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Ischemic tolerance is a phenomenon whereby transient exposure to a non-injurious preconditioning stimulus triggers resistance to a subsequent lethal ischemic insult. Despite the fact that not only neurons but also astrocytes and endothelial cells have a unique response to preconditioning stimuli, current research has been focused mostly on the effect of preconditioning on neuronal death. Thus, it is unclear if the blood-brain barrier (BBB) can be preconditioned independently of an effect on neuronal survival. The release of tissue-type plasminogen activator (tPA) from perivascular astrocytes in response to an ischemic insult increases the permeability of the BBB. In line with these observations, treatment with recombinant tPA increases the permeability of the BBB and genetic deficiency of tPA attenuates the development of post-ischemic edema. Here we show that tPA induces ischemic tolerance in the BBB independently of an effect on neuronal survival. We found that tPA renders the BBB resistant to an ischemic injury by inducing TNF-α-mediated astrocytic activation and increasing the abundance of aquaporin-4-immunoreactive astrocytic end-feet processes in the neurovascular unit. This is a new role for tPA, that does not require plasmin generation, and with potential therapeutic implications for patients with cerebrovascular disease.

INTRODUCTION: proper detection and analysis of the biological evidence is a crucial matter for crime scene reconstruction and the most promising method for tissue determination is the analysis of differential DNA methylation patterns according to cell or tissue type. These patterns are chemically and biologically more stable thus representing a more reliable identification tool. Identification of semen and sperms are particularly important, especially in sexual assaults, where sperms are the source of DNA in the human ejaculate. In practice, sperms isolated from sexual assault evidence lose most of their distinctive sub-cellular organelles upon which morphological identification depends. AIM OF THE RESEARCH: to identify stability of the FGF7 marker in stored seminal specimens. MATERIAL and METHODS: 15 seminal specimens stored for over 10 years were used along with 5 fresh samples for FGF7 assessment. RESULTS and CONCLUSION: DNA methylation proved to be a stable marker for both fresh semen and stored semen samples.

OBJECTIVES We used laser ablation inductively coupled plasma mass spectrometry to quantify gadolinium in hair samples from autopsy cases with gadolinium-based contrast agent (GBCA) exposure. Hair gadolinium data were correlated with gadolinium concentrations in brain, skin, and bone tissues from the same case to investigate a potential noninvasive method for gadolinium quantification and monitoring. MATERIALS AND METHODS Medical records from autopsy cases at our institution were screened for history of GBCA exposure. Cases with exposure to a single type of GBCA with the most recent injection occurring within 1 year were identified and included in the study. The concentration of gadolinium in hair samples was analyzed by laser ablation inductively coupled plasma mass spectrometry, and brain (globus pallidus, dentate nucleus, white matter), bone, and skin tissues were analyzed by bulk inductively coupled plasma mass spectrometry. The mean of the maximum value in the hair samples was used to generate a representative measurement of the hair gadolinium concentration for each case. A linear regression analysis between each tissue type and hair was conducted to assess for possible correlation. RESULTS Tissue and hair samples from 18 autopsies (16 cases with exposure to GBCA, 2 controls) were included in the study. Comparing the different tissues revealed good correlation between some tissue types. The best model fit occurred between white matter and hair (R = 0.83; P < 0.0001) followed by the comparison between dentate nucleus and hair (R = 0.72; P < 0.0001) and dentate nucleus and skin (R = 0.70; P < 0.0001). CONCLUSIONS A significant correlation in this study between hair gadolinium concentrations and brain and skin gadolinium concentrations suggests that hair may serve as a safe and effective biomonitoring tissue for patients who receive GBCA injections.

Fatty acid profiles increasingly are being used to quantify foraging patterns of consumers, but the associated interpretation may vary with the tissue type and its lipid content. For salmonids, lipid deposits can be found in both dorsal and ventral (“belly flap”) areas of muscle tissues. However, it is uncertain whether belly flap and dorsal muscle fatty acid profiles are similar in natural populations of fish. We examined how fatty acid profiles of belly flap compared to those of dorsal muscle and the consequent impacts on dietary inferences. Fatty acid profiles were derived from lake trout (Salvelinus namaycush) caught in five North American lakes: Champlain, Flathead, Michigan, Ontario, and Swan. Fatty acid profiles were most similar between tissues when lipid content of muscle was > ~10%, the threshold below which similarities decreased and thus increasingly affected dietary inference. Some fatty acids commonly used as trophic indicators preferentially accrued in one tissue over the other depending on lipid content of the tissues. Regardless of tissue type, fatty acid profiles were specific to each lake indicating that food web structures were distinctive over a broad geographic range. Fatty acid profiles of tissues from lakes Michigan and Ontario were highly similar, so were those from Flathead and Swan lakes, whereas those from Lake Champlain were distinct, having comparatively high proportions of 18:1n-9. We conclude that lipid storage areas like belly flaps likely provide a more accurate signal than muscle when using fatty acids to investigate dietary patterns, particularly when muscle lipid levels are low.

The effects of thrombo-prevention, such as antiplatelet and anticoagulant activity, have been reported with the usage of Ginkgo biloba extract (GbE); however, the detailed mechanism has not yet been fully investigated, especially the role of Krüppel-like factor 2 (KLF2). This study aimed to investigate whether GbE can activate KLF2 and then induce thrombomodulin (TM) and tissue-type plasminogen activator (t-PA) secretion to enhance the effects of thrombo-prevention. Different concentrations of GbE were incubated with human umbilical vein endothelial cells (HUVECs) to evaluate its effect on endothelial cells. We found that KLF2 expression is correlated to the risk of atherosclerosis and venous thromboembolism in clinical practice. In the HUVEC cell model, GbE stimulated the expression of KLF2 in a dose-dependent manner. Moreover, TM and t-PA secretion increased when the cells were cultured with GbE. Both the expressions and activities of TM and t-PA in the GbE-treated cells declined after KLF2 was blocked by shKLF2. In sum, with GbE treatment, KLF2 expression in human endothelial cells was significantly activated, which in turn induced an increase in the protein expression and activity of TM and t-PA. After shRNA inhibited the KLF2 expression, GbE stopped inducing the expression and activity of TM and t-PA. These findings suggest that GbE exerts an antithrombotic effect on endothelial cells by increasing the TM expression and t-PA secretion; further, KLF2 is a key factor in this mechanism.

This study aimed to investigate accuracy in different sectional planes of the TMA Grand Master (3DHISTECH) Workstation in various soft tissue samples collected from Wistar rats. A total of 108 animals were sacrificed and 963 tissue specimens collected from 12 soft-tissue types. A total of 3307 tissue cores were punched and transferred into 40 recipient TMA blocks. Digital image analysis was performed. Core loss showed a significant correlation with tissue type and was highest in skin tissue (p &lt, 0.001), renal medulla and femoral artery, nerve, and vein bundle (p &lt, 0.01). Overall, 231 of 3307 tissue cores (7.0%) were lost. Hit rate analysis was performed in 1852 punches. The target was hit completely, partially and missed totally by 89.4%, 7.2% and 2.2%. A total of 54.5% of punches had good accuracy with less than 200 µm deviation from the centre of the targeted region and 92.6% less than 500 µm. Accuracy decreases with greater sectional depth. In the deepest sectional plane of roughly 0.5 mm median depth, almost 90% of cores had a deviation below 500 µm. Recommendations for automated TMA creation are given in this article. The ngTMA®-method has proven accurate and reliable in different soft tissues, even in deeper sectional layers.

Platelets play a key role in hemostasis as cofactors of thrombin generation, fibrin polymerization centers­, and initiators of clot retraction, while their ability to modulate clot dissolution remains less understood. The aim of this study was to investigate the interaction of plasminogen and tissue plasminogen activator with native and activated platelets, to determine the amount of plasmin generated by various activators in the presence of platelets, and the ability of platelets to modulate the rate of polymer fibrin hydrolysis. Spectrometric and immunofluorometric methods were used in the study. It was shown that intact circulating platelets carry a small amount of plasminogen on their surface, whereas thrombin-induced activation led to the exposure of plasminogen-binding sites on their plasma membrane. Activated platelets stimulated plasminogen activation by tissue plasminogen activator, urokinase, and streptokinase. Components of prothrombin complex enhanced plasminogen activation by tissue plasminogen activator on the surface of activated platelets. Model system with desAB-fibrin revealed the ability of platelets to stimulate fibrinolysis. These results suggest that the regulation of fibrinolysis by platelets is provided by the binding of plasminogen and plasminogen activators on their surface, acceleration of plasmin generation and, consequently, acceleration of the onset of fibrin lysis and reducing of the clot lifetime, which is important to maintain hemostatic balance.

The development of precision testing for disease diagnosis has advanced medicine by specifically matching patients with drugs to treat specific diseases. High-quality diagnostics start with high-quality tissue specimens. The development and optimization of tissue handling and processing have lagged behind bioassay development. Ultrasound time-of-flight (TOF) technology has been successfully used to monitor the critical processing step of tissue fixation with formalin. In this study, we expand the use of this technology to monitor tissue dehydration and clearing by analyzing TOF signals from 270 different specimens, representing 13 different tissue types obtained through surgical resections. We determined the time constant τ90 for each tissue type for the following tissue processing solvents: 70% ethanol, 90% ethanol, 100% ethanol, and xylene. The TOF signals were correlated with tissue morphology to ensure that high-quality tissue was produced. Tissues can be grouped into those exhibiting fast and slow reagent diffusion. We monitored incomplete dehydration of tissue by skipping a key processing step, dehydration in absolute ethanol, and then correlated the τ90 with poor histomorphology, demonstrating that the technique can detect significant processing errors. Ultrasound TOF technology can therefore be used to monitor all phases of tissue processing cycle and yields an important preanalytical quality metric.

Characterization of brain tumours requires neuropathological expertise and is generally performed by histological evaluation and molecular analysis. One emerging technique to assist pathologists in future tumour diagnostics is multimodal optical spectroscopy. In the current clinical routine, tissue preprocessing with formalin is widely established and suitable for spectroscopic investigations since degradation processes impede the measurement of native tissue. However, formalin fixation results in alterations of the tissue chemistry and morphology for example by protein cross-linking. As optical spectroscopy is sensitive to these variations, we evaluate the effects of formalin fixation on multimodal brain tumour data in this proof-of-concept study. Nonfixed and formalin-fixed cross sections of different common human brain tumours were subjected to analysis of chemical variations using ultraviolet and Fourier-transform infrared microspectroscopy. Morphological changes were assessed by elastic light scattering microspectroscopy in the visible wavelength range. Data were analysed with multivariate data analysis and compared with histopathology. Tissue type classifications deduced by optical spectroscopy are highly comparable and independent from the preparation and the fixation protocol. However, formalin fixation leads to slightly better classification models due to improved stability of the tissue. As a consequence, spectroscopic methods represent an appropriate additional contrast for chemical and morphological information in neuropathological diagnosis and should be investigated to a greater extent. Furthermore, they can be included in the clinical workflow even after formalin fixation.

Objective Humans consume low quantities of cadmium (Cd), a non-nutritive and potentially toxic heavy metal, primarily via the dietary intake of grains. A trial experiment was conducted to investigate physiological and developmental differences in Cd content in four flax cultivars (‘AC Emerson’, ‘Flanders’, ‘CDC Bethune’, and ‘AC McDuff’) as part of a study to provide information that will assist in the breeding of low Cd-accumulating flax cultivars. Our objective was to identify varietal differences in the uptake and distribution of Cd in various tissues among flax cultivars grown in naturally Cd-containing soil in a controlled environment. Results Cadmium concentration was dependent on genotype, developmental stage, and tissue type, as well as their interaction. Cadmium concentration was higher in roots and leaves, relative to all other tissues, with a general trend of decreasing Cd content over time within leaves and stems. Notably, the concentration of Cd was higher in ‘AC Emerson’ relative to ‘AC McDuff’ across tissues and ages, including the seeds, while the concentration of ‘Flanders’ was higher than in ‘AC McDuff’ in seeds and other reproductive organs but similar in roots and leaves. The results suggest varietal differences in the mechanisms that determine Cd content in seeds.

HOXB13, the most posterior HOX B gene, is primarily expressed in the prostate. Using immunohistochemical staining, we evaluated the expression of HOXB13 in prostatic tissues. We found that HOXB13 was expressed in both benign prostatic tissues and prostate adenocarcinoma (AdPCa) but its expression was reduced or lost in neuroendocrine prostate cancer (NEPCa). Additionally, using RNAseq data of HOXs in cell lines derived from various tissue origins, we established HOX codes for these tissue types. We applied the HOX codes to PCa samples and found that the majority of AdPCa samples sustained the prostate-specific HOX code but the majority of NEPCa samples lost it. Our analysis also showed that the NEPCa samples did not correlate well with the HOX codes of any other tissue type. This indicates that NEPCa tumors lose prostate identity but do not gain a clear-cut new tissue identity. Finally, we treated PCa cells with all trans retinoic acid and found that the reduced/lost HOXB13 expression can be reverted, as can AR and AR targets. Taken together, our data indicate that HOXB13 expression is reduced or lost in NEPCa and a loss of prostate-specific HOX code in NEPCa represents a loss of prostatic identity.

Carotid atherosclerosis is one of the main causes of stroke, mortality and disability worldwide. The disease is characterized by plaques, heterogeneous depositions of lipids and necrotic debris in the vascular wall, which grow gradually and may remain asymptomatic for decades. However, at some point a plaque can evolve to a high-risk plaque phenotype, which may trigger a cerebrovascular event. Lipids play a key role in the development and progression of atherosclerosis. Using matrix-assisted laser desorption/ionization mass spectrometry imaging, we visualized the distribution of approximately 200 lipids in 106 tissue sections of 12 human carotid atherosclerotic plaques. We performed unsupervised classification of the mass spectrometry dataset, as well as a histology-driven multivariate analysis. These data allowed us to compare the spatial lipid patterns with morphological plaque features that have been associated with enhanced risk of symptoms. The abundances of sphingomyelin and oxidized cholesteryl ester species were elevated specifically in necrotic intima areas, while diacylglycerols and triacylglycerols were spatially correlated to areas containing the coagulation protein fibrin. This is the first study to systematically investigate spatial lipid patterns in atherosclerosis, analyze their relation to histological tissue type, and to demonstrate a clear co-localization between plaque features and specific lipid classes and individual lipid molecules in high-risk atherosclerotic plaques.

An appropriate identification of the tissue type that is in front of the excision surgical tool is critical, for either tumoral tissue removal, or even accessory healthy tissues whose destruction could cause severe collateral damage. Healthy tissue distinction is an unsolved task in real surgical praxis, due for instance to alterations or hemorrhages. Many approaches have tried to solve the problem of healthy tissue discrimination, such as spectroscopy. Diffuse Reflectance Spectroscopy (DRS), an easy to implement and reliable optical diagnostic technique, has shown great potential in tumoral tissue discrimination, as long as in healthy tissue distinction. However, the identification of nerve tissue in particular is challenging. What is more, nerve tissue distinction is critical in surgical interventions, due to the severe irreversible dysfunctions in patient mobility that collateral damage could cause. Fluorescence is an optical technique that could be of relevance for nerve identification. The need of specific fluorophores that tend to accumulate preferentially on nerve tissue is essential for this task. Image quality requires also an analysis of the optical source, filters employed, fluorophore concentration or inoculation method. Together with those aspects, additional open questions include incubation time or cytotoxicity, critical for clinical translation. In this work, fluorescence imaging for nerve identification is applied on in vivo rats. The approach uses several fluorophore concentrations, based on Oxazine, and combines topical and intravascular inoculation. Optical irradiance is considered for image contrast. The results show an appreciable nerve contrast for optimized parameters. This work has been partially supported by the project “High-pressure driven plasmonic and luminescence properties ofnaked and core/shell metal-oxide nanocomposites” (PGC2018-101464-B-I00) of the Spanish Ministry of Science, Research and Universities, cofunded by FEDER funds, and by the San Cándido Foundation.

Magnetization transfer (MT)–magnetic resonance imaging (MRI) techniques can provide access to visualizing macromolecular content of tissues, otherwise not assessable using conventional MRI. MT contrast is generated by the interaction between mobile water protons and the protons associated with tissue macromolecules. Given substantial variation between macromolecular compositions of tissues, this interaction differs by tissue type and results in macromolecular-sensitive contrast in MT-weighted images. MT contrast is generated by applying radiofrequency pulses to selectively saturate the macromolecular protons, and this saturation is transferred to the water protons resulting in an observed signal attenuation. The MT effect can be characterized via the MT ratio (MTR) and, more recently, via quantitative MT (qMT) methods. MT-MRI indices may be useful to detect and quantify macromolecular alterations, potentially offering unique opportunities to link pathophysiologic and clinical outcomes in disorders of the central nervous system (CNS), and response to treatments.

The decellularization method efficacy varies depending on the tissue type. There are commonly used physical, chemical and enzymatic methods. Most of the methods affect the extracellular matrix properties that will in turn alter the cell fate when the biological scaffold is recellularised. We aimed to optimize the decellularization of ram cardiac via scCO2 extraction to preserve the moisture and the components of the ECM during the decellularization procedure. In literature decellularization studies focus on heart vessels. In this study, we aimed to decellularize and recellularize ram cardiac tissue for the first time. Besides we obtained a decellularized tissue without cells on it with a mechanical strength similar to native ECM. Decellularized ram cardiac tissue is expected to be animal-based biomaterial for candidate biomedical applications in vitro and in vivo further studies.

Gulf Menhaden, Brevoortia patronus, in the northern Gulf of Mexico support a large commercial fishery and are thought to play an important trophic role in the coastal ecosystem. The temporal dynamics of both fatty acid and oil content have a direct impact on the value of Gulf Menhaden to predators and to the fishery. In this work, we describe how oil content of Gulf Menhaden varies with season, sex, age, condition, and tissue and investigate how fatty acid composition of mature ( ≥ 137.5 mm FL) female tissues varies with season, month, and tissue type. We found pronounced temporal (January to October) variation in mean oil content ranging from 0.062 to 0.579 mg g−1 that exhibited a significant (p

Single-chain tissue-type plasminogen activator (sctPA) and single-chain urokinase plasminogen activator (scuPA) have attracted interest as enzymes for the treatment of inhalational smoke-induced acute lung injury (ISALI). In this study, the pulmonary delivery of commercial human sctPA and lyophilized scuPA and their reconstituted solution forms were demonstrated using vibrating mesh nebulizers (Aeroneb® Pro (active) and EZ Breathe® (passive)). Both the Aeroneb® Pro and EZ Breathe® vibrating mesh nebulizers produced atomized droplets of protein solution of similar size of less than about 5 μm, which is appropriate for pulmonary delivery. Enzymatic activities of scuPA and of sctPA were determined after nebulization and both remained stable (88.0% and 93.9%). Additionally, the enzymatic activities of sctPA and tcuPA were not significantly affected by excipients, lyophilization or reconstitution conditions. The results of these studies support further development of inhaled formulations of fibrinolysins for delivery to the lungs following smoke-induced acute pulmonary injury.

Appropriate timing of cold deacclimation is an important component of winter survival of perennial plants, such as trees, in temperate and boreal zones. Recently, concerns about predicted global climate change disturbing deacclimation timing have been increasing. The relationship between ambient temperatures and the manner by which cells' freezing resistance changes is essential for forecasting the timing of deacclimation. In this study, Japanese white birch twigs that underwent deacclimation treatment at a constant temperature of −2, 0, 4, 10, or 20 °C were separated into bark in which cells adapted to subfreezing temperatures by extracellular freezing and xylem in which cells adapted to subfreezing temperatures by deep supercooling, and the freezing resistance of cells in each tissue type was investigated by measuring percentage electrolyte leakage. Birch cells deacclimated in a different manner according to tissue type. Within 7 days under deacclimation treatment, xylem cells decreased their freezing resistance significantly at a high subfreezing temperature (−2 °C). In contrast, bark cells required a temperature of 10 or 20 °C for a detectable decrease in freezing resistance to occur within the same period. At a temperature lower than 0 °C, bark cells did not decrease their freezing resistance, even after 28 days of treatment. The difference in freezing behavior of cells might involve the difference in how deacclimation occurred in bark and xylem cells.

Total mercury concentrations ([THg]) were determined in muscle and liver of the bat ray (Myliobatis californica), shovelnose guitarfish (Pseudobatos productus) and banded guitarfish (Zapteryx exasperata). Generalized linear models (GLM) were used to determine the effects of size and sex in [THg] and showed that both are determinants of [THg] in these species. The [THg] in both tissues significantly increased with length especially in sexually mature organisms with a steeper slope for mature male than mature female. This may relate to elasmobranchs sexual dimorphism driven variation in growth rates. Median muscle [THg] was significantly greater than liver in each ray species but there were some individuals with higher liver [THg] than muscle. There were individuals with muscle [THg] higher than the advisory thresholds of 0.2 and 0.5mgkg-1ww (2.4 and 11% of the bat ray; 2.1 and 10% of the shovelnose guitarfish; 12.6 and 45% of the banded guitarfish, respectively).

In this paper, we characterize and discriminate between normal and cancer cells from three different tissue types, liver, lung, and breast, using capacitance-voltage-based extracted set of parameters. Cells from each type of cancer cell line were suspended in a liquid media either individually or as mixtures with their normal counterparts. Empirically, normal cells were observed to exhibit higher dielectric constants when compared to cancer cells from the same tissue. Moreover, adding cancer cells to normal cells was observed to increase the capacitance of normal cells, and the extent of this increase varied with the type of tissue tested with the lung cells causing the greatest change. This shows that the cancer cells of different cell origin possess their own signature electrical parameters, especially when compared with their normal counterparts, and that cancer cell seems to affect normal cells in a different manner, depending upon the tissue type. It was also noticed that the cells (both cancer and normal) exhibited a higher dielectric value as per the following order (from least to most): breast, lung, and liver. The changes in electrical parameters from normal to cancer state were explained not only by the modification of its physiological and biochemical properties but also by the morphological changes. This approach paves the way for exploring unique electrical signatures of normal and their corresponding cancer cells to enable their detection and discrimination.

Background After resection, specimens are subjected to formalin fixation during histological processing. This procedure can result in tissue shrinkage, with the amount of shrinkage related to tissue composition and tissue type. Aims To evaluate the shrinkage of nasal mucosa and cartilage tissue and compare differences in shrinkage after resection, after formalin fixation, and during microscopic examination to understand differences in the rate of shrinkage of different tissue types. Study design Animal experimentation. Methods Fresh nasal septa were excised from sheep (10 mm diameter in 40 sheep and 20 mm diameter in 40 sheep). The mucosa was separated from one side of the cartilage, with the contralateral mucosa remaining attached to the cartilage. Specimen diameters were measured in situ, after resection, after fixation for 6 or 24 hours (10% formalin), and during microscopic examination. Results There were no differences between the in situ and after resection diameters of any tissue components (free mucosa, mucosa attached to cartilage, and cartilage) of all nasal specimens (10- or 20-mm diameter and 6- or 24-hour fixation). However, significant shrinkage occurred between resection and after-fixation. Regarding tissue specimens that were fixed for different durations (6 or 24 hours), we observed a significantly smaller mean tissue diameter in specimens fixed for 24 hours versus those fixed for 6 hours for mucosa attached to cartilage (in the 10-mm diameter after-fixation samples), free mucosa (in the 20-mm diameter after-fixation samples), mucosa attached to cartilage (in the 20-mm diameter after-fixation and microscopic measurement samples), and cartilage (in the 20-mm diameter after-fixation samples). Tissue shrinkage was greatest in free mucosal tissue and least in cartilage. Conclusion These results should be considered when evaluating patients undergoing surgical procedures for nasal cavity and paranasal sinus malignancies. Surgical margins should be measured before fixation or evaluated if possible before fixation and shrinkage.

Creatine/phosphorylcreatine (PCr) responses to creatine supplementation may be modulated by age, diet, and tissue, but studies assessing this possibility are lacking. Therefore we aimed to determine whether PCr responses vary as a function of age, diet, and tissue. Fifteen children, 17 omnivorous and 14 vegetarian adults, and 18 elderly individuals (“elderly”) participated in this study. Participants were given placebo and subsequently creatine (0.3 g·kg−1·day−1) for 7 days in a single-blind fashion. PCr was measured through phosphorus magnetic resonance spectroscopy (31P-MRS) in muscle and brain. Creatine supplementation increased muscle PCr in children ( P < 0.0003) and elderly ( P < 0.001), whereas the increase in omnivores did not reach statistically significant difference ( P = 0.3348). Elderly had greater PCr increases than children and omnivores ( P < 0.0001 for both), whereas children experienced greater PCr increases than omnivores ( P = 0.0022). In relation to diet, vegetarians ( P < 0.0001), but not omnivores, had significant increases in muscle PCr content. Brain PCr content was not affected by creatine supplementation in any group, and delta changes in brain PCr (−0.7 to +3.9%) were inferior to those in muscle PCr content (+10.3 to +27.6%; P < 0.0001 for all comparisons). PCr responses to a standardized creatine protocol (0.3 g·kg−1·day−1 for 7 days) may be affected by age, diet, and tissue. Whereas creatine supplementation was able to increase muscle PCr in all groups, although to different extents, brain PCr was shown to be unresponsive overall. These findings demonstrate the need to tailor creatine protocols to optimize creatine/PCr accumulation both in muscle and in brain, enabling a better appreciation of the pleiotropic properties of creatine. NEW & NOTEWORTHY A standardized creatine supplementation protocol (0.3 g·kg−1·day−1 for 7 days) effectively increased muscle, but not brain, phosphorylcreatine. Older participants responded better than younger participants whereas vegetarians responded better than omnivores. Responses to supplementation are thus dependent on age, tissue, and diet. This suggests that a single “universal” protocol, originally designed for increasing muscle creatine in young individuals, may lead to heterogeneous muscle responses in different populations or even no responses in tissues other than skeletal muscle.

Background and Purpose— We investigated whether administration of edaravone, a free radical scavenger, before or during tissue-type plasminogen activator (tPA) can enhance early recanalization in a major arterial occlusion. Methods— The YAMATO study (Tissue-Type Plasminogen Activator and Edaravone Combination Therapy) is an investigator-initiated, multicenter (17 hospitals in Japan), prospective, randomized, and open-label study. Patients with stroke secondary to occlusion of the M1 or M2 portion of the middle cerebral artery and within 4.5 hours of the onset were studied. The subjects were randomly allocated to the early group (intravenous edaravone [30 mg] was started before or during tPA) and the late group (edaravone was started after tPA and the assessment of early recanalization). Results— One-hundred sixty-five patients (96 men; median age [interquartile range], of 78 [69–85] years) were randomized 1:1 to either the early group (82 patients) or the late group (83 patients). Primary outcome, defined as an early recanalization 1.5 hour after tPA, was observed in 53% of the early group and in 53% of the late group ( P =1.000). About secondary outcomes, the rate of significant recanalization of ≥50% was not different between the 2 groups (28% versus 34%; P =0.393). The symptomatic intracerebral hemorrhage has occurred in 4 patients (5%) in the early group and in 2 patients (2%) in the late group ( P =0.443). The favorable outcome (modified Rankin Scale score of 0–2) at 3 months was also similar between the groups (53% versus 57%; P =0.738). Conclusions— The timing of edaravone infusion does not affect the rate of early recanalization, symptomatic intracerebral hemorrhage, or favorable outcome after tPA therapy. Clinical Trial Registration— URL: http://www.umin.ac.jp/ctr/index-j.htm . Unique identifier: UMIN000006330.

Developmental changes occur in maize (Zea mays L.) as it transitions from juvenile stages to the mature plant. Changes also occur as newly formed cells mature into adult cells. Maize leaf blades including the midribs and sheaths undergo cell wall changes as cells transition to fully mature cell types. As is common in grasses during cell wall maturation, the lignin in the plant tissue is acylated with p-coumarates (pCA). This work characterizes cell walls in maize that make up leaf blade, leaf midrib, and sheath tissues corresponding to tissue development. Maize plants grown in the greenhouse were harvested; leaf, leaf midrib, and sheath tissues from nodes 9 through 14 tissues were analyzed for cell wall composition. Cell wall carbohydrates varied with the type of maize tissue, but there was little change within a tissue type among the different nodes. Lignin concentrations were lowest in the leaf blade (70 - 88 g&#183kg-1 CW) followed by the sheath (123 - 140 g&#183kg-1 CW) and highest in the midrib (140 - 168 g&#183kg-1 CW). Incorporation of pCA into cell walls paralleled the lignification. Ferulates (FA) remained relatively constant as a proportion of the cell wall (3.1 - 6.4 g&#183kg-1 CW) across nodes and across all tissue types. The range of FA was 3.8 vs 5.7 g&#183kg-1 CW averaged over all nodes with leaf blades being the lowest. Lignin composition did not change significantly with cell wall maturation within a given tissue type. The aerial portions of maize plants excluding the stem showed little difference in cell wall composition along the different nodes. Higher levels of ferulates were found in the sheath and leaf midrib compared to the leaf blade tissues. Leaf midribs from the upper nodes of the plant contained the highest levels of lignin. Perhaps a reflection of the function to keep leaves extended and in an upward angle to help maximize photosynthetic capacity.

© 2019, King Abdulaziz City for Science and Technology. Blood and the linings of blood vessels may be regarded as a fifth tissue type. The human body contains 5 × 109 red blood cells (RBCs) per ml, a total of 2.5 × 1013 cells in the 5 l of blood present in the body. With an average lifetime of 125 days, human RBCs are destroyed by leukocytes in the spleen and liver. Nowadays red blood cells are extensively used to study various metabolic functions. Nanoparticles (NP) are being widely accepted for drug delivery system. This review summarizes the red blood cells, NPs and their characteristics on the basis of the RBC components along with drug delivery systems through RBCs. Further, we also discussed that how erythrocytes can be used as an efficient in vitro model for evaluating the efficacy of various nanocomposite materials.

The congenital disorders of glycosylation are a diverse group of disorders, which present both common and unique challenges in the diagnosis of rare disorders. These disorders affect a variety of structures and processes in their synthesis. Studies by Himmelreich and by Ng and their coworkers are discussed as they exemplify the extremes of such challenges. These include ascertainment bias associated with the recognition of only extreme phenotypes, variant classification limited by the rarity of the observed variant, limitations in glycan methodology, and expression patterns that can change with time and tissue type. The continuing importance of functional studies to help sort out these challenges is highlighted.

Multidomain biotherapeutic proteins present additional behavioral and analytical challenges for the optimization of their kinetic stability by formulation. Tissue-type plasminogen activator (tPA) comprises six protein domains that exhibit a complex and pH-dependent thermal unfolding profile, due to partially independent domain unfolding. Here we have used tPA as a model for evaluating the relationships between various thermal unfolding and aggregation parameters in multidomain proteins. We show that changes in the thermal unfolding profile of tPA were parametrized by the overall thermal midpoint transition temperature, Tm, and the Van’t Hoff entropy for unfolding, ΔSvh, which is a measure of unfolding cooperativity. The kinetics of degradation at 45 °C, leading to aggregation, were measured as rates of monomer and activity loss. These two rates were found to be coincident at all pH. Aggregation accelerated at pH 4 due to the early unfolding of the serine protease N-terminal domain (SP-N), whereas at pH 5–...

Brain bioenergetic abnormalities have been observed frequently in adults with major depressive disorder (MDD); however, results have been inconsistent regarding whether decreased or increased metabolism was observed. Phosphorus-31 magnetic resonance spectroscopy (31P MRS) allows for the quantification of bioenergetic molecules, containing high-energy phosphates, over the whole brain as well as measuring the differences between gray matter and white matter. We recruited 50 subjects with a current diagnosis of MDD, not currently treated with psychotropic medication, between ages of 18 and 65 (mean±SD age: 43.4±13.6; 46% female) and 30 healthy volunteers, matched for age and gender (39.0±12.5 years of age; 36.6% female). All subjects received a T1 MP-FLASH scan for tissue segmentation followed by 31P MRS, chemical shift imaging scan with 84 voxels of data collected over the entire brain utilizing a dual-tuned, proton-phosphorus coil to minimize subject movement. Phosphocreatine and inorganic phosphate (Pi) varied in opposite directions across gray matter and white matter when MDD subjects were compared with controls. This finding suggests alterations in high-energy phosphate metabolism and regulation of oxidative phosphorylation in MDD patients. In addition, within the MDD group, gray matter Pi, a regulator of oxidative phosphorylation, correlated positively with severity of depression. These data support a model that includes changes in brain bioenergetic function in subjects with major depression.

We reported that tissue-type plasminogen activator (tPA) secreted from vascular endothelial cells (VECs) is retained on the cell surface and effectively evokes both plasminogen activation and fibrin clot dissolution (fibrinolysis) on VECs. Here, to evaluate possibly different behaviors of variants of tPA, we quantitatively assessed these two events separately using green fluorescent protein (GFP)-conjugated tPA in cultured human VECs. The amount of secreted wild-type tPA-GFP correlated well with both the activities of plasminogen activation (r = 0.66) and fibrinolysis (r = -0.93). A variant of tPA-GFP, with a lower affinity to the surface of VECs but higher affinity to fibrin, showed higher fibrinolysis and lower plasminogen activation activity compared to the wild-type.

This protocol details protein extraction from mouse tissues for immunoprecipitation purposes and has been applied for the performance of large-scale immunoprecipitations of target proteins from various tissues for the identification of associated proteins by mass spectroscopy. The key factors in performing a successful immunoprecipitation directly relate to the abundance of target protein in a particular tissue type and whether or not the embryonic, newborn, or adult mouse-derived tissues contain fibrous and other insoluble material. Several tissue types, including lung and liver as well as carcinomas, contain significant amounts of fibrous tissue that can interfere with an immunoprecipitation.

Four LC-MS/MS methods were developed to quantify melamine (MEL) and cyanuric acid (CYA) in various pig tissues at or above the level of concern (2.5 mg/kg). Pigs treated with 200 mg/kg bw/day CYA daily for 7 days did not accumulate significant residue concentrations in muscle, liver or kidney. Pigs treated with 200 mg/kg bw MEL daily for 7 or 28 days had MEL residues in muscles (3–13 ppm), liver (2.8–14.1 ppm) and kidney (9.4–27.2 ppm). Treatment with MEL and CYA at 100 mg/kg bw of each triazine daily for 7 days resulted in MEL (26–59 ppm in muscle, 30–49 ppm in liver and 367–6300 ppm in kidney) and CYA (1.8–5.8 ppm in muscle, 2.6–6.5 ppm in liver and 303–7100 ppm in kidney). Treatment with MEL and CYA at 1, 3 or 10 mg/kg bw/day for 7 days did not result in residues greater than the level of concern in all tissues tested. Pigs dosed with 33 mg/kg bw/day of MEL + CYA for 7 days contained residues above the level of concern only in kidney. Deposition of MEL and CYA depends on the tissue type (muscles, liver and kidney), dosage and whether the triazines are given alone or in combination.

Resveratrol is well known for its strong antioxidant properties. To accelerate research on resveratrol metabolism and to evaluate the genetic potential for breeding, the stilbenoid contents of four tissues (leaf and berry skin, flesh and seed) were examined in 21 accessions of six wild Chinese grapevine species (Vitis quinquangularis, V. romanetii, V. piasezkii, V. shenxiensis, V. amurensis and V. davidii). Resveratrol derivatives in V. quinquangularis, V. romanetii, V. piasezkii and V. shenxiensis are reported for the first time. Three kinds of stilbenoid (trans-resveratrol, cis-piceid and trans-piceid) were detected in the berry skins, flesh, seeds and leaves but cis-resveratrol was found to be absent. Stilbenoid content was predominantly affected by genetic background and by tissue type. In most accessions, berry skins produced the highest total stilbenoid, followed by leaves, seeds and flesh. As a proportion, cis-piceid was the major stilbenoid component in berry skins, seeds and leaves, whereas trans...

The renal glomeruli in lower vertebrates display mesangium-like cells and matrices interposed between the capillary endothelium and the basement membrane, while those in mammals reportedly lack such interpositions except in pathological conditions. By combined scanning and transmission electron microscopic observations, the pericapillary mesangial tissues were comparatively analyzed in four vertebrate classes: mammals (rats and rabbits), reptiles (green iguanas), amphibians (bullfrogs), and teleosts (carps). The observations discriminated three types of pericapillary interposition. The first, acellular interpositions, occurred universally, with mammalians displaying rudimental ones. This tissue type corresponded with extracellular matrices held in subendothelial grooves which were supported by fine endothelial projections anchored to the basement membrane. In lower vertebrates these grooves constituted an anastomosed system of subendothelial channels that communicated with the mesangial region, to favor cleaning of the glomerular filter. The second, compound type was specific to reptiles and amphibians, affecting the entire capillary circumference in the latter. In this tissue type, fine mesangial processes--which accompanied considerable amounts of fibrillar matrices--were loosely associated with the endothelial bases, indicating their possible nature as a kind of myofibroblast. Occurrence of the third, cellular interpositions was confined to small incidental loci in mammalian and teleost glomeruli. This tissue type was mostly occupied by thick processes or main bodies of the mesangial cells that tightly interlocked their short marginal microvilli with corresponding indentations on the endothelial bases.

Lipid and fatty acid (FA) analysis is commonly used to describe the trophic ecology of an increasing number of taxa. However, the applicability of these analyses is contingent upon the collection and storage of sufficient high quality tissue, the limitations of which are previously unexplored in elasmobranchs. Using samples from 110 white sharks, Carcharodon carcharias, collected throughout Australia, we investigated the importance of tissue type, sample quantity, and quality for reliable lipid class and FA analysis. We determined that muscle and sub-dermal tissue contain distinct lipid class and FA profiles, and were not directly comparable. Muscle samples as small as 12 mg dry weight (49 mg wet weight), provided reliable and consistent FA profiles, while sub-dermal tissue samples of 40 mg dry weight (186 mg wet weight) or greater were required to yield consistent profiles. This validates the suitability of minimally invasive sampling methods such as punch biopsies. The integrity of FA profiles in muscle was compromised after 24 hours at ambient temperature (~20°C), making these degraded samples unreliable for accurate determination of dietary sources, yet sub-dermal tissue retained stable FA profiles under the same conditions, suggesting it may be a more robust tissue for trophic ecology work with potentially degraded samples. However, muscle samples archived for up to 16 years in -20°C retain their FA profiles, highlighting that tissue from museum or private collections can yield valid insights into the trophic ecology of marine elasmobranchs.

High-quality biosamples are valuable resources for biomedical research. However, some tissues are stored without being sectioned into small aliquots and have to undergo repeated freeze-thaw cycles throughout prolonged experimentation. Little is known regarding the effects of repeated freeze-thaw cycles on the quality of biomolecules in tissues. The aim of this study was to evaluate the impact of repeated freeze-thaw (at room temperature or on ice) cycles on biomolecules and gene expression in four different types of tissues. Each fresh tissue was sectioned into seven aliquots and snap-frozen before undergoing repeated freeze-thaw cycles at room temperature or on ice. Biomolecules were extracted and analyzed. Both relative and absolute quantification were used to detect the changes in gene expression. The results indicated that the impact of repeated freeze-thaw cycles on RNA integrity varied by tissue type. Gene expression, including the housekeeping gene, was affected in RNA-degraded samples according to absolute quantification rather than relative quantification. Furthermore, our results suggest that thawing on ice could protect RNA integrity compared with thawing at room temperature. No obvious degradation of protein or DNA was observed with repeated freeze-thaw cycles either at room temperature or on ice. This research provides ample evidence for the necessity of sectioning fresh tissues into small aliquots before snap-freezing, thus avoiding degradation of RNA and alteration of gene expression resulting from repeated freeze-thaw cycles. For frozen tissue samples that were already in storage and had to be used repeatedly during their lifecycle, thawing on ice or sectioned at ultralow temperature is recommended.

ObjectivesThe tropics harbor a large part of the world’s biodiversity and have a long history of human habitation. However, paleogenomics research in these climates has been constrained so far by poor ancient DNA yields. Here we compare the performance of two DNA extraction methods on ancient samples of teeth and petrous portions excavated from tropical and semitropical sites in Tanzania, Mexico, and Puerto Rico (N=12).Materials and MethodsAll samples were extracted twice, built into double-stranded sequencing libraries, and shotgun sequenced on the Illumina HiSeq 2500. The first extraction protocol, Method D, was previously designed for recovery of ultrashort DNA fragments from skeletal remains. The second, Method H, modifies the first by adding an initial EDTA wash and an extended digestion and decalcification step.ResultsNo significant difference was found in overall ancient DNA yields or post-mortem damage patterns recovered from samples extracted with either method, irrespective of tissue type. However, Method H samples had higher endogenous content and more mapped reads after quality-filtering, but also higher clonality. In contrast, samples extracted with Method D had shorter average DNA fragments.DiscussionBoth methods successfully recovered endogenous ancient DNA. But, since surviving DNA in ancient or historic remains from tropical contexts is extremely fragmented, our results suggest that Method D is the optimal choice for working with samples from warm and humid environments. Additional optimization of extraction conditions and further testing of Method H with different types of samples may allow for improvement of this protocol in the future.

Mitochondrial respiration in the dark (R dark) is a critical plant physiological process, and hence a reliable, efficient and high-throughput method of measuring variation in rates of R dark is essential for agronomic and ecological studies. However, currently methods used to measure R dark in plant tissues are typically low throughput. We assessed a high-throughput automated fluorophore system of detecting multiple O2 consumption rates. The fluorophore technique was compared with O2-electrodes, infrared gas analysers (IRGA), and membrane inlet mass spectrometry, to determine accuracy and speed of detecting respiratory fluxes. The high-throughput fluorophore system provided stable measurements of R dark in detached leaf and root tissues over many hours. High-throughput potential was evident in that the fluorophore system was 10 to 26-fold faster per sample measurement than other conventional methods. The versatility of the technique was evident in its enabling: (1) rapid screening of R dark in 138 genotypes of wheat; and, (2) quantification of rarely-assessed whole-plant R dark through dissection and simultaneous measurements of above- and below-ground organs. Variation in absolute R dark was observed between techniques, likely due to variation in sample conditions (i.e. liquid vs. gas-phase, open vs. closed systems), indicating that comparisons between studies using different measuring apparatus may not be feasible. However, the high-throughput protocol we present provided similar values of R dark to the most commonly used IRGA instrument currently employed by plant scientists. Together with the greater than tenfold increase in sample processing speed, we conclude that the high-throughput protocol enables reliable, stable and reproducible measurements of R dark on multiple samples simultaneously, irrespective of plant or tissue type.

This chapter examines the use of the fluorescence spectroscopy and microscopy in the study of biological systems. The fluorescence technique is attractive since it offers less tedious preparation, high detection sensitivity, and the ability to give spectrotemporal as well as spectrospatial imaging of cells, which allows real-time monitoring of reactive species production in live cells. Detection techniques using fluorescent probes are employed through the aid of optical microscopes, spectrofluorimetric microplate readers, flow cytometric systems allowing high-throughput studies, and high-performance liquid chromatography (HPLC) analysis providing a more specific detection. The chapter discusses the chemistry of RS detection using fluorescence techniques and the methods employed by RS probes to assess overall cellular redox states through direct detection of reactive species generation as well as biomarkers of oxidative state are described. The chapter describes the classification of fluorescent RS probes by specificity, including by global redox state or total ROS detection and by selective detection, and also details the cell/tissue type or biological system for which each type of probe is most appropriate. The chapter details the specific uses of each type of probe, the advantages and limitations of each type of probe, and the factors that must be considered, such as the type of instrumentation, the type of expected radicals, and possible interferences.

International audience; Raman spectroscopy is a non-destructive, non-invasive, rapid and economical technique which has the potential to be an excellent method for the diagnosis of cancer and understanding disease progression through retrospective studies of archived tissue samples. Historically, biobanks are generally comprised of formalin fixed paraffin preserved tissue and as a result these specimens are often used in spectroscopic research. Tissue in this state has to be dewaxed prior to Raman analysis to reduce paraffin contributions in the spectra. However, although the procedures are derived from histopathological clinical practice, the efficacy of the dewaxing procedures that are currently employed is questionable. Ineffective removal of paraffin results in corruption of the spectra and previous experiments have shown that the efficacy can depend on the dewaxing medium and processing time. The aim of this study was to investigate the influence of commonly used spectroscopic substrates (CaF2, Spectrosil quartz and low-E slides) and the influence of different histological tissue types (normal, cancerous and metastatic) on tissue preparation and to assess their use for spectral histopathology. Results show that CaF2 followed by Spectrosil contribute the least to the spectral background. However, both substrates retain paraffin after dewaxing. Low-E substrates, which exhibit the most intense spectral background, do not retain wax and resulting spectra are not affected by paraffin peaks. We also show a disparity in paraffin retention depending upon the histological identity of the tissue with abnormal tissue retaining more paraffin than normal.

Background: The field of immuno-oncology research has enthusiastically adopted multiplex immunohistochemistry (IHC) techniques to establish the spatial relationships between various immune cell populations in a tumor biology in context. Multiplexing enables researchers to gain a deeper understanding and insight into the tumor microenvironment. Unfortunately, many multiplexing technologies face several challenges, specifically in generating highly robust, reproducible, and easily quantifiable data sets. Ultivue’s UltiMapper I/O APC and T-act kits utilize InSituPlex® technology, a new method of multiplexed IHC that utilizes a streamlined workflow with single antigen retrieval, staining, amplification, and detection steps, allowing for the completion of the assay in 5 hours. Here we assess these kits for intra- and inter-run reproducibility and quantification. Methods: Intra-run reproducibility and quantification was accomplished by manually staining 5 serial sections from three tissue types (deidentified samples of tonsil, melanoma, NSCLC) with one set for each of the UltiMapper I/O APC (CD11c, CD20, CD68/CD163, and MHC Class II) and T-act (CD3, Granzyme B, Ki67, and pan-Cytokeratin/SOX10) kits. Inter-run assessment was determined by staining each slide from a set of five serial sections of each tissue type independently. Images were acquired using the ZEISS® Axio Scan.Z1, without the need for linear unmixing allowing for direct whole slide imaging. Analysis was accomplished using Indica Labs HALO® software. Coefficients of variation (CVs) were calculated based on resulting data. Results: Analysis of intra-run serial section images revealed that cell counts from section to section had CVs of Conclusions: The results presented here indicate that InSituPlex technology is potentially much more reproducible than other tissue multiplexing techniques currently available. Histological standards for coefficients of variation in IHC based assays are typically Note: This abstract was not presented at the meeting. Citation Format: Bonnie Phillips, Katir Patel, Courtney Hebert, Aditi Sharma, Jamie Buell, Sean Downing. Intra- and inter-run assessment of reproducibility and quantification of UltiMapper™ I/O APC and T-act kits for tissue multiplexing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4569.

The research work presented in this paper focuses on qualitative tissue differentiation by monitoring the intensity ratios of atomic emissions using ‘Laser Induced Breakdown Spectroscopy’ (LIBS) on the plasma plume created during laser tissue ablation. The background of this study is to establish a real time feedback control mechanism for clinical laser surgery systems during the laser ablation process. Ex-vivo domestic pig tissue samples (muscle, fat, nerve and skin) were used in this experiment. Atomic emission intensity ratios were analyzed to find a characteristic spectral line for each tissue. The results showed characteristic elemental emission intensity ratios for the respective tissues. The spectral lines and intensity ratios of these specific elements varied among the different tissue types. The main goal of this study is to qualitatively and precisely identify different tissue types for tissue specific laser surgery. (© 2013 WILEY-VCH Verlag GmbH &Co. KGaA, Weinheim)

In the mid-1940s, the United States began producing atomic weapon components at the Los Alamos National Laboratory (LANL). In an attempt to better understand historical exposure to nearby residents, this study evaluates plutonium activity in human tissue relative to residential location and length of time at residence. Data on plutonium activity in the lung, vertebrae, and liver of nearby residents were obtained during autopsies as a part of the Los Alamos Tissue Program. Participant residential histories and the distance from each residence to the primary plutonium processing buildings at LANL were evaluated in the analysis. Summary statistics, including Student t-tests and simple regressions, were calculated. Because the biological half-life of plutonium can vary significantly by organ, data were analyzed separately by tissue type (lung, liver, vertebrae). The ratios of plutonium activity (vertebrae:liver; liver:lung) were also analyzed in order to evaluate the importance of timing of exposure. Tissue data were available for 236 participants who lived in a total of 809 locations, of which 677 were verified postal addresses. Residents of Los Alamos were found to have higher plutonium activities in the lung than non-residents. Further, those who moved to Los Alamos before 1955 had higher lung activities than those who moved there later. These trends were not observed with the liver, vertebrae, or vertebrae:liver and liver:lung ratio data, however, and should be interpreted with caution. Although there are many limitations to this study, including the amount of available data and the analytical methods used to analyze the tissue, the overall results indicate that residence (defined as the year that the individual moved to Los Alamos) may have had a strong correlation to plutonium activity in human tissue. This study is the first to present the results of Los Alamos Autopsy Program in relation to residential status and location in Los Alamos.

Tissue-type plasminogen activator (t-PA) has shown significant effects on the treatment of common thrombosis. In this work, molecular dynamics simulations are used in protein–ligand interaction analysis to investigate the affinity of ligands for affinity chromatography. A hydrogel matrix grafted with amine (positively charged), carboxyl (negatively charged) and hydroxyl (neutral) ligands separately is designed, and its adsorption–desorption dynamics are studied in detail. The residues on the surface of t-PA, on which the S1 pocket is located, could be more easily adsorbed by charged ligands grafted onto the hydrogel matrix than neutral ligands. The findings offer new insights into the affinity of various ligands for t-PA, and could be of potential use in t-PA purification.

Summary Background Thrombolysis with tissue-type plasminogen activator (t-PA) is the only treatment approved for acute ischemic stroke. Although t-PA is an efficient clot lysis enzyme, it also causes damage to the neurovascular unit, including hemorrhagic transformations and neurotoxicity. Objectives On the basis of the mechanism of action of t-PA on neurotoxicity, we aimed at studying the molecular requirements to generate safer thrombolytics. Methods We produced original t-PA-related mutants, including a non-cleavable single-chain form with restored zymogenicity (sc*-t-PA) and a t-PA modified in the kringle 2 lysine-binding site (K2*-t-PA). Both sc*-t-PA and K2*-t-PA showed fibrinolytic activities similar to that of wild-type t-PA on both euglobulin-containing and plasma-containing clots. In contrast to wild-type t-PA, the two mutants did not promote N-methyl-d-aspartate receptor-mediated neurotoxicity. Conclusions We designed t-PA mutants with molecular properties that, in contrast to t-PA, do not induce neurotoxicity.

Introduction&Hypothesis: The aim of the present study was to investigate whether administration of edaravone, a free radical scavenger, before or during t-PA administration can increase the rate of early recanalization and improve the clinical outcome in stroke patients with major arterial occlusion. Methods: YAMATO study is an investigator initiated, multicenter (17 hospitals in Japan), prospective, randomized, open labeled study. Acute stroke patients with horizontal (M1) or vertical (M2) portion of the middle cerebral artery occlusion within 4.5 h of onset were studied. The subjects were randomly allocated to the early edaravone (early-E) group (intravenous edaravone [30 mg] was started before or during t-PA administration) and the late edaravone (late-E) group (edaravone was started after t-PA administration). Primary outcome, defined as any early recanalization 1h after t-PA therapy. Secondary outcomes included the rate of the significant recanalization, defined as ≥50% of the territory of the occluded artery on magnetic resonance angiography, or the thrombolysis in cerebral infarction score ≥2b on digital subtraction angiography as well as the incidence of symptomatic intracerebral hemorrhage (sICH), and the favorable clinical outcome (modified Rankin scale [mRS] of 0-2) at 3 months after onset. Results: One-hundred and sixty-six patients (96 men; median age [interquartile range], 78 [69-85] years) were randomized 1:1 to either the early-E group or the late-E group. Twenty-three (13.9%) had proximal M1 occlusion; 60 (36.1%), distal M1 occlusion; 83 (50%), M2 occlusion. Early recanalization was similarly observed in the early-E group and in the late-E group (53.1% vs. 53.0%, P=1.000). The rate of significant recanalization was also similar between the 2 groups (27.2% vs. 33.7%, p=0.399). sICH was occurred in 4 (4.8%) patients in the early-E group and in 2 (2.4%) in the late-E group (p=0.682). Among the 144 patients who were pre-morbid mRS of 0-2 and eligible for 3 months assessment, favorable outcome was seen in 53.9% in the early-E group and 57.4% in the late-E group (p=0.738) Conclusions: The timing of the edaravone infusion should not affect the rate of early recanalization, sICH, or favorable outcome after t-PA therapy.