The N-Terminal Region of Soybean PM1 Protein Protects Liposomes during Freeze-Thaw.

Late embryogenesis abundant (LEA) group 1 (LEA_1) proteins are intrinsically disordered proteins (IDPs) that play important roles in protecting plants from abiotic stress. Their protective function, at a molecular level, has not yet been fully elucidated, but several studies suggest their involvement in membrane stabilization under stress conditions. In this paper, the soybean LEA_1 protein PM1 and its truncated forms (PM1-N: N-terminal half; PM1-C: C-terminal half) were tested for the ability to protect liposomes against damage induced by freeze-thaw stress. Turbidity measurement and light microscopy showed that full-length PM1 and PM1-N, but not PM1-C, can prevent freeze-thaw-induced aggregation of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) liposomes and native thylakoid membranes, isolated from spinach leaves (Spinacia oleracea). Particle size distribution analysis by dynamic light scattering (DLS) further confirmed that PM1 and PM1-N can prevent liposome aggregation during freeze-thaw. Furthermore, PM1 or PM1-N could significantly inhibit membrane fusion of liposomes, but not reduce the leakage of their contents following freezing stress. The results of proteolytic digestion and circular dichroism experiments suggest that PM1 and PM1-N proteins bind mainly on the surface of the POPC liposome. We propose that, through its N-terminal region, PM1 functions as a membrane-stabilizing protein during abiotic stress, and might inhibit membrane fusion and aggregation of vesicles or other endomembrane structures within the plant cell.

In Situ Salt Formation during Melt Extrusion for Improved Chemical Stability and Dissolution Performance of a Meloxicam-Copovidone Amorphous Solid Dispersion.

As the pipeline for poorly soluble compounds continues to grow, drug degradation during melt extrusion must be addressed. We present a novel method for stabilizing a thermally labile drug substance while preserving its physical stability and even improving its dissolution performance. In a previous study, we found that incorporating meglumine during extrusion of meloxicam results in chemical stabilization that cannot be achieved using process optimization alone. The purpose of this study is to understand the mechanism behind this stabilization and its impact on the performance of a meloxicam-Kollidon VA64 amorphous solid dispersion. The meloxicam concentration was maintained at 10% (w/w) for blends with and without meglumine. The optimal meglumine blend contained an equimolar amount of meloxicam to meglumine with the remainder consisting of Kollidon VA64. Both formulations were processed with optimized extrusion conditions and analyzed by HPLC for purity. Meglumine at a 1:1 molar ratio with meloxicam results in 100% purity of meloxicam after melt extrusion. Solid-state NMR revealed a proton transfer between the meloxicam and meglumine indicating an in situ salt formation. During non-sink dissolution, the meglumine ASD enables meloxicam to maintain supersaturatation (≅50 times more than meloxicam free acid) for >7.25 h. The ASD without meglumine began precipitating 2.25 h following the pH shift. The ASDs were placed at 40 °C/75% RH for 6 months, and their stability was assessed. No significant chemical degradation, recrystallization, or significant moisture uptake was observed after six months' storage at 40 °C/75% RH.

Evaluation of viral contamination in a baculovirus expression system.

Insect expression systems based on baculovirus are widely used for generating recombinant proteins. Here, the infectivity of baculoviruses under the physiological stresses of 'freeze-thaw' and sonication and the baculoviral contamination of recombinant proteins after protein purification were evaluated. Our findings suggest that Nonidet P-40 (NP-40) treatment of baculoviruses completely abolishes their infectivity and that recombinant proteins purified with affinity beads do not include infectious baculoviruses. We therefore suggest that baculovirus is completely inactivated by NP-40 treatment and that recombinant proteins are unlikely to be contaminated with infectious baculoviruses after their affinity purification.

Cryopreservation of putative pre-pubertal bovine spermatogonial stem cells by slow freezing.

Development of techniques for the preservation of mammalian spermatogonial stem cells (SSCs) is a critical step in commercial application of SSC based technologies, including species preservation, amplification of agriculturally valuable germ lines, and human fertility preservations. The objective of this study was to develop an efficient cryopreservation protocol for preservation of bovine SSCs using a slow freezing technique. To maximize the efficiency of SSC cryopreservation, the effects of various methods (tissue vs. cell freezing) and cryoprotective agents (trehalose, sucrose, and polyethylene glycol [PEG]) were tested. Following thawing, cells were enriched for undifferentiated spermatogonia by differential plating and evaluated for recovery rate, proliferation capacity, and apoptosis. Additionally, putative stem cell activity was assessed using SSC xenotransplantation. The recovery rate, and proliferation capacity of undifferentiated spermatogonia were significantly greater for germ cells frozen using tissue freezing methods compared to cell freezing methods. Cryopreservation in the presence of 200 mM trehalose resulted in significantly greater recovery rate, proliferation capacity, and apoptosis of germ cells compared to control. Furthermore, cryopreservation using the tissue freezing method in the presence of 200 mM trehalose resulted in the production of colonies of donor-derived germ cells after xenotransplantation into recipient mouse testes, indicating putative stem cell function. Collectively, these data indicate that cryopreservation using tissue freezing methods in the presence of 200 mM trehalose is an efficient cryopreservation protocol for bovine SSCs.

Response of the cell membrane-cytoskeleton complex to osmotic and freeze/thaw stresses. Part 2: The link between the state of the membrane-cytoskeleton complex and the cellular damage.

In an earlier paper [35], we examined the mutual interaction between the actin cytoskeleton and the cell membrane and explored the role this interaction plays during freeze/thaw. In this follow-up paper, we investigate the physical and chemical stresses induced by freeze/thaw and explore the different mechanisms of damage caused by these stresses. Our results showed that changes in cell volume during freeze/thaw and the unfrozen water content in the solution alter the cytoskeleton stiffness, and the available membrane material. Combined with unfavorable ice-membrane interactions and increasing membrane stiffness, increased de-structuring of the membrane (such as bleb and microvilli formation) synergistically act on the membrane-cytoskeleton system generating irreversible damage.

[Potential exposure to carcinogens in low-melting alloys processing]. / Potenziale esposizione a cancerogeni nella lavorazione di leghe bassofondenti.

The objective in our study was to evaluate the worker's exposition to lead and cadmium in 32 radiology technicians in an eastern Sicily hospital in workers of low melting point alloy of lead, tin, cadmium and bismuth (league that can be melted at 73 degrees C as CERROBEND). Such alloy is used for the fabrication of objects used for the personal protection of cancer patients subject to high energy treatment. The parameters taken into consideration for this study were sex, age and smoking habits. In the test subject's working cycle reported in our case, there were traces of smoke formation containing lead, tin, bismuth and cadmium. Cadmium is a substance considered by IARC to be cancerous and can be found in both work and living environments, therefore it is often difficult to establish rather its presence in the organism is due to working activities and/or the living environment. In these cases it is necessary to evaluate whether the work represents an added risk to develop neoplasia, compared to the consequences due to normal environmental exposure. The added risk linked to work is evaluated comparing the concentration of toxic substances found in the living environments (Environmental Reference Value) with the toxic and/or metabolite found in the working environment, and comparing the biological reports of the population not directly exposed by work (Biological Reference Value) and those exposed. We performed a biological monitoring for lead and cadmium on the workers examined. The Italian Legislature, aside from lead, has not yet issued guidelines pertaining to professional exposure to cadmium, and therefore it is mandatory to take reference to the American Hygienist's charts both for environmental exposure (TLVs) andfor biological monitoring (BEI). Biological monitoring, which allows to evaluate the absorption by both inhalation and gastrointestinally, was performed through measuring the levels of lend and cadmium in the bloodstream (PbB and CdB) and the Cd in urine (CdU). The results show that in no case the levels of lead in the bloodstream (PbB) were above the reference value and BEI. The levels of cadmium urine (CdU) weren't above the reference level and the BEI, while the haematic levels of cadmium (CdB) were higher than the reference value in 8 subjects, each long time smokers, each of about 20 cigarettes a day. This data shows how, in the evaluation of exposition to cadmium, aside from the exam of data pertaining to work, the study of ways of absorption and the interpretation of the results of environmental and biological monitoring, it is important to consider the possibility of intoxication outside of the workplace. Cigarette smoke, as already indicated by other authors, is also confirmed in our studies as one of the major fonts of non professionally linked inhalation of cadmium.

A pharmacodynamic investigation into the efficacy of osteoprotegerin during aseptic inflammation.

BACKGROUND: Osteoprotegerin (OPG), as an osteoclast antagonist, limits mineralised tissue resorption under physiological conditions. Previous work investigating OPG in a rat periodontal ligament (PDL) ankylosis model found no inhibitory effect on osteoclasts when OPG was administered at a dosage of 2.5mg/kg. AIMS: The object of this study was to determine whether dosages higher than 2.5 mg/kg of OPG were required to limit osteoclastic activity in an aseptic inflammatory model in rats. MATERIALS AND METHODS: Dry ice was applied for 15 minutes to the upper right first molar crown of eighteen, 8-week-old, male Sprague-Dawley rats. Three groups of 3 were injected with OPG at dosages of 2.5, 5.0 and 7.5 mg/kg of body weight immediately following the thermal insult. After 7 days, the rats were sacrificed and each maxilla processed for histological examination and stained for osteoclastic activity using tartrate-resistant acid phosphatase (TRAP). Osteoclast population numbers were estimated via light microscopy and results were analysed using a comparative mixed model statistical analysis. RESULTS: Results showed OPG inhibited osteoclastic activity in a dose-dependent manner. From 2.5 mg/kg to 7.5 mg/kg, osteoclast populations were linearly reduced by 39.78% (p < 0.05). OPG did not appear to affect the inflammatory process and had varied efficacy in different regions of individual teeth. CONCLUSION: Although osteoclastic activity reduced, it was not completely eliminated, perhaps because dosages were still inadequate, or additional factors might influence OPG and osteoclast activation in the aseptic inflammatory model.

Pre-treatment of sperm reduces success of ICSI in the pig.

In pigs, although ICSI is a feasible fertilization technique, its efficiency is low. In general, injected pig sperm are insufficient to induce oocyte activation and embryonic development. Pretreatments for disrupting sperm membranes have been applied to improve the fertility of ICSI oocytes; however, we hypothesize that such pretreatment(s) may reduce the ability of the sperm to induce oocyte activation. We first evaluated the effects of sperm pretreatments (sonication (SO) to isolate the sperm heads from the tails, Triton X-100 (TX), and three cycles of repeated freezing/thawing (3×-FT) for disrupting sperm membranes) on the rate of pronucleus (PN) formation after ICSI. We found that oocytes injected with control (whole) sperm had higher rates of PN formation than those obtained after subjecting the sperm to SO, TX, and 3×-FT. The amounts of phospholipase Cζ (PLCζ), which is thought to be the oocyte-activating factor in mammalian sperm, in sperm treated by each method was significantly lower than that in whole untreated sperm. Furthermore, using immunofluorescence, it was found that in pig sperm, PLCζ was localized to both the post-acrosomal region and the tail area. Thus we demonstrated for the first time that sperm pretreatment leads to a reduction of oocyte-activating capacity. Our data also show that in addition to its expected localization to the sperm head, PLCζ is also localized in the tail of pig sperm, thus raising the possibility that injection of whole sperm may be required to attain successful activation in pigs.

Intracellular ice formation in confluent monolayers of human dental stem cells and membrane damage.

Dental pulp stem cells (DPSCs) are of interest to researchers and clinicians due to their ability to differentiate into various tissue types and potential uses in cell-mediated therapies and tissue engineering. Currently DPSCs are cryopreserved in suspension using Me(2)SO. However, preservation as two and three dimensional constructs, along with the elimination of toxic Me(2)SO, may be required. It was shown that intracellular ice formation (IIF), lethal to cells in suspensions, may be innocuous in cell monolayers due to ice propagation between cells through gap junctions that results in improved post-thaw recovery. We hypothesized that innocuous IIF protects confluent DPSC monolayers against injury during cryopreservation. The objective was to examine the effects of IIF on post-thaw viability of both confluent monolayers and suspensions of DPSCs. Confluent DPSC monolayers were assessed for the expression of gap junction protein Connexin-43. IIF was induced on the cryostage and in the methanol bath at different subzero temperatures. Membrane integrity and colony-forming ability were assessed post-thaw. Confluent DPSC monolayers expressed Connexin-43. In cell suspensions, 85.9+/-1.7% of cells were damaged after 100% IIF. In cell monolayers, after 100% IIF, only 25.5+/-5.5% and 14.8+/-3.3% of cells were damaged on the cryostage and in the methanol bath respectively. However, DPSC monolayers exposed to 100% IIF showed no colony-forming ability. We conclude that confluent monolayers of DPSCs express the gap junction-forming protein Connexin-43 and upon IIF retain membrane integrity, however lose the ability to proliferate.

A multi-model, large range and anti-freezing sensor based on a multi-crosslinked poly(vinyl alcohol) hydrogel for human-motion monitoring.

Conductive hydrogels are capturing intensive attention for versatile applications in flexible wearable devices on account of their unique combination of softness, stretchability, conductivity and biocompatibility. However, most of the hydrogel sensors can only serve as single-type sensors to detect strain or pressure, accompanied by a limited detection range. Moreover, the poor anti-freezing performance is also a serious problem to be addressed for their practical applications. Herein, a multi-model, large range and anti-freezing hydrogel sensor was constructed from a high-mechanical and ionic conductive multi-crosslinked poly(vinyl alcohol) (M-PVA) hydrogel, which was prepared via incorporating chain entanglement interaction and complexation between Fe3+ ions and hydroxyl groups into the microcrystalline network through immersion treatment in Fe2(SO4)3 solution. The three reversible and reconstructable crosslinks within the M-PVA hydrogel worked in tandem to achieve ultra-stretchability (1120%), supercompressibility (98%), high toughness, fast self-recoverability and excellent fatigue resistance. Meanwhile, the introduction of Fe3+ and SO42- ions endowed the M-PVA hydrogel with good ionic conductivity and remarkable anti-freezing properties (-50 °C), which benefited the M-PVA hydrogel to act as a freezing-tolerant dressing. The assembled multi-model hydrogel sensor can sensitively and stably detect large range elongation (∼900%), compression (∼70%), bend and pressure (up to 4.60 MPa) concurrently, as well as various human activities including speaking, finger bending and treading behavior. Notably, the hydrogel sensor was capable of maintaining excellent mechanical flexibility and sensitive sensing capacity at low temperature. The M-PVA hydrogel is a promising flexible sensing material for versatile applications in ionic skin, motion recognition and intelligent wearable devices.

Assessment of Post-thaw Quality of Dental Mesenchymal Stromal Cells After Long-Term Cryopreservation by Uncontrolled Freezing.

Cryopreservation abilities of dental tissue-derived mesenchymal stromal cells (DMSCs) including dental pulp stem cells (DPSCs) and dental follicle stem cells (DFSC) play an important role in the applications of these cells in clinical settings. In this context, we checked whether storage at - 80 °C in 10% DMSO for a longer period has any adverse effect on the functionality and genetic stability. We carried our studies on DPSC and DFSC samples that were revived after a maximum of 5 years of cryopreservation. We observed that even after long-term uncontrolled freezing at - 80 °C, these cells survived and proliferated efficiently. The assessment was made based on their post-thaw morphology, immunophenotypes, differentiation potential, growth kinetics, and genetic features. These cells retained the expression of stemness markers, differentiation ability and maintained their normal karyotype. Our results indicated no significant morphological or immunophenotypic differences between the cryopreserved DMSCs and the fresh DMSCs. Our study implies that mesenchymal stromal cells derived from the dental tissue origin are very robust and do not require any sophisticated preservation protocols. Thus, these can be an ideal source for research, stem cell banking, as well as successful clinical applications in tissue engineering and cell-based therapeutics. Graphical Abstract Schematic diagram showing the cryopreservation of DMSCs by uncontrolled freezing at -80 c has no adverse effects on their functionality and genetic stability.

Evaluation of the Effect of Crosslinking Method of Poly(Vinyl Alcohol) Hydrogels on Thrombogenicity.

PURPOSE: Crosslinked poly(vinyl alcohol) (PVA) is a biomaterial that can be used for multiple cardiovascular applications. The success of implanted biomaterials is contingent on the properties of the material. A crucial consideration for blood-contacting devices is their potential to incite thrombus formation, which is dependent on the material surface properties. The goal of this study was to quantify the effect of different crosslinking methods of PVA hydrogels on in vitro thrombogenicity. METHODS: PVA was manufactured using three different crosslinking methods: 30% sodium trimetaphosphate (STMP), three 24 h freeze-thaw cycles (FT), and 2% glutaraldehyde-crosslinked (GA) to produce STMP-PVA, FT-PVA and GA-PVA, respectively. Expanded polytetrafluoroethylene (ePTFE) was used as a clinical control. As markers of thrombus formation, the degree of coagulation factor (F) XII activation, fibrin formation, and platelet adhesion were measured. RESULTS: The GA-PVA material increased FXII activation in the presence of cofactors compared to vehicle and increase platelet adhesion compared to other PVA surfaces. The STMP-PVA and FT-PVA materials had equivalent degrees of FXII activation, fibrin formation and platelet adhesion. CONCLUSION: This work supports crosslinker dependent thrombogenicity of PVA hydrogels and advances our understanding of how the manufacturing of a PVA hydrogel affects its hemocompatibility.

Preserving the Efficacy of Glycopeptide Antibiotics during Nanoencapsulation in Liposomes.

Liposome nanovesicles are attractive vehicles for encapsulation and localized delivery of antibiotics. Most liposomal batch preparation processes involve numerous freeze-thaw cycles and heating or sonication steps, all of which can potentially deactivate or degrade antibiotics. We investigated the extent of antibiotic deactivation during various liposomal preparation methods using two glycopeptide antibiotics clinically administered for Staphylococcus infections, namely, vancomycin hydrochloride and teicoplanin. Both antibiotics, in the nonencapsulated state, were found to be highly sensitive to the freeze-thaw/sonication; vancomycin completely lost efficacy after only three cycles of freeze-thaw, and teicoplanin lost efficacy after 20 min of sonication. When the antibiotics were encapsulated in liposomes, vancomycin retained full potency against bacterial cultures of Staphylococcus aureus but encapsulated teicoplanin suffered a decrease in activity. Differential scanning calorimetry and mass spectrometry suggest that liposomes have a protective effect on the encapsulated antibiotic, the extent of which was found to differ on the basis of the processing conditions.

Long-term cold acclimation extends survival time at 0°C and modifies the metabolomic profiles of the larvae of the fruit fly Drosophila melanogaster.

BACKGROUND: Drosophila melanogaster is a chill-susceptible insect. Previous studies on this fly focused on acute direct chilling injury during cold shock and showed that lower lethal temperature (LLT, approximately -5°C) exhibits relatively low plasticity and that acclimations, both rapid cold hardening (RCH) and long-term cold acclimation, shift the LLT by only a few degrees at the maximum. PRINCIPAL FINDINGS: We found that long-term cold acclimation considerably improved cold tolerance in fully grown third-instar larvae of D. melanogaster. A comparison of the larvae acclimated at constant 25°C with those acclimated at constant 15°C followed by constant 6°C for 2 d (15°C→6°C) showed that long-term cold acclimation extended the lethal time for 50% of the population (Lt(50)) during exposure to constant 0°C as much as 630-fold (from 0.137 h to 86.658 h). Such marked physiological plasticity in Lt(50) (in contrast to LLT) suggested that chronic indirect chilling injury at 0°C differs from that caused by cold shock. Long-term cold acclimation modified the metabolomic profiles of the larvae. Accumulations of proline (up to 17.7 mM) and trehalose (up to 36.5 mM) were the two most prominent responses. In addition, restructuring of the glycerophospholipid composition of biological membranes was observed. The relative proportion of glycerophosphoethanolamines (especially those with linoleic acid at the sn-2 position) increased at the expense of glycerophosphocholines. CONCLUSION: Third-instar larvae of D. melanogaster improved their cold tolerance in response to long-term cold acclimation and showed metabolic potential for the accumulation of proline and trehalose and for membrane restructuring.