Wetlands regulate the balance of global organic carbon. Small changes in the carbon stocks of wetland ecosystem play a crucial role in the regional soil carbon cycle. However, an accurate estimation of carbon stocks is still be debated for China’s wetlands ecosystem due to the limitation of data collection and methodology. Here, we investigate the soil organic carbon (SOC) storage in a 1-m depth in China’s palustrine wetlands. A total of 1383 sample data were collected from palustrine wetlands in China. The data sources are divided into three parts, respectively, data collection from published literature, data from books, and actual measurement data of sample points. The results demonstrate that there is considerable SOC storage in China’s palustrine wetlands (9.945 Pg C), primarily abundant in the northeast, northwest arid and semi-arid as well as Qinghai-Tibet Plateau regions. The SOC density in per unit area soil was higher in the wetland area of northeast, southwest and Qinghai-Tibet plateau. Within China terrestrial scale, the temperature and precipitation differences caused by latitude were the main environmental factors affecting the organic carbon content. Furthermore, except for the southeast and south wetland region, SOC content decreased with depth. Although, many studies have been conducted to identify the physical, chemical and biological factors on SOC biogeochemical circulation, only a few parameters in these analyses could account for the correlation between SOC and soil properties on a national-scale [Wetlands are important ecosystems located at the water-land interface, and have a significant effect on regulating the source-sink mechanism of soil organic carbon (SOC) . With real-scale ,15,16. TAt present, there are still many uncertainties in SOC estimations, which limits the understanding of the important role of wetlands in the global carbon circulation and climate change mitigation. Firstly, the total wetland area has been reduced by 68% due to climate change and anthropogenic activities ,17, resuDespite considerable efforts directed at understanding SOC distribution and storage on global ,21, counIn order to ensure the validity and accuracy of the data, the data sources of this paper are divided into three parts: literature data collection, field survey and measurement data, and preliminary data sorting. A systematic literature search was conducted with Web of Science, Elsevier Science Direct, Google Scholar and China National Knowledge Infrastructure using the following keyword combinations ‘marsh wetland’, ‘swamp wetland’, bogs’, ‘fens’, and ‘soil organic carbon OR soil organic matter’ in the title, abstract and keywords. The research on carbon storage in China’s palustrine wetland began in the late 1990’s, limiting this study to a more recent timespan which was represented in the literature. The oldest relevant paper was published in 2000, while the most recent research was published in 2019. A total of 1267 studies were entered into databases and analyzed for relevance. Articles which were deemed irrelevant or repetitive were exclude during a preliminary screening procedure, while the remaining publications were evaluated to extract data on SOC or SOM and the influencing environmental factors, including longitude, latitude, altitude, mean annual precipitation (AMP), mean annual temperature (AMT), moisture content (MC) and acid-base property (pH). To improve the spatial distribution of data points extracted from the literature, historical data and additional measured datapoints are used in supplement to this paper. Historical data was obtained from the book “China Marsh Annals”, from which data including relevant information of longitude, latitude, soil organic carbon content, soil bulk density, water content and pH of each point was extracted, totaling 67 sample points. However, the distribution of data from literature and historical resources is considerably lower in wetlands in the Great Hing’an Mountains, in the northeast of China. The Great Hing’an Mountains area is located along the southern margin of the Eurasian tundra, and is sensitive to climate change. Therefore, study on the carbon storage of palustrine wetlands in this area is of great importance for the estimation of SOC in China’s palustrine wetlands. Given this, 49 effective samples were collected in the Great Hing’an Mountains to improve the spatial distribution of the data. In addition, in order to verify the accuracy of the mathematical model use in this paper, three 1-m soil cores were collected in each wet area for error analysis. The distribution of sample points was shown in Based on the distribution of wetlands in China, and considering the natural variation in these characteristics across different regions , China’s wetland resources are divided into eight wetland regions, the northeast wetland region (C1), the southwest wetland region (C2), wetlands in the middle and lower reaches of the Yangtze River region (C3), the southeast and south wetland regions (C4), coastal wetland region (C5), wetlands in the Qinghai-Tibet plateau region (C6), wetlands in the middle and lower reaches of the Yellow River regions (C7) and wetlands in the northwest arid and the semi-arid regions (C8). Further information on geography of these regions is showed in 3), SOM (g/kg) and H (m), represent soil bulk density, soil organic matter content and soil depth. In this study, the downcore was divided into five layers: 0–20, 20–40, 40–60, 60–80 and 80–100 cm).As an indicator of soil physical structure, bulk density (BD), which is related to the water holding capacity of soil and SOC biogeochemical circulation, was used in the construction of soil mathematical model ,33. In ti) per unit area is estimated for each layer of i [i and BDi represent SOC content and BD within a layer of i, and Hi represents the depth of the soil in that layer.Soil organic carbon density Sa, Cb, and K were constants calculated from exponential function to calculate a region’s SOC storage  per unit area . The specific information about each area is shown in In this study, only SOC or SOM content in the soil surface layer was available, while the vertical distribution of SOC content is also essential information for estimating SOC storage. Numerous studies demonstrated that SOC content decreased rapidly with depth as a function of soil types, land-use, and vegetation communities . In receMost of the SOC data collected was from surface layers with a few datapoints from deeper layers (1 m). To ensure the reliability of the mathematical model, we selected some data to verify the mathematical model. The verification data were divided into two parts: one was the measured SOC content at 1 m depth collected from published literature; the second part was from the survey undertaken at the sample site. We sampled each wetland regions, collecting 1 m core of undisturbed soil which was analyzed in the laboratory for measurement.Several measured values were selected to verify the applicability of equation, and demonstrated a maximum error of 18.61%, a minimum of 1.31%, and a mean error of 8.93% between measured and estimated SOC values .Experimental data was processed with Excel software Ver. 2007  to calculate BD. Redundancy analysis (RDA) was completed with Canoco software Ver. 4.5  to identify the explanatory capacity of environmental variables to SOC content. Using the statistical software SPSS Ver. 20.0 , the direct and indirect influence of environmental variables on SOC distribution were assessed with path analysis (PA). Structural equation models (SEMs) was conducted with Amos Ver. 20.0. All figures were generated using Origin Ver. 9.0 .Results showed there is a considerable SOC storage in China’s palustrine wetlands, with a total of 9.945 Pg . However2, respectively, followed by the C4 (20.31 kg/m2), C3 (17.04 kg/m2), C7 (10.98 kg/m2), and C5 (10.84 kg/m2) geographic regions. The average SOC density was 27.17 ± 16.54 kg/m2.SOC density showed clear variation between geographic regions . In the With respect to SOC storage and content, the C6 and C1 geographic regions showed higher values, particularly C6 . SOC stoRDA showed that the first two axis components explained 76.10% of the variance in the correlation between SOC content and environmental controls . More thp < 0.05) and significantly (p < 0.01) correlated with SOC content in both C1 and C6 geographic regions.Best-fit stepwise regression results indicated that when combined, these selected environmental controls can account for 99.9% variation in SOC content . LatitudDue to the significant differences in climate characteristics, topography and geomorphology in the different geographical areas, the migration and transformation of SOC are affected by different environmental factors. In this study, the typical wetland regions of northeast China and Qinghai-Tibet plateau were selected for analysis . The resWith respect to wetland formation, palustrine wetlands is the manifestation of bidirectional ecological evolution processes between the land and surface water, which is formed under the comprehensive action of various natural factors, such as regional climate, hydrology, geological landform, and vegetation . Climate2) from the atmosphere [Climatic factors, especially hydrothermal conditions, are an important influence on the formation and development of palustrine wetlands and indirectly regulate SOC source-sink processes through affecting vegetation community assemblage and growth, as well as plant-residue decomposition quality and intensity . The conmosphere , enhancimosphere . China’smosphere .2 emissions [Soil physical properties, such as MC and BD ,52, and missions . AdditioGenerally, MC plays an important role in the SOC source-sink process through regulating the oxidation-reduction potential (ORP), microbial biomass and vegetation types ,59,60. HThere was a considerable SOC storage in China’s palustrine wetlands with a value of 9.945 Pg C, primarily abundant in the northeast and northwest arid and semi-arid regions as well as Qinghai-Tibet Plateau regions, account for 99.11% of total SOC storage. Specially, within China terrestrial scale a total of 3.456 Pg C was estimated with a depth profile of 0–0.3 m. The variation in MAP and MAT as a result of latitude was primary reason for the spatial differences in SOC content. Under the influence of climatic conditions and soil parent material, SOC content in per-unit volume was the highest in the Qinghai-Tibet plateau region (C6 geographic region), while that of coastal wetlands (C5 geographic region) was the lowest.


Streptococcus mutans and Rothia dentocariosa. The chemical-physical parameters of acacia honey and the RP-HPLC polyphenolic profile of pomegranate peel extract have been previously described in our studies, while the characterization of myrtle extract, performed by HPLC analysis, is reported here. All the extracts were used singly and in binary combinations to highlight any synergistic effects. Moreover, the extracts were tested in association with amoxicillin to evaluate their ability to reduce the effective dose of this drug in vitro. The values of minimal inhibitory concentrations and minimal bactericidal concentrations have been used to quantitatively measure the antibacterial activity of the single extracts, while the fractional inhibitory concentration index has been considered as predictor of in vitro anticariogenic synergistic effects. Finally, a time-kill curve method allowed for the evaluation of the bactericidal efficacy of the combined extracts. The microbiological tests suggest that acacia honey, myrtle, and pomegranate extracts are able to inhibit the cariogenic bacteria, also with synergistic effects. This study provides useful and encouraging results for the use of natural extract combinations alone or in association with antibiotics (adjuvant therapy) as a valid alternative for the prevention and treatment of oral infectious diseases.The increasing incidence rate of oral diseases, the wide spread of antimicrobial resistance, and the adverse effects of conventional antibiotics mean alternative prevention and treatment options are needed to counteract oral pathogens. In this regard, our study aims to evaluate the antibacterial activity of polyphenolic extracts prepared from acacia honey, myrtle leaves, and pomegranate peel against cariogenic bacteria, such as  Dysbiosis of oral microbiota, followed by the transition of several oral microorganisms from a commensal to pathogenic lifestyle, is a significant cause of dental caries . Tooth dStreptococcus mutans is an acidogenic and aciduric bacterial species, considered as the primary pathogen in human dental caries , dry myrtle (Myrtus communis L) leaves, and dry pomegranate (Punica granatum L.) peel fruit, with solid-liquid solvent-extraction method. The dilution ratio was 1:10 (w/v). The procedure of the hydro-alcoholic polyphenolic extracts production of acacia honey was similar to that described by Robinia pseudoacacia honey, including the concentration of polyphenols, flavonoids, and mineral elements, as well as the identification of major volatile molecules, was previously described in The extracts for the microbiological assays were prepared from samples of fresh acacia honey . After drying at 40°C in an incubator for 5 days, the leaves were fine-powdered using a mixing grinder. Leaf powder was mixed with a hydro-alcoholic buffer, composed of ethanol and distilled water at 50% (v/v), reaching a final concentration of 0.1 g/mL. The extraction method consisted of stirring the solid matrix into the selected solvent at room temperature for 30 min, using a rotary shaker. After extract centrifugation at 10000 rpm for 15 min at room temperature, the double supernatant filtration with Whatman N°1 filter paper and membrane filters Millex-GS (Merk-Millipore) with a porosity of 0.22 μm, was carried out.The hydro-alcoholic polyphenolic extracts of pomegranate peels used in this study were prepared according to –1) were mixed. After storing each sample away from light for 5 min, 2 mL of 7.5% Na2CO3 were added to the mix. The incubation of samples for 2 h in the dark at room temperature was carried out and subsequently the absorbance was determined by spectrophotometry (UV/Vis-6715 Jenway Spectrophotometer) at a wavelength of 760 nm. The Gallic acid standard  (0–200 mg L–1) was used to produce the calibration curve, in order to express the total phenolic content in mg of GAE for g of each sample. In particular, aliquots of three separately prepared extracts from each natural matrix were analyzed.The amount of total polyphenols in the hydro-alcoholic extracts from honey, myrtle, and pomegranate, was determined by a colorimetric assay of Folin-Ciocalteu, as reported in Myrtus communis L. leaves was performed using LC-4000 series integrated HPLC systems . A C18 Luna column 5 μm particle size, 25 cm × 3.00 mm i.d.  was used. The mobile phase composed of water with 0.5% formic acid (v/v) (solvent A) and HPLC grade methanol (solvent B) was used and the linear gradient started with 5% B in A to reach 20% B in A at 5 min, 25% B in A at 45 min, 45% B in A at 47 min, 55% B in A at 57 min, and 80% B in A at 67 min, with some modifications to the Lorach method  and literature data.The identification and quantification of the main polyphenolic compounds in hydro-alcoholic extracts prepared from h method . The floStreptococcus mutans ATCC 25175  strain, isolated from carious dentin, and the Rothia dentocariosa Rd1 clinical isolate from samples of dental plaque provided by the Pediatric Dentistry Department of “Federico II” University of Naples , where it was identified by MALDI-MS and BD PhoenixTM systems as described in in vitro anticariogenic activity of the hydro-alcoholic polyphenolic extracts. Bacteria were cultured in aerobic conditions at a temperature of 37°C in Brain Heart Infusion (BHI) agar/broth medium  and on Columbia CNA agar medium (5% Sheep Blood) supplemented with Colistin and Nalidixic Acid .The Bacterial strains were maintained at 4°C on agar media. The isolates were stored frozen at −80°C in BHI broth supplemented with 10% glycerol (v/v)  until use and the working cultures were activated in the respective broth at 37°C for 15–18 h.in vitro anticariogenic effects of the hydro-alcoholic polyphenolic extracts from honey, myrtle, and pomegranate, individually and in binary combination, with or without amoxicillin, against cariogenic microorganisms, an in vitro antimicrobial activity assay was performed using the agar diffusion method, as reported in in vitro anticariogenic activities against the tested microorganisms.To evaluate the ∗105 CFU/mL (Colonies Forming Units/mL), according to Clinical and Laboratory Standards Institute (CLSI) 2017 guidelines  was defined as the minimum concentration of agent or binary combination of in vitro anticariogenic agents that killed 99% of bacteria from the initial inoculum.The susceptibility of cariogenic strains to different concentrations of single polyphenolic extracts and in binary combination, with or without AMX, was determined by the tube dilution method with standard inoculum 1idelines . AMX wasin vitro .The effects of honey, myrtle, and pomegranate polyphenolic extracts, alone or in combination, with or without AMX, were deemed synergistic, indifferent, or antagonistic against the two oral pathogens, thanks to the measuring of the fractional inhibitory concentration index (FICI) of binary combinations. In particular, the following formulae, in accordance with the Odds’ interpretation , were usS. mutans and R. dentocariosa, a time-killing assay was performed, which allowed for the investigation of the bactericidal efficacy of this antimicrobial combination over time. In particular, an assay of bacterial survival, with single and mixed cultures of two microorganisms, was carried out, with increasing concentrations of the binary combination of the two extracts in a 1:1 ratio . To evaluate the survival of each strain, during the observation period of 168 h, aliquots of serial dilutions of the bacterial suspensions were spread on BHI agar, and the plates were incubated at 37°C for 48 h to evaluate the viable bacterial colony counts.To verify the inhibitory synergistic effect of the binary combination of pomegranate and myrtle polyphenolic extracts on the survival of post hoc correction for multiple comparisons against a control, indicating the statistical significance of treated bacterial cultures with extracts against monitored bacterial cultures in absence of the extracts. Statistical significance was assigned to all P-values < 0.05.Three replicates were carried out for all analyses and the antimicrobials tests were performed with independent microbial cultures. Results were graphically reported by “GraphPad Prism 6” software and statistical significance was validated by parametric and non-parametric tests. In particular, the TPC  data were analyzed using a Mann–Whitney test comparing two groups at a time. The two-way ANOVA test was used to perform significance analysis of Log CFU (Colony Forming Unit) data, with Bonferroni –1 for acacia honey extracts, 54.86 mg GAE g–1 for myrtle leaves extracts, and 45.9 mg GAE g–1 for pomegranate peel extracts.The total polyphenol content (TPC) in honey, myrtle, and pomegranate extracts is reported in The HPLC profile of myrtle leaves extract is reported in S. mutans ATCC 25175 and R. dentocariosa Rd1 strains, as demonstrated by the evident inhibition zone of bacterial growth estimated through the agar diffusion method. The mean diameters of the inhibition zones (MDIZ) of bacterial growth exerted by the extracts, used individually and in binary combination, with or without amoxicillin (AMX), against the two cariogenic bacteria was reported in S. mutans) and 29.0 ± 1.9 mm . AMX, tested as a positive control, showed antibacterial efficacy against both cariogenic isolates, while no effects were observed for the negative control, i.e., the hydro-alcoholic buffer used for the extraction.Hydro-alcoholic polyphenolic extracts from honey, myrtle, and pomegranate exhibited an appreciable inhibitory activity against both The inhibition zone (mm) is reported as mean of triplicate assay ± standard deviation. MDIZ, mean diameter inhibition zone; PE, pomegranate extracts; ME, myrtle extracts; HE, honey extracts; AMX, amoxicillin.in vitro anticariogenic activity of the extracts was also confirmed by quantitative assays. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values are reported in The in vitro anticariogenic activity of the binary combination of acacia honey, myrtle leaves, and pomegranate peel extracts, with or without AMX, was measured by the determination of fractional inhibitory concentration (FIC) value, for each extract and for the AMX, and of the FIC index (FICI) for each binary combination. FIC and FICI values are reported in in vitro anticariogenic effects against both S. mutans and R. dentocariosa  honey, Myrtus communis leaves, and Punica granatum L. fruit peel are able to counteract cariogenic bacteria of dental plaque. In particular, the extracts showed inhibitory activity against S. mutans ATCC 25175 and R. dentocariosa Rd1 clinical isolates, which are considered main etiological agents of dental caries. Our results are in agreement with several studies that demonstrated how polyphenols can slow down the growth of pathogenic microorganisms, affect the external morphology and membrane integrity, or inhibit some cellular processes that are involved in microbial growth  in line with literature data. An experimental study investigated the antibacterial effects of myrtle extracts against several human pathogens by dilution methods. Particularly, the myrtle MBC was 0.5 mg mL–1 for S. aureus, 2.5 mg mL–1 for Proteus mirabilis and Proteus vulgaris, 15 mg mL–1 for Klebsiella spp. and Salmonella typhi, and 20 mg mL–1 for Pseudomonas aeruginosa. The MBC of myrtle for the two relatively least sensitive species, Shigella sp. and E. coli, was 40 mg mL–1 and 45 mg mL–1 (Myrtus communis oil (MCO) on some oral pathogens, including S. mutans, has been determined. The MICs of MCO for S. pyogenes, S. mutans, C. albicans, and P. gingivalis were 29.68 ± 4.8, 31.25 ± 0, 46.9 ± 16, and 62.5 ± 0 μg mL–1, respectively , as well as the low pH (3.5) of acacia honey, contribute to its antimicrobial activity  against oral microorganisms  when compared with antibiotics (0.031–512 g mL–1), in the past few years the effects of combinations between antibiotics and phytochemicals have been studied. Several scientific evidences showed that phytochemicals can modulate bacterial mechanisms of antimicrobial resistance  were particularly remarkable. Amoxicillin is not only heavily used in medicine but is also frequently prescribed in dentistry , togethe. mutans . Both poathogens .The demonstrated ability of plant extracts to act in synergy with antibiotics could represent an effective approach in solving the problem of antimicrobial resistance .in vitro microbiological tests showed a strong anticariogenic effect of the hydro-alcoholic polyphenolic extracts from acacia honey, myrtle leaves, and pomegranate peel, alone and in synergy, against S. mutans and R. dentocariosa strains.Results of our in vitro studies on biofilm models for the possible development of natural medical tools in the treatment and prophylaxis of dental caries. Furthermore, the synergistic activity of myrtle and pomegranate extracts in combination with AMX suggests that the natural extracts can be used as adjuvants of conventional antibiotic therapy and as an alternative therapeutic strategy against oral pathogens. Therefore, the polyphenols from honey, myrtle, and pomegranate represent nutraceutical agents to recommend in the prevention and treatment of oral diseases as a valid alternative to synthetic drugs, especially if they are detrimental and ineffective. This knowledge opens up a wide range of possibilities for the combined antimicrobial therapy against cariogenic bacteria and other oral pathogens (in vivo testing and clinical trials, could allow the development of new antimicrobials as alternative agents in combination with existing antibiotics in clinical use, taking another step forward to overcome the problem of the antimicrobial resistance phenomenon.Although this study is limited to the analysis of these effects against only two of the main bacterial species that compose the complex oral plaque community, these data encourage the promotion of athogens . More reThe datasets generated for this study are available on request to the corresponding author.DS and CaP conceived and planned the work. SF played part to experimental carrying out. RC, ChP, EV, MP, MV, and PS contributed to the design and implementation of the research, to the analysis of the results, and to the writing of the manuscript. All the authors agree to be accountable for the content of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Autoantibodies against tumor-associated antigens (TAAbs) can be used as potential biomarkers in the detection of cancer. Our study aims to identify novel TAAbs for gastric cancer (GC) based on human proteomic chips and construct a diagnostic model to distinguish GC from healthy controls (HCs) based on serum TAAbs. The human proteomic chips were used to screen the candidate TAAbs. Enzyme-linked immunosorbent assay (ELISA) was used to verify and validate the titer of the candidate TAAbs in the verification cohort (80 GC cases and 80 HCs) and validation cohort , respectively. Then, the diagnostic model was established by Logistic regression analysis based on OD values of candidate autoantibodies with diagnostic value. Eleven candidate TAAbs were identified, including autoantibodies against INPP5A, F8, NRAS, MFGE8, PTP4A1, RRAS2, RGS4, RHOG, SRARP, RAC1, and TMEM243 by proteomic chips. The titer of autoantibodies against INPP5A, F8, NRAS, MFGE8, PTP4A1, and RRAS2 were significantly higher in GC cases while the titer of autoantibodies against RGS4, RHOG, SRARP, RAC1, and TMEM243 showed no difference in the verification group. Next, six potential TAAbs were validated in the validation cohort. The titer of autoantibodies against F8, NRAS, MFGE8, RRAS2, and PTP4A1 was significantly higher in GC cases. Finally, an optimal prediction model with four TAAbs  showed an optimal diagnostic performance of GC with AUC of 0.87 in the training group and 0.83 in the testing group. The proteomic chip approach is a feasible method to identify TAAbs for the detection of cancer. Moreover, the panel consisting of anti-NRAS, anti-MFGE8, anti-PTP4A1, and anti-RRAS2 may be useful to distinguish GC cases from HCs. Gastric cancer (GC) is one of the most important causes of cancer death in the world . It was With the development of cancer, the abnormal expression of tumor-associated antigen (TAAs) can trigger an autoimmune response, and the corresponding antibodies are called autoantibodies against the tumor-associated antigen (TAAbs) –12. ManyProteomic chip is a high-throughput technology for cancer biomarker development. It can simultaneously analyze serum autoantibodies against many proteins for further screening and identifying novel TAAbs –21. TherA total of 692 samples were included in this study, including 282 GC cases, 282 healthy controls (HCs), and 128 benign gastric diseases (BGD) cases. GC and BGD serum samples were collected from a tertiary Level A hospital of Zhengzhou (January 2012 to June 2017). All patients were histopathologically confirmed and had not received any treatments. All HCs were selected from the biobank of Henan Key Laboratory of Tumor Epidemiology and were free of any digestive tract diseases and autoimmune diseases. In the discovery phase, 10 GC cases and 10 HCs were selected, and in the verification and validation phase, 672 subjects were selected, among which the verification cohort included 80 GC cases and 80 HCs, while the validation cohort included 192 GC cases, 192 HCs, and 128 BGD cases. All GC cases and HCs were matched by frequency matching method. The serums were collected according to the standard protocol. This study was approved by the Institutional Review Board of Zhengzhou University and informed consent forms were received from all participants.https://cdi.bio/huprot/) available. It contains more than 21,000 recombinant proteins and covers all human recombinant proteins that can be purified, covering 81% of the human proteome. It is the most comprehensive chip available at present. More detailed information about proteomic chip can be found at https://cdi.bio/huprot/. Theoretically, it is the most comprehensive protein-chip to select possible TAAbs specific to gastric cancer. The human proteomic chips were used to detect the titer of TAAbs in serum samples from 10 GC cases and 10 HCs, to screen candidate TAAbs for GC detection. The experiment was carried out according to the manufacturer’s protocol and their previous publication . HuProt™ human proteomic chip used in this study is by far the world’s largest collection of full-length human proteins (lication .The titer of TAAbs was detected in serum samples by indirect enzyme-linked immunosorbent assay (ELISA). The protocol was described in detail in our previous study . In thisP values were determined based on two-tailed, and P<0.05 was defined to be significant.GenePix Pro 6.0 was used to acquire the original data from proteomic chips. IBM SPSS statistical software (version 21.0), GraphPad Prism 6.0, and MedCalc 11 were used to analyze the data. Nonparametric test was used to analyze the difference of TAAbs level between different groups. The sensitivity, specificity, and AUC were calculated by receiver operating characteristic (ROC) curve analysis. Meanwhile, the positive predictive value (PPV), negative predictive value (NPV), and Yoden index were used to evaluate the validity and reliability of the diagnostic tests using serum autoantibodies as biomarkers. When the specificity was greater than 85%, the maximum Yoden index (YI) was used to set the cutoff value of TAAbs to determine the positive reaction. Logistic regression analysis was used to establish the optimal model to distinguish GC from NC. All The experiment was divided into the discovery phase, the verification phase, and the validation phase. In the discovery phase, the titer of TAAbs in serum samples from 10 GC cases and 10 HCs were measured by human proteomic chips. In the verification phase and validation phase, ELISA was applied to test the title of 11 candidate TAAbs in serums from 272 GC cases, 272 HCs, and 128 BGD cases. The clinical characteristics of all participants were shown in P value of <0.05 (two sided) was considered to be significant. (2). Fold change (FC) of GC cases against HCs was calculated, and FC ≥ 1.2 was used as a cutoff value to select potential TAAbs. (3). When the difference of positive rate (cut off = mean + standard error) of SNR between GC cases and HCs was more than 80%, the protein was identified as a candidate TAAb. (4). KEGG analysis was performed to select cancer-associated proteins as candidate TAAs. Finally, 11 candidate TAAs were identified, and autoantibodies against these 11 TAAs, including INPP5A, F8, NRAS, MFGE8, PTP4A1, RRAS2, RGS4, RHOG, SRARP, RAC1, and TMEM243 were evaluated as potential markers in GC. The basic characteristics of 11 TAAs were shown in Based on the SNRs of 20 serum samples in the proteomic chips, four criteria were used to screen the candidate TAAbs. (1). Mann-Whitney U test was used to compare whether there was a statistical difference in SNR between GC cases and HCs. A To determine the diagnostic value of the 11 aforementioned candidate TAAbs, two independent cohorts were selected to detect their titer. First, in the verification cohort, 160 serum samples were detected by ELISA. The OD values of the 11 TAAbs in the verification cohort were shown in Further, six potential TAAbs were validated by ELISA in the validation cohort, including 192 GC cases, 128 BGD cases, and 192 HCs. P < 0.001) (P = 0.325).The cohort with a larger sample size was used as the training group to construct a model by logistic regression analysis, and another cohort was used as the testing group to evaluate the model. Based on the OD value of five significant TAAbs  in the validation cohort (192 GC cases and 192 HCs), logistic regression analysis was used to generate a diagnostic model. The diagnostic model was completely consistent by forward or backward logistic regression methods. Finally, autoantibodies against NRAS, MFGE8, PTP4A1, and RRAS2 entered the model. The predicted possibility for classification as GC was PRE  = 1/{1+ EXP [-(−2.517 + 25.928 × anti-NRAS + 14.823 × anti-MFGE8 + 5.862 × anti-PTP4A1 – 32.91 × anti-RRAS2)]}. The AUC of the diagnostic model was 0.87 (95% CI: 0.83–0.90), sensitivity, specificity, and accuracy rates were 70.8, 85.9, and 78.4%, respectively , 80 hepatocellular carcinoma cases (HCCs), 80 lung cancer cases (LCs), and 80 healthy controls by ELISA. The OD values of four TAAbs were shown in To explore the progression pattern of healthy cases to BGD cases to GC cases, we compared the diagnostic value of single TAAb for BGD cases and established a diagnostic model. Based on the results of ELISA in the validation cohort , the titer of four TAAbs  were significantly higher in BGD cases , sensitivity, specificity, and accuracy rates were 70.8, 85.9, and 78.4%, respectively, in the training group. The results indicated that the model was stable. Although many studies have reported that optimal combinations of autoantibodies could aid in the diagnosis of GC, the results from the current study showed better performance in distinguishing GC and HC.Several approaches have been used to identify valuable TAAbs in cancers, among which serological analysis of expression cDNA libraries (SEREX) and serological proteome analysis (SERPA) are the most commonly used technologies –31. HoweBased on the human proteomic chips, 11 candidate TAAbs  were identified. Then, two independent cohorts were used to further identify TAAbs. Finally, five potential TAAbs  were identified by ELISA. The five potential biomarkers have not been reported in GC. F8 belongs to the coagulation factor family and plays a vital role in the coagulation cascade . Some stMany studies have shown that TAAbs can be used to detect cancer. However, previous studies also have shown that the diagnostic performance of a single anti-TAA autoantibody was not sufficient to be used in the screening of cancers , 49. In In addition, we selected the serum of 80 ECs, 80 HCCs, and 80 LCs and measured the titer of four TAAbs  by ELISA to verify the specificity of four TAAbs in gastric cancer. The results showed that the titer of anti-PTP4A1 in HCCs was higher than that in healthy controls. Many studies shown that PTP4A1 is highly correlated with the occurrence, development, and prognosis of HCC , 54. MorThere are some advantages in the current study. Firstly, the human proteomic chips were used to screen candidate TAAbs associated with GC and had yielded promising results. Secondly, the diagnostic model was established by logistic regression analysis in the training group, and another independent group was used to test this model’s performance. However, some limitations also need to be mentioned. Firstly, all proteins on the proteomic chips were homogeneously expressed from normal human coding genes, so it is hard to identify the TAAbs with structural changes and post-translational modification aberrance. Secondly, further validations are warranted to confirm the results from the current study.In summary, the proteomic chip approach is a feasible method to identify TAAbs for the detection of cancer. Moreover, the diagnostic panel  may be useful to distinguish GC cases from HCs.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by The Institution Review Board of Zhengzhou University. The patients/participants provided their written informed consent to participate in this study.JZ and JS conceived the design of the current study. CC and YD conducted experiments and drafted the manuscript. CQ, GS, and ZH participated in the data analysis. PW, HY, KW, LD, and CS helped to draft the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the Major Project of Science and Technology in Henan Province (No.161100311400), the Zhengzhou Major Project for Collaborative Innovation (No.18XTZX12007), the National Science and Technology Major Project of China (2018ZX10302205), and the Project of Basic Research Fund of Henan Institute of Medical and Pharmacological Sciences (2019BP0202).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Objectives: The aim of this study was to make a comprehensive economic assessment of the costs of hospital-acquired C. difficile infections (CDI).Methods: We carried out a retrospective matched cohort study utilizing Danish registry data with national coverage to identify CDI cases and matched reference patients without CDI (controls) for economic burden assessment in Denmark covering 2011–2014. Health care costs and public transfer costs were obtained from national registries, and calculated for 1 year prior to, and 2 years after index admission using descriptive statistics and regression analysis.Results: The study included 12,768 CDI patients and 23,272 matched controls. The total health care cost was significantly larger for CDI cases than controls throughout all periods. During the index admission period, cost was €12,867 per CDI case compared to €4,522 (p < 0.001) for controls, which increased to an average of €31,388 and €19,512 (p < 0.001) in Year 1 for the two groups, respectively. Excess costs were found both among infections with onset in hospitals and in the community. Diagnosis compatible with complications increased costs to on average >€91,000 per case. The regression analysis showed that CDI adds a substantial economic burden, but only explains about 1/3 of the crude difference observed in the matched analysis.Discussion: The major economic impact of hospital-acquired CDI with complications underlines the importance of preventing complications in these patients. Our study provides an informed estimate of the potential economic gain per patient by successful intervention, which is likely to be relatively comparable across countries. Clostridioides difficile is the leading cause of infectious diarrhea in hospitalized patients  has over the past decades been recognized as an increasing problem with rising mortality rates (https://www.ssi.dk/-/media/arkiv/subsites/miba-og-haiba/dokument/haiba_2018-rsrapport.pdf?la=da), and this possible trend highlights the need for differentiation of the cost estimations for HOHA and COHA CDI. With the implementation of surveillance and more insight into the health burden of CDI, attention is drawn to improved prevention and control of CDI. To determine the cost-effectiveness of potential preventive measures and stimulate research, an economic assessment of the cost associated with CDI is needed. The aim of the present study was to assess the economic burden attributable to CDI during and after hospitalization for both HOHA and COHA CDI, using a population based matched cohort design.patients , and occpatients , 3. C. d systems , 5. C. dty rates , 7. Due ty rates . This hyThe study was approved under the general agreement for non-interventional database studies between the Danish Data Protection Agency and Statens Serum Institut, reference number 2008-54-0474 and reported according to the STROBE statement .We carried out a retrospective matched cohort study utilizing Danish registry data with national coverage to acquire CDI cases and matched reference patients without CDI for economic burden assessment. The sampling population was the entire Danish population from 2011–2014, provided through the Civil Registry System (CRS) containing unique Personal Identification Numbers (PIN), which is linkable to various service records.We identified cases as patients with a confirmed first episode CDI, through the Hospital-Acquired Infections database (HAIBA). As part of the Danish surveillance system, CDI cases are registered in HAIBA and classified according to the ECDC case definition  with a fThe reference population (hereafter termed controls) were sampled from all admissions recorded during the study period in the DNPR and linked with the CRS to obtain sex and age, but excluding patients from HAIBA with CDI and those without a PIN. We matched each CDI case to two non-CDI controls, but cases with only one match were also retained. The controls were matched according to hospital, region, age, sex, action diagnosis, index year, and index month (that is the month of admission where the first CDI was registered). The action diagnosis was matched on the first three digits and age was matched in groups of intervals of 10 years until ≥81. Furthermore, an outpatient match was additionally performed for COHA patients with a pool of controls with no hospital admission 6 months prior to the end date of their ambulatory treatment. They were matched by sex, age, month, and year of their treatment end date. See the To determine the periods for cost estimations we used the index date in HAIBA as index, but for Year-1, Year 1, and Year 2 we used the dates from DNPR to match with the diagnosis-related groups in the Danish National Cost Database. This database provided the total cost for every in- and outpatient discharged from a public hospital in Denmark based on the patient's actual utilization of hospital services, with Diagnose Related Groups (DRG) for inpatients and Danish Ambulant Grouping System (DAGS) for outpatients. DRG-costs are linked to the discharge date and DAGS-costs are on the date of visit. Due to high mortality in the study population, the number of cases and controls declined over time. We therefore constructed the cost data over time as a gross dataset including all periods for all patients whether they were alive or not, but calculated the average cost of the patients by period, whilst taking the declining population into account. Apart from direct costs, we included derived costs to public transfer from the DREAM database containing all social transfer payments for all citizens in Denmark . Cost anCost data from DRG and DAGS were prices including rehabilitation. Index admission costs for HOHA were from the cost database. For transfer payment , we calculated the number of months, since we did not have prices for these transfers. Only transfers for people in the workforce were in the data, thus age and pension were unavailable. Complications were identified as the action diagnosis for outpatients and as the main action or secondary diagnosis during admission for inpatients. Patients only contributed cost until the date of death, as cost only occur while patients are alive. The cost were deflated to 2016 prices using the Danish Consumer Price index.t-test with no additional explanatory variables in the model. As cost may be affected before onset of CDI, and CDI may result in long term complications, the costs where compared across different cost periods, to be able to compare cost over time. Cost were calculated for pre-index period (Year-1), index, and post-index period (Year 1 and Year 2). The pre-period was divided into two sub-periods, month−12 to −7 and month−6 to −1. The 24 months post-index period was split into the sub-periods month 1–3, month 4–6, month 7–9, month 10–12, and month 13–24. Patients were included if they were alive at least part of the sub-period, but excluded in future sub-periods after death occurred. Means of cost data for cases and controls was calculated with 95% confidence intervals (CI), with all costs adjusted to 2016 prices.The first analysis included average cost and a test for significant difference between cases and controls. Since cases and controls were matched, we used a bootstrapped To control for residual confounding, we used a regression analysis for specific patients groups in order to determine the added cost of CDI. We used a 2-step gamma distributed analysis for all health cost . A gamma distribution link rather than a logarithmic transformation was motivated by the occurrence of 0's in the cost variables, which did not follow a normal distribution . As indeCost analysis stratified on complications were run to determine to what extent excess costs in patients could be related to the range of defined clinical complications. Complications were defined as having at least one of the following diagnoses in Year 1 or Year 2 : septicaemia, dehydration, renal failure, ileus, hypotension, shock, thromboembolic episodes, toxic megacolon, bowel perforation, colectomy, and isolation regimes. Cases with complications were allocated to the complication group for the whole study period and then stratified regardless of when the complication occurred. For a detailed description of the methodology of the matching and the cost analysis, please see the The study included 12,768 patients with hospital-acquired CDI and 23,272 matched controls . Of the Prior to the index date, cases and matched controls were incomparable on a number of parameters. Cases had on average spent 16.3 days in hospital compared with 6.1 days spent among the controls. The share of long-term hospitalization admissions (7+ days) 6 months prior was 46.9 and 18.6% among the cases and the controls, respectively. The Charlson score 12 months prior to inclusion was on average 1.4 and 0.9 for cases and controls, respectively. Among the cases, 11.5% had a diagnosis of IBD 12 months prior to inclusion, whereas 3.9% of the controls had IBD. Taken together, even with a careful matching procedure based on underlying disease codes, cases of CDI had an excess of severe illness and were hospitalized longer, and had higher frequencies of IBD before index date. Thus, matching did not account for the differences in morbidity between the cases and controls.Typical complications for COHA and HOHA were septicaemia, renal failure, dehydration, and isolation regimes . During The mortality among CDI patients was high, especially among the age group 71+ years, where 38% survived until 24 months, compared to 58% of the matched controls . The difp < 0.001) for the controls, which increased to €21,795 compared to €10,188 (p < 0.001) for the two groups during month−6 to −1. At index, cost was €12,867 for cases compared to €4,522 (p < 0.001) for controls. In Year 1, the cost was €31,388 for CDI and €19,512 for the controls (p < 0.001), and €17,590 and €11,260 (p < 0.001) for the two groups, respectively in Year 2. During the index period and Year 1 and 2, CDI cases spent on average 1.1 month longer on public transfer income compared to the controls (p < 0.001). We found increased costs both for COHA and for HOHA CDI cases compared with the matched controls (p < 0.001) for a control, and €16,640 vs. €9,850 (p < 0.001) during Year 2 for cases and controls, respectively. HOHA CDI cases on average were estimated to cost €19,992 at index compared to €5,714 (p < 0.001) for controls. During Year 2 the costs were estimated to €18,553 and €12,523 (p < 0.001) for cases and controls, respectively. Based on the cumulative health care cost from index to Year 2, HOHA CDI cases were more costly with a mean economic burden of >€70,000, whereas, a COHA CDI case had a mean economic burden of ~€50,000  . AlthougA recent retrospective analysis using individual-level data from from Apr. 1, 2005 to Mar. 31, 2015, in Ontario Canada health databases, found the median costs attributable to C. difficile infection to be $1051 for that associated with long-term care facilities, $13 249 for community-associated infection and $11 917 for ACH-associated/community-onset infection  were not associated with increased costs in the regression analysis. Finally, complications were defined based on data from administrative registries and not a detailed clinical assessment, which was beyond the scope of this study.In conclusion, our study estimates the attributable economic burden of hospital-acquired CDI in Danish patients and provides an informed estimate of the potential economic gain per patient by successful intervention. We emphasize the need to include COHA CDI in order to make comprehensive estimates of the overall economic burden of CDI on health care systems. At present, only few national surveillance systems have the capacity to disentangle HOHA from COHA, and often COHA cases may be ignored because the onset is in the community where they may not be categorized as hospital-acquired cases. Furthermore, the results highlight the importance of identifying and preventing complications in patients with hospital-acquired CDI, and the need to investigate strategies to prevent CDI in susceptible patients.The datasets presented in this article are not readily available because the authors confirm that, for approved reasons, some access restrictions apply to the data underlying the findings. We used population-register based data including personal identifiers. To access this data it is necessary to apply to the Danish National Board of Health by completing an extensive data approval application.RI and JK generated and analyzed the data. UB, FM, SE, and KM interpreted the data. UB wrote the first manuscript draft. All authors critically reviewed the manuscript.RI was employed by the company i2minds. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Digital elevation model (DEM) plays a vital role in hydrological modelling and environmental studies. Many essential layers can be extracted from this land surface information, including slope, aspect, rivers, and curvature. Therefore, DEM quality and accuracy will affect the extracted features and the whole process of modeling. Despite freely available DEMs from various sources, many researchers generate this information for their areas from various observations. Sentinal-1 synthetic aperture radar (SAR) images are among the best Earth observations for DEM generation thanks to their availabilities, high-resolution, and C-band sensitivity to surface structure. This paper presents a comparative study, from a hydrological point of view, on the quality and reliability of the DEMs generated from Sentinel-1 data and DEMs from other sources such as AIRSAR, ALOS-PALSAR, TanDEM-X, and SRTM. To this end, pair of Sentinel-1 data were acquired and processed using the SAR interferometry technique to produce a DEM for two different study areas of a part of the Cameron Highlands, Pahang, Malaysia, a part of Sanandaj, Iran. Based on the estimated linear regression and standard errors, generating DEM from Sentinel-1 did not yield promising results. The river streams for all DEMs were extracted using geospatial analysis tool in a geographic information system (GIS) environment. The results indicated that because of the higher spatial resolution (compared to SRTM and TanDEM-X), more stream orders were delineated from AIRSAR and Sentinel-1 DEMs. Due to the shorter perpendicular baseline, the phase decorrelation in the created DEM resulted in a lot of noise. At the same time, results from ground control points (GCPs) showed that the created DEM from Sentinel-1 is not promising. Therefore, other DEMs’ performance, such as 90-meters’ TanDEM-X and 30-meters’ SRTM, are better than Sentinel-1 DEM . Digital elevation model (DEM) is one of the essential geospatial data tools used in geological, geographical, geomorphological, and environmental applications. It allows extracting essential terrain features such as slope, aspect, elevation, and many more ,2,3,4. TAmong the RS sensor technologies, SAR Interferometry has shown its advantages for DEM generation, such as sensitivity of SAR signal to the surface physical and topographic properties, independence of weather and illumination conditions, and robust and accurate analytical approaches ,9. With Because of the different product types and sensor modes, Sentinel-1 has many practical applications such as subsidence, landslide and, land cover flood detection ,12,13. SA DEM can be acquired from different sources, including ground-based surveys ,16,17,18As a very successful example, many researchers worldwide tend to create DEMs from Sentinel-1 SAR data. Braun  created The main objective of the current study was to generate a DEM from Sentinel-1 satellite imagery by using the InSAR technique and also to compare it with DEMs of airborne synthetic aperture Radar (AIRSAR) product (with 10 × 10 cell size), advanced land observing satellite phase array L-band synthetic aperture radar (ALOS-PALSAR) imagery , TanDEM-X (with 90 × 90 cell size) and SRTM (with 30 × 30 cell size) for two different study areas.2 and is placed in Peninsular Malaysia’s central part and the main range of geological units of Malaysia [2, and the second formation (Silurian-Ordovician) consists of slate, schist, limestone with minor intercalation of sandstone, and phyllite [When a comparative study is done, every aspect, even the study area’s selection, must be considered. For example, regarding SAR data and DEM generation, collecting data on hot days and dense vegetation coverage  result in low coherence extraction. In this study, two study areas of a part of the Cameron Highlands, Pahang, Malaysia, and Sanandaj county, Kurdistan, Iran, were selected to carry out this comparative study. The Cameron Highlands is mostly covered by dense and sparse vegetation, while vegetation coverage for Sanandaj county is negligible. The Cameron Highlands is located between Longitudes 101°20′00″ to 101°27′10″ East and Latitudes 4°23′30″ to 4°31′10″ North . This scMalaysia ,48. Therphyllite . Based ophyllite ,50.With a population of almost 432 thousand, the city of Sanandaj is the biggest in Kurdistan Province, Iran. Sanandaj is located in the Longitudes 46°24′00″ to 47°19′00″ East and Latitudes 35°3′00″ to 35°39′00″ North . The citSentinel-1 satellite data are the first program by Copernicus, conducted by the ESA. Sentinel-1A and Sentinel-1B are two platforms for it . This prInSAR technique is the comparison of the phase values for a pair of SAR images ,55. The SLC product has 3 Sub-Swaths of IW1, IW2, and IW3, which each sub swath is for an adjacent acquisition by the TOPS mode . SentineThe back geocoding (co-registration) operator in SNAP software was used to co-register the two split satellite data based on the orbital information. In order to extract the phase difference, two acquisitions must be stacked first. This is an essential step in interferogram formation, as it ensures that each ground object has the same range and azimuth pixel in either image ,46. EnhaAn interferogram is calculated by cross multiplication of the master and slave images . Here, tThe interferogram consists of phase variation Interferogram formation represents a raw DEM resolution ion (2)) :(2)Ωr=naCoherence is a useful source to know about the quality of an interferogram . CoherenTOPS data has 9 bursts, which is typically separated by some specific zones . HoweverFor a proper unwrapping process, phase filtering and multi-looking are two essential steps. Noise from geometric and temporal decorrelation and volume scattering can corrupt the interferometric phase ,57. Restψ can be eventually obtained for each pixel (Equation (3)):Unwrapping was applied in this study by using SNAPHU software . Phase uThe interferometric phase is somewhat ambiguous and within the scale of own only . In ordeown only .The unwrapped phase is not a metric measure yet. To convert the radian into the meter unit, the phase to elevation operator should be applied. The acquisition geometry for both images (master and slave), phase noise and possible unwrapping errors significantly affect this step .TC will geocode the final bands by correcting geometric distortions of SAR images using a ready to download DEM and, lastly, produce a projected map. On the other hand, TC (geocoding) converts an image from either slant range (SR) or ground range (GR) into a coordinate system. Finally, this step using a DEM corrects geometric distortions of foreshortening, layover, and shadow.Y is an actual variable, and N is the number of GCPs. The numerator is squared differences between the actual variables and the estimated one. As a whole, the lower the numerator, the higher the accuracy will be [Generally, the accuracy assessment is vital in approving the quality of extracted information from remotely sensed data . Regress will be ,66.By delineating the stream networks for the created DEM from Sentinel-1 and the other products of AIRSAR, TanDEM-X, ALOS-PALSAR and SRTM in the GIS environment using the hydrology toolbox, the study was well-validated and justified as well. Based on the quality of the decorrelation of phase and spatial resolution for each DEM, many stream orders can be extracted in this method. The higher the spatial resolution, the more the stream orders can be delineated. Based on the system’s structure, rivers of the order number “1” are the highest tributaries. Thus, if two streams of the outermost tributaries merge, it gives a higher order. Therefore, whenever two streams with different orders merge, it gives a higher-order number than them.The DEMs were used to delineate streams one by one. For example, the created DEM from Sentinel-1 was imported into GIS, and a hydrology toolbox was employed for extracting stream orders for it. In the first step, the filling operator was utilized for filling any possible gap in the DEM. In the second step, the flow direction operator was applied for obtaining all directions of water flow in the DEM. At the third stage, using the flow accumulation raster threshold, those in the same direction or path were connected. Next, by using a stream link, the streams in the same directions were linked together. Then, a stream order operator was applied to order the streams from 1 to whatever. Finally, the extracted rivers were converted to feature using stream to feature operator. It is worth mentioning that these processes were applied for all the DEMs.The vertical accuracy of the DEMs was validated via comparing the interpolated elevations from each DEM and global navigation satellite system (GNSS) ground control points (GCPs). All DEMs of the both study areas were validated through available GCPs. The static GCPs  were measured using a GPS (geodetic receivers). Trimble device in Malaysia and Leica device in Iran were placed for minimum three hours until the signals were received from the satellites (based on the nearest continuously operating reference stations). The horizontal and vertical accuracy of each gcpare not exceeded ±2 cm and ±5 cm, respectively.2 meaning 1 GCP per 4400 km2) for a study performed in East of Malaysia. And in another study done in Perlis region in Malaysia, Pa’suya, et al. [2 meaning 1 GCP per 21 km2). The obtained elevations from DEMs compared with the corresponding elevation values of the GCPs. The outlier test using interquartile range (IQR) was done for the discrepancy between each DEM and the GCPs based on Equation 5 [IQR is Interquartile range, Q1 represents the lower quartile, and Q3 is the upper quartile.Available eight GCPs for Malaysia and six for Iran were used for validation. Normally, available GCPs are rare for example, Halim, et al.  used 45 , et al. , used 38uation 5 .(5)OutlAccordingly, the Root Mean Square Error (RMSE) for the discrepancy between each DEM and the GCPs were also computed using Equation 6 .(6)RMSEWhere, Using InSAR technique, for each study area, a DEM was extracted from Sentinel-1 data. Because of low coherence, vegetated areas typically will produce poor elevation value in C-band ,71. Our DEMs vary in quality and pixel sizes depending on the creation methods and data used . The linThe linear regression and standard errors of the estimate were employed to validate the extracted DEM’s relative accuracy. For this reason, 8120 and 5334 systematic points for Malaysia and Iran were selected, respectively  were creTo assess the results’ quality and validity, several available GCPs points were collected to be compared with all DEMs including the created DEM from Sentinel-1 SAR satellite imagery. The results showed differences between them, meaning that based on the GCP points, the DEM created from Sentinel-1 could not be reliable. Therefore, the researchers should be cautious about using it as a base DEM for their studies. The outlier test results show that there are no outlier concerning the obtaining elevation values from the DEMs for both study areas .In fact, establishing GCPs is a time-consuming, hard job, costly, and even impossible sometimes . The GCPAccuracy assessment using the available GCPs was performed for the created DEM from Sentinel-1 based on the land cover types . The intOverall, RMSE values are quite high and ranges from five to thirteen meters in the study area of Malaysia, were mostly covered by dense forest and vegetation. In Malaysia, the highest RMSE of 13 m can be seen in township (this maybe because there are high rising hotels in the Cameron Highlands with sparse trees coverages). Then, the highest value was calculated for forests of 11.7 m, followed by vegetation and florification with 5.6 m. Because of low vegetation coverages these values were quite lower in Iran than Malaysia, but because of the technical issues the RMSE for Iran also showing that Sentinel-1 failed to create an accurate DEM. In Iran, sparse and bare lands are contributed to lower RMSE meaning that agriculture, dryfarming, and rangeland showed lower RMSE of 7, 5, and 3.5 m, respectively. The township has RMSE of 9 m.Hydrological units are used for the planning and management of natural resources. To better understand the low accuracy of the extracted DEM, the hydrological analysis was performed as well. Therefore, the created DEMs were hydrologically compared with AIRSAR, ALOS-PALSAR. TanDEM-X, and SRTM products. The hydrological networks were delineated for all four DEMs using the hydrology toolbox in the GIS environment. The concern with the extracting stream networks, given the same accumulation raster layer, the higher threshold value contributes to less dense stream orders and vice versa .The nature of the InSAR technique is noisy and this approach is limited to coarse scene descriptions. At the same time, in built-up areas it result in layover and shadow . InSAR lFor such areas like the Cameron Highlands , where mostly covered by dense vegetation coverages, multi-temporal InSAR technique, which integrates different estimators, resulting in a self-adaptive and reliable data processing ,82. AnotShort temporal baseline, suitable perpendicular baseline, and suitable atmospheric conditions for data acquisition are very essential and should be considered ,88,89. IDue to the good temporal characteristics of the data used in this study, we did not apply atmospheric delays correction. Date of the data used for the study area  are in months of late February and early March. In addition, for the study area (Iran) the data are in months of late July and mid-August. Refer to the weather condition in the study areas, we can find out that the images were not much affected by hot days (water vapor) and rainfall (wet days). Because there are two main monsoon seasons in Malaysia from late May to September and from October to February . In IranAlthough, based on the above-mentioned statements, this paper did not used any method for removing atmospheric path delays, but suggests a few models to be used in the future studies , including direct observations of atmospheric properties , empirical corrections , weatherThe X-, C- and L-band SAR signals have a different interaction with surface objects. Typically, X-band signals backscatter from the top of the trees canopy, while C-band signals penetrate deeper into the canopy . In contDifferent DEMs only with common coordinate systems can be compared ,106,107.The optimum perpendicular baseline (PB) for DEM creation should be between 150 to 300 m ,46. The In terms of data acquisition and generation processes, creating a DEM using SAR data is a time-consuming issue . It is wBased on findings using evaluation techniques of the linear regression, ground control points and hydrological models, the created DEM has low accuracy. The linear regression showed more error for dense vegetated areas. Moreover, the hydrological networks for the created DEM, showed bad results, especially in stream orders number one. Additionally, results by GCPs were also confirmed that creating DEM from Sentinel-1 data is not giving promising result, especially over the vegetated areas.It is noted that DEM generation due to numerous usages for extracting different attributes, such as slope, aspect, elevation, curvature, etc., is one of the essential spatial information tools used in the earth sciences. Therefore, optical and radar satellite images are the two main sources to produce DEM. DEM generation with the SAR interferometry approach is one of the most common methods and It is very interesting for geosciences researchers. Furthermore, one of the most recent Sentinel-1 missions is SAR imagery. Sentinal-1 images are among the best earth observations for DEM generation due to acquire images from the whole earth surface once every six-day, availabilities, high-resolution, and C-band sensitivity to the surface structure.The objective of this study was to produce a DEM from Sentinel-1 satellite imagery using the InSAR technique along with to compare it with DEMs of AIRSAR, ALOS-PALSAR, TanDEM-X, and SRTM. For this reason, Sentinel-1 products for two different study areas were acquired, from which DEM with 14-m pixel size was obtained for a part of the Cameron Highlands, Pahang, Malaysia, and a part of Sanandaj, Iran. Based on the analysis and comparisons with the other proven DEMs using Linear regression and hydrological models, the quality of derived DEM for the two study areas was less than the other DEMs, even 90- and 30-m cell size DEM of TanDEM-X and SRTM, respectively. At the same time, results of RMSE using a few available GCPs showed that the created DEMs were not giving promising results. The validation results showed that Sentinel-1 could not produce accurate DEMs for two reasons. Firstly, the C-band signals cannot penetrate the denser vegetation coverage, especially canopies and trees. Secondly, the relatively low accuracy can be due to the small perpendicular baseline of Sentinel-1 data, which is rarely longer than 100 m could be found. For creating a DEM, the baseline should generally be between 150 to 300 m. This study recommends researchers to use freely available DEMs  instead of creating DEM from Sentinel-1. Finally, if a DEM with a higher spatial resolution is required, they can use other products such as ALOS-PALSAR and LiDAR (Light Detection and Ranging) data, which is a technique to measure distances by target illuminating using a laser and calculating the backscatters with a sensor.


Background: Pulmonary hypertension is the major cause of morbidity and mortality in congenital diaphragmatic hernia (CDH). Mutations in several genes that encode signaling molecules of the transforming growth factor β (TGFβ) and bone morphogenetic protein (BMP) pathways have previously been associated with CDH. Since studies on the activation of these pathways in CDH are scarce, and have yielded inconsistent conclusions, the downstream activity of both pathways was assessed in the nitrofen-CDH rat model.Methods and Results: Pregnant Sprague-Dawley rats were treated with nitrofen at embryonic day (E) 9.5 to induce CDH in offspring. At E21, lungs were screened for the expression of key factors of both signaling pathways, at both the mRNA transcript and protein levels. Subsequently, paying particular attention to the pulmonary vasculature, increased phosphorylation of SMAD2, and decreased phosphorylation of Smad5 was noted in the muscular walls of small pulmonary vessels, by immunohistochemistry. This was accompanied by increased proliferation of constituent cells of the smooth muscle layer of these vessels.Conclusions: Increased activation of the TGFβ pathway and decreased activation of the BMP pathway in the pulmonary vasculature of rats with experimentally-induced CDH, suggesting that the deregulated of these important signaling pathways may underlie the development of pulmonary hypertension in CDH. Congenital diaphragmatic hernia (CDH) is a severe developmental anomaly characterized by a diaphragmatic defect. The concomitant pulmonary hypertension (PH) that develops in affected lungs can cause severe problems in the newborn, and is responsible for the high morbidity and mortality in these patients. Although the muscularization of the pulmonary vessels has been demonstrated to be increased in CDH , the patTGFβ is a negative regulator of airway branching in early lung development. However, TGFβ signaling is also active in the vascular and airway smooth muscle and alveolar and airway epithelium during late lung development. Both up- and down-regulation of TGFβ signaling impairs the alveolarization process , 4, depeStudies on the TGFβ pathway in CDH have not yielded consistent conclusions. Decreased expression of TGFβ1 was found at the mRNA level in the hearts of the nitrofen-exposed rat pups with CDH , where iIn contrast to the TGFβ pathway, conclusions drawn in several reports on components of the BMP pathway in CDH are consistent. Reduced expression of BMPR2 , 12 and Investigations conducted to date have focused largely on the expression of receptors in both the TGFβ and BMP pathways, but little is known about the actual activation of these pathways. Therefore, we hypothesized that the analysis of downstream mediators would identify changes in TGFβ and BMP signaling pathways in the lungs of rats in which CDH was induced by nitrofen exposure.Pregnant Sprague-Dawley rats received either 100 mg nitrofen dissolved in 1 ml olive oil or just 1 ml olive oil by gavage on gestational age day E9.5. Nitrofen induces CDH in ~70% of the offspring, while all pups have pulmonary hypertension , 19. At RNA isolation, cDNA synthesis and subsequent qPCR analysis on right lung lobes was performed as previously . The genImmunohistochemistry (IHC) was performed on 5-μm paraffin sections of the left lobe according to standard protocols, using the Envision™ detection system  . PrimarySnap-frozen right lung lobes were homogenized on ice in Carin buffer , containing protease inhibitor Complete . Samples were centrifuged at 14,200 r.p.m. for 15 min and protein concentration in the supernatant was measured using the Bradford method. Subsequently 50 μg of protein per lane was loaded onto an SDS-PAGE and transferred to nitrocellulose membranes using wet blotting. Antigens were detected with TGFβ , pSMAD2 , SMAD2 , pSMAD5 , SMAD5 , and Zeb2 . Cofilit-tests for normally distributed variables. The analyses were performed using SPSS 21.0 for Windows . All statistical tests were two-sided and used a significance level of 0.05.Data are presented as percentages, means (SD) for normally distributed variables. Univariate analyses were performed using independent samples Tgfbr1 and Tgfbr2 receptors, but no difference in the abundance of the ligand Tgfb1 mRNA transcript was noted. The abundance of mRNA transcripts encoding both the receptor-activated SMADs, Smad2, and Smad3, as well as the co-SMAD, Smad4, which together form a signaling complex for translocation into the cell nucleus, was increased in CDH , another receptor in the BMP/TGFβ pathway which mediates the signal of Bmp9 and Bmp10, was slightly increased in CDH. Bmp4, one of the important ligands in this pathway, and the receptor-activated Smad1 and Smad5 showed an increase in CDH  , and incStudies available in the literature have reported conflicting trends in expression of different signaling factors in CDH, which might be explained by the differences in the gestation of the animals under study. In the present study, some variability between samples was also noted, suggesting that small differences in gestational age may have an appreciable impact on trends in the expression of signaling molecules under study.We initially analyzed the TGF-β and BMP pathways in whole lungs, and given our previous report on vascular abnormalities in CDH , we focuIn conclusion, increased phosphorylation of SMAD2 and decreased phosphorylation of SMAD5 was noted in the in the vessel walls of small pulmonary vessels of nitrofen-CDH pups. These data indicate increased activation of the TGFβ pathway and decreased activation of the BMP pathway in the pulmonary vasculature of these animals at day 21 of gestation, possibly leading to increased proliferation of the muscularized vessel wall. Since the different factors in these pathways are differently expressed during gestation and might differ from the human situation, further research must be conducted at different developmental stages, and most importantly, in material of human patients.The original contributions presented in the study are included in the article/The animal study was reviewed and approved by independent animal ethical committee (EMC).DT, RM, and RR: conceptualization and writing—original draft. DM, MB-vK, RM, and RR: data curation. DM, MB-vK, and RR: formal analysis and methodology. RM and RR: funding acquisition and supervision. DM and MB-vK: investigation and validation. DT and RW: resources. DM, MB-vK, RW, DT, RM, and RR: writing—review and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Alzheimer's disease (AD) is an age-related neurodegenerative disease that is slowly becoming a global problem. Salvia miltiorrhiza (SM) has a history of thousands of years of use in China. In recent years, SM has been reported to have the effect of improving Alzheimer's disease. However, there is no systematic review of its efficacy and safety yet. Therefore, we propose a systematic review to evaluate the efficacy and safety of SM for AD patients.Six databases will be searched: China National Knowledge Infrastructure (CNKI), China Biological Medicine (CBM), China Scientific Journals Database (CSJD), Wanfang database, PubMed, and EMBASE. The information is searched from January 2010 to July 2020. Languages are limited to English and Chinese. The primary outcomes include changes in the Mini-Mental State Examination (MMSE), Alzheimer's Disease Assessment Scale-cognitive subscale (ADAS-Cog) and Activities of Daily Living scale (ADL). Additional outcomes include clinical effective rate and adverse event rate. The Grading of Recommendations Assessment, Development and Evaluation (GRADE) system will be used to assess the strength of the evidence.This systematic review will evaluate the efficacy and safety of SM in the treatment of Alzheimer's disease.This systematic review provides evidence as to whether SM is effective and safe for Alzheimer's disease patients.INPLASY202070066. The pathogenesis of AD is very complicated, and the current mainstream view believes that it involves cholinergic system, cell autophagy, neuroinflammation, β-amyloid precursor protein and oxidative stress.–5Alzheimer's disease (AD) is an age-related neurodegenerative disease characterized by progressive cognitive and memory disorders. It is estimated that there are nearly 47 million AD cases globally, with substantial new cases each year.–8 Many clinical trials have found that SM has a significant improvement effect on Alzheimer's disease.–11Salvia miltiorrhiza (SM) is a medicinal plant with a history of more than 2000 years of use in China. Traditional Chinese medicine (TCM) uses SM to treat palpitation, stroke, perimenopausal syndrome, anemia, and other diseases.Unfortunately, there is currently no systematic review of the safety and effectiveness of SM in the treatment of AD. Therefore, we propose a protocol for a systematic review to evaluate the effectiveness and safety of SM in the treatment of AD patients, so as to provide a rigorous evaluation of the existing evidence.22.1https://inplasy.com/) with number INPLASY202070066. Ethical approval is unnecessary because this study only involves the data of published previous studies.This protocol of systematic and meta-analysis review has been registered on Inplasy  can be included. Observation studies, animal research, case report, review, and meta-analysis are excluded.2.2.2Patients diagnosed with AD (using any recognized diagnostic criteria), such as Diagnostic and Statistical Manual of Mental Disorders (DSM-IV), or Recommendations from the National Institute on Aging-Alzheimer's Association work groups on diagnostic guidelines for AD (NIA-AA), or Chinese Guidelines for the Diagnosis and Treatment of Alzheimer's Disease or Other Dementia. There is no restriction on age, gender, nationality of the patient, and the duration and severity of the disease.Include:1.vascular dementia, frontotemporal, or any other forms of dementia,2.other disorders such as Parkinson's disease, traumatic brain injury, stroke, and cancer that may impact cognitive function will be excluded.2.2.3Analyzed interventions included Salvia miltiorrhiza used as monotherapy, Chinese herbal compound prescription and related alternative therapies.2.2.4In the same study, AD patients received other treatments.2.2.5The primary outcomes include changes in the Mini-Mental State Examination (MMSE), Alzheimer's Disease Assessment Scale-cognitive subscale (ADAS-Cog), and Activities of Daily Living (ADL) scale. Additional outcomes are clinical effective rate and adverse event rate.2.3We search the following databases from January 2010 to July 1, 2020: China National Knowledge Infrastructure, China Biological Medicine, China Scientific Journals Database, Wanfang database, PubMed, and EMBASE. We will search English and Chinese articles for review, and collect additional references from review references and original research articles.2.4Two review authors will search the literature independently with cross-check. Any inconsistency will be solved by a third reviewer. Manual search will be performed if relevant literatures are found in the included studies. The electronic search will be conducted using a combination of following keywords: AD, Alzheimer's disease, dementia, senile dementia, cognitive impairment, neurocognitive disorder, cognitive, decline, salvia miltiorrhiza, salvia, dan shen, randomized controlled trial, controlled clinical trial, trial, RCT, randomized, randomly. The search strategy for PubMed is presented in Table 2.52.5.1Two reviewers will perform literature screening, study selection, and data extraction independently. The literature obtained will be imported into EndnoteX9 to screen the title and abstract, the duplication and studies failing to meet the pre-specified inclusion criteria will be excluded. After reading the full text of the remained literature and discussing within the group, the final included studies will be determined. The corresponding author of original RCT will be contacted when the full text is unavailable. Disagreements will be solved by consulting a third-party arbitrator or discussing within a group. The flowchart of studies searching process is shown in Figure 2.5.2For each RCT, we extracted the following information:1.general information, including name of the first author and year of publication;2.participant characteristics, including sample size, AD severity, gender composition, mean age, and diagnostic criteria;3.intervention details, including the experimental medicine and control group care, doses of medications, duration of treatment; and4.outcome measures and intergroup differences.Extract data from the primary and secondary results for further evaluation. The inconsistency between the two reviewers will be resolved by the third reviewer.2.5.3The quality of the studies will be assessed by using the Cochrane Handbook 5.1.0 (Cochrane Handbook 5.1.0). The assessment will include random sequence generation, randomization correctness, allocation scheme hiding, blinding of patients and implementers, accuracy of data results, and other risk of bias. The risk of low bias is expressed as “low risk” and the risk of high bias is expressed as “high risk.” The information provided in the studies is inaccurate or does not provide sufficient information for the bias assessment to be expressed as “unclear risk.” The above content evaluation was independently evaluated by 2 researchers, and any differences will be resolved through discussions with the third reviewer.2.5.4Two reviewers will analyze the data independently using RevMan 5.3.5 Risk ratio with 95% confidence interval will be adopted for the dichotomous data, whereas the mean difference or standardized mean difference with 95% CI will be utilized for the continuous data.2.5.5A funnel plot will be performed to assess any publication bias when more than 10 RCTs are included. In additional, Egger regression and Begg correlation test will also be performed to identify the funnel plot asymmetry.2.5.6I2 and Q tests will be applied to evaluate the heterogeneity with the cut-off value of I2 = 50. If I2 > 50% and/or Q test < 0.10, the heterogeneity will be deemed significant.The Cochrane 2.5.7I2 < 50, and a random-effect model will be employed if I2 ≥ 50. Subgroup analysis or meta-regression will be performed to assess the potential sources and present reasonable explanations for the heterogeneity.In line with the Cochrane guideline, a fixed-effect model will be utilized to pool and analyze the outcome data if 2.5.8Sensitivity analysis will be applied to evaluate the stability of the pooled results of included RCTs according to the methodological quality, sample size, and missing data.2.5.9The Grading of Recommendations Assessment, Development and Evaluation (GRADE) guidelines will be utilized to grade the quality of evidence as very low, low, moderate, or high.3 According to the Alzheimer's disease report, there are more than 40 million AD patients in the world, and as the problem of aging increases, the number of AD patients will continue to increase. AD as the main cause of dementia accounts for the total 70% of patients. However, effective cure method for AD and dementia is still absent, and mainstream research currently focuses on reducing progressive clinical symptoms rather than its cure. TCM is a unique treatment, which has been in use since 2000 years ago in China. It differs from modern medicine and has its own distinctive advantages.,17 The use of natural herb is a characteristic of TCM. Salvia miltiorrhiza first recorded in the Shennong's Classic of Materia Medica, is widely distributed in East Asia. TCM uses SM to treat palpitations, stroke, perimenopausal syndrome, anemia, and other diseases. Previous research found that Salvia miltiorrhiza has anti-inflammatory, anti-oxidation, and anti-fibrosis effects.–20 A growing body of studies have shown that Salvia miltiorrhiza can alleviate cognitive dysfunction of AD patients, indicating that it may be a useful therapeutic agent for AD.–23 The way SM improves AD is to reduce the accumulation of β-amyloid protein, reduce the oxidative stress response, and improve the energy system of the cholinergic system.AD is an age-related neurodegenerative disease characterized by progressive cognitive and memory disorders, senile plaques, neurofibrillary tangles, and neuronal cell death.However, currently no systematic review and meta-analysis have been conducted regarding the efficacy and safety of SM for the treatment of patient with AD. The findings of this study may yield helpful evidence for the clinicians and investigators concerned in decision-making process about the efficacy and safety of SM for patients with AD.The authors would like to thank SanYin Zhang for critically reviewing the manuscript.Data collection: Ying Guo, Xing Dong.Statistical analysis: Renyan Zhang, Yanmei Zhong.Supervision: SanYin Zhang, Peng Yang.Writing – original draft: Ying Guo.Writing – review & editing: SanYin Zhang.When originally published, Dr. Sanyin Zhang's name appeared incorrectly as San Ying Zhang. This has since been corrected.


Background: We aimed to investigate the difference in the total length of hospital stay (LOS) after intraperitoneal vs. intravenous antibiotic treatment in patients with complicated appendicitis.Methods: We conducted a quasi-randomized prospective clinical trial. The intervention group received 4 g fosfomycin, 1 g metronidazole, and 50 μg recombinant human granulocyte-macrophage colony-stimulating factor intraperitoneally, which was left in the abdominal cavity, immediately after laparoscopic appendectomy. Postoperatively, this group received antibiotics orally. The control group received intravenous antibiotics both during surgery and postoperatively. We primarily evaluated total LOS within 30 days. Furthermore, we evaluated harms and adverse events, Gastrointestinal Quality of Life Index, postoperative complications, and convalescence. Participants were followed for 30 days postoperatively.Results: A total of 12 participants concluded the trial. The total LOS was significantly shorter in the intervention group  than in the control group , p = 0.017. Comparable harms and Gastrointestinal Quality of Life Index scores were found in the two groups. The time to return to normal activities was median 6 and 10 days for the intervention and the control group, respectively. There were no serious adverse events related to the trial nor any complications in the intervention group. In the control group, two patients developed intraabdominal abscesses.Conclusions: The intervention group had a significantly shorter total LOS. The study was not powered to assess differences in complications, but the results indicate that the intervention seems to be a safe regimen, which can be investigated further to treat patients with complicated appendicitis.Identifiers: EudraCT no. 2017-004753-16.ClinicalTrials:https://clinicaltrials.gov/ct2/show/NCT03435900?term=NCT03435900&draw=2&rank=1">draw=2&rank=1. Enterobacterales, Enterococcus faecalis, and Bacteroides species  and monitored by the Good Clinical Practice Unit, Copenhagen University Hospital. It is reported according to the CONSORT statement . The trial protocol was approved prior to initiation by the Danish Medicines Health Authority 2017113663), the local Ethics Committee (H-17037698), and the Danish Data Protection Agency HGH-2017-124). The trial was registered at tatement  and its 3663, the4. The trThe inclusion criteria were: age≥18 years, laparoscopic appendectomy for perforated appendix, and written informed consent. If the participant was a fertile woman a negative urine pregnancy test was required. A perforated appendix was defined as an appendectomy during which the operating surgeon or the supervisor determined the need for postoperative intravenous antibiotic treatment. This usually included a visible appendix perforation, free intra-abdominal pus, visible feces, and/or an abscess.Exclusion criteria were: inability to understand, read or speak Danish; previous allergic reaction to fosfomycin, metronidazole, rhGM-CSF, or penicillins; diagnostic laparoscopy revealing a normal appendix not requiring an appendectomy or appendicitis without a perforated appendix; other intra-abdominal pathology requiring surgical intervention (diagnosed either during surgery or at a preoperative CT-scan); renal, hepatic, hematological disease; American Society of Anesthesiologists (ASA) physical status >3 ; body weEscherichia coli , which is purified prior to clinical use, corresponding to a volume of 0.2 ml. All the drugs were combined prior to instillation but administered immediately after mixing to ensure full antimicrobial effect  diluted in 300 ml of sterile water for injections , 1 g metronidazole  corresponding to a volume of 200 ml and 50 μg molgramostim, which is rhGM-CSF expressed in l effect . PostopeThe control group received standard intravenous antibiotic agents during surgery (either 1.5 g cefuroxime or 4 g piperacillin/500 mg tazobactam and 1 g metronidazole) and a minimum of 500 ml saline was used for irrigation of the abdominal cavity. Postoperatively, participants received 3 days of intravenously administered antibiotic agents: 4 g piperacillin/500 mg tazobactam and 500 mg metronidazole. These doses were administered three times daily for a minimum of 3 days.All study participants could receive preoperative intravenously administered antibiotic agents; however, for the intervention group, intravenous administration immediately before initiation of surgery was avoided.All participants could be discharged when the Postanesthesia Recovery Score for Ambulatory Patients (PARSAP score)  was ≥18.The primary outcome was total LOS in the two groups, defined as the number of hours in hospital after the end of the surgery and until 30-day follow-up including any readmissions related to the laparoscopic appendectomy or appendicitis. The secondary outcomes included the following:The questionnaire Gastrointestinal Quality of Life Index (GIQLI)  was fillPostoperative complications were graded according to the Clavien-Dindo classification . FurtherParticipants were asked both when they could return to normal activities and how long the period of sick leave (absence from work) had lasted. Data were collected up to 30 days (±3 days) postoperatively.These were registered by trial personnel during admission, 10 days (±2 days) postoperatively when the patients had their sutures removed by the trial personnel and 30 days (±3 days) postoperatively through review of the patient's medical records and at a planned telephone interview. Adverse events were defined as any untoward medical occurrence including unfavorable and unintended signs , symptoms, and disease as defined by ICH-GCP . SeriousWe performed a power calculation for the trial. The cumulative 30-day total LOS was expected to be 72 h with a standard deviation of 12 h in the control group. When alpha was set at 0.05, beta was set at 0.80, and a minimal relevant difference between the groups was set at 48 h, a randomized design required four patients in each group, a total of eight patients. However, as we expected data not to be normally distributed and planned to use a quasi-randomized design, we decided to include six patients in each group, a total of 12 patients, to ensure sufficient power. Participants were included until data on the primary outcome at day 30 were secured for six patients in each group. The intervention group was recruited at one hospital  and the control group at another hospital . The two hospitals are located 9 km apart. Thus, the allocation was not concealed, and participants, care providers, and outcome assessment were not blinded to the intervention.Supplements. In short, continuous numerical values were reported as median and range if not normally distributed. Not normally distributed continuous data were analyzed with non-parametric statistics: Mann-Whitney U-test. Binary, categorical data were reported as numbers and proportions in %. We analyzed these with Chi-square-test, and if any of the expected cell counts were <5, then p-values from the Fisher's exact test is reported. A p-value ≤ 0.05 was considered statistically significant.Data were analyzed using SAS Enterprise Guide 7.1 . Details on the statistical methods can be seen in the statistical analysis plan in Eligible patients were recruited at the Department of Surgery, Herlev Hospital (intervention group) and the Digestive Disease Centre, Bispebjerg Hospital (control group) from 14th February 2018 to 17th June 2018. A total of 13 participants were included, as one patient withdrew consent 3 days after surgery. Therefore, data on the primary outcome could not be retrieved and another patient was included according to the predefined protocol. The screening process is depicted in p-value = 0.017 (Mann-Whitney U-test).The total LOS was significantly lower in the intervention group  than in the control group , The total GIQLI score seemed to increase from 10th to 30th postoperative day in both groups . The scoAn overview of the reported harms is presented in Of the 12 evaluable participants, there were no complications in the intervention group and two complications in the control group within 30 days after surgery. These complications consisted of two intraabdominal abscesses in two participants (33%). The Clavien-Dindo grade of both complications was 3a, which are complications requiring radiological intervention that are not under general anesthesia. The complications meant that one participant had a prolonged hospital stay and one participant was readmitted.In the intervention group, one participant had not returned to normal activity at day 30. Median time to return to normal activities was 6 days [range 1 to >30 days]. In the control group, the median time to return to normal activities was 10 days [range 4–28 days].One participant in the intervention group was still on sick leave at 30 days postoperatively. The period of sick leave for the intervention group was median 13 days [range 1 to >30 days]. The control group had a median sick leave of 12 days [range 4–28 days].There were no unexpected adverse events in either the intervention or control group. There were six adverse events (0–2 per participant) in the intervention group. These included nausea (17%), diarrhea (67%), and pain in the right lower quadrant (17%). Six adverse events (0–3 per participant) were found in the control group. These included diarrhea (50%), nausea and vomiting (17%), vomiting (17%), and haematoma (17%).n = 1) and readmission within 30 days due to an intraabdominal abscess requiring drainage (n = 1). The unrelated SAE was readmission within 30 days due to diverticulitis.Four SAE were reported: two unrelated and two potentially related to the trial. One unrelated SAE occurred in the intervention group. The admission for appendicitis was prolonged due to influenza, and this participant withdrew consent. There were two potentially related and one unrelated SAE in the control group. The potentially related SAEs included prolonged admission due to an intraabdominal abscess that required both drainage and prolonged antibiotic treatment  , intravein vitro investigations .SF, BH, MA, and JR: substantial contributions to the conception or design of the work. SR, CO, JO, LS, RA, SG, TR, and LJ: acquisition. SF, BH, MA, LJ, and JR: analysis or interpretation of data for the work. SF: drafting the work. SR, BH, CO, JO, LS, RA, SG, TR, MA, LJ, and JR: revising it critically for important intellectual content. SF, SR, BH, CO, JO, LS, RA, SG, TR, MA, LJ, and JR: provide approval for publication of the content, agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy, or integrity of any part of the work are appropriately investigated and resolved.This study was funded by a grant from Reponex Pharmaceuticals ApS to the Department of Surgery, Herlev Hospital. The company did not have any influence on the study design, study conduct, or writing of the manuscript. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.