99 research outputs found
Raman spectroscopic analysis of cell differentiation and death modes
Raman spectroscopy provides opportunities for non-invasive, non-destructive, label-free analysis of cell states based on changes in the biochemical composition of cells. We are investigating the suitability of Raman spectroscopy to assess the stages of human embryonic stem cell (hESC) differentiation towards pancreatic insulin-positive cells. Raman microspectrometry analysis has revealed macromolecular composition differences over time that distinguished cell populations differentiating to pancreatic cell types, such as by an increase in the protein-to-nucleic acid signal ratio and to distinguish the presence of insulin. Added insight into these macromolecular changes were provided by principal component analysis (PCA) of the data. However, the application of PCA can be difficult to interpret. The usefulness of non-negative matrix factorization was explored to improve the interpretability of overlapping Raman bands. We demonstrated the utility of this procedure by analyzing spectra to determine the cellular insulin or glucagon content. Thus, Raman spectroscopy can detect such differences in cells to detect the desired product as well as the potential to detect residual hESCs or the emergence of unwanted cells.
We also investigated the suitability of Raman spectroscopy to detect the onset and types of cell death. Apoptotic, necrotic or autophagic Chinese Hamster Ovary cells were compared to uninduced cultures using Raman spectroscopy and PCA. Furthermore, uninduced cells were compared to cells sorted at different stages of apoptosis to determine how early the onset of apoptosis could be detected. Changes were observed in several peaks during the course of cell death, with repeated changes observed in nucleic acid- and lipid-associated peaks, enabling the distinction of cell death modes. Application of such death monitoring capabilities to cellular therapy cultures should be even more useful, given the need for more process analytical technologies to address the often more variable performance of these cultures, especially when adaptive control is needed for primary cell derived manufacturing
Acoustic cell washing and raman spectroscopy technologies To address cell therapy bioprocess challenges
Many organizations are confronting the challenges of economically ensuring the manufacture of safe and efficacious cell therapy products. These processes often depend on devices and methods that were developed for only related applications, such as blood cell processing or scientific research. Thus, we are in a window of opportunity to tailor innovative technologies to address the emerging specialized needs of cell therapy manufacturing.
The most frequent unit operation is to wash cells between process stages, such as from DMSO containing cryopreservation medium to culture expansion medium. In particular for relatively small-scale autologous cell therapy processing, cell washing is imperfectly performed by closed system blood cell centrifuges or filters. We previously developed an acoustic cell separation device, widely used for over 15 years in CHO cell perfusion cultures. This technology acts as a non-fouling filter for months of operation, by using the forces generated in ultrasonic standing wave fields. These forces separate cells from medium based on differences in density and compressibility. Greater than 99.9% cell washing with 95% washed cell recovery efficiencies have been provided by our device. We also have recently enhanced the acoustic technology to perfuse 100 million cell/mL cultures, maintaining \u3e99% cell separation efficiencies. This provides an alternative high performance closed manufacturing system, to perfuse, concentrate and wash cells, with no physical filter barrier or mechanical moving parts.
While many clinical trials have had few adverse events, the great promise of cellular therapies comes with grave risks, such as from potentially oncogenic pluripotent cells present in embryonic stem cell derived populations. There is an urgent need for process analytical technologies to non-invasively monitor mammalian cell populations and improve the reliability of manufactured cell products. This includes to monitor both the expected differentiation as well as to detect unexpected cells in the process. Recently, technological advances have led to an explosive growth in the capabilities of Raman spectroscopy, increasing the potential for novel applications. We are developing the use of this spectroscopic technique to track cell development, by measuring macromolecular changes in cell samples from cultures where stem cells are differentiated towards insulin-producing cells for the treatment of diabetes. Raman spectroscopy has great potential to provide continuous on-line assessment of cell quality during the manufacture of cell-derived therapeutic cells
Attentive Learning of Sequential Handwriting Movements: A Neural Network Model
Defense Advanced research Projects Agency and the Office of Naval Research (N00014-95-1-0409, N00014-92-J-1309); National Science Foundation (IRI-97-20333); National Institutes of Health (I-R29-DC02952-01)
Gravitational Radiation From Cosmological Turbulence
An injection of energy into the early Universe on a given characteristic
length scale will result in turbulent motions of the primordial plasma. We
calculate the stochastic background of gravitational radiation arising from a
period of cosmological turbulence, using a simple model of isotropic
Kolmogoroff turbulence produced in a cosmological phase transition. We also
derive the gravitational radiation generated by magnetic fields arising from a
dynamo operating during the period of turbulence. The resulting gravitational
radiation background has a maximum amplitude comparable to the radiation
background from the collision of bubbles in a first-order phase transition, but
at a lower frequency, while the radiation from the induced magnetic fields is
always subdominant to that from the turbulence itself. We briefly discuss the
detectability of such a signal.Comment: 20 pages. Corrections for an errant factor of 2 in all the gravity
wave characteristic amplitudes. Accepted for publication in Phys. Rev.
Interleukin-6 Receptor Antagonists in Critically Ill Patients with Covid-19.
BACKGROUND: The efficacy of interleukin-6 receptor antagonists in critically ill patients with coronavirus disease 2019 (Covid-19) is unclear. METHODS: We evaluated tocilizumab and sarilumab in an ongoing international, multifactorial, adaptive platform trial. Adult patients with Covid-19, within 24 hours after starting organ support in the intensive care unit (ICU), were randomly assigned to receive tocilizumab (8 mg per kilogram of body weight), sarilumab (400 mg), or standard care (control). The primary outcome was respiratory and cardiovascular organ support-free days, on an ordinal scale combining in-hospital death (assigned a value of -1) and days free of organ support to day 21. The trial uses a Bayesian statistical model with predefined criteria for superiority, efficacy, equivalence, or futility. An odds ratio greater than 1 represented improved survival, more organ support-free days, or both. RESULTS: Both tocilizumab and sarilumab met the predefined criteria for efficacy. At that time, 353 patients had been assigned to tocilizumab, 48 to sarilumab, and 402 to control. The median number of organ support-free days was 10 (interquartile range, -1 to 16) in the tocilizumab group, 11 (interquartile range, 0 to 16) in the sarilumab group, and 0 (interquartile range, -1 to 15) in the control group. The median adjusted cumulative odds ratios were 1.64 (95% credible interval, 1.25 to 2.14) for tocilizumab and 1.76 (95% credible interval, 1.17 to 2.91) for sarilumab as compared with control, yielding posterior probabilities of superiority to control of more than 99.9% and of 99.5%, respectively. An analysis of 90-day survival showed improved survival in the pooled interleukin-6 receptor antagonist groups, yielding a hazard ratio for the comparison with the control group of 1.61 (95% credible interval, 1.25 to 2.08) and a posterior probability of superiority of more than 99.9%. All secondary analyses supported efficacy of these interleukin-6 receptor antagonists. CONCLUSIONS: In critically ill patients with Covid-19 receiving organ support in ICUs, treatment with the interleukin-6 receptor antagonists tocilizumab and sarilumab improved outcomes, including survival. (REMAP-CAP ClinicalTrials.gov number, NCT02735707.)
Broad-scale patterns of body size in squamate reptiles of Europe and North America
Aim To document geographical interspecific patterns of body size of European and North American squamate reptile assemblages and explore the relationship between body size patterns and environmental gradients. Location North America and western Europe. Methods We processed distribution maps for native species of squamate reptiles to document interspecific spatial variation of body size at a grain size of 110 x 110 km. We also examined seven environmental variables linked to four hypotheses possibly influencing body size gradients. We used simple and multiple regression, evaluated using information theory, to identify the set of models best supported by the data. Results Europe is characterized by clear latitudinal trends in body size, whereas geographical variation in body size in North America is complex. There is a consistent association of mean body size with measures of ambient energy in both regions, although lizards increase in size northwards whereas snakes show the opposite pattern. Our best models accounted for almost 60% of the variation in body size of lizards and snakes within Europe, but the proportions of variance explained in North America were less than 20%. Main conclusions Although body size influences the energy balance of thermoregulating ectotherms, inconsistent biogeographical patterns and contrasting associations with energy in lizards and snakes suggest that no single mechanism can explain variation of reptile body size in the northern temperate zone
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Environmental effects of solar thermal power systems: ecological observations during construction of the Barstow 10 MWe pilot STPS
The environmental monitoring plan used consists of comparisons of a few meteorological variables and changes in the states of a limited array of indicator species or assemblages of species of plants and animals. Observations inlude aerial photography of the site, saltation meter measurements downwind from the site to measure fluxes of windblown sand, measurements of airborne particulates and atmospheric pollutants, and baseline temperature profiles made at two sites near the heliostat field to measure micro-meteorological patterns. Observations were made of annual plants both in off-field plots and in heliostat field, of shrubs, birds, rodents, reptiles, and sensitive species listed as rare or endangered. (LEW
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