The treatment of uveal melanoma and its metastases has not evolved sufficiently over the last decades in comparison to other tumour entities, posing a great challenge in the field of ocular oncology. Despite improvements in the conventional treatment regime and new discoveries about the genetic and molecular background of the primary tumour, effective treatment strategies to either prevent tumours or treat patients with advanced or metastatic disease are still lacking. New therapeutic options are necessary in order to achieve satisfactory local tumour control, reduce the risk of metastasis development, and preserve the eyeball and possibly the visual function of the eye. The development of in vivo model systems remains crucial for the identification and investigation of potential novel treatment modalities. The aim of this study was the optimisation of the chorioallantoic membrane (CAM) model for uveal melanoma research. We analysed the established CAM assay and its modification after the implantation of three-dimensional spheroids. The chorioallantoic membrane of a chick embryo was used to implant uveal melanoma-cell-line-derived spheroids in order to study their growth rate, angiogenic potential, and metastatic capability. Using the UM 92.1, UPMD2, UPMM3, and Mel270 cell lines, we were able to improve the viability of the embryos from 20% to >80% and to achieve up to a fourfold volume increase of the transplanted spheroid masses. The results point to the value of an optimised chicken embryo assay as an in vivo model for testing novel therapies for uveal melanoma by simplifying the research conditions and by contributing to a considerable reduction in animal experiments. Uveal melanoma (UM) belong to the group of most common primary intraocular malignant tumours in adults. Six to seven new cases per 1 million residents in Europe are diagnosed each year, and it, therefore, remains a rare tumour disease ,3,4. TheThe molecular pathogenesis of UM is characterised by specific chromosome alterations and gene mutations. The most common abnormalities include loss of the 1p, 3, 6q, and 8p chromosomes as well as a gain of the 1q, 6p, and 8q chromosomes. Inactivating mutations in BAP1, a tumour suppressor gene located on chromosome 3p, are found in approximately 47% of primary UM and 84% of metastatic UM cases, consistent with the association between BAP1 mutations and poor prognosis . MoreoveTreatment options for UM include radiotherapy (brachytherapy or teletherapy) and tumour surgery  ,15. OculThe chorioallantoic membrane (CAM) model is considered to be a reliable in vivo experimental model for studying tumour biology and can be advantageous due to its immunodeficiency properties, allowing transplantation of various tissues without triggering an immune response or rejection of the implant. The CAM constitutes a biologically faithful model and is an increasingly valuable tool in oncological research, enabling the transplantation of various types of cancer cell lines or even human tumour tissue and providing information regarding tumour formation, angiogenesis, and metastasis. The presence of T cells can first be detected on day 11, B cells are present on day 12, and by day 18, the chicken embryos become immunocompetent . MoreoveTo our knowledge, the optimisation of the chicken CAM as a reliable in vivo model for the analysis of choroidal melanoma has not yet been described in the literature. In this study, we analysed the established CAM assay using UM 92.1, UPMD2, UPMM3, and Mel270 cell lines as references and optimised the chick embryo viability in order to evaluate the tumour spheroid attachment, the implantation/growth rate, the angiogenic potential, and the changes in the tumour spheroid’s volume and metastatic capability.Various parameters were tested to improve the viability of the chicken embryo and the growth of the implanted spheroid tumours .3 or 20 × 103 living cells in round-bottomed 96-well ultra-low attachment plates  in 100 µL of the cell culture medium.UM cell lines UM92.1, UPMD2, and UPMM3 were provided by M. Zeschnigk , and Mel 270 was provided by K.G. Griewank . The UM cell lines were maintained in an RPMI 1640 medium supplemented with 10% foetal calf serum and 1% penicillin-streptomycin (5000 U/mL). The spheroids were generated by seeding 10 × 102) for the indicated period of time. Compact spheroids could be generated on day 4 of the culture, and on day 9, the spheroids were transplanted on the CAM.The medium was refreshed two times per week. The cell and spheroid cultures were incubated in a humidified incubator . In order to avoid contamination and to evaluate the effect on embryo viability, the eggs were cleaned with either distilled water or 70% ethanol. After approximately 12 h of preservation at a temperature of 21 °C, the eggs were placed in an incubator . The eggs were incubated in a horizontal position at a temperature of 37.8 °C and a humidity of 70% with permanent agitation (at least 3 times/day). On day four of embryonic development (ED 4), approximately 4–5 mL of albumin were removed from the apical side of the egg by positioning it vertically and using a 20-gauge needle without detaching the embryo or the yolk. This step was performed using diaphanoscopy for an easier localisation of the embryo and the yolk sac. The puncture hole was covered with Leukosilk tape. At ED 5, Leukosilk tape was applied on the upper side of the egg before an oval window of 1 × 1 cm was cut out of the eggshell to access the CAM. The window was resealed with adhesive tape.To implant tumour spheroids, the window was reopened, and a sterile plastic ring (2 mm inner diameter) was placed into the CAM. The small plastic ring was made of various standard lab plastic pipettes . The CAM area within the ring was gently lacerated using different techniques for the removal of the epithelium layer, including the use of a 30-gauge cannula, Nr. 11 scalpels , and a 25-gauge vitrectomy membrane scraper before the spheroids were inoculated. Before transplantation, the tumour spheroids were covered in different matrices and transplanted into the CAM at ED 5, 7, 9, or 11. To improve the spheroid attachment into the CAM, we compared spheroids covered in Matrigel as well as various other viscoelastic substances. Tumour spheroids covered with either Matrigel or the viscoelastic substance were placed in the middle of the ring between two large chicken vessels B. The wiThe Matrigel  was thawed at 4 °C, and 80 µL of the respective cell culture medium was removed; 20 µL of it was mixed with 20 µL of matrix material and transplanted into the CAM after Matrigel polymerisation. The polymerisation of the matrix material occurred within 30 min at 37 °C.Apart from Matrigel, various other viscoelastic substances were tested. We applied Hyalon , the most commonly used viscoelastic  with a molecular weight of 4 million daltons and a viscosity of 229,000 cP), Hyalon GV (Johnson & Johnson Vision), the modification of the Hyalon , Provisc , and Viscoat .Excised tumour spheroids were fixed in buffered formalin (Histofix). Formalin-fixed paraffin-embedded sections were cut at a thickness of 3 µm. The Melan A  and SOX10  immunohistochemistry analysis was performed using the Ventana BenchMark ULTRA system . The immunohistochemistry (IHC) analysis for Melan A was performed using the following protocol: pretreatment CC1 was performed for 36 min at 95 °C, and incubation occurred for 24 min at 27 °C. For SOX10, CC2 pretreatment was performed for 40 min at 90 °C, and incubation occurred for 32 min at 36 °C. Visualisation was conducted using the OptiView DAB System . Thereafter, slides were scanned using a Leica DM4000B microscope with a Leica DFC290 camera and analysed using the Leica Suite version 2.8.1 . The slides were reviewed by a board-certified pathologist.The vessels of the CAM were detected in ovo after the intravascular injection of 50 µL Fluorescein (Fluorescite 10% Firma Alcon) and were imaged with an Olympus BX51 microscope at 20× magnification.2).The excised tumour spheroids were imaged at ED 5, ED 11, and ED 17 using a Zeiss Primovert bright-field microscope at 40× magnification. Images were recorded using a Zeiss Axiocam 105 and ZENcore software and analysed using ImageJ Fiji  image processing software. The sizes of the tumour spheroids and nodule explants were determined by calculating the cross-sectional area of the spheroids or explants . A value of We compared the effect of the cleansing method using either distilled water or a 70% ethanol disinfectant on the embryo viability. Ethanol significantly reduced the viability within the first five embryonic development days (ED 5) to approximately 65% (ED 13–17) compared to 85% in the sterile-water treated group A.The rich vascular system of the CAM develops within the intermediate mesodermal layer that is located between the outer chorionic epithelium and the inner allantoic epithelium. To facilitate tumour spheroid engraftment and neovascularisation, the outer epithelium must be partially removed using gentle laceration. We compared different techniques for the removal of the epithelium layer including using a 30-gauge cannula, an Nr. 11 scalpel, and a 25-gauge vitrectomy membrane scraper. The resulting vascularisation of the tumour mass was higher  when a 25-gauge membrane scraper or a scalpel was used to scratch the CAM surface. Furthermore, the removal using a 25-gauge membrane scraper enabled gentler scratching, and serious injuries of the blood vessels, as well as excessive haemorrhages, could be avoided.The critical conditions for successful positioning and maintenance of the tumour spheroid within a defined area of the CAM were the use of a plastic ring and an extracellular matrix . The mai3 cells for all four tested cell lines. The most noticeable growth development during the experiment could be documented for the UM 92.1 spheroids and is presented in We investigated the influence of the incubation time on the tumour attachment rate and viability of the spheroids. A transplantation with a ring plus Matrigel at ED 5 reduced the tumour attachment rate to 60%. Similarly, the viability of the chicken embryos and the volumes of the developing tumours increased when transplantation took place at a later time. Transplantation on ED 11 and resection on ED 17 were the optimal conditions. The best results concerning tumour growth were observed after the implantation of tumour spheroids formed by 20 × 10Histological analysis of the grafted tumour spheroids at ED 17 confirmed that they were completely integrated into the CAM and had formed a nodule-like structure . The tumThe tumour spheroid nodules showed an optimal and rapid vascularisation after transplantation on ED 11. The intravascular injection of Fluorescein revealed tumour spheroid vascularisation at day ED 17 . Haemato³ cells as the optimal cell number increased the overall tumour attachment rate to 80%.The application of Matrigel as a matrix, the use of a plastic ring for the positioning of the tumour spheroids between large vessels on the CAM, the laceration of the CAM using a 25-gauge membrane scraper, and the use of 20 × 10The significant similarity of the CAM to the human embryo at cellular and anatomical levels, including its rapid development, its comparatively large structures during the early developmental stages, and the accessibility of the CAM for experimental manipulation, constitute its establishment as a unique and crucial research model. There are no available literature sources for the optimisation of pre-established CAM assays for the analysis of UM tumours. Different groups report a high mortality rate of the embryos and varying success rates concerning the implantation of the tumour cells . To optiA standardised protocol for the pretreatments of the CAM played an important role. We observed that gently scratching the CAM surface using a 25-gauge membrane scraper, putting a plastic ring on the CAM, and applying Matrigel and a cell medium to the CAM delivered the best results. Although viscoelastic substances have an advantage over other mediums due to their rheological properties, including viscosity, viscoelasticity, pseudo-plasticity, cohesiveness, and coatability, the achieved tumour mass volumes and the attachment rates were higher using Matrigel . These rThe external application of ethanol or other disinfectants may have a significant impact on the viability of the embryos due to the permeability of the eggshell and should be avoided. The use of distilled water in our study significantly reduced the mortality rate without any increase in contaminations. A further improvement of the survival rate of the embryos could be achieved by comparing various surgical instruments for the fenestration, thereby minimising the risk of damage during the opening of the eggs. In our study, we found that further damage could be avoided during the removal of albumen by using diaphanoscopy. Moreover, the optimisation of the tumour cell count for the spheroids to form CAM tumours may crucially affect the viability and growth potency, as shown in our results. A CAM protocol for osteosarcoma cell lines reports an improvement of the tumour take rate from 51% to 94% and an improvement in the rate of the viability of the embryos from 40% to >80% after optimisation. The results are similar to the data presented in our study, underlining the importance of every step and each procedural detail during the preparation and growth of the embryos .The optimisation of the CAM assay for UM tumours and a higher rate of viability and growth of tumour masses may enable the implantation not only of tumour spheroids such as those used in our study but also of human tumour tissue for research purposes. The differences between the various cell lines tested in our study pose a challenge depending on the origin of the implanted tumour spheroids. The UM92.1 cell line showed a more robust growth development in the CAM that can likely be accredited to its emergence from a massive tumour mass that had destroyed the eye and orbit and led to extraorbital metastases . In addiThe ability of the UM cell lines UM92.1 and Mel270 to form solid tumour spheroids and induce metastasis has already been discussed ,29,30. TThe chick CAM assay is an important model in cancer research. The main advantages of this assay pertain to economy and reliability. The use of this model supports a considerable replacement of animal experiments. A disadvantage is the high mortality rate of the embryos and the rejection of tumours after transplantation into the CAM. Moreover, not all tumour cells can produce macroscopically visible colonies over the short time of the experiment. However, we were able to improve the viability rate of the embryos by up to 80% by using a standardised protocol. We established strategies to raise the stability of the spheroids in the CAM, improved the vascularisation of the spheroid, and achieved a threefold increase of the transplanted spheroid volume. The optimisation of the chicken CAM in UM research may expedite preclinical tests and facilitate the development of personalised methods in oncology.


Although the field of liquid biopsy is clearly having an effect on other tumour types, in endometrial cancer (EC) there is important work to do to implement the analysis of circulating biomarkers into the clinical routine. One of the most evident contexts of application is the disease follow-up in both localized and advanced diseases, which at present is primarily made by imaging techniques. In the present review, we conducted an overview of the circulating biomarkers with the potential to be used as monitoring biomarkers in endometrial tumours and highlighted the key challenges for their translation into the patients’ management in order to help researchers to better focus their work in this field.Endometrial cancer (EC) is the most frequent gynecological cancer in developed countries and its incidence shows an increasing trend. Fortunately, the prognosis of the disease is good when the tumour is diagnosed in an early phase, but some patients recur after surgery and develop distant metastasis. The therapy options for EC for advanced disease are more limited than for other tumours. Therefore, the application of non-invasive strategies to anticipate the recurrence of localized tumours and guide the treatment in advanced stages represents a clear requirement to improve the survival and quality of life of patients with EC. To achieve this desired precision oncology, it is necessary to invest in the identification and validation of circulating markers that allow a more effective stratification and monitoring of patients. We here review the main advances made for the evaluation of circulating tumour DNA (ctDNA), circulating tumour cells (CTCs), circulating extracellular vesicles (cEVs), and other non-invasive biomarkers as a monitoring tool in the context of localized and advanced endometrial tumours, with the aim of providing a global perspective of the achievements and the key areas in which the use of these markers can be developed into a real clinical tool. Endometrial cancer (EC) is one of the most common malignancies in the developed world, and its incidence has been increasing during over last decade . Up to 1POLE ultramutated, includes tumours with inactivating mutations in POLE exonuclease. MSI hypermutated corresponds to the hypermutated/microsatellite unstable (MSI) group, while the copy-number low (CN low) corresponds to those tumours with low copy-number aberrations, and copy-number high (CN-high) to those with high copy-number alterations [p53 alterations and a poor outcome [Traditionally this tumour has been classified according to tumour histopathology in two types of endometrial tumours . Type I,erations . The las outcome .In EC, total hysterectomy and bilateral salpingo-oophorectomy are usually per-formed as primary treatment. In a few cases, omentectomy and retroperitoneal lymph-node dissection are also applied . Other tMolecular studies, such as that performed by the TCGA, have revealed the landscape of genomic alterations present in EC, and have provided valuable insight into the pathogenesis of this disease ,21. HoweA key factor to really personalize the management of EC patients is to complement the tissue-based analyses, which require invasive surgical procedures associated with mortality and patient anxiety, with the analysis of circulating biomarkers. Thus, the present review is directed to provide an overview of different circulating biomarkers described as potential tools to monitor EC patients at the different stages of the disease, from diagnosis to the therapy follow-up of advanced tumours.Current strategies performed to analyse and monitor EC rely on traditional biopsy, an invasive method, which is an essential part of the diagnostic and prognostic workup. Although traditional biopsy has been considered as the gold-standard approach for several years , despiteThe term “liquid biopsy” was coined by Pantel & Panabiéres in 2010 for the analysis of circulating tumour cells (CTCs) in blood from cancer patients . Today, Currently, liquid biopsy-based tests directed to characterize cell free DNA (cfDNA) are approved as companion diagnostics to select targeted therapies in a reduced number of tumours, such as lung or breast cancer ,30. ThisDespite the advances reached in these tumour types for the clinical application of circulating biomarkers to improve the patient management, in gynecological tumours, such as ovarian and endometrial cancer, there is still important work to do in order to implement the use of fluid samples into the clinics . In thisThe majority of endometrial tumours are diagnosed early, and patients usually have a five-year survival rate of 90%. Unfortunately, up to 20% of the lesions progress to an advanced stage carcinoma, whose 5-year survival rate drops to 15% . TherefoAs of today, there are two main serum circulating biomarkers proposed to follow-up the evolution of EC after surgery, which correspond to the human epididymis protein 4 (HE4) and CA125 . Serum ln = 9), Feng et al. associated the presence of ctDNA at surgery with late FIGO stages and the presence of lymph node affectation. They monitored ctDNA levels after surgery and found ctDNA in 44% (four of nine) of the patients, showing positive disease relapse in three of these cases [The clearest alternative to serum proteins among other circulating biomarkers is the cfDNA. Although most of the research directed to the study of cfDNA in non-metastatic EC focuses on the search for diagnostic biomarkers, there are a growing number of studies directed to validate cfDNA assessment as a prognostic and monitoring tool. Cicchillitti et al. analyzed cfDNA levels in EC patients and benign controls by qRT-PCR and observed a high correlation between high cfDNA levels and high-risk stages . Recent se cases . NotablyAlthough endometrial tumours are not considered as important CTC shedders, the presence of this tumour population has been evaluated by applying different technologies in patients with EC. Ni et al. analyzed the Epithelial Cell Adhesion Molecule (EpCAM) positive CTCs levels in patients with high or intermediate risk endometrial tumours using the CellSearch technology at time of surgery. They found an association between the presence of CTCs and cervical involvement, while no correlation was found between CTCs and serum CA125/HE4. CTC levels did not show an increment after the first cycle of standard chemotherapy, although the monitoring cohort was not enough to generate any robust conclusion about the value of CTCs as a follow-up tool . Our groOn the other side, the analysis of circulating EVs represents a promising alternative for improving the detection of protein and genetic tumour biomarkers in gynecologic cancers . HoweverFinally, circulating miRNAs such as miR-100, miR-151a-5p, miRNA205, miR484, miR23a or miR-135b have also been proposed as potential tools for EC diagnosis and monitoring ,71,72,73Advanced or recurrent EC is not amenable to curative therapies, with the prognosis of patients being quite poor due to the limited treatment options when they progress to chemotherapy. Although new targeted therapies are being assayed in different clinical trials, their current clinical application is in the minority mainly because of the lack of predictive biomarkers that can be interrogated within the disease evolution .PTEN mutations (mTOR inhibitors or PARP inhibitors), HRD (PARP inhibitors), ARID1 mutations  and FGFR2 mutations (FGFR inhibitors) [Endocrine therapy is considered for the treatment of recurrent/metastatic low-grade EEC with expression of estrogen (ER) and progesterone (PR) receptors, and low disease burden. However, tumours without receptor expression may also respond to this therapeutic strategy . Among tibitors) . Taking PIK3CA, BRAF and CTNNB1 genes in the uterine aspirates may serve as an indicator of the tumour burden [PIK3CA, we generated a PDX model which resembled the original tumour characteristics and successfully tested the activity of BYL719 (PI3KCA inhibitor) in these preclinical models, showing the clinical potential of ctDNA monitoring to find therapeutic alternatives for advanced disease [PIK3CA and KRAS were also analyzed by ddPCR on cfDNA from EC in another recent work, where two cases of advanced EC were monitored based on ctDNA content during chemotherapy. In both patients, ctDNA levels served as an accurate tool to follow-up the therapy response and the tumour burden, improving the information obtained with CA125 measurements [PIK3CA was also analyzed in cfDNA from patients with advanced EC in a clinical trial which interrogated the activity response of the PI3K inhibitor Pilaralisib, finding a good concordance with the results obtained in the primary tumour [HE4 levels have been proposed as a useful tool for surveillance of cancer recurrence, but their value in monitoring the tumour burden in advanced disease is unknown . Apart fr burden . In the  disease . Frequenurements . The stay tumour . Moss ety tumour  also lonOther circulating biomarkers, such as CTCs or cEVs, are even less explored in advanced stages of EC. CTCs longitudinal assessment was reported during the disease evolution in a cohort of 30 patients with advanced EC using an EpCAM dependent strategy. They detected CTCs in 13 patients during treatment, mainly based on chemotherapy, and showed a CTC dynamic in accordance with their clinical and radiological evolution. This study demonstrated the potential of the CTC population as a follow-up tool, although the rate of CTCs positivity was moderate .TBX2, CHST11, NID2, ZNF154, PCDH, TNXB and DPP6 in tissue samples from patients with EC has been associated with a poor prognosis in low-risk and high-risk EC patients [HOXA11 methylation status has shown value to predict recurrence in stage I and II EC [The application of minimally invasive follow-up strategies in the context of EC constitutes a key element for advancing in the application of personalized medicine in this tumour. As we summarized in this review, there is scientific evidence about the potential of different circulating biomarkers for improving the surveillance and disease monitoring in the context of non-advanced and advanced tumours . Most ofpatients ,86. Besind II EC . TherefoIn the same line, the study of cEVs is emerging as a valuable source of protein, and genetic biomarkers with increasing data supporting their potential for the diagnosis of gynecologic tumours including EC, but with important work to do for clarifying their potential to anticipate tumour recurrence, guide therapy selection or identify the appearance of resistance to treatment prior to image evaluation. The identification of tumour-specific EVs biomarkers and the development of new isolation technologies suitable for their clinical implementation, such as EXoGAG, successfully applied to characterize ANXAII in plasma from EC patients , are impOn the other hand, the application of CTCs monitoring and characterization with clinical intention in patients with EC has as a principal limitation the low rate of CTCs that normally characterize this tumour. This fact does not impact on the biological significance of the CTCs dynamics but implies that the majority of non-advanced tumours will present no CTCs during the clinical course of the disease. Therefore, the future of CTCs in EC will probably continue to be associated more with the translational research that tries to understand the mechanisms behind tumour dissemination.In the context of localized disease in young women, fertility-sparing treatment is of increasing interest in young women. Thus, Casadio et al. showed the convenience of conservative treatment to preserve the fertility opportunities of young patients . The selIn the era of immunotherapy, which is also changing the therapeutic options for treatment of re-current/advanced endometrial tumours, oncologists need to complement the current selection biomarker, MSI or MMR deficiency analyzed in tissue samples, with other accurate biomarkers that can improve the patients’ stratification for receiving this therapy and the response assessment. For this purpose, cfDNA- and cEVs-based studies will be of great relevance in the near future due to the important information about the tumour and its microenvironment that can be obtained through their analysis. In this sense, the characterization of circulating immune cells would be also relevant for the evaluation checkpoints-inhibition response.In addition, the development of robust preclinical models able to recapitulate the tumour molecular characteristics taking into account the heterogeneity of EC is essential to validate the new targeted therapies in a personalized approach. Different pre-clinical models have been developed for drug screening in EC, ranging from the conventional 2D cultures to the patient--derived xenograft (PDXs). However, 2D models are not representative enough of intra-tumour heterogeneity and fail to mimic specific interactions between the tumour and stroma . NotablyWe are enjoying exciting advances in the field of EC management with the arrival of targeted therapies and immunotherapy, but there is a need to apply more accurate and non-invasive markers to better stratify, follow-up and treat endometrial tumours. The present review shows the great potential of different circulating markers for the tumour burden evaluation, MRD detection, recurrence surveillance and treatment monitoring . Among tIn this regard, some general questions that should be answered before the implementation of liquid biopsy to monitor EC and other tumours are the optimal timepoints for fluid sample collection, the requirements of the tests employed or the threshold criteria to select or change the treatment based on biomarkers that are normally present in very low concentrations. The inclusion of a longitudinal collection of fluid samples as part of ongoing and future clinical trials will be critical for answering these important questions.


Nutrients comparing different drink composition on fluid balance [Our lab read with interest the recent article published in  balance , as we h balance . It appeNoticeably, the one statistically significant finding of fluid retention is misleadingly presented. In the text, values are presented as 30%, 10% and −4% for DD, SD and WA, respectively. Yet, in Figure 1b, these numbers are presented as 42%, 25% and 16% for DD, SD and WA, respectively. When one completes the math from the means, it appears the numbers presented in the text are correct. This became obvious when the mean “-“ in.Figure 1b does not appear to represent the mean of the individual values. We also would have appreciated the level of dehydration that occurred during exercise, as this is how their fluid recovery scheme was determined. From the data presented (1.5 mL/kg/15 min during exercise and 150% post-exercise), the participants received ~400 mL during the 75 min cycling bout, became ~1.5% dehydrated, leaving ~1400 mL as a bolus post-exercise. This would certainly present a challenge to retention, which is supported by the resulting urine output of >1.5 L.Although this brings into question the data presentation, we do not doubt the modest, yet significant, effect of DD on rehydration as this confirms previous literature. However, it is also telling that this was the only outcome that reached statistical significance. Not mentioned, but understood, is the powerful effect of the exercise induced antidiuretic hormones on fluid retention. Indeed, it was only during the thermoneutral 5 h of recovery, once these hormones were attenuated, that a small difference in fluid retention was captured. In our previous study, we did not include a long post-exercise recovery as our interest was fluid retention during exercise, which had not been previously compared between SD and DD. Oral rehydration solutions were originally formulated by the WHO to promote rehydration following diarrhea in the absence of intravenous saline . AlthougPractically, it is important to note that only under the conditions of thermoneutral rest for 5 hours, following a large bolus of fluid, in the absence of ingestion of any other nutrition, were small differences in fluid retention found. For workers or athletes in the field, this is not a realistic scenario and, thus, lacks external validity.


In the current investigation, the thermal and thermodynamic behavior of a buoyancy-driven evacuated tube solar collector (ETSC) has undergone precise evaluation, and the efficacy of nanoparticle dispersion in the testing fluid was scrutinized. The natural convection process was analyzed in different vertical sections of the absorber tube. The outputs for water and the utilized nanofluid were compared at various cutting planes along the tube during the simulation time. In this problem, CuO nanoparticles with optimum thermal properties were distributed in the base fluid. According to the surveyed results, the temperature distribution analysis illustrates that the mean wall temperature experiences more enhancement when the nanofluid is used. The comparison of the heat transfer coefficient between the two simulated cases show the competency of utilizing CuO- Solar water and air heaters, solar dryers, desalination systems, etc. are exemplar of solar energy applications in industrial and residential sectors3. Solar water heating systems are sweepingly utilized as one of the most prevalent solar energy technologies, which can supply approximately 70% of residential or commercial water heating demand4. FPSC and ETSC are including immovable solar collectors by which solar energy can be captured to produce hot water. From a comparative point of view, ETSCs play a more significant role than flat plate collectors because they provide higher thermal efficiency at operating temperatures below 100 ℃5. ETSCs are composed of an absorber tube surrounded by two concentric glass tubes that are directly exposed to solar radiation. The vacuum envelope region between the absorber and external tube leads to minimization of the heat loss originating from radiation and convection heat transfer6. Moreover, the transmission of the absorbed energy toward the absorber tube occurs by the use of various heat extraction methods7. ETSCs are assorted into three types in terms of heat extraction techniques: heat-pipe ETSC, U-pipe ETSC, and water-in-glass ETSC8. Water-in-glass or thermosyphon models have been the center of attention of researchers by virtue of low maintenance cost, simplicity, and appropriate thermal performance9. Copious numerical and empirical studies were fixated on the treatment of ETSCs. The collector slope, type of working fluids, inlet fluid temperature, and geometrical parameters are the most efficacious factors affecting the collector efficiency10. Alfaro-Ayala et al.11 carried out optimization for WIGETCs with steady-state and laminar regime using the CFD model and the simulated annealing method. They concluded form optimization analysis that absorber area of the collector’s commercial geometry has been 19.4% lower than case of the optimal geometry with a minimum absorber area of approximately 2.5 12 employed twisted tape inside a single-ended evacuated tube for various initial temperatures. The obtained results illustrated that using twist tape is an efficacious method for the system development at the high range of initial temperatures. The intensification of solar flux and its incident angle on the collector performance was performed by Essa and Mostafa13 in a numerical transient simulation considering a single-ended tube with a storage manifold. Based on the simulation results, alteration of solar flux intensity leads the flow array to change from the linear profile to the helical form, that originates from the movement of the sun rays. Alfaro-Ayala et al.14 performed an investigation changes of the features approaches to evaluate the outlet fluid temperature within the manifold of a WIGETC using CFD simulation. It was achieved that the collector efficiency and outlet fluid temperature were in closer concordance to the experimental data by utilization of the BA method compared to the VPT model. Mazarron et al.15 evaluated the fluid flow, design, and installation of a SWH system operating with ETSC at various operational temperatures. Authors proved that the greatest productivity of the proposed unit was obtained for water with T = 50 16 who found that, using 17 analyzed the productivity of a HPETC using silver-water nanofluid. They reported that the proposed system witnessed the efficiency increment about 20–40%, while using nanofluid in place of pure water. Gan et al.18 found that employing 19 conducted an experimental study to evaluate the efficiency of HPETC by using MgO nanofluid at different volume fractions. They found that MgO/water nanofluid can enhance the productivity of the collector more than case with water. According to the observations of Yan et al.20 in their empirical experiment, 21 demonstrated the significance and the efficacy of Cu/water nanofluid in ETSC, by examining the nanoparticles at various volume concentrations. The results have divulged that a 50% increment took place in Tout while employing the nanofluid at 0.03%, and 0.8 L/min of volume concentration and volume flow rate, respectively.The last decades have witnessed a burgeoning demand for energy consumption produced by fossil fuels, which in turn has led to growing concerns about environmental issues such as air pollution and other serious problems. Take the limited resources of fossil fuels and their high cost as an example. Based on this fact, to alleviate deleterious efficacies in connection with it, researchers have focused their attention and dedicated their study to renewable energy resources. More specifically, solar energy, as an auspicious type of clean energy, is applied in a wide range of applications owing to its accessibility and innocuous usage22 as an efficacious way to justify the performance improvement in thermal systems, where there are limitations for energy analysis23. Employing nanofluids leads the entropy generation of the system to reduce24. There are few kinds of literature about ETSCs, which put their main focus on entropy generation and irreversibility. Leong et al.25 examined a comparison in terms of entropy generation has been drawn between 26 appraised the rate of entropy generation for FPC and ETSC using CFD simulation. The obtained results revealed that ETSC has higher thermal entropy generation than FPC by virtue of more heat transmission, which takes place in a vacuum tube collector. In contrast, FPC has shown higher fluid friction entropy generation.Regarding the 2nd law, the thermal efficiency of unit will experience a reduction over time, for the reason that the heat energy conversion process is irreversible. The assessment of entropy generation in engineering applications was proposed by Bejangen are illustrated throughout the model. For thermal evaluation of the proposed system, temperature and velocity distributions and contours inside the three cutting sections of collector along with the heat transfer coefficient were analyzed for water and nanofluid, by which the merits of using the CuO nanofluid are demonstrated.According to the lack of numerical researches concentrating on the appraisal of irreversibility in ETSCs, this study is fixated on analyzing the irreversibly inside an open thermosyphon solar collector aim to extract the efficacy of entropy generation on the productivity of suggested unit. Furthermore, a comparative evaluation between the two cases by utilization of pure water and nanofluid are presented over 1 h of the simulation process. CuO-The modeling technique has been employed for a close-ended thermosyphon ETSC along with the manifold investigating its thermal behavior and entropy generation. Figure The mesh structure is effective elements in CFD simulations, on which the correctness of outcome is based. In this investigation, the average tank temperature in different cases with various numbers of nodes varying from 258,743 to approximately 500,000 was compared to reach the optimum number of nodes to reduce computational cost and time. When the number of nodes surpasses 380,000, the changes in the results are trivial and negligible. Hence, 380,675 nodes were specified as the extra fine mesh for modeling. In addition, the value of skewness was 0.17, which shows the high quality of the mesh structure. The shape of the grid applied in current article was illustrated in Fig. h = 8 W/28.The components of the described model are comprised of the manifold and the absorber tube. There is a heat exchange between reservoir tank and ambient with mentclass2pt{minim7 has been considered to verify the simulation in the present model. As it is depicted in Fig. Proving the accuracy of the results is an important part of a numerical simulation. Because of this, the proposed model of Jowzi et al.tube and Ttank witnesses a significant difference from the outcomes reported at two other time steps after 25 and 15 min of flow time, respectively. Thus, a time step size of 2 s is acceptable for use in the numerical simulation process.Choosing an appropriate size for the time step can be helpful to reduce the computational time and simultaneously give accurate results. Figure 29:7:Aiming to consider lifting efficacy in the modeling, the Boussinesq approximation model has been taken into account. In this approach, density is considered as the variable parameter, while all other thermophysical and transport characteristics are assumed constant. It is stated in terms of temperature (T) and thermal expansion coefficient , each of which was surrounded by cutting planes. To reduce the computational cost, the numerical simulation results have been considered for 1 h of flow time, which can be extended to a one-day duration. In the first step, the velocity distributions of fluid flow with plain water and nanofluid are compared in the entire tube and each cutting section after 30 and 60 min, respectively. In the second step, the same comparative analysis has been conducted for the temperature range within the unit for both fluid flows. Finally, entropy generation assessment is carried out for both cases.V for water at 0.5 and 1 h after the flow time. To elaborate, the fluid flow inside the tube is divided into layers of fluid with high and low temperatures at the top half and bottom half, which is separated. Figure V of different vertical planes of lines 1, 2, and 3 during the passage of flow time. At t = 0.5 h after the flow time, it is observable that a slight discrepancy in velocity values occurs between the two simulated cases in the regions of  and  along the vertical sections for all three lines. The negative velocity values denote the fluid motion toward the pipe’s close end, while positive values refer to the outflow which is moving toward the manifold in the form of natural convection. Among all the cutting sections, the least difference in velocity magnitudes for both cases exists in line 3, which is near the stagnant region on the close-ended side of the absorber, which leads the fluid flow to experience the least velocity value compared to the first line nearby the entry region. On the contrary, the utmost velocity difference of water and nanofluid appertain to line 1, where the fluid flows with the maximum velocity. The last point which can be deduced from Fig. h" factor was measured as depicted in Fig. h" was discorved to be higher with the utilization of nanofluid than that of the conventional fluid over the simulation time.To elaborate on the entire natural circulation procedure taking place inside the tube, at the beginning of the process, cold fluid flows from the storage tank toward the absorber’s close-ended region. Meanwhile, the top wall of the absorber, which is at the direct experience to solar radiation, is involved in absorbing and transferring the radiation to the fluid, which migrates inside the collector pipe. After the absorbed heat transmission to the base fluid, the change in density engenders the buoyancy effect, which eventually leads the fluid to experience a free circulation process. Figure Besides the thermal assessment aimed at exploring the impact of nanofluid in the collector, an entropy generation analysis is provided for both cases over the simulated flow time to compare the irreversibility in the system under the simulated cases. Fluid friction (n of Eq. . Figure mentclasspt{minimamentclasspt{minima2O starts to enhance. Second, the velocity magnitude for the nanofluid was found to be 40 mm/s in line 1 at the peak point in an hour, which was the highest value among all other cutting sections. In addition, to authenticate the productivity improvement of unit by the employ of nanomaterial, a comparison was drawn in terms of "h" between the two simulated cases which denote the thermal competency of the provided nanomaterial in proposed system. The convective coefficient was obtained to have higher values over the simulation process with the utilization of CuO by virtue of the effectual thermal characteristics of the prepared fluid when coalesced with the nanoparticles, homogenously. Ultimately, the entropy generation examination as the main objective of this study has shown that adding CuO nanoparticles is a contributing factor, affecting the heat transfer irreversibility to endure a reduction when compared to the conventional fluid. In detail, the irreversibility originated from the heat transfer reduces around 6% with the existence of CuO-gen owing to the fluid viscosity was found to have minuscule values in both simulated cases, which shows the irrelevancy of the fluid flow viscous stresses in light of the buoyancy-driven nature of the fluid flow within the solar unit. All in all, a higher total entropy generation rate was obtained for the conventional type of collector with pure water.The lack of meticulous studies fixated on the thermodynamic scrutinization of evacuated tube collectors by the presence of nanofluids as an efficacious working fluid for improving the thermal treatment of the system was the main reason this study was carried out. Moreover, the proposed system has undergone thermal evaluation to analyze the fluid structure inside the system and the desirability of the nanofluid flow to enhance the functionality of the collector. The collector tube’s inclination angle tube was fixed at 45


P > 0.05). The volume of drainage in the improved group and the regular group within 24 hours after surgery was 234 ± 26.3 ml and 307 ± 31.8 ml, respectively, P < 0.01, and the difference was statistically significant. The incidence of adverse reactions in the improved group was much lower than that in the regular group, P < 0.01. The administration route of the improved group was beneficial to maintain the stability of hemodynamics when using the protamine sulfate to neutralize heparin, which is worthy of clinical nursing promotion.In this study, we have evaluated and examined various nursing effects of improved administration of protamine sulfate neutralizing heparin after cardiopulmonary bypass. For this purpose, retrospective analysis was made about the nursing records and clinical data of 216 patients who underwent cardiac operation under cardiopulmonary bypass in our hospital from January 2018 to December 2020. Among the enrolled patients, 118 patients were given subinterval administration of protamine sulfate neutralizing heparin via aortic root with the assistance of the scrub nurse at the end of cardiac surgery (improved group). A total of 98 patients were administered by the circulating nurse via the central vein (regular group). The changes of body temperature, blood pressure, oxygen saturation before and after heparin neutralization, and the total volume of thoracic drainage within 24 hours after operation were observed in the two groups, so as to evaluate the application effect of the improved administration of protamine sulfate neutralizing heparin from the perspective of nursing. There was no significant difference in age, gender, and other basic characteristics between the two groups ( Most cardiac operations require cardiopulmonary bypass (CPB). Heparin is still the preferred anticoagulant for cardiopulmonary bypass . ProtamiIn this study, we have evaluated and examined various nursing effects of improved administration of protamine sulfate neutralizing heparin after cardiopulmonary bypass. For this purpose, retrospective analysis was made about the nursing records and clinical data of 216 patients who underwent cardiac operation under cardiopulmonary bypass in our hospital from January 2018 to December 2020. Among the enrolled patients, 118 patients were given subinterval administration of protamine sulfate neutralizing heparin via aortic root with the assistance of the scrub nurse at the end of cardiac surgery (improved group). A total of 98 patients were administered by the circulating nurse via the central vein (regular group). The changes of body temperature, blood pressure, oxygen saturation before and after heparin neutralization, and the total volume of thoracic drainage within 24 hours after operation were observed in the two groups, so as to evaluate the application effect of the improved administration of protamine sulfate neutralizing heparin from the perspective of nursing.The remaining sections of this manuscript are arranged according to the following agenda items or paragraph which is given as follows.In subsequent section entitled as Materials and Methods, the proposed mechanism is described in detail such as collection of data, nursing administration methods, observation, and statistical indexing methods. In From January 2018 to December 2020, a total of 216 patients underwent cardiopulmonary bypass surgery. The clinical data of the patients were studied and analyzed. The patients were divided into two groups according to the different ways of nursing and administration of protamine sulfate neutralizing heparin. The scrub nurse assisted the surgeon to inject protamine sulfate through the aortic root intermittently (improved group). There were 118 patients in the improved group. There were 98 patients administered with protamine sulfate by the circulating nurse via the central vein (regular group).Routine thoracotomy or thoracoscopic incision was performed, and heparin intravenous injection was done at a dose of 3 mg/kg bodyweight before the establishment of cardiopulmonary bypass. After the completion of heart surgery, the cardiopulmonary bypass was gradually stopped. After observing the stability of hemodynamic indexes, heparin was neutralized with protamine sulfate in a ratio of 1.2 : 1. In the improved group, protamine sulfate was administered at intervals. The surgical instrument nurse drew the medicine in the syringe and connected to the scalp needle. First, we slowly injected 20% protamine sulfate through the aortic root; then, we observed patient's blood pressure, heart rate, and blood oxygen saturation for 120 seconds. In the stable range, we continued to slowly inject the remaining 80% of protamine sulfate. In the routine group, protamine sulfate was injected slowly by the circulating nurse through the subclavian vein or internal jugular vein through the superior vena cava. Heparin  and protamine sulfate  have the same source of supply and the same dosage ratio (protamine sulfate: heparin was 1.2 : 1).t-test was used for measurement data, and the chi-square test was used for enumeration data. P < 0.05 was considered as statistically significant.The hemodynamic index of heparin neutralization was recorded during the whole administration of protamine sulfate. The patient's hemodynamic index included the changes of extremity temperature, blood pressure, oxygen saturation, and total amount of drainage fluid within 24 hours after operation and so on. The paired The ages of the modified group and the conventional group were 17–76 years old (median age 58 years old) and 21–79 years old (medium age 59 years old), respectively. There were 48 males  and 57 females . The body mass index (BMI) of the patients in the two groups was 22 ± 3 and 22 ± 4, respectively. There was no statistical difference in the above indicators between the two groups, as given in P < 0.01. There were 48 cases of mild reaction and 54 cases of mild reaction, respectively, and there was no statistical difference between the two groups (P > 0.05). Among the patients with severe protamine adverse reactions, 3 cases were in the modified group. They had significant decrease in blood pressure and slow heart rates. In the conventional group, there were 9 patients with severe protamine adverse reactions, including 5 cases with decreased blood pressure, 2 cases with bradycardia, and 2 cases with airway resistance increased, and the difference was statistically significant, P < 0.05, as given in In the administration process of protamine sulfate, nurses should closely observe the patient's limb temperature, blood pressure, and saturation of pulse oxygen. Drug-induced reactions can be divided into mild and severe levels according to the changes of the above indicators , 4. In tIn this group, we slowly injected 20% protamine sulfate through the aortic root first; then, we observed patient's blood pressure, heart rate, and blood oxygen saturation for 120 seconds and continued to slowly inject the remaining 80% of protamine sulfate. In the routine group, protamine sulfate was injected slowly by the circulating nurse through the subclavian vein or internal jugular vein through the superior vena cava. The ACT values of patients in the two groups were recorded 10 minutes before operation (basic ACT), 5 minutes after heparinization, and 5 minutes, 30 minutes, 60 minutes, and 180 minutes after neutralization, and the results are given in P < 0.01), as given in P < 0.01), the higher LVEF 24 hours after surgery (P < 0.05), as given in We recorded the length of operation time and the median drainage volume within 24 hours. The retention time of drainage tube and thoracotomy for hemostasis were also recorded. In the improved group, the median 24 hour drainage volume after the neutralization of heparin with protamine sulfate was 261 ml, significantly less than the conventional group . At the same time, we found that the median amount of drainage fluid 24 hours after surgery in the conventional group was 307 ml, which was significantly higher than that in the improved group (P < 0.001), and the possible reason was analyzed to be related to the drug treatment after the increase of pulmonary artery pressure caused by protamine.In our study, the incidence of severe adverse reactions in the modified protamine sulfate administration pathway group was reduced to 3.4%, while that in the conventional group was 17%, suggesting that improved nursing measures including optimizing the protamine administration pathway can reduce the occurrence of adverse reactions in the protamine sulfate group . A total of 98 patients were administered by the circulating nurse via the central vein (regular group). The changes of body temperature, blood pressure, oxygen saturation before and after heparin neutralization, and the total volume of thoracic drainage within 24 hours after operation were observed in the two groups, so as to evaluate the application effect of the improved administration of protamine sulfate neutralizing heparin from the perspective of nursing. Finally, this study found that the modified administration of intermittent administration of protamine sulfate and heparin via the aortic route resulted in a lower incidence of protamine-related adverse reactions and less postoperative drainage volume in patients, which was a safer administration method for protamine sulfate and heparin.


The pandemic has challenged training programs in numerous ways, specifically in the ability to conduct group based teaching sessions. To overcome this challenge Twitter was examined as a vehicle for engaging Geriatric Fellows in education about critical appraisal of clinical research. A secondary objective was to develop educational synergy among university-based programs. To achieve these aims, 5 Midwestern Geriatric and Palliative Medicine Fellowship programs agreed to enroll their fellows into a monthly Geriatrics Twitter Journal Club, that commences on Twitter Tuesday and lasts a week. Each month, an assigned fellow selects an article to discuss and creates a short video to introduce it. A Twitter meister deliveres structured questions to guide fellows’ collective input on the article being critiqued. Over a 3 month roll out of @GeriatricJC, the twitter account of the journal club has gained 144 followers that includes 20 fellows, 63 geriatricians/geriatric faculty, 28 organizational accounts, 5 students and around 28 other providers and experts. From December 2020- February 2021, account generated tweets resulted in an average of 397 impressions/day with 2548 visits to the account profile per month. Videos posted have averaged 73 views/video. Discussion in journal club using #GeriJC has garnered 178 tweets from participants. This project shows that Twitter is a feasible platform for a fellowship journal club among several training programs, thus expanding expertise in evidence-based medicine while lowering the administrative burden of preparing journal club within a single program and increasing both faculty and trainee convenience of learning.


Following publication of the original article , it was The declaration has been corrected in the published article and may be seen below:“This work was supported by the Consortium for Advanced Research Training in Africa (CARTA), awarded to MFF. CARTA is jointly led by the African Population and Health Research Center and the University of the Witwatersrand and funded by the Carnegie Corporation of New York (Grant No: G-19-57145), Sida (Grant No: 54100113), Uppsala Monitoring Centre and the DELTAS Africa Initiative (Grant No: 107768/Z/15/Z). The DELTAS Africa Initiative is an independent funding scheme of the African Academy of Sciences (AAS)’s Alliance for Accelerating Excellence in Science in Africa (AESA) and supported by the New Partnership for Africa’s Development Planning and Coordinating Agency (NEPAD Agency) with funding from the Wellcome Trust (UK) and the UK government. The statements made and views expressed are solely the responsibility of the Fellow. This work was also supported by the Wellcome trust International Masters Fellowship in Tropical Medicine and Hygiene (Grant No: 212633/Z/18/Z), Bill and Melinda Gates Foundation (Grant Number: OPP1177156), Howard Hughes Medical Institute (Grant Number: OPP1099295) and by Application of Novel Transgenic technology & Inherited Symbionts to Vector Control (ANTI-VeC) (Grant Number: AVPP0027/1), all awarded to Ifakara Health Institute.”The authors apologize for any inconvenience caused.


In traditional Chinese medicine, it is believed that the “tongue coating is produced by fumigation of stomach gas”, and that tongue coating can reflect the health status of humans, especially stomach health. Therefore, studying the relationship between the microbiome of the tongue coating and the gastric fluid is of great significance for understanding the biological basis of tongue diagnosis.This paper detected the microbiomes of the tongue coating and the gastric fluid in 35 gastritis patients using metagenomic sequencing technology, systematically constructed the microbial atlas of tongue coating and gastric juice, and first described the similar characteristics between the two sites.There was a significant correlation between tongue coating and gastric juice in terms of microbial species composition and overall diversity. In terms of species composition, it was found that the two sites were dominated by five phyla, namely, Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria and Proteobacteria, and that most of the gastric microbial species could be detected from the patient's own tongue coating. In terms of overall diversity, a significant correlation was found between the alpha diversity of the tongue coating microbiome and the gastric juice microbiome. Furthermore, in terms of abundance, 4 classes, 2 orders, 4 families, 18 genera and 46 species were found to significantly correlate between the tongue coating and the gastric fluid.The results provide microbiome-based scientific evidence for tongue diagnosis, and offer a new perspective for understanding the biological basis of tongue diagnosis. Bacillus only exists in chronic erosive gastritis patients with yellow tongue coatings [Traditional Chinese medicine (TCM) believes that the “tongue coating is produced by the fumigation of stomach gas”. The tongue coating as the initial part of the digestive tract, can reflect the health status of humans, especially the status of the stomach. Some studies have characterized the correlation between the tongue coating microbiome and the status of the host, especially with regard to stomach disease. Our group has revealed variations in tongue coating microbiomes in gastritis patients with different TCM subtypes by 16S rRNA gene sequencing, while 123 OTUs enriched in cold patients with typical white-greasy tongue coating, and 258 OTUs enriched in hot patients with typical yellow-dense tongue coating [coatings . Han et Prevotella, Fusobacterium, and Leptotrichia, and an increase in abundance of Lactobacillus. Through transcriptional activity analysis, it was found that the activity of Actinobacteria was decreased in gastric juice, and Campylobacter activity was increased in gastric juice [The gastric microbiome has also been repeatedly shown to be associated with the health of the stomach. Chang Soo Eun et al. revealed a significant difference in the microbial composition of the gastric mucosa between patients with chronic gastritis, intestinal metaplasia and gastric cancer through 16S sequencing . Paola Mic juice . Francisic juice . Olabisiic juice . Rui M Fic juice .Although the tongue coating and gastric fluid microbiome each have a strong correlation with the health status of the stomach, the association analysis of the microbiome between the two sites is still deficient, especially in terms of an in-depth systematic analysis and similar characteristics. This paper constructed the microbial map of the tongue coating and gastric juice and first established the systematic association between two sites through metagenomic sequencing, taking gastritis patients as an example.Gastritis patients, including 16 with superficial gastritis, 7 with atrophic gastritis, 4 with intestinal metaplasia and 8 with dysplasia were recruited for this study  –15 databGastric fluid samples generated 13,573,094–38,965,184 reads after metagenomic sequencing. After data analysis, bacteria, viruses, archaea and eukaryotes were found in the gastric fluid samples. Bacteria were found in all gastric fluid samples and were the most abundant microbes with abundances of 83.89–100% in all samples. Viruses existed in 13 samples, with abundances of 0.14–16.11%. Archaea existed in 1 sample with an abundance of 0.082%. Eukaryotes existed in 1 sample with an abundance of 0.35%. Finally, 13 phyla, 21 classes, 31 orders, 58 families, 88 genera, and 194 species of bacteria were detected in the gastric fluid samples. The cladogram of the gastric fluid microbiome is constructed in Fig. Haemophilus parainfluenzae existed in all 35 gastric fluid samples, while 57 species were present in only one gastric fluid sample. Twenty-six species were present in more than half of the gastric fluid samples, and most species existed in less than 5 gastric fluid samples , Shannon index (P = 0.012) and Simpson index (P = 0.048) were significantly correlated between the tongue coating and the gastric fluid in the same patient Fig. d, which Beta diversity was computed based on Bray–Curtis distance and Jaccard distance. The distances between the tongue coating microbiome samples and the gastric fluid samples from the same patient were significantly smaller than those from different patients based on the Bray–Curtis distance after the Wilcoxon rank-sum test (P = 0.0007) Fig. e, which The Spearman correlation test was used to analyze the microbes that were significantly correlated between the tongue coating and the gastric fluid; after false discovery rate correction, 4 classes, 2 orders, 4 families, 18 genera and 46 species were found to have a strong correlation between the gastric fluid and the tongue coating (P < 0.05). Associated microbes were plotted according to taxonomic classification Fig. , and assAfter comparing genes and pathways, in top 10 most abundant genes and pathways, 4 KOs, namely, K02078, K02358, K02874, K03088, and 7 pathways, namely, PWY-6386, PWY-6387, PWY-6700, PWY-7219, PWY-7221, PWY-7229, PEPTIDOGLYCANSYN-PWY were found both in tongue coating and gastric fluid. These discoveries indicated that there was an overall consistency of genes and pathways in the tongue coating and gastric fluid samples.Helicobacter pylori could be detected in the gastric fluid samples of patients whose pathological results for H. pylori (Helicobacter pylori) were positive, and could not be detected in gastric fluid samples of patients whose pathological results for H. pylori were negative; H. pylori was not found in the tongue coating of any patients. The tongue coating microbiomes of HP (Helicobacter pylori)-positive and HP-negative patients were compared, and alpha diversity was not significantly different between the two groups. For beta diversity, the Bray–Curtis distance and Jaccard distance between HP-positive patients were both higher than those between HP-negative patients   Fig. a, which For gastric fluid microbes, in terms of alpha diversity, the richness, Shannon index and Simpson index were not significantly different between the HP-positive and HP-negative groups . For beta diversity, Bray–Curtis distances and Jaccard distances between HP-negative patients were significantly higher than those between HP-positive patients  Fig. b. There Epsilonproteobacteria, Campylobacterales, Helicobacteraceae, Helicobacter and Helicobacter pylori were found to be significantly different between the two groups  tubes marked with 1, 2, and 3 were prepared, and 1 ml of phosphate buffered saline was added to each tube. Second, one tongue coating swab was used to scrape the tongue coating from the base to the tip of the tongue 30 times, while simultaneously turning the swab. Then, the swab was placed into EP tube 1, the swab was shaken to wash off the sample, and the samples were washed in tubes 2 and 3 in turn. The second swab was used to scrape the tongue coating the same way as stated above, and then the samples were washed in tubes 2 and 3 in turn. The third swab was used to scrape the tongue coating and wash the sample in tube 3. The three tubes of suspension were centrifuged at 2000×g for 5 min. The supernatant was transferred to a new EP tube and centrifuged at 20,000×g for 15 min, and then the precipitate was stored at − 80 °C.Gastric fluid was collected during gastroscopy examination into a collector. Gastric fluid was moved into a 15-ml EP tube, and an equal amount of PBS was added. The mixture was divided into 1.5-ml EP tubes and centrifuged at 3000×The microbial DNA of the tongue coating and the gastric fluid samples was extracted using an MO-BIO PowerSoil DNA Isolation Kit  following the procedure in the instruction manual.The microbial DNA was cut into segments with lengths of 400–500 bp using a Biouptor Pico nucleic acid interrupter, and the microbial genome DNA library was constructed by an ND604-VAHTS Universal DNA Library Prep Kit. Then, the DNA was sequenced using the Illumina 2500 sequencing platform with a reading length of PE125.SolexaQA  was usedHigh-quality data was mapped to the human genome  using SOAPAligner2  to removMetaPhlAn2  was usedThe correlated microbes between gastric fluid and tongue coating samples were tested by the Spearman correlation coefficient test followed by FDR correction. The Wilcoxon rank-sum test was used to compare the distance between the tongue coating and gastric fluid samples from the same patient and for the different patients.In traditional Chinese medicine, the variation of the tongue coating is related to physical condition. However, the underlying mechanism of this relationship is unclear. This paper attempts to explain the scientific mechanism from the perspective of microorganisms. We detected the microbiome of the tongue coating and the gastric fluid using shotgun metagenomic sequencing, using gastritis patients as an example.H. pylori infection, until other microbes were found in later studies [Campylobacter jejuni in the gastric biopsy of a women by culturing, biochemical tests and PCR amplification [Evidence from many clinical studies indicates that variation in the tongue coating microbiome is associated with the occurrence and development of gastric disease , 21–23.  studies , 24, 25.fication . Elias Hfication . Erik C.fication , 28–33.H. pylori+/CagA+ samples [Other studies have found both gastric juice and tongue coating microbiome variation under states of disease. Yubin Zhao profiled the gastric mucosa and tongue coating microbiome of chronic gastritis patients using 16S rRNA gene sequencing, and found that the number of bacterial interactions was greatly reduced in both the tongue coating and gastric microbiota of the  samples . HoweverThis paper constructed a microbiome atlas of tongue coatings and gastric juices using metagenomics. We found that both the tongue coating and gastric fluid microbiomes were dominated by Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria, and Proteobacteria, which is consistent with previous research , 9, 24, In the theory of Traditional Chinese Medicine, tongue coating is produced by Qi of stomach, and the health state of the stomach could be inferred by observing the shape of the tongue coating, such as color, thickness, dryness, wetness, and so on. Our research found that there is a strong correlation between the tongue coating and the gastric fluid in terms of the composition and diversity of microbiome. On the one hand, microbes entering the mouth are carried to the stomach by swallowing and eating; on the other hand, microbes in the stomach can enter the mouth through reflux, burping, etc. It has been shown that microbiome in the stomach is correlated with the health status of the stomach. We speculate that metabolites produced by microbes accumulated in the tongue could cause changes in the morphology of the tongue coating. This may be one of the biological bases for inferring the health status of the stomach through tongue diagnosis in TCM.Porphyromonas gingivalis, Streptococcus salivarius, and Neisseria subflava, which were correlated between tongue coating and gastric fluid, were shown to overcome changes in pH and survive in the stomach [This study found some species correlated between tongue coating and gastric fluid. We speculate that species that could be resistant to the low pH environment in the stomach may be shared in two locations. Some species were validated by other research. For example,  stomach –37. The H. pylori could be detected by metagenomic sequencing in the gastric fluid of HP-positive patients. Furthermore, the abundances of Betaproteobacteria and Gammaproteobacteria, which belong to the same phylum as H. pylori, were found to be higher in the gastric fluid of HP-negative patients, which was similar to the research finding by Llorca [H. pylori [H. pylori infection are diagnosed with gastritis [H. pylori infection are not cured by eliminating H. pylori. We speculate that microbes belonging to the same phylum as H. pylori may play a similar role as H. pylori in gastritis patients. Further studies are needed to reveal the mechanism.As a critical microbe in the stomach –53, H. py Llorca . Betaproy Llorca . From th. pylori –58. Thisastritis , while iIn future research on larger samples, taking pH value information into consideration in the study may reveal more specific results. At the sample processing stage, the Bead-beating step could be taken before DNA extraction to compare the results and get a more accurate assessment of the abundance and composition of the microbiome. Furthermore, more samples which including healthy cohort are needed to verify the reliability of the results, and confounding factors that could influence on the results should be verified.This study elucidates the health relationship between tongue coating and stomach from the perspective of microorganisms, which provide a scientific basis for tongue diagnosis. A better understanding of tongue diagnosis could lead to the development of non-invasive diagnosis and better treatment of gastric diseases.


Intravenous fluids are an essential component of shock management in human and veterinary emergency and critical care to increase cardiac output and improve tissue perfusion. Unfortunately, there are very few evidence-based guidelines to help direct fluid therapy in the clinical setting. Giving insufficient fluids and/or administering fluids too slowly to hypotensive patients with hypovolemia can contribute to continued hypoperfusion and increased morbidity and mortality. Similarly, giving excessive fluids to a volume unresponsive patient can contribute to volume overload and can equally increase morbidity and mortality. Therefore, assessing a patient's volume status and fluid responsiveness, and monitoring patient's response to fluid administration is critical in maintaining the balance between meeting a patient's fluid needs vs. contributing to complications of volume overload. This article will focus on the physiology behind fluid responsiveness and the methodologies used to estimate volume status and fluid responsiveness in the clinical setting. Although the route of administration is not always specified within this review, it should be presumed all references to fluids refer to intravenous (IV) administration unless otherwise stated. In hemodynamically unstable patients, IV fluids are administered to increase cardiac output (CO) and improve tissue perfusion . Howeverthe positive reaction of a physiologic parameter of a certain size to a standardized volume of a certain type of fluid (or other type of induced preload change) within a certain amount of time and measured within a certain interval”  setting, 20% of patients receive resuscitation fluids, with this number increasing to >30% on the first day of ICU admission . ResultsMerely assessing volume status does not predict a patient's response to fluid loading. A patient may be hypovolemic and non-fluid responsive due to the complex interaction of the glycocalyx, vascular permeability, capillary leak, vascular tone, and cardiac function. At the same time, the fact that a patient is fluid responsive does not always imply hypovolemia is present. For example, healthy euvolemic patients given a fluid bolus may meet the definition of being fluid responsive. Finally, a patient that is hypovolemic and fluid responsive may still develop complications of volume overload, particularly if increased vascular permeability is present , 41. ForGiven there is a lack of data regarding evidence-based fluid management in hypotensive human and veterinary ECC patients it is not surprising there is considerable heterogeneity in the way IV fluids are administered in shock states, including the criteria used as triggers, targets, volume, and safety limits for fluid input , 39, 42.Understanding circulatory physiology and pathophysiology helps understand how different scenarios mandate different therapeutic and fluid management strategies. Two general assumptions are expected in response to a preload challenge; (1) increased venous volume leads to increased cardiac preload (venous loading = cardiac loading), (2) increased preload leads to increased stroke volume/cardiac output (VR = CO).It may seem intuitive that an IV fluid bolus will lead to increased VR. However, a clear understanding of circulatory physiology may explain why increased VR is not always observed.Preload, or VR, is determined by the pressure gradient between capacitance veins and the right atrium (RA). The pressure within the capacitance veins is termed mean circulatory filling pressure (MCFP) , 29, 30.MCFP [or mean systemic pressure (MSP)] varies with changes in venous blood volume or vessel tone, which determines the balance of venous blood contained within the unstressed and stressed blood volume. As a consequence, MCFP may only be increased by changes in blood volume, or by changes in vessel tone  29). Th. Th29). MCFP is predominantly regulated by the effects of the sympathetic nervous system on the splanchnic venous system . DependiMCFP, right atrial pressure (RAP) and the resistance to venous flow (Rv) are the main driving forces of VR as expressed through the following formula: VR = [(MCFP – RAP)/Rv],  13). Th. Th13). From a physiologic standpoint, given VR is determined by the pressure gradient between capacitance veins and the right atrium, decreasing the right atrial pressure should increase VR, provided all other variables are held constant. However, techniques to decrease RAP are limited, and if RAP falls too low, venous collapse at the thoracic inlet may occur . Venous Resistance to venous flow may be decreased by increasing venous compliance through venodilation . HoweverMCFP can be increased through venoconstriction or fluid loading  13, 29,, 45. Ven2) to the tissues. The relationship between these parameters can be expressed through the following 2 formulas: (1) DO2 = CaO2 × CO, and (2) CO = HR × SV. Moreover, MAP = CO × SVR.The Frank-Starling curve  describe2 is oxygen delivery (mL/min), CaO2 is the arterial oxygen content (mL/dL), CO is cardiac output (mL/min), HR is heart rate (beats/min), SV is stroke volume (mL), MAP is mean arterial pressure (mmHg), and SVR is systemic vascular resistance (dynes/seconds/cm−5).Where DOThis is the foundation for the concept of fluid responsiveness.Stroke volume is the amount of blood ejected from the heart with each beat and is dependent on preload , contractility and afterload . According to the Frank-Starkling curve, within certain limits an increase in VR will lead to an increase in preload . This inFor a patient to be fluid responsive both the right and left ventricles must be functioning on the steep part of the Frank-Starling curve  .Cardiac contractility can be affected by a variety of processes, fluid overload and myocardial hibernation to name a few. During fluid overload, myocytes get stretched beyond a certain level, rendering them unable to contract more forcefully, with SV subsequently failing to increase any further (fluid “unresponsiveness”). At this point fluid administration is likely to increase the stressed volume and thus MCFP, but to a lesser extent than it increases RAP, leading to volume overload  44). In. In44). The Marik-Philips curve describes the relationship between increasing preload and extravascular lung water (EVLW). When the Marik-Philips curve is superimposed over the Frank-Starling curve the risk of a patient developing edema during fluid loading is nicely illustrated  45). A . A 45). It should be remembered that every patient has its own unique set of individual Frank-Starling and Marik-Philips curves, and that these curves are influenced by factors associated with specific disease states. Knowing the factors that govern these equations will allow the clinician to estimate the effect of fluid administration, but should never be considered an absolute certainty.The underlying cause of shock will influence an individual patient's Frank-Starling and Marik-Philips curves, and subsequently fluid responsiveness. The incidence of hypotension in hemodynamically unstable human ICU patients is as high as 33%. Considering Weil's classification of shock, it is reported in humans that 62–71% of cases are due to septic shock, 16% cardiogenic, 16% hypovolemic, 4% other distributive shock, and 2% obstructive shock . In contHypovolemic shock patients are usually located on the ascending portion of the Frank Startling curve (fluid responders). These patients are likely to benefit from fluid administration. Administration of fluid boluses will increase SV, which typically results in improved clinical cardiovascular parameters such as HR and MAP. At the same time, their Marik-Philips curve will not be as steep, and the administered fluid boluses are unlikely to cause a dramatic increase in EVLW . This coSeptic shock patients are more often located on the flat portion of the Frank-Starling curve (non-fluid responders), particularly following initial fluid resuscitation. These patients are more likely to benefit from vasopressors and positive inotropes as additional fluid boluses will not increase SV and will likely lead to volume overload and increased EVLW . This isAs the majority of ECC veterinary patients have non-distributive hypovolemic shock, they are more likely to respond to volume resuscitation , 39 and Roughly 50% of preload challenges performed in critically ill human patients do not result in an increase in CO or SV, exposing these patients to the potential harm of fluid overload . ResearcA major clinical challenge that all veterinarians must address when confronted with a hemodynamically unstable patient is to successfully identify patients that will increase CO in response to fluid therapy (fluid responsive) vs. those who will develop adverse events from unnecessary administration of IV fluids (non-fluid responsive). The previous paragraph indicates not every patient, human or veterinary, will be a fluid responder. In severely hypovolemic patients additional fluid boluses have a high benefit to risk ratio . Conversely, additional fluid boluses will be detrimental in markedly hypervolemic patients. Between the extremes of severe hypovolemia and marked hypervolemia, predicting if the patient will benefit from fluid therapy without developing complications of fluid overload  is more challenging. Answering whether an additional fluid bolus will be beneficial (improve CO) or not (cause volume overload) with 100% accuracy is not always possible. The gray-zone approach, which is a form of statistical analysis, suggests a “three level decision tree” when assessing if a patient might be fluid responsive or not: “Yes—Maybe—No.” The gray-zone approach can be calculated for any physiologic parameter used to assess a patient's response to a preload challenge . More specifically, it is defined as the interval of values (for the physiologic parameter assessed) between a sensitivity or specificity lower than 90% to classify patients as fluid responders or non-responders , 17, 19.Static markers, such as central venous pressure (CVP), MAP, inferior vena cava (IVC) diameter, and end-diastolic ventricular area (LVEDA) are measures of pressure and/or volume that estimate the amount of fluid in the entire cardiovascular system at a specific point in time. They are intended to assess volume status and are most useful as “alarm” signals indicating severe hypo- or hypervolemia. Static markers are subject to numerous confounding factors, which limit their precision in the clinical setting. Even measuring baseline CO as a one-time static marker possesses only moderate predictive value to estimate volume status because the cardiac function curve characteristics differ among patients as well as within patients due to continuously changing pathophysiologic conditions. Moreover, any single value does not discriminate where a patient is located on the Frank-Starling curve; on the ascending limb or near the plateau of the curve. Although static markers often help assess a patient's hemodynamic status they do not predict if the patient will be fluid responsive or if vasopressors and/or positive inotropes should be initiated, limiting their value to tailor optimal treatment strategies in the individual patient .Dynamic markers try to determine a patient's location along the curve by inducing a short-term change in preload  and measuring the magnitude of response to the change in preload in real time. Therefore, dynamic markers assess at least two points on the curve to estimate a patient's fluid responsiveness. The magnitude of response is usually measured as a change in CO, SV, or their surrogates such as pulse pressure or other clinical parameters. Dynamic markers do not assess volume status, yet assess fluid responsiveness in order to decrease the incidence of fluid overload, as well as the risk of recurring hypotension.CI), because of heart-lung interactions, is a dynamic marker (see below). Although many parameters can be assessed dynamically in response to a change in preload, many perform poorly because they are influenced by factors independent of preload. These markers are therefore more commonly employed as indicators of circulatory shock or used as static “alarm” markers . Moreover, many of these typical static markers are not sufficiently sensitive, or their measurement is not sufficiently precise to allow detection of small changes in preload, limiting their use as dynamic markers . This means that most parameters, including clinical and point of care ultrasound (POCUS) can be considered both static and dynamic, depending whether a measurement is taken at a fixed time point or over a period of time in response to a preload challenge. For example, HR in isolation is a static marker, while a change in HR following a fluid bolus is a dynamic marker; the caudal vena cava (CVC) diameter in isolation is a static marker, while the CVC collapsibility index  phase of fluid therapy is complete. However, depending on the level of invasiveness and skill required to assess the dynamic parameter, the applications will vary. For example, the authors commonly assess parameters such as the left atrial to aortic (LA:Ao) ratio and caudal vena cava diameter (CVCd) to assess volume status and the CVCThe preferred techniques used to assess fluid responsiveness may also differ depending on the clinical setting. Some techniques are less precise, yet less invasive, more rapid, and more easily applicable in a less controlled environment, making them perfectly suited for the triage or ER setting . More acAs mentioned above, many parameters can be measured in a static or dynamic fashion. However, the following section discusses clinical exam findings, MAP, shock index (SI), CVP, and lactate as primarily static markers in the clinical setting as evidence suggests they are not as accurate as some markers when assessed dynamically in response to a preload challenge.The purpose of the clinical examination in the patient with circulatory failure is to identify patients that are most likely to benefit from IV fluid therapy. The initial assessment of volume status begins with interpretation of clinical signs and symptoms including HR, pulse quality, mental status, jugular vein distention, urine production, skin turgor, temperature, capillary refill time (CRT), and the presence of peripheral edema , 25, 39.During circulatory dysfunction, the compensatory neurohumoral response redistributes blood flow to vital organs by reducing blood flow to non-vital structures such as the skin and mucous membranes. Subjective assessment of the temperature of the extremities –56, centAnother marker that may be reflective of hypoperfusion is urinary output . Hypersta posteriori suggests a positive response, but does not predict the effect of a subsequent fluid bolus.A specific HR that can be used to guide fluid resuscitation has not been well-studied in either the human or veterinary profession. Tachycardia is an important early sign of shock and is often considered a static marker indicative of hypovolemia . AlthougEnsuring MAP remains above a target value is an established goal in maintaining adequate perfusion of vital organs, although it has not been studied extensively as a predictor of fluid responsiveness, particularly in veterinary medicine. Fluid loading is indicated if tachycardia and hypotension are attributed to fluid depletion . As an iAlthough HR and MAP individually are considered rather poor indices to guide fluid resuscitation, the identification of tachycardia coupled with hypotension should trigger the clinician to strongly consider fluid resuscitation in the absence of cardiac failure , 59. TheCentral venous pressure (CVP) and pulmonary artery occlusion pressure (PAOP) are still two of the most commonly used hemodynamic monitoring tools in the assessment of volume status in human ICU's . Central2 or shock, regardless of volume status  is an automatic measure of the dynamic change in perfusion index (PI), as determined by a pulse oximeter, occurring during a complete respiratory cycle. PI demonstrates the pulse strength at the sensor site and represents a relative value of tissue perfusion and blood flow at the site of measurement, which can change based on the patient's clinical status. The pulse oximeter derived pulsatile signal is indexed against the non-pulsatile infrared signal and expressed as a percentage reflecting the amplitude of the pulse oximeter waveform. More precisely, the measurement of PVI involves mathematical calculations using PI which are automatically performed by the internal software installed in the pulse oximeter. The PVI is calculated as [(PImax – PImin)/PImax] × 100  see Tab. It is cSimilar to several parameters already discussed, assessment of point of care ultrasound (POCUS) parameters can be assessed in a static fashion to estimate volume status or applied dynamically to assess fluid responsiveness. POCUS for volume status and fluid responsiveness includes both cardiac and vascular components.Cardiac POCUS, also referred to as abbreviated echocardiography, is commonplace in many human ICU settings, predominantly because of its rapid, non-invasive, real time ability to assess patient volume status . ClinicaLeft atrial size is probably the easiest clinical cardiac POCUS parameter to assess for volume status estimation in dogs and cats . It is nIn addition to changes in lumen size, the ventricular wall thickness should be considered relative to the dimensions of the left ventricular lumen in dogs and cats , 101. AlSubjective and objective assessment of contractility of the left ventricle can also be assessed in relation to volume status, with increased contractility being seen in response to compensatory shock states and as an adaptive response to ventricular filling , 111. Ho2 in humans) suggests significant hypovolemia, while an enlarged LVEDA (>20 cm2 in humans) suggests volume overload (Measurement of left ventricular end-diastolic area (LVEDA) has been used for assessment of volume status in the human critical care setting due to its simplicity of measurement . It is moverload . Obliteroverload . It shouoverload , 116. Fuoverload  and a reoverload .At a more advanced level CO can be directly calculated based on the cross-sectional area of the descending aorta and the left ventricular outflow tract volume time integral (LVOT VTI), which has been used to predict fluid responsiveness in both humans and dogs , 116. Thd) has become well-established in many human ECC settings as a means of assessing volume status  correlates with hypovolemia and poor IVC dilation following fluid resuscitation suggests inadequate intravascular volume, independent of arterial blood pressure. Similar to humans, CVC diameter (CVCd) has been shown to correlate with volume status and CVP in dogs . The CVCCI expresses the percentage change in the diameter of the CVC during the respiratory cycle based on the following formula: CVCCI = (CVCd max – CVCd min)/CVCd max  in spontaneously breathing dogs with compromised hemodynamics or tissue hypoperfusion suggested that CVCCI can accurately predict fluid responsiveness, although the authors also concluded research is necessary to extrapolate their results to larger populations of hospitalized dogs  protocol in human ICU settings to help physicians detect a safety threshold for fluid resuscitation in patients presenting in shock . The FALAdditional POCUS findings that may be identified in patients developing volume overload secondary to excessive fluid administration include fluid accumulation in body cavities and development of gall bladder wall edema, often referred to as a gall bladder “halo sign”  122). A. A122). CI, increased number of B lines, gall bladder wall edema and body cavity fluid accumulation. As these findings are not specific, it is important to interpret POCUS findings in light of the entire clinical picture.In summary, POCUS findings suggestive of volume overload include enlarged left atrial and ventricular lumen size, thinning of the ventricular walls, a distended CVC with a decreased CVCOptimizing individual fluid therapy is complex with very little veterinary evidence available to guide clinical decision-making. However, a greater knowledge and understanding of the differences between volume status and fluid responsiveness, and the factors influencing both allow the clinician to interpret clinical findings with greater confidence. The decisions regarding estimation of fluid volume and fluid responsiveness vary depending on the clinical scenario and how well the patient's environment can be controlled. POCUS provides rapid real time patient assessment that can guide volume status and fluid responsiveness regardless of the setting. The clinician should however consider a gray-zone approach in answering the question of volume status and fluid responsiveness to minimize clinically relevant hypo- or hypervolemic states and ensure optimal fluid management in critical care patients. Within the gray-zone approach more precise titration of fluids becomes paramount and fluid responsiveness should be assessed through techniques that minimize the risk of volume overload; small IV bolus administration, and/or assessment of parameters that respond to heart-lung interactions in the absence of administering fluids.Both authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.