Chitin and chitosan remodeling defines vegetative development and Trichoderma biocontrol.

Fungal parasitism depends on the ability to invade host organisms and mandates adaptive cell wall remodeling to avoid detection and defense reactions by the host. All plant and human pathogens share invasive strategies, which aid to escape the chitin-triggered and chitin-targeted host immune system. Here we describe the full spectrum of the chitin/chitosan-modifying enzymes in the mycoparasite Trichoderma atroviride with a central role in cell wall remodeling. Rapid adaption to a variety of growth conditions, environmental stresses and host defense mechanisms such as oxidative stress depend on the concerted interplay of these enzymes and, ultimately, are necessary for the success of the mycoparasitic attack. To our knowledge, we provide the first in class description of chitin and associated glycopolymer synthesis in a mycoparasite and demonstrate that they are essential for biocontrol. Eight chitin synthases, six chitin deacetylases, additional chitinolytic enzymes, including six chitosanases, transglycosylases as well as accessory proteins are involved in this intricately regulated process. Systematic and biochemical classification, phenotypic characterization and mycoparasitic confrontation assays emphasize the importance of chitin and chitosan assembly in vegetative development and biocontrol in T. atroviride. Our findings critically contribute to understanding the molecular mechanism of chitin synthesis in filamentous fungi and mycoparasites with the overarching goal to selectively exploit the discovered biocontrol strategies.

IL-6 trans-signaling is essential for the development of hepatocellular carcinoma in mice.

Hepatocellular carcinoma (HCC) is one of the most frequent tumors worldwide with rising incidence. The inflammatory cytokine, interleukin-6 (IL-6), is a critical mediator of HCC development. It can signal through two distinct pathways: the IL-6 classic and the IL-6 trans-signaling pathway. Whereas IL-6 classic signaling is important for innate and acquired immunity, IL-6 trans-signaling has been linked to accelerated liver regeneration and several chronic inflammatory pathologies. However, its implication in liver tumorigenesis has not been addressed yet. Here, we show that IL-6 trans-signaling, but not IL-6 classic signaling, is essential to promote hepatocellular carcinogenesis by two mechanisms: First, it prevents DNA-damage-induced hepatocyte apoptosis through suppression of p53 and enhances ß-catenin activation and tumor proliferation. Second, IL-6 trans-signaling directly induces endothelial cell proliferation to promote tumor angiogenesis. Consequently, soluble gp130 fused to Fc transgenic mice lacking IL-6 trans-signaling are largely protected from tumor formation in a diethylnitrosamine/3,3',5,5'-tetrachloro-1,4-bis(pyridyloxy)benzene model of HCC. CONCLUSION: IL-6 trans-signaling, and not IL-6 classic signaling, is mandatory for development of hepatocellular carcinogenesis. Therefore, specific inhibition of IL-6 trans-signaling, rather than total inhibition of IL-6 signaling, is sufficient to blunt tumor initiation and impair tumor progression without compromising IL-6 classic signaling-driven protective immune responses. (Hepatology 2017;65:89-103).

Visualizing fungal metabolites during mycoparasitic interaction by MALDI mass spectrometry imaging.

Studying microbial interactions by MALDI mass spectrometry imaging (MSI) directly from growing media is a difficult task if high sensitivity is demanded. We present a quick and robust sample preparation strategy for growing fungi (Trichoderma atroviride, Rhizoctonia solani) on glass slides to establish a miniaturized confrontation assay. By this we were able to visualize metabolite distributions by MALDI MSI after matrix deposition with a home-built sublimation device and thorough recrystallization. We present for the first time MALDI MSI data for secondary metabolite release during active mycoparasitism.

Self-assembly at air/water interfaces and carbohydrate binding properties of the small secreted protein EPL1 from the fungus Trichoderma atroviride.

The protein EPL1 from the fungus Trichoderma atroviride belongs to the cerato-platanin protein family. These proteins occur only in filamentous fungi and are associated with the induction of defense responses in plants and allergic reactions in humans. However, fungi with other lifestyles also express cerato-platanin proteins, and the primary function of this protein family has not yet been elucidated. In this study, we investigated the biochemical properties of the cerato-platanin protein EPL1 from T. atroviride. Our results showed that EPL1 readily self-assembles at air/water interfaces and forms protein layers that can be redissolved in water. These properties are reminiscent of hydrophobins, which are amphiphilic fungal proteins that accumulate at interfaces. Atomic force microscopy imaging showed that EPL1 assembles into irregular meshwork-like substructures. Furthermore, surface activity measurements with EPL1 revealed that, in contrast to hydrophobins, EPL1 increases the polarity of aqueous solutions and surfaces. In addition, EPL1 was found to bind to various forms of polymeric chitin. The T. atroviride genome contains three epl genes. epl1 was predominantly expressed during hyphal growth, whereas epl2 was mainly expressed during spore formation, suggesting that the respective proteins are involved in different biological processes. For epl3, no gene expression was detected under most growth conditions. Single and double gene knock-out strains of epl1 and epl2 did not reveal a detectable phenotype, showing that these proteins are not essential for fungal growth and development despite their abundant expression.

A comparative cell wall analysis of Trichoderma spp. confirms a conserved polysaccharide scaffold and suggests an important role for chitosan in mycoparasitism.

Fungal cell walls are dynamic extracellular matrices that enable efficient adaptation to changing environments. While the cell wall compositions of yeasts, human, and plant pathogenic fungi have been studied to some extent, the cell walls of mycoparasites remain poorly characterized. Trichoderma species comprise a diverse group of soil fungi with different survival strategies and lifestyles. The comparative study of cell wall carbohydrate-active enzymes in 13 Trichoderma spp. revealed that the types of enzymes involved in chitin and chitosan metabolism are phylogenetically distant between mycoparasitic and saprotrophic species. Here, we compare the carbohydrate composition and function of the cell wall of a saprotrophic strain Trichoderma reesei with that of the mycoparasitic, biological control agent Trichoderma atroviride. Monosaccharide and glycosidic linkage analyses as well as dual in situ interaction assays showed that the cell wall polysaccharide composition is conserved between both species, except for the amounts of chitin detected. The results suggest that the observed accumulation of chitosan during mycoparasitism may prevent host recognition. Remarkably, Trichoderma atroviride undergoes dynamic cell wall adaptations during both vegetative development and mycoparasitism, which appears to be confirmed by an evolutionarily expanded group of specialized enzymes. Overall, our analyses support the notion that habitat specialization is reflected in cell wall architecture and that plastic chitin remodeling may confer an advantage to mycoparasites, ultimately enabling the successful invasion and parasitism of plant pathogens. This information may potentially be exploited for the control of crop diseases using biological agents. IMPORTANCE: Trichoderma species are emerging model fungi for the development of biocontrol agents and are used in industrial biotechnology as efficient enzyme producers. Fungal cell walls are complex structures that differ in carbohydrate, protein, and enzyme composition across taxa. Here, we present a chemical characterization of the cell walls of two Trichoderma spp., namely the predominantly saprotrophic Trichoderma reesei and the mycoparasite Trichoderma atroviride. Chemical profiling revealed that Trichoderma spp. remodel their cell wall to adapt to particular lifestyles, with dynamic changes during vegetative development. Importantly, we found that chitosan accumulation during mycoparasitism of a fungal host emerged as a sophisticated strategy underpinning an effective attack. These insights shed light on the molecular mechanisms that allow mycoparasites to overcome host defenses and can be exploited to improve the application of T. atroviride in biological pest control. Moreover, our results provide valuable information for targeting the fungal cell wall for therapeutic purposes.

Comparative transcriptomics reveals different strategies of Trichoderma mycoparasitism.

BACKGROUND: Trichoderma is a genus of mycotrophic filamentous fungi (teleomorph Hypocrea) which possess a bright variety of biotrophic and saprotrophic lifestyles. The ability to parasitize and/or kill other fungi (mycoparasitism) is used in plant protection against soil-borne fungal diseases (biological control, or biocontrol). To investigate mechanisms of mycoparasitism, we compared the transcriptional responses of cosmopolitan opportunistic species and powerful biocontrol agents Trichoderma atroviride and T. virens with tropical ecologically restricted species T. reesei during confrontations with a plant pathogenic fungus Rhizoctonia solani. RESULTS: The three Trichoderma spp. exhibited a strikingly different transcriptomic response already before physical contact with alien hyphae. T. atroviride expressed an array of genes involved in production of secondary metabolites, GH16 ß-glucanases, various proteases and small secreted cysteine rich proteins. T. virens, on the other hand, expressed mainly the genes for biosynthesis of gliotoxin, respective precursors and also glutathione, which is necessary for gliotoxin biosynthesis. In contrast, T. reesei increased the expression of genes encoding cellulases and hemicellulases, and of the genes involved in solute transport. The majority of differentially regulated genes were orthologues present in all three species or both in T. atroviride and T. virens, indicating that the regulation of expression of these genes is different in the three Trichoderma spp. The genes expressed in all three fungi exhibited a nonrandom genomic distribution, indicating a possibility for their regulation via chromatin modification. CONCLUSION: This genome-wide expression study demonstrates that the initial Trichoderma mycotrophy has differentiated into several alternative ecological strategies ranging from parasitism to predation and saprotrophy. It provides first insights into the mechanisms of interactions between Trichoderma and other fungi that may be exploited for further development of biofungicides.

Comparative analysis of the repertoire of G protein-coupled receptors of three species of the fungal genus Trichoderma.

BACKGROUND: Eukaryotic organisms employ cell surface receptors such as the seven-transmembrane G protein-coupled receptors (GPCRs) as sensors to connect to the environment. GPCRs react to a variety of extracellular cues and are considered to play central roles in the signal transduction in fungi. Several species of the filamentous ascomycete Trichoderma are potent mycoparasites, i.e. can attack and parasitize other fungi, which turns them into successful bio-fungicides for the protection of plants against fungal phytopathogens. The identification and characterization of GPCRs will provide insights into how Trichoderma communicates with its environment and senses the presence of host fungi. RESULTS: We mined the recently published genomes of the two mycoparasitic biocontrol agents Trichoderma atroviride and Trichoderma virens and compared the identified GPCR-like proteins to those of the saprophyte Trichoderma reesei. Phylogenetic analyses resulted in 14 classes and revealed differences not only among the three Trichoderma species but also between Trichoderma and other fungi. The class comprising proteins of the PAQR family was significantly expanded both in Trichoderma compared to other fungi as well as in the two mycoparasites compared to T. reesei. Expression analysis of the PAQR-encoding genes of the three Trichoderma species revealed that all except one were actually transcribed. Furthermore, the class of receptors with a DUF300 domain was expanded in T. atroviride, and T. virens showed an expansion of PTH11-like receptors compared to T. atroviride and T. reesei. CONCLUSIONS: Comparative genome analyses of three Trichoderma species revealed a great diversity of putative GPCRs with genus- and species- specific differences. The expansion of certain classes in the mycoparasites T. atroviride and T. virens is likely to reflect the capability of these fungi to establish various ecological niches and interactions with other organisms such as fungi and plants. These GPCRs consequently represent interesting candidates for future research on the mechanisms underlying mycoparasitism and biocontrol.

Self versus non-self: fungal cell wall degradation in Trichoderma.

Lysis of the prey's cell wall is one of the key steps during mycoparasitism. Genome analysis of two mycoparasitic Trichoderma species, T. atroviride and T. virens, revealed an expanded arsenal of genes encoding enzymes potentially involved in cell wall hydrolysis. Glycoside hydrolase family 18, which contains all fungal chitinases, is the largest family of carbohydrate-active enzymes in mycoparasitic Trichoderma species. However, in addition to their aggressive functions during mycoparasitism, the roles of chitinases and other cell wall degrading enzymes also include remodelling and recycling of the fungus's own cell wall. In this review we discuss current knowledge about fungal cell wall degrading enzymes in Trichoderma and how the fungus distinguishes between self- and non-self fungal cell wall degradation. In the past few years, the chitinolytic enzyme machinery of Trichoderma has been used as a model system to address this question. Gene expression profiles of most investigated chitinases indicate an overlap of functions of the respective enzymes and an involvement in both self- and non-self fungal cell wall degradation. Similar sets of enzymes appear to be involved in mycoparasitism, exogenous chitin decomposition and recycling of the fungus's own cell wall. Thus, we hypothesize that the regulation of self and non-self fungal cell wall degradation is not due to a speciation of individual chitinases. Rather, we hypothesize that it is regulated by substrate accessibility due to cell wall protection in healthy hyphae vs deprotection during mycoparasitic attack, hyphal ageing and autolysis.

The Multilateral Efficacy of Chitosan and Trichoderma on Sugar Beet.

The majority of all fungal formulations contain Trichoderma spp., making them effective biological control agents for agriculture. Chitosan, one of the most effective natural biopolymers, was also reported as a plant resistance enhancer and as a biocide against a variety of plant pathogens. An in vitro three-way interaction assay of T. atroviride, chitosan, and important plant pathogens (such as Cercospora beticola and Fusarium oxysporum) revealed a synergistic effect on fungistasis. Furthermore, chitosan coating on Beta vulgaris ssp. vulgaris seeds positively affected the onset and efficiency of germination. We show that priming with T. atroviride spores or chitosan leads to the induced expression of a pathogenesis-related gene (PR-3), but only supplementation of chitosan led to significant upregulation of phytoalexin synthesis (PAL) and oxidative stress-related genes (GST) as a defense response. Repeated foliar application of either agent promoted growth, triggered defense reactions, and reduced incidence of Cercospora leaf spot (CLS) disease in B. vulgaris. Our data suggest that both agents are excellent candidates to replace or assist common fungicides in use. Chitosan triggered the systemic resistance and had a biocidal effect, while T. atroviride mainly induced stress-related defense genes in B. vulgaris. We assume that both agents act synergistically across different signaling pathways, which could be of high relevance for their combinatorial and thus beneficial application on field.

Analysis of subgroup C of fungal chitinases containing chitin-binding and LysM modules in the mycoparasite Trichoderma atroviride.

Fungi have a plethora of chitinases, which can be phylogenetically divided into three subgroups (A, B and C). Subgroup C (sgC) chitinases are especially interesting due to their multiple carbohydrate-binding modules, but they have not been investigated in detail yet. In this study, we analyzed sgC chitinases in the mycoparasites Trichoderma atroviride and Trichoderma virens. The expression of sgC chitinase genes in T. atroviride was induced during mycoparasitism of the fungal prey Botrytis cinerea, but not Rhizoctonia solani and correspondingly only by fungal cell walls of the former. Interestingly, only few sgC chitinase genes were inducible by chitin, suggesting that non-chitinous cell wall components can act as inducers. In contrast, the transcriptional profile of the most abundantly expressed sgC chitinase gene tac6 indicated a role of the protein in hyphal network formation. This shows that sgC chitinases have diverse functions and are not only involved in the mycoparasitic attack. However, sequence analysis and 3D modelling revealed that TAC6 and also its ortholog in T. virens have potentially detrimental deletions in the substrate-binding site and are thus probably not catalytically active enzymes. Genomic analysis showed that the genes neighboring sgC chitinases often encode proteins that are solely composed of multiple LysM modules, which were induced by similar stimuli as their neighboring sgC chitinase genes. This study provides first insights into fungal sgC chitinases and their associated LysM proteins.

Novel hydrophobins from Trichoderma define a new hydrophobin subclass: protein properties, evolution, regulation and processing.

Hydrophobins are small proteins, characterised by the presence of eight positionally conserved cysteine residues, and are present in all filamentous asco- and basidiomycetes. They are found on the outer surfaces of cell walls of hyphae and conidia, where they mediate interactions between the fungus and the environment. Hydrophobins are conventionally grouped into two classes (class I and II) according to their solubility in solvents, hydropathy profiles and spacing between the conserved cysteines. Here we describe a novel set of hydrophobins from Trichoderma spp. that deviate from this classification in their hydropathy, cysteine spacing and protein surface pattern. Phylogenetic analysis shows that they form separate clades within ascomycete class I hydrophobins. Using T. atroviride as a model, the novel hydrophobins were found to be expressed under conditions of glucose limitation and to be regulated by differential splicing.

Differential regulation of orthologous chitinase genes in mycoparasitic Trichoderma species.

Mycoparasitic Trichoderma species have expanded numbers of fungal subgroup C chitinases that contain multiple carbohydrate binding modules and could thus be important for fungal cell wall degradation during the mycoparasitic attack. In this study, we analyzed the gene regulation of subgroup C chitinases in the mycoparasite Trichoderma virens. In addition to regulation by nutritional stimuli, we found complex expression patterns in different parts of the fungal colony, and also, the mode of cultivation strongly influenced subgroup C chitinase transcript levels. Thus, the regulation of these genes is governed by a combination of colony-internal and -external signals. Our results showed completely different expression profiles of subgroup C chitinase genes in T. virens than in a previous study with T. atroviride, although both fungi are potent mycoparasites. Only a few subgroup C chitinase orthologues were found in T. atroviride and T. virens, and even those showed substantially divergent gene expression patterns. Microscopic analysis revealed morphogenetic differences between T. atroviride and T. virens, which could be connected to differential subgroup C chitinase gene expression. The biological function of fungal subgroup C chitinases therefore might not be as clear-cut as previously anticipated. They could have pleiotropic roles and might be involved in both degradation of exogenous chitinous carbon sources, including other fungal cell walls, and recycling of their own cell walls during hyphal development and colony formation.

Personal Trust and System Trust in the Sharing Economy: A Comparison of Community- and Platform-Based Models.

Currently, new business models created in the sharing economy differ considerably and they differ in the formation of trust as well. If and how trust can be created is shown by a comparison of two examples which diverge in their founding philosophy. The chosen example of community-based economy, Community Supported Agriculture (CSA), no longer trusts the capitalist system and therefore distances itself and creates its own environment including a new business model. It is implemented within rather small groups where trust is created by personal relations and face-to-face communication. On the contrary, the example of a platform economy, the accommodation-provider company Airbnb, shows trust in the system and pushes technological innovations through the use of platform applications. It promotes trust and confidence in the progress of technology. For the conceptual analysis, the distinction between personal trust and system trust defined by Niklas Luhmann is adopted. The analysis describes two different modes of trust formation and how they push distrust or improve trust. Grounded on these analyses, assumptions on the process of trust formation within varying models of the sharing economy are formulated as well as a hypothesis about possible developments is introduced for further research.

The Effect of Sprouting in Lentil (Lens culinaris) Nutritional and Microbiological Profile.

Biological and vegetarian raw food products, in particular based on legume sprouts, are an increasing food trend, due to their improved nutritional value when compared to seeds. Herein, protein and mineral profiles were studied in 12 lentil varieties, with varieties Du Puy, Kleine Schwarze, Rosana, Flora, Große Rote and Kleine Späths II demonstrating the highest protein percentages. After sprouting, protein percentages increased significantly in 10 of the 12 varieties, with the highest increases ranging between 20-23% in Dunkelgrün Marmorierte, Du Puy, Große Rote and Kleine Späths II varieties. While Fe concentration was significantly decreased in three varieties (Samos, Große Rote and Kleine Späths II), Zn and Mn were positively impacted by sprouting (p ≤ 0.05). Magnesium concentration was not affected by sprouting, while Ca and K had percentage increases between 41% and 58%, and 28% and 30%, respectively, in the best performing varieties (Kleine Schwarze, Dunkelgrün Marmorierte, Samos and Rosana). Regardless of the associated nutritional benefits, issues pertaining to sprouts microbiological safety must be ensured. The best results for the disinfection protocols were obtained when combining the seed treatment with SDS reagent followed by an Amukine application on the sprouts, which did not affect germination rates or sprout length. The increasing levels of sprout consumption throughout the world require efficient implementation of safety measures, as well as a knowledge-based selection for the nutritional quality of the seeds.

Relationship between volume and outcome for surgery on congenital diaphragmatic hernia: A systematic review.

BACKGROUND: Congenital diaphragmatic hernia (CDH) is a rare and life-threatening anomaly that needs surgical therapy after clinical stabilization of the neonate. Given an existing volume-outcome relationship for other high-risk, low volume procedures, we aimed at examining the relationship between hospital or surgeon volume and outcomes for surgery on CDH. METHODS: We conducted a systematic search in multiple databases in September 2019 and searched for additional literature. We assessed risk of bias of included studies using ROBINS-I and synthesized results in a structured narrative way using GRADE. RESULTS: We included 5 cohort studies on hospital volume. Results for in-hospital mortality, one-year mortality and length of stay are inconclusive. The certainty of the evidence was very low for all outcomes, due to risk of bias, inconsistency and imprecision. We did not identify any study on surgeon volume. CONCLUSION: Due to the very low certainty of the evidence it is uncertain whether higher hospital volume is associated with favorable outcomes for neonates undergoing surgery for CDH. There is no evidence on the relationship between surgeon volume and outcomes. Future studies should use more rigorous methodology and analyze additional outcomes to allow for more meaningful inferences. LEVEL OF EVIDENCE: III SYSTEMATIC REVIEW REGISTRATION: PROSPERO (CRD42018090231).

Transcriptomic response of the mycoparasitic fungus Trichoderma atroviride to the presence of a fungal prey.

BACKGROUND: Combating the action of plant pathogenic microorganisms by mycoparasitic fungi has been announced as an attractive biological alternative to the use of chemical fungicides since two decades. The fungal genus Trichoderma includes a high number of taxa which are able to recognize, combat and finally besiege and kill their prey. Only fragments of the biochemical processes related to this ability have been uncovered so far, however. RESULTS: We analyzed genome-wide gene expression changes during the begin of physical contact between Trichoderma atroviride and two plant pathogens Botrytis cinerea and Rhizoctonia solani, and compared with gene expression patterns of mycelial and conidiating cultures, respectively. About 3000 ESTs, representing about 900 genes, were obtained from each of these three growth conditions. 66 genes, represented by 442 ESTs, were specifically and significantly overexpressed during onset of mycoparasitism, and the expression of a subset thereof was verified by expression analysis. The upregulated genes comprised 18 KOG groups, but were most abundant from the groups representing posttranslational processing, and amino acid metabolism, and included components of the stress response, reaction to nitrogen shortage, signal transduction and lipid catabolism. Metabolic network analysis confirmed the upregulation of the genes for amino acid biosynthesis and of those involved in the catabolism of lipids and aminosugars. CONCLUSION: The analysis of the genes overexpressed during the onset of mycoparasitism in T. atroviride has revealed that the fungus reacts to this condition with several previously undetected physiological reactions. These data enable a new and more comprehensive interpretation of the physiology of mycoparasitism, and will aid in the selection of traits for improvement of biocontrol strains by recombinant techniques.

Differential signaling of cmvIL-10 through common variants of the IL-10 receptor 1.

Human IL-10 (hIL-10) signaling is mediated by receptors consisting of two subunits, IL-10 receptor 1 (IL-10R1) and IL-10 receptor 2. Two common variants of the IL-10R1 (Ser 138 Gly (single-nucleotide polymorphism 3, SNP3) and Gly 330 Arg (SNP4)) are associated with diverse disease phenotypes. Viral homologs to hIL-10, such as cmvIL-10, utilize the same IL-10 receptor complex as part of viral immune evasion strategies. For the present study we hypothesized that IL-10R1 variants alter the ability of viral IL-10 to utilize the IL-10R1 signaling pathway. HeLa cell clones expressing different IL-10R1 haplotypes (WT or any variant) were incubated with hIL-10 or cmvIL-10. In cells expressing IL-10R1-WT, cmvIL-10 (both non-glycosylated- and HeLa-expressed) resulted in equal or slightly stronger STAT3 phosphorylation compared with hIL-10. In clones expressing IL-10R1-SNP3, IL-10R1-SNP4 or IL-10R1-SNP3+4, the cmvIL-10 showed significantly less STAT3 phosphorylation, especially when HeLa-expressed cytokines were used. Time course experiments demonstrated a slower kinetic of cmvIL-10 STAT3 activation through the variant IL-10R1. Similarly, IL-10R1 variants decreased the cmvIL-10-induced SOCS3 and signaling lymphocytic activation molecule mRNA expression. These data suggest that the IL-10R1 variants differentially reduce the signaling activity of cmvIL-10 and thereby may affect CMV's ability to escape from the host's immune surveillance.

Reconstruction of soft tissue defects overlying the Achilles tendon using the super extended abductor hallucis muscle flap.

BACKGROUND: Soft tissue defects overlying the Achilles tendon often occur after traumatic ruptures of the Achilles tendon or because of pressure ulcers in patients suffering from circulatory problems. Ideally, reconstruction should be achieved in as few stages as possible. Currently, there are different methods used to treat these defects. Here, we examined whether reconstruction of this region could benefit from the super extended abductor hallucis muscle flap. METHODS: In 12 cadaver feet, the vascular supply and mobilization radius of the abductor hallucis muscle were studied to clarify the possible clinical utilization of this flap. A technique for Achilles defect reconstruction using this flap, along with the functional and cosmetic results in six patients are presented. RESULTS: Our anatomic findings agree with those available in the literature and the adaptation in pedicle preparation allowed an increase in rotation of the flap for successful coverage of defects overlying the Achilles tendon. Using the super extended abductor hallucis muscle flap, the functionality and the anatomic shape were successfully reconstructed. Six weeks after surgery all patients were fully mobile, unless they suffered from Achilles tendon ruptures then they were mobile after 12 weeks. CONCLUSIONS: The super extended abductor hallucis muscle flap might represent an alternative to established methods because of ease of handling and a shorter anesthesia compared with a free flap procedure.

Relationship between volume and outcome for congenital diaphragmatic hernia: a systematic review protocol.

BACKGROUND: Congenital diaphragmatic hernia is a rare and life-threatening anomaly that occurs during fetal development and results in an incomplete or incorrect formation of the diaphragm. Surgical therapy of the diaphragm should be performed after clinical stabilization of the neonate. Higher hospital or surgeon volume has previously been found to be associated with better clinical outcomes for different especially high-risk, low-volume procedures. Therefore, we aim to examine the relationship between hospital or surgeon volume and outcomes for congenital diaphragmatic hernia. METHODS: This systematic review protocol has been designed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis Protocol. We will perform a systematic literature search in MEDLINE, Embase, CINAHL and Biosis Previews without applying any limitations. In addition, we will search for relevant conference abstracts. We will screen titles and abstracts of retrieved studies, obtain potentially relevant full texts, and assess the eligibility of those full texts against our inclusion criteria. We will include comparative studies analyzing the relationship between hospital or surgeon volume and clinical outcomes. We will systematically assess risk of bias of included studies and extract data on the study design, patient characteristics, case-mix adjustments, statistical methods, hospital and surgeon volume, and outcomes into standardized tables. Title and abstract screening, full-text screening, critical appraisal, and data extraction of results will be conducted by two reviewers independently. Other data will be extracted by one reviewer and checked for accuracy by a second one. Any disagreements will be resolved by discussion. We will not perform a meta-analysis as we expect included studies to be clinically and methodologically very diverse. We will synthesize findings from primary studies in a structured narrative way and using GRADE. DISCUSSION: Given the lack of a comprehensive summary of findings on the relationship between hospital or surgeon volume and outcomes for congenital diaphragmatic hernia, this systematic review will put things right. Results can be used to inform decision makers or clinicians and to adapt medical care. SYSTEMATIC REVIEW REGISTRATION: PROSPERO ( CRD42018090231 ).

Hepatic leptin receptor expression can partially compensate for IL-6Rα deficiency in DEN-induced hepatocellular carcinoma.

OBJECTIVE: The current obesity pandemic represents a major health burden, given that it predisposes to the development of numerous obesity-associated disorders. The obesity-derived adipokines not only impair systemic insulin action but also increase the incidence of hepatocellular carcinoma (HCC), a highly prevalent cancer with poor prognosis. Thus, worldwide incidences of HCC are expected to further increase, and defining the molecular as well as cellular mechanisms will allow for establishing new potential treatment options. The adipose tissue of obese individuals increases circulating leptin and interleukin-6 (IL-6) levels, which both share similar signaling capacities such as Signal Transducer and Activator of Transcription 3 (STAT3) and Phosphoinositide 3-kinase (PI3K)/Akt activation. While mouse models with deficient IL-6 signaling show an ameliorated but not absent Diethylnitrosamine (DEN)-induced HCC development, the morbid obesity in mice with mutant leptin signaling complicates the dissection of hepatic leptin receptor (LEPR) and IL-6 signaling in HCC development. Here we have investigated the function of compensating hepatic LEPR expression in HCC development of IL-6Rα-deficient mice. METHODS: We generated and characterized a mouse model of hepatic LEPR deficiency that was intercrossed with IL-6Rα-deficient mice. Cohorts of single and double knockout mice were subjected to the DEN-HCC model to ascertain liver cancer development and characterize metabolic alterations. RESULTS: We demonstrate that both high-fat diet (HFD)-induced obesity and IL-6Rα deficiency induce hepatic Lepr expression. Consistently, double knockout mice show a further reduction in tumor burden in DEN-induced HCC when compared to control and single LepRL-KO/IL-6Rα knock out mice, whereas metabolism remained largely unaltered between the genotypes. CONCLUSIONS: Our findings reveal a compensatory role for hepatic LEPR in HCC development of IL-6Rα-deficient mice and suggest hepatocyte-specific leptin signaling as promoter of HCC under obese conditions.