Hypothermic Conditions Research Articles

Effects of ATP and adenosine on contraction amplitude of rat soleus muscle at different temperatures.

The aim of this study was to evaluate the effects of adenosine 5'-triphosphate (ATP) and adenosine on the contractility of mammalian skeletal muscle under hypothermic conditions. Contractions of isolated rat soleus muscle were induced by either electrical stimulation (ES) or carbachol at physiological temperatures (37°C) and hypothermic conditions (30-14°C) and recorded in the presence of ATP, adenosine, suramin, and 8-(p-sulfophenyl)-theophylline (8-SPT). At 37°C, incubation of the muscles with ATP inhibited ES-induced contractions; the inhibitory effect of ATP disappeared at 14°C. Adenosine inhibited ES-induced contractions at all temperature levels; 8-SPT fully prevented the action of adenosine. ATP and adenosine did not significantly affect carbachol-induced contractions at 37°C, while at lower temperatures ATP potentiated them. Suramin fully prevented effects of ATP. ATP is involved in both pre- and postsynaptic regulation of rat soleus muscle contractility, and these processes are significantly more pronounced at low temperatures. Muscle Nerve 55: 417-423, 2017.

Aggressive re-warming at 38.5°C following deep hypothermia at 21°C increases neutrophil membrane bound elastase activity and pro-inflammatory factor release.

BackgroundCardiopulmonary bypass (CPB) is often performed under hypothermic condition. The effects of hypothermia and re-warming on neutrophil activity are unclear. This study aimed to compare the effects of different hypothermia and re-warming regimens on neutrophil membrane bound elastase (MBE) activity and the release of pro-inflammatory factors from neutrophils.MethodsHuman neutrophils were exposed to different hypothermia and re-warming regimens. MBE activity and the release of interleukin (IL)-β1, IL-6, IL-8, and tumor necrosis factor (TNF)-α were measured.ResultsNeutrophil MBE activity was significantly reduced after 60-min moderate (28 °C) or deep (21 °C) hypothermic treatment. Compared with normothermic (37 °C) re-warming, aggressive re-warming (38.5°) for 120 min following deep hypothermia (21 °C) dramatically increased neutrophil MBE activity (P < 0.05). Co-incubation of neutrophils with platelet-rich plasma further increased MBE activity significantly under all the tested temperature regimens. IL-β1 release from neutrophils was significantly higher after deep hypothermia (21 °C) followed by normothermic (37 °C) re-warming than after moderate hypothermia (28 °C) followed by normothermic re-warming (P < 0.05). Aggressive re-warming (38.5°) following deep hypothermia significantly increased the release of IL-β1, IL-8, and TNF-α from neutrophil compared with moderate re-warming (37 °C) (all P < 0.05).ConclusionAggressive re-warming following deep hypothermia may contribute to CPB-associated tissue injury by increasing neutrophil MBE activity and stimulating pro-inflammatory factor release, thus, should be avoided. The optimal hypothermic temperature of CPB should be determined based on patient clinical characteristics and surgery type.

Collectin-11 detects stress-induced L-fucose pattern to trigger renal epithelial injury.

Physiochemical stress induces tissue injury as a result of the detection of abnormal molecular patterns by sensory molecules of the innate immune system. Here, we have described how the recently discovered C-type lectin collectin-11 (CL-11, also known as CL-K1 and encoded by COLEC11) recognizes an abnormal pattern of L-fucose on postischemic renal tubule cells and activates a destructive inflammatory response. We found that intrarenal expression of CL-11 rapidly increases in the postischemic period and colocalizes with complement deposited along the basolateral surface of the proximal renal tubule in association with L-fucose, the potential binding ligand for CL-11. Mice with either generalized or kidney-specific deficiency of CL-11 were strongly protected against loss of renal function and tubule injury due to reduced complement deposition. Ex vivo renal tubule cells showed a marked capacity for CL-11 binding that was induced by cell stress under hypoxic or hypothermic conditions and prevented by specific removal of L-fucose. Further analysis revealed that cell-bound CL-11 required the lectin complement pathway-associated protease MASP-2 to trigger complement deposition. Given these results, we conclude that lectin complement pathway activation triggered by ligand-CL-11 interaction in postischemic tissue is a potent source of acute kidney injury and is amenable to sugar-specific blockade.

STRUCTURAL-METABOLIC CHARACTERISTICS OF BONES AND CARTILAGE UNDER THE INFLUENCE OF HYPOTHERMIA (THE REVIEW OF LITERATURE)

The goal: to analyze the data presented in the scientific literature concerning the structural and metabolic features of bone and cartilage tissue after the action of total induced hypothermia. General idea of hypothermia and its existing classifications for humans and animals are given in the first section of the literature review. This section also contains the data concerning the influence of hypothermia on the human body, in particular older people. The second section presents the results of experimental research on the effect of hypothermic conditions on cultured bone cells (osteoblasts and osteoclasts) and articular cartilage (chondrocytes). The research results show the negative impact of induced total hypothermia of varying intensity on the structural-metabolic parameters of these cultured cells. Ultrastructural changes were noted in the cytoplasm and cells nuclei, as well as disturbances in the metabolism of matrix macromolecules. The third section deals with general questions of influence of total induced hypothermia on bone in normal and osteoporosis. It has been established that hypothermia directly affects bone tissue, destroying its structural-metabolic characteristics. Severe destructive changes in cells, matrix and micro elemental composition of bones have been noticed. It is shown that hypothermia causes systemic changes in the body (of the antioxidant system, hormone balance, blood supply, and others). Under the influence of hypothermia significant violations of osteoblasts ultrastructure, increased osteoclastogenesis, reduced osteoblastogenesis and activation of bone resorption were recorded in bone. The fourth section presents the data about structural-metabolic condition of articular cartilage after the action of severe hypothermia early after exposure. It is shown that severe hypothermia leads to pronounced ultra structural changes of articular cartilage cells and extracellular matrix.

CHANGES IN ERYTHROCYTE SURFACE MARKER CD44 DURING HYPOTHERMIC AND LOW TEMPERATURE STORAGE.

We studied the changes in surface marker CD44 in erythrocytes, cryopreserved under the protection of glycerol and PEG–1500, or stored in hypothermic conditions. It was shown that during hypothermic storage the CD44 characteristics in erythrocyte suspension were unchanged within 10 days. In cryopreserved erythrocytes a reduction in CD44–positive cells and in the level of expression of the surface marker were marked. Using PEG–1500 resulted in more pronounced change in erythrocyte CD44 characteristics after freeze–thawing in comparison with glycerol. Removal of cryoprotectants and the loss of a part of cells during the washing process led to the restoration of the CD44 characteristics in freeze–thawed erythrocytes suspension which successfully survived after the stresses. The results indicate that revealed changes in cryopreserved erythrocytes cover only a part of the cells, and they are associated with the instability of the population of erythrocytes with altered CD44 characteristics wherethrough after the removal of cryoprotectants with concomitant hemolysis of unstable cells the CD44 parameters in erythrocyte suspensions recovered. The mechanisms underlying the changes in the parameters of the surface marker CD44 in freeze–thawed erythrocyte may be related to the disruption of intermolecular interactions in the membrane under the influence of physical and chemical environmental factors, followed by the membrane vesiculation with the inclusion of the CD44 into the vesicles.

The role of the equatorial ligands for the redox behavior, mode of cellular accumulation and cytotoxicity of platinum(IV) prodrugs

The current study aims to elucidate the possible reasons for the significantly different pharmacological behavior of platinum(IV) complexes with cisplatin-, carboplatin- or nedaplatin-like cores and how this difference can be related to their main physicochemical properties. Chlorido-containing complexes are reduced fast (within hours) by ascorbate and are able to unwind plasmid DNA in the presence of ascorbate, while their tri- and tetracarboxylato analogs are generally inert under the same conditions. Comparison of the lipophilicity, cellular accumulation and cytotoxicity of the investigated platinum compounds revealed the necessity to define new structure-property/activity relationships (SPRs and SARs). The higher activity and improved accumulation of platinum(IV) complexes bearing Cl− in equatorial position cannot only be attributed to passive diffusion facilitated by their lipophilicity. Therefore, further platinum accumulation experiments under conditions where active/facilitated transport mechanisms are suppressed were performed. Under hypothermic conditions (4°C), accumulation of dichloridoplatinum(IV) complexes is reduced down to 10% of the amount determined at 37°C. These findings suggest the involvement of active and/or facilitated transport in cellular uptake of platinum(IV) complexes with a cisplatin-like core. Studies with ATP depletion mediated by oligomycin and low glucose partially confirmed these observations, but their feasibility was severely limited in the adherent cell culture setting.

Effect of Continuous Propofol Infusion in Rat on Tau Phosphorylation with or without Temperature Control.

Several studies suggest a relationship between anesthesia-induced tau hyperphosphorylation and the development of postoperative cognitive dysfunction. This study further characterized the effects of continuous propofol infusion on tau protein phosphorylation in rats, with or without temperature control. Propofol was administered intravenously to 8-10-week-old male Sprague-Dawley rats and infused to the loss of the righting reflex for 2 h continuously. Proteins from cortex and hippocampus were examined by western blot and immunohistochemistry. Rectal temperature was significantly decreased during propofol infusion. Propofol with hypothermia significantly increased phosphorylation of tau at AT8, AT180, Thr205, and Ser199 in cortex and hippocampus except Ser396. With temperature maintenance, propofol still induced significant elevation of AT8, Thr205, and Ser199 in cortex and hippocampus; however, increase of AT180 and Ser396 was only found in hippocampus and cortex, respectively. Differential effects of propofol with or without hypothermia on multiple tau related kinases, such as Akt/GSK3β, MAPK pathways, or phosphatase (PP2A), were demonstrated in region-specific manner. These findings indicated that propofol increased tau phosphorylation under both normothermic and hypothermic conditions, and temperature control could partially attenuate the hyperphosphorylation of tau. Further studies are warranted to determine the long-term impact of propofol on the tau pathology and cognitive functions.

Influence of hypothermia and subsequent rewarming upon leukocyte-endothelial interactions and expression of Junctional-Adhesion-Molecules A and B.

Patients with risks of ischemic injury, e.g. during circulatory arrest in cardiac surgery, or after resuscitation are subjected to therapeutic hypothermia. For aortic surgery, the body is traditionally cooled down to 18 °C and then rewarmed to body temperature. The role of hypothermia and the subsequent rewarming process on leukocyte-endothelial interactions and expression of junctional-adhesion-molecules is not clarified yet. Thus, we investigated in an in-vitro model the influence of temperature modulation during activation and transendothelial migration of leukocytes through human endothelial cells. Additionally, we investigated the expression of JAMs in the rewarming phase. Exposure to low temperatures alone during transmigration scarcely affects leukocyte extravasation, whereas hypothermia during treatment and transendothelial migration improves leukocyte-endothelial interactions. Rewarming causes a significant up-regulation of transmigration with falling temperatures. JAM-A is significantly modulated during rewarming. Our data suggest that transendothelial migration of leukocytes is not only modulated by cell-activation itself. Activation temperatures and the rewarming process are essential. Continued hypothermia significantly inhibits transendothelial migration, whereas the rewarming process enhances transmigration strongly. The expression of JAMs, especially JAM-A, is strongly modulated during the rewarming process. Endothelial protection prior to warm reperfusion and mild hypothermic conditions reducing the difference between hypothermia and rewarming temperatures should be considered.

Hypothermic modulation of human cortical neurons to explore a role for tau protein in neuroprotection

BackgroundCooling is the single most effective treatment for acute neuronal injury. Understanding the molecular mechanisms that mediate cooling-induced neuroprotection might yield novel therapeutic targets for neurodegenerative disease. Many of these disorders involve modulation of tau, a protein that is enriched in neurons and becomes hyperphosphorylated in hypothermic, injury-resistant rodent brains as well as in Alzheimer's disease. We sought to establish a new model for exploring human tau physiology and its role in neuroprotection in the context of therapeutic hypothermia. MethodsFunctional cortical neurons (hCNs) were differentiated from three independent human pluripotent stem-cell lines and validated for regional identity and tau status. Matched cultures were incubated at 37°C or clinically targeted temperatures for therapeutic hypothermia (32°C) and suspended animation (28°C). Effects of cooling on tau status and neuronal injury elicited by common neurotoxic stressors were established. Injury experiments were repeated while manipulating tau phosphorylation. FindingshCN differentiation featured transitions in tau status, recapitulating transcriptional and post-translational human in-vivo cortical tau development. Key aspects of this development were reversed by cooling. Notably, cooling induced tau hyperphosphorylation via rapid inhibition of the major tau phosphatase, protein phosphatase 2A (PP2A). Multiplexed injury analysis confirmed that hypothermia robustly protected hCNs from oxidative (100 μM hydrogen peroxide) and excitotoxic (30 μM glutamate) stress (at 28°C, injury was reduced by 78% and 56%, respectively; p<0·0005). Selective block of a major tau kinase reduced hCN tau phosphorylation and abrogated hypothermic protection (p=0·001) against oxidative injury at 28°C (injury reduction diminished to 9% at 100 μM hydrogen peroxide), whereas pharmacological inhibition of PP2A mimicked cooling-induced tau phosphorylation and protected hCNs from oxidative injury at 37°C (up to 22% injury reduction [p=0·008], with no benefit under hypothermic conditions). InterpretationTo our knowledge, this is the first study of human neuronal tau physiology under hypothermic conditions. Although definitive experiments are needed, our findings support previous work linking phospho-tau to neuroprotection. Furthermore, cold-induced tau hyperphosphorylation is a potential trigger for proteostatic priming, a recently discovered mechanism of hypothermic preconditioning in hCNs. Exploiting these cryobiological effects might lead to new treatments for acute and chronic neuronal injury. FundingWellcome Trust, Anne Rowling Regenerative Neurology Clinic, Euan MacDonald Centre for Motor Neurone Disease Research.

Prolonged hypothermia exposure diminishes neuroprotection for severe ischemic-hypoxic primary neurons

This study aimed to identify optimal mild hypothermic (MH) condition that would provide the best protection for neuronal cells undergoing severe ischemia and hypoxia. We also sought to determine if longer exposure to mild hypothermia would confer greater protection to severe ischemia and hypoxia in these cells. We designed a primary neuronal cell model for severe glucose and oxygen deprivation/reoxygenation (OGD/R) to simulate the hypoxic-ischemic condition of patients with severe stroke, trauma, or hypoxic-ischemic encephalopathy. We evaluated the viability of these neurons following 3 h of OGD/R and variable MH conditions including different temperatures and durations of OGD/R exposure. We further explored the effects of the optimal MH condition on several parts which are associated with mitochondrial apoptosis pathway: intracellular calcium, reactive oxygen species (ROS), and mitochondrial transmembrane potential (MTP). The results of this study showed that the apoptosis proportion (AP) and cell viability proportion (CVP) after OGD/R significantly varied depending on which MH condition cells were exposed to (p < 0.001). Further, our findings showed that prolonged MH reduced the neuroprotection to AP and CVP. We also determined that the optimal MH conditions (34 °C for 4.5 h) reduced intracellular calcium, ROS, and recovered MTP. These findings indicate that there is an optimal MH treatment strategy for severely hypoxia-ischemic neurons, prolonged duration might diminish the neuroprotection, and that MH treatment likely initiates neuroprotection by inhibiting the mitochondrial apoptosis pathway.

Avoidance of Profound Hypothermia During Initial Reperfusion Improves the Functional Recovery of Hearts Donated After Circulatory Death.

The resuscitation of hearts donated after circulatory death (DCD) is gaining widespread interest; however, the method of initial reperfusion (IR) that optimizes functional recovery has not been elucidated. We sought to determine the impact of IR temperature on the recovery of myocardial function during ex vivo heart perfusion (EVHP). Eighteen pigs were anesthetized, mechanical ventilation was discontinued, and cardiac arrest ensued. A 15-min standoff period was observed and then hearts were reperfused for 3 min at three different temperatures (5°C; N = 6, 25°C; N = 5, and 35°C; N = 7) with a normokalemic adenosine-lidocaine crystalloid cardioplegia. Hearts then underwent normothermic EVHP for 6 h during which time myocardial function was assessed in a working mode. We found that IR coronary blood flow differed among treatment groups (5°C = 483 ± 53, 25°C = 722 ± 60, 35°C = 906 ± 36 mL/min, p < 0.01). During subsequent EVHP, less myocardial injury (troponin I: 5°C = 91 ± 6, 25°C = 64 ± 16, 35°C = 57 ± 7 pg/mL/g, p = 0.04) and greater preservation of endothelial cell integrity (electron microscopy injury score: 5°C = 3.2 ± 0.5, 25°C = 1.8 ± 0.2, 35°C = 1.7 ± 0.3, p = 0.01) were evident in hearts initially reperfused at warmer temperatures. IR under profoundly hypothermic conditions impaired the recovery of myocardial function (cardiac index: 5°C = 3.9 ± 0.8, 25°C = 6.2 ± 0.4, 35°C = 6.5 ± 0.6 mL/minute/g, p = 0.03) during EVHP. We conclude that the avoidance of profound hypothermia during IR minimizes injury and improves the functional recovery of DCD hearts.

Zinc Finger Nuclease: A New Approach to Overcome Beta-Lactam Antibiotic Resistance.

Background:The evolution of antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs) has been accelerated recently by the indiscriminate application of antibiotics. Antibiotic resistance has challenged the success of medical interventions and therefore is considered a hazardous threat to human health.Objectives:The present study aimed to describe the use of zinc finger nuclease (ZFN) technology to target and disrupt a plasmid-encoded β-lactamase, which prevents horizontal gene transfer-mediated evolution of ARBs.Materials and Methods:An engineered ZFN was designed to target a specific sequence in the ampicillin resistance gene (ampR) of the pTZ57R plasmid. The Escherichia coli bacteria already contained the pZFN kanamycin-resistant (kanaR) plasmid as the case or the pP15A, kanaR empty vector as the control, were transformed with the pTZ57R; the ability of the designed ZFN to disrupt the β-lactamase gene was evaluated with the subsequent disturbed ability of the bacteria to grow on ampicillin (amp) and ampicillin-kanamycin (amp-kana)-containing media. The effect of mild hypothermia on the ZFN gene targeting efficiency was also evaluated.Results:The growth of bacteria in the case group on the amp and amp-kana-containing media was significantly lower compared with the control group at 37°C (P < 0.001). Despite being more efficient in hypothermic conditions at 30°C (P < 0.001), there were no significant associations between the incubation temperature and the ZFN gene targeting efficiency.Conclusions:Our findings revealed that the ZFN technology could be employed to overcome ampicillin resistance by the targeted disruption of the ampicillin resistance gene, which leads to inactivation of β-lactam synthesis. Therefore, ZFN technology could be engaged to decrease the antibiotic resistance issue with the construction of a ZFN archive against different ARGs. To tackle the resistance issue at the environmental level, recombinant phages expressing ZFNs against different ARGs could be constructed and released into both hospital and urban wastewater systems.

Effects of Brain Temperature on Cerebrovascular Autoregulation During the Acute Stage of Severe Traumatic Brain Injury.

The pressure reactivity index (PRx) is calculated as a moving correlation coefficient between intracranial pressure (ICP) and mean arterial blood pressure (MABP), and this analytical value is viewed as reflecting a vasomotor response to MABP variability. At present, the factors influencing the PRx value during the acute stage of traumatic brain injury (TBI) are not known. We observed significant cases where changes in the calculated value of PRx seemed to be influenced by changes in brain temperature during the course of acute stage TBI. In one case, a patient was treated for 72 h with therapeutic brain hypothermia after a decompressive hemicraniectomy. During the hypothermic condition, the mean value of PRx was -0.019; however, after gradual rewarming, the value of PRx increased drastically, and the mean value during the rewarming period, when the brain temperature exceeded 35 °C, was 0.331. Similarly, in another case where the patient underwent therapeutic brain hypothermia, the PRx showed a mean value of -0.038 during the hypothermic condition, and a mean value of 0.052 during the rewarming period. In both cases, a trend toward a negative correlation between ICP and MABP during brain hypothermia shifted to a positive correlation upon rewarming.

Effects of Sex and Mild Intrainsult Hypothermia on Neuropathology and Neural Reorganization following Neonatal Hypoxic Ischemic Brain Injury in Rats.

Hypoxia ischemia (HI) is a recognized risk factor among late-preterm infants, with HI events leading to varied neuropathology and cognitive/behavioral deficits. Studies suggest a sex difference in the incidence of HI and in the severity of subsequent behavioral deficits (with better outcomes in females). Mechanisms of a female advantage remain unknown but could involve sex-specific patterns of compensation to injury. Neuroprotective hypothermia is also used to ameliorate HI damage and attenuate behavioral deficits. Though currently prescribed only for HI in term infants, cooling has potential intrainsult applications to high-risk late-preterm infants as well. To address this important clinical issue, we conducted a study using male and female rats with a postnatal (P) day 7 HI injury induced under normothermic and hypothermic conditions. The current study reports patterns of neuropathology evident in postmortem tissue. Results showed a potent benefit of intrainsult hypothermia that was comparable for both sexes. Findings also show surprisingly different patterns of compensation in the contralateral hemisphere, with increases in hippocampal thickness in HI females contrasting reduced thickness in HI males. Findings provide a framework for future research to compare and contrast mechanisms of neuroprotection and postinjury plasticity in both sexes following a late-preterm HI insult.

δV1-1 Reduces Pulmonary Ischemia Reperfusion-Induced Lung Injury by Inhibiting Necrosis and Mitochondrial Localization of PKCδ and p53

Ischemia-reperfusion (IR)-induced lung injury is one of the major contributing factors of morbidity and mortality after lung transplantation. To determine the IR-induced molecular changes in lung epithelial cells, we developed a cell-culture model that simulates lung preservation and transplantation. Six hours of cold ischemic time (CIT) and reperfusion elicited production of multiple inflammatory cytokines and chemokines and increased expression of endoplasmic reticulum (ER) proteins. Prolonged hypothermic condition (18 h CIT) reduced ER stress protein levels, and induced apoptosis and necrosis (via mechanisms related to mitochondrial permeability transition pore opening). Protein kinase C (PKCδ) was activated during CIT, and its downregulation via small interference (si) (in siRNA) RNA reduced IR-induced cytokine production and apoptotic cell death. δV1-1, a PKCδ peptide inhibitor, reduced translocation of PKCδ and p53 to the mitochondria after 18 h CIT, rescued ER stress protein expression, and converted the major mode of cell death from necrosis to apoptosis. Administration of δV1-1 effectively reduced lung transplantation and IR-induced pulmonary injury in rats. Therefore, inhibition of PKCδ by δV1-1 could be an effective strategy to ameliorate IR-induced lung injury by inhibiting the signaling pathways leading to necrosis.

Spontaneous Packaging and Hypothermic Storage of Mammalian Cells with a Cell-Membrane-Mimetic Polymer Hydrogel in a Microchip.

Currently, continuous culture/passage and cryopreservation are two major, well-established methods to provide cultivated mammalian cells for experiments in laboratories. Due to the lack of flexibility, however, both laboratory-oriented methods are unable to meet the need for rapidly growing cell-based applications, which require cell supply in a variety of occasions outside of laboratories. Herein, we report spontaneous packaging and hypothermic storage of mammalian cells under refrigerated (4 °C) and ambient conditions (25 °C) using a cell-membrane-mimetic methacryloyloxyethyl phosphorylcholine (MPC) polymer hydrogel incorporated within a glass microchip. Its capability for hypothermic storage of cells was comparatively evaluated over 16 days. The results reveal that the cytocompatible MPC polymer hydrogel, in combination with the microchip structure, enabled hypothermic storage of cells with quite high viability, high intracellular esterase activity, maintained cell membrane integrity, and small morphological change for more than 1 week at 4 °C and at least 4 days at 25 °C. Furthermore, the stored cells could be released from the hydrogel and exhibited the ability to adhere to a surface and achieve confluence under standard cell culture conditions. Both hypothermic storage conditions are ordinary flexible conditions which can be easily established in places outside of laboratories. Therefore, cell packaging and storage using the hydrogel incorporated within the microchip would be a promising miniature and portable solution for flexible supply and delivery of small amounts of cells from bench to bedside.

Short-term Hypothermia Induces Beta-catenin-interacting Protein 1 Gene Expression in PC12 Cells

The effects of hypothermic treatment (32℃) on recovery from ischemia are controversial because the precise mechanisms of hypothermia remain unclear. We demonstrated previously that hypothermia induces beta-catenin-interacting protein 1 (CTNNBIP1) gene expression in vitro. In this study, we evaluated the effects of various hypothermic conditions, including lithium chloride treatment, on CTNNBIP1 gene expression. The results show that short-term hypothermic treatment resulted in relatively higher CTNNBIP1 gene expression than that of a longer treatment. These findings indicate that hypothermia controls CTNNBIP1 gene expression, which may provide clues to develop treatments to recover from and diagnose ischemia.

The prospects of the application of gases and gas hydrates in cryopreservation

In the present review, we attempt to evaluate the known properties of gas hydrates and gases that form them from the point of view of the mechanisms of cryoinjury and cryoprotection, to consider the publications on the freezing of biological materials in the presence of inert gases, and to assess the prospects of development in this field. For this purpose, we searched for information on the physical properties of gases and gas hydrates, compared the processes that occur during the formation of gas hydrates and water ice, and analyzed the effects of the formation and growth of gas hydrates on the structures of biological objects. We prepared a short review on the biological effects of xenon, krypton, argon, carbon dioxide, hydrogen sulfide, and carbon monoxide with emphasis on hypothermal conditions and the probable application of these properties in cryopreservation technology. The descriptions of the existing experiments on cryopreservation of biological objects using gases are analyzed. Based on the information we found, the most promising directions of work in the field of cryopreservation of biological objects using gases are outlined; potential problems in this field are anticipated.

Structural plasticity of green fluorescent protein to amino acid deletions and fluorescence rescue by folding-enhancing mutations.

BackgroundGreen fluorescent protein (GFP) and its derivative fluorescent proteins (FPs) are among the most commonly used reporter systems for studying gene expression and protein interaction in biomedical research. Most commercially available FPs have been optimized for their oligomerization state to prevent potential structural constraints that may interfere with the native function of fused proteins. Other approach to reducing structural constraints may include minimizing the structure of GFPs. Previous studies in an enhanced GFP variant (EGFP) identified a series of deletions that can retain GFP fluorescence. In this study, we interrogated the structural plasticity of a UV-optimized GFP variant (GFPUV) to amino acid deletions, characterized the effects of deletions and explored the feasibility of rescuing the fluorescence of deletion mutants using folding-enhancing mutations.MethodsTransposon mutagenesis was used to screen amino acid deletions in GFP that led to fluorescent and nonfluorescent phenotypes. The fluorescent GFP mutants were characterized for their whole-cell fluorescence and fraction soluble. Fluorescent GFP mutants with internal deletions were purified and characterized for their spectral and folding properties. Folding-ehancing mutations were introduced to deletion mutants to rescue their compromised fluorescence.ResultsWe identified twelve amino acid deletions that can retain the fluorescence of GFPUV. Seven of these deletions are either at the N- or C- terminus, while the other five are located at internal helices or strands. Further analysis suggested that the five internal deletions diminished the efficiency of protein folding and chromophore maturation. Protein expression under hypothermic condition or incorporation of folding-enhancing mutations could rescue the compromised fluorescence of deletion mutants. In addition, we generated dual deletion mutants that can retain GFP fluorescence.ConclusionOur results suggested that a “size-minimized” GFP may be developed by iterative incorporation of amino acid deletions, followed by fluorescence rescue with folding-enhancing mutations.Electronic supplementary materialThe online version of this article (doi:10.1186/s12858-015-0046-5) contains supplementary material, which is available to authorized users.

Evaluation of hypothermia on the in vitro metabolism and binding and in vivo disposition of midazolam in rats.

The effect of hypothermia on the in vivo pharmacokinetics of midazolam was evaluated, with a focus on altered metabolism in the liver and binding to serum proteins. Rat primary hepatocytes were incubated with midazolam (which is metabolized mainly by CYP3A2) at 37, 32 or 28 °C. The Michaelis-Menten constant (Km) and maximum velocity (Vmax) of midazolam were estimated using the Michaelis-Menten equation. The Km of CYP3A2 midazolam remained unchanged, but the Vmax decreased at 28 °C. In rats, whose temperature was maintained at 37, 32 or 28 °C by a heat lamp or ice pack, the plasma concentrations of midazolam were higher, whereas those in the brain and liver were unchanged at 28 °C. The tissue/plasma concentration ratios were, however, increased significantly. The unbound fraction of midazolam in serum at 28 °C was half that at 37 °C. These pharmacokinetic changes associated with hypothermic conditions were due to reductions in CYP3A2 activity and protein binding.