhibited a contrast-absorbing infiltration of the temporo-mesial structures, whereas 14 of the 17 patients had a FLAIR-correlate in the area of the temporo-mesial structures. In selected patients, additional amygdalohippocampectomy was performed in order to achieve GTR, better relief of space-occupying effect and/or a more favorable seizure outcome. Therefore, 12 of 13 patients (92%) with additional amygdalohippocampectomy achieved favorable seizure outcome compared to 12 of 20 patients (60%) without additional amygdalohippocampectomy (p = 0.06).The groups of ATL and temporal GTR did not significantly differ in regard of age, sex, tumor localization and MGMT promoter methylation status Table .Table 3C20 patients (61%) with temporal glioblastoma and preoperative TRE underwent GTR, whilst ATL was performed in 13 patients (39%). Patients who underwent ATL for temporal glioblastoma achieved significantly more often favorable seizure outcome (ILAE 1) compared to patients with temporal GTR .The concept of supra-total resection strategies extending considerably beyond tumor-enhancing MRI lesions is gaining a growing evidence base in neurosurgical oncology for the surgical treatment of malignant primary brain tumors. In addition to improved overall survival and prolonged progression-free survival interval in previous studies, the present case study also indicates an advantage of a supra-marginal resection strategy with regard to seizure outcome in temporally localized glioblastoma associated with tumor-related epilepsy.In brain tumors, epilepsy often represents the first symptom and is considered to be a favorable prognostic factor with regard to survival in both low and high-grade gliomas –16. HoweNevertheless, in addition to pharmacological therapy, surgical treatment—as evidenced by epilepsy surgery and experiences with low-grade glioma surgery—can also make a decisive contribution to seizure outcome , 23, 24.Anterior temporal lobectomy has already been established in epilepsy surgical research as a useful surgical strategy for alleviating seizures in patients with an epileptogenic focus in the temporal lobe area , 26. In Infiltration of the temporo-mesial structures seems to make a significant contribution to the postoperative seizure outcome. In this context, an additional amygdalohippocampectomy was performed in patients with preoperatively proven contrast agent enriching lesions in the area of the temporo-mesial structures. In patients who had a FLAIR component without any contrast enhancement in the temporo-mesial structures, the removal of these structures was at the discretion of the surgeon and was usually not carried out for epileptological considerations, but rather for neuro-oncological considerations regarding a reduction of the local mass in view of an imminent further therapy such as radiation with the potential danger of subsequent reactive tissue swelling. In the literature, especially in the case of low-grade gliomas with accompanying epilepsy, surgical resection of the temporo-mesial structures is associated with an improved seizure outcome , 27. BesThe present study has several limitations. The main limitations of this study are the small sample size and the retrospective study design. The surgical procedure of ATL as a paradigm for supratotal resection regimes entails a highly selected cohort of glioblastoma patients with a tumor manifestation within 4 to 5 cm from the temporal pole on the dominant hemisphere and 5 to 6 cm on the non dominant hemisphere. Therefore, a greater sample size is instantly needed in order to increase the validity of our findings. For this purpose, rather multicenter prospective studies will be capable to cope with the restriction of a low incidence of precisely temporal located glioblastoma and thus might create a safe prerequisite to sufficiently assess ATL as a supramarginal resection strategy in temporal glioblastoma disease. Furthermore, estimating the potential effects of radiation and chemotherapy on seizure outcome is complex. However, there are only a few studies assessing the outcome of seizures in patients undergoing glioblastoma resection. The vast majority of studies on glioblastoma resection focus on factors that influence survival or recurrence. Seizure control represents an aspect that has not yet been studied in depth, and has so far been limited to patients with low-grade gliomas. Therefore, the homogeneity of patient/tumor characteristics, methods and results provide an encouraging clinical background for future studies focusing on the influence of the extent of resection on the control of preoperative tumor-related epilepsy in patients suffering from glioblastoma.The present study indicates that anterior temporal lobectomy might be associated with a more favorable seizure outcome after temporal glioblastoma resection in terms of a supra-marginal resection strategy compared to GTR. In addition to the described survival advantage, ATL for surgical treatment of temporally localized glioblastoma henceforth seems to be the therapy of first choice given the potential increased seizure freedom.