Cell Damage Mechanisms during Cryopreservation in a Zwitterion Solution and Its Alleviation by DMSO.

Recently, zwitterions have been proposed as novel cryoprotectants. However, some cells are difficult to cryopreserve using aqueous zwitterion solutions alone. We investigated here the reason for cell damage in such cells, and it was the osmotic pressure after freeze concentration. Furthermore, the addition of dimethyl sulfoxide (DMSO) has been reported to improve the cryoprotective effect in such cells: the zwitterion/DMSO aqueous solution shows a higher cryoprotective effect than the commercial cryoprotectant. This study also clarified the mechanisms underlying the improvement in a cryoprotective effect. The addition of cell-permeable DMSO alleviated the osmotic pressure after the freeze concentration. This alleviation was also found to be a key factor for cryopreserving cell spheroids, while there has been no insight into this phenomenon.

Optimization of Zwitterionic Polymers for Cell Cryopreservation.

Cryopreservation techniques are valuable for the preservation of genetic properties in cells, and the development of this technology contributes to various fields. In a previous study, an isotonic freezing medium composed of poly(zwitterion) (polyZI) has been reported, which alleviates osmotic shock, unlike typical hypertonic freezing media. In this study, the primitive freezing medium composed of emerging polyZI is optimized. Imidazolium/carboxylate-type polyZI (VimC3 C) is the optimal chemical structure. The molecular weight and degree of ion substitution (DSion ) are not significant factors. There is an impediment with the primitive polyZI freezing media. While the polyZI forms a matrix around the cell membrane to protect cells, the matrix is difficult to remove after thawing, resulting in low cell proliferation. Unexpectedly, increasing the poly(VimC3 C) concentration from 10% to 20% (w/v) improves cell proliferation. The optimized freezing medium, 20% (w/v) poly(VimC3 C)_DSion(100%) /1% (w/v) NaCl aqueous solution, exhibited a better cryoprotective effect.

Protein Cryoprotectant Ability of the Aqueous Zwitterionic Solution.

Protein cryopreservation is important for the long-term storage of unstable proteins. Recently, we found that N-acetylglucosaminyltransferase-V (GnT-V) can be cryopreserved in a deep freezer without temperature control using a dilute binary aqueous solution of 3-(1-(2-(2-methoxyethoxy)ethyl)imidazol-3-io)butane-1-carboxylate (OE2imC3C) [10 wt %, mole fraction of solute (x) = 7.75 × 10-3], an artificial zwitterion. However, it is unclear which solvent properties are required in these media to preserve unstable proteins, such as GnT-V. In this study, we investigated the melting phenomena and solution structure of dilute binary aqueous OE2imC3C solutions [x = 0-2.96 × 10-2 (0-30 wt %)] using differential scanning calorimetry (DSC) and Raman and Fourier transform infrared (FTIR) spectroscopies combined with molecular dynamics (MD) simulation to compare the cryoprotectant ability of OE2imC3C with two general cryoprotectants (CPAs), glycerol and dimethyl sulfoxide. DSC results indicated that aqueous OE2imC3C solutions can be melted at lower temperatures with less energy than the control CPA solution, with increasing x, primarily due to OE2imC3C having a higher content of unfrozen water molecules. Moreover, Raman and FTIR results showed that the high content of unfrozen water molecules in aqueous OE2imC3C solutions was due to the hydration around the ionic parts (the COO- group and imidazolium ring) and the OCH2CH2O segment. In addition, the MD simulation results showed that there were fewer structured water molecules around the OCH2CH2O segment than the hydration water molecules around the ionic parts. These solvent properties suggest that dilute aqueous OE2imC3C solutions are effective in preventing freezing, even in a deep freezer. Therefore, this medium has the potential to act as a novel cryoprotectant for proteins in biotechnology and biomedical fields.

Cell-compatible isotonic freezing media enabled by thermo-responsive osmolyte-adsorption/exclusion polymer matrices.

During the long-term storage of cells, it is necessary to inhibit ice crystal formation by adding cryoprotectants. Non-cell-permeable cryoprotectants have high osmotic pressure which dehydrates cells, indirectly suppressing intracellular ice crystal formation. However, the high osmotic pressure and dehydration often damage cells. Emerging polymer-type non-cell-permeable cryoprotectants form matrices surrounding cells. These matrices inhibit the influx of extracellular ice nuclei that trigger intracellular ice crystal formation. However, these polymer-type cryoprotectants also require high osmotic pressure to exert an effective cryoprotecting effect. In this study, we designed a poly(zwitterion) (polyZI) that forms firm matrices around cells based on their high affinity to cell membranes. The polyZI successfully cryopreserved freeze-vulnerable cells under isotonic conditions. These matrices also controlled osmotic pressure by adsorbing and desorbing NaCl depending on the temperature, which is a suitable feature for isotonic cryopreservation. Although cell proliferation was delayed by the cellular matrices, washing with a sucrose solution improved proliferation.

Consistently low levels of histidine-rich glycoprotein as a new prognostic biomarker for sepsis: A multicenter prospective observational study.

BACKGROUND: Few sepsis biomarkers accurately predict severity and mortality. Previously, we had reported that first-day histidine-rich glycoprotein (HRG) levels were significantly lower in patients with sepsis and were associated with mortality. Since the time trends of HRG are unknown, this study focused on the time course of HRG in patients with sepsis and evaluated the differences between survivors and non-survivors. METHODS: A multicenter prospective observational study was conducted involving 200 patients with sepsis in 16 Japanese hospitals. Blood samples were collected on days 1, 3, 5, and 7, and 28-day mortality was used for survival analysis. Plasma HRG levels were determined using a modified quantitative sandwich enzyme-linked immunosorbent assay. RESULTS: First-day HRG levels in non-survivors were significantly lower than those in survivors (mean, 15.7 [95% confidence interval (CI), 13.4-18.1] vs 20.7 [19.5-21.9] µg/mL; P = 0.006). Although there was no time × survivors/non-survivors interaction in the time courses of HRG (P = 0.34), the main effect of generalized linear mixed models was significant (P < 0.001). In a univariate Cox proportional hazards model with each variable as a time-dependent covariate, higher HRG levels were significantly associated with a lower risk of mortality (hazard ratio, 0.85 [95% CI, 0.78-0.92]; P < 0.001). Furthermore, presepsin levels (P = 0.02) and Sequential Organ Function Assessment scores (P < 0.001) were significantly associated with mortality. Harrell's C-index values for the 28-day mortality effect of HRG, presepsin, procalcitonin, and C-reactive protein were 0.72, 0.70, 0.63, and 0.59, respectively. CONCLUSIONS: HRG levels in non-survivors were consistently lower than those in survivors during the first seven days of sepsis. Repeatedly measured HRG levels were significantly associated with mortality. Furthermore, the predictive power of HRG for mortality may be superior to that of other singular biomarkers, including presepsin, procalcitonin, and C-reactive protein.

The Effect of Postinduction Blood Glucose on Intraoperative Hypothermia.

Background and Objectives: Hypothermia frequently occurs in patients undergoing surgery and is associated with adverse complications. Therefore, this study aimed to investigate the postinduction blood glucose and occurrence of intraoperative hypothermia in patients undergoing laparoscopic surgery. Materials and Methods: This retrospective observational study included 334 patients aged ≥20 years who had undergone elective laparoscopic surgery. The primary outcome of this study was the incidence of intraoperative hypothermia. Stratified analysis revealed differences between patients with and without diabetes. Results: Hypothermia occurred in 200 (59.9%) patients. In multivariate analysis, out-of-range postinduction glucose was independently associated with hypothermia (>150 mg/dL: odds ratio 2.17, 95% confidence interval (1.02, 4.61), p = 0.045; <110 mg/dL: odds ratio 2.02, 95% confidence interval (1.15, 3.55), p = 0.015), whereas preoperative HbA1c >6% was not significantly associated with hypothermia (odds ratio 1.02, 95% confidence interval (0.56, 1.84), p = 0.961). Considering only patients with diabetes, the incidence of hypothermia was lower (p = 0.002), the duration of hypothermia was shorter (p = 0.007), and the minimum temperature was higher (p = 0.006) in those with a postinduction glucose level of 110-150 mg/dL. Conclusions: The postinduction glucose level is independently associated with intraoperative hypothermia. Out-of-range postinduction glucose appeared to have an impact on the development of hypothermia in patients with diabetes, especially those with a postinduction glucose level <110 mg/dL.

Therapeutic plasma exchange in postpartum HELLP syndrome: a case report.

BACKGROUND: Postpartum hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome is more difficult to treat than HELLP syndrome during pregnancy. We describe a case of postpartum HELLP syndrome that responded to plasma exchange (PE) therapy. CASE PRESENTATION: A 30-year-old primipara woman was hospitalized for gestational hypertension at 33 weeks of gestation and underwent an emergent cesarean section at 36 weeks and 6 days of gestation due to rapidly progressing pulmonary edema. After delivery, liver dysfunction and a rapid decrease in platelet count were observed, and the patient was diagnosed with severe HELLP syndrome. She experienced multiple organ failure despite intensive care, and PE therapy was initiated. Her general condition dramatically stabilized within a few hours of PE therapy. CONCLUSION: It is controversial whether PE therapy should be used primarily in the management of HELLP syndrome, but early initiation of PE therapy could be effective for severe HELLP syndrome.

Cryopreservation of tissues by slow-freezing using an emerging zwitterionic cryoprotectant.

Cryopreservation of tissues is a tough challenge. Cryopreservation is categorized into slow-freezing and vitrification, and vitrification has recently been recognized as a suitable method for tissue cryopreservation. On the contrary, some researchers have reported that slow-freezing also has potential for tissue cryopreservation. Although conventional cryoprotectants have been studied well, some novel ones may efficiently cryopreserve tissues via slow-freezing. In this study, we used aqueous solutions of an emerging cryoprotectant, an artificial zwitterion supplemented with a conventional cryoprotectant, dimethyl sulfoxide (DMSO), for cell spheroids. The zwitterion/DMSO aqueous solutions produced a better cryoprotective effect on cell spheroids, which are the smallest units of tissues, compared to that of a commercial cryoprotectant. Cryopreservation with the zwitterion/DMSO solutions not only exhibited better cell recovery but also maintained the functions of the spheroids effectively. The optimized composition of the solution was 10 wt% zwitterion, 15 wt% DMSO, and 75 wt% water. The zwitterion/DMSO solution gave a higher number of living cells for the cryopreservation of mouse tumor tissues than a commercial cryoprotectant. The zwitterion/DMSO solution was also able to cryopreserve human tumor tissue, a patient-derived xenograft.

Aryloxy Ionic Liquid-Catalyzed Homogenous Esterification of Cellulose with Low-Reactive Acyl Donors.

Ionic liquids (ILs) are recyclable, non-volatile, and can dissolve cellulose, a natural polymer that is insoluble in versatile solvents. Therefore, ILs have been used to modify cellulose. However, 1-ethyl-3-methylimidazolium acetate (EmimOAc), a commercially available IL often used to dissolve and modify cellulose to prepare cellulose-based materials, causes the undesired introduction of an acetyl group derived from the acetate anion of EmimOAc onto the hydroxy group of cellulose during esterification. In this study, for cellulose esterification, we prepared aryloxy ILs as non-carboxylate-type and basic ILs, which can theoretically prevent the undesired introduction of an acyl group from the IL onto the hydroxy group of cellulose. The optimized 1-ethyl-3-methylimidazolium 2-pyridinolate (Emim2OPy) and mixed solvent system achieved rapid cellulose esterification (within 30 min) with an excellent degree of substitution (DS) value (up to >2.9) derived from the employed low-reactive vinyl esters and bio-based unsaturated aldehydes, without any undesired substituent introduction from side reactions.

Histidine-rich glycoprotein as a novel predictive biomarker of postoperative complications in intensive care unit patients: a prospective observational study.

BACKGROUND: Decrease in histidine-rich glycoprotein (HRG) was reported as a cause of dysregulation of the coagulation-fibrinolysis and immune systems, leading to multi-organ failure, and it may be a biomarker for sepsis, ventilator-associated pneumonia, preeclampsia, and coronavirus disease 2019. However, the usefulness of HRG in perioperative management remains unclear. This study aimed to assess the usefulness of HRG as a biomarker for predicting postoperative complications. METHODS: This was a single-center, prospective, observational study of 150 adult patients who were admitted to the intensive care unit after surgery. Postoperative complications were defined as those having a grade II or higher in the Clavien-Dindo classification, occurring within 7 days after surgery. The primary outcome was HRG levels in the patients with and without postoperative complications. The secondary outcome was the ability of HRG, white blood cell, C-reactive protein, procalcitonin, and presepsin to predict postoperative complications. Data are presented as number and median (interquartile range). RESULTS: The incidence of postoperative complications was 40%. The HRG levels on postoperative day 1 were significantly lower in patients who developed postoperative complications (n = 60; 21.50 [18.12-25.74] µg/mL) than in those who did not develop postoperative complications (n = 90; 25.46 [21.05-31.63] µg/mL). The Harrell C-index scores for postoperative complications were HRG, 0.65; white blood cell, 0.50; C-reactive protein, 0.59; procalcitonin, 0.73; and presepsin, 0.73. HRG was independent predictor of postoperative complications when adjusted for age, the presence of preoperative cardiovascular comorbidities, American Society of Anesthesiologists Physical Status Classification, operative time, and the volume of intraoperative bleeding (adjusted hazard ratio = 0.94; 95% confidence interval, 0.90-0.99). CONCLUSIONS: The HRG levels on postoperative day 1 could predict postoperative complications. Hence, HRG may be a useful biomarker for predicting postoperative complications.

Cryostorage of unstable N-acetylglucosaminyltransferase-V by synthetic zwitterions.

We report biocompatible materials for cryostorage of unstable proteins such as cancer-related enzyme, N-acetylglucosaminyltransferase-V (GnT-V). GnT-V activity and the amount of protein after freezing were better retained in synthetic zwitterion solutions than in the glycerol solution. This study highlights the potential utility of synthetic zwitterions as novel cryoprotectants.

Current status of delirium assessment tools in the intensive care unit: a prospective multicenter observational survey.

Delirium is a critical challenge in the intensive care unit (ICU) or high care unit (HCU) setting and is associated with poor outcomes. There is not much literature on how many patients in this setting are assessed for delirium and what tools are used. This study investigated the status of delirium assessment tools of patients in the ICU/HCU. We conducted a multicenter prospective observational study among 20 institutions. Data for patients who were admitted to and discharged from the ICU/HCU during a 1-month study period were collected from each institution using a survey sheet. The primary outcome was the usage rate of delirium assessment tools on an institution- and patient-basis. Secondary outcomes were the delirium prevalence assessed by each institution's assessment tool, comparison of delirium prevalence between delirium assessment tools, delirium prevalence at the end of ICH/HCU stay, and the relationship between potential factors related to delirium and the development of delirium. Result showed that 95% of institutions used the Intensive Care Delirium Screening Checklist (ICDSC) or the Confusion Assessment Method for the ICU (CAM-ICU) to assess delirium in their ICU/HCU, and the remaining one used another assessment scale. The usage rate (at least once during the ICU/HCU stay) of the ICDSC and the CAM-ICU among individual patients were 64.5% and 25.1%, and only 8.2% of enrolled patients were not assessed by any delirium assessment tool. The prevalence of delirium during ICU/HCU stay was 17.9%, and the prevalence of delirium at the end of the ICU/HCU stay was 5.9%. In conclusion, all institutions used delirium assessment tools in the ICU/HCU, and most patients received delirium assessment. The prevalence of delirium was 17.9%, and two-thirds of patients had recovered at discharge from ICU/HCU.Trial registration number: UMIN000037834.

Polar zwitterion/saccharide-based deep eutectic solvents for cellulose processing.

Liquid zwitterions are biocompatible cellulose solvents and have enabled successive ethanol production from plant biomass in the same reaction pot. However, only a few carboxylate-type liquid zwitterions have been reported since almost all zwitterions are solid. Here, we propose zwitterion-based deep eutectic solvents (DESs) to expand the choices of zwitterionic solvents for cellulose dissolution and the subsequent processing. Zwitterion-based DESs were prepared by mixing four types of saccharide at various ratios. Twenty-two combinations of zwitterion/saccharide mixtures formed DESs, that is, liquid state below 100 °C. Two of them, whose saccharide ratio were 5 wt%, successfully dissolved cellulose because the low saccharide load was sufficient for liquefaction but did not disrupt the intrinsic cellulose dissolution ability of zwitterions.

High loading of trimethylglycine promotes aqueous solubility of poorly water-soluble cisplatin.

Trimethylglycine (TMG) is a cheap, natural, and highly biocompatible compound. Therefore, it has been used in the fields of food and life sciences, but the application of solid TMG is limited to utilisation as an "additive". In the present study, we focussed on the high solubility of TMG in water, derived from the aprotic zwitterionic structure, and proposed TMG as the chemical accounting for a major portion of the aqueous solution (e.g., 50 wt%). High loading of TMG shifted the properties of water and enabled the dissolution of poorly water-soluble cisplatin, an anticancer agent, at high concentration (solubility of cisplatin: 0.15 wt% in water vs 1.7 wt% in TMG aqueous solution). For hepatic arterial infusion, this can reduce the amount of cisplatin administered from 40 to 4 mL. It enables simple injection using a syringe, without the need for catheters and automatic pumps, leading to critical alleviation of the risk to patients. Furthermore, we produced a dry powder from a cisplatin-containing TMG aqueous solution via freeze-drying. Powders can be conveniently stored and transported. Furthermore, cisplatin is often used as a mixture with other drugs, and cisplatin aqueous solutions are not preferred as they dilute the other drugs.

Histidine-rich glycoprotein as a prognostic biomarker for sepsis.

Various biomarkers have been proposed for sepsis; however, only a few become the standard. We previously reported that plasma histidine-rich glycoprotein (HRG) levels decreased in septic mice, and supplemental infusion of HRG improved survival in mice model of sepsis. Moreover, our previous clinical study demonstrated that HRG levels in septic patients were lower than those in noninfective systemic inflammatory response syndrome patients, and it could be a biomarker for sepsis. In this study, we focused on septic patients and assessed the differences in HRG levels between the non-survivors and survivors. We studied ICU patients newly diagnosed with sepsis. Blood samples were collected within 24 h of ICU admission, and HRG levels were determined using an enzyme-linked immunosorbent assay. Ninety-nine septic patients from 11 institutes in Japan were included. HRG levels were significantly lower in non-survivors (n = 16) than in survivors (n = 83) (median, 15.1 [interquartile ranges, 12.7-16.6] vs. 30.6 [22.1-39.6] µg/ml; p < 0.01). Survival analysis revealed that HRG levels were associated with mortality (hazard ratio 0.79, p < 0.01), and the Harrell C-index (predictive power) for HRG was 0.90. These results suggested that HRG could be a novel prognostic biomarker for sepsis.

Synthetic zwitterions as efficient non-permeable cryoprotectants.

Cryopreservation of cells is necessary for long periods of storage. However, some cell lines cannot be efficiently cryopreserved, even when optimized commercial cryoprotectants are employed. Previously, we found that a low-toxic synthetic zwitterion aqueous solution enabled good cryopreservation. However, this zwitterion solution could not cryopreserve some cells, such as human kidney BOSC cells, with good efficiency. Therefore, details of the cryoprotective effect of the zwitterions and optimization based on its mechanisms are required. Herein, we synthesized 18 zwitterion species and assessed the effects of the physical properties of water/zwitterion mixtures. Non-cell-permeable zwitterions can inhibit ice crystal formation extracellularly via direct interaction with water and intracellularly via dehydration of cells. However, cells that could not be cryopreserved by zwitterions were insufficiently dehydrated in the zwitterion solution. Dimethyl sulfoxide (DMSO) was combined as a cell-permeable cryoprotectant to compensate for the shortcomings of non-cell-permeable zwitterions. The water/zwitterion/DMSO (90/10/15, v/w/w) could cryopreserve different cells, for example freezing-vulnerable K562 and OVMANA cells; yielding ~1.8-fold cell viability compared to the case using a commercial cryoprotectant. Furthermore, molecular dynamics simulation indicated that the zwitterions protected the cell membrane from the collapse induced by DMSO.

Non-aqueous, zwitterionic solvent as an alternative for dimethyl sulfoxide in the life sciences.

Dimethyl sulfoxide (DMSO) is widely used as a solvent in the life sciences, however, it is somewhat toxic and affects cell behaviours in a range of ways. Here, we propose a zwitterionic liquid (ZIL), a zwitterion-type ionic liquid containing histidine-like module, as a new alternative to DMSO. ZIL is not cell permeable, less toxic to cells and tissues, and has great potential as a vehicle for various hydrophobic drugs. Notably, ZIL can serve as a solvent for stock solutions of platinating agents, whose anticancer effects are completely abolished by dissolution in DMSO. Furthermore, ZIL possesses suitable affinity to the plasma membrane and acts as a cryoprotectant. Our results suggest that ZIL is a potent, multifunctional and biocompatible solvent that compensates for many shortcomings of DMSO.

Local Anesthetic Systemic Toxicity Following General and Epidural Anesthesia in A patient with a History of Muscle Relaxant-induced Anaphylaxis.

We here report that a 71-year-old Japanese woman with a history of anaphylaxis induced by muscle relaxants had local anesthetic systemic toxicity (LAST) following an abdominal surgery under general anesthesia with combined spinal-epidural anesthesia without muscle relaxants. The total dosages of local anesthetics reached 0.67 mg/kg of ropivacaine and 11.5 mg/kg of lidocaine over 12.5 h to obtain sufficient muscle relaxation for surgery. Regional anesthesia is useful in cases in which muscle relaxants are to be avoided during a surgery. However, especially for a patient with risk factors and prolonged surgery, precautions should be taken to prevent LAST.

Examining the unique retention behavior of volatile carboxylic acids in gas chromatography using zwitterionic liquid stationary phases.

For the first time, gas chromatographic (GC) stationary phases consisting of zwitterionic liquids (ZILs) possessing sulfonate functional groups were utilized for the analysis of volatile carboxylic acids (VCAs). The highly polar and acidic nature of VCAs significantly limits the number of currently available GC stationary phases, which are all largely based on acid-modified polyethylene glycol. In this study, it is shown that this class of ZILs exhibit strong retention of VCAs with excellent peak symmetry. Unique chromatographic selectivity toward VCAs is also demonstrated by tuning the structural features of the ZILs. The solvation properties of the three ZILs as well as a structurally similar conventional monocationic IL were characterized using the Abraham solvation parameter model.

Zwitterionic polymeric ionic liquid-based sorbent coatings in solid phase microextraction for the determination of short chain free fatty acids.

Five different zwitterionic sorbent coatings based on polymeric ionic liquids (PILs) were developed by the on fiber UV co-polymerization of the zwitterionic monomers 1-vinyl-3-(alkylsulfonate)imidazolium or 1-vinyl-3-(alkylcarboxylate)imidazolium and different dicationic ionic liquid (IL) crosslinkers. The developed sorbent coatings were applied in headspace solid-phase microextraction in combination with gas chromatography-mass spectrometry for the determination of short chain free fatty acids in wine. The sorbent coatings were found to extract these analytes via a non-competitive extraction mechanism. The methodology was optimized for the two best zwitterionic PIL coatings and compared to the commercially-available carboxen/polydimethylsiloxane (CAR/PDMS) and polyacrylate (PA) fibers. The sorbent coating based on the 1-vinyl-3-(propanesulfonate)imidazolium IL (Fiber 1) was more sensitive than PA while providing similar limits of detection to CAR/PDMS for the determination of analytes in a diluted synthetic wine sample. At the same time, Fiber 1 required lower extraction times (only 20 min versus 60 min for CAR/PDMS and 40 min for PA), exhibited higher reproducibility (with relative standard deviation lower than 8.9% for a spiked level of 7 µM) and was more tolerant to ethanol present within the sample. The zwitterionic PILs were also applied for the analysis of red wine, and the results were in agreement with those obtained for CAR/PDMS. The analytes were detected and quantified in the concentration range from 0.18 ±â€¯0.03 mg L-1 to 4.8 ±â€¯0.9 mg L-1, depending on the analyte and fiber.