Cooling was induced by cold intravenous saline infusion, ice packs and nasal cooling to achieve a body temperature of 33–35 °C. After 6 h, animals were rewarmed to baseline temperature (within 5 h). The cerebral microvascular network was evaluated  using sidestream dark-field (SDF) video-microscopy. Cerebral blood flow , oxygenation  and lactate/pyruvate ratio (LPR) using brain microdialysis  were measured hourly. Results: In HT animals, cerebral functional capillary density (FCD) and proportion of small-perfused vessels (PSPV) significantly decreased over time during the cooling phase; concomitantly, PbtO2 increased and LPR decreased. After rewarming, all microcirculatory variables returned to normal values, except LPR, which increased during the rewarming phase in the two groups subjected to HT when compared to the group maintained at normothermia. Conclusions: In healthy animals, TTM can be associated with alterations in cerebral microcirculation during cooling and altered metabolism at rewarming.Purpose: Target temperature management (TTM) is often used in patients after cardiac arrest, but the effects of cooling on cerebral microcirculation, oxygenation and metabolism are poorly understood. We studied the time course of these variables in a healthy swine model.Methods: Fifteen invasively monitored, mechanically ventilated pigs were allocated to sham procedure (normothermia, NT;  Targeted temperature management (TTM) is often used for brain protection after cardiac arrest (CA) or after traumatic brain injury ,2,3. DesIn 1962, Ashton et al. showed that temperature influences vascular waterfalls and possibly microvascular flow ; hypotheIn TTM after CA, experimental evidence remains controversial. Additionally, favorable results on brain cell survival were published on an experimental model of hypothermia after ischemic and traumatic injury in rats and mice ,29,30; aThe aim of this study was, therefore, to evaluate the effects of TTM on brain microcirculation, oxygenation and metabolism in a healthy pig model.The study protocol was approved by the Institutional Review Board for Animal Care of the Free University of Brussels . Care and handling of the animals were in agreement with the “Guide for Care and Use of Laboratory Animals” published by the U.S. National Institute of Health  and the National Institutes of Health Guidelines .Sus Scropha), weighing between 36 and 44 kgs, were fasted in a ventilated and controlled temperature chamber (20–24 °C) for 24 h before the study, with free access to water. On the day of the experiment, the animals were premedicated with intramuscular midazolam  and ketamine hydrochloride  and placed in the supine position on an operating table. A superficial ear vein was cannulated with a peripheral venous 20-gauge catheter . An intravenous administration of 30 mcg/kg fentanyl citrate , 5 mg/kg thiopental  and 0.5 mg/kg rocuronium bromide  was used for endotracheal intubation . Thereafter, all pigs were anesthetized with a continuous intravenous administration of thiopental (3 mg/kg·h), ketamine hydrochloride (0.5 mg/kg·h) and morphine (0.2 mg/kg·h). In order to avoid movement artifacts, a muscular blockade was achieved with a continuous infusion of 10 mcg/kg·h of rocuronium throughout the experiment. Boluses of fentanyl (15 mcg/kg) and of thiopental (2 mg/kg) were administered if needed in case of tachycardia and/or hypertension, suggesting insufficient anesthesia; boluses of rocuronium (0.5 mg/kg) were given to avoid shivering. Mechanical ventilation  was initiated with a tidal volume of 10 mL/kg, respiratory rate of 12–16 breaths/minute, positive end-expiratory pressure (PEEP) of 5 cm H2O, an inspired oxygen fraction of 1 (FiO2), inspiratory time to expiratory time ratio (I/E) of 1:2 and a square-wave pattern. Respiratory rate was adjusted to maintain end-tidal carbon dioxide pressure  between 35 and 45 mmHg before arterial cannulation and blood gas analyses were available.Fifteen domestic pigs  was invasively introduced into the femoral artery; an introducer was inserted through the femoral vein, and a 7F pulmonary artery (PA) catheter  was advanced under the monitoring of pressure waveforms and connected to pressure transducers , zeroed at mid-chest level. Core temperature, cardiac output and PA pressures were measured by Sirecust 404  and Vigilance monitor . Both catheters were flushed intermittently with a heparinized solution . A small midline laparotomy was performed to expose the bladder, and a 14F Foley catheter  was placed to record urinary output throughout the experiment. The abdomen was then closed in two layers by end-knot continuous sutures. During the surgical operation, Ringer’s lactate and 6% hydroxyethyl starch (HES) solutions were titrated to prevent hypovolemia . After vascular and abdominal surgery, the animals were turned to the prone position.2) measurement and a catheter to measure brain temperature . All catheters were placed under sterile conditions at a depth of 0.8–1.0 cm into the brain parenchyma so that their tips were located in the frontal grey matter. Finally, catheters were sutured to the skin to avoid displacement during the experiment (the correct positioning of the catheters was further confirmed in post-mortem examination). A third tiny hole was eventually performed through the skull with a needle-like drill so as to insert a laser-doppler catheter ; its right position catheter was confirmed by the presence of values at least 800 Units; the catheter was then fixed with wax to the skull to prevent any additional movements. The craniotomy holes were then protected by wet sterile gauzes, avoiding any contact with the brain cortex. Brain desiccation was prevented by local hourly administration of 1–2 mL saline solution. All animals were then allowed to stabilize before baseline measurements were recorded.The scalp was opened with a cross-shape incision using an electric bistoury . The ski2), oxygen consumption (VO2) and oxygen extraction (OER) were calculated using standard formulas. The total amount of blood withdrawn for analyses was less than 50 mL .Arterial and mixed venous blood samples were analyzed hourly with automated analyzers . Hemoglobin concentration and oxygen saturation were measured with an analyzer calibrated for animals . All monitored variables were recorded every 60 min. Measurements of mean arterial pressure (MAP), pulmonary arterial pressure (PAP), right atrial pressure (RAP) and pulmonary occlusion arterial pressure (PAOP) were obtained at the end of expiration . Core temperature and cardiac output  were continuously monitored. Body surface area , cardiacRinger’s lactate solution (RL) and 6% hydroxyethyl starch solution  were initially infused via the cephalic vein catheter at a rate of 2 and 1 mL/kg·hour, respectively; fluid administration was then titrated to maintain PAOP similar to baseline values. Potassium chloride and glucose were administered in case of hypokalaemia (<3.5 mEq/L) and hypoglycaemia (<40 mg/dL), respectively.TM, MicroVisionMedical Inc., Amsterdam, The Netherlands) with a 5× imaging objective living 326× magnification. The lens of the imaging device was covered with a disposable sterile cap and was applied without pressure to the cerebral frontoparietal cortex. Because of brain pulsatility, this was best accomplished by placing the animal head and the device on a metallic arm for stabilization . The absence of external superimposed pressure was confirmed by the preservation of flow in large vessels [2 catheters were connected to specific monitoring , where the initial temperature was manually adjusted to blood temperature, and data were collected hourly. The probe function was confirmed by an oxygen challenge (FiO2 1.0 for 2 min). In order to allow probe equilibration, data from the first hour after placement were not used. The CMA 20 catheter was perfused with CNS perfusion fluid  at a flow rate of 1.0 μL/min by a miniaturized infusion pump . This flow rate guaranteed almost 30–50% recovery rate for molecules of less than 20 kDa and was selected to provide additional fluid for further research on brain metabolites. After one hour of stabilization, the perfusate was collected every 60 min in specific microvials. Samples were analyzed for lactate, pyruvate, glycerol, glutamate and glucose by an automatic analyzer . The lactate/pyruvate ratio (LPR) was automatically calculated.The microvascular network of the cerebral cortex was visualized using an SDF videomicroscopy system , which may have affected our final results. Thus, after approval of the protocol modification from the animal Ethics Committee, we added a third study group, which underwent HT with PaO2 concentrations kept constant (HT-Oxy) through repeated initial blood gas analyses . In the absence of previous studies to calculate the effects of HT on brain microcirculation, we estimated a convenient sample size of 5 animals for each group, and the study protocol was designed on another experimental study evaluating mild hypothermia performed in our institution [2 was adjusted according to the temperature-corrected blood gas values to maintain a PaO2 at baseline values during the entire study period. All variables were recorded hourly over the study period. At the end of the study, the animals were euthanized using the injection of a high dose of intravenous potassium chloride.After the surgical procedures, baseline measurements, including cerebral microcirculation and cerebral dynamic data collection, were obtained. Animals were randomly allocated to groups according to pre-sealed envelopes. At first, the study was conceived to compare normothermia (NT) with induced hypothermia (HT). Nevertheless, we observed after the first animal submitted to HT a significant increase in brain and systemic oxygenation . Data are presented as mean ± SD or median (range). Normal distribution was confirmed with the Kolmogorov–Smirnov test. Variables were compared with parametric Student’s t-test or the Mann–Whitney U test for non-parametric data. The significance of differences in the measured variables between groups was analyzed using a two-way (time and groups) analysis of variance for repeated measures, followed by a Bonferroni post hoc analysis. In order to estimate the correlation (expressed as “r” coefficient) between different variables in the presence of repeated measurements, we used a mixed model with the SAS system . A At baseline, there were no significant differences between groups in hemodynamic and respiratory variables, cerebral microcirculation, oxygenation and metabolic data .During the cooling phase, core and brain temperatures remained stable in the NT group, while they decreased significantly in the HT and HT-Oxy groups .In particular, body temperature decreased below 35 °C within the first 2 h, while brain temperature was around 32 °C within the same time period .2 remained stable in the NT group and increased in the two others .In all animals, cerebral glucose levels decreased progressively together with plasma glucose levels  as well The key findings of this experimental study are that cortical microvascular flow is progressively reduced during cooling, in particular when HT is associated with increased systemic oxygenation. The reduction in cerebral capillary perfusion was associated with stable or increased oxygen concentrations and preserved metabolism during cooling. During rewarming, altered cerebral metabolism persisted but with different patterns that could be related to different tissular oxygen concentrations.Microcirculation may play a crucial role when cellular metabolism adapts to temperature variations: in particular, when the metabolic rate slows down, the precapillary resistance vessels constrict to reduce blood flow, resulting in less effective microcirculation perfusion capability. This is indicated by a reduction in FCD, PPV and MFI during hypothermia . In swin2 values exceeded 160 mmHg in the three groups over time, so it remains unclear whether the differences in PaO2 in our study might explain the potentially detrimental effects of oxygen.Cerebral effects of cooling, when associated with hyperoxia, have never been specifically addressed in the literature. Despite its known adverse effects , normobaSince the beginning of the cooling process, we were able to identify microcirculatory abnormalities in both groups treated with hypothermia. Interestingly, those alterations were not associated with significant changes in MAP or CI, despite changes in systemic blood pressure could be recorded among groups. On the one hand, those findings are consistent with previous studies in clinical and experimental settings, in which similar microcirculatory alterations were described in the brain and sublingual area, despite adequate global hemodynamics ,23,24,37+ should be performed.These microcirculatory alterations were not associated with significant increases in blood lactate concentrations. Neurochemical disturbances can also be assessed by microdialysis, which typically includes elevated glutamate (excitotoxicity), glycerol (degradation of cellular membrane) and LPR , as well as low glucose concentrations  ,56. AugmThere are some limitations to this study. First, we studied healthy animals, and no conclusions on brain injury situations can be drawn; this study is part of a wider project that we have been conducting with the aim of evaluating brain dynamics during and after cardiac arrest, along with various therapeutic options, mild hypothermia included. This study represents the beginning of our project, the groups of healthy animals to be compared with ill/injured ones; this choice limits the applicability of our results to other areas of research on brain damage. Second, since full anesthesia was necessary, no clinical correlation between reduced microvascular blood flow and clinical abnormalities could be established. Third, any potential effects on microcirculation due to changes in intracranial pressure were prevented by the chosen surgical technique. Fourth, we analyzed limited regions of the brain microvasculature that could not be representative of deeper structures, such as the white matter or the brain stem. Fifth, we did not analyze the release of oxygen species nor anatomo-pathological correlates, limiting the interpretability of our findings. We also could not assess whether microcirculatory disturbances would have any impact on brain function, as evaluated by electroencephalography or neuropathologic examination of the frontal lobe. Moreover, no mechanistic explanations of the observed phenomena were provided. Sixth, the cerebral temperature was lower than the core, probably related to the use of the Rhinochill system and to the craniotomy, and this aspect does not enable us to translate our results into the clinical application of TH. Finally, the study timeline, despite being in line with the available experimental literature, is shorter than the ones applied in clinics, and rewarming was performed at a 1 °C/h speed (as per protocol design); this could have underestimated the risks related to both cooling and rewarming procedures. We cannot exclude that the excess in both cooling depth and rewarming rate might have partially been responsible for the observed phenomena.In healthy animals treated with TTM, cerebral microvascular perfusion was reduced during cooling, and the metabolism was altered at rewarming, potentially enhanced by concomitant hyperoxia.


Substandard and falsified pharmaceuticals can present a major health risk, particularly for low- and middle-income countries. In a Sub-Saharan African market, United States Agency for International Development (USAID) staff found an oral contraceptive product (0.15 mg levonorgestrel/0.03 mg ethinyl estradiol) labeled with a logo (and brand name) visually matching one historically used by USAID but purportedly manufactured by an unauthorized source. Additional assessment was conducted to determine if more evidence of falsification was present to better understand the public health impact.Relative to a control, the suspect sample was assessed visually for tablet features and with United States and International Pharmacopeia methods for levonorgestrel/ethinyl estradiol tablets. Diffuse reflectance spectra (350–2500 nm) were collected to further characterize the sample.Although the suspect sample aligned with pharmacopeia tests, visual examination of tablet characteristics and diffuse reflectance spectroscopy (350–2500 nm) assessments supported the tablets were not the same as control samples, confirming the suspect sample was falsified. These results raised concerns for the overall regulatory oversight available for the market with uncertainty regarding the true clinical bioequivalence levels .Comprehensive characterization of suspect pharmaceuticals from the field can often be required depending on the nature of the sample and can have dramatic implications for understanding the public health risks to the end user within the local market. Simple visual assessment and spectroscopic techniques to screen a sample can help to supplement traditional pharmacopeia approaches.Proper characterization of suspect pharmaceuticals is necessary to best understand their potential public health impact. Situations can occur where traditional pharmacopeial techniques may not adequately characterize a sample. Visual assessments and diffuse reflectance spectroscopy can be supplemented to provide a more holistic analysis.  Falsified pharmaceutical; Rapid screening; Oral contraceptives; Public health. PromineCommonly, poor quality medicines are assessed with more traditional pharmacopeia methods (often HPLC based). A variety of alternative techniques are available that involve spectrometers and devices that are often more portable than traditional techniques , 11. TheThis work presents the characterization of oral contraceptives, specifically tablets containing both levonorgestrel and ethinyl estradiol, found in the market that were suspected of being falsified . To22.1The oral contraceptives are comprised of 21 active tablets and 7 hormone free tablets per blister pack with levonorgestrel (0.15 mg) and ethinyl estradiol (0.03 mg) in each active tablet. One blister pack (with outer box) of suspect sample was able to be purchased from a local pharmacy in Sub-Saharan Africa and made available for testing. Further details disclosing product brand name, manufacturer, and purchasing location of the suspect sample will not be provided for legal reasons but have been provided to USAID for investigation with the authentic manufacturer and Sub-Saharan African country’s regulatory authority. Multiple blister packs of authentic product (from the same lot) were obtained from USAID’s retain supply as a control sample.2.2Digital images of the front and back of both outer boxes and the blister packs were captured for the control and suspect samples and provided to USAID for review (not provided here to protect the manufacturer). From these images, the general features of the packaging and tablets for the suspect sample was visually compared to the control samples.2.3Chemical testing for quantifying active ingredient and dissolution was based on the USP monograph for Levonorgestrel and Ethinyl Estradiol tablets . With liActive ingredient quantification was conducted with HPLC using a Phenomenex Luna C8 4.6-mm x 15-cm with L7 packing of 5 μm 100 Å particle size . The chromatography used an isocratic mobile phase of acetonitrile: methanol: water 35:15:45 v/v under ambient temperature with a 1 mL/min flow rate, 215 nm detection, and 50 μL injection. Standards contained both levonorgestrel RS (0.015 mg/mL) and ethinyl estradiol RS (0.003 mg/mL) in mobile phase. Samples were prepared by placing 1 tablet in a 40 mL centrifuge tube with 10 mL of mobile phase, sonicating (30 min), mechanically shaking (20 min), and centrifuging (2000–3000 RPM for 10–15 min) to obtain the supernatant. Individual tablets were quantified as % label claim (LC) per active.Dissolution was conducted with a VK 7000 Varian apparatus , where each vessel contained 500 mL of medium, polysorbate 80 (5 μg/g) in water, at 37 °C with paddles operating at 75 RPM. An aliquot of ∼15 mL was removed 60 min after tablets were dropped in separate vessels. Final working standards contained both levonorgestrel RS (0.0003 mg/mL) and ethinyl estradiol RS (0.00006 mg/mL) in dissolution medium (containing ∼0.3% volume of ethanol). Standards and samples were analyzed with HPLC using a Phenomenex Luna C8 4-mm x 15-cm with L7 packing of 5 μm 100 Å particle size . The chromatographic conditions used an isocratic mobile phase of acetonitrile:water (60:40) v/v operating under ambient temperature with a 1 mL/min flow rate, 247 nm UV detection for levonorgestrel, 285 nm excitation/310 nm emission fluorescence detection for ethinyl estradiol, and 100 μL injection. Dissolution (%Q) was calculated for individual tablets based on the percent active ingredient released relative to the theoretical amount in the tablet.2.4To provide evidence the tablets contained levonorgestrel (not norgestrel), chiral chromatography was conducted , 22. Nor2.5Like other approaches , diffusemax threshold of 4.73 was calculated at a 95% confidence level [max of 4.73 . The results from the positive/negative controls showed the method could discriminant tablets manufactured by other sources relative to the reference spectra. With this method, five active tablets from control and suspect samples were assessed for Mahalanobis distances relative to reference spectra.To build the method, scans from 78 different active tablets (across 15 lots previously tested for active ingredient quantity and dissolution ) of USAIce level , 24. To ce level , spectra33.1Visual differences first identified the suspect sample. A replica of USAID’s logo historically used for their brand was visible on the box but stated a different manufacturer than used by USAID. Further inspection of the box and blister packs  found other distinctions from the authentic product. An image of example tablets are provided in 3.23.3d-norgestrel, (−)-norgestrel) and dextronorgestrel -norgestrel) [From the HPLC analysis for content and dissolution, identification of ethinyl estradiol and levonorgestrel was supported based on comparable retention times between standards and samples. However, levonorgestrel is the biologically active enantiomer  of norgegestrel) , 26, and3.4max for the method, while the suspect sample was well outside the limit. The exact source of these differences cannot be determined from the spectra, but could be due to differences in excipients, particle size, moisture and/or hardness. Comparison of the diffuse reflectance spectra (covering visible and NIR) indicated differences in the suspect sample relative to the authentic sample. Lower Mahalanobis distances (D) indicate a closer spectral agreement with the authentic product. The control sample was within the D4Although this falsified product had appropriate levels of active ingredient and dissolution, the reflectance spectrum was distinguishable from the authentic product, which was consistent with the visual assessments. There is no indication that the suspect sample is the authentic product, however the sample appears to be made by a source that is experienced in the production of hormonal contraceptive tablets. However, this does not alleviate the potential risk for this sample. The sample may not have been deemed suspect if only the chemical tests presented here were relied upon. For this work, the visual and diffuse reflectance spectroscopy assessments were vital to the sample analysis and highlights the need for multi-faceted approaches to characterize suspect samples. The results from this analysis were provided to USAID and the manufacturer so that necessary legal and regulatory actions could be pursued. With this sample being presented as another registered product’s brand name, the integrity of the regulatory oversight that the end users rely upon was compromised and points to the continued need to further assist regulatory programs in the global market.With an end user’s reliance on accurate packaging and labeling, this research highlights the potential risk for other product suppliers that may attempt to present their product (which may be substandard) as that of another well-known brand. Furthermore, the prominence of this sample is unknown in the general market regarding the number of blister packs available or the number of times this has occurred with other products. Although dissolution levels appeared adequate, the clinical level of bioequivalence of this suspect sample relative to the product provided by USAID is unclear. Additionally, there could be other tablets in the market that may have no active ingredient.Utilization of quality medicines is important for universal health coverage , 32 due David Jenkins: Conceived and designed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.Cherif Diallo, Michael Payne: Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.10.13039/100000200United States Agency for International Development (AID-OAA-C-15-00001).This work was supported by Data included in article/supplementary material/referenced in article.The authors declare no conflict of interest.No additional information is available for this paper.


Pneumonia is a major cause of death in the elderly population. Considering body weight loss, muscle loss, and reflux after gastrectomy, elderly patients are considered to be at very high risk for pneumonia, which could decrease overall survival because early gastric cancer is mostly curable only by surgery. We aimed to clarify the incidence of pneumonia in the long-term period after gastrectomy in elderly patients who were diagnosed with early gastric cancer and its risk factors.We retrospectively examined patients of > 75 years of age who underwent R0 gastrectomy for gastric cancer and who were diagnosed with T1 disease at National Cancer Center Hospital between 2005 and 2012. Long-term postoperative pneumonia was diagnosed by chest computed tomography every year until 2 years after surgery. The presence of preoperative sarcopenia was assessed using preoperative L3 skeletal muscle index.167 patients were included in this study. Long-term postoperative pneumonia was observed in 44 (26%) patients. Of the 44 people diagnosed with long-term postoperative pneumonia, 33 were diagnosed in the 1st year and 11 in the 2nd year. 117 patients (70%) were diagnosed with sarcopenia which was significantly frequently found in the patients who developed long-term postoperative pneumonia (91%) than those without (63%). Preoperative sarcopenia was the only independent risk factor in multivariate analysis. Type of gastrectomy was not a significant risk factor.Long-term postoperative pneumonia was frequently observed in the elderly patients. Preoperative sarcopenia was associated with long-term postoperative pneumonia in elderly patients who underwent curative surgery for gastric cancer. After gastrectomy, long-term special care would be required for elderly patients, especially with sarcopenia. In Japan, gastric cancer is characterized by increased rate of early-stage disease and occurrence in elderly patients , 2. EarlPneumonia in the elderly population is associated with frailty caused by muscle depletion . On the There have been no reports focusing on LTPP in elderly patients. Its incidence and risk factors remain unclear. Physicians may select endoscopic treatment instead of gastrectomy, even in cases that endoscopic treatment is not oncologically justified, if the frequency of LTPP is high or if physicians can identify patients who are at high risk for LTPP.In this background, we investigated the frequency of LTPP in the elderly patients who received gastrectomy and were diagnosed with stage I gastric cancer and its risk factors.Patients were selected from the clinical database of consecutive patients who underwent gastrectomy for gastric cancer at National Cancer Center Hospital from January 2005 to December 2012, based on the following criteria: (1) pathological diagnosis of T1, (2) age ≥ 75 years, (3) R0 resection achieved, and (4) chest computed tomography (CT) performed before surgery and within 2 years after surgery.Surgery was basically determined by the Japanese Gastric Cancer Treatment Guideline version 2 or version 3 depending on the date of the surgery , 11. In LTPP was diagnosed by chest CT findings performed every year until 2 years after surgery. According to the guidelines for diagnostic imaging of adult community-acquired pneumonia 2007, diagnostic imaging of pneumonia in CT was classified into three types Fig. : consoliAfter the certificated radiologists checked the radiological findings, two surgeons evaluated the images for the diagnosis of LTPP. If the diagnosis of the two surgeons differed, the images were evaluated again and final judgments were made. The patients were classified into those with LTPP (P group) and without LTPP (C group).2/m2)] [Preoperative sarcopenia was evaluated by measuring the skeletal muscle area by the SYNAPSE VINCENT system based on CT . The eva2/m2)] . We inve2], % vital capacity (%VC) and forced expiratory volume in 1 s (FEV1%) were converted to binary data. Statistical comparisons of the differences in each variable between the C and the P groups were made using the Chi-squared test. Variables were also investigated by a multivariate logistic regression analysis to assess the risk factors associated with LTPP. p values of < 0.05 were considered to indicate statistical significance.SPSS version 15.0  was used to perform the statistical analyses. To identify risk factors for LTPP, age, surgical procedure, Charlson score , Body MaAmong the 3412 patients who underwent gastrectomy for gastric cancer at National Cancer Center Hospital from 2005 to 2012, a total of 167 patients were included in the present study. Figure 2/m2, respectively. By this cut-off, 117 patients (70%) were diagnosed with sarcopenia which was significantly frequently found in the P group (91%) than the C group (63%). During the follow-up period, 1 patient in the C group died of recurrent disease, and 28 patients (12 patients of the C and 16 patients of the P group) died of causes other than gastric cancer.Table To identify risk factors for LTPP, variables were converted to binary data Table . The surIn the present study, we called the pneumonia shadow found in the long-term period after gastrectomy “LTPP” and examined its incidence and risk factors in elderly patients who received curative gastrectomy and who were diagnosed with early gastric cancer. We firstly clarified that more than one quarter of the elderly patients developed LTPP within 2 years after gastrectomy. This high incidence must impact the decision on whether the patient should receive surgery or not. Moreover, preoperative sarcopenia was the only significant risk factor for LTPP. After gastrectomy, special care through the long-term period is required for elderly patients, especially with sarcopenia.In this study, LTPP was observed in 26% of elderly patients. There are no reports on the frequency of LTPP in any age group. On the other hand, the incidence of hospitalized community-acquired pneumonia in the elderly was reported to be approximately 2% in the United States . Kaplan Aging is associated with loss of muscle mass and physical function, that often leads to progressive disability . The amoSeveral studies have shown that gastric cancer patients with sarcopenia at the time of surgery experienced worse long-term outcomes than non-sarcopenic patients –30. Pneu2/m2, respectively. They were similar to the previous reports examining the preoperative sarcopenia of gastric cancer patients, in which the cut-off values for males and females were 40.8–55 and 34.9–41 cm2/m2, respectively [Originally, diagnosis of sarcopenia required low muscle mass plus either low muscle strength or low physical performance and the diagnosis criteria was defined for aging patients , 24. Howectively –29.Previously, surgical procedure, comorbidity, and preoperative respiratory function have been reported as risk factors for perioperative pneumonia –37, whilThe present study was associated with some limitations. First, this was a retrospective single-center study. Our results should be further validated in a multicenter study. Second, the frequency of LTPP may be underestimated because our hospital specializes in oncology and often performs surgery for patients with few comorbidities. In the community hospital setting, where surgeons operate on elderly patients with severe comorbidities, LTPP would be more frequent because such patients would be sarcopenic.LTPP was frequently observed in elderly patients regardless of the type of gastrectomy. Preoperative sarcopenia was associated with LTPP in elderly patients who underwent curative surgery for gastric cancer. We expect our findings to help inform the better treatment strategies for elderly gastric cancer patients. After gastrectomy, special care, such as nutritional support, respiratory rehabilitation, oral care and swallowing rehabilitation, through the long-term period is required for elderly patients, especially with sarcopenia.


Melanoma is a lethal skin malignant tumor, and its formation or development is regulated by various genetic and epigenetic molecules. Although there are traditional methods provided for the doctors to evaluate the patients' prognosis or make the diagnosis, the novel method based on epigenetic markers is still needed to make the early diagnosis. P < 0.0001) and divided patients into seven methylation subgroups. Methylation levels and survival time in the C2 subgroup were lower than that of other clusters (P < 0.05). We established the predicted model of prognosis risk for melanoma using the significantly changed methylation sites in C2. The model efficiently divided patients into high- and low-risk groups . Risk scores and patient survival time were negatively correlated . Genes corresponding to the independent prognosis-associated methylation sites were enriched in cancer- and immunology-related pathways. We identified 35 hub genes. DOK2, GBP4, PSMB9, and NLRC5 were significantly changed according to methylation subgroups, survival, tumor stages, and T categories and were positively correlated, which was validated in the testing group (P < 0.05). The levels of DOK2, GBP4, PSMB9, and NLRC5 had an opposite trend to their methylation sites in patients with poor prognosis.  We identified 256 melanoma-independent prognosis-related methylation sites ( We identified seven DNA methylation subtypes and constructed a highly effective prognosis risk assessment model. The transcript levels of key genes corresponding to the independent prognosis-related methylation sites were significantly changed in patients according to prognosis and positively correlated with each other, indicating they may collaboratively promote melanoma formation. These findings further our understanding of the mechanism of melanoma and provide new targets for diagnosis and treatment. Melanoma is the most aggressive skin cancer and originates from malignant melanocytes . Once meDNA methylation is a common epigenetic change involved in multiple cellular processes. Three DNA methyltransferases (DNMTs) have been identified in advanced eukaryotes, and several studies have linked aberrant protein structures of DNMTs with abnormal embryonic development and cancer development , 10. PreCancer genomics studies identified a recurrent mutation in DNMT3a in 25% of patients with acute myeloid leukemia  that affIn this study, the methylation levels were examined in samples from 475 melanoma patients and the patients were divided into different methylation subtypes. A prognostic model was built based on the prognosis-related DNA methylation sites. These results may help provide a new method to assess the prognosis of patients and lay a theoretical foundation for researchers to understand this disease from a uniquely epigenetic perspective.https://portal.gdc.cancer.gov/) on May 7, 2021. The platform of transcriptome file was Illumina. Detailed information of patients is shown in https://xena.ucsc.edu/) on May 8, 2021. The testing data were from GSE98394. The exclusion criteria for the DNA methylation sites were as follows: there were more than 70% missing data in the whole sample; the sites located on sex chromosome; single nucleotide polymorphisms; the sites were not on the gene promoter region (2 kb upstream to 0.5 kb downstream of the transcription start site); and they were cross-reactive genome sites [The study flow chart is shown in me sites . Clinicame sites –19.The univariate Cox proportional risk regression model was built by DNA methylation sites, patients' age, stage, gender, TNM classifications, grade, and the follow-up data; after calculating, we got the prognosis-related sites . Those sWe selected the subcluster with dramatically high or low survival probability and significantly changed methylation levels compared with other subclusters; if there were more sites in the targeted subcluster, those sites would be chosen to build the Cox proportional hazard model by coxph function in R software , 23.https://string-db.org) and imported it into CytoHubba to calculate the scores of the total genes. The main parameters in this research were maximal clique centrality (MCC), depth, edge percolated component (EPC), and maximum neighborhood component (MNC). We selected the top 50 genes in each method and constructed a Venn diagram. The overlapping genes were identified as key genes and used for subsequent analysis.We used CytoHubba to predict or discover important genes. We drew the interaction map of genes using the String tool  and the C2 subcluster had a lower survival rate than the others. These results indicated that these could distinguish patients with different prognostic status.The study flow chart is shown in  Figures . Therefo Figures , indicat Figures . As showWe further compared the clinical parameters of the different subclusters and found no significant differences in patient age and gender among the subclusters .Comparison of the methylation levels of the 256 independent prognostic DNA methylation sites in the seven subclusters revealed that the methylation levels of DNA methylation sites in the C2 subcluster were lower than those of the other subclusters . We idenAs the C2 subtype had the lowest survival rate and methylation levels compared with other subclusters, and as this subcluster had the most significantly changed sites, we selected the significantly changed sites in the C2 subtype to draw the boxplot and construct the prognosis prediction model.We used the coxph function in the R software to process the significantly changed methylation sites in the C2 subcluster and built the prediction model of clinical prognosis risk for patients with melanoma using the formula: risk score = Id1 × Co.1 + Id2 × Co.2 + Id3 × Co.3……+Idn × Co.n; the Id value and Co. are shown in P = 1.11 × 10−15, rs = −0.325, 95% confidence interval, −0.420 to 0.224, P < 0.0001, We then divided patients according to the median risk score: patients with a risk score higher than the median were high-risk cases, and those with lower risk scores than the median were low-risk cases. The survival rate of high-risk patients was significantly lower than that of patients with a low-risk score (P < 0.05), demonstrating that this model could distinguish patients in the high-risk group from patients in the low-risk group  was less than 0.05 in almost all the 100 trials  curve analysis to evaluate the efficiency of this method and found that the method had high efficiency. Area under the curve (AUC) was 0.833 , guanylate-binding protein 4 (GBP4), proteasome 20S subunit beta 9 (PSMB9), and NLR family CARD domain containing 5 (NLRC5) were significantly altered in different DNA methylation subgroups, patients with different survival status, different stages, and T categories (P < 0.05). To explore the correlation of the 35 hub genes, we conducted Pearson correlation analysis and found that there was positive correlation between DOK2, GPBAR1, GBP4, PSMB9, and NLRC5; further, we validated their correlation in the testing group (GSE98394) and confirmed the positive correlation between DOK2, GBP4, PSMB9, and NLRC5 (P < 0.05, Tables The expression levels of the hub genes in the seven subgroups are shown in Melanoma is a malignant cancer with an increasing incidence worldwide . A patieThe pathogenesis of melanoma is mediated by a series of genetic and epigenetic changes . EpigeneIn this study, we successfully built a prognostic risk assessment model based on the independent prognosis-associated DNA methylation sites, and this model could efficiently distinguish low-risk patient from high-risk patients . PatientThe enriched pathways of genes corresponding to the prognostic methylation sites were mainly in autoimmune or immune-related disease and tumor-related pathways. There was a correlation between the melanoma pathway and pathways such as glioma and colorectal cancer. The tumor pathways may be connected to each other through signaling pathways such as the central carbon metabolism, phospholipase D signaling, or sphingolipid signaling pathways. Alternatively, they may affect the immune homeostasis of melanoma patients and further contribute to the development of this disease by pathways such as the B cell receptor signaling pathway Figures . The immFurther, we found that the gene expression levels of DOK2, GBP4, PSMB9, and NLRC5 were changed according to different methylation subclusters, patient state, tumor stage, and clinical T categories; the transcript level of PSMB8 was changed in different methylation subgroups and patients with different survival states Figures . DOK2 isFurthermore, we found a positive correlation between the gene expression levels of DOK2, GBP4, PSMB9, and NLRC5, and their correlation had been confirmed in the testing group , indicatDOK2, GBP4, PSMB9, and NLRC5 were critical and positively correlated with each other, which was validated in the testing group, but there are few data sets to be found to retest the efficiency of the predicted model. In the future, we will reexamine them clinically.There are also some limitations in this research, which are as follows: we used 475 methylation files to build the predicted model of patients' prognosis and found the independent prognosis sites' corresponding genes as Here, we established a prediction model for the prognosis of patients with melanoma based on prognosis-related DNA methylation sites in gene promoter regions. This model efficiently distinguished high-risk patient from low-risk patients. Among the 35 hub genes corresponding to the prognosis-related DNA methylation sites, the gene expression levels of DOK2, GBP4, PSMB9, and NLRC5 were significantly changed in different patient subgroups according to DNA methylation subtypes, patient states, tumor stages, and T categories. The genes were positively correlated with each other, and the altered DNA methylation levels on the gene promoter regions of these genes might affect their expression. Furthermore, these genes might be involved in the pathological process of melanoma. We plan to focus future studies on these five identified hub genes and explore their potential mechanisms in DNA methylation and their interactions. Our findings may help provide new targets for clinicians to treat patients with melanoma and enable early diagnosis based on these critical DNA methylation sites.


This study is aimed at comparing cardiac computed tomographic angiography (CTA) with echocardiography in the assessment of ventricular septal perforation diameter.  A total of 44 ventricular septal rupture (VSR) patients undertaking transcatheter occlusion were included and randomly divided into the CTA group and echocardiography group with a 1 : 1 ratio. Clinical data, operation-related data, and 30 d follow-up data were collected and analyzed. p value of 0.005. However, no significant differences were found in all-cause mortality and incidence of operative complications within 30 days after surgery. Within the CTA group, the correlation was strongest between postoperative occluder diameter and long diameter measured by CTA with a correlation coefficient of 0.799 and p < 0.001, followed by the correlation between postoperative occluder diameter and mean diameter measured by CTA with a correlation coefficient of 0.740 and p < 0.001. The diameter measured by echocardiography was not correlated to postoperative occlude diameter.  Incidence of closure failure, occluder displacement, poor occluder molding, and occluder waist diameter shrinkage between the two groups were not statistically different. The mean residual shunt volume in the echocardiography group  mm) was significantly higher than that in the CTA group  mm) with a  Assessment of VSR diameter by cardiac CTA is more accurate than by echocardiography. Ventricular septal rupture (VSR) is a rare mechanical complication of acute myocardial infarction (AMI) with a high mortality rate. Mortality within 1 week ranges from 67% to 82% with conservative medical treatment and the one-year survival rate is only 7% . HoweverWith the development of computed tomography (CT), scanning speed and resolution have been greatly improved, mainly reflected in the field of structural heart disease such as transcatheter aortic valve replacement . In clinThis is a prospective, single-center, randomized controlled study. A total of 67 VSR patients undertaking transcatheter occlusion in Fuwai Central China Cardiovascular Hospital were screened from June 2020 to December 2021. The inclusion criteria were as follows: (1) diagnosis of AMI according to the fourth edition of the Global Definition of Myocardial Infarction and diagnosis of VSR by echocardiography or ventriculography after AMI, (2) patients with stable vital signs after drug therapy including vasoactive drugs or cardiac assist device therapy and with tolerance for surgery until 3 weeks after MI, and (3) patients or their legal guardian have signed the informed consent. Patients were excluded if (1) they had a VSR diameter greater than 22 mm as assessed by echocardiography or cardiac CTA, or they had comorbid ventricular septal dissection and were unsuitable for interventional occlusion therapy; (2) they had uncontrolled preoperative 24-hour cardiac insufficiency and were unable to lie supine for more than half an hour; and (3) they had comorbid with other diseases requiring surgical treatments. Of all 67 VSR patients, 2 cases had three-vessel coronary heart disease and needed surgical thoracotomy, 1 case had comorbidity of giant ventricular septal dissection and was not suitable for interventional therapy, and 20 cases deteriorated and family members asked for discharge or inhospital death. A total of 44 patients were finally included for further analysis and were treated with interventional therapy 3 weeks after MI. The institutional review board of Fuwai Central China Cardiovascular Hospital approved this study ((2020) Ethical Review No. (3)), and informed consent was obtained from all patients.Patients were divided into the echocardiography group and CTA group with a 1 : 1 ratio. Both groups received interventional occlusion 3 weeks after MI and transthoracic echocardiography within 24 hours before the operation. In addition, cardiac CTA was conducted within 72 hours before the operation in the CTA group. Echocardiography and chest X-ray were reexamined after the operation, or cardiac CTA was performed if the chest X-ray could not show the image of the occlude clearly.All procedures referred to the Chinese expert consensus on interventional therapy for common congenital heart diseases . The VSRPatients' data included (1) demographic features such as age and gender; (2) medical history: coronary heart disease, hypertension, diabetes, cerebral infarction, hyperlipidemia, etc.; (3) heart rate, blood pressure, and laboratory test indicators at admission; (4) preoperative VSR diameter measured by echocardiography or CTA; (5) postoperative residual shunt and occluder position; and (6) postoperative occluder diameter (POD).Methods for measuring VSR diameter by CTA: under the horizontal and coronal plane, the VSR was scanned layer by layer to find the section that could show the largest defect. Measure the diameter of the defect and obtained it in the two sections. The larger one was the long diameter by CT (CTLD), the smaller one was the short diameter by CT (CTSD), and the average value of the two was the mean diameter measured by CT (CTMD).Methods for measuring POD by CT: using 3D-MPR mode, adjust the occlude to the center of three-dimensional section so that one section tangentially cut the finest part of the short axis waist of occluder, while the other two sections vertically tangent the center of the long axis of occluder, respectively. Then, measure the diameter of the short axis waist as shown in Patients were followed up by telephone 30 days after surgery for their survival status. Primary endpoint indicators were the rates of closure failure, occluder displacement, poor occluder molding, and compression rate of the occlude waist diameter (CROWD). Secondary endpoint indicators were all-cause mortality and incidence of procedural complications within 30 days after surgery. Procedural complications included significant residual shunt , valvular injury, reoperation , major adverse cardiovascular events (MACE), vascular injury, hemolysis, pericardial tamponade, and high-grade atrioventricular block.2.Occluder displacement was defined as the edge of either side of the occluder partially dislodged into the interventricular septum with a residual shunt > 5 mm. Poor occluder molding was defined as either side of the occluder cannot fully expand, or the CROWD is >50%. CROWD was calculated as /occluder original diameter. Valvular injury was identified if any of the following appeared: (1) new valve tendon rupture, leaflet prolapse, or obstruction of the valve by the occluder indicated by echocardiography; (2) presence of severe postoperative valve regurgitation, with mild/no valve regurgitation before the operation; and (3) presence of severe postoperative valve regurgitation, with moderate valve regurgitation before the operation, along with the regurgitation area increased by more than 5 cmt-test or Mann–Whitney U test for continuous variables. Scatterplots and Pearson correlation analysis were used to compare the correlation between the postoperative occluder diameter and VSR diameter measured by echocardiography or CTA. The threshold for significance was p = 0.05. All statistical analyses were conducted using SPSS, version 22.0 .Continuous variables were expressed as mean ± standard deviation or median (interquartile range) for normally distributed and nonnormally distributed ones, separately. Numerical differences between the two groups were assessed by chi-square test for categorical variables and p value 0.023. No other significant differences were found between the two groups in the baseline, see A total of 44 VSR patients were included in this study. There were 15 (68.2%) females in echocardiography group with an age of 68.6 ± 6.3 years and 11 (50.0%) females in the CTA group with an age of 66.7 ± 10.0 years. Compared with the CTA group ), the echocardiography group ) had significant higher platelet count with p = 0.822). Anterior descending branch was a large majority of target vascular lesions, accounting for 86.4% and 68.2% in the echocardiography and CTA groups, respectively (p = 0.150). The time from AMI to closure is 22 days for the echocardiography group and 23.5 days for the CTA group, but there is no statistical difference (p = 0.092). In addition, no significant differences were found in other variables between the two groups, see Coronary artery lesions were mainly double-vessel and triple-vessel lesions and fewer single-vessel lesions in both groups without significant difference (p = 0.226). Among them, 2 cases of closure failure in the echocardiography group were due to the fact that the occluder could not be fixed after release, and the operation was abandoned after retrieval, without occluder detachment. The CROWD between the two groups was also not statistically different (p = 0.546).12 (54.5%) cases and 8 (36.4%) cases in echocardiography and CTA groups had primary outcomes, respectively, resulting from the lower rate of closure failure and occluder displacement in the CTA group, but the difference was not significant (p = 0.070). Although the incidence of massive residual shunt was not significantly different between the two groups (p = 0.082), the mean residual shunt was significantly higher in the echocardiography group (4.2 mm) than in the CTA group (2.05 mm) with a p value of 0.005. No statistical differences were found in 30 d all-cause mortality, valvular injury, reoperation, MACE, and vascular injury between the two groups, see As for secondary endpoint indicators, 14 (63.6%) cases and 8 (36.4%) cases in the echocardiography and CTA groups had secondary outcomes, respectively, but the difference was not significant .We analyzed the relationship between procedural complication and 30 d mortality. A total of 17 procedural complications (11 in echocardiography group and 6 in CTA group) occurred within 30 days after the operation, with 8 cases dying. However, among 27 cases without surgical complications, only 5 cases died. The presence of procedural complications could significantly increase 30 d mortality with a p < 0.001), followed by CTMD with a correlation coefficient of 0.740 (p < 0.001), shown in As POD could best accurately reflect the true size of VSR, the accuracy criteria of VSR diameter should be based on its correlation with POD. We collected both CTA and echocardiographic evaluations of all 22 patients in the CTA group except 1 case with poor occluder molding due to distortion. The mean PDO of the remaining 21 patients was 14.9 ± 5.4 mm. Scatter diagram was developed for the relationship between POD and ECD/CTLD/CTSD/CTMD . The Pean = 15) and posterior septal perforation group (n = 6). Furthermore, according to whether there was an obstruction such as an adjustable bundle at the outlet of right ventricular surface defect, they were divided into the right ventricular surface obstruction group (n = 7) and the no right ventricular surface obstruction group (n = 8).As shown in p < 0.001), although the size of the occluder was chosen by the same principle. It is because that if the right ventricular surface was obstructed, the waist of the occluder POD could not be fully stretched and resulting in the POD being much smaller even smaller than the CTLD. We can see that there is a significant difference in different values of POD and CTLD , as When comparing the effect of different perforation sites and right ventricular surface obstruction on occluder molding, no significant differences were found between anterior or posterior septal perforation in POD/CTLD/POD-CTLD/occluder diameter and CROWD. However, in the intra-anterior septal group, right ventricular surface obstruction patients had a higher CROWD compared with no right ventricular surface obstruction patients , followed by the CTMD with a correlation coefficient of 0.740 (p < 0.001). There was no significant correlation between ECD and POD. Therefore, CTA is more accurate in the assessment of VSR diameter, and when selecting the occluder size, CTLD should be used as the standard parameter while CTMD should be referenced as well.The advantages of clinical outcome measures in the CTA group were mainly due to a more precise assessment of VSR diameter. In this study, the POD was used as the criterion for judging the true diameter of VSR. Since the myocardial tissue itself has certain elasticity and the traction effect of occluder, the POD may be slightly larger than the true diameter of VSR, so we considered the correlation between each measurement value and the POD as the evaluation criteria rather than the numerical proximity to ensure the reliability of. Through the scatter plot and Pearson correlation test, it was found that CTLD had the highest correlation with POD with a correlation coefficient of 0.799 (The choice of occluder size should also pay attention to the VSR site and the obstruction of right ventricular surface. This study proved that although there is no difference between anterior and posterior septal ruptures in POD or CROWD, right ventricular surface obstruction patients had higher CROWD and smaller POD. Therefore, when selecting occluder size for the anterior septal perforation, it is necessary to focus on observing whether there is a thick moderator band obstructing the right ventricular surface. If so, it is feasible to select the occluder with a size of 3-4 mm smaller than the conventional choice, to avoid poor occluder molding. Moreover, we observed that the POD in patients without right ventricular surface obstruction in the anterior septal perforation was 1.3 mm larger than the CTLD, and the POD in patients with posterior septal perforation was basically consistent with CTLD, suggesting that the anterior septal perforation without right ventricular surface obstruction had better elasticity; thus, attention should be paid to such characteristics when selecting the occluder size for these two types of VSR patients.There are some limitations to this study. First, the sample size was relatively small, which might lead to many nonsignificant differences. Second, the study had a short follow-up time and no long-term data was collected. Thus, further study on verification of the current results is required with a larger sample size and a longer follow-up time.In conclusion, this study preliminarily verified the accuracy of cardiac CTA in evaluating VSR diameter compared with echocardiography, while the evaluation needed to be in combination with different VSR sites and right ventricular surface obstruction.


This study aims to evaluate the incidence of postoperative ileus (POI) following cytoreductive surgery in epithelial ovarian cancer (EOC) patients and its impact on anastomotic leakage occurrence and postoperative complications.A total of 357 surgeries were performed on 346 ovarian cancer patients between 1/2010 and 12/2020 at our institution. The postoperative course regarding paralytic ileus, anastomotic leakage, and postoperative complications was analyzed by Fisher's exact test and through ordinal logistic regression.p 0.001), stoma creation (p 0.0001), and early start of laxatives (p 0.0048) significantly decreased POI, while anastomoses in general (p 0.0465) and especially low anastomoses (p 0.0143) showed increased POI rates. Intraoperative positive fluid excess >5,000 cc was associated with a higher risk for POI (p 0.0063), anastomotic leakage (p 0.0254), and severe complications (p 0.0012).A total of 233 patients (65.3%) returned to normal gastrointestinal functions within 3 days after surgery. A total of 123 patients (34.5%) developed POI. There were 199 anastomoses in 165 patients and 24 leakages (12.1%). Postoperative antibiotics (Postoperative antibiotics, an early start with laxatives, and stoma creation were associated with reduced POI rates. Patients with anastomoses showed an increased risk for POI. Severe complications, anastomotic leakages, and POI were more common in the case of intraoperative fluid balance exceeding 5,000 cc. Among gynecologic malignancies, epithelial ovarian cancer (EOC) is the most frequent cause of death in women . The mosThe POI relates to severe patient discomfort, including a lack of flatus, abdominal distension, nausea and vomiting, absence of normal bowel sounds, and a delay in the passage of stool, causing prolonged hospital stays, readmissions, reoperations, and possibly anastomotic leakages –13. POI This study was conducted in accordance with the standards of the ethics committee of the Faculty of Medicine at the University of Bonn, Germany No.: 14/22). The institutional record database was screened for ovarian cancer patients with cytoreductive surgery between January 2010 and December 2020. Pre-, intra-, and postoperative patient information was recorded from patient's charts, surgery reports, and pathologic findings. All data were evaluated regarding the following postoperative outcomes: postoperative paralytic ileus, anastomotic leakage, and general postoperative complications. In consideration of the I-FEED scoring system, POI was considered in case of an I-FEED score of equal or more than 3 points . The per. The insAll variables were analyzed by Fisher's exact test to identify significant correlations of pre-, intra-, and postoperative findings regarding the outcomes of POI, general morbidity, and anastomotic leakage. Differences were considered to be significant at a threshold of ≤0.05. Ordinal logistic regression was used to analyze morbidity in the context of comorbidity and surgical complexity. The test of all slopes equal to zero indicated that complications had a significant association with comorbidity and surgical complexity. The goodness of fit was tested by Pearson’s test and deviance test to verify that the model using link function logit was appropriate. All statistical analyses were performed using Minitab, version 18 .There were 357 surgeries in a total of 346 patients. Eleven patients received their primary surgery in our department, came back for recurrency, and received surgery again in our department. Fifteen patients received HIPEC  at the end of surgery. The median age was 61 years (range: 16–86 years). The physical status classification by the American Society of Anaesthesiologists (ASA) showed 1 ASA-4 patient and 104 ASA-3 patients. The rest of the patients scored ASA-1 and -2. The median PCI was 8 (range: 1–30). The median duration of surgery was 357 min (range: 24–695 min). The median amount of intraoperative kristalloid infusion was 7,500 ml . The median positive fluid excess was 6,580 ml . The median number of erythrocyte concentrates was 2 (range: 0–30). The median number of fresh frozen plasma was 0 (range: 0–19). The median highest intraoperative noradrenalin dosing during surgery was 5 µg/ml (range: 0–80 µg/ml). The median number of days in the intensive care unit was 1 day (range: 0–50 days). The median duration of postoperative ventilation was 4 h (range: 0–567 h). The median surgical complexity score was 6 (range: 0–15). Further general patient characteristics are presented in Of all patients, 233 (65.3%) returned to normal gastrointestinal functions within 3 days after surgery. A total of 123 patients (34.5%) were considered as experiencing POI due to a lack of flatus and defecation, nausea, and abdominal distension at least. Fifteen patients needed the placement of a nasogatric tube due to irresistible vomiting. p-value: 0.1634) and between severe postoperative morbidity (G4–G5) and POI . The comorbidity index did not correlate with increasing severe postoperative morbidity . A significant increase in severe postoperative morbidity was seen in the case of increasing surgical complexity .There was no significant correlation between morbidity in general (G0 vs. G1–G5) and POI , anastomotic leakage , and POI . A significant prolongation of the hospital stay was seen in the case of anastomotic leckage , severe postoperative complications (G3–G5) , and anastomosis in general  but not in the case of a prolonged POI only , as seen in A positive fluid excess of more than 5,000 ml showed a significant increase in severe complications (G3–G5) . The test of all slopes equal to zero indicated that severe complications had a significant association with comorbidity and surgical complexity. The goodness of fit was tested by Pearson’s test with a p-value of 0.249 and deviance test with a p-value of 0.243, indicating that the model using link function logit is appropriate. Odds ratios are seen in Considering surgical complexity, complications, and comorbidity index only, ordinal logistic regression showed significantly more severe postoperative complications (G4–G5) as comorbidity and surgical complexity increased (Anastomoses in general and especially low rectal anastomoses led to a significant increase in POI. No correlation was found between POI and anastomotic leakage or POI and small vs. large bowel anastomoses or POI in the case of anastomoses and increasing surgical complexity without stoma creation, as seen in There is a broad consensus that intestinal manipulation leads to intestinal inflammation causing impaired gastrointestinal motility , 23, 24.Although there is strong evidence for the use of preoperative antibiotics in colorectal surgery to prevent POI, surgical site infections, and anastomotic leckage, there is little evidence for the same outcomes for the use of postoperative i.v. antibiotics , 28. PosFluid overload in colorectal surgery has been identified as a promoter of POI and increased complications such as anastomotic leakage. Especially crystalloid fluids are suspected of producing splanchnic edema and thus escalating abdominal pressure, which in turn reduces mesenteric blood flow and contributes to tissue hypoxemia and impaired anastomotic healing –36. FluiDiverting ileostomies may reduce the clinical severity of anastomotic leakage, but no reduction of leakages is seen in the case of rectal cancer, remaining a controversial topic . EspeciaMost likely due to a different policy between the USA and Germany regarding the duration of in-hospital length of stay after major abdominal surgeries, we saw no significant association between POI and prolonged in-hospital length of stay .Our study has some limitations. Due to the retrospective evaluation of POI, a clear distinction between gastrointestinal intolerance and gastrointestinal dysfunction was not possible, although it may be important, especially with regard to POI-associated morbidity. Therefore, over- and underestimation of the true postoperative gastrointestinal impairment may be possible.Additionally, the fluid status depends on a number of factors such as the preoperative fluid status, preoperative bowel preparation, duration of surgery, blood loss, and patient fluid output by urine and intra-abdominally with respect to the amount of resected tumor. Therefore, more accurate measurements are needed in further studies to further define fluid overload in ovarian cancer patients.The strength of our study is the homogenous patient population, of only patients undergoing cytoreduction for epithelial ovarian cancer. Furthermore, we are the first to show that increasing surgical complexity does relate to increased morbidity but does not affect POI.The postoperative paralytic ileus may be reduced in the case of postoperative antibiotic use, early initiation of laxatives, and stoma creation. While antibiotics and laxatives are easy to implement, no recommendation for stoma creation can be drawn from our data due to the negative side effects of a stoma and POI will be overcome without a stoma. Optimal fluid management in ovarian cancer patients continues to be challenging as postoperative complications, postoperative paralytic ileus, and anastomotic leakage increase considerably with an increase in fluid excess. Duration of hospitalization is prolonged in the case of anastomotic leakage and severe morbidity but not in the case of a postoperative ileus.


A lower iAUC for insulin was observed after consumption of BCAAsB (p < 0.05 compared to CB and ArgB), while ArgB elicited higher iAUC for GLP-1 accompanied by higher glicentin response (p < 0.05 compared to CB). BCAAsB and ArgB increased postprandial amino acid concentrations and caused stronger satiety effects compared to CB. Increasing protein content of wheat biscuits with supplementation of plant flours originating from legumes/seeds decreases postprandial glycemia and provides with healthier snack alternatives which can easily be incorporated into diet.The study investigates the effects of wheat biscuits supplemented with plant flours originating from legumes/seeds enriched either in L-arginine (L-arg) or branched-chain amino acids (BCAAs) on postprandial glucose response of healthy subjects. Gastrointestinal hormone and amino acid responses as well as subjective appetite sensations are also evaluated. Subjects consumed wheat-based biscuits, enriched either in L-arg (ArgB) or BCAAs (BCAAsB) or a conventional wheat biscuit (CB) or a glucose solution (GS) in an acute randomized crossover design. Responses of glucose, insulin, ghrelin, glucagon-like peptide-1 (GLP-1), peptide YY (PYY) and glicentin, as well as those of L-arginine, L-leucine, L-isoleucine and L-valine, were evaluated over 180 min. Consumption of ArgB and BCAAsB elicited lower glucose iAUC compared to GS ( Glucose responses after a meal are related with appetite and there is evidence from acute studies that low-glycemic foods or meals have higher satietogenic effect than high-glycemic foods or meals . It is aProteins stimulate insulin secretion and reduce glucose concentration, depending on the amino acid profile of the ingested protein . Amino aBoth animal and plant proteins have been trending towards increased recognition in consumers’ minds; however, demand for healthier and sustainable nutrition drive research efforts to the development of plant protein products with enhanced protein quality. Plant proteins contain lower amounts of essential amino acids compared to animal proteins and are less digestible . StrategBranched chain amino acids (BCAAs) and especially L-leucine (L-leu) activate key-signaling systems within hypothalamic neurons  which regulate glucose metabolism and other essential physiological processes . StudiesBiscuits enjoy high popularity in the human diet and can be excellent snack alternatives as well as potential carriers of ingredients with glucose- and appetite-regulating properties ,20. HoweProcedures involving human subjects were carried out in the Diabetes Laboratory of the 1st Department of Propaedeutic and Internal Medicine, Laiko General Hospital, Athens School of Medicine, in collaboration with the Laboratory of Chemistry-Biochemistry-Physical Chemistry of Foods, Department of Nutrition and Dietetics, Harokopio University of Athens, Greece. The study was approved by the Institutional Review Board/Ethics Committee of Laiko General Hospital and the Bioethics Committee of Harokopio University. The study was registered in clinicaltrials.gov as NCT03974165.−2 were recruited by poster advertisements and personal communication. Subjects who were currently modifying diet or exercise patterns to lose weight or with known food allergies or intolerances, restrictive eaters, excessive exercisers or trained athletes, as well as candidates taking any medications or food supplements, were excluded. Pregnant, breast feeding and postmenopausal women were also excluded. Thirty healthy subjects , mean age 26.5 ± 5.3 years with body mass index (BMI) 25.7 ± 4.6 kg mBefore entering the study, participants were informed about the time and efforts required, gave voluntary informed written consent for participation and completed questionnaires related to diet, exercise and lifestyle habits. Two different wheat-based biscuits were designed, with the same protein content originating from plant flours (legumes and seeds) but with different amino acid compositions especially regarding BCAAs (BCAAsB) and L-arg (ArgB). More specifically, BCAAsB had higher BCAAs content while ArgB higher L-arg content. A common wheat biscuit (CB) with the same basic recipe was also prepared. Flours originating from legumes which are used in the Mediterranean cuisine, such as white beans and lentils as well as seeds such as millet and sesame, were examined regarding their amino acid composition. Flours were combined in a way to achieve the enrichment of the final products with the specific amino acids as well as to differentiate BCAAs and L-arg content between the two types of biscuits. The other amino acids and dietary fiber content remained as close as possible. The composition of flours of the test biscuits  to achieve desired amino acids content was 28.8% wheat flour, 18.9% millet flour, 4.7% white bean flour and 10.5% yellow lentil protein concentrate (which consists of 55% protein on dry basis) for BCAAsB. For ArgB the composition was 41.2% wheat flour, 11.8% defatted sesame flour and 5.9% yellow lentil flour. CB contained only wheat flour . The biscuits were prepared by a food manufacturer  and flours were supplied by the manufacturer’s partners.Nitrogen (protein: Nx6.25) was measured by Kjeldahl (ISO 1871)  and fat The study was designed as an acute, single-blind randomized controlled cross-over study with the three test biscuits  and a solution containing 50 g of glucose as reference food (GS), provided in random order with an in-between washout period of 1 week. Randomization was performed using a computer-generated randomization schedule. Subjects arrived at the Diabetes Laboratory of the 1st Department of Propaedeutic and Internal Medicine of Laiko University Hospital between 08:00 and 09:00 after having fasted overnight. On arrival, subjects underwent anthropometric and baseline blood pressure measurements. Body weight was measured in the fasted state with light clothing on a scale  and height using a stadiometer . Body fat was determined by bioelectrical impedance analysis . Waist circumference was measured at the midpoint between the lower margin of the least palpable rib and the top of the iliac crest in a standing position at the end of gentle expiration. Hip circumference measurement was taken around the widest portion of the buttocks. Blood pressure was performed by conventional methods. After 10 min of rest, subjects were asked to fill out the first set of visual analogue scales (VAS) questions assessing hunger, fullness and desire to eat. Then, an intravenous catheter was placed in a forearm vein and baseline samples were drawn. Subjects were then given one of the four test meals to consume within 15 min. Biscuit amounts yielding 50 g of available carbohydrates were served with 250 mL of tap water. Glucose solution contained 50 g of glucose (anhydrous) diluted in 250 mL water. Blood samples were collected and VAS were filled at 15, 30, 45, 60, 90, 120 and 180 min after food ingestion. Participants were instructed to eat a meal of their preference the previous evening and repeat it prior to each study session. Twenty-four-hour dietary recalls and a physical activity questionnaire were collected for the day before each study visit. Participants did not exercise regularly and were highly encouraged to maintain their regular lifestyle habits throughout the entire experimental period.3EDTA-coated vacutainers and centrifuged immediately (1000× g for 10 min at 4 °C) for plasma separation. For serum, blood was collected in plain tubes, allowed to clot at room temperature for 30 min and then centrifuged (1000× g for 10 min at 4 °C). After isolation, plasma and serum were stored at −80 °C until further analysis.Blood samples were collected in KGlucose concentrations at 0, 15, 30, 45, 60, 90, 120 and 180 min were determined in plasma by an electrochemical method in an automated analyzer . Basal biochemical measurements were performed in serum, at the beginning of the study, on an automated biochemical analyzer  using commercially available diagnostic kits.For the determination of ghrelin, plasma was pre-treated as previously described . Ghreling, 10 μL of the supernatant was transferred into a new tube and mixed with 40 μL of labelling buffer. The samples were vortexed and centrifuged again and 10 μL of the supernatant were mixed by vortex with 5 μL of aTRAQ® labelling reagent. Samples were incubated at room temperature for 30 min and at the end of the incubation, and 5 μL of hydroxylamine were added. Samples were vortexed and then dried for 45 min with no temperature applied. The dried samples were reconstituted to 32 μL of the 2.5 μL/L aTRAQ® internal standard. A total of 40 μL of plasma was placed inside a 0.5 mL tube and 10 μL of 10% sulfosalicylic acid was added for protein precipitation. After vortexing and centrifugation for 2 min at 10,000× ® H Class system from Waters company  system (Waters) Detection was performed on an AB Sciex TripleTOF® 5600+ mass spectrometer from AB Sciex company  equipped with a DuoSpray™ ion source operated in the positive ESI mode. Separation was achieved on a C18 column , using a gradient containing water (mobile phase A) and methanol (mobile phase B), both containing 0.1% formic and 0.01% heptafluorobutyric acid. Column temperature was maintained at 50 °C and the flow rate was set to 0.8 mL/min. The run time, injection-to-injection, was 22 min. Liquid chromatography analysis on an Acquity UPLCm/z windows (accumulation time 0.075 s) and a full scan mass experiment (accumulation time 0.25 s). Declustering potential was set to 30, while collision energy (CE) was set to 18 V for up to 150 m/z analytes and to 30 V for the higher masses, with a spread of 5 V. For the ESI source, temperature was set to 600 °C and ion spray voltage was 4500 V. Source gas and exhaust gas were set to 60 psi while curtain gas was set to 30 psi. Data acquisition was performed using the Analyst® 1.7.1 software  and spectral interpretation was performed using the Sciex OS software platform . The variable windows for the Swath method resulted from the Sciex’s Swath Variable Window Calculator.The analytes’ determination was carried out with a Swath acquisition method with 12 variable Quantification was achieved by dividing the analyte area by the IS area and then multiplying by the IS concentration. The full scan spectrum was used for analytes’ quantification while for the identification both the full scan spectrum and the spectrum MS/MS were used, as well the elution time of each amino acid in comparison with its respective internal standard. Additionally, a library with pure MS/MS spectra was created with the implementation of Information Dependent Acquisition (IDA) methods to standard solutions for aTraq amino acid derivatives and their corresponding Internal Standards in order to enhance the identification confidence of the analytes with %Fit Scores of samples’ spectrums in comparison to the standard solutions’ ones.The method was validated in accordance with the EMEA/CHMP/EWP/192217/2009 Rev. 1 Corr. 2. . The proStudy participants were provided with VAS booklets in order to assess appetite and satiety regarding the four different meals. VAS were filled before meal and then at 15, 30, 45, 60, 90, 120, and 180 min postprandially just before blood samples were obtained, in order to reduce the stress of sampling. Three main questions were included; “How hungry do you feel”, “How full do you feel” and “How great is your desire to eat.” A 10 cm line scale ranging from 0  to 10 (“extremely”) expressing the most negative and the most positive rating, respectively, was used. Subjects were instructed to place a vertical mark on the line to correspond with their feelings and were not allowed to discuss their ratings with each other or refer to the previous ratings when marking the scale. Quantification was made by measuring the distance in cm from the left end of the line to the mark. Sensory evaluation of the three biscuits was performed in order to investigate the effect of different composition on organoleptic characteristics. Twenty-five trained testers participated. Specifically, appearance, color intensity, texture, odor, taste, aftertaste and overall acceptance were evaluated on a 9-point hedonic scale . The sensory evaluation took place at the manufacturer’s facilities.x axis, while AUCs for GLP-1, PYY, glicentin and amino acids  were expressed as increase from preprandial values and were calculated using iAUC ignoring area below the x axis. Accordingly, iAUCs for VAS were estimated. AUCs of glycemic and insulinemic responses were calculated applying the trapezoidal rule using the incremental AUC (iAUC), ignoring area under the baseline . AUC forDescriptive statistics are presented as mean ± SD and results as mean ± SEM. ANOVA for repeated measures, followed by Bonferroni’s post hoc test was used to compare the postprandial changes in blood variables and subjective appetite ratings between treatments and identify significant differences at specific time points as well as differences between iAUC values. Regarding sensory characteristics, one-way ANOVA was applied.p < 0.05 was considered statistically significant. The SPSS 21.0  statistical software package was used for the analysis.All subjects completed the four sessions of the study and no adverse reactions were reported. Subjects’ characteristics are presented on Participants complied with the requirements of the study including continuation of dietary and physical exercise habits and maintenance of consistent body weight throughout the entire experimental period.Nutritional composition of each test biscuit and amino acids content are presented in No significant treatment × weight interactions were detected after the application of a linear mixed model, with patients fitted at random; iAUC of each parameter was considered as a dependent variable and treatment and weight as fixed factors.p < 0.05). A significant difference between ArgB, BCAAsB and CB compared to GS was noticed at all the rest time points i.e., 15, 45, 60, 120 and 180 min postprandially (p < 0.05). GS also exerted the highest iAUC value followed by CB, ArgB and BCAAsB while BCAAsB and ArgB caused lower glucose iAUC compared to CB and to GS  and at 60 min compared to CB and ArgB (p < 0.05). iAUC caused by BCAAsB was also significantly lower compared to those caused by CB and ArgB (p < 0.05 for both biscuits).BCAAsB elicited a milder insulinemic response compared to the other two biscuits B. Specifp < 0.05). No differences between iAUCs were observed.Total ghrelin concentrations remained at low levels after the ingestion of the three biscuits reaching significant difference after BCAAsB and ArgB compared to CB at 180 min  while the difference between BCAAsB and CB iAUCs was marginally significant . Glicentin iAUC was higher after ArgB compared to CB (p = 0.009) while the difference between BCAAsB and CB was marginally significant . L-leu concentrations were significantly elevated compared to CB at 60, 90 and 180 min after ingestion of BCAAsB and at 90 and 180 min after ingestion of ArgB . ArgB caused lower values at 60, 90, 120 and 180 min also compared to CB (p < 0.05). Hunger iAUCs of the three biscuits were significantly decreased compared to GS while that of ArgB was also lower compared to that of CB  at time points 30, 45, 60, 90, 120 and 180 min. ArgB caused significantly higher values at 30, 45, 60, 90, 120 and 180 min also compared to CB . ArgB consumption showed significant effects also compared to CB at all time points after 45 min  in contrast to their appearance, which did not seem to be affected (p < 0.05) compared to BCAAsB and ArgB. Furthermore, no differentiation was observed in terms of odor of the evaluated samples, while the control biscuit appeared to be characterized by a clearly stronger taste, compared to the other two biscuits. Overall acceptance showed that both biscuits were more preferred than the control biscuit.Different composition of the biscuits had a significant effect on some organoleptic characteristics. In particular, color intensity was different between the enriched samples and the control sample  were examined. Sensory evaluation was also performed. Enrichment with specific amino acids was obtained by using flours originating from common legumes and seeds of the Mediterranean cuisine . The main effort was the enrichment of the final products through the addition of a combination of plant flours with high content of these amino acids. The target was to differentiate BCAAs and L-arg content between the two types of biscuits and keep as close as possible the content of total/other amino acids, macronutrients and dietary fibers, thus enabling the distinction of postprandial effects to be attributed to BCAAs or L-arg. Both biscuits, BCAAsB and ArgB, caused a lower glycemic response compared to the common wheat biscuit and to the reference food (glucose solution). BCAAsB also elicited a lower insulinemic response than that of ArgB and CB, whereas ArgB consumption led to higher insulin levels similar to those caused by CB. This could be attributed to the high L-arg content of ArgB (~29% higher than that of BCAAsB). L-Arg exhibits strong insulinotropic characteristics and these results are in accordance with previous studies which have shown that substitution of wheat flour for legume flour rich in L-arg resulted in lower GI but also elicited a higher insulin response .Consumption of the three biscuits caused a reduction in total plasma ghrelin concentration, which remained at low levels until 120 min postprandially. BCAAsB and ArgB consumption contributed to maintenance of low hormone concentrations until 180 min, reaching statistical significance compared to CB at this time point. Both biscuits helped subjects to maintain lower concentrations of ghrelin for a longer time.GLP-1 iAUC after ArgB consumption was significantly higher compared to CB while BCAAsB also caused a higher iAUC with marginally significant difference. Both biscuits enhanced plasma L-arg response. L-arg is a GLP-1 secretagogue  and possIn addition to GLP-1, glicentin is another proglucagon peptide which is also secreted by L-cells in response to food intake and recent studies have shown that similar to GLP-1, glicentin exerts insulinotropic effects . GlicentThe results of the present study revealed that both ArgB and BCAAsB caused a postprandial increase in glicentin response after ingestion, inducing stronger effects which remained for a longer time than those of the common wheat biscuit. Significantly higher levels of the hormone were observed at certain time points. iAUC after ArgB consumption was significantly higher compared to CB while BCAAsB also caused a higher iAUC with marginally significant difference. The role of specific amino acids in glicentin response has not been evaluated so far except for one older study . EffectsPlasma concentrations of BCAAs remained at higher levels for a longer time after consumption of BCAAsB. BCAAs act in the brain as signals of dietary protein intake. L-Leu suppresses food intake and influences signaling systems associated with feeding behavior  and reguConsumption of both biscuits caused stronger feelings of satiety compared to the control biscuit as revealed after evaluation of their effects on subjective appetite sensations. Specifically, BCAAsB and ArgB elicited significantly higher iAUCs for “fullness” and lower iAUCs for “desire for the next meal” compared to CB. ArgB consumption exerted also a significantly lower iAUC for “hunger” compared to that of CB. Increasing the number of questions and performing VAS measurements in different sessions from those of blood sampling could provide additional, clearer information. The two biscuits had very good organoleptic characteristics, were well-tolerated by the subjects and were not less preferred than the control biscuit.13C] acetic acid [The evaluation of hormone responses led to better conclusions regarding the satiating effect of the enriched biscuits. However, a factor which has to be taken into consideration is that amino acids produce a slowing down of stomach emptying that could explain, in part, the observed metabolic responses. Certainly, intragastric administration of L-leu and L-ile in healthy men lowers postprandial blood glucose, at least in part by slowing gastric emptying . Additiotic acid .ATP channels and subsequent membrane depolarization in GLUTag cells. Membrane depolarization results in Ca2+ influx, which induces vesicular exocytosis and secretion of GLP-1 into the circulation [Beyond amino acid and carbohydrate content, differences in energy intake could account for the differences in subjective appetite and gut hormone responses. Since the primary outcome of the study was to examine the effects on glucose concentrations, biscuit meals with the same content of available carbohydrates were provided to the subjects. In addition, the provided amount of carbohydrates affects GLP-1 and different carbohydrates’ loads would lead to different GLP-1 responses. Indeed, ingestion of carbohydrates causes a rapid increase in circulating GLP-1 levels within 30–60 min in healthy humans. Glucose and fructose increase GLP-1 secretion in a dose-dependent manner and a possible mechanism involves the closure of ATP-sensitive Kculation ,50.It has to be reported that ArgB was further examined in a long-term study conducted in subjects with overweight/obesity following a hypocaloric diet. The results showed that participants who consumed the ArgB exerted significantly higher body weight reduction and lower energy intake compared to the participants who consumed isocaloric amounts of the control wheat biscuit . Animal A limitation of the present study is that amino acids content of the two designed biscuits could not be more differentiated regarding BCAAs and L-arg amounts since they were developed using flours and not pure substances. This fact may possibly make it difficult to clearly distinguish their results or highlight statistical differences. The significant results seem to be related more to L-arg content than BCAAs and the ideal case would be to have a biscuit enriched in proteins but with very low BCAAs vs L-arg content. In addition, the role of other biscuit components on the observed differences in metabolic responses and appetite ratings, such as the different amounts and composition of fat and fibers, cannot be underestimated.In conclusion, the results of the present study showed that supplementation of wheat biscuits with plant flours originating from legumes and seeds, enriched either in L-arg or BCAAs, improved postprandial glucose responses compared to a common wheat biscuit. Additional analysis revealed stronger effects after consumption of the enriched biscuits regarding gastrointestinal hormone responses and subjective appetite sensations. Further investigation concerning enrichment with L-arg, an insulinotropic amino acid and GLP-1 secretagogue, becomes challenging especially if the cardioprotective effects of L-arg from the one side and the incriminating roles of BCAAs regarding their association with type 2 diabetes and insulin resistance on the other, are taken into consideration. The new products had very good organoleptic characteristics and were not less preferred than the common wheat biscuit.In the present study, better and healthier alternatives in terms of snacking were made by simple materials with high nutritional quality and easy to find. Enrichment of foods with specific components of plant origin which positively affect glucose and gastrointestinal appetite-regulating hormone responses could provide a novel strategy for metabolic regulation and prevention of obesity.Patent No: GR1010123 B1/ELBISCO S.A., HAROKOPIO UNIVERSITY OF ATHENS.