A novel simplified ultra-rapid freezing technique for cryopreservation of tissue slices

Cryopreservation offers the potential to maximize the use and availability of biological materials that have a limited supply. This study demonstrates an enhanced technique for the parallel cryopreservation of a series of liver tissue slices using a tray modeled from aluminium foil and low concentrations of a cryoprotectant. Cooling and warming rates of approximately 2000 and 3900 degrees C min(-1), respectively, were achieved as the thermal capacity of the foil-tray was significantly reduced compared to the aluminium sandwich device introduced by Day et al. [S.H. Day, D.A. Nicoll-Griffith, J.M. Silva, Cryopreservation of rat and human liver slices by rapid freezing, Cryobiology 38 (1999) 154-159]. Additionally, the two critical steps involved in the sandwich approach, i.e., clamping the plates and complete filling of the entire space between the plates with liquid, can be omitted using the foil tray. The viability of the slices was verified by measuring tetrazolium salt reduction capacity, cytosolic enzyme lactate dehydrogenase leakage, and ethoxycoumarin metabolism.     

Controlling the freezing process: a robotic device for rapidly freezing biological tissues with millisecond time resolution

A robotic cryogenic device was developed which allows freezing of thick biological tissues with millisecond time resolution. The device consists of two horizontally oriented hammers (pre-cooled with liquid N(2)) driven by two linear servo-motors. The tissue sample is bathed in Ringers contained in a chamber which drops rapidly out of the way just as the hammers approach. A third linear motor is vertically oriented, and permits the rapidly dropping chamber to smoothly decelerate. All movements were performed by the three motors and four solenoids controlled by a PC. Mechanical adjustments, that change the size of the gap between the hammers at the end position, permit the final thickness of the frozen tissue to be varied. Here we show that the freezing time increased with the square of the final thickness of the frozen bundle. However, when bundles of different original thicknesses (up to at least 1mm) were compressed to the same final thickness (e.g., 0.2mm), they exhibited nearly equal freezing times. Hence, by being able to adjust the final thickness of the frozen bundles, the device not only speeds the rate of freezing, but standardizes the freezing time for different diameter samples. This permits the use of freezing for accurate determination of the kinetics of cellular processes in biological tissue.     

Examining the freezing process of an intermediate bulk containing an industrially relevant protein

Numerous biopharmaceuticals are produced in recombinant microorganisms in the controlled environment of a bioreactor, a process known as Upstream Process. To minimize product loss due to physico-chemical and enzymatic degradation, the Upstream Process should be directly followed by product purification, known as Downstream Process. However, the Downstream Process can be technologically complex and time-consuming which is why Upstream and Downstream Process usually have to be decoupled temporally and spatially. Consequently, the product obtained after the Upstream Process, known as intermediate bulk, has to be stored. In those circumstances, a freezing procedure is often performed to prevent product loss. However, the freezing process itself is inseparably linked to physico-chemical changes of the intermediate bulk which may in turn damage the product.The present study analysed the behaviour of a Tris-buffered intermediate bulk containing a biopharmaceutically relevant protein during a bottle freezing process. Major damaging mechanisms, like the spatiotemporal redistribution of ion concentrations and pH, and their influence on product stability were investigated. Summarizing, we show the complex events which happen in an intermediate bulk during freezing and explain the different causes for product loss.     

Effects of prolonged freezing stress on the photosynthetic apparatus of moderately hardy leaves as assayed by chlorophyll fluorescence kinetics

Abstract Moderately frost-hardy leaves of the wintergreen broadleaf woody shrubs Pyracantha coccinea and Ligustrum ovalifolium and the winter annual herb Spinacia oleracea were subjected to extended freezing stress up to 15 d at temperatures 2–8°C above the mean lethal temperature (LT₅₀). After thawing, the fast kinetics of in vivo chlorophyll fluorescence of photosystem II (PSII) and the potential of linear photosynthetic electron transport of isolated thylakoid membranes was measured at room temperature. The lower the minimum freezing temperature and the longer the time of exposure, the greater was the suppression of the fluorescence signals of the leaves and decrease of the electron transport capacity of the thylakoid membranes. The pattern of inactivation of PSII -mediated electron flow, i.e. inhibition of photoreaction to photochemistry and/or electron donation to the photochemical reaction, during long-term freezing at temperatures somewhat above the LT₅₀ of the leaves was similar to that observed earlier after relatively brief exposure of leaves and isolated thylakoid membranes to more severe freezing stress. As injury occurred during freezing in complete darkness, it is likely that prolonged winter stress under natural environmental conditions causes changes in the photosynthetic apparatus of moderately hardy leaves which are not due to photoinhibition.     

Ultrastructure of infected cells in the actinorhizal root nodules ofGymnostoma papuanum (Casuarinaceae) prepared by high pressure freezing and chemical fixation

Summary Actinorhizal root nodules ofGymnostoma papuanuum (Casuarinaceae) were examined with transmission electron microscopy after being either fixed with glutalaldehyde and osmium tetroxide or frozen with liquid nitrogen at high pressure and freeze-substituted. Much better preservation was obtained by the cryopreservation method. Mitochondria, plastids, membranes und ribosomes were much better preserved in the frozen specimens than in the chemically fixed tissues. No nucleoids were observed in the microsymbiont in frozen specimens. In contrast nucleoid regions were present in chemically fixed specimens. The actinomycete microsymbiont differentiates small spherical-shaped symbiotic vesicles after the hyphae have grown and penetrated into most regions of the cytoplasm.Dedicated to the memory of Professor Oswald Kiermayer     

Simulation of in situ freezing damage of the photosynthetic apparatus by freezing in vitro of thylakoids suspended in complex media

Chloroplast thylakoid membranes were isolated from leaves of unhardened and cold-acclimated spinach (Spinacia oleracea L.). For freezethaw treatment, the membranes were suspended in complex media composed to simulate the solute concentrations in the chloroplast stroma in the unhardened and hardened states of the leaves. In particular, high concentrations of amino acids were applied for simulating the hardened state. After frost treatment, photosynthetic activities and chlorophyll fluorescence parameters of the thylakoids were tested to determine the degree of freezing damage. The results revealed a pattern of freezing injury similar to that observed upon frost treatment of thylakoids in situ. A major manifestation of damage was the inhibition of photosynthetic electron transport. Uncoupling of photophosphorylation, which is the dominating effect of freezing of thylakoids suspended in binary solutions (e.g., containing one sugar and one inorganic salt), was also visible but less pronounced in the complex media. Thylakoids obtained from cold-acclimated leaves did not exhibit an increased frost tolerance in vitro, as compared with thylakoids from unhardened plants. The results, furthermore, indicated a strong protective effect of free amino acids at the concentrations and composition found in chloroplasts of hardened leaves. The presence of inorganic salts in the complex media slightly stabilized rather than damaged the membranes during freezing. It is concluded that inactivation of thylakoids in situ may be understood as the destabilizing action of the combined solutes surrounding the thylakoids, occurring when solute concentration is raised due to freezing of water.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Hepes 4-(2-hydroxyethyl)-1-piper-azineethanesulfonic acid - PSI photosystem I - PSII photosystem II     

Improving the tablet characteristics and dissolution profile of ibuprofen by using a novel coprocessed superdisintegrant: A technical note

Summary and Conclusion  The coprocessed superdisintegrant proved to be superior to the physical blend in terms of flow due to size enlargement. Furthermore, the coprocessed superdisintegrant displayed superiority in terms of crushing strength, disintegration time, and drug dissolution. The advantages of the proposed method are easy adaptability in industry and the possibility of bypassing the existing patents in the ereas of quick disintegration and dissolution. Published: February 16, 2007     

Efficiency improvement of an antibody production process by increasing the inoculum density

Increasing economic pressure is the main driving force to enhance the efficiency of existing processes. We developed a perfusion strategy for a seed train reactor to generate a higher inoculum density for a subsequent fed batch production culture. A higher inoculum density can reduce culture duration without compromising product titers. Hence, a better capacity utilization can be achieved. The perfusion strategy was planned to be implemented in an existing large scale antibody production process. Therefore, facility and process constraints had to be considered. This article describes the initial development steps. Using a proprietary medium and a Chinese hamster ovary cell line expressing an IgG antibody, four different cell retention devices were compared in regard to retention efficiency and reliability. Two devices were selected for further process refinement, a centrifuge and an inclined gravitational settler. A concentrated feed medium was developed to meet facility constraints regarding maximum accumulated perfundate volume. A 2‐day batch phase followed by 5 days of perfusion resulted in cell densities of 1.6 × 10¹⁰ cells L^?1, a 3.5 fold increase compared to batch cultivations. Two reactor volumes of concentrated feed medium were needed to achieve this goal. Eleven cultivations were carried out in bench and 50 L reactors showing acceptable reproducibility and ease of scale up. In addition, it was shown that at least three perfusion phases can be combined within a repeated perfusion strategy. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:607–615, 2014     

一株铁皮石斛内生真菌的色素稳定性研究

本研究以铁皮石斛体内分离筛选得到一株产橙红色色素的内生真菌X1为研究对象,为明确该菌株的种属及其所产天然色素的稳定性,采用分子生物学的方法,鉴定该菌株归属于Neurospora sp.一种。该菌株所产橙色素在400 nm波长处有最大吸收峰;从温度、pH、光照、氧化剂与还原剂四个单因子,对该株菌进行色素稳定性研究。结果表明该色素在紫外光下放置6 h橙色素光吸收值增大,说明有增色效应;在pH 2～8,20～100℃下不受太大影响;H_20_2和Na_2SO_3对该色素有较强的破坏力;Fe³⁺、Zn²⁺、Cu²⁺、Ca²⁺和Mn²⁺对该色素具有保色或增色作用;10%NaCl和15%葡萄糖溶液对色素均有增色效应。该研究对内生真菌分离及真菌色素提取工艺具有一定的理论指导意义。     

Recovery of lignocelluloses from pre-hydrolysis liquor in the lime kiln of kraft-based dissolving pulp production process by adsorption to lime mud

Dissolved lignocelluloses from the pre-hydrolysis liquor (PHL) of kraft-based dissolving pulp production process were recovered by adsorption to lime mud produced in the causticizing plant of the kraft process. The adsorption of lignocelluloses was a fast process, and could be completed within one hour. The addition of polydiallyldimethylammonium chloride (PDADMAC) significantly increased the amounts of adsorbed lignin and hemicelluloses, which more than doubled at the PDADMAC dosage of 0.1% (based on the weight of PHL). The measured heating values of the adsorbed lignocelluloses indicate that adsorption of lignocelluloses to lime mud may result in the energy saving of the lime kiln. The process proposed in this study could also be adapted to decrease inhibitor concentrations (lignin and acetic acid) if the dissolved hemicelluloses in the PHL were used to produce value-added products, e.g., ethanol, xylitol, based on the fermentation process.     

Enhancement of the activated sludge process by activated carbon produced from surplus biological sludge

Surplus biological sludge from wastewater treatment operations was converted into activated carbon and then added to the aerated vessel of an activated sludge process treating phenol and glucose. The addition of activated carbon, either sludge-based or commercial, enhanced phenol removal from 58 to 98.7% and from 87 to 93% for COD with feed concentrations of 100 mg phenol l^–1 and 2500 mg COD l^–1. No differences were found between the activated sludge-activated carbon bench scale continuous reactors operating with either commercial or sludge-based activated carbon in spite of the higher adsorption capacity of the former.     

Structural basis of the destabilization produced by an amino-terminal tag in the beta-glycosidase from the hyperthermophilic archeon Sulfolobus solfataricus

We have previously shown that the major ion-pairs network of the tetrameric beta-glycosidase from the hyperthermophilic archeon Sulfolobus solfataricus involves more than 16 ion-pairs and hydrogen bonds between several residues from the four subunits and protects the protein from thermal unfolding by sewing the carboxy-termini of the enzyme. We show here that the amino-terminal of the enzyme also plays a relevant role in the thermostabilization of the protein. In fact, the addition of four extra amino acids at the amino-terminal of the beta-glycosidase, though not affecting the catalytic machinery of the enzyme and its thermophilicity, produced a faster enzyme inactivation in the temperature range 85-95 degrees C and decreased the Tm of the protein of 6 degrees C, measured by infrared spectroscopy. In addition, detailed two-dimensional IR correlation analysis revealed that the quaternary structure of the tagged enzyme is destabilized at 85 degrees C whilst that of the wild type enzyme is stable up to 98 degrees C. Molecular models allowed the rationalization of the experimental data indicating that the longer amino-terminal tail may destabilize the beta-glycosidase by enhancing the molecular fraying of the polypeptide and loosening the dimeric interfaces. The data support the hypothesis that fraying of the polypeptide chain termini is a relevant event in protein unfolding.     

The expression of a rab-related gene,rab18, is induced by abscisic acid during the cold acclimation process of Arabidopsis thaliana (L.) Heynh

We have isolated a rab-related (responsive to ABA) gene, rab18 from Arabidopsis thaliana. The gene encodes a hydrophilic, glycine-rich protein (18.5 kDa), which contains the conserved serine- and lysine-rich domains characteristic of similar RAB proteins in other plant species. The rab18 mRNA accumulates in plants exposed to low temperature, water stress or exogenous ABA but not in plants subjected to heat shock. This stress-related accumulation of the rab18 mRNA is markedly decreased in the ABA-synthesis mutant aba-1, the ABA-response mutant abi-1 or in wild-type plants treated with the carotenoid synthesis inhibitor, fluridone. Exogenous ABA treatment can induce the rab18 mRNA in the aba-1 mutant but not in the abi-1 mutant. These results provide direct genetic evidence for the ABA-dependent regulation of the rab18 gene in A. thaliana.     

Seasonal ultrastructural alterations in the plasma membrane produced by slow freezing in cortical tissues of mulberry (Morus bombyciz Koidz. cv. Goroji)

Seasonal alterations in the ultrastructure of the plasma membrane produced by slow freezing were examined in cortical parenchyma cells of mulberry twigs (Morus bombyciz Koidz. cv. Goroji) grown in northern Japan. In freezing-sensitive summer, freezing produced distinct aparticulate domains with accompanying inverted hexagonal_II (H_II) phase transitions in the plasma membrane. In autumn and spring, during cold acclimation and deacclimation, freezing produced aparticulate domains in the plasma membrane without accompanying H_ii phase transitions. In winter, when the twigs were freezing-tolerant, freezing did not produce ultrastructural alterations in the plasma membrane. A significant relationship was recognized between the percentages of cells with aparticulate domains in the plasma membrane, regardless of the presence or absence of H_II phase transitions, and the occurrence of freezing injury throughout all seasons and at all freezing temperatures tested in each season. The aparticulate domains in the plasma membranes were shown to be produced by the close apposition of membranes due to freezing-induced dehydration and deformation of cells. Although the precise mechanisms that cause injury as a result of the formation of aparticulate domains in the plasma membrane remain unclear, our results indicate that the development of cold acclimation paralleled the process whereby cells developed the ability to reduce and finally to prevent the formation of aparticulate domains in the plasma membrane that would otherwise result from freezing-induced cellular dehydration and deformation that brings membranes into close proximity with one another.     

A noncoding RNA produced by arthropod-borne flaviviruses inhibits the cellular exoribonuclease XRN1 and alters host mRNA stability

All arthropod-borne flaviviruses generate a short noncoding RNA (sfRNA) from the viral 3′ untranslated region during infection due to stalling of the cellular 5′-to-3′ exonuclease XRN1. We show here that formation of sfRNA also inhibits XRN1 activity. Cells infected with Dengue or Kunjin viruses accumulate uncapped mRNAs, decay intermediates normally targeted by XRN1. XRN1 repression also resulted in the increased overall stability of cellular mRNAs in flavivirus-infected cells. Importantly, a mutant Kunjin virus that cannot form sfRNA but replicates to normal levels failed to affect host mRNA stability or XRN1 activity. Expression of sfRNA in the absence of viral infection demonstrated that sfRNA formation was directly responsible for the stabilization of cellular mRNAs. Finally, numerous cellular mRNAs were differentially expressed in an sfRNA-dependent fashion in a Kunjin virus infection. We conclude that flaviviruses incapacitate XRN1 during infection and dysregulate host mRNA stability as a result of sfRNA formation.     

Evaluation of the effect of process variables on the fatty acid profile of single cell oil produced by Mortierella using solid-state fermentation

This article reviews some of the aspects of single cell oil (SCO) production using solid-state fermentation (SSF) by fungi of the genus Mortierella. This article provides an overview of the advantages of SSF for SCO formation by the aforementioned fungus and demonstrates that the content of the polyunsaturated fatty acids (PUFA) depend on the type of fermentation media and culture conditions. Process variables that influence lipid accumulation by Mortierella spp. and the profile of the fatty acids are discussed, including incubation temperature, time, aeration, growth phase of the mycelium, particle size of the substrate, carbon to nitrogen ratio, initial moisture content and pH as well as supplementation of the substrate with nitrogen and oil. Finally, the article highlights future research trends for the scaled-up production of PUFAs in SSF.     

Rapid isolation of muscle and heart mitochondria, the lability of oxidative phosphorylation and attempts to stabilize the process in vitro by taurine, carnitine and other compounds

We modified the isolation procedure of muscle and heart mitochondria. In human muscle, this resulted in a 3.4 fold higher yield of better coupled mitochondria in half the isolation time. In a preparation from rat muscle we studied factors that affected the stability of oxidative phosphorylation (oxphos) and found that it decreased by shaking the preparation on a Vortex machine, by exposure to light and by an increase in storage temperature. The decay was found to be different for each substrate tested. The oxidation of ascorbate was most stable and less sensitive to the treatments.When mitochondria were stored in the dark and the cold, the decrease in oxidative phosphorylation followed first order kinetics. In individual preparations of muscle and heart mitochondria, protection of oxidative phosphorylation was found by adding candidate stabilizers, such as desferrioxamine, lazaroids, taurine, carnitine, phosphocreatine, N-acetylcysteine, Trolox-C and ruthenium red, implying a role for reactive oxygen species and calcium-ions in the in vitro damage at low temperature to oxidative phosphorylation.In heart mitochondria oxphos with pyruvate and palmitoylcarnitine was most labile followed by glutamate, succinate and ascorbate.We studied the effect of taurine, hypotaurine, carnitine, and desferrioxamine on the decay of oxphos with these substrates. 1 mM taurine (n = 6) caused a significant protection of oxphos with pyruvate, glutamate and palmitoylcarnitine, but not with the other substrates. 5 mM L-carnitine (n = 6), 1 mM hypotaurine (n = 3) and 0.1 mM desferrioxamine (n = 3) did not protect oxphos with any of the substrates at a significant level.These experiments were undertaken in the hope that the in vitro stabilizers can be used in future treatment of patients with defects in oxidative phosphorylation. (Mol Cell Biochem 174: 61–66, 1997)     

Activation process of the mosquitocidal delta-endotoxin Cry39A produced by Bacillus thuringiensis subsp. aizawai BUN1-14 and binding property to Anopheles stephensi BBMV

Most delta-endotoxins produced by Bacillus thuringiensis require proteolytic processing in order to become active. The in vitro and in vivo activation processes of Cry39A, a delta-endotoxin that is highly toxic to Anopheles stephensi, were investigated. Cry39A with a molecular mass of 72 kDa was processed in vitro into a 60 kDa fragment by trypsin and gut extract from A. stephensi larvae. N-terminal amino acid sequencing of the 60 kDa fragment revealed that trypsin and the protease(s) in the gut extract cleaved Cry39A between Arg(61) and Gly(62). In contrast, 40 and 25 kDa polypeptides were generated in vivo by intramolecular cleavage of the 60 kDa fragment in A. stephensi larvae. Further, a co-precipitation assay was used to investigate the binding property of the activated Cry39A to A. stephensi BBMV. Cry39A bound to A. stephensi BBMV specifically and did not compete with the Cry4Aa toxin. This indicated that the binding molecule(s) for Cry39A might differ from those for Cry4A. In addition, Cry39A preferentially bound to the Triton X-100-insoluble membrane fraction.