Phosphate Buffer Saline Wash Research Articles

Poster 112: Toxicity of Chlorhexidine and Povidone-Iodine to Rotator Cuff Tendon

Objectives: Antimicrobial wound lavages containing chlorhexidine gluconate (CHG) or povidone-iodine (PI) are commonly used to prevent periprosthetic infection during shoulder arthroplasty. However, the potential toxicity of these solutions to the native surrounding rotator cuff tendon is not well understood. In total shoulder arthroplasty, the rotator cuff tendon is often concomitantly repaired, and any toxic effects from antimicrobial wound lavage during surgery may be detrimental to postoperative rotator cuff tendon healing. The purpose of this study was to determine the in vitro effects of a 1-minute CHG exposure and a 3-minute 0.35% PI exposure on the viability and biochemical content of rotator cuff tendon. Methods: Infraspinatus tendons (n=6 per group) were isolated from shoulders of Nubian goats (n=3; 2 female and 1 male). Tendons were submerged in Irrisept (0.05% CHG in sterile water) for 1 minute or 0.35% Betadine (PI) for 3 minutes per manufacturer guidelines, followed by phosphate-buffered saline wash and culture in tissue culture medium. Control tendon specimens were similarly exposed to tissue culture medium, followed by phosphate-buffered saline wash and culture in tissue culture medium. After seven days of culture, isolates from both the tendinous and enthesis portions of the rotator cuff were analyzed for viability, collagen content (hydroxyproline assay), and glycosaminoglycan (GAG) content (dimethylmethylene blue assay). Statistical analyses were performed via one-way ANOVA with post hoc Tukey’s test. Results: Within the tendinous portion of the rotator cuff, CHG-treated specimens demonstrated a decrease in mean tenocyte viability compared to controls (p=0.045) (Fig.1). While PI-treated specimens exhibited lower mean viability compared to controls, this difference was not statistically significant. Within the enthesis portion of the rotator cuff, tenocyte viability decreased in both CHG- and PI-treated groups; however, these decreases were not statistically significant (Fig. 2). There were no significant differences in mean collagen content/wet weight or GAG/wet weight among groups within the tendinous and enthesis portion of the rotator cuff (Fig. 3). Conclusions: Due to the potential catastrophic consequences associated with periprosthetic infection, CHG and PI antimicrobial lavages are commonly used to reduce the risk of periprosthetic infection during shoulder arthroplasty. However, in this study, brief 0.05% CHG and 0.35% PI exposures to rotator cuff tendon resulted in lower tenocyte viability 7 days after treatment. These results are consistent with the toxicity reported with exposure of CHG and PI to other musculoskeletal tissues, including articular cartilage. The toxicity of antimicrobial irrigation to the surrounding native rotator cuff tendon may be detrimental to the postoperative healing of concomitant rotator cuff repair in the setting of shoulder arthroplasty.

Cell detachment rates and confluence of fibroblast and osteoblast cell culture using different washing solutions.

The advancements in the cell culture studies have led to the development of regenerative medicine concept. The aim of this study is to compare the effectiveness of some washing solutions, including phosphate buffered saline (PBS), sodium chloride (NaCl), and ringer's lactate (RL) on the rate of detachment and confluency in fibroblast and osteoblast cell culture. Baby Hamster Kidney 21 clone 13 (BHK21/C13) fibroblast cells and 7F2 osteoblast were cultured on T25 flasks for 3-4 days. Three treatment groups were classified on the basis of different washing solutions used in the moment before trypsinization: PBS, 0.9% NaCl, and RL. Each group was measured for the detachment rate and cell confluence. The measurement was done in 2 passage numbers. The use of PBS, NaCl, and RL washing solution showed that detachment time was less than 5 minutes for the fibroblasts and 3 minutes for the osteoblasts. There was a significant difference in the rate of fibroblast cell detachment (p=0.006) and osteoblast (p=0.016). The capability of fibroblasts and osteoblasts to achieve a confluence of 106 cells/well on the first and second measurements was almost the same between the washing solution groups. The use of physiological 0.9% NaCl solution as a washing solution in fibroblast and osteoblast cell culture has almost the same effectiveness as PBS to help accelerate cell detachment in less than 5 minutes without influencing the capability of cells to proliferate.

Processing of boar spermatozoa with phosphate-buffered saline at 4˚C induces an increase in 32 kDa tyrosine-phosphorylated protein (p32)

We aimed to investigate the impact of processing boar spermatozoa with phosphate-buffered saline (PBS) at 4 ˚C on acrosomal integrity and increase in 32 kDa tyrosine-phosphorylated protein (p32). Following cooled PBS washing, we observed a significant increase in p32 levels and in the proportion of dead spermatozoa with compromised acrosomal integrity compared to sperm washing using PBS at room temperature. Interestingly, this increase in p32 was effectively inhibited when cooled PBS was supplemented with 1 mM AEBSF, a serine protease inhibitor. Our findings suggest that the increase of p32 in response to cooled PBS washing in boar spermatozoa is associated with enhanced protease activity in dead spermatozoa.

Process Development of Recombinant Adeno-Associated Virus Production Platform Results in High Production Yield and Purity.

Optimization of recombinant adeno-associated virus (rAAV) production has important clinical implications, as manufacturing is one of the major challenges for rAAV gene therapy. In this study, we optimized upstream and downstream processing of the rAAV production platform created by an earlier design-of-experiment approach. Our results showed that adding peptones (yeastolate, Trypton N1 or both) increased production yield by 2.8- to 3.4-folds. For downstream processing, a variety of wash buffers for an affinity resin, POROS™ CaptureSelect™ (PCS)-AAVX, were tested for their effects on rAAV8 purity, including NaCl, MgCl2, arginine, Triton X-100, CHAPS, Tween 20, octyl β-d-1-thioglucopyranoside (OTG), and low pH. The results showed that the OTG wash significantly improved the rAAV purity to 97% and reduced endotoxins to an undetectable level (<0.5 EU/mL), while retaining the yield at 92.3% of the phosphate-buffered saline (PBS) wash. The OTG wash was successfully applied to purifications of rAAV1, rAAV2, and rAAV5 using PCS-AAVX, and rAAV9 using PCS-AAV9. rAAV8 purified with OTG wash showed comparable transduction efficiency in HEK 293T cells to the rAAV8 purified with PBS wash. The optimized rAAV production process yielded 5.5-6.0 × 1014 and 7.6 × 1014 vector genome per liter of HEK 293T cells for purified rAAV8- and rAAV5-EF1α-EGFP (enhanced green fluorescent protein), respectively. The platform described in this study is simple with high yields and purity, which will be beneficial to both research and clinical gene therapy.

64Cu-labeling of small extracellular vesicle surfaces via a cross-bridged macrocyclic chelator for pharmacokinetic study by positron emission tomography imaging

We developed a method of labeling the surfaces of small extracellular vesicles (sEVs) with 64Cu using a cross-bridged, macrocyclic chelator (CB-TE1A1P) and applied to pharmacokinetics study with positron emission tomography (PET). After incubation in 20% plasma for 10 min, approximately a half of the 64Cu was desorbed from 64Cu-labeled sEVs purified by phosphate-buffered saline wash, suggesting partly weak interaction without coordinating to CB-TE1A1P. After subsequent purification with albumin, 64Cu desorption was greatly reduced, resulting in a radiochemical stability of 95.7%. Notably, labeling did not alter the physicochemical and biological properties of sEVs. After intravenous injection, 64Cu-labeled sEVs rapidly disappeared from the systemic blood circulation and accumulated mainly in the liver and spleen of macrophage-competent mice. In macrophage-depleted mice, 64Cu-labeled sEVs remained in the blood circulation for a longer period and gradually accumulated in the liver and spleen, suggesting mechanisms of hepatic and splenic accumulation other than macrophage-dependent phagocytosis. The comparison of tissue uptake clearance between macrophage-competent and macrophage-depleted mice suggests that macrophages contributed to 67% and 76% of sEV uptake in the liver and spleen, respectively. The application of this method in pharmacokinetics PET studies can be useful in preclinical and clinical research and the development of sEV treatment modalities.

Quantitative sperm characteristics of Tankwa goats with special reference to hyperactivated motility

Computer-assisted semen analysis (CASA) is an automated and objective method of evaluating structural (e.g. morphology) and functional sperm parameters (e.g. motility and hyperactivation). Sperm hyperactivation is essential for successful fertilization and is thus an important aspect in determining the fertility potential of a male. In the current study, CASA was used for standard semen analysis and for comparison of the ability of phosphate buffered saline (PBS), BO sperm wash (10 mM caffeine), 4% lignocaine, and 5 mM procaine hydrochloride to induce hyperactivation in Tankwa goat spermatozoa. Twenty-nine ejaculates were collected from randomly selected male goats by electroejaculation. Although none of the four media affected percentage total sperm motility, lignocaine caused a significant decrease (P &gt;0.05) in percentage progressive motility. Exposure to procaine resulted in an increase in swimming speed (P ≤0.05) and star-spin motility tracks, which are typical of sperm hyperactivation. Using PBS and procaine motility data from individually selected spermatozoa, receiver operator characteristic curves were constructed to distinguish the kinematic parameters employed as cut-off values for sperm hyperactivation. PBS and BO sperm wash did not induce hyperactivation (0.1 + 0.2% and 0.04  0.2% respectively), while lignocaine induced little hyperactivation (3.4 + 3.0%) and procaine hydrochloride had the highest percentage hyperactivation (25.3 + 13.6%). The large variation in hyperactivation (0–54.5%) may reflect inter-individual differences in sperm quality among these males. This study indicated procaine hydrochloride was the most promising hyperactivation-inducing medium for Tankwa goat spermatozoa and should be considered for similar assessments in other animal species&#x0D; Keywords: computer-aided sperm analysis, procaine hydrochloride, sperm kinematics, sperm morphometry, sperm motility

Fluorescent Visualization of Nucleolar G-Quadruplex RNA and Dynamics of Cytoplasm and Intranuclear Viscosity

The nucleolus, the locus of ribosome biogenesis, was found to be the predominant intracellular target of a new fluorescent probe, V-P1. In solution, the probe demonstrated both a selectivity to RNA...

Impact of CD138 Magnetic Bead-based Positive Selection on Bone Marrow Plasma Cell Surface Markers

BackgroundIsolation of malignant plasma cells from bone marrow of patients with monoclonal gammopathies is critical for studies into the disease biology. The plasma cells are typically isolated by positive selection using plasma cell markers such as CD138. Here we have examined the effect of CD138 magnetic bead selection on the expression of other surface phenotypic markers on plasma cells. Materials and MethodsBone marrow aspirates from 16 patients were split and prepared using 2 methods before staining for flow cytometric evaluation. The first method (whole bone marrow) used an ammonium-chloride-potassium lyse of whole bone marrow followed by 2 phosphate buffered saline washes. The second method used CD138-positive magnetic sorting technology (Stem Cell Technology). The cells were run on the FACSCanto flow cytometer after staining for CD38, CD45, CD56, activation markers CD71, CD69, CD154, adhesion markers CD49d, CD49e, CD11a, CD11b, and CD66, B cell markers CD19 and CD20, and for clonality. ResultsThere was a substantial loss in the expression of CD71, CD11b, CD11a, CD69, and CD49e on plasma cells following CD138-based sorting. Moreover, in 8 of the 16 cases, there was a nearly complete loss of the CD45-positive subset with a loss of discrimination between CD45-negative and CD45-positive plasma cell subsets in the remaining CD138-sorted preparations. ConclusionsThe change in immunophenotype of the plasma cells on magnetic sorting should be kept in mind when isolating plasma cells using CD138-positive selection for analysis of plasma cells. The technique for characterizing plasma cells should be selected based on the study design to prevent loss of crucial and valuable information.

Adipose-derived stromal cell therapy combined with a short course nonmyeloablative conditioning promotes long-term graft tolerance in vascularized composite allotransplantation.

The risks of chronic immunosuppression limit the utility of vascularized composite allotransplantation (VCA) as a reconstructive option in complex tissue defects. We evaluated a novel, clinically translatable, radiation-free conditioning protocol that combines anti-lymphocyte serum (ALS), tacrolimus, and cytotoxic T-lymphocyte-associated protein 4 immunoglobulin (CTLA4-Ig) with adipose-derived stromal cells (ASCs) to allow VCA survival without long-term systemic immunosuppression. Full-mismatched rat hind-limb-transplant recipients received tacrolimus (0.5mg/kg) for 14days and were assigned to 4 groups: controls (CTRL) received no conditioning; ASC-group received CTLA4-Ig (10mg/kg body weight i.p. postoperative day [POD] 2, 4, 7) and donor ASCs (1×106 iv, POD 2, 4, 7, 15, 28); the ASC-cyclophosphamide (CYP)-group received CTLA4-Ig, ASC plus cyclophosphamide (50mg/kg ip, POD 3); the ASC-ALS-group received CTLA4-Ig, ASCs plus ALS (500µL ip, POD 1, 5). Banff grade III or 120days were endpoints. ASCs suppressed alloresponse in vitro. Median rejection-free VCA survival was 28days in CTRL (n=7), 34 in ASC (n=6), and 27.5 in ASC-CYP (n=4). In contrast, ASC-ALS achieved significantly longer, rejection-free VCA survival in 6/7 animals (86%), with persistent mixed donor-cell chimerism, and elevated systemic and allograft skin Tregs , with no signs of acute cellular rejection. Taken together, a regimen comprised of short-course tacrolimus, repeated CTLA4-Ig and ASC administration, combined with ALS, promotes long-term VCA survival without chronic immunosuppression.

Short-Term Deposition of PM2.5 Particles on Contact Lens Surfaces: Effect on Oxygen Permeability and Refractive Index

ABSTRACTPurposes: To identify the deposition of fine (≤2.5 μm diameter) particulate matter (PM) particles (PM2.5) on contact lens surfaces and to investigate the effects of such deposition on the oxygen permeability (OP) and refractive index (RI) of contact lenses.Methods: A total of 36 contact lenses, including rigid gas permeable (RGP) lens and soft contact lens (SCL), were investigated. RGP lens (n=12) and SCL (n=12) (experimental group) were incubated in a PM2.5 solution for 24 h, after which PM2.5-treated RGP lens (n=6) and SCL (n=6) were further washed for 1 h in phosphate-buffered saline (PBS). All lenses were examined by field emission scanning electron microscopy. OP and RI of all lenses were measured.Results: Average-sized PM2.5 particles deposited on RGP contact lens and SCL surfaces after immersion in the PM2.5 solution were 3.192 ± 1.637 and 2.158 ± 1.187/100 μm2, respectively. On RGP lens surfaces, we observed both large (≥2.5 µm diameter) and small (PM2.5) particles. PM2.5 particles were deposited in diffuse patterns, primarily along the honeycomb structural border of SCL, while no PM2.5 particles were found in the honeycomb hole of SCL surfaces. Washing in PBS removed the larger PM particles from RGP lens surfaces, but left copious amounts of PM2.5 particles. In contrast, nearly all PM particles were removed from SCL surfaces after PBS washing. OP values of RGP lens and SCL appeared to be unchanged by PM2.5 deposition. RI values increased in both RGP lens and SCL groups after PM2.5 deposition. However, these increases were not statistically significant, suggesting that PM2.5 deposition itself does not cause fluctuations in contact lens RI.Conclusions: Deposition of PM2.5 particles on contact lens surfaces varies according to lens material. PM2.5 particles deposited on SCL, but only large particles on RGP surfaces were able to be removed by washing in PBS and did not appear to alter OP and RI of either lens type.

Preservation Method and Phosphate Buffered Saline Washing Affect the Acute Myeloid Leukemia Proteome.

Acute myeloid leukemia (AML) primary cells can be isolated from peripheral blood, suspended with media containing bovine serum and cryoprotectant, and stored in liquid nitrogen before being processed for proteomic analysis by mass spectrometry (MS). The presence of bovine serum and human blood proteins in AML samples can hamper the identifications of proteins, and thereby reduce the proteome coverage of the study. Herein, we have established the effect of phosphate buffered saline (PBS) washing on AML patient samples stored in media. Although PBS washes effectively removed serum and blood contaminants, the saline wash resulted in cell burst and remarkable protein material loss. We also compared different methods to preserve the AML proteome from THP-1 and Molm-13 cell lines before MS analysis: (1) stored in media containing bovine serum and dimethyl sulfoxide (DMSO); (2) stored as dried cell pellets; and (3) stored as cell lysates in 4% sodium dodecyl sulfate (SDS). MS analysis of differently preserved AML cell samples shows that preservation with DMSO produce a high number of fragile cells that will burst during freezing and thawing. Our studies encourage the use of alternative preservation methods for future MS analysis of the AML proteome.

Comparison of phytochemical profiles, antioxidant and cellular antioxidant activities of different varieties of blueberry (Vaccinium spp.)

Numerous reports have demonstrated that the consumption of fruits and vegetables is beneficial for the human health. Blueberries, in particular, are rich in phytochemicals including free and bound forming. Phytochemical profiles of 14 varieties of blueberry were compared in this study. 12 compounds were analyzed and had significant changes in blueberry fruits. Total antioxidant activities in different blueberry varieties varied about 2.6times by oxygen radical absorbance capacity (ORAC) assay, and 2times by peroxyl radical scavenging capacity (PSC) assay. The cellular antioxidant activities (CAA) in different varieties varied about 3.9times without phosphate buffer saline (PBS) wash, and 4.7times with PBS wash by CAA assay. Blueberry extracts had potent antiproliferative activities against HepG2 human liver cancer cells, indicating the potential protective benefits associated with their use as functional foods. The anti-proliferative activity was observed to be dose-dependent in blueberry extracts.

Optimization of Metabolite Extraction Protocols for the Identification and Profiling of Small Molecule Metabolites from Planktonic and Biofilm Pseudomonas aeruginosa Cultures.

Metabolomics aims to characterize the metabolic phenotype and metabolic pathways utilized by microorganisms or other cellular systems. A crucial component to metabolomics research as it applies to microbial metabolism is the development of robust and reproducible methods for extraction of intracellular metabolites. The goal is to extract all metabolites in a non-biased and consistent manner; however, most methods used thus far are targeted to specific metabolite classes and use harsh conditions that may contribute to metabolite degradation. Metabolite extraction methodologies need to be optimized for each microorganism of interest due to different cellular characteristics contributing to lysis resistance. Three cell pellet wash solutions were compared for the potential to influence intracellular metabolite leakage of P. aeruginosa. We also compared four different extraction methods using (i) methanol:chloroform (2:1); (ii) 50% methanol; (iii) 100% methanol; or (iv) 100% water to extract intracellular metabolites from P. aeruginosa planktonic and biofilm cultures. Intracellular metabolite extraction efficiency was found to be dependent on the extraction method and varies between microbial modes of growth. Methods using the 60% methanol wash produced the greatest amount of intracellular material leakage. Quantification of intracellular metabolites via 1H NMR showed that extraction protocols using 100% water or 50% methanol achieved the greatest extraction efficiencies, while addition of sonication to facilitate cell lysis to the 50% methanol extraction method resulted in at least a two-fold increase in signal intensities for approximately half of the metabolites identified. Phosphate buffered saline (PBS) was determined to be the most appropriate wash solution, yielding little intracellular metabolite leakage from cells. We determined that washing in 1X PBS and extracting intracellular metabolites with 50% methanol is the most appropriate metabolite extraction protocol because (a) leakage is minimal; (b) a broad range of metabolites present at sufficiently high concentrations is detectable by NMR; and (c) this method proved suitable for metabolite extraction of both planktonic and biofilm P. aeruginosa cultures.

Evaluation of Cellular Antioxidant and Antiproliferative Activities of Five Main Phyllanthus Emblica L. Cultivars in China.

The cell-based antioxidant activity assay as more biological relevant assay was considered to be more accurate to predict antioxidant activity in vivo than chemical activity assays. In the present study, the five main Phyllanthus emblica L. cultivars in China were subjected for cellular antioxidant activity based on HepG2 cells as well as antiproliferative activity. Total phenolics, total flavonoids and oxygen radical absorbance capacity were also measured. The results showed that Qingyougan, Binggan and Boligan (832±100, 774±52 and 704±28 μmol of quercetin equivalents/100 g) had higher cellular antioxidant activity than Tianyougan and Yougan (553±50 and 457±24 μmol of quercetin equivalents/100 g) in phosphate buffered saline wash protocol whereas, Boligan (3735±217 μmol of quercetin equivalents/100 g) had the highest cellular antioxidant activity and Tianyougan (2025±171 μmol of quercetin equivalents/100 g) had the lowest cellular antioxidant activity in no phosphate buffered saline wash protocol. The highest and lowest antiproliferative activities were observed in Binggan and Tianyougan (median effective dose: 6.95±0.11 and 14.03±0.10 mg/ml), respectively. The significant correlation was only observed between total flavonoids and cellular antioxidant activity from no phosphate buffered saline wash protocol (R(2) =0.908, P<0.05), and total flavonoids and antiproliferative activity (R(2) =0.887, P<0.05), suggesting the major contribution of flavonoids to the bioactivities of emblica. Overall, the data obtained revealed that different Phyllanthus emblica L. cultivars had strong cellular antioxidant and antiproliferative activities, thus should be recommended to increase consumption for health.

Phenolic Compounds and Antioxidant Capacities of 10 Common Edible Flowers from China

The free and bound phenolic compounds in 10 common Chinese edible flowers were investigated using reversed phase high-performance liquid chromatography. Their antioxidant capacities were evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical-scavenging activity, oxygen radical absorption capacity (ORAC), ferric reducing antioxidant power (FRAP), and cellular antioxidant activity (CAA). Free factions were more prominent in phenolic content and antioxidant capacity than bound fractions. Paeonia suffruticosa and Flos lonicerae showed the highest total phenolic content (TPC) 235.5 mg chlorogenic acid equivalents/g of dry weight and total flavonoid content 89.38 mg rutin equivalents/g of dry weight. The major phenolic compounds identified were gallic acid, chlorogenic acid, and rutin. P. suffruticosa had the highest antioxidant capacity in the DPPH, ABTS, and ORAC assays, which were 1028, 2065, 990 μmol Trolox equivalents/g of dry weight, respectively, whereas Rosa chinensis had the highest FRAP value (2645 μmol Fe(2+) equivalents /g of dry weight). The P. suffruticosa soluble phenolics had the highest CAA, with the median effective dose (EC50 ) 26.7 and 153 μmol quercetin equivalents/100 g of dry weight in the phosphate buffered saline (PBS) and no PBS wash protocol, respectively. TPC was strongly correlated with antioxidant capacity (R = 0.8443 to 0.9978, P < 0.01), which indicated that phenolics were the major contributors to the antioxidant activity of the selected edible flowers.

Enhancement of nano-hydroxyapatite bonding to dentin through a collagen/calcium dual-affinitive peptide for dentinal tubule occlusion

Calcium phosphate crystals, as the main component of dentin and enamel, have been widely used for the occlusion of dentinal tubules. However, the low bond strength and poor sealing effect limit their clinical practicality. In this study, a collagen/calcium dual-affinitive peptide E8DS (EEEEEEEEDSpESpSpEEDR) and nano-hydroxyapatite (nano-HAp) flocculi were developed to seal dentin tubules for reducing dentin hypersensitivity, whereas the E8DS peptides were pre-applied to improve the adhesion of occlusive hydroxyapatite coating on dentin collagen matrix for the long-lasting sealing effect and relief from hypersensitivity. Our study showed that E8DS peptides had a strong affinity with dentin collageneous matrix that almost 43.7% of initial E8DS peptides immobilized on exposed dentin samples remained detained after continuous washing by distilled water for four weeks at a rate of 1 mL/min. Nano-HAp flocculi were obtained by re-neutralization of HAp-HCl solution and then brushed onto the surfaces of pre-treated human dentin disks with E8DS peptides, which showed a perfect occlusion of exposed dentinal tubules, as compared with Nano-HAp only and a commercial desensitizer, Green Or. With only around 10-min E8DS peptide pre-treatment, the occlusive mineral layers remained intact against consecutively stirred washing in phosphate-buffered saline or coke for 15 min, and 6 min of tooth-brushing, which implied that our E8DS peptide could comparatively improve the durability of sealant-dentin interface bonds for long-lasting dentine desensitization.

Larynx Decellularization: Combining Freeze-Drying and Sonication as an Effective Method

Ideal methods for the reconstruction of the laryngeal structure and restoration of the laryngeal function once the larynx has been damaged or removed have not yet been developed. Thus, larynx tissue engineering practices have recently been extensively investigated. A scaffold may be generated using biocompatible or artificial materials. Decellularization methods, which use preexisting tissues as material sources, have also been used to manufacture larynx scaffolds with promising results. In this study, we developed a novel decellularization method that combines freezing, drying, and sonication. Porcine model study. Fresh porcine larynxes were used for decellularization. The process of the decellularization cycle comprised overnight freeze-drying, defreezing in phosphate-buffered saline (PBS) for 30 minutes, and washing in PBS for another 30 minutes. Sonication treatment was further added during the defreezing process. The decellularized tissue was then evaluated through histologic sections under hematoxylin and eosin staining. The results showed that a single use of the freeze-drying modality has little effect with regards to removing cellular components, even with increased decellularization cycles. However, when sonication was added to the defreezing process, the cellular contents were removed significantly (the residual nucleus ratios of freeze-drying:freeze-drying and defreezing one cycle:freeze-drying and defreezing three cycles:freeze-drying and defreezing under sonication three cycles were 91%:70%:47%:16%, respectively). However, the processed scaffold became structurally more fragile through the procedure. Combining freeze-drying and sonication during the defreezing process could be a promising method of decellularizing laryngeal tissues. However, this purely physical method may also induce structural damage to the scaffold.

Development for Dynamic Live Cell Imaging by Cryo-Electron Tomography and Stem

Cryo-EM tomography of intact cells is an emerging technology that compliments crystallography, NMR and single molecule imaging techniques. Its strength is in that it reveals the spatial arrangements of key proteins and complexes during intracellular signaling and mechanical events like motility and division. In this meeting, we describe dynamical Live Cell imaging by Cryo-EM tomography using the Titan Krios microscope with 300 kV electron column and HAADEF detector. Observed living cells were grown directly on Holey Carbon Support Film. In order to make sure that the cells are suitable for observation we analyzed the some parameters of cell movement and cell division on Quantifoil using Fiji and compared these properties with those of cells grown on a normal cell culture plastic plate. After phosphate-buffered saline washing, the cells were done rapid freeze fixation (vitrification) by dropping in liquid ethane using VitrobotTM Mark IV instead of the usual chemical fixation and were transferred onto the microscope immediately while keeping the environment under liquid N2. Cells were imaged over an angular range from −70 degrees to 70 degrees at 2 degrees x cos θ tilt increments automatically and analyzed with Inspect 3D and Amira software to provide 3D images and Volume rendering respectively.We observed some unique architectures of a part of Lamellipodium in GFP-Myosin X expressed COS 7 cell and them from near the nuclear membrane to plasma membrane in COS 7 and several eukaryotic cells (HeLa, NIH3T3⋯ etc.) by Cryo-EM tomography using STEM using intact cells and vitreous cell sections.At future, our system can provide any new information about many kinds of cells and organelles during important events.

Proteomic Profiling of S-acylated Macrophage Proteins Identifies a Role for Palmitoylation in Mitochondrial Targeting of Phospholipid Scramblase 3

S-Palmitoylation, the reversible post-translational acylation of specific cysteine residues with the fatty acid palmitate, promotes the membrane tethering and subcellular localization of proteins in several biological pathways. Although inhibiting palmitoylation holds promise as a means for manipulating protein targeting, advances in the field have been hampered by limited understanding of palmitoylation enzymology and consensus motifs. In order to define the complement of S-acylated proteins in the macrophage, we treated RAW 264.7 macrophage membranes with hydroxylamine to cleave acyl thioesters, followed by biotinylation of newly exposed sulfhydryls and streptavidin-agarose affinity chromatography. Among proteins identified by LC-MS/MS, S-acylation status was established by spectral counting to assess enrichment under hydroxylamine versus mock treatment conditions. Of 1183 proteins identified in four independent experiments, 80 proteins were significant for S-acylation at false discovery rate = 0.05, and 101 significant at false discovery rate = 0.10. Candidate S-acylproteins were identified from several functional categories, including membrane trafficking, signaling, transporters, and receptors. Among these were 29 proteins previously biochemically confirmed as palmitoylated, 45 previously reported as putative S-acylproteins in proteomic screens, 24 not previously associated with palmitoylation, and three presumed false-positives. Nearly half of the candidates were previously identified by us in macrophage detergent-resistant membranes, suggesting that palmitoylation promotes lipid raft-localization of proteins in the macrophage. Among the candidate novel S-acylproteins was phospholipid scramblase 3 (Plscr3), a protein that regulates apoptosis through remodeling the mitochondrial membrane. Palmitoylation of Plscr3 was confirmed through (3)H-palmitate labeling. Moreover, site-directed mutagenesis of a cluster of five cysteines (Cys159-161-163-164-166) abolished palmitoylation, caused Plscr3 mislocalization from mitochondrion to nucleus, and reduced macrophage apoptosis in response to etoposide, together suggesting a role for palmitoylation at this site for mitochondrial targeting and pro-apoptotic function of Plscr3. Taken together, we propose that manipulation of protein palmitoylation carries great potential for intervention in macrophage biology via reprogramming of protein localization.

Peroxiredoxin II Restrains DNA Damage-induced Death in Cancer Cells by Positively Regulating JNK-dependent DNA Repair

The 2-Cys peroxiredoxins (Prx) belong to a family of antioxidant enzymes that detoxify reactive oxygen and nitrogen species and are distributed throughout the intracellular and extracellular compartments. However, the presence and role of 2-Cys Prxs in the nucleus have not been studied. This study demonstrates that the PrxII located in the nucleus protects cancer cells from DNA damage-induced cell death. Although the two cytosolic 2-Cys Prxs, PrxI and PrxII, were found in the nucleus, only PrxII knockdown selectively and markedly increased cell death in the cancer cells treated with DNA-damaging agents. The increased death was completely reverted by the nuclearly targeted expression of PrxII in an activity-independent manner. Furthermore, the antioxidant butylated hydroxyanisole did not influence the etoposide-induced cell death. Mechanistically, the knockdown of Prx II expression impaired the DNA repair process by reducing the activation of the JNK/c-Jun pathway. These results suggest that PrxII is likely to be attributed to a tumor survival factor positively regulating JNK-dependent DNA repair with its inhibition possibly sensitizing cancer cells to chemotherapeutic agents.