The technology in cryotechnology.

The process of freezing biological material at extremely low temperatures is known as cryopreservation. To ensure the preservation of cells and tissues over an extended period of time, low temperatures are applied since biological processes, including the biochemical ones, come to a halt under cryogenic conditions and thus it is possible to maintain their structural and functional integrity. The field of cryopreservation gained more prominence in the 20th century and emerged as an unavoidable technology for different applications such as cell therapy, tissue engineering, or assisted fertilization. In this work we provide an overview of various technologies in the field of cryotechnology with regard to the freezing, storage and thawing of living cells. The first part covers the freezing process, starting with cryoprotective agents regarding their protection mechanisms and compositions, passing by cryo-imaging, micro-fluidic systems, and the currently available freezing and biobanking equipment. The second part focusses on the thawing process as well as the hypothermic preservation for the short-term storage of biological materials and constructs. Doi.org/10.54680/fr23610110112.

pH regulation of the kinetic stability of the lipase from Thermomyces lanuginosus.

Thermomyces lanuginosus lipase (TlL) is a kinetically stable protein, resistant toward both denaturation and refolding in the presence of the ionic surfactant sodium dodecyl sulfate (SDS) and the nonionic surfactant decyl maltoside (DecM). We investigate the pH dependence of this kinetic stability. At pH 8, TlL remains folded and enzymatically active at multimillimolar surfactant concentrations but fails to refold from the acid urea-denatured state at submillimolar concentrations of SDS and DecM, indicating a broad concentration range of kinetic trapping or hysteresis. At pH 8, very few SDS molecules bind to TlL. The hysteresis SDS concentration range shrinks when moving to pH 4-6; in this pH range, SDS binds as micellelike clusters. Although hysteresis can be eliminated by reducing disulfide bonds, destabilizing the native state, and lowering the unfolding activation barrier, SDS sensitivity is not directly linked to intrinsic kinetic stability [its resistance to the general chemical denaturant guanidinium chloride (GdmCl)], because TlL unfolds more slowly in GdmCl at pH 6.0 than at pH 8.0. However, the estimated net charge drops from approximately -12 to approximately -5 between pH 8 and 6. SDS denatures TlL at pH 6.0 by nucleating via a critical number of bound SDS molecules on the surface of native TlL to form clusters. These results imply that SDS sensitivity is connected to the availability of appropriately charged regions on the protein. We suggest that conformational rigidity is a necessary but not sufficient feature of SDS resistance, because this has to be combined with sufficient negative electrostatic potential to avoid extensive SDS binding.

High performance flow injection analysis of recombinant protein G.

Chromatographic discs were investigated for their potential to substitute for the hitherto used cartridges in heterogeneous flow injection analysis. Originally designed for fast high performance liquid chromatography (HPLC) of biopolymers, the discs combine reliability with speed and resolution. This together with their price and their long-standing time made them attractive for use in flow injection analysis. The base material of the discs is a glycidyl methacrylate-co-ethylene dimethacrylate (GMA-EDMA) co-polymer. The epoxy groups inherent to this base structure can be used for immobilization purposes. In this first demonstration, antibodies were immobilized and the resulting affinity discs used for the fast analysis (< 5 min) of protein G from cell lysate of recombinant Escherichia coli. A linear calibration curve over several orders of magnitude as well as excellent reproducibility and correlation with data produced by conventional protein assay were obtained.

Effect of progestins on androgen delta4-3-ketosteroid-5alpha-A-ring reductase system in rat oral mucosa.

Rat oral mucosa microsomal delta4-3-ketosteroid-5alpha-A-ring reductase enzyme system, reducing testosterone and 4-androstenedione, was found to be inducible by systemic administration of medroxyprogesterone acetate (MPA). MPA, when used in a mixture with testosterone and/or 4-androstenedione in vitro, acted as a competitive inhibitor of the reduction of these substrates.

Production of monoclonal antibodies by hybridoma cells in a flat sheet membrane bioreactor.

The purpose of this study was to investigate hybridoma growth and monoclonal antibody formation in a flat sheet membrane bioreactor. The effects of several different molecular weight cutoff membranes on growth and antibody formation were investigated. Nutrient and toxic product diffusion through the membranes were quantified, and the kinetic and physical constants of the system were determined.

Coiled tube membrane bioreactor for cultivation of hybridoma cells producing monoclonal antibodies.

A coiled tube membrane reactor was developed for the cultivation of mouse-mouse hybridoma cells producing monoclonal antibodies. The cell and antibody concentrations obtained in the membrane reactor were higher than that obtained in a batch spinner flask without a membrane. A mathematical model has been developed to describe the membrane transport coupled growth and product formation, and the physical and kinetic constants of the system were determined.

IMPI: Making MPI Interoperable.

The Message Passing Interface (MPI) is the de facto standard for writing parallel scientific applications in the message passing programming paradigm. Implementations of MPI were not designed to interoperate, thereby limiting the environments in which parallel jobs could be run. We briefly describe a set of protocols, designed by a steering committee of current implementors of MPI, that enable two or more implementations of MPI to interoperate within a single application. Specifically, we introduce the set of protocols collectively called Interoperable MPI (IMPI). These protocols make use of novel techniques to handle difficult requirements such as maintaining interoperability among all IMPI implementations while also allowing for the independent evolution of the collective communication algorithms used in IMPI. Our contribution to this effort has been as a facilitator for meetings, editor of the IMPI Specification document, and as an early testbed for implementations of IMPI. This testbed is in the form of an IMPI conformance tester, a system that can verify the correct operation of an IMPI-enabled version of MPI.

Accelerating Scientific Discovery Through Computation and Visualization.

The rate of scientific discovery can be accelerated through computation and visualization. This acceleration results from the synergy of expertise, computing tools, and hardware for enabling high-performance computation, information science, and visualization that is provided by a team of computation and visualization scientists collaborating in a peer-to-peer effort with the research scientists. In the context of this discussion, high performance refers to capabilities beyond the current state of the art in desktop computing. To be effective in this arena, a team comprising a critical mass of talent, parallel computing techniques, visualization algorithms, advanced visualization hardware, and a recurring investment is required to stay beyond the desktop capabilities. This article describes, through examples, how the Scientific Applications and Visualization Group (SAVG) at NIST has utilized high performance parallel computing and visualization to accelerate condensate modeling, (2) fluid flow in porous materials and in other complex geometries, (3) flows in suspensions, (4) x-ray absorption, (5) dielectric breakdown modeling, and (6) dendritic growth in alloys.

Variant of the Thermomyces lanuginosus lipase with improved kinetic stability: a candidate for enzyme replacement therapy.

Lipases with high kinetic stability and enzymatic efficiency in the human gastro-intestinal tract may help against exocrine pancreatic insufficiency. Here we mimic gastric conditions to study how bile salts and pH affect the stability and activity of Thermomyces lanuginosus lipase (TlL) and its stabler variant StL using spectroscopy, calorimetry and gel electrophoresis. Both enzymes resist trypsin digestion with and without bile salts. Bile salts activate native TlL and StL equally well, bind weakly to denatured TlL and StL at lower pH and precipitate native TlL and StL at pH 4. StL refolds more efficiently than TlL from gastric pH in bile salts, regaining activity when refolding from pH as low as 1.8 and above while TlL cannot go below pH 2.6. StL also unfolds 10-40 fold more slowly in the denaturant guanidinium chloride and the anionic surfactant SDS. We ascribe StL's superior performance to general alterations in its electrostatic potential which makes it more acid-resistant. These superior properties make StL a good candidate for pancreatic enzyme replacement therapy.

[Maxillofacial tumors subject to registration in the district of Frankfurt (Oder) during the years 1963-1973]. / Die meldepflichtigen Tumoren des Kiefer-Gesichts-Bereiches im Bezirk Frankfurt (Oder) von 1963 bis 1973.

Prior to the opening of the Clinic of Maxillofacial Surgery of the District Hospital at Franfort-on-the-Oder, important groups of diseases belonging to this speciality were studied epidemiologically on the district level to obtain a maximum of information of the future tasks. From the viewpoints of detection, prevention, diagnosis and therapy, the epidemiology of notifiable tumours in the maxillofacial region was of paramount interest in a district without university and clinic specialized in maxillofacial surgery. The results obtained were used in various ways in preparing the opening of our special clinic.

Color appearance of surfaces viewed through fog.

How do the colors of surfaces seen through fog depend on the chromatic properties of the fog? Prior work (e.g. Chen and D'Zmura, 1998 Perception 27 595-608) shows that the colors of surfaces seen through a transparent filter can be described by a convergence model. The convergence model takes into account color shift and change in contrast. Whether the convergence model can also be applied to fog was tested experimentally with an asymmetric matching task. In computer graphic simulation, observers adjusted the color of a surface seen through fog in order to match the color of a surface seen in the absence of fog. The convergence model fits the data well. The results suggest that the color constancy revealed in this task with fog is described best by a model that takes into account both shift in color and change in contrast.

[Results of experimental animal studies on pervascular influences of indwelling intravasal catheters upon free tissue grafts (author's transl)]. / Ergebnisse tierexperimenteller Untersuchungen zur perivaskulären Beeinflussung freier Gewebstransplantate mittels intravasaler Dauerkatheter.

Free tissue grafting procedures were performed on sixteen experimental animals on the basis of previously described experiments (first communication). The objective of finding a suitable operating method for pervascular perfusion of epidermal grafts in animal experiments has been accomplished. At the same time, several possible ways of objectifying the results of blood gas analyses, temperature measurements, colorimetric determinations, and histological studies were subjected to tests.

[First results of experimental animal studies on pervascular effects of indwelling intravasal catheters upon free tissue grafts. First communication (author's transl)]. / Erste Ergebnisse tierexperimenteller Untersuchungen zur pervaskulären Beeinflussung freier Gewebstransplantate mittels intravasaler Dauerkatheter.

This is the first in a series of two papers reporting first results of studies made to set up a basic model of intraarterial perfusions in free tissue grafts. Epidermal tissue grafts were obtained from thirty-two domestic pigs prior to slaughtering and pervascularly perfused for twelve to ninety-six hours. Histological examinations showed that it is possible for tissue necrosis to be arrested as a prerequisite of subsequent in vivo experiments performed with the object of pervascularly supporting the healing-in of tissue grafts.

Low IgG4 concentrations in infants with Haemophilus influenzae type b infections.

Haemophilus influenzae type b causes considerable morbidity and mortality in infants and young children. Immunity to this organism has been attributed in part to the formation, and increase with age, of antibodies to the capsular polysaccharide of the bacteria. A degree of immunodeficiency could explain why some infants and young children develop invasive haemophilus disease. In this study we investigated immunocompetence in ten infants with invasive haemophilus disease. We found normal lymphocyte mitogen responses, neutrophil iodination and bactericidal and fungicidal capacities in this group. While we found no deficiencies of any of the immunoglobulin classes, three patients had low concentrations of IgG4 and two of them had low concentrations of IgG2 as well. These findings suggest that in some infants who develop haemophilus infections, the measurement of IgG subclasses may reveal immune defects that would not otherwise be apparent.

Flow-injection sandwich ELISA for bioprocess monitoring.

A fully automated flow-injection immunoassay based on sandwich enzyme-linked immunosorbent assay (ELISA) is described for the model system: protein G-sepharose, rabbit IgG and horseradish peroxidase (HRP)-labelled protein A. After injecting rabbit IgG and HRP-labelled protein A into a cartridge containing protein G-sepharose sequentially, a mixture of hydrogen peroxide and the redox indicator, 2.2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) is passed through the cartridge. The HRP-labelled protein A bound in the cartridge is directly proportional to the concentration of rabbit IgG. The colour variation of ABTS caused during the reaction between HRP and H202 in the cartridge is detected photometrically. The whole assay procedure is controlled and evaluated by a computer. Rabbit IgG and HRP-labelled protein A are also detected by a fluorometer, which is introduced into the flow system. In the flow-injection sandwich ELISA, the slope of the calibration curve is 0.4491 in the range of 0 and 300 microg ml(-1) rabbit IgG, while it is 0.1274 in the heterogeneous immunoassay. So the flow-injection sandwich ELISA system is found to be more sensitive than a heterogeneous immunoassay for the monitoring of the model protein.

The enzymatic basis for the rat liver 6-hydroxymelatonin sulfotransferase activity.

Understanding 6-hydroxymelatonin (6HM) sulfation is deemed important to explaining normal and oncostatic actions of the pineal gland. Here we identify the enzymatic basis for this sulfation in rats. First, a quantitative assay was designed for measuring hepatic 6HM sulfotransferase (6HMST) activity. The assay was then used to identify a male dominant sexual dimorphism wherein liver from males contains double the 6HMST per g or per 100 g body weight seen in females. Examination of other rat tissues showed that most in vivo 6HM sulfation was likely to occur in liver. In addition, DEAE-Sephadex chromatography of liver cytosol indicated that 80-90% of the 6HMST activity in both sexes was due to an enzyme we named 6HMST II. A minor 6HM sulfotransferase (6HMST I) eluted from the columns prior to the main enzyme. 6HMST II, purified additionally, was shown to convert 6HM to a product that appeared to be 6HM sulfate (6-sulfa-toxymelatonin). The enzyme was inhibited by Na+, K+, Zn2+, and Cd2+. Its pH optimum was 7.80 +/- 0.30. Comparisons are made between 6HMST II, dopamine sulfotransferase II, and aryl sulfotransferase IV.

Two novel gene expression systems based on the yeasts Schwanniomyces occidentalis and Pichia stipitis.

Two non-Saccharomyces yeasts have been developed as hosts for heterologous gene expression. The celD gene from Clostridium thermocellum, encoding a heat-stable cellulase, served as the test sequence. The first system is based on the amylolytic species Schwanniomyces occidentalis, the second on the xylolytic species Pichia stipitis. The systems comprise auxotrophic host strains (trp5 in the case of S. occidentalis; trp5-10, his3 in the case of P. stipitis) and suitable transformation vectors. Vector components consist of an S. occidentalis-derived autonomously replicating sequence (SwARS) and the Saccharomyces cerevisiae-derived TRP5 sequence for plasmid propagation and selection in the yeast hosts, an ori and an ampicillin-resistance sequence for propagation and selection in a bacterial host. A range of vectors has been engineered employing different promoter elements for heterologous gene expression control in both species. Homologous elements derived from highly expressed genes of the respective hosts appeared to be of superior quality: in the case of S. occidentalis that of the GAM1 gene, in the case of P. stipitis that of the XYL1 gene. Further elements tested are the S. cerevisiae-derived ADH1 and PDC1 promoter sequences.

Recurrent herpes simplex encephalitis: recovery of virus after Ara-A treatment.

A 54-year-old man developed clinical findings consistent with herpes simplex virus (HSV) encephalitis. These signs included an abrupt onset of focal central nervous system disease, cerebrospinal fluid pleocytosis, localized electroencephalographic abnormalities, and a computerized tomographic scan showing right temporal lobe involvement. Treatment with adenine arabinoside (Ara-A) resulted in improvement. Two months later he again became confused, and a left hemiparesis developed. Although biopsy revealed extensive necrosis and inflammatory response, HSV antigens and herpesvirus particles were not detected. Culture of biopsy tissue yielded HSV type 1 only after 18 days. A second course of Ara-A was administered but the patient failed to improve and died four months later. Extensive inflammatory necrosis of both temporal lobes involving gray and white matter was found. Cultures were negative for HSV. The recovery of virus from our patient during the second encephalitic episode should raise concerns regarding the efficacy of Ara-A treatment and the role of the virus in recurrent disease. In addition, the importance of maintaining biopsy tissue in culture for prolonged periods is emphasized.