At the end of life oral fluid intake is often reduced. Consensus about the most appropriate management for terminally ill patients with limited oral fluid intake is lacking. The objective of this study is to investigate to what extent the amount of fluid intake, preceding and during the dying phase, is related to the occurrence of death rattle and terminal restlessness.A multicentre prospective observational study was performed. Data on the occurrence of death rattle and terminal restlessness, fluid intake and opioid use of patients expected to die within a few days or hours were collected.371 patients were included. Death rattle was reported at least once in 40% (n=149) of patients during the dying phase. Death rattle occurrence was not associated with the amount of fluid intake during the days before dying. Terminal restlessness was reported in 26% of patients (n=96). Terminal restlessness was not associated with a lower amount of fluid intake during the days before dying. Terminal restlessness during the last 24 hours of life was associated with a higher amount of fluid  during 48–25 hours before death.Caution with fluid intake to prevent development of death rattle does not seem to be necessary. Our study suggests that a higher amount of fluid intake during 48–25 hours before death may be associated with the occurrence of terminal restlessness during the last 24 hours of life. These results suggest that actively providing dying patients with artificial fluid may not be beneficial. Most patients with a deteriorating chronic illness have a reduced oral intake at the end of life. This may be due to illness or treatment-related symptoms or complications, such as dysphagia, nausea or vomiting, generalised weakness and, in the last days of life, to a decreased level of consciousness or a loss of desire to drink.The debate about possible benefits of AH has focused especially on two distinct symptoms in particular: death rattle and terminal restlessness. Death rattle due to respiratory tract secretion is a common symptom with a prevalence of 35% among dying patients and has been linked to overhydration at the end of life.We performed a multicentre prospective observational study in patients, 18 years or older, who were, according to the multidisciplinary care team, likely to die within a few days. Data were collected in eight hospitals  and five hospices, including three palliative care units in nursing homes, in the Netherlands. Data collection took place between November 2012 and November 2013.Anonymous data were collected using a digital version of the Care Program for the Dying (CPD), a Dutch instrument for multidisciplinary care for patients in the dying phase that was originally based on the Liverpool Care Pathway for the dying patient.10.1136/bmjspcare-2018-001729.supp1Supplementary dataPatients were excluded from the study if data were missing on the date and time of the start of the CPD, the date and time of death or for more than six consecutive measurements .Duration of the dying phase was calculated using the start date and time of the CPD and the date and time of the patient’s death. Total fluid intake was based on the intake of oral fluid, intravenous fluid as well as fluid intake via a feeding tube. Nurses estimated patient’s oral intake during the week before the start of the CPD, during the last day before the start of the CPD and during the dying phase. The fluid intake by different routes was added per period of time and scored into three categories: 0–499 mL/day, 500–999 mL/day and ≥1000 mL/day for the periods preceding the start of the CPD; and 0–249 mL/day, 250–499 mL/day and ≥500 mL/day during the dying phase. Opioid doses were recalculated to the morphine equivalent daily dose (mg/day) according to published equianalgesic dose tables;In this study, the dying phase was defined as the phase when death is expected to occur within hours or days.fluid intake and opioid dose during 48–25 hours before death  were calculated using the four hourly consecutive measurements from part 2 of the CPD. In case the dying phase had a duration between 24 and 48 hours, fluid intake and opioid dose were calculated using data concerning the intake/dose during the last day before the start of the CPD and the four hourly information from the consecutive measurements from part 2 of the CPD. In case the dying phase had a duration shorter than 24 hours, fluid intake and opioid dose were based on the data concerning the intake/dose during the last day before the start of the CPD .In case the dying phase had a duration longer than 48 hours,  the CPD . Death rThe primary outcome of this study was the association between death rattle or terminal restlessness and patients’ fluid intake during the week and day before the start of the CPD and during 48–25 hours before death. The statistical significance of this association was tested using the non-parametric Mann-Whitney tests. The secondary outcomes were associations between death rattle or terminal restlessness and other characteristics, including patients’ gender, age at death, diagnosis, place of death, duration of the dying phase, level of consciousness at the recognition of the dying phase and opioid use . These were analysed using Chi-Square test or Mann-Whitney test, where appropriate. Non-parametric tests were used when data were not normally distributed. All analyses were performed using SPSS for Windows V.22.0 (SPSS).During the study period 631 patients died in the participating care settings. The CPD was initiated for 476 patients , 371 of whom were included in this study . One hundred and five patients could not be included; 49 due to missing data about the date and time of the start of the CPD and/or death and 56 due to missing data for more than six consecutive four hourly measurements (part 2 of the CPD) .The included patients had a mean age of 72 years, almost half of them were male and 79% had been diagnosed with cancer . Forty-fPatients’ total fluid intake decreased during the last phase of life . During No significant association between death rattle and the amount of fluid intake was found . A higheWe found no significant association between the amount of fluid intake and the occurrence of death rattle. We did not find an association between a lower amount of fluid intake and terminal restlessness either. Terminal restlessness during the last 24 hours of life was however associated with a higher amount of fluid intake during the time period of 48–25 hours before death.et alet alet alet alet alThree previous studies have assessed death rattle occurrence and its association with AH.et alet alet alet alWe did not find an association between a lower amount of fluid intake during the days before dying and the occurrence of terminal restlessness. Terminal restlessness during the last 24 hours of life was however associated with a higher amount of fluid during the time period of 48–25 hours before death. Previous studies on the relation between fluid intake and occurrence of terminal restlessness or delirium showed diverse results. Morita Patients’ total fluid intake overall decreased during the last phase of life. However, in the hospital population the percentage of patients receiving parenteral fluids increased with death approaching. It is unclear why, but it is possible that healthcare professionals in hospital are more inclined or used to provide parenteral hydration at the end of life compared to professionals working in a hospice. It is also possible that both patient groups differed, eg in diagnosis. However, this did not result in a different occurrence of terminal restlessness and death rattle.et alet alThis study has some limitations. First, the optimal design to study the effects of fluid intake would be a randomised controlled trial, which would however pose ethical challenges. We conducted a prospective observational study to explore variations in fluid intake in daily practice. A recent study by Davies In conclusion, we found that a higher amount of fluid intake , preceding and during the dying phase, was not associated with the occurrence of death rattle. Further, a lower amount of fluid intake , preceding and during the dying phase, was not associated with the occurrence of terminal restlessness. Caution with fluid intake to prevent development of death rattle does not seem to be necessary. Our study suggests that a higher amount of fluid intake during the period of 48–25 hours before death may be related to occurrence of terminal restlessness during the last 24 hours of life. These results suggest that actively providing dying patients with artificial fluid may not be beneficial, although intervention studies are needed to test this assumption.


Staphylococcus aureus is the most frequently isolated organism associated with periprosthetic joint infections of the knee or hip, and due to the emergence of antibiotic-resistant strains, treatment options are limited. Here, we show that synovial fluid from osteoarthritic patients is iron restrictive toward S. aureus and, for strains representing the clonal lineages USA100, USA200, USA400, and USA600, bactericidal. Remarkably, community-associated methicillin-resistant S. aureus (CA-MRSA) strain USA300-LAC was highly resistant to synovial fluid killing but could be sensitized to killing by mutation of the GraXRS regulatory system and GraXRS-regulated mprF gene or by small-molecule inhibition of GraR. Thus, we propose the GraXRS-VraFG regulatory system and mprF as targets for future therapeutics for treatment of S. aureus bone and joint infections.Osteoarthritis is the most prevalent joint disease in the United States, with many patients requiring surgical replacement of the affected joint. The number of joint arthroplasty procedures performed each year is increasing, and infection is a leading cause of implant failure. IMPORTANCE Osteoarthritis, a degenerative disease that results in the breakdown of joint cartilage and underlying bone, is the most prevalent joint disease in the United States. Surgical intervention, including total joint replacement, is a clinically effective procedure that can help to restore the patient’s quality of life. Unfortunately, joint replacement procedures come with a risk of infection that is estimated to occur in 1 to 2% of cases, and periprosthetic joint infection (PJI) is a leading cause of implant failure, requiring revision surgery. Staphylococcus aureus is well known for its ability to cause PJIs and was found to be the most frequently isolated organism from PJIs of the knee or hip. Antibiotic-resistant strains can often limit treatment options. In this study, we demonstrate that the MRSA strain LAC can resist killing and grow in human synovial fluid from osteoarthritic knees. Furthermore, we show that the GraXRS regulatory system is required for the displayed synovial fluid resistance. We further demonstrate that a small-molecule inhibitor of GraR sensitizes LAC to synovial fluid, validating the Gra system as a therapeutic target for the treatment of PJIs in humans. Osteoarthritis is the most prevalent joint disease in the United States and other developed countries, with osteoarthritis of the knee accounting for over 80% of the total disease burden , 2. OsteStaphylococcus aureus is a Gram-positive facultative anaerobe that is found as a commensal species, colonizing the anterior nares of approximately 30% of the human population, but it is also an opportunistic pathogen  poses a serious risk not only to the future success of the implanted joint but also to the patient’s life. Despite this, treatment options are severely limited, and therefore new therapeutics are desperately needed.pathogen . S. auremyelitis . Furthere or hip . Additioesistant . PJI by S. aureus, S. epidermidis, and S. pyogenes, which are all common causes of bone and joint infections  in human colonization and infection, including CC5 (USA100), CC30 (USA200), CC8 (USA300), CC1 (USA400), and CC45 (USA600), along with the osteomyelitis isolate UAMS-1. The bacteria were incubated at 37°C in RPMI 1640 with, or without, synovial fluid at various concentrations and then enumerated after 24 h. We found that the presence of synovial fluid significantly impacted the growth of all strains tested, although the levels of sensitivity varied greatly from strain to strain  strain LAC exhibiting a high level of resistance.To initially explore the effect of synovial fluid taken from osteoarthritic knees against o strain . Strainso strain  and B. Uo strain . MW2  gel was run to determine the protein profiles of synovial fluids from different osteoarthritic donors. Various bands were excised and identified by matrix-associated laser desorption–ionization mass spectrometry (MALDI-MS), which confirmed the presence in synovial fluid of transferrin and several other proteins, which are listed in 16–S. aureus. To determine this, we examined the relative expression levels of several iron-regulated genes, with and without exposure to synovial fluid, in LAC and UAMS-1, which were chosen as representative resistant and sensitive strains and because of their relevance to bone and joint infections  LAC in the presence or absence of synovial fluid. We observed that the LAC graS mutant was sensitive to killing in as little as 25% synovial fluid, whereas WT LAC was completely resistant in 50% synovial fluid, the highest concentration tested  LL-37, human β defensin-2, and human β defensin-3 were previously identified in joint aspirates of patients with periprosthetic joint infections , 28. S. G system –33. Thusn tested . Notablysistance , 35. Theal fluid . When weal fluid . We nextal fluid . Indeed,al fluid ; this leS mutant . These dgraS-regulated mprF for survival in synovial fluid, we next measured the expression levels of mprF, dltA, sbnA, and sfaA for LAC  compared to those in UAMS-1  when cultured with and without synovial fluid. Strains were grown to an optical density at 600 nm (OD600) of 0.5 in RPMI 1640, which was then supplemented with 5% synovial fluid, or not, for 2 h, after which the expression levels of the aforementioned genes were measured using quantitative PCR (qPCR) and compared between strains. We found that LAC expressed higher levels of sbnA than UAMS-1, with and without exposure to synovial fluid, and that synovial fluid led to significantly increased expression of mprF and sfaA by LAC relative to that in UAMS-1 , or a diagnosis of fibromyalgia were excluded. All samples were tested individually and were not pooled unless otherwise stated. Patients were monitored at their standard follow-up appointments with the surgeon for adverse events or complications according to the standard of care at the University Hospital. There was no follow-up monitoring for this study.The fluid was then spun in a centrifuge for 10 min at 4,700 × S. aureus strains used in this study are listed in S. aureus. Overnight cultures of the bacteria were grown in 5-ml cultures of TSB and normalized to an OD600 of 1.0 in sterile saline. Tubes containing a 2-fold dilution series of synovial fluid in RPMI 1640 were inoculated with 10 μl of the bacterial suspension at an OD600 of 1.0 to start each tube at an initial OD600 of 0.01, equivalent to ∼2 × 106 to 4 × 106 CFU/ml. The samples were incubated at 37°C with shaking at 200 rpm for 24 h, after which the samples were serially diluted and 10 μl was drop plated on TSA plates. After overnight incubation, the colonies were counted to determine the final number of CFU per milliliter for each sample.Aliquots of frozen human synovial fluid samples from individual donors were thawed and diluted in the chemically defined medium RPMI 1640 immediately prior to use for growth assays with S. aureus bacteria to survive in synovial fluid. As before, aliquots of synovial fluid were thawed immediately prior to use and were diluted in saline in order to assess bacterial survival/death in the absence of replication. FeCl3, transferrin (Sigma-Aldrich), or the GraR inhibitor Mac-545496 (Cedarlane) were added when needed at the concentrations indicated in the figure legends. Bacteria were grown overnight in tubes containing 5 ml of TSB and with 1 μM Mac-545496 when needed and normalized to an OD600 of 1.0 prior to inoculation at an initial OD600 equivalent to 0.01 in growth medium containing synovial fluid. All samples were plated immediately after inoculation to determine the starting number of bacteria, expressed as CFU per milliliter. Samples were again plated after a 24-h incubation at 37°C to determine the number of bacteria remaining.Viability assays were performed to determine the ability of various strains and mutants of Synovial fluid samples from random patients were thawed and diluted to an initial concentration of 20% (vol/vol) in saline. Then, 10 μl of the diluted fluid from different donors was mixed 1:1 with Laemmli buffer and boiled for 10 min. After cooling, 7 μl of each sample was loaded and run on a 12% sodium dodecyl sulfate-polyacrylamide gel in Tris-glycine buffer at 150 V and 500 mA for 90 min. Upon completion, the gel was stained with InstantBlue, and an automated spot picker at the London Regional Proteomics Centre was used to excise individual protein bands from the gel. The samples were digested with trypsin, and MALDI-MS was performed on the digested samples.3, to an OD600 of 0.5 and then either supplemented with 5% (vol/vol) synovial fluid or kept as no-synovial-fluid controls and incubated for 2 h at 37°C with shaking. A volume of each culture containing the equivalent number of cells as a 1-ml culture at an OD600 of 3 was mixed with a 2× volume of RNAprotect (Qiagen) and incubated at room temperature for 5 min before being pelleted and frozen. RNA was isolated using an RNeasy minikit (Qiagen), and 500 ng of RNA was reverse transcribed using a one-step reverse transcription (RT)-PCR kit (Bio-Rad) according to manufacturer’s instructions. cDNA was PCR amplified using iTaq SYBR green (Bio-Rad), and data were normalized to data for the reference gene, rpoB. Primers used are listed in t method (where Ct is the cycle threshold)  , and staData are presented as means ± standard deviations (SD). All statistical analyses and graph production were done through GraphPad Prism .Prior to patient enrollment for the synovial fluid collection, this research was approved by the Health Sciences Research Ethics Board of the University of Western Ontario and Lawson Health Research Institute. An informed consent was obtained from each patient prior to the procedure.


The surfactant concentrations varied from 0.2 mmol/L to 3.0 mmol/L. All of the hydrogels presented viscous behavior (G″ > G′). Hydrogels containing AOT (c > 2.0 mmol/L) led to cryogels with the lowest compressive modulus (13 ± 1 kPa), the highest specific surface area (2.31 m2/g), the lowest thermal conductivity (0.030 W/(m·°C)), and less hygroscopic walls. The addition of Kolliphor® EL to the hydrogels yielded the stiffest cryogels (320 ± 32 kPa) with the lowest specific surface area (1.11 m2/g) and the highest thermal conductivity (0.055 W/(m·°C)). Density functional theory (DFT) calculations indicated an interaction energy of −31.8 kcal/mol due to the interaction between the AOT sulfonate group and the HPMC hydroxyl group and the hydrogen bond between the AOT carbonyl group and the HPMC hydroxyl group. The interaction energy between the HPMC hydroxyl group and the Kolliphor® EL hydroxyl group was calculated as −7.91 kcal/mol. A model was proposed to describe the effects of AOT or Kolliphor® EL on the microstructures and the mechanical/thermal properties of HPMC cryogels.The mechanical and thermal properties of cryogels depend on their microstructure. In this study, the microstructure of hydroxypropyl methylcellulose (HPMC) cryogels was modified by the addition of ionic (bis (2-ethylhexyl) sodium sulfosuccinate, AOT) and non-ionic (Kolliphor There are different methods for preparing 3D porous polymeric structures. One of them is the addition of porogenic agents to the polymerization process or to the polymer system. The choice of porogens requires a careful analysis on their chemical structure, physical state, solubility, and miscibility with other chemical constituents  . Other sructures . Cryogelructures . On the −3 and surface area larger than several hundreds of m2 g−1. For instance, chitosan aerogels presented a thermal conductivity of 0.022 W/(m K) and a specific surface area of 545 of m2 g−1 [2 based aerogels presented a thermal conductivity of (0.022–0.025 W m−1 K−1) [−1 K−1) and density values below 0.15 g cm−3 [Polysaccharide-based porous structures are sustainable materials because they stem from renewable sources, they are biodegradable and nontoxic, and their preparation does not generate hazardous waste. Aerogels and cryogels have interconnected micro- and macropores and low density and low thermal conductivity, respectively. Recently, Zou and Budtova presented an overview on the correlation between the thermal conductivity, density, and surface area of polysaccharide-based aerogels and cryogels reported in the literature . They cof m2 g−1 . Pectin-m−1 K−1) , whereas5 g cm−3 . However5 g cm−3 ; the dis5 g cm−3 .co-polyethylene glycol diacrylate) [One strategy to control the microstructure of cryogels and therefore their properties is the addition of surfactants to the hydrogel precursor. Ni et al. prepared Konjac glucomannan-based cryogels from hydrogel precursors containing different glyceryl monostearate contents . A high crylate) . However® EL (Polyoxyl-35 castor oil), a non-ionic surfactant with long alkyl chains that is frequently used as emulsifier in cosmetic and drug formulations [Hydroxypropyl methylcellulose (HPMC) is a cellulose ether with amphiphilic character controlled by the degree of substitution (DS) of the methyl groups and the molar substitution (MS) of the hydroxypropyl groups in the repeated unit . Due itsulations , or dioculations , to HPMC3 (AOT5) to 39 ± 5 kg/m3 (K7.25). The gel content, which indicates the efficiency of the esterification reaction between citric acid carboxyl groups and HPMC hydroxyl groups, decreased from 86 ± 1% (HPMC~K0.5) to 61 ± 1% (K7.25) as the Kolliphor® EL concentration in the precursor hydrogels increased. This finding indicates that the Kolliphor® EL hydroxyl groups might compete with the HPMC hydroxyl groups for esterification with the citric acid carboxylic acid groups [−1 assigned as the ester -C=O stretching vibrations in all of the cryogels evidenced crosslinking between HPMC and citric acid [−1.The sample codes and their compositions are listed in d groups . In the ric acid ,22. Noteric acid ,22, but Y) at which the material starts to plastically deform for all of the cryogels. All of the cryogels prepared with Kolliphor® EL were considerably stiffer than pure HPMC cryogels. On the other hand, the addition of a low content (less than 1.0 mM) of AOT in the hydrogel precursors resulted in the cryogels being slightly stiffer than HPMC cryogels, whereas the addition of a higher content (more than 2.0 mM) of AOT yielded very soft cryogels. AOT7.25 presented a mean E value one order of magnitude smaller than that of the HPMC cryogel. A plausible explanation for this effect is that the addition of AOT yielded cryogels with thinner cell walls than those of pure HPMC cryogels.® EL stiffened the HPMC cell walls, whereas the presence of AOT (at concentration higher than the cmc) yielded softer cryogels.The type or the amount of surfactant had no substantial effect on the rheological behavior of the precursor hydrogel at 25.0 ± 0.5 °C. All of the hydrogels showed viscous behavior (G″ > G′) , in agre® EL presented large pores in a lesser amount, implying cell wall thickening. On the other hand, the cryogels prepared with AOT presented small pores in larger quantities, implying cell wall thinning. These trends explain the high values of Young’s moduli determined for cryogels prepared with Kolliphor and the low values of Young’s moduli determined for cryogels prepared with AOT , and av2 g−1 and ~1.8 m2 g−1 determined for AOT and HPMC are of the same order of magnitude of those determined for cellulose nanofibrils/nonionic polyoxamer-based foams [® EL hydroxyl groups compete with the HPMC hydroxyl groups for esterification with citric acid. One should notice that the connectivity density correlates to the amount of the connected polymeric material to form the cell walls [ed foams  and nanoed foams ,28. Furtll walls . Cryogelll walls  might ink) values obtained using the THS method at 23.7 ± 0.7 °C and 60% relative humidity. The linear region between 46 s and 164 s of each curve was fitted to linear regression  although there was no statistically significant difference (pn = 3) among this k value and those determined for the AOT2.75, AOT5, K0.5, and K7.25 cryogels; it was statically different from the HPMC (0.038 W m−1 °C−1) cryogel. Cellulose nanofibrils crosslinked with 1,2,3,4-butanetetracarboxylic acid and N-methylol dimethylphosphonopropionamide led to sponges with a k value (0.0326 W m−1 °C−1) and an E value of ~5 kPa [k value (0.033± 0.001 W m−1 °C−1) and E value of ~13 ± 1 kPa. The highest k values were observed for K2.75 (0.055 W m−1 °C−1), K5 (0.045 W m−1 °C−1), and AOT0.5 (0.043 W m−1 °C−1); similar values were reported for nanofibrillated cellulose sponges [gression ; the lowf ~5 kPa ; these p sponges . Materia sponges , becausek values obtained are consistent with the range observed by Jimenez-Saelices et al. [The s et al. . The heall voids .3). Among all samples, AOT7.25 presented the smallest lowest k value because it contains the largest surface area (Asp) and the smallest pore size ; the compressive modulus can be tuned by the amount of AOT added to the precursor hydrogel, keeping low thermal conductivity. Cryogels prepared with Kolliphor® EL occupied a region (magenta) characteristic of higher compressive modulus and thermal conductivity values; the thermal conductivity can be tuned by the content of Kolliphor® EL in the precursor hydrogel.® EL, and AOT was evaluated using TG/DTG curves  between 30 °C  of the HPMC cryogels, K0.5, AOT 0.5 (lowest surfactant concentration), and K7.25 (highest surfactant concentration) were similar, ranging from 32 ± 1 ggwater/g . However® EL system, the calculated interaction energy was −7.91 kcal/mol. ® EL, where only one H bond between the Kolliphor® EL hydroxyl and the HPMC hydroxyl groups drives the interaction.Density Functional Theory (DFT) calculations were performed in order to gain insight into the molecular interactions between the surfactants and the HPMC in vacuum, which might be correlated to the molecular interactions in the cryogels. The optimized geometries indicated an interaction energy of −31.8 kcal/mol for the HPMC–AOT system, driven by the strong interaction between the AOT sulfonate group (ion) and the HPMC hydroxyl group (dipole) and the hydrogen bond between the AOT carbonyl group and the HPMC hydroxyl group, as depicted in n < 20) [The relation between the microstructure and the mechanical/thermal properties of HPMC cryogels could be tuned by the concentration of AOT added to the precursor HPMC hydrogels; the highest AOT concentrations (AOT5 or AOT7.25) led to soft cryogels with a relatively low thermal conductivity, which were associated to a microstructure of many small pores and thin cell walls. Furthermore, these cryogels were less hydrophilic than the control (HPMC cryogel). AOT is frequently used as an emulsifier in water/oil emulsion, building reverse micelles, where the aggregation number is usually low (n < 20) . In HPMCn < 20) . Based on < 20) . Therefo® EL added to the precursor hydrogels led to cryogels with a high compressive modulus and high thermal conductivity compared to HPMC cryogels. DFT calculations evidenced that the interactions between Kolliphor® EL and HPMC are not as strong as those between AOT and HPMC are. The presence of the Kollipho® EL micelles [The concentration of Kolliphormicelles  possibly−1 °C−1) and low hygroscopicity. The interactions between Kolliphor® EL and HPMC are weak; the Kolliphor® EL micelles probably increased the ice crystal size, yielding stiffer cryogels. The stiffest cryogels (E = 55 kPa) presented the highest thermal conductivity value (0.055 W m−1 °C−1). By changing the type and concentration of the surfactant in the precursor hydrogels, a wide range of compressive modulus and thermal conductivity values could be achieved  cryogels with relatively low thermal conductivity , dioctyl sulfosuccinate salt sodium or AOT , citric acid , and sodium hypophosphite  were used as received. MilliQ water was employed in all experiments. The critical micelle concentration (cmc) of AOT and Kolliphor® EL in water were determined as 1.9 mM and 0.02 mM, respectively, at 21 ± 1 °C  of 1.5 and a molar substitution (MS) of 0.75 (USP 1828) was kindly supplied by The Dow Chemical Company ; the viscometric average molar mass  and sodium hypophosphite  were then added at 1.0 g·L−1 and 0.5 g·L−1; these concentrations were chosen based on previous reports [−1 and was homogenized for 1 h. The resulting hydrogels were coded as HPMC , K0.5 and AOT0.5 (0.2 mM), K2.75 and AOT2.75 (1.0 mM), K5 and AOT5 (2.0 mM), and K7.25 and AOT7.25 (3.0 mM), as shown in ® EL in all gels was above of its cmc (0.02 m)M, whereas the concentration of AOT in the AOT0.5 and AOT2.75 gels was below its cmc, and in the AOT5 and AOT7.25 gels, it was above its cmc. Aqueous solutions of AOT and Kolliphor reports . After 3The precursor hydrogels were transferred to acrylic molds of different geometries: A , B , C , D , and E . The molds were covered in order to avoid significant volume variation due to ice crystallization during freezing. They were kept inside three Styrofoam boxes  for 24 h® rheometer from Anton Paar at 25.0 ± 0.5 °C under a dry nitrogen atmosphere. A cone and plate geometry (angle of 0.992°) with a gap size of 0.101 mm and a diameter of 50 mm was used. For DSST, pure HPMC (blank), K7.25, and AOT7.25 hydrogels were tested at the frequencies of 1 Hz and 10 Hz, measuring G′, G″ as a function of the strain amplitude (Dynamic strain sweep tests (DSST), dynamic time sweep tests (DTST), and small amplitude oscillatory shear (SAOS) tests were performed for the hydrated samples using a stress-controlled MCR 501mplitude . In orde at 0.5% . The obt−1 in order to remove unreacted molecules. The cryogels were then frozen and freeze-dried again. The apparent density (apρ) of six dried cryogels (mold B) was determined at 23 ± 1 °C and relative air humidity of 65 ± 10%. The ratio between their masses  until the rinsing water achieved the conductivity of ~5 µS·cmTo determine the gel content (percentage of crosslinked HPMC chains), the cryogels (mold C) were swollen with MilliQ water, freeze-dried, and weighed (waterm) divided by driedm, as shown by Equation (3): The swelling degree (SD) was determined for HPMC, K0.5, AOT0.5, K7.25, and AOT7.25 (mold C) samples using a force tensiometer Krüss K100 at 21.0 ± 0.5 °C as the mass of sorbed MilliQ water (pH 5.5) at equilibrium (®) with a diamond crystal and an accumulation of 128 scans (2 cm−1 resolution). Thermogravimetric analyses (TGA) were performed for the HPMC, K0.5, AOT0.5, K7.25, and AOT7.25 samples (mold C) in alumina crucibles using the Netzsch STA 449 F3 Jupiter (Netzsch®) equipment under a N2 atmosphere. The temperature program had three segments: 1 (from 30 °C to 200 °C at a heating rate of 20 °C/min), 2 (from 200 °C to 30 °C at a cooling rate of −20 °C/min), and 3 (from 30 °C to 500 °C at a heating rate of 10 °C/min). The first run up to 200 °C or well below the degradation point was performed in order to remove thermal history and moisture. Afterwards, the second run was performed slowly at the heating rate of 10 °C/min to observe the actual thermal stability in TGA and its char yield.FTIR-ATR vibrational spectra of the HPMC, K0.5, AOT0.5, K7.25, and AOT7.25 samples (mold C) were obtained using an Alpha FTIR-ATR spectrometer  method ,39,40,41.5 kg/m3 ). The co® in order to obtain the electric power supplied to the strip over time. An Arduino Uno R3 board was used to transfer the acquired data to the computer .All measurements of thermal conductivity were performed with a low cost (~USD 100) homemade device. It comprises an isothermal aluminum case with a NiCr resistive strip (2.0 mm × 0.1 mm × 110.0 mm) positioned at the center of the case, and one of the sample faces was against the strip. One piece of PU foam (reference) was placed against the other side of the strip, and another piece of PU foam was placed against the sample, as shown in computer b. The te−1 and at an air temperature of 23 ± 1 °C (60% relative humidity). The samples remained close to the dynamometer under 60% relative humidity for 24 h as recommended by ISO 3386-1 [The compressive tests were performed in quadruplicate for the cryogel samples (mold B) using an Impac Digital Dynamometer IP-90DI with a 1 kN load cell at strain rate of 0.01 sO 3386-1 . ® software were the specific area 3); and average pore size. The pore size distributions in the HPMC, K7.25, and AOT7.25 samples were calculated with CTan® software.VOI (volume of interest) is the reconstructed volume from which the software performed the calculations, VOI, and ρ is the material density, which was considered as 34 kg/m3 (Freeze-dried cryogels (mold D) coated with a gold layer (~10 nm) were analyzed in a JEOL Neoscope JCM-5000 microscope operating at voltage of 10 kV. The cryogels (mold E) were analyzed using X-ray microtomography (micro-CT) with Bruker Skyscan 1272 equipment with the X-ray source operating at 20 kV and a current of 175 μA. The samples were rotated 0.6° per step, obtaining a resolution of 4 μm. The NRecon  and CTVOx  software programs were used for the 3D reconstruction from cross-sectional images and for the visualization and image acquisition, respectively. Specific surface area values of volumes of interest (VOI) were calculated using the software CTan . The analyses of the VOIs with the CTan34 kg/m3 .® EL–HPMC in vacuum. For the calculations, an HPMC tetramer containing 6 -CH3, 3 -CH2-CHOH-CH3, and 5 OH groups and one third of the Kolliphor® EL molecule were drawn (the -O-CH2- bond from the precursor glycerol was considered as -O-CH3), as shown in Density Functional Theory (DFT) calculations were performed using the software Gaussian 09  with the


Fluid overload has a high prevalence in haemodialysis patients and is an important risk factor for excess mortality and hospitalisations. Despite the risks associated with chronic fluid overload, it is clinically difficult to assess and maintain fluid status adequately. Current methods of fluid status assessment are either imprecise or time intensive. In particular, to date, no method exists to accurately assess fluid status during the interdialytic interval.This pilot study aimed to evaluate whether a prototype wearable hydration monitor can accurately and reproducibly detect fluid overload in the haemodialysis population when compared to haemodialysis and bioimpedance data.A prospective, open-label, single-arm observational trial of 20 patients commenced in January 2021 in a single haemodialysis centre in Ireland, with a wearable hydration monitor, the Sixty device. The Sixty device uses diffuse reflectance spectroscopy to measure fluid levels at the level of the subdermis and uses machine learning to develop an algorithm that can determine fluid status. The Sixty device was worn at every dialysis session and nocturnally over a three-week observational period. Haemodialysis parameters including interdialytic weight gain, ultrafiltration volume, blood pressure, and relative blood volume were collected from each session, and bioimpedance measurements using the Fresenius body composition monitor were performed on 4 occasions as a comparator. The primary objective of this trial was to determine the accuracy and reproducibility of the Sixty device compared to bioimpedance measurements.If the accuracy of the wearable hydration monitor is validated, further studies will be conducted to integrate the device output into a multi-parameter machine learning algorithm that can provide patients with actionable insights to manage fluid overload in the interdialytic period.www.clinicaltrials.govNCT04623281. Registered November 10th, 2020. Haemodialysis (HD) patients experience an excess of cardiovascular morbidity and mortality . Volume Maintaining fluid is an integral aspect of haemodialysis care, but is difficult to perform adequately, as reflected in the high prevalence of fluid overload . TraditiBioimpedance is a validated method of estimating volume overload by directly assessing the extracellular water (ECW), intracellular water (ICW), and total body water (TBW) compartments, and compares well against gold standard methods , but is To date, no specific outpatient wearable hydration monitor for haemodialysis patients is available and validated. The purpose of this trial is to compare the reproducibility and accuracy of a wearable hydration monitor, the Sixty device, against standard haemodialysis parameters and bioimpedance measurements. If this hydration monitor is validated, it will contribute to the development of a multivariate algorithm to assist haemodialysis patients in monitoring and adjusting their fluid intake. To our knowledge, this approach has not yet been explored in haemodialysis patients.This trial was conducted to evaluate if a wearable hydration monitor (the Sixty device) has validity and reproducibility against traditional and validated methods of volume assessment in haemodialysis patients. If the device is validated, further studies will be conducted to integrate the Sixty device data into a multi-parameter machine learning algorithm that can provide patients with actionable insights to manage fluid overload in the interdialytic period.This was a pilot-scale study to assess if this approach has merit and could be tested and developed further. As no specific wearable hydration monitor for haemodialysis patients is available and validated, there was sufficient justification in testing the effectiveness and acceptability of the Sixty device in a controlled observational setting.The primary objective was to evaluate the accuracy of the Sixty device in assessing volume in haemodialysis patients compared to bioimpedance measurements using the Fresenius body composition monitor (BCM).Comparison of fluid status as measured by the Sixty device compared to bioimpedance measurements performed pre-dialysis by a body composition monitor .Comparison of changes in fluid status as determined by the Sixty device versus ultrafiltration millilitres/per unit time during a haemodialysis sessionComparison of changes in fluid status as determined by the Sixty device versus change in weight pre and post dialysis (kg)Comparison of changes in fluid status as determined by the Sixty device versus change in blood volume monitoring (relative blood volume %)Comparison of changes in fluid status as determined by the Sixty device versus blood pressure (mmHg)The patient's opinion of the acceptability of the Sixty device as assessed by their response to a questionnaire.The outcome measures were chosen as proxies of fluid status used in clinical practice.This was a prospective, open-label, single-arm observational study with wearable hydration, the Sixty device, occurring over 3 weeks in a single centre at Beaumont Hospital, a tertiary care centre for nephrology in Dublin, Ireland Fig. .Fig. 1DiAn estimated 20 patients were recruited. All patients were allocated to the same observation sequence. Patients followed their usual haemodialysis regimen throughout the study. Patients’ dry weights were optimised using bioimpedance measurements during the study.Patients were asked to wear a wearable hydration monitor on their forearm during haemodialysis sessions and nocturnally. Clinical assessments  were assessed at each dialysis visit. Bioimpedance measurements using the body composition monitor (Fresenius BCM) were performed at the start of the observation period, and once a week before the mid-week dialysis session.Approximately 300 patients attend for haemodialysis in an ambulatory care setting under the governance of Beaumont Hospital. Eligible individuals were invited to participate. Patients with a range of baseline fluid status, age, and gender were considered for enrolment. Written informed consent was obtained for each patient prior to starting the study. Twenty  people wParticipants were able to enrol in the study if they were attending the haemodialysis unit for haemodialysis at least two to three times a week, and if all of the following applied at initial consent: aged at least 18 years; demonstrates understanding of correct use of the Sixty device; willing to give written informed consent.Participants were excluded from the study if there were conditions precluding accurate use of bioimpedance such as the presence of an implantable cardioverter defibrillator, pacemaker, hearing aids, or pregnancy. Patients were furthermore excluded if there was significant confusion or any concomitant medical condition, which would limit the ability of the patient to record symptoms or other parameters.Patients’ consent was sought during the normal haemodialysis treatment sessions. Patients were provided with a plain-language patient information leaflet related to the Sixty device, as well as a consent form explaining the study rationale, processes, data storage procedure, and the contact details for researchers. Consent was obtained from patients by the co-investigators of the trial, or a suitable health research personnel nominee. Recruitment was continued until the planned sample size was achieved.Patients were free to withdraw from the study at any time without any impact on their ongoing medical care.The investigator could withdraw a patient from the study at any time if they believed that further participation in the study was not in the best interests of the patients. The study could be terminated early if recruitment was significantly behind schedule or if for any other reason, it was unlikely that the study would be completed.The Sixty device is a wearable hydration monitor that is currently in development by Design to Value Ltd. Photographs of the current prototype and expected final designs are shown in Fig. The prototype device (left-hand image) measures 66mm × 48mm × 10mm. The on/off button and charging port are visible on the top edge. The surface facing the camera was in contact with the dorsal surface of the user’s wrist. The expected final design is shown in the right-hand image. This is expected to be approximately the size of a commercially available smartwatch.The Sixty device combines photonics sensors and the principle of diffuse reflectance spectroscopy to determine fluid status. The sensors include red, green, and infrared wavelengths in the range of 530 to 950nm. The amount of light reflected back can be correlated with the concentration of molecules within the tissue (e.g. water). The algorithm has been trained using a calibrated dataset of known fluid states that allows the device to interpret the data and provide feedback on fluid status in kilogrammes and/or litres. As the algorithm has previously been trained on healthy volunteers, a new algorithm was developed for the prediction of fluid states in haemodialysis patients.The prototype device used in this study does not reflect the final aesthetic, but provides the necessary functionality to establish proof of principle in fluid overloaded patients. The prototype device only collected data and did not provide feedback to the user on their hydration status. Data was collected locally on a memory card (i.e. SD card) which was removed periodically to recover the data.The device and battery are designed to run for an extended period and can be charged via a USB cable. Charging the device and collection of data were coordinated at the haemodialysis study centre to minimise data gaps.The body composition monitor is a whole-body bioimpedance device that uses a multi-frequency current to measure body compartments, such as intracellular water (ICW), extracellular water (ECW), and total body water (TBW) through electrodes placed on a parallel wrist and ankle . In addiBCM is a reliable, validated method of fluid assessment that has been validated against gold standard dilution methods of volume assessment across multicentre studies in both healthy patients and haemodialysis patients, such as sodium bromide (ECW), deuterium and tritium (TBW) and total body potassium (ICW) . Good agThe trial procedures at each visit are summarized in the flow chart Table .Table 1FFollowing consent, demographic data, medical history and concomitant medications were obtained directly from the patient or from their electronic record (eMED).At the baseline visit and at each haemodialysis visit during the study the following usual care parameters were assessed and recorded directly from the patient and nursing staff and from their dialysis electronic record (eMED). Haemodialysis session data was downloaded directly from the dialysis online documentation system; the Fresenius Therapy Monitor (TMON). Symptoms, adverse events, and requirement for additional unscheduled dialysis were captured on case report forms, filed in the trial documentation on-site.Total fluid removal IDWGProportion of haemodialysis sessions in which IDWG is ≤ 4%Pre-dialysis and post-dialysis weightPre-dialysis, intradialytic, and post-dialysis BPPulse rateRelative Blood Volume (RBV %)Requirement for additional nursing intervention Adverse events related to the patient’s underlying condition Requirement for additional unscheduled dialysis, or need for hospitalisationNumber and types of BP medication(s)Haemodialysis session variables:The research team allocated a Sixty device to the patient and instructed them on the correct use of the device. The patient wore the Sixty device and switched it on before the start of haemodialysis at this clinic visit. The patient wore the Sixty device throughout the haemodialysis session at this and all subsequent haemodialysis sessions during the study observation period as well as nocturnally.At each clinic visit, before the patient started haemodialysis, the research team removed the SD card from the patient’s Sixty device and downloaded the data collected in the previous 2–3 nights. The SD card was re-inserted into the Sixty device and the patient wore the Sixty device during the haemodialysis procedure. If necessary, the Sixty device was recharged during haemodialysis and continued to collect data while recharging.The measurements related to haemodialysis and described previously  were recorded at each haemodialysis visit during the study observation period.Fluid status was assessed by bioimpedance immediately before haemodialysis on four occasions, once on the most fluid overloaded day following the long interdialytic interval. Thereafter, bioimpedance measurements were performed once a week prior to the mid-week dialysis session.Symptoms of hyper and hypovolaemia occurring within the past week were recorded once a week.Thirst directly after haemodialysisSymptomatic hypotensionNausea and vomitingMuscle crampsLimpness/tiredness between dialysis sessionsDizziness between dialysis sessionsSymptoms of hypovolaemia:Chronic coughing (new)Dyspnea on exertionDyspnea at rest; 1 pillowDyspnea at rest: 2 pillowDyspnea at rest: 3 pillowsPretibial oedemaParoxysmal nocturnal dyspnea (PND)Symptoms of hypervolaemia:The patients completed a questionnaire to give their opinion on the acceptability of the Sixty device. This was assessed and recorded following the end of the week 3 observation period.How do you currently manage your fluid intake in between dialysis sessions?On a scale of 1 to 5, how well do you feel in control of fluid status during this interdialytic period? (1 meaning “no control”... with 5 meaning “fully in control with good understanding what triggers fluid overload”)Would you wear a device similar in looks to a FitBit/Apple watch, if it could continuously monitor your fluid status and prompt you when and how much to drink to stay hydrated yet limit fluid overload?Tells me my hydration level and prompts me on how to manage fluid intakeTells the timeMeasures heart rateMeasures steps/activityMeasures sleep qualityWhat is the minimum acceptable functionality that this device would need to have in order for you to wear it (check box list of features such as):Examples of questions in the acceptability questionnaire include:The study procedures were concluded at this visit.The primary objective was to evaluate the accuracy and reproducibility of the Sixty device in assessing volume in haemodialysis patients compared to bioimpedance using the Fresenius body composition monitor (BCM).Volume overload as defined by bioimpedanceRelative overhydration: Overhydration (OH)/Extracellular water (ECW) > 7%Overhydration index: ≥ 1.1LComparison of fluid status as measured by Sixty device compared to bioimpedance measurements using BCM.Normohydration is defined as OH/ECW − 7 to + 7% (corresponding to OH index of − 1.1 L to + 1 L)Relative OH < − 7%OH index ≤ − 1.1LDehydration as defined by bioimpedanceThe primary endpoint variables included:Comparison of changes in fluid status as determined by the Sixty device versus ultrafiltration millilitres/per unit time during a haemodialysis session.Comparison of changes in fluid status as determined by the Sixty device versus change in weight pre and post dialysis (kg)Comparison of changes in fluid status as determined by the Sixty device versus blood pressure (mmHg)Comparison of changes in fluid status as determined by the Sixty device versus change in blood volume monitoring (Relative blood volume %)The patient’s opinion of the acceptability of the Sixty device as assessed by their response to a questionnaire.Secondary outcome measures:An accuracy of > 70% in predicting fluid status categories as measured by BCM and/or evidence of good correlation with BCM readings as indicated by a correlation coefficient > 0.70 will be considered sufficient justification for further trials. In the absence of the above, evidence of high correlation with ultrafiltration volume (> 0.70) will be considered reasonable grounds for further assessment of the device.All endpoint data was stored on a central database for analysis. The data as reported by the patients was queried before descriptive statistical analysis tables were prepared. Patients are identified by a unique identification number on the study database (i.e. data is pseudonymised). Each patient’s data is linked to their unique identification number.The original electronic data and relevant medical records were the source documents. The research team ensured that all data entered into the central database was a true record of events. Source data verification was not performed. Medical data relating to patient care has been stored in the medical records according to the usual procedures of the treatment site(s).The data processor for this study is patientMpower Ltd., a digital health company. The endpoint data recorded on the Sixty device, haemodialysis parameters (including bioimpedance data), and symptoms data is stored on a central database managed by patientMpower Ltd. Pseudonmyised data was transferred to patientMpower via a secure Amazon Web Service server. The pseudonymised data was shared with the Beaumont Hospital research team, Design to Value Ltd. (developer and owner of the Sixty device), and patientMpower Ltd.The trial was completed when 20 patients completed the 3-week observation period.All data was analysed on an intention to treat basis without regard to protocol violations.Patient demographic data will be reported as means and standard deviations or frequencies and proportions. “A proprietary algorithm is used by Design to Value Ltd. (developer of the Sixty device) to develop personalised determination of fluid status based upon the raw data from the Sixty device. The algorithm is capable of outputting an outcome concordant with fluid status in kilogrammes and/or litres. Haemodialysis sessions per patient will be divided into training (approx. 2/3) and test (1/3) sets to develop and test the Sixty machine learning algorithm. Reproducibility of the algorithm will be evaluated by evaluating the algorithm’s performance on the test dataset. Training of the algorithm will be performed by Design to Value Ltd., developers of the Sixty device. Evaluation of the algorithm on the test set will be performed by the clinical research team. The primary and secondary objectives will be analysed by the clinical research team.The primary objective will be assessed by examining the agreement or correlation between the Sixty hydration score within the first 20 min and last 20 min and BCM measures of fluid status including the overhydration index (OH) pre and post dialysis. This will assess the accuracy of the device algorithm.t-test will be calculated to determine if the fluid changes are statistically different.The change in Sixty hydration readings from start to end in dialysis (kg) will be calculated and compared with the change in ultrafiltration volume (l). The correlation and root mean squared error (RMSE) for the fluid change  will be calculated on training and test data separately. A The interdialytic change in the Sixty hydration value in kilogrammes for the start of the current dialysis session minus the end of the previous dialysis session will be calculated and compared to the interdialytic weight gain. The correlation and RMSE for the interdialytic change  will be calculated and a t-test will be applied to assess for statistical significance.t-test for statistical difference.The Sixty hydration value in kilogrammes for the start of dialysis, end of dialysis, and the change  will be compared to the pre-dialysis weight, post-dialysis weight and pre- minus post-dialysis weight respectively using correlation and RMSE, and a Nocturnal Sixty data will be evaluated by (1) assessing if there is a statistical difference between the means and standard deviations of nights one to three; (2) assessing if there is a statistical difference between the Sixty value at the end of a dialysis session and the Sixty value start of the nearest night and, similarly, for Sixty values at the start of a dialysis session versus Sixty values in the morning, following nocturnal wear; and (3) assessing if there is a statistical difference between the change in Sixty hydration values from start to end of dialysis versus change in Sixty hydration values overnight.A secondary analysis will determine the association between the Sixty hydration score and other correlates of volume including BP, RPV, and symptoms.The acceptability and utility of the Sixty device will be assessed by analysis of the responses to a patient questionnaire.Patient-reported symptoms and adverse events will be tabulated and displayed. Any additional healthcare resource utilisation  will be tabulated and displayed. No imputations of missing data will be made.No safety issues were anticipated from the use of the Sixty device or bioimpedance assessments. Issues regarding abnormal fluid assessments were addressed by the nephrology department at Beaumont Hospital.Any adverse events observed with medical treatments were reported to the manufacturers or suppliers of those treatments.Fluid overload, fluid depletion, and fluid status variability are independent risk factors for cardiovascular events and mortality in hemodialysis patients , 20. MaiNo universal standard for evaluating fluid status in dialysis patients exists. Clinical assessments of fluid, such as signs, symptoms, and BP measurements, lack the precision to detect subtle changes in volume status , 18. TheTo our knowledge, no device exists yet that can monitor patients’ fluid status interdialytically, during the outpatient period. If this wearable hydration monitor is validated, further studies will be conducted to integrate the Sixty device output into a multi-parameter machine learning algorithm that can provide patients with actionable insights into their fluid status. This algorithm will be tested prospectively in an observational study examining the use of digital health in fluid maintenance in dialysis patients.


Iatrogenic fluid overload is a potential side effect of intravenous fluid therapy in the hospital. Little attention has been paid to sodium administration as a separate cause of harm. With this narrative review, we aim to substantiate the hypothesis that a considerable amount of fluid-induced harm is caused not only by fluid volume, but also by the sodium that is administered to hospitalized patients.We show how a regular dietary sodium intake is easily surpassed by the substantial amounts of sodium that are administered during typical hospital stays. The most significant sodium burdens are caused by isotonic maintenance fluid therapy and by fluid creep, defined as the large volume unintentionally administered to patients in the form of dissolved medication. In a section on physiology, we elaborate on the limited renal handling of an acute sodium load. We demonstrate how the subsequent retention of water is an energy-demanding, catabolic process and how free water is needed to excrete large burdens of sodium. We quantify the effect size of sodium-induced fluid retention and discuss its potential clinical impact. Finally, we propose preventive measures, discuss the benefits and risks of low-sodium maintenance fluid therapy, and explore options for reducing the amount of sodium caused by fluid creep.The sodium burdens caused by isotonic maintenance fluids and fluid creep are responsible for an additional and avoidable derailment of fluid balance, with presumed clinical consequences. Moreover, the handling of sodium overload is characterized by increased catabolism. Easy and effective measures for reducing sodium load and fluid retention include choosing a hypotonic rather than isotonic maintenance fluid strategy (or avoiding these fluids when enough free water is provided through other sources) and dissolving as many medications as possible in glucose 5%. Well-known examples are the different clinical problems associated with the use of NaCl 0.9% and specific colloid solutions –4. The bIn the ICU, the largest burden of sodium is caused by maintenance fluid therapy prescribed to cover patients’ daily needs for fluids and electrolytes –11. FurtThe aim of this narrative review is to draw attention to fluid retention caused by the large sodium amounts typically administered to hospitalized patients. We summarize the physiological background and explain why renal sodium handling is inefficient and energy-intensive. We attempt to quantify sodium-induced fluid retention and assess its clinical impact , 8. FinaThe Intersalt study evaluated sodium intake in 52 populations throughout the world and found a wide range of sodium excretion (and thus intake) with medians from 1 to 246 mmol per day . AccordiA significant source of sodium in the hospital is the extensive use of maintenance fluid therapy. A prospective single-day point prevalence survey, conducted in 46 Australian and New Zealand ICUs, demonstrated that maintenance and replacement fluids are responsible for 30.9% of the total daily sodium administration of 220 mmol (5 g) and—contrary to common belief—a much more significant source of sodium than resuscitation fluids . We confA second major—this time unintentional—source of sodium in the hospital is the custom of dissolving medications in NaCl 0.9% to enable intravenous administration. Bihari et al. demonstrated that drug boluses accounted for 12.3% of total sodium administration and drug infusions for 8.6% . We prevAs early as the nineteenth century, it was demonstrated that there is a delay of 3 days before the kidneys are able to excrete sodium at the rate of intake following abrupt sodium load , 19. LatAfter an episode of sodium gain, total body water osmolality needs to be restored by the accrual of free water. Counterintuitively, it is not water ingestion (thirst-induced or otherwise) that is the most important effector in realigning osmolality following increased salt intake , 24. HumEventually, superfluous sodium will need to be excreted. In view of the evolutionary adaptations discussed above, the renal ability to concentrate sodium is limited to about two times the plasma sodium concentration. This is in sharp contrast to other solutes that can be concentrated 10 to 1000 times above their plasma value . When thMoreover, apart from sodium, other solutes also need to be excreted. Many critically ill patients exert osmotic diuresis due to a disturbed glucose metabolism, osmotic therapies or increased urea levels caused by (hyper)alimentation and ongoing catabolism , 28. TheAn important aspect is that sodium is usually co-administered with chloride. It is well-recognized that sodium chloride—but not other sodium salts such as sodium bicarbonate—causes renal vasoconstriction, leading to a decrease in renal blood flow and glomerular filtration rate , 31. ThiQuantifying the effect size of sodium-induced fluid retention due to fluid therapy is challenging. Different experiments showed that NaCl 0.9% is excreted more slowly than more hypotonic solutions. However, these studies only assessed the short-term effect of a single-fluid bolus , 34. In Two of our own studies, the MIHMoSA and the TOPMAST studies, had the primary goal of measuring the effect size of sodium-induced fluid retention using two common maintenance fluid regimens. During the MIHMoSA crossover experiment, cumulative fluid balance was assessed in 12 healthy volunteers who refrained from any oral intake during two separate study periods of 48 h. They were administered maintenance fluids with a sodium concentration of 154 (Na154) or 54 mmol/L (Na54), administered at 25 mL/kg/day . The est2/FiO2 and increased duration of mechanical ventilation [p = 0.05) [A positive cumulative fluid balance has long been associated with undesirable outcomes in septic and critically ill patients . In the tilation . Interes = 0.05) . Even leIt cannot be ignored that a positive fluid balance is often a marker of illness severity rather than the cause of harm itself. Indeed, even a markedly positive fluid balance is frequently unavoidable, as it is not merely the result of imprudent fluid therapy, but also a combination of physiological processes responding to certain clinical realities such as hypovolemia, vasodilation, and capillary leakage. As such, a patient’s fluid balance can be positive in the presence of euvolemia, or even intravascular hypovolemia. Figure Preventing fluid overload is notoriously difficult, and determining the optimal amount of fluid is one of the most challenging clinical decisions in critically ill and surgical patients. In contrast, avoiding unnecessary sodium overload might be the low-hanging fruit. Reducing the sodium burden could thus be a straightforward and feasible option in the battle against iatrogenic fluid overload. Fortunately, the search for avoidable sources of sodium is not extremely challenging, as two of them are hidden in plain sight.Dissolving as many medications as possible in glucose or dextrose 5% can dramatically reduce unintentional sodium administration. Oral intake or enteral administration could be valuable alternatives to drugs with high oral bioavailabilities. Table First, we want to emphasize that the necessary amounts of water and electrolytes should ideally be administered orally or enterally. Intravenous maintenance fluid therapy should only be prescribed in situations where patients are able neither to ingest food or fluids nor to receive (par)enteral nutrition. As soon as other sources provide enough free water to excrete sodium and other solutes, maintenance fluids become redundant. On the other hand, banning all maintenance fluids from the hospital or the ICU will lead to many patients being unable to excrete the necessary solutes, especially because of the displacement of intravascular fluid towards the interstitial space. The ensuing risk of acute kidney injury, encountered after a too-restrictive fluid policy, is unacceptable . We wantp = 0.07), but all instances occurred in patients with known seizure disorders. In the adult patients of the TOPMAST trial, most instances of hyponatremia were mild and none was symptomatic. Bihari et al. showed that a reduction of sodium and chloride in maintenance fluids and fluid creep led to no differences in either the rate or severity of hyponatremia [The other side of the coin and the most feared trade-off of a hypotonic maintenance fluid strategy is the occurrence of hyponatremia. This is the reason why maintenance fluids’ ideal sodium content remains a matter of heated debate –47. In-hnatremia .In order to take all the elements above into account when prescribing maintenance fluid therapy, we propose the algorithm in Fig. We again point out that popular balanced solutions such as Ringer’s lactate are efficient in preventing hyperchloremia but have little added value over NaCl 0.9% in terms of sodium content Table . On the On a therapeutic level, it is difficult to remove sodium once administered. This is especially true as many diuretics result in diuresis in excess of natriuresis, causing hypernatremia . Just liAlthough well-known in experimental research and in the field of hypertension, the dangers of unnecessary or unintentional sodium intake have not yet entered the clinical arena. In the light of the abovementioned findings, this seems unjustified. Evidence supports the hypothesis that high sodium burdens are a separate risk factor for fluid overload and induce a catabolic hormone profile. We therefore call for increased attention to be paid to sodium overload, especially since many of the preventive measures are easy to implement in clinical practice. Ideal starting points include adopting a maintenance fluid strategy that is low in sodium and provides enough free water, and avoiding NaCl 0.9% as the diluent for medication. Caution remains warranted with regard to the development of hyponatremia and hypovolemia. Now that the awareness of iatrogenic volume overload is being incorporated in daily practice and the measures for avoiding it are being pushed to their limits, we are convinced that the battle against sodium-induced fluid overload is the logical next step.Unphysiological amounts of sodium, surpassing normal dietary intake, are commonly administered to hospitalized patients through ill-considered maintenance fluid therapy and fluid creep. The notoriously difficult renal handling of this sodium overload leads to potentially harmful fluid retention. We call for increased attention to be paid to this this avoidable problem and suggest the use of low-sodium maintenance fluids (or their avoidance whenever possible) and recommend “cutting the creep” by avoiding NaCl 0.9% as the diluent for medication.


Phil. Trans. R. Soc. B376, 20200297. (Published 4 October 2021). (doi:10.1098/rstb.2020.0297)Funding information for one author was omitted from the funding statement.S.L. is part of the relationship programme supported by The Medical Research Council and Scotland's Chief Scientist Office (grant no. MC_UU_00022/3) and with CSO funding of the Relationships programme (grant no. SPHSU18).This has been updated on the publisher's website.


The objective of this study was to investigate whether the use of live attenuated varicella zoster virus (VZV) MAV vaccination can efficiently induce VZV antibody production in naive rhesus monkeys as an approach to prevent simian varicella virus (SVV) reactivation in animals immunosuppressed for transplantation studies.Clinically available human VZV vaccine was used to induce the production of anti-VZV antibodies in rhesus monkeys. A vial of the vaccine was subcutaneously injected at 0 week, and the second and third vaccination was performed at 5 and 6 weeks by intratracheal inoculation. The titer of anti-VZV IgG was assessed at 0, 2, 4, 6, and 7 weeks. At 2 weeks, 3/16 were seropositive for VZV IgG. At 6 weeks, 9/16 were shown to be seropositive. At 7 weeks, 16/16 were found to be seropositive.The VZV vaccine via intratrachael inoculation was shown to induce VZV IgG humoral immunity in rhesus monkeys and may be important immunosuppressed macaques for transplantation studies. Although the humoral immunity produced is an important finding, further studies will be necessary to confirm possible protection and it could protect probably against SVV infection in rhesus monkey. Simian varicella virus (SVV) in Old World monkeys causes epizootic erythematous disease, which is characterized by fever, vesicular skin rash, hepatitis, and high morbidity and mortality . OutbreaR1 (ID of rhesus monkey number 1), an immunosuppressed rhesus monkey, for porcine islet transplantation among 14 rhesus monkeys , housed in Nonhuman Primate Research Center at Seoul National University Hospital exhibited clinical symptoms, such as systemic vesicular rash, fever, anorexia, and vomiting  were seropositive at 2 weeks. At 6 weeks , 9 out of 16 monkeys (56.2%) were shown to be seropositive. At 7 weeks , 16 out of 16 monkeys (100%) were found to be seropositive , especially via intratracheal inoculation, was able to induce VZV IgG production. Meyer et al. demonstrated a lack of varicella and viral replication in either the lungs or whole blood by wild type VZV intratracheal inoculation in rhesus monkeys. All four rhesus monkeys generated an immune response characterized by the generation of VZV-specific antibodies and T-cells in their study. VZV-immune rhesus monkeys displayed no varicella rash and had lower SVV viral loads as well as earlier and stronger humoral and cellular immune responses than controls in the SVV challenge . Our datThe seropositivity of SuduVax (VZV MAV vaccine) by subcutaneous inoculation in humans was shown to be 51 ~ 69% in 1- to 6-year-olds , and theThere were a few limitations of this study. We were not able to did not confirm that VZV IgG could neutralize the SVV antigen due to the absence of the SVV antigen. The SVV IgG level could not be measured because an ELISA kit, which measures only SVV, was not available. A challenge test of SVV in VZV-vaccinated rhesus monkeys was not performed due to lack of pathogenic SVV. In the future, SVV can be obtained from leaders in the SVV field for the needed challenge studies. However, because various studies, especially Meyer et al.’s report , have coWe used 0.7 ml of SuduVax as an intratracheal inoculation. This volume might be negligible in terms of safety concerns. A typical volume of 1–2 mL/kg body weight was safely used to avoid any potential coughing (> 2 ml/kg) and subsequent loss of instilled test material .Our results showed that clinically available SuduVax was able to induce VZV IgG production, especially after two intratracheal inoculations at one-week intervals. These data show a strong humoral immune response was produced by VZV vaccination via intratracheal inoculation. They suggest further efficacy testing against SVV challenge is needed to confirm the use of VZV vaccination to protect against SVV reactivation and disease in rhesus monkeys utilized for immunosuppressive and organ transplantation studies.All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Biomedical Research Institute at the Seoul National University Hospital, an AAALAC accredited facility .After the SVV outbreak and follow-up in our NHP facility, we planned to immunize newly imported macaques using clinically available human VZV vaccine. SuduVax  was used to immunize rhesus monkeys. Sixteen naïve female rhesus monkeys (age: 3–5 years) were used. Sedation of the monkeys was induced with medetomidine  and ketamine . The injection site was prepared aseptically. A vial of the vaccine solution 0.7 ml, 1400 pfu) was subcutaneously injected at week 0 and administered via intratracheal inoculation at week 5 and 6 due to poor VZV IgG seropositive after 0 week subcutaneous VZV inoculation. The trachea of the rhesus monkey was grasped by the thumb and index finger of the nondominant hand. The needle was inserted into the trachea in a 30-degree cephalad or caudal orientation to the skin and directed cranially or caudally after penetration of the anterior tracheal wall. Before inoculation, air was aspirated to ensure that the needle was in the proper position. The complications related to subcutaneous or intratracheal injection were monitored by observer’s subjective judgement for pain  and observer’s objective judgement for swelling and redness at the injection site, fever, and rash by systemic reaction. VZV IgG titers were monitored at week 0, 2, 4, 6, and 7 . VZV IgG titers greater than 150 mIU/ml were considered seropositive VZV IgG titers greater than 150 mIU/ml were considered seropositive which is suggested by chemiluminescent immunoassay  for human VZV , cutoff To determine how the VZV IgG antibody titer waned over time, we analyzed five monkeys among 16 at one of three time points among 19, 23, 31, and 55 weeks.


E. cloacae species complex (ECL), S. marcescens (SM), C. freundii species complex (CF), and P. aeruginosa (PSA) are common pathogens in a variety of clinical infections. These organisms can overexpress the chromosomal AmpC that encodes resistance to several β-lactams. We evaluated the activity of ceftazidime-avibactam (CAZ-AVI) and comparators against these organisms.Enterobacterales [ENT] and ≥16 mg/L for PSA) and considered probable AmpC hyperproducers. Isolates were susceptibility tested by broth microdilution method. 17,650 isolates, including 4,400 ECL, 2,074 SM, 1,644 CF, and 9,532 PSA, were consecutively collected from 88 US medical centers in 2016-2020. Among these isolates, 3,127 were ceftazidime-nonsusceptible  and CAZ-AVI was more active than MEM  against these organisms. C-T  and PIP-TAZ  exhibited limited activity against CAZ-NS ENT. Among comparator agents, only amikacin (99.0%S), tigecycline (95.6%S), and imipenem (92.1%S) showed good activity against CAZ-NS ENT. Also, CAZ-AVI retained activity against 86.7% of ENT isolates that were NS to CAZ and MEM (n=113). CAZ-AVI  and C-T  were the most active compounds tested against PSA and both retained activity against CAZ-NS PSA. CAZ-AVI  and C-T  activity against CAZ-NS PSA was comparable to tobramycin . Susceptibility to CAZ ranged from 73.6% (ECL) to 97.5% . Overall, 99.8% of ENT (99.7-99.9%) and 97.1% of PSA were CAZ-AVI-S; whereas 84.3% (79.0-97.7%) of ENT and 97.4% of PSA were ceftolozane-tazobactam (C-T)-S, 83.0% (78.5-94.8%) of ENT and 80.0% of PSA were piperacillin-tazobactam (PIP-TAZ)-S, and 98.4% (98.3-98.7%) of ENT and 79.5% of PSA were meropenem (MEM)-S. CAZ-AVI retained potent activity and broad spectrum against CAZ-NS ENT  (Research Grant or Support)Basilea Pharmaceutica International, Ltd. (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)Department of Health and Human Services Melinta Therapeutics, LLC (Research Grant or Support)Nabriva Therapeutics (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Cecilia G. Carvalhaes, MD, PhD, AbbVie (formerly Allergan) (Research Grant or Support)Cidara Therapeutics, Inc. (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Pfizer, Inc. (Research Grant or Support) Rodrigo E. Mendes, PhD, AbbVie (Research Grant or Support)AbbVie (formerly Allergan) (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)ContraFect Corporation (Research Grant or Support)GlaxoSmithKline, LLC (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Nabriva Therapeutics (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Mariana Castanheira, PhD, AbbVie (formerly Allergan) (Research Grant or Support)Bravos Biosciences (Research Grant or Support)Cidara Therapeutics, Inc. (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Qpex Biopharma (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Mariana Castanheira, PhD, Affinity Biosensors  Involved: Self): Research Grant or Support; Allergan  Involved: Self): Research Grant or Support; Amicrobe, Inc  Involved: Self): Research Grant or Support; Amplyx Pharma  Involved: Self): Research Grant or Support; Artugen Therapeutics USA, Inc.  Involved: Self): Research Grant or Support; Astellas  Involved: Self): Research Grant or Support; Basilea  Involved: Self): Research Grant or Support; Beth Israel Deaconess Medical Center  Involved: Self): Research Grant or Support; BIDMC  Involved: Self): Research Grant or Support; bioMerieux Inc.  Involved: Self): Research Grant or Support; BioVersys Ag  Involved: Self): Research Grant or Support; Bugworks  Involved: Self): Research Grant or Support; Cidara  Involved: Self): Research Grant or Support; Cipla  Involved: Self): Research Grant or Support; Contrafect  Involved: Self): Research Grant or Support; Cormedix  Involved: Self): Research Grant or Support; Crestone, Inc.  Involved: Self): Research Grant or Support; Curza  Involved: Self): Research Grant or Support; CXC7  Involved: Self): Research Grant or Support; Entasis  Involved: Self): Research Grant or Support; Fedora Pharmaceutical  Involved: Self): Research Grant or Support; Fimbrion Therapeutics  Involved: Self): Research Grant or Support; Fox Chase  Involved: Self): Research Grant or Support; GlaxoSmithKline  Involved: Self): Research Grant or Support; Guardian Therapeutics  Involved: Self): Research Grant or Support; Hardy Diagnostics  Involved: Self): Research Grant or Support; IHMA  Involved: Self): Research Grant or Support; Janssen Research & Development  Involved: Self): Research Grant or Support; Johnson & Johnson  Involved: Self): Research Grant or Support; Kaleido Biosceinces  Involved: Self): Research Grant or Support; KBP Biosciences  Involved: Self): Research Grant or Support; Luminex  Involved: Self): Research Grant or Support; Matrivax  Involved: Self): Research Grant or Support; Mayo Clinic  Involved: Self): Research Grant or Support; Medpace  Involved: Self): Research Grant or Support; Meiji Seika Pharma Co., Ltd.  Involved: Self): Research Grant or Support; Melinta  Involved: Self): Research Grant or Support; Menarini  Involved: Self): Research Grant or Support; Merck  Involved: Self): Research Grant or Support; Meridian Bioscience Inc.  Involved: Self): Research Grant or Support; Micromyx  Involved: Self): Research Grant or Support; MicuRx  Involved: Self): Research Grant or Support; N8 Medical  Involved: Self): Research Grant or Support; Nabriva  Involved: Self): Research Grant or Support; National Institutes of Health  Involved: Self): Research Grant or Support; National University of Singapore  Involved: Self): Research Grant or Support; North Bristol NHS Trust  Involved: Self): Research Grant or Support; Novome Biotechnologies  Involved: Self): Research Grant or Support; Paratek  Involved: Self): Research Grant or Support; Pfizer  Involved: Self): Research Grant or Support; Prokaryotics Inc.  Involved: Self): Research Grant or Support; QPEX Biopharma  Involved: Self): Research Grant or Support; Rhode Island Hospital  Involved: Self): Research Grant or Support; RIHML  Involved: Self): Research Grant or Support; Roche  Involved: Self): Research Grant or Support; Roivant  Involved: Self): Research Grant or Support; Salvat  Involved: Self): Research Grant or Support; Scynexis  Involved: Self): Research Grant or Support; SeLux Diagnostics  Involved: Self): Research Grant or Support; Shionogi  Involved: Self): Research Grant or Support; Specific Diagnostics  Involved: Self): Research Grant or Support; Spero  Involved: Self): Research Grant or Support; SuperTrans Medical LT  Involved: Self): Research Grant or Support; T2 Biosystems  Involved: Self): Research Grant or Support; The University of Queensland  Involved: Self): Research Grant or Support; Thermo Fisher Scientific  Involved: Self): Research Grant or Support; Tufts Medical Center  Involved: Self): Research Grant or Support; Universite de Sherbrooke  Involved: Self): Research Grant or Support; University of Iowa  Involved: Self): Research Grant or Support; University of Iowa Hospitals and Clinics  Involved: Self): Research Grant or Support; University of Wisconsin  Involved: Self): Research Grant or Support; UNT System College of Pharmacy  Involved: Self): Research Grant or Support; URMC  Involved: Self): Research Grant or Support; UT Southwestern  Involved: Self): Research Grant or Support; VenatoRx  Involved: Self): Research Grant or Support; Viosera Therapeutics  Involved: Self): Research Grant or Support; Wayne State University  Involved: Self): Research Grant or Support


This study investigates the morphological and rheological properties of blended gelatin ) and hydroxypropyl methylcellulose ) systems using a fluorescence microscope, small angle X-ray scattering (SAXS), and a rheometer. The results clearly indicate that the two biopolymers are immiscible and have low compatibility. Moreover, the rheological behavior and morphology of the GA/HPMC blends significantly depend on the blending ratio and concentration. Higher polysaccharide contents decrease the gelling temperature and improve the gel viscoelasticity character of GA/HPMC blended gels. The SAXS results reveal that the correlation length (ξ) of the blended gels decreases from 5.16 to 1.89 nm as the HPMC concentration increases from 1 to 6%, which suggests that much denser networks are formed in blended gels with higher HPMC concentrations. Overall, the data reported herein indicate that the gel properties of gelatin can be enhanced by blending with a heating-induced gel. Gelatin, derived from the partial hydrolysis of collagen, has a wide range of application in the food, pharmaceutical, cosmetic, and photographic fields ,2,3,4. TInterestingly, the physiochemical properties of hydrogels can be improved simply by blending a cooling-induced gel (cool-gel) with a heating-induced gel (thermo-gel). This processing method is relatively easy and does not require any chemical crosslinking agent to enhance product performance, which renders it important on both scientific and commercial levels. The most popular and widely available thermo-gel used to improve the properties of hydrogels is hydroxypropyl methylcellulose (HPMC) . In partIt is well known that the properties of a blending system depend on its morphology, which is mainly controlled by compatibility and phase separation behavior. In a system containing two opposite thermo-reversible gels, compatibility and phase separation are complex and strongly dynamic. For example, the phase behavior of a gelatin/HPMC composite gel depends on the concentration, blending ratio, and temperature. Considering that HPMC is a thermo-gel and gelatin is a cool-gel, the gelation and phase separation behaviors of the gelatin/HPMC composite gel are expected to reach equilibrium at different temperatures. In this work, we focus on how the compatibility and phase behaviors of the cool-gel (gelatin) and thermo-gel (HPMC) affect their solution and gel properties, in particular the effects of the blending ratio and solution concentration. The phase diagram of the GA/HPMC blends was established based on blending ratio and solution concentration. Rheometers, fluorescence microscopes, and SAXS were used to characterize the blending system, and the relationship between microstructures and gel performance was determined.3, NaOH, fluorescein 5(6)-isothiocyanate, and DMSO chemicals were bought from Aladdin, Shanghai, China, and they were used as received.Food-grade gelatin  (GA) was supplied by Rousselot Company, Giangzhou, China, and pharmaceutical-grade hydroxypropyl methyl cellulose (HPMC)  was purchased from Hopetop Pharmaceutical Company, Huzhou, China. Analytical-grade NaN3) was added to the GA solutions to inhibit the growth of microorganisms. As for the HPMC solutions, they were prepared by dispersing HPMC powder in hot water and stirring for 30 min. Afterward, the solutions were cooled to ambient temperature with continuous stirring to achieve complete dissolution. All the solutions were stored in a refrigerator at 4 °C prior to use.GA and HPMC stock solutions of varying concentrations  were prepared with a pH5 according to the previous study . GA  were prepared by mixing 0, 2, 4, 6, 8, 10, 12, and 14 wt% gelatin stock solutions with 0, 2, 4, 6, 8, 10, 12, and 14 wt% HPMC stock solutions, respectively. The solutions were mixed at 55 °C with a continuous stirring for 1 h. Subsequently, 5 g of each blended solution were transferred to plastic centrifuge tubes that were sealed to prevent water evaporation. The tubes were held in a water bath at 25 °C for 24 h to determine whether phase transition (from gel to liquid state) or phase separation would occur. The different phase behaviors of the mixed systems were recorded.3 (0.02 wt%) was added to inhibit the growth of bacteria. The deionized water was changed every 2 h during dialysis. The FITC-labeled GA was lyophilized, and the yellow powder obtained was mixed with HPMC. The sample solutions were poured onto glass slides, covered with cover slips, and hermetically sealed with oil, then they were observed at the excitation wavelength of 460–495 nm. The emission of samples was recorded between 510 and 550 nm.The micro-networks of 6/0, 6/1 6/2, 6/3, 6/4, 6/5, and 6/6 GA/HPMC blends  equipped with a color and monochrome camera . GA was covalently labelled with fluorescein isothiocyanate (FITC) before mixing with HPMC solutions. First, 10 mg of FITC were dissolved in 10 mL DMSO solution. Then, 100 μL of the FITC solution were added to 100 mL of the GA stock solution . The mixture was allowed to react for 1 h at 40 °C with continuous stirring. Then, the reaction was stopped by adding ethanolamine. Finally, the mixture was dialyzed against deionized water for 48 h to remove unreacted FITC, and NaNA rheometer  with a concentric cylinder cell (28 mm inner diameter) was used to investigate the gel property of GA/HPMC blends  by analyzing the variation of viscosity as a function of temperature. The solution was first heated from 25 to 85 °C at the rate of 2 °C/min. Then, the temperature was decreased back to 25 °C at the same rate. The rotor speed was set to 25 rad/s during the entire heating and cooling process.−1 in 1 min.A parallel-plate geometry with a diameter of 40 mm was used to measure the flow pattern and study the effect of shear rate on the viscosity of the pure and blending solution at 25 °C. The shear rate was increased from 0 to 1000 sThe dynamic rheological properties were explored using small-amplitude oscillatory shear test. The frequency was set at 1 Hz and the strain at 1% . To evaluate the effect of temperature on the mixed systems, temperature ramps were implemented at the scan rate of 2 °C/min, with heating from 5 to 85 °C, followed by holding at 85 °C for 5 min, then cooling back to 5 °C with the same scan rate of 2 °C/min. The samples were placed between parallel plates, and a small amount of silicone oil was spread over the periphery of each sample to prevent moisture evaporation. Changes in the storage modulus (G′) and loss modulus (G″) were recorded as a function of temperature. The gelation and melting temperatures of GA and HPMC are defined as the temperature at which G′ is equal to G″ (the G′/G″ crossover point) ,23.To assess the dynamic mechanical behavior of GA/HPMC blends, 6% gelatin and 6% HPMC were prepared according to A SAXSess small-angle X-ray scattering system  equipped with a PW3830 X-ray generator  was used to study the gel structure. Measurements were performed according to a previously described method ,29. SampThe relationship between q and θ was determined according to the following equation :q = 4πsiThe light and black zones that appear in the fluorescence micrographs shown in The phase separation of the gels was induced by the immiscibility of the two polymers, similar to many other protein-polysaccharide systems ,32,33. SThe viscosity of GA/HPMC solutions of varying blending ratios was studied at different temperatures and a specific shear rate. As shown in The effect of shear velocity on the viscosity of different blends at room temperature was also investigated. As shown in mg) and the gelling temperature of HPMC (Tgh) in the heating stage, and the melting temperature of the HPMC gel (Tmh) and the gelling temperature of gelatin (Tgg) in the cooling stage. The sol-gel transition points of gelatin and HPMC were ~30 and ~56 °C, respectively. Based on the data listed in mg = 31 °C), showing a slight increase. This may be due to the fact that the protein aggregation induced by HPMC improved the gel network, which can only be destroyed by high energy. Moreover, the increase in Tmg value at higher HPMC content is likely associated with the higher local protein concentration resulting from biopolymer phase separation [gh), it was lower for the G6H0, G6H1, and G6H2 samples than for the G6H4, G6H5, and G6H6 samples. The increased Tgh at higher HPMC contents may be attributed to the complete phase separation between GA and HPMC and the relatively low viscosity of HPMC at these conditions. These phenomena promote the self-aggregation of HPMC molecules, resulting in increased local HPMC concentration and lower gelling temperatures. In the cooling stage, hysteresis [The sol-gel transition points (G′ = G″) for the GA/HPMC blends are listed in paration , as descsteresis  and/or csteresis ,23. TherThe increase in the gelation temperature and thermal stability of gelatin in GA/HPMC systems compared to the GA system is related to phase distribution  and locaFrequency sweep analyses were conducted on the co-hydrogels at room temperature (25 °C), in the frequency (ω) range of 0.1–100 rad/s. The G (ω) versus ω plots shown in n. The n and S parameters are both sensitive to the crosslinking density inside the gel, with higher S values signifying “harder” gels. The data presented in The variation in storage modulus at low frequencies follows the power law given by G′ = SωTo investigate the structural differences between the GA/HPMC hydrogels investigated herein, their SAXS profiles were recorded at room temperature. The gel network of each sample was determined based on the viscosity measurements and rheology analysis. As shown in tructure ,45. Basetructure .In this work, the gelation and phase behaviors of blended gelatin (cool-gel) and HPMC (thermo-gel) systems were assessed by analyzing the phase diagram of these blends. The obtained results show that the behavior of binary GA/HPMC systems changes from water-in-water emulsion-like to a bi-continuous network whose microstructure clearly depends on the ratio of the two polymers. The formation of phase-separated networks is attributed to the incompatibility between gelatin and HPMC, as well as to their reversed gelation behavior. The solid character of the mixed gels is correlated with the component constituting the continuous phase, and the gel stability of gelatin gels has no significant changes with increasing HPMC concentration. Based on the SAXS results obtained herein, larger HPMC concentrations lead to structural changes in GA/HPMC mixed gels at various length scales. These structural differences probably induce variations in rheological properties. The elucidation of GA/HPMC interactions is a key element in formulating and controlling the quality of manufactured foods, as well as in developing novel food processes and edible film materials.


Grallina cyanoleuca). The circular genome has a size of 16,933 bp and contains 13 protein-coding genes, 22 tRNA genes, and 2 rRNA genes. This study provides a reference mitochondrial genome of magpie-lark for further molecular studies.Here, we report the complete mitochondrial genome sequence of an Australian passerine bird, magpie-lark ( Grallina cyanoleuca) is native to Australia, Timor, and southern New Guinea. Molecular studies on the magpie-lark (G. cyanoleuca) are limited, and only partial mitochondrial sequences of this species are available in the NCBI database  is the largest order of birds and the dominant avian group on Earth today. With more than 140 families and some of 6,500 identified species, Passeriformes is one of the most diverse orders of terrestrial vertebrates  to remove possible bacterial contamination. Trimmed and unmapped clean reads were used as input data for de novo assembly in CLC Genomics Workbench (version 9.5.4). This resulted in the generation of a 16,933-bp mitogenome obtained from G. cyanoleuca. A total of 9.10 million clean reads were mapped back to the mitogenome of G. cyanoleuca that resulted in an average coverage of 53.76×. Annotation of the assembled mitogenome of G. cyanoleuca was performed using default parameters under the genetic code of vertebrate mitochondria (transl_table 2) in Geneious (version 10.2.2). The final mitogenome of G. cyanoleuca was circularized using Geneious software under default parameters.A cutaneous tissue sample from a wild magpie-lark was collected at Canley Heights Veterinary Clinic, NSW, Australia , and was deposited at Charles Sturt University. Animal sampling was performed following approved guidelines set by the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and approved by the Charles Sturt University Animal Ethics Committee (research authority permit 09/046). The genomic DNA (gDNA) was isolated using a ReliaPrep genomic DNA tissue miniprep system  69. SequenG. cyanoleuca had a circular genome of 16,933 bp with a G+C content of 44.1%. Our annotation identified 1 control region (D-loop), 2 rRNA regions (12S and 16S), 22 tRNA genes, and 13 protein-coding genes (PCGs), and the gene content of the mitogenome of G. cyanoleuca was consistent with that of the other members of the order Passeriformes  (100% bootstrap support) and demonstrated a 90.36% pairwise nucleotide identity between them. We concluded that the complete mitogenome of G. cyanoleuca will be a useful database among the family Monarchidae for further studying the host-phylogenetic relationship.The assembled complete mitogenome of  regions S and 16SG. cyanoleuca has been deposited in DDBJ/ENA/GenBank under the accession number MW133233. The version described in this paper is the first version, MW133233.1. Raw sequencing data from this study have been deposited in the NCBI Sequence Read Archive (SRA) under the accession number SRR14381441 .The complete mitochondrial genome sequence of