Molecular profiling and specific targeting of gemcitabine-resistant subclones in heterogeneous pancreatic cancer cell populations.

Purpose: Chemotherapy is pivotal in the multimodal treatment of pancreatic ductal adenocarcinoma (PDAC). Technical advances unveiled a high degree of inter- and intratumoral heterogeneity. We hypothesized that intratumoral heterogeneity (ITH) impacts response to gemcitabine treatment and demands specific targeting of resistant subclones. Methods: Using single cell-derived cell lines (SCDCLs) from the classical cell line BxPC3 and the basal-like cell line Panc-1, we addressed the effect of ITH on response to gemcitabine treatment. Results: Individual SCDCLs of both parental tumor cell populations showed considerable heterogeneity in response to gemcitabine. Unsupervised PCA including the 1,000 most variably expressed genes showed a clustering of the SCDCLs according to their respective sensitivity to gemcitabine treatment for BxPC3, while this was less clear for Panc-1. In BxPC3 SCDCLs, enriched signaling pathways EMT, TNF signaling via NfKB, and IL2STAT5 signaling correlated with more resistant behavior to gemcitabine. In Panc-1 SCDCLs MYC targets V1 and V2 as well as E2F targets were associated with stronger resistance. We used recursive feature elimination for Feature Selection in order to compute sets of proteins that showed strong association with the response to gemcitabine. The optimal protein set calculated for Panc-1 comprised fewer proteins in comparison to the protein set determined for BxPC3. Based on molecular profiles, we could show that the gemcitabine-resistant SCDCLs of both BxPC3 and Panc-1 are more sensitive to the BET inhibitor JQ1 compared to the respective gemcitabine-sensitive SCDCLs. Conclusion: Our model system of SCDCLs identified gemcitabine-resistant subclones and provides evidence for the critical role of ITH for treatment response in PDAC. We exploited molecular differences as the basis for differential response and used these for more targeted therapy of resistant subclones.

Panomics reveals patient individuality as the major driver of colorectal cancer progression.

BACKGROUND: Colorectal cancer (CRC) is one of the most prevalent cancers, with over one million new cases per year. Overall, prognosis of CRC largely depends on the disease stage and metastatic status. As precision oncology for patients with CRC continues to improve, this study aimed to integrate genomic, transcriptomic, and proteomic analyses to identify significant differences in expression during CRC progression using a unique set of paired patient samples while considering tumour heterogeneity. METHODS: We analysed fresh-frozen tissue samples prepared under strict cryogenic conditions of matched healthy colon mucosa, colorectal carcinoma, and liver metastasis from the same patients. Somatic mutations of known cancer-related genes were analysed using Illumina's TruSeq Amplicon Cancer Panel; the transcriptome was assessed comprehensively using Clariom D microarrays. The global proteome was evaluated by liquid chromatography-coupled mass spectrometry (LC‒MS/MS) and validated by two-dimensional difference in-gel electrophoresis. Subsequent unsupervised principal component clustering, statistical comparisons, and gene set enrichment analyses were calculated based on differential expression results. RESULTS: Although panomics revealed low RNA and protein expression of CA1, CLCA1, MATN2, AHCYL2, and FCGBP in malignant tissues compared to healthy colon mucosa, no differentially expressed RNA or protein targets were detected between tumour and metastatic tissues. Subsequent intra-patient comparisons revealed highly specific expression differences (e.g., SRSF3, OLFM4, and CEACAM5) associated with patient-specific transcriptomes and proteomes. CONCLUSION: Our research results highlight the importance of inter- and intra-tumour heterogeneity as well as individual, patient-paired evaluations for clinical studies. In addition to changes among groups reflecting CRC progression, we identified significant expression differences between normal colon mucosa, primary tumour, and liver metastasis samples from individuals, which might accelerate implementation of precision oncology in the future.

Protein Expression of AEBP1, MCM4, and FABP4 Differentiate Osteogenic, Adipogenic, and Mesenchymal Stromal Stem Cells.

Mesenchymal stem cells (MSCs) gain an increasing focus in the field of regenerative medicine due to their differentiation abilities into chondrocytes, adipocytes, and osteoblastic cells. However, it is apparent that the transformation processes are extremely complex and cause cellular heterogeneity. The study aimed to characterize differences between MSCs and cells after adipogenic (AD) or osteoblastic (OB) differentiation at the proteome level. Comparative proteomic profiling was performed using tandem mass spectrometry in data-independent acquisition mode. Proteins were quantified by deep neural networks in library-free mode and correlated to the Molecular Signature Database (MSigDB) hallmark gene set collections for functional annotation. We analyzed 4108 proteins across all samples, which revealed a distinct clustering between MSCs and cell differentiation states. Protein expression profiling identified activation of the Peroxisome proliferator-activated receptors (PPARs) signaling pathway after AD. In addition, two distinct protein marker panels could be defined for osteoblastic and adipocytic cell lineages. Hereby, overexpression of AEBP1 and MCM4 for OB as well as of FABP4 for AD was detected as the most promising molecular markers. Combination of deep neural network and machine-learning algorithms with data-independent mass spectrometry distinguish MSCs and cell lineages after adipogenic or osteoblastic differentiation. We identified specific proteins as the molecular basis for bone formation, which could be used for regenerative medicine in the future.

Contribution of Interneuron Subtype-Specific GABAergic Signaling to Emergent Sensory Processing in Mouse Somatosensory Whisker Barrel Cortex.

Mammalian neocortex is important for conscious processing of sensory information with balanced glutamatergic and GABAergic signaling fundamental to this function. Yet little is known about how this interaction arises despite increasing insight into early GABAergic interneuron (IN) circuits. To study this, we assessed the contribution of specific INs to the development of sensory processing in the mouse whisker barrel cortex, specifically the role of INs in early speed coding and sensory adaptation. In wild-type animals, both speed processing and adaptation were present as early as the layer 4 critical period of plasticity and showed refinement over the period leading to active whisking onset. To test the contribution of IN subtypes, we conditionally silenced action-potential-dependent GABA release in either somatostatin (SST) or vasoactive intestinal peptide (VIP) INs. These genetic manipulations influenced both spontaneous and sensory-evoked cortical activity in an age- and layer-dependent manner. Silencing SST + INs reduced early spontaneous activity and abolished facilitation in sensory adaptation observed in control pups. In contrast, VIP + IN silencing had an effect towards the onset of active whisking. Silencing either IN subtype had no effect on speed coding. Our results show that these IN subtypes contribute to early sensory processing over the first few postnatal weeks.

Interaction Effect between Elevated CO2 and Fertilization on Biomass, Gas Exchange and C/N Ratio of European Beech (Fagus sylvatica L.).

The effects of elevated CO2 and interaction effects between elevated CO2 and nutrient supplies on growth and the C/N ratio of European beech (Fagus sylvatica L.) saplings were studied. One-year-old beech saplings were grown in a greenhouse at ambient (385 ppm) and elevated CO2 (770 ppm/950 ppm), with or without fertilization for two growing seasons. In this study, emphasis is placed on the combined fertilization including phosphorus, potassium and nitrogen with two level of elevated CO2. The fertilized plants grown under elevated CO2 had the highest net leaf photosynthesis rate (Ac). The saplings grown under elevated CO2 had a significantly lower stomatal conductance (gs) than saplings grown under ambient air. No interaction effect was found between elevated CO2 and fertilization on Ac. A interaction effect between CO2 and fertilization, as well as between date and fertilization and between date and CO2 was detected on gs. Leaf chlorophyll content index (CCI) and leaf nitrogen content were strongly positively correlated to each other and both of them decreased under elevated CO2. At the end of both growing seasons, stem dry weight was greater under elevated CO2 and root dry weight was not affected by different treatments. No interaction effect was detected between elevated CO2 and nutrient supplies on the dry weight of different plant tissues (stems and roots). However, elevated CO2 caused a significant decrease in the nitrogen content of plant tissues. Nitrogen reduction in the leaves under elevated CO2 was about 10% and distinctly higher than in the stem and root. The interaction effect of elevated CO2 and fertilization on C/N ratio in plants tissues was significant. The results led to the conclusion that photosynthesis and the C/N ratio increased while stomatal conductance and leaf nitrogen content decreased under elevated CO2 and nutrient-limited conditions. In general, under nutrient-limited conditions, the plant responses to elevated CO2 were decreased.

Quantitative Tissue Proteomics Analysis Reveals Versican as Potential Biomarker for Early-Stage Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most aggressive tumors, and the treatment outcome of this disease is improved when the cancer is diagnosed at an early stage. This requires biomarkers allowing an accurate and early tumor diagnosis. To identify potential markers for such applications, we analyzed a patient cohort consisting of 50 patients (50 HCC and 50 adjacent nontumorous tissue samples as controls) using two independent proteomics approaches. We performed label-free discovery analysis on 19 HCC and corresponding tissue samples. The data were analyzed considering events known to take place in early events of HCC development, such as abnormal regulation of Wnt/b-catenin and activation of receptor tyrosine kinases (RTKs). 31 proteins were selected for verification experiments. For this analysis, the second set of the patient cohort (31 HCC and corresponding tissue samples) was analyzed using selected (multiple) reaction monitoring (SRM/MRM). We present the overexpression of ATP-dependent RNA helicase (DDX39), Fibulin-5 (FBLN5), myristoylated alanine-rich C-kinase substrate (MARCKS), and Serpin H1 (SERPINH1) in HCC for the first time. We demonstrate Versican core protein (VCAN) to be significantly associated with well differentiated and low-stage HCC. We revealed for the first time the evidence of VCAN as a potential biomarker for early-HCC diagnosis.

Analysis of disease-associated protein expression using quantitative proteomics­fibulin-5 is expressed in association with hepatic fibrosis.

Hepatic fibrosis and cirrhosis are major health problems worldwide. Until now, highly invasive biopsy remains the diagnostic gold standard despite many disadvantages. To develop noninvasive diagnostic assays for the assessment of liver fibrosis, it is urgently necessary to identify molecules that are robustly expressed in association with the disease. We analyzed biopsied tissue samples from 95 patients with HBV/HCV-associated hepatic fibrosis using three different quantification methods. We performed a label-free proteomics discovery study to identify novel disease-associated proteins using a subset of the cohort (n = 27). Subsequently, gene expression data from all available clinical samples were analyzed (n = 77). Finally, we performed a targeted proteomics approach, multiple reaction monitoring (MRM), to verify the disease-associated expression in samples independent from the discovery approach (n = 68). We identified fibulin-5 (FBLN5) as a novel protein expressed in relation to hepatic fibrosis. Furthermore, we confirmed the altered expression of microfibril-associated glycoprotein 4 (MFAP4), lumican (LUM), and collagen alpha-1(XIV) chain (COL14A1) in association to hepatic fibrosis. To our knowledge, no tissue-based quantitative proteomics study for hepatic fibrosis has been performed using a cohort of comparable size. By this means, we add substantial evidence for the disease-related expression of the proteins examined in this study.

Left-right dissociation of hippocampal memory processes in mice.

Left-right asymmetries have likely evolved to make optimal use of bilaterian nervous systems; however, little is known about the synaptic and circuit mechanisms that support divergence of function between equivalent structures in each hemisphere. Here we examined whether lateralized hippocampal memory processing is present in mice, where hemispheric asymmetry at the CA3-CA1 pyramidal neuron synapse has recently been demonstrated, with different spine morphology, glutamate receptor content, and synaptic plasticity, depending on whether afferents originate in the left or right CA3. To address this question, we used optogenetics to acutely silence CA3 pyramidal neurons in either the left or right dorsal hippocampus while mice performed hippocampus-dependent memory tasks. We found that unilateral silencing of either the left or right CA3 was sufficient to impair short-term memory. However, a striking asymmetry emerged in long-term memory, wherein only left CA3 silencing impaired performance on an associative spatial long-term memory task, whereas right CA3 silencing had no effect. To explore whether synaptic properties intrinsic to the hippocampus might contribute to this left-right behavioral asymmetry, we investigated the expression of hippocampal long-term potentiation. Following the induction of long-term potentiation by high-frequency electrical stimulation, synapses between CA3 and CA1 pyramidal neurons were strengthened only when presynaptic input originated in the left CA3, confirming an asymmetry in synaptic properties. The dissociation of hippocampal long-term memory function between hemispheres suggests that memory is routed via distinct left-right pathways within the mouse hippocampus, and provides a promising approach to help elucidate the synaptic basis of long-term memory.

Identification of novel biomarker candidates for the immunohistochemical diagnosis of cholangiocellular carcinoma.

The aim of this study was the identification of novel biomarker candidates for the diagnosis of cholangiocellular carcinoma (CCC) and its immunohistochemical differentiation from benign liver and bile duct cells. CCC is a primary cancer that arises from the epithelial cells of bile ducts and is characterized by high mortality rates due to its late clinical presentation and limited treatment options. Tumorous tissue and adjacent non-tumorous liver tissue from eight CCC patients were analyzed by means of two-dimensional differential in-gel electrophoresis and mass-spectrometry-based label-free proteomics. After data analysis and statistical evaluation of the proteins found to be differentially regulated between the two experimental groups (fold change ≥ 1.5; p value ≤ 0.05), 14 candidate proteins were chosen for determination of the cell-type-specific expression profile via immunohistochemistry in a cohort of 14 patients. This confirmed the significant up-regulation of serpin H1, 14-3-3 protein sigma, and stress-induced phosphoprotein 1 in tumorous cholangiocytes relative to normal hepatocytes and non-tumorous cholangiocytes, whereas some proteins were detectable specifically in hepatocytes. Because stress-induced phosphoprotein 1 exhibited both sensitivity and specificity of 100%, an immunohistochemical verification examining tissue sections of 60 CCC patients was performed. This resulted in a specificity of 98% and a sensitivity of 64%. We therefore conclude that this protein should be considered as a potential diagnostic biomarker for CCC in an immunohistochemical application, possibly in combination with other candidates from this study in the form of a biomarker panel. This could improve the differential diagnosis of CCC and benign bile duct diseases, as well as metastatic malignancies in the liver.

A practical data processing workflow for multi-OMICS projects.

Multi-OMICS approaches aim on the integration of quantitative data obtained for different biological molecules in order to understand their interrelation and the functioning of larger systems. This paper deals with several data integration and data processing issues that frequently occur within this context. To this end, the data processing workflow within the PROFILE project is presented, a multi-OMICS project that aims on identification of novel biomarkers and the development of new therapeutic targets for seven important liver diseases. Furthermore, a software called CrossPlatformCommander is sketched, which facilitates several steps of the proposed workflow in a semi-automatic manner. Application of the software is presented for the detection of novel biomarkers, their ranking and annotation with existing knowledge using the example of corresponding Transcriptomics and Proteomics data sets obtained from patients suffering from hepatocellular carcinoma. Additionally, a linear regression analysis of Transcriptomics vs. Proteomics data is presented and its performance assessed. It was shown, that for capturing profound relations between Transcriptomics and Proteomics data, a simple linear regression analysis is not sufficient and implementation and evaluation of alternative statistical approaches are needed. Additionally, the integration of multivariate variable selection and classification approaches is intended for further development of the software. Although this paper focuses only on the combination of data obtained from quantitative Proteomics and Transcriptomics experiments, several approaches and data integration steps are also applicable for other OMICS technologies. Keeping specific restrictions in mind the suggested workflow (or at least parts of it) may be used as a template for similar projects that make use of different high throughput techniques. This article is part of a Special Issue entitled: Computational Proteomics in the Post-Identification Era. Guest Editors: Martin Eisenacher and Christian Stephan.

Proteomic differences between hepatocellular carcinoma and nontumorous liver tissue investigated by a combined gel-based and label-free quantitative proteomics study.

Proteomics-based clinical studies have been shown to be promising strategies for the discovery of novel biomarkers of a particular disease. Here, we present a study of hepatocellular carcinoma (HCC) that combines complementary two-dimensional difference in gel electrophoresis (2D-DIGE) and liquid chromatography (LC-MS)-based approaches of quantitative proteomics. In our proteomic experiments, we analyzed a set of 14 samples (7 × HCC versus 7 × nontumorous liver tissue) with both techniques. Thereby we identified 573 proteins that were differentially expressed between the experimental groups. Among these, only 51 differentially expressed proteins were identified irrespective of the applied approach. Using Western blotting and immunohistochemical analysis the regulation patterns of six selected proteins from the study overlap (inorganic pyrophosphatase 1 (PPA1), tumor necrosis factor type 1 receptor-associated protein 1 (TRAP1), betaine-homocysteine S-methyltransferase 1 (BHMT)) were successfully verified within the same sample set. In addition, the up-regulations of selected proteins from the complements of both approaches (major vault protein (MVP), gelsolin (GSN), chloride intracellular channel protein 1 (CLIC1)) were also reproducible. Within a second independent verification set (n = 33) the altered protein expression levels of major vault protein and betaine-homocysteine S-methyltransferase were further confirmed by Western blots quantitatively analyzed via densitometry. For the other candidates slight but nonsignificant trends were detectable in this independent cohort. Based on these results we assume that major vault protein and betaine-homocysteine S-methyltransferase have the potential to act as diagnostic HCC biomarker candidates that are worth to be followed in further validation studies.

Scale invariance and viscosity of a two-dimensional Fermi gas.

We investigate collective excitations of a harmonically trapped two-dimensional Fermi gas from the collisionless (zero sound) to the hydrodynamic (first sound) regime. The breathing mode, which is sensitive to the equation of state, is observed with an undamped amplitude at a frequency 2 times the dipole mode frequency for a large range of interaction strengths and different temperatures. This provides evidence for a dynamical SO(2,1) scaling symmetry of the two-dimensional Fermi gas. Moreover, we investigate the quadrupole mode to measure the shear viscosity of the two-dimensional gas and study its temperature dependence.

Observation of a pairing pseudogap in a two-dimensional Fermi gas.

Pairing of fermions is ubiquitous in nature, underlying many phenomena. Examples include superconductivity, superfluidity of (3)He, the anomalous rotation of neutron stars, and the crossover between Bose-Einstein condensation of dimers and the BCS (Bardeen, Cooper and Schrieffer) regime in strongly interacting Fermi gases. When confined to two dimensions, interacting many-body systems show even more subtle effects, many of which are not understood at a fundamental level. Most striking is the (as yet unexplained) phenomenon of high-temperature superconductivity in copper oxides, which is intimately related to the two-dimensional geometry of the crystal structure. In particular, it is not understood how the many-body pairing is established at high temperature, and whether it precedes superconductivity. Here we report the observation of a many-body pairing gap above the superfluid transition temperature in a harmonically trapped, two-dimensional atomic Fermi gas in the regime of strong coupling. Our measurements of the spectral function of the gas are performed using momentum-resolved photoemission spectroscopy, analogous to angle-resolved photoemission spectroscopy in the solid state. Our observations mark a significant step in the emulation of layered two-dimensional strongly correlated superconductors using ultracold atomic gases.

Radio-frequency spectroscopy of a strongly interacting two-dimensional Fermi gas.

We realize and study a strongly interacting two-component atomic Fermi gas confined to two dimensions in an optical lattice. Using radio-frequency spectroscopy we measure the interaction energy of the strongly interacting gas. We observe the confinement-induced Feshbach resonance on the attractive side of the 3D Feshbach resonance and find the existence of confinement-induced molecules in very good agreement with theoretical predictions.

Familial recurrence of gastroschisis: literature review and data from the population-based birth registry "Mainz Model".

BACKGROUND: Familial forms of gastroschisis are considered rare. A search for these forms in a population-based birth registry in 1993 found a recurrence risk of 3.5% among first-degree relatives. Since then, similar investigations in population-based registries have led to contradictory results. METHODS: A search of the population-based birth registry "Mainz Model" for familial cases of gastroschisis and a systematic review of the literature were performed. RESULTS: The Mainz Model database yielded 1 familial recurrence out of 27 gastroschisis cases. From the literature, 37 affected families could be retrieved. Among 412 gastroschises from population-based registries, 10 familial recurrences have been found. These translate into a recurrence risk of 2.4%, with a strong tendency toward underestimation. CONCLUSION: The existing data support the hypothesis that familial recurrence of gastroschisis is much more likely than previously thought.

Peek a peak: a glance at statistics for quantitative label-free proteomics.

Today, label-free mass spectrometry methods are frequently used for quantification of proteins and peptides. There have been several proposals of measurable parameters that best reflect quantities, such as peak areas as well as spectral counts. This review provides a systematic overview of the proposed methods. Owing to the shotgun proteomics approach generally used today for label-free mass spectrometry, any quantitative measure in the first place is a measure of peptide quantity. There has been no systematic research on how to best infer protein quantity from its measured peptides' quantities. The way peptide identifications are assembled to protein lists may especially lead to significantly different results in protein quantification. A further focus of this review will thus be the assembly of measured peptide quantities to a protein quantity.

Measuring temperature-dependent water vapor and gas permeation through high barrier films.

A new test device for temperature-dependent permeation measurement, existing of a mass spectrometer and sample holders inside a climatic chamber was developed. The front face of a sample is loaded with the atmosphere in the cabinet or a test gas mixture, respectively. The permeated species are accumulated in a cell behind the sample. The increasing partial pressures of the permeants are measured by the mass spectrometer and than transferred into a transmission rate. The time-lag technique enables the determination of the diffusion coefficient. Results are given for atmospheric components as O(2), N(2), and water vapor permeated through different barrier films and laminates at temperatures from 23 to 80 degrees C. The limits of the detection of the transmission rates are in the range of 10(-6) g/m(2) d.

Feasibility of microlaparoscopy for surgical procedures of advanced complexity in children.

UNLABELLED: Abstract Purpose: We conducted a prospective study to determine the value and the feasibility of the microlaparoscopic approach for surgical procedures of advanced complexity in children. We report our experience with these small instruments. We also report the preliminary results of a recently developed 2.4 mm prototype scope, specifically developed for the present study. METHODS AND PATIENTS: This study includes 15 children (aged two weeks to 11 years; median, 2.8 years). Eighteen procedures with advanced complexity were carried out: Nissen fundoplication, thoracoscopic congenital diaphragmatic hernia repair in a newborn, hiatoplasty with repair of an upside-down-stomach, laparoscopically assisted pull-through for Hirschsprung's disease in a newborn, laparoscopic transperitoneal pyeloplasty and laparoscopy for acute abdominal illness. RESULTS: No complications occurred due to the exclusive use of 2 mm instruments. All microlaparoscopy procedures were performed successfully except one fundoplication that was converted to an open procedure due to anatomical deformities. The average operative times for the 5 mm and 2 mm groups were similar compared across a similar age and type-of-intervention population. CONCLUSION: The microlaparoscopic approach for surgical procedures of advanced complexity in pediatric surgery is feasible, safe, and effective in conditions where they are indicated and are practical in terms of the surgeon's experience and the facilities. The advantages of microlaparoscopy are obvious: minimum access trauma, "nearly scarless" healing, less risk of damage to abdominal organs, the possibility of fewer adhesions and less postoperative pain. We suggest the further consideration of the use of the microlaparoscope for advanced laparoscopic procedures in children.

Quantum transport through a Tonks-Girardeau gas.

We investigate the propagation of spin impurity atoms through a strongly interacting one-dimensional Bose gas. The initially well localized impurities are accelerated by a constant force, very much analogous to electrons subject to a bias voltage, and propagate as a one-dimensional impurity spin wave packet. We follow the motion of the impurities in situ and characterize the interaction induced dynamics. We observe a very complex nonequilibrium dynamics, including the emergence of large density fluctuations in the remaining Bose gas, and multiple scattering events leading to dissipation of the impurity's motion.

Microlaparoscopic-assisted gastrostomy in children: early experiences with our technique.

INTRODUCTION: The aim of this prospective study was to evaluate the feasibility and safety of a new technique for microlaparoscopic-assisted gastrostomy (MAG) in children and infants by using a 1.9-mm scope only. In this paper, we report our early experiences with this method. MATERIALS AND METHODS: In a prospective study, MAG was performed on 12 children (7 boys and 5 girls) after a primary percutaneous endoscopic gastrostomy procedure was considered not feasible or unsafe. The median patient age at the time of surgery was 1.2 years (range, 4 months to 4 years). A 1.7- or 1.9-mm 0-degree scope was introduced at the umbilicus. A 5-mm port was placed over the stomach at the designated site for the tube placement. The stomach was grasped and exteriorized. The gastrostomy was opened, and a balloon gastrostomy tube was placed. RESULTS: In 12 children, the procedure was successfully completed without conversions. Mean operative time was 19 minutes (range, 13-35). No intraoperative complications occurred. Feeds were instituted 5 hours postoperatively and quickly advanced the following day in 11 of 12 children. The procedure may be performed as day surgery. CONCLUSIONS: In our opinion, MAG is a feasible, safe procedure in children. Technically, it is a simple method, particularly in cases where percutaneous tube placement is not feasible. It combines the advantages of microlaparoscopy and open surgery. It is easy to perform in children and allows a primary gastrostomy tube placement. Parents and nursing staff also seem satisfied.