Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Amyloidogenesis via interfacial shear in a containerless biochemical reactor aboard the International Space Station

Amyloidogenesis via interfacial shear in a containerless biochemical reactor aboard the... www.nature.com/npjmgrav ARTICLE OPEN Amyloidogenesis via interfacial shear in a containerless biochemical reactor aboard the International Space Station 1 1,2,3 1,3 1,3✉ Patrick McMackin , Joe Adam , Shannon Griffin and Amir Hirsa Fluid interfaces significantly influence the dynamics of protein solutions, effects that can be isolated by performing experiments in microgravity, greatly reducing the amount of solid boundaries present, allowing air-liquid interfaces to become dominant. This investigation examined the effects of protein concentration on interfacial shear-induced fibrillization of insulin in microgravity within a containerless biochemical reactor, the ring-sheared drop (RSD), aboard the international space station (ISS). Human insulin was used as a model amyloidogenic protein for studying protein kinetics with applications to in situ pharmaceutical production, tissue engineering, and diseases such as Alzheimer’s, Parkinson’s, infectious prions, and type 2 diabetes. Experiments investigated three main stages of amyloidogenesis: nucleation studied by seeding native solutions with fibril aggregates, fibrillization quantified using intrinsic fibrillization rate after fitting measured solution intensity to a sigmoidal function, and gelation observed by detection of solidification fronts. Results demonstrated that in surface-dominated amyloidogenic protein solutions: seeding with fibrils induces fibrillization of native protein, intrinsic fibrillization rate is independent of concentration, and that there is a minimum fibril concentration for gelation with gelation rate and rapidity of onset increasing monotonically with increasing protein concentration. These findings matched well with results of previous studies within ground-based analogs. npj Microgravity (2022) 8:41 ; https://doi.org/10.1038/s41526-022-00227-2 INTRODUCTION other amyloidogenic proteins besides insulin exist , some of 35–37 which are functional amyloids which support natural biolo- Protein biology in spaceflight is a field of research that has been gical functions while others are related to disease, such as the beta expanding along with the advancement of space exploration and 38–41 42,43 1–5 amyloid and tau proteins of Alzheimer’s disease, alpha- human habitation in altered gravity . Studying the biophysical 44,45 46,47 synuclein of Parkinson’s disease, infectious prion proteins, and fluid dynamic behavior of liquid protein solutions in 48–50 and the islet protein involved in type 2 diabetes. Compara- microgravity can provide insight into fundamental physical tively, insulin is a relatively safe model amyloidogenic protein for phenomena in space, as well as within biochemical systems on space studies, as dangerous proteins such as infectious prions are Earth. In space, the absence of gravity increases the prominence of not allowed on the ISS due to safety regulations . Overall, insulin the air-liquid interface, material properties such as surface tension, is a multifaceted model for studying protein interfacial rheology surface viscosities, and molecular adsorption becoming even more 1,2 and kinetics with relevance to biophysics, fluid physics, medicine, impactful to the behavior of a liquid system . On Earth many and spaceflight. biochemical systems exist where fluid interfaces have major The process of amyloidogenesis applicable to human insulin effects of key importance, including environmental surfactant 6 7 and other amyloidogenic proteins, progresses in three key layers , industrial bioprocessing , and physiological tissue sur- biophysical stages: nucleation, fibrillization, and gela- faces within the body. Such systems with fluid interfaces, both in 10,29,32 tion . Nucleation is the joining of two monomers, a space and on Earth, can exhibit unique alterations in fluid and 9 10 molecular association that often changes secondary and protein behavior due to protein adsorption , fibrillization , 11 12 tertiary protein structure, to form a pre-fibril aggregate, or biopolymer dynamics , and gelation , all dependent upon the nucleate, with quaternary structure that can accept additional fluid system’s geometry and the specific type of proteins present. monomeric subunits, seeding the system with starting points Human insulin was selected as the model protein for this study 10,34 for fibrillization . Fibrillization, or more specifically elonga- based on two main points of significance: relevance to studies in tion, is the addition of a monomer to an existing fibril, a microgravity and relevance to protein biophysics with interfacial polymerization process which lengthens protein polymers from hydrodynamics. Insulin’s relevance to spaceflight originates from pre-fibril aggregates, to fibrils, to fibers, with the distribution of the protein’s history of kinetics and crystallization in micrograv- 10,12 1,13–15 ity , medical applications to diabetogenic effects in human fibril size changing as the process progresses .Gelationis 2,5,16–18 spaceflight , and application as a model pharmaceuti- the linking of protein fibers to form a polymer network, a 7,19,20 cal for studies of protein stability and in situ resource structure filled with solvent which maintains a defined shape, a utilization in spaceflight. From a biophysics and fluid physics process that can occur concurrently with fibrillization if 21–23 12,29,32 perspective, insulin displays rich bulk hydrodynamic , inter- sufficiently large fibril sizes are present .These defining 24–28 29–33 facial , and protein kinetics behavior. Moreover, insulin is protein kinetic processes of amyloidogenesis are governed by an amyloidogenic protein that can undergo a fibrillization process, asystem’s dynamic microenvironment, and lead to changes in 32,52–55 termed amyloidogenesis, to produce amyloid fibrils which possess protein mechanical properties ,cytotoxic effectsin 10,32,34 39,40,42,45,46,48–50,55–57 a durable beta-cross quaternary protein structure . Many amyloid diseases ,and formationof 1 2 Mechanical, Aerospace, and Nuclear Engineering, Rensselaer Polytechnic Institute, 110 8th St, Troy 12180 NY, USA. Department of Biological Sciences, Rensselaer Polytechnic Institute, 110 8th St, Troy 12180 NY, USA. Chemical and Biological Engineering, Rensselaer Polytechnic Institute, 110 8th St, Troy 12180 NY, USA. email: hirsaa@rpi.edu Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; P. McMackin et al. Fig. 1 The ring-sheared drop (RSD) with the bottom ring rotating. a Image of the ring-sheared drop aboard the ISS showing a pre-sheared 8 mg/mL human insulin solution spinning at 30 rpm. b Axisymmetric computation of primary (color map of normalized azimuthal velocity, ν) and secondary (black arrowed streamlines in the azimuthal plane) flow of a Newtonian fluid in the RSD sheared at 30 rpm. 58,59 intricate nanostructure , potentially adaptable to tissue engineering. The chemical and thermodynamic state of a system governs protein kinetics, biophysical processes of the constituent mole- 10,12 cules progressing toward a point of lower free energy . Fluid transport and associated hydrodynamic stresses influence the thermodynamic state of amyloid systems, both bulk shear 21–23,60–62 24–28,63–67 flow and air-liquid interface activity affecting the number of molecular collisions, with more frequent collisions increasing the probability for interactions including nucleation, fibrillization, and gelation. Geometries with fluid interfaces are well-suited to the study of physiological systems as most interfaces within the body are fluidic in nature, flow of cerebrospinal fluid (CSF) within the brain being of specific 8,62,68–72 importance for many neurodegenerative diseases . Fig. 2 Intensity of scattered light verses time for pre-sheared Furthermore, brain structure and CSF have been observed to insulin cases. ± 1 standard deviation error bars represent measure- 73–78 ment uncertainty and dashed lines represent sigmoidal fits to a undergo alterations due to spaceflight , making the study of theoretical fibrillization function (Eq. 1) presented in the Methods such systems imperative to long-term space habitation. Along section. with these fluid effects, protein kinetics are also affected by the quantity of protein present, which can alter molecular dynamics RESULTS and the overall interactions with bulk fluid and fluid interfaces. Pre-sheared fibrillization Microgravity provides a unique environment for the experi- Three pre-sheared trials were performed in this investigation at mental study of systems with solely fluid interfaces, the total protein concentrations of 2, 4, and 8 mg/mL, each subject to dominance of free surfaces in the absence of gravity facilitating steady interfacial shear at 30 rpm for 3.5 days. Image data was the removal of solid boundaries which often introduce nucleation captured every 0.5 days for characterization of fibrillization sites and unintended wall effects. The ring-sheared drop (RSD) is a kinetics. Measured intensity of image data was used to construct containerless surface tension-contained microgravity biochemical fibrillization curves of intensity versus time, as depicted in Fig. 2. reactor consisting of a 2.54 cm diameter drop pinned between Measured intensity increased monotonically with time for all two rings, one stationary and one shearing, that transfer interfacial samples, indicative of the presence of fibrils with larger shear to the bulk fluid by surface shear viscosity and mix the liquid aggregates producing increased scattering and higher image 79–83 using inertial flow with secondary motion (Fig. 1) . The RSD intensity. Curves in Fig. 2 represent nonlinear least-square fits to a was first deployed to the international space station (ISS) in the theoretical sigmoidal fibrillization function (Eq. 1) that align well Fall of 2019 and again for a second operations campaign in the with experimentally measured data. Summer and Fall of 2021. A ground-based preliminary study has been performed with human insulin in the Earth analog of the Pre-sheared fibrillization kinetics RSD, the knife-edge viscometer (KEV) , an apparatus which Curves in Fig. 2 were quantified by fitting measured data to a requires a glass dish for containment under gravity yet, like the theoretical fibrillization function with empirical parameters of RSD, produces shear flow using surface shear viscosity and mixes biophysical relevance (Eq. 1). Importantly, this equation does not via secondary flow. The present investigation examined human directly model fibrillization when applied to intensity data (Fig. 2), insulin within the RSD aboard the ISS to measure the effects of as this theoretical function typically applies to fibril content steady interfacial shear on the amyloidogenesis processes of measured using spectroscopy as opposed to solution intensity. nucleation, fibrillization, and gelation in a microgravity, air-liquid The first adjustment required for intensity fitting was a horizontal interface dominated system. Specifically, the hypothesis tested offset to account for pre-shearing of these fibrillization trials, as was that if protein solutions in microgravity are seeded with fibrils these trials did not begin as completely native solutions. The and subject to steady interfacial shear, then amyloidogenesis will second adjustment was that the fitting parameter typically occur with the extent of gelation depending on total protein describing total protein concentration, I , instead described the concentration. intensity of a fully monomeric protein solution. This value was npj Microgravity (2022) 41 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; P. McMackin et al. have drawn particles into areas of the flow occluded from the Table 1. Sigmoidal fit (Eq. 1) parameters of native intensity (I ) and camera’s field of view. normalized fit root-means-square (rms) error. C (mg/mL) I rms Error (%) Pre-sheared gelation 0 0 Protein gelation was observed by the presence of a turbid 4 0.49 ± 0.03 1.3 stationary solidification front moving southward from the 2 0.33 ± 0.02 2.1 stationary ring. Figure 6 depicts the progression of this gelation 8 0.88 ± 0.02 2.7 front with time. The 2 mg/mL sample did not reach a sufficiently high fibril concentration in order to produce gelation. This requirement of a minimum fibril concentration for gelation is 28,29 consistent with previous ground-based studies . Figure 7 quantifies the progression of these gelation fronts with time in terms of a gelation front polar angle, θ . Both the rate of progression and rapidity of onset of gelation increase with increasing protein concentration. To further this observation, the 8 mg/mL case had transitioned entirely to a linked polymer gel by the trial’s conclusion, preventing liquid extraction and remaining affixed to the rings even after test cell removal. DISCUSSION Fluid interfaces produce significant effects on the biophysics of protein solutions, defining the microenvironment and energetic 10,12 9 landscape through processes such as molecular adsorption and imparted forces such as interfacial shear affecting biological 10,11 12 behaviors including the fibril dynamics and gelation of Fig. 3 Intrinsic fibrillization rate k versus protein concentration proteins. This investigation was the space continuation of an C . Values of k were determined by fitting to a fibrillization model Earth-based study , this work studying amyloidogenesis, produc- (Eq. 1). Error bars represent fit uncertainty. tion of amyloid fibrils and plaques from native amyloidogenic proteins, of human insulin in an air-liquid interface dominated used to improve fits by providing a measureable vertical offset biochemical reactor in microgravity. This fluid system, the RSD due to background monomer intensity. Fit rms error (Table 1 aboard the ISS, offered a platform for studying both the effects of column 3) was < 3% for all cases. Larger fit errors (8 mg/mL) fluid interfaces and microgravity on protein fibrillization. Amyloi- resulted from measurement accuracy reduction due to high dogenesis of human insulin was used as a model biophysical sample intensity, which can slightly effect curve shape due to system due to its applicability to biotechnology, physiology, near-upper limit sensor values. The altered application of this medicine, and spaceflight. equation to optical intensity as opposed to spectroscopic fibril Three stages of amyloidogenesis were quantified in this study, content remained suitable for the study of fibrillization kinetics including seeding, fibrillization, and gelation of insulin. A native with a focus on intrinsic fibrillization rate. The fit-determined solution of insulin serendipitously seeded with insulin fibrils native intensity values, I , are displayed in Table 1. Furthermore, (Fig. 4) displayed an earlier onset of fibrillization (Fig. 5). This sigmoidal fits allowed for relation between intrinsic fibrillization accelerated onset of fibrillization due to seeding is applicable to rate and total protein concentration (Fig. 3). Intrinsic rate was 46,47 diseases such as infectious prions or biotechnological shown to be independent of concentration and thus, the time processes that introduce a portion of fibrils to a native solution scale of fibrillization not dependant on protein concentration, a and in turn promote fibrillization of the native solution. Fibrilliza- result consistent with the ground study . tion experiments with pre-sheared insulin samples (Fig. 2) displayed the induction of amyloidogenesis via steady axisym- Seeded fibrillization metric interfacial shear, with intrinsic fibrillization rates (Fig. 3) The seeding trial occurred during measurement of a native (fully independent of total protein concentration. This concentration monomeric) 2 mg/mL sample. After 5.6 days an additional independence matches results of the ground study and injection of 0.4 mL of fluid from the deployment tube was made indicates that forces imposed at the fluid boundary lead to to account for evaporative losses and return the drop to a changes in the protein microenvironment that govern the spherical shape. Serendipitously, this injection provided a means timescale of fibrillization. The average value of this rate constant, to measure the effect of seeding with fibril aggregates. The inside 0.43 ± 0.07 1/days, matches the expected value (0.47 1/days) of the deployment tube, where remnants of the injection volume obtained by a logarithmic extrapolation using Re of fibrillization had resided for 5.5 days, was a rough unfinished stainless steel rates from Fig. 4a of the ground study . Gelation was observed surface that provided ample nucleation sites for fibrils to form, (Fig. 6) in the form of gelation fronts, only in cases with sufficiently which were subsequently transported into the bulk of the RSD high protein fibril concentration to from crosslinked polymer during the seeding injection. The turbidity of this fibril seeding networks, and the rapidity of gelation onset and rate of gelation injection was readily observable, and upon shear restart, the (Fig. 7) increased monotonically with increasing protein resulting mixing within the drop allowed for visualization of the concentration. RSD’s secondary inertial flow as displayed in Fig. 4 (see also This study marks the first successful use of the RSD fluid Supplement1.mp4). Following the initial increase in intensity due apparatus (Fig. 1) with protein solutions in microgravity aboard to added fibril content, intensity continued to increase, as the the ISS. Hardware and biological samples were transported to the solution had begun fibrillizing due to the previous seeding event ISS and installed in the MSG without fault. During operation, this (Fig. 5). The slight decrease in measured intensity observable at device successfully deployed, pinned, steadily sheared, and 9.0 and 9.5 days was unexpected and may be due to fibril extracted fluid drops of protein solutions, demonstrating perfor- adsorption to liquid-solid interfaces of the rings , which could mance of a novel method for producing air-liquid interface Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 41 P. McMackin et al. Fig. 4 Images of the native 2 mg/mL insulin case being seeded at 5.6 days with 0.4 mL of insulin fibrils. Panels show the injected volume (a) before and (b, c) after restarting ring rotation. Mixing of the turbid fibrillized volume allowed for visualization of the RSD's secondary flow (Fig. 1b and Supplement1.mp4). effects of flow in physiological systems such as the gastrointest- 104,105 73–78 106,107 108,109 inal , glymphatic , circulatory , or respiratory systems and the effects of spaceflight on these systems and the 110,111 cells within. The behavior of microbial biofluids is important for understanding the effects of spaceflight on microbiol- 112–121 ogy and potential applications to pharmaceutical produc- 4,117,122,123 tion and bio-engineering in support of space exploration. Future investigations of interfacial hydrodynamics in microgravity could offer insight into fluid systems that better facilitate spaceflight. METHODS RSD The RSD consists of a 2.54 cm diameter spherical liquid drop Fig. 5 Intensity of scattered light versus time for the 2 mg/mL pinned between two thin titanium contact rings (Fig. 1). The top native insulin case with seeding by insulin fibrils at 5.6 days. ring is connected via four prongs to a 10-gauge stainless-steel ±1 standard deviation error bars represent measurement deployment tube used to grow the liquid drop. The lower ring uncertainty. rotates to produce interfacial shearing of the drop, shear being transmitted to the bulk by means of surface shear viscosity and dominated systems in microgravity. Optics, deployment, shearing, 79–83 mixing occurring due to secondary flow . The RSD was and complete operation of the apparatus were successfully conceived in 2013, and the hardware was developed and performed using real-time remote control from the ground. With launched to the ISS on SpaceX CRS-18 July 2019 after a series of minimal impact of solid boundaries, sole components being the parabolic flights. Following these engineering missions, the thin contact rings used to transmit interfacial shear, this device is science mission (including biological samples and hardware well-suited to the study of interfacial phenomena and the modifications presented in this study) was launched on Cygnus dynamics of fluid interfaces. NG-16 in August 2021, operations being performed in the Results of this investigation center on the three main aspects of following months concluding in December 2021. The RSD amyloidogenesis: nucleation, fibrillization, and gelation. Seeding hardware was operated within the Microgravity Science Glovebox of protein solutions in microgravity was shown to promote earlier (MSG), located in the Destiny module of the ISS, providing 3 levels onset of fibrillization by bolstering the nucleation process. of containment (test cell, MSG airflow, and MSG wall) between the Fibrillization was demonstrated to be promoted by interfacial astronauts and protein samples. Due to the low 1.6 pH, samples shear, with the intrinsic rate of fibrillization being independent of were classified as a hazard response level 2 material (HRL), which protein concentration. Gelation was found to require a critical necessitated at least 3 levels of containment in accordance to concentration of protein fibrils with gelation onset and rate NASA crew-safety requirements . becoming more rapid with increasing protein concentration. Furthermore, findings presented here demonstrate the successful Protein samples performance of the microgravity biochemical reactor, the RSD, Protein samples of human insulin were prepared by dissolving utilized in this investigation. A multitude of future space lyophilized recombinant human insulin (Sigma-Aldrich, 91077C) in investigations exist that could make use of an interfacial a 0.1 M NaCl 1.6 pH buffer solution (deionized water, pH cycled biochemical reactor such as the RSD, including studies on drop 124,125 with HCL and NaOH) to pharmaceutical-relevant concentra- rheology, interface creation and substrate interaction, different tions of 2, 4 and 8 mg/mL. The low pH of the buffer allowed for interfacial flow regimes, and microbial biofluids. Drop rheology in contamination resistance in addition to pH control. Each sample space is applicable to fields ranging from fundamental changes in 85–88 89–91 was pre-sheared for 1 day at a Reynolds number of 6000 in a fluid behavior to the study of planetary bodies and their 26,126 deep-channel surface viscometer which produced partial material properties. Studies of interface creation and substrate fibrillization that allowed for earlier onset of fibrillization during interaction can be used to describe fundamental contact line 92–100 87,101 102,103 dynamics , 3D printing , and combustion in micro- operations in space. Additionally, a native (fully un-fibrillized gravity. Use of select interfacial flow regimes such as steady, monomeric, or dimeric at 1.6 pH) 2 mg/mL sample was also pulsatile, or oscillatory flows, allows fluid devices to mimic the prepared, ultimately used in a serendipitous seeding trial. After npj Microgravity (2022) 41 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA P. McMackin et al. Fig. 6 Images of gelation front progression in pre-sheared insulin cases. If present, southward-moving gelation fronts are indicated by yellow dashed lines. Experimental trials Experimental trials began after crew members installed the 12 mL sample syringe and test cell (test cells containing the RSD’s pinning rings and deployment tube) into the experimental hardware and sealed the MSG. Drop deployment followed installation by 0.5 days to dissipate any static charge accumulated during installation and to place deployment during crew sleep, avoiding deleterious accelerations. Drops were deployed at a rate of 10 mL/min in two stages to the total volume of 8.58 mL (volume of a 2.54 cm diameter sphere). Steady shear commenced after deployment with the lower ring rotating at 30 rpm, corresponding to a Reynolds number of 180 (where Re = Ωa /ν, where Ω is ring rotation rate, a is ring radius, and ν is the kinematic viscosity of water). Wide-field image data (example in Fig. 1a) of steadily- Fig. 7 Gelation front polar position, θ , vs time for pre-sheared sheared drops was collected every 0.5 days, with pre-sheared trials insulin cases. This data was extracted using the videos from which shearing for 3.5 days and the native trial shearing for 9.5 days with Fig. 6 was generated. a midpoint seeding injection of 0.4 mL of insulin fibrils after 5.6 days. LED light modules used for illumination were deactivated outside of sampling to maintain ambient conditions within the test cell. preparation, all samples were degassed under a 710 mmHg vacuum for 0.5 days to minimize air bubbles and subsequently frozen at −20 C before transportation and launch to the ISS. On Protein amyloidogenesis orbit, sample syringes were thawed 1 day at ambient temperature Protein amyloidogenesis was quantified using the measured before installation in the RSD hardware. After each experimental intensity of light from a drop’s bulk fluid in both the native trial, each protein solution was withdrawn back from the drop into seeding trial and the pre-sheared fibrillization trials. As fibrillization the syringe and placed in cold stowage (4 C) until returning to proceeded the size of particles within a solution increased leading Earth. Each sample syringe had two controls, a flight control which to increased scattering of light and visible increases in turbidity. accompanied samples to the ISS and a ground control that While such optical methods of measurement lag behind spectro- remained on Earth. Both controls underwent no shear experi- scopic measurements of monomer extinction (as sufficiently mentation and were identically degassed, frozen, thawed and small fibrils and pre-fibril aggregates will not scatter light), trends subsequently refrigerated. in fibrillization remain observable. Measured intensity was defined Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 41 P. McMackin et al. as the average of normalized red, green, and blue intensity values 6. Jenkinson, I. R., Seuront, L., Ding, H. & Elias, F. Biological modification of mechanical properties of the sea surface microlayer, influencing waves, ripples, within an interrogation area centered on the drop, calculated foam and air-sea fluxes. Elem. Sci. Anth. 6,1–32 (2018). using a MATLAB script summing over the 120 frames (30 fps) 7. Li, J. et al. Interfacial stress in the development of biologics: Fundamental recorded at each time point. Measurement uncertainty was understanding, current practice, and future perspective. AAPS J. 21,1–17 (2019). defined as the standard deviation of these intensity values within 8. Thomas, J. H. Fluid dynamics of cerebrospinal cluid flow in perivascular spaces. J. the interrogation window over each of the 120 frames of the video R. Soc. Interface 16,1–11 (2019). camera. This average intensity was then normalized to produce 9. Adamson, A. W. & Gast, A. P. Physical Chemistry of Surfaces. 6th edn. (John Wiley the measured intensity, I, which ranged between 0 and 1 and Sons Inc, NY, USA, 1997). corresponding to no detected light and a fully saturated camera 10. Otzen, D. E. Amyloid Fibrils ad Prefibrillar Aggregates. (Wiley-VCH, Weinheim, sensor. Furthermore, optical measurement was also utilized for the Germany, 2013). quantification of the final stage of amyloidogenesis, protein 11. Morrison, F. A. Understanding Rheology. (Oxford University Press, NY, USA, 2001). 12. Doi, M. Soft Matter Physics. (Oxford University Press, Oxford, United Kingdom, gelation. As a protein solution transitioned from a liquid 2013). suspension of fibers to a linked network of fibers, a noticeable 13. Snell, E. H. & Helliwell, J. R. Macromolecular crystallization in microgravity. Rep. change occurred in the optical properties of regions undergoing Prog. Phys. 68, 799–853 (2005). this phase transition. Differences in turbidity, highly turbid 14. Timofeev, V. I. et al. X-ray investigation of gene-engineered human insulin unmoving regions indicative of gel, allowed for the measurement crystallized from a solution containing polysialic acid. Acta Cryst. 66, 259–263 of gelation progression using optical tracking of gelation fronts. (2010). 15. Snell, E. H. & Helliwell, J. R. Microgravity as an environment for macromolecular crystallization - an outlook in the era of space stations and commercial space Model fit flight. Crystallogr. Rev. 10, 1080 (2021). A three-parameter sigmoidal function was utilized to obtain I (t), 16. Tobin, B. W., Uchakin, P. N. & Leeper-Woodford, S. K. Insulin secretion and representing a solution’s intensity based on a specific fibril sensitivity in space flight: Diabetogenic effects. Nutrition 18, 842–848 (2002). content as a function of time: 17. Bergouignan, A. et al. Towards human exploration of space: The theseus review series on nutrition and metabolism research priorities. npj Microgravity 2,1–8 1 1 (2016). I ðtÞ¼ I  : (1) f 0 kðt tÞ kt h h 1 þ e 1 þ e 18. Hughson, R. L. et al. Increased postflight carotid artery stiffness and inflight insulin resistance resulting from 6-mo spaceflight in male and female astro- This three-parameter function originated from protein fibrillization nauts. Am. J. Physiol. Heart Circ. Physiol. 310, 628–638 (2016). theory and contains constants that are of relevance to biophysical 19. D’Souza, A., Theis, J. D., Vrana, J. A. & Dogan, A. Pharmaceutical amyloidosis 10,25,29 properties . I is the initial intensity describing a fully monomeric associated with subcutaneous insulin and enfuvirtide administration. Amyloid 21,71–75 (2014). protein solution, t is the time (days) required to reach a half fibrillized 20. Zapadka, K. L., Becher, F. J., dos Santos, A. L. G. & Jackson, S. E. Factors affecting solution, and k is the intrinsic rate coefficient(1/days)thatdepends on the physical stability (aggregation) of peptide thereputics. Interface Focus 7, both the processes of nucleation and fibril elongation. Measured 1–18 (2017). intensity values of the pre-sheared trials were fittoEq. (1)using a 21. Szymczak, P. & Cieplak, M. Hydrodynamic effects in proteins. J. Phys. Condens. nonlinear least-squares MATLAB algorithm to obtain these biophysical Matter 23,1–14 (2010). parameters as functions of protein concentration. 22. Bekard, I. B., Asimakis, P., Bertolini, J. & Dunstan, D. E. The effects of shear flow on protein structure and function. Biopolymers 95, 733–745 (2011). 23. McBride, S. A., Tilger, C. F., Sanford, S. P., Tessier, P. M. & Hirsa, A. H. Comparison Reporting summary of human and bovine insulin amyloidogenesis under uniform shear. J. Phys. Further information on research design is available in the Nature Chem. B 119, 10426–10433 (2015). Research Reporting Summary linked to this article. 24. Pandey, L. M. et al. Surface chemistry at the nanometer scale influences insulin aggregation. Colloids Surf. B 100,69–76 (2012). 25. McBride, S. A., Sanford, S. P., Lopez, J. M. & Hirsa, A. H. Shear-induced amyloid DATA AVAILABILITY fibrillization: The role of inertia. Soft Matter 12, 3461–3467 (2016). 26. Balaraj, V. S. et al. Surface shear viscosity as a macroscopic probe of amyloid The data collected during this study is available from the corresponding authors fibril formation at a fluid interface. Soft Matter 13, 1780–1787 (2017). upon reasonable request. 27. Grigolato, F. & Arosio, P. The role of surfaces on amyloid formation. Biophys. Chem. 270, 106533–106546 (2021). 28. Adam, J. A., Middlestead, H. R., Debono, N. E. & Hirsa, A. H. Effects of shear rate CODE AVAILABILITY and protein concentration on amyloidogenesis via interfacial shear. J. Phys. The code written during this study is available from the corresponding authors upon Chem. B 125, 10355–10363 (2021). reasonable request. 29. Nielsen, L. et al. Affect of environmental factors on the kinetics of insulin fibril formation: Elucidation of the molecular mechanism. Biochemistry 40, 6036–6046 Received: 23 May 2022; Accepted: 23 August 2022; (2001). 30. Krebs, M. R. H. et al. The formation of spherulites by amyloid fibrils of bovine insulin. Proc. Natl Acad. Sci. Usa. 101, 14420–14424 (2004). 31. Pasternack, R. F. et al. Formation kinetics of insulin-based amyloid gels and the effect of added metalloporphyrins. Biophysical J. 90, 1033–1042 (2006). 32. Schleeger, M. et al. Amyloids: From molecular structure to mechanical proper- REFERENCES ties. Polymer 54, 2473–2488 (2013). 1. Clement, G. & Slenzka, K. Fundamentals of Space Biology. (Springer Science 33. Surmacz-Chwedoruk, W., Babenko, V., Dec, R., Szymczak, P. & Dzwolak, W. The +Business Media LLC, NY, USA, 2006). emergence of superstructural order in insulin amyloid fibrils upon multiple 2. Council, N. R. Recapturing a Future for Space Exploration: Life and Physical Sci- rounds of self-seeding. Sci. Rep. 6,1–12 (2016). ences Research for a New Era. (The National Academic Press, Washington DC, 34. Iadanza, M. G. et al. A new era forunderstanding amyloid structures and disease. USA, 2011). Nat. Rev. Mol. Cell Biol. 19, 755–773 (2018). 3. Barzegari, A. & Saei, A. A. An update to space biomedical research: Tissue 35. Fowler, D. M., Koulov, A. V., Balch, W. E. & Kelly, J. W. Functional amyloid - from engineering in microgravity bioreactors. BioImpacts 2,23–32 (2012). bacteria to humans. Trends Biochem. Science 32, 217–224 (2007). 4. Blue, R. S. et al. Supplying a pharmacy for nasa exploration spaceflight: Chal- 36. Otzen, D. & Riek, R. Functional amyloids. Cold Spring Harb. Perspect. Biol. 11, lenges and current understanding. npj Microgravity 5,1–12 (2019). 1–29 (2019). 5. Smith, S. M., Zwart, S. R., Douglas, G. L. & Heer, M. Human Adaptation to 37. Balistreri, A., Goetzler, E. & Chapman, M. Functional amyloids are the rule rather Spaceflight: The Role of Food and Nutrition. 2nd edn (National Aeronautics and than the exception in cellular biology. Microorganisms 8,1–13 (2020). Space Administration, TX, USA, 2021). npj Microgravity (2022) 41 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA P. McMackin et al. 38. Hortschansky, P., Schroeckh, V., Christopeit, T., Zandomeneghi, G. & Fandrich, M. 69. Iliff, J. J. et al. The emerging relationship between interstitial fluid-cerebrospinal The aggregation kinetics of alzheimer’s β-amyloid peptide is controlled by fluid exchange, amyloid β and sleep. Biol. Psychiatry 83, 328–336 (2018). stochastic nucleation. Protein Sci. 14, 1753–1759 (2005). 70. Rasmussen, M. K., Mestre, H. & Nedergaard, M. The glymphatic pathway in 39. Murphy, M. P. & LeVine(III), H. Alzheimer’s disease and the β-amyloid peptide. J. neurological disorders. Lancet Neural 17, 1016–1024 (2018). Alzheimers Dis. 19, 311–323 (2010). 71. Frankel, R. et al. Autocatalytic amplification of alzheimer-associated aβ42 pep- 40. Chen, G. et al. Amyloid beta: Structure, biology and structure-based therapeutic tide aggregation in human cerebrospinal fluid. Commun. Biol. 2,1–11 (2019). development. Acta Pharmacologica Sin. 38, 1205–1235 (2017). fang. 72. Kylkilahti, T. M. et al. Achieving brain clearance and preventing neurodegen- 41. Yagi-Utsumi, M. et al. Characterization of amyloid β fibril formation under erative diseases - a glymphatic perspective. J. Cereb. Blood Flow. Metab. 0,1–13 microgravity conditions. npj Microgravity 6,1–6 (2020). (2021). 42. Nizynski, B., Dzwolak, W. & Nieznanski, K. Amyloidogenesis of tau protein. Pro- 73. Roberts, D. R. et al. Effects of spaceflight on astronaut brain structure as indi- tein Sci. 26, 2126–2150 (2017). cated on mri. N. Engl. J. Med. 377, 1746–1753 (2017). 43. Joie, R. L. et al. Rospective longitudinal atrophy in alzheimer’s disease correlates 74. Zhang, L.-F. & Hargens, A. R. Spaceflight-induced intracranial hypertension and with the intensity and topography of baseline tau-pet. Sci. Transl. Med. 12,1–13 visual impairment: Pathophysiology and countermeasures. Physiol. Rev. 98, (2020). 59–87 (2018). 44. Araki, K. et al. Parkinson’s disease is a type of amyloidosis featuring accumula- 75. Kramer, L. A. et al. Intracranial effects of microgravity: A prospective longitudinal tion of amyloid fibrils of α-synuclein. Proc. Natl Acad. Sci. U. S. A. 116, mri study. Radiology 295, 640–648 (2020). 17963–17969 (2019). 76. ichi Iwasaki, K. et al. Long-duration spaceflight alters estimated intracranial 45. de Oliveira, G. A. P. & Silva, J. L. Alpha-synuclein stepwise aggregation reveals pressure and cerebral blood velocity. J. Physiol. 599, 1067–1081 (2021). features of an early onset mutation in parkinson’s disease. Commun. Biol. 2,1–13 77. Roy-O’Reilly, M., Mulavara, A. & Williams, T. A review of alterations to the brain (2019). during spaceflight and the potential relevance to crew in long-duration space 46. Ghetti, B. et al. Prion protein amyloidosis. Brain Pathol. 6, 127–145 (1996). exploration. npj Microgravity 7,1–9 (2021). 47. Choi, J.-K. et al. Amyloid fibrils from the n-terminal prion protein fragment are 78. Barisano, G. et al. The effect of prolonged spaceflight on cerebrospinal fluid and infectious. Proc. Natl Acad. Sci. U. S. A. 113, 13851–13856 (2016). perivascular spaces of astronauts and cosmonauts. Proc. Natl Acad. Sci. U. S. A. 48. Marzban, L., Park, K. & Verchere, C. B. Islet amyloid polypeptide and type 1 119,1–3 (2022). diabetes. Exp. Gerontol. 38, 347–351 (2003). 79. Gulati, S., Raghunandan, A., Rasheed, F., McBride, S. A. & Hirsa, A. H. Ring-sheared 49. Hull, R. L., Westermarl, G. T., Westermark, P. & Kahn, S. E. Islet amyloid: A critical drop (rsd): Microgravity module for containerless flow studies. Microgravity Sci. entity in the pathogenesis of type 2 diabetes. J. Clin. Endocrinol. Metab. 89, Technol. 29,81–89 (2017). 3629–3643 (2004). 80. Gulati, S., Riley, F. P., Lopez, J. M. & Hirsa, A. H. Mixing within drops via surface 50. Abedini, A. & Schmidt, A. M. Mechanisms of islet amyloidosis toxicity in type 2 shear viscosity. Int. J. Heat. Mass Trans. 125, 559–568 (2018). diabetes. FEBS Lett. 587, 1119–1127 (2013). 81. Gulati, S., Riley, F. P., Hirsa, A. H. & Lopez, J. M. Flow in a containerless liquid 51. ISS safety requirements document. Tech. Rep. SSP 51721, National Aeronautics system: Ring-sheared drop with finite surface shear viscosity. Phys. Rev. Fluids 4, and Space Administration, Houston, Texas (2019). 1–9 (2019). 52. Amin, S., Barnett, G. V., Pathak, J. A., Roberts, C. J. & Sarangapani, P. Protein 82. McMackin, P. M. et al. Simulated microgravity in the ring-sheared drop. npj aggregation,particle formation, characterization and rheology. Curr. Opin. Colloid Microgravity 6,1–7 (2020). Interface Sci. 19, 438–449 (2014). 83. Riley, F. P., McMackin, P. M., Lopez, J. M. & Hirsa, A. H. Flow in a ring-sheared 53. Gong, Z., You, R., Chang, R. C.-C. & Lin, Y. Viscoelastic response of neural cells drop: Drop deformation. Phys. Fluids 33,1–12 (2021). governed by the deposition of amyloid-β peptides (aβ). J. Appl. Phys. 119,1–7 84. Lopez, J. M. & Hirsa, A. H. Coupling of the interfacial and bulk flow in kinfe-edge (2016). viscometers. Phys. Fluids 27,1–13 (2015). 54. Mattana, S., Caponi, S., Tamagnini, F., Fioretto, D. & Palombo, F. Viscoelas- 85. A Pojman, J., Bessonov, N., Volpert, V. & Paley, M. S. Miscible fluids in micogravity ticity of amyloid plaques in transgenic mouse brain studied by brillouin (mfmg): A zero-upmass investigation on the international space station. microspectroscopy and correlative raman analysis. J. Innov. Opt. Health Sci. Microgravity Sci. Technol. XIX-1,33–41 (2007). 10,1–24 (2017). 86. Derkach, S. R., Kragel, J. & Miller, R. Methods of measuring rheological properties 55. Wang, R., Yang, X., Cui, L., Yin, H. & Xu, S. Gels of amyloid fibers. Biomolecules 9, of interfacial layers (experimental methods of 2d rheology). Colloid J. 71,1–17 1–12 (2019). (2009). 56. Woodard, D. et al. Gel formation in protein amyloid aggregation: A physical 87. Tamim, S. I. & Bostwick, J. B. Oscillations of a soft viscoelastic drop. npj Micro- mechanism for cytotoxicity. PLoS ONE 9,1–8 (2014). gravity 7,1–8 (2021). 57. Jean, L., Lee, C. F., Hodder, P., Hawkins, N. & Vaux, D. J. Dynamics of the for- 88. Guo, X., Chen, X., Zhou, W. & Wei, J. Effect of polymer drag reducer on rheo- mation of a hydrogel by a pathogenic amyloid peptide: Islet amyloid poly- logical properties of rocket kerosene solutions. Materials 15,1–14 (2022). peptide. Sci. Rep. 6,1–10 (2016). 89. Correia, A. C. M., Boue, G., Laskar, J. & Rodriguez, A. Deformation and tidal 58. Courchesne, N.-M. D., Duraj-Thatte, A., Tay, P. K. R., Nguyen, P. Q. & Joshi, N. S. evolution of close-in planets and satellites using a maxwell viscoelastic rheol- Scalable production of genetically engineered nanofibrous macroscopic mate- ogy. Astron. Astrophys. 571,1–16 (2014). rials via filtration. ACS Biomater. Sci. Eng. 3, 733–741 (2017). 90. Samuel, H., Lognonne, P., Panning, M. & Lainey, V. The rheology and thermal 59. Reynolds, N. P. Amyloid-like peptide nanofibrils as scaffolds for tissue engi- histroy of mars revealed by the orbital evolution of phobos. Nature 569, neering: Progress and challenges (review). Biointerphases 14,1–8 (2019). 523–527 (2019). 60. Hill, E. K., Krebs, B., Goodall, D. G., Howlett, G. J. & Dunstan, D. E. Shear flow 91. Suresh, R. & Simranjeet, S. Exoplanets and their structure, rheology and induces amyloid fibril formation. Biomacromolecules 7,10–13 (2006). dynamics. Int. Res. J. Eng. Technol. 7,44–49 (2020). 61. Dunstan, D. E., Hamilton-Brown, P., Asimakis, P., Ducker, W. & Bertolini, J. Shear 92. Trinh, E. H. & Depew, J. Solid surface wetting and the deployment of drops in flow promotes amyloid-β fibrilization. Protein Eng. Des. Sel. 22, 741–746 (2009). microgravity. Microgravity Sci. Technol. 7, 299–306 (1993). 62. Trumbore, C. N. Progress in Molecular Biology and Translational Science. (Elsevier 93. Brutin, D. et al. Sessile drop in microgravity: Creation, contact angle and inter- Inc, Amsterdam Netherlands, 2019). face. Microgravity Sci. Technol. 21, S67–S76 (2009). 63. Schladitz, C., Vieira, E. P., Hermel, H. & Mohwald, H. Amyloid-β-sheet formation at 94. Xu, S.-H., Wang, C.-X., Sun, Z.-W. & Hu, W.-R. The influence of contact line velocity the air-water interface. Biophys. J. 77, 3305–3310 (1999). and acceleration on the dynamic contact angle: An experimental study in 64. Jean, L., Lee, C. F. & Vaux, D. J. Enrichment of amyloidogenesis at an air-water microgravity. Int. J. Heat. Mass Trans. 54, 2222–2225 (2011). interface. Biophys. J. 102, 1154–1162 (2012). 95. Rizzardi, L. F. et al. Evaluation of techniques for performing cellular isolation and 65. Campioni, S. et al. The presence of an air-water interface affects formation and preservation during microgravity conditions. npj Microgravity 2,1–10 (2016). elongation of alpha-synuclein fibrils. J. Am. Chem. Soc. 136, 2866–2875 (2014). 96. Baba, P., Toth, A. & Horvath, D. Surface-tension-driven dynamic contact line in 66. Duerkop, M., Berger, E., Durauer, A. & Jungbauer, A. Impact of cavitation, high microgravity. Langmuir 35, 406–412 (2019). shear stress and air/liquid interfaces on protein aggregation. Biotechnol. J. 13, 97. Amberg, G. Detailed modelling of contact line motion in oscillatory wetting. npj 1–9 (2018). Microgravity 8,1–8 (2022). 67. Zhou, J. et al. Effects of sedimentation, microgravity, hydrodynamic mixing and 98. Ludwicki, J. M. et al. Is contact-line mobility a material parameter? npj Micro- air-water interface on α-synuclein amyloid formation. Chem. Sci. 11, 3687–3693 gravity 8,1–8 (2022). (2020). 99. Torres, L. J. & Weislogel, M. M. The ejection of large non-oscillating droplets from 68. Padayachee, E. R. et al. Cerebrospinal fluid-induced retardation of amyloid β a hydrophobic wedge in microgravity. npj Microgravity 7,1–10 (2021). aggregation correlates with alzheimer’s disease and the apoe ϵ4 allele. Brain Res 100. Weislogel, M. M. et al. How advances in low-g plumbing enable space 1651,11–16 (2016). exploration. npj Microgravity 8,1–11 (2022). Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 41 P. McMackin et al. 101. Reitz, B. et al. Additive manufacturing under lunar gravity and microgravity. 126. Edwards, D. A., Brenner, H. & Wasan, D. T. Interfacial Transport Processes and Microgravity Sci. Technol. 33,1–12 (2021). Rheology. (Butterwortj-Heinemann, MA, USA, 1991). 102. Dietrich, D. L. et al. Droplet combustion experiments aboard the international space station. Microgravity Sci. Technol. 26,65–76 (2014). 103. Meyer, F. et al. Oxygen droplet combustion in hydrogen under microgravity ACKNOWLEDGEMENTS conditions. Combust. Flame 241,1–11 (2022). The authors would like to thank Louise Littles, Sridhar Gorti, Hong Q. Yang, Kevin 104. Amidon, G. L., DeBrincat, G. A. & Najib, N. Effects of gravity on gastric emptying, Depew, Michael Hall, James McClellan, Heidi Parris, Shawn Reagan, Ryan Reeves, intestinal transit, and drug absorption. J. Clin. Pharmacol. 31, 968–973 (1991). Shawn Stephens, Paul Galloway, Ben Murphy, and Fran Chiramonte for their 105. Yang, J.-Q. et al. The effects of microgravity on the digestive system and the continued support of both the RSD project and the operations team at Rensselaer new insights it brings to the life sciences. Life Sci. Space Res 27,74–82 (2020). Polytechnic Institute. The authors also thank astronauts Raja Chari, Shane Kimbrough, 106. Bureau, L. et al. Blood flow and microgravity. C. R. Mecanique 345,78–85 (2017). Christina Koch, Akihiko Hoshide, Megan McArthur, Luca Parmitano, Thomas Pasquet, 107. Navasiolava, N. et al. Vacsular and microvascular dysfunction induced by and Mark Vande Hei for their excellence and flexibility during real-time space microgravity and its analogs in humans: Mechanisms and countermeasures. operations. The authors are also grateful for the support to this study given by NASA Front. Phys. 11, 420–428 (2020). BPS, NASA MSFC, NASA JSC, NSF-CASIS, and Teledyne-Brown Engineering. This work 108. Galvin, I., Drummond, G. B. & Nirmalan, M. Distribution of blood flow and ventilation was supported by NASA Grant 80NSSC20K1726 and NSF Grant 1929134. in the lung: gravity is not the only factor. Br. J. Anaesth. 98,420–428 (2007). 109. Prisk, G. K. Microgravity and the respiratory system. Eur. Respir. J. 43, 1459–1471 (2014). AUTHOR CONTRIBUTIONS 110. Janmaleki, M., Pachenari, M., Seyedpour, S. M., Shahghadami, R. & Sanati-Nez- P.M., J.A., and A.H. conceptualized and designed the experiment. J.A. prepared insulin had, A. Impact of simulated microgravity on ctoskeleton and viscoelastic solutions. P.M. and J.A. performed the remote ISS operations and experimental properties of endothelial cell. Sci. Rep. 6,1–11 (2016). measurements. P.M. performed the analysis of experimental measurements. S.G. 111. Bradbury, P. et al. Modeling microgravity at the cellular level: Implications for performed supporting computational fluid dynamics simulations. P.M. produced all human disease. Front. Cell Dev. Biol. 8,1–11 (2020). the figures and tables. P.M., J.A., and A.H. wrote the manuscript. P.M., J.A., S.G., and 112. Kennedy, M. J. & Volz, P. A. The effect of space flight irradiation on sacchar- A.H. critically evaluated data and results, including data interpretation, figure omyces cerevisiae growth and respiration. FEMS Microbiol. Lett. 19, 125–128 development, and manuscript editing. (1983). 113. Bouloc, P. & D’Ari, R. Escherichia coli metabolism in space. J. Gen. Microbiol. 137, 2839–2843 (1991). COMPETING INTERESTS 114. Kobayashi, Y., Kikuchi, M., Nagaoka, S. & Watanabe, H. Recovery of deinococcus radiodurans from radiation damage was enhanced under microgravity. Biol. Sci. The authors declare no competing interests. Space 10,97–101 (1996). 115. Walther, I., Bechler, B., Muller, O., Hunzinger, E. & Cogoli, A. Cultivation of sac- charomyces cerevisiae in a bioreactor in microgravity. J. Biotechnol. 47, 113–127 ADDITIONAL INFORMATION (1996). Supplementary information The online version contains supplementary material 116. Saffary, R. et al. Microbial survival of space vacuum and extreme ultraviolet available at https://doi.org/10.1038/s41526-022-00227-2. irradiation: strain isolation and analysis during a rocket flight. FEMS Microbiol. Lett. 215, 163–168 (2002). Correspondence and requests for materials should be addressed to Amir Hirsa. 117. Horneck, G., Klaus, D. M. & Mancinelli, R. L. Space microbiology. Microbiol. Mol. Biol. Rev. 74, 121–156 (2010). Reprints and permission information is available at http://www.nature.com/ 118. Nislow, C. et al. Genes required for survival in microgravity revealed by genome- reprints wide yeast deletion collections cultured during spaceflight. BioMed. Res. Intern. 10,1–10 (2015). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims 119. Ott, E. et al. Molecular repertoire of deinococcus radiodurans after 1 year of in published maps and institutional affiliations. exposure outside the international space station within the tanpopo mission. Microbiome 8,1–16 (2020). 120. Padgen, M. R. et al. Ecamsat spaceflight measurements of the role of σsin antibiotic resistance of stationary phase escherichia coli in microgravity. Life Sci. Open Access This article is licensed under a Creative Commons Space Res 24,18–24 (2020). Attribution 4.0 International License, which permits use, sharing, 121. McMackin, P. M. et al. Effects of microorganisms on drop formation in micro- adaptation, distribution and reproduction in any medium or format, as long as you give gravity during a parabolic flight with residual gravity and jitter. Microgravity Sci. appropriate credit to the original author(s) and the source, provide a link to the Creative Technol. 34,1–9 (2022). Commons license, and indicate if changes were made. The images or other third party 122. Walther, I. Space bioreactors and their applications. Adv. Space Biol. Med. 8, material in this article are included in the article’s Creative Commons license, unless 197–213 (2002). indicated otherwise in a credit line to the material. If material is not included in the 123. Menezes, A. A., Cumbers, J., Hogan, J. A. & Arkin, A. P. Towards synthetic bio- article’s Creative Commons license and your intended use is not permitted by statutory logical approaches to resource utilization on space missions. J. R. Soc. Interface regulation or exceeds the permitted use, you will need to obtain permission directly 12,1–20 (2015). from the copyright holder. To view a copy of this license, visit http:// 124. Painter, N. A. & Sisson, E. An overview of concentrated insulin products. Diabetes creativecommons.org/licenses/by/4.0/. Spectr. 29, 136–140 (2016). 125. Knopp, J. L., Holder-Pearson, L. & Chase, J. G. Insulin units and conversion factors: A story of truth, boots, and faster half-truths. J. Diabetes Tech. Soc. 13, © The Author(s) 2022 597–600 (2019). npj Microgravity (2022) 41 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png npj Microgravity Springer Journals

Amyloidogenesis via interfacial shear in a containerless biochemical reactor aboard the International Space Station

Loading next page...
 
/lp/springer-journals/amyloidogenesis-via-interfacial-shear-in-a-containerless-biochemical-L7XNG0HAdt

References (141)

Publisher
Springer Journals
Copyright
Copyright © The Author(s) 2022
eISSN
2373-8065
DOI
10.1038/s41526-022-00227-2
Publisher site
See Article on Publisher Site

Abstract

www.nature.com/npjmgrav ARTICLE OPEN Amyloidogenesis via interfacial shear in a containerless biochemical reactor aboard the International Space Station 1 1,2,3 1,3 1,3✉ Patrick McMackin , Joe Adam , Shannon Griffin and Amir Hirsa Fluid interfaces significantly influence the dynamics of protein solutions, effects that can be isolated by performing experiments in microgravity, greatly reducing the amount of solid boundaries present, allowing air-liquid interfaces to become dominant. This investigation examined the effects of protein concentration on interfacial shear-induced fibrillization of insulin in microgravity within a containerless biochemical reactor, the ring-sheared drop (RSD), aboard the international space station (ISS). Human insulin was used as a model amyloidogenic protein for studying protein kinetics with applications to in situ pharmaceutical production, tissue engineering, and diseases such as Alzheimer’s, Parkinson’s, infectious prions, and type 2 diabetes. Experiments investigated three main stages of amyloidogenesis: nucleation studied by seeding native solutions with fibril aggregates, fibrillization quantified using intrinsic fibrillization rate after fitting measured solution intensity to a sigmoidal function, and gelation observed by detection of solidification fronts. Results demonstrated that in surface-dominated amyloidogenic protein solutions: seeding with fibrils induces fibrillization of native protein, intrinsic fibrillization rate is independent of concentration, and that there is a minimum fibril concentration for gelation with gelation rate and rapidity of onset increasing monotonically with increasing protein concentration. These findings matched well with results of previous studies within ground-based analogs. npj Microgravity (2022) 8:41 ; https://doi.org/10.1038/s41526-022-00227-2 INTRODUCTION other amyloidogenic proteins besides insulin exist , some of 35–37 which are functional amyloids which support natural biolo- Protein biology in spaceflight is a field of research that has been gical functions while others are related to disease, such as the beta expanding along with the advancement of space exploration and 38–41 42,43 1–5 amyloid and tau proteins of Alzheimer’s disease, alpha- human habitation in altered gravity . Studying the biophysical 44,45 46,47 synuclein of Parkinson’s disease, infectious prion proteins, and fluid dynamic behavior of liquid protein solutions in 48–50 and the islet protein involved in type 2 diabetes. Compara- microgravity can provide insight into fundamental physical tively, insulin is a relatively safe model amyloidogenic protein for phenomena in space, as well as within biochemical systems on space studies, as dangerous proteins such as infectious prions are Earth. In space, the absence of gravity increases the prominence of not allowed on the ISS due to safety regulations . Overall, insulin the air-liquid interface, material properties such as surface tension, is a multifaceted model for studying protein interfacial rheology surface viscosities, and molecular adsorption becoming even more 1,2 and kinetics with relevance to biophysics, fluid physics, medicine, impactful to the behavior of a liquid system . On Earth many and spaceflight. biochemical systems exist where fluid interfaces have major The process of amyloidogenesis applicable to human insulin effects of key importance, including environmental surfactant 6 7 and other amyloidogenic proteins, progresses in three key layers , industrial bioprocessing , and physiological tissue sur- biophysical stages: nucleation, fibrillization, and gela- faces within the body. Such systems with fluid interfaces, both in 10,29,32 tion . Nucleation is the joining of two monomers, a space and on Earth, can exhibit unique alterations in fluid and 9 10 molecular association that often changes secondary and protein behavior due to protein adsorption , fibrillization , 11 12 tertiary protein structure, to form a pre-fibril aggregate, or biopolymer dynamics , and gelation , all dependent upon the nucleate, with quaternary structure that can accept additional fluid system’s geometry and the specific type of proteins present. monomeric subunits, seeding the system with starting points Human insulin was selected as the model protein for this study 10,34 for fibrillization . Fibrillization, or more specifically elonga- based on two main points of significance: relevance to studies in tion, is the addition of a monomer to an existing fibril, a microgravity and relevance to protein biophysics with interfacial polymerization process which lengthens protein polymers from hydrodynamics. Insulin’s relevance to spaceflight originates from pre-fibril aggregates, to fibrils, to fibers, with the distribution of the protein’s history of kinetics and crystallization in micrograv- 10,12 1,13–15 ity , medical applications to diabetogenic effects in human fibril size changing as the process progresses .Gelationis 2,5,16–18 spaceflight , and application as a model pharmaceuti- the linking of protein fibers to form a polymer network, a 7,19,20 cal for studies of protein stability and in situ resource structure filled with solvent which maintains a defined shape, a utilization in spaceflight. From a biophysics and fluid physics process that can occur concurrently with fibrillization if 21–23 12,29,32 perspective, insulin displays rich bulk hydrodynamic , inter- sufficiently large fibril sizes are present .These defining 24–28 29–33 facial , and protein kinetics behavior. Moreover, insulin is protein kinetic processes of amyloidogenesis are governed by an amyloidogenic protein that can undergo a fibrillization process, asystem’s dynamic microenvironment, and lead to changes in 32,52–55 termed amyloidogenesis, to produce amyloid fibrils which possess protein mechanical properties ,cytotoxic effectsin 10,32,34 39,40,42,45,46,48–50,55–57 a durable beta-cross quaternary protein structure . Many amyloid diseases ,and formationof 1 2 Mechanical, Aerospace, and Nuclear Engineering, Rensselaer Polytechnic Institute, 110 8th St, Troy 12180 NY, USA. Department of Biological Sciences, Rensselaer Polytechnic Institute, 110 8th St, Troy 12180 NY, USA. Chemical and Biological Engineering, Rensselaer Polytechnic Institute, 110 8th St, Troy 12180 NY, USA. email: hirsaa@rpi.edu Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; P. McMackin et al. Fig. 1 The ring-sheared drop (RSD) with the bottom ring rotating. a Image of the ring-sheared drop aboard the ISS showing a pre-sheared 8 mg/mL human insulin solution spinning at 30 rpm. b Axisymmetric computation of primary (color map of normalized azimuthal velocity, ν) and secondary (black arrowed streamlines in the azimuthal plane) flow of a Newtonian fluid in the RSD sheared at 30 rpm. 58,59 intricate nanostructure , potentially adaptable to tissue engineering. The chemical and thermodynamic state of a system governs protein kinetics, biophysical processes of the constituent mole- 10,12 cules progressing toward a point of lower free energy . Fluid transport and associated hydrodynamic stresses influence the thermodynamic state of amyloid systems, both bulk shear 21–23,60–62 24–28,63–67 flow and air-liquid interface activity affecting the number of molecular collisions, with more frequent collisions increasing the probability for interactions including nucleation, fibrillization, and gelation. Geometries with fluid interfaces are well-suited to the study of physiological systems as most interfaces within the body are fluidic in nature, flow of cerebrospinal fluid (CSF) within the brain being of specific 8,62,68–72 importance for many neurodegenerative diseases . Fig. 2 Intensity of scattered light verses time for pre-sheared Furthermore, brain structure and CSF have been observed to insulin cases. ± 1 standard deviation error bars represent measure- 73–78 ment uncertainty and dashed lines represent sigmoidal fits to a undergo alterations due to spaceflight , making the study of theoretical fibrillization function (Eq. 1) presented in the Methods such systems imperative to long-term space habitation. Along section. with these fluid effects, protein kinetics are also affected by the quantity of protein present, which can alter molecular dynamics RESULTS and the overall interactions with bulk fluid and fluid interfaces. Pre-sheared fibrillization Microgravity provides a unique environment for the experi- Three pre-sheared trials were performed in this investigation at mental study of systems with solely fluid interfaces, the total protein concentrations of 2, 4, and 8 mg/mL, each subject to dominance of free surfaces in the absence of gravity facilitating steady interfacial shear at 30 rpm for 3.5 days. Image data was the removal of solid boundaries which often introduce nucleation captured every 0.5 days for characterization of fibrillization sites and unintended wall effects. The ring-sheared drop (RSD) is a kinetics. Measured intensity of image data was used to construct containerless surface tension-contained microgravity biochemical fibrillization curves of intensity versus time, as depicted in Fig. 2. reactor consisting of a 2.54 cm diameter drop pinned between Measured intensity increased monotonically with time for all two rings, one stationary and one shearing, that transfer interfacial samples, indicative of the presence of fibrils with larger shear to the bulk fluid by surface shear viscosity and mix the liquid aggregates producing increased scattering and higher image 79–83 using inertial flow with secondary motion (Fig. 1) . The RSD intensity. Curves in Fig. 2 represent nonlinear least-square fits to a was first deployed to the international space station (ISS) in the theoretical sigmoidal fibrillization function (Eq. 1) that align well Fall of 2019 and again for a second operations campaign in the with experimentally measured data. Summer and Fall of 2021. A ground-based preliminary study has been performed with human insulin in the Earth analog of the Pre-sheared fibrillization kinetics RSD, the knife-edge viscometer (KEV) , an apparatus which Curves in Fig. 2 were quantified by fitting measured data to a requires a glass dish for containment under gravity yet, like the theoretical fibrillization function with empirical parameters of RSD, produces shear flow using surface shear viscosity and mixes biophysical relevance (Eq. 1). Importantly, this equation does not via secondary flow. The present investigation examined human directly model fibrillization when applied to intensity data (Fig. 2), insulin within the RSD aboard the ISS to measure the effects of as this theoretical function typically applies to fibril content steady interfacial shear on the amyloidogenesis processes of measured using spectroscopy as opposed to solution intensity. nucleation, fibrillization, and gelation in a microgravity, air-liquid The first adjustment required for intensity fitting was a horizontal interface dominated system. Specifically, the hypothesis tested offset to account for pre-shearing of these fibrillization trials, as was that if protein solutions in microgravity are seeded with fibrils these trials did not begin as completely native solutions. The and subject to steady interfacial shear, then amyloidogenesis will second adjustment was that the fitting parameter typically occur with the extent of gelation depending on total protein describing total protein concentration, I , instead described the concentration. intensity of a fully monomeric protein solution. This value was npj Microgravity (2022) 41 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; P. McMackin et al. have drawn particles into areas of the flow occluded from the Table 1. Sigmoidal fit (Eq. 1) parameters of native intensity (I ) and camera’s field of view. normalized fit root-means-square (rms) error. C (mg/mL) I rms Error (%) Pre-sheared gelation 0 0 Protein gelation was observed by the presence of a turbid 4 0.49 ± 0.03 1.3 stationary solidification front moving southward from the 2 0.33 ± 0.02 2.1 stationary ring. Figure 6 depicts the progression of this gelation 8 0.88 ± 0.02 2.7 front with time. The 2 mg/mL sample did not reach a sufficiently high fibril concentration in order to produce gelation. This requirement of a minimum fibril concentration for gelation is 28,29 consistent with previous ground-based studies . Figure 7 quantifies the progression of these gelation fronts with time in terms of a gelation front polar angle, θ . Both the rate of progression and rapidity of onset of gelation increase with increasing protein concentration. To further this observation, the 8 mg/mL case had transitioned entirely to a linked polymer gel by the trial’s conclusion, preventing liquid extraction and remaining affixed to the rings even after test cell removal. DISCUSSION Fluid interfaces produce significant effects on the biophysics of protein solutions, defining the microenvironment and energetic 10,12 9 landscape through processes such as molecular adsorption and imparted forces such as interfacial shear affecting biological 10,11 12 behaviors including the fibril dynamics and gelation of Fig. 3 Intrinsic fibrillization rate k versus protein concentration proteins. This investigation was the space continuation of an C . Values of k were determined by fitting to a fibrillization model Earth-based study , this work studying amyloidogenesis, produc- (Eq. 1). Error bars represent fit uncertainty. tion of amyloid fibrils and plaques from native amyloidogenic proteins, of human insulin in an air-liquid interface dominated used to improve fits by providing a measureable vertical offset biochemical reactor in microgravity. This fluid system, the RSD due to background monomer intensity. Fit rms error (Table 1 aboard the ISS, offered a platform for studying both the effects of column 3) was < 3% for all cases. Larger fit errors (8 mg/mL) fluid interfaces and microgravity on protein fibrillization. Amyloi- resulted from measurement accuracy reduction due to high dogenesis of human insulin was used as a model biophysical sample intensity, which can slightly effect curve shape due to system due to its applicability to biotechnology, physiology, near-upper limit sensor values. The altered application of this medicine, and spaceflight. equation to optical intensity as opposed to spectroscopic fibril Three stages of amyloidogenesis were quantified in this study, content remained suitable for the study of fibrillization kinetics including seeding, fibrillization, and gelation of insulin. A native with a focus on intrinsic fibrillization rate. The fit-determined solution of insulin serendipitously seeded with insulin fibrils native intensity values, I , are displayed in Table 1. Furthermore, (Fig. 4) displayed an earlier onset of fibrillization (Fig. 5). This sigmoidal fits allowed for relation between intrinsic fibrillization accelerated onset of fibrillization due to seeding is applicable to rate and total protein concentration (Fig. 3). Intrinsic rate was 46,47 diseases such as infectious prions or biotechnological shown to be independent of concentration and thus, the time processes that introduce a portion of fibrils to a native solution scale of fibrillization not dependant on protein concentration, a and in turn promote fibrillization of the native solution. Fibrilliza- result consistent with the ground study . tion experiments with pre-sheared insulin samples (Fig. 2) displayed the induction of amyloidogenesis via steady axisym- Seeded fibrillization metric interfacial shear, with intrinsic fibrillization rates (Fig. 3) The seeding trial occurred during measurement of a native (fully independent of total protein concentration. This concentration monomeric) 2 mg/mL sample. After 5.6 days an additional independence matches results of the ground study and injection of 0.4 mL of fluid from the deployment tube was made indicates that forces imposed at the fluid boundary lead to to account for evaporative losses and return the drop to a changes in the protein microenvironment that govern the spherical shape. Serendipitously, this injection provided a means timescale of fibrillization. The average value of this rate constant, to measure the effect of seeding with fibril aggregates. The inside 0.43 ± 0.07 1/days, matches the expected value (0.47 1/days) of the deployment tube, where remnants of the injection volume obtained by a logarithmic extrapolation using Re of fibrillization had resided for 5.5 days, was a rough unfinished stainless steel rates from Fig. 4a of the ground study . Gelation was observed surface that provided ample nucleation sites for fibrils to form, (Fig. 6) in the form of gelation fronts, only in cases with sufficiently which were subsequently transported into the bulk of the RSD high protein fibril concentration to from crosslinked polymer during the seeding injection. The turbidity of this fibril seeding networks, and the rapidity of gelation onset and rate of gelation injection was readily observable, and upon shear restart, the (Fig. 7) increased monotonically with increasing protein resulting mixing within the drop allowed for visualization of the concentration. RSD’s secondary inertial flow as displayed in Fig. 4 (see also This study marks the first successful use of the RSD fluid Supplement1.mp4). Following the initial increase in intensity due apparatus (Fig. 1) with protein solutions in microgravity aboard to added fibril content, intensity continued to increase, as the the ISS. Hardware and biological samples were transported to the solution had begun fibrillizing due to the previous seeding event ISS and installed in the MSG without fault. During operation, this (Fig. 5). The slight decrease in measured intensity observable at device successfully deployed, pinned, steadily sheared, and 9.0 and 9.5 days was unexpected and may be due to fibril extracted fluid drops of protein solutions, demonstrating perfor- adsorption to liquid-solid interfaces of the rings , which could mance of a novel method for producing air-liquid interface Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 41 P. McMackin et al. Fig. 4 Images of the native 2 mg/mL insulin case being seeded at 5.6 days with 0.4 mL of insulin fibrils. Panels show the injected volume (a) before and (b, c) after restarting ring rotation. Mixing of the turbid fibrillized volume allowed for visualization of the RSD's secondary flow (Fig. 1b and Supplement1.mp4). effects of flow in physiological systems such as the gastrointest- 104,105 73–78 106,107 108,109 inal , glymphatic , circulatory , or respiratory systems and the effects of spaceflight on these systems and the 110,111 cells within. The behavior of microbial biofluids is important for understanding the effects of spaceflight on microbiol- 112–121 ogy and potential applications to pharmaceutical produc- 4,117,122,123 tion and bio-engineering in support of space exploration. Future investigations of interfacial hydrodynamics in microgravity could offer insight into fluid systems that better facilitate spaceflight. METHODS RSD The RSD consists of a 2.54 cm diameter spherical liquid drop Fig. 5 Intensity of scattered light versus time for the 2 mg/mL pinned between two thin titanium contact rings (Fig. 1). The top native insulin case with seeding by insulin fibrils at 5.6 days. ring is connected via four prongs to a 10-gauge stainless-steel ±1 standard deviation error bars represent measurement deployment tube used to grow the liquid drop. The lower ring uncertainty. rotates to produce interfacial shearing of the drop, shear being transmitted to the bulk by means of surface shear viscosity and dominated systems in microgravity. Optics, deployment, shearing, 79–83 mixing occurring due to secondary flow . The RSD was and complete operation of the apparatus were successfully conceived in 2013, and the hardware was developed and performed using real-time remote control from the ground. With launched to the ISS on SpaceX CRS-18 July 2019 after a series of minimal impact of solid boundaries, sole components being the parabolic flights. Following these engineering missions, the thin contact rings used to transmit interfacial shear, this device is science mission (including biological samples and hardware well-suited to the study of interfacial phenomena and the modifications presented in this study) was launched on Cygnus dynamics of fluid interfaces. NG-16 in August 2021, operations being performed in the Results of this investigation center on the three main aspects of following months concluding in December 2021. The RSD amyloidogenesis: nucleation, fibrillization, and gelation. Seeding hardware was operated within the Microgravity Science Glovebox of protein solutions in microgravity was shown to promote earlier (MSG), located in the Destiny module of the ISS, providing 3 levels onset of fibrillization by bolstering the nucleation process. of containment (test cell, MSG airflow, and MSG wall) between the Fibrillization was demonstrated to be promoted by interfacial astronauts and protein samples. Due to the low 1.6 pH, samples shear, with the intrinsic rate of fibrillization being independent of were classified as a hazard response level 2 material (HRL), which protein concentration. Gelation was found to require a critical necessitated at least 3 levels of containment in accordance to concentration of protein fibrils with gelation onset and rate NASA crew-safety requirements . becoming more rapid with increasing protein concentration. Furthermore, findings presented here demonstrate the successful Protein samples performance of the microgravity biochemical reactor, the RSD, Protein samples of human insulin were prepared by dissolving utilized in this investigation. A multitude of future space lyophilized recombinant human insulin (Sigma-Aldrich, 91077C) in investigations exist that could make use of an interfacial a 0.1 M NaCl 1.6 pH buffer solution (deionized water, pH cycled biochemical reactor such as the RSD, including studies on drop 124,125 with HCL and NaOH) to pharmaceutical-relevant concentra- rheology, interface creation and substrate interaction, different tions of 2, 4 and 8 mg/mL. The low pH of the buffer allowed for interfacial flow regimes, and microbial biofluids. Drop rheology in contamination resistance in addition to pH control. Each sample space is applicable to fields ranging from fundamental changes in 85–88 89–91 was pre-sheared for 1 day at a Reynolds number of 6000 in a fluid behavior to the study of planetary bodies and their 26,126 deep-channel surface viscometer which produced partial material properties. Studies of interface creation and substrate fibrillization that allowed for earlier onset of fibrillization during interaction can be used to describe fundamental contact line 92–100 87,101 102,103 dynamics , 3D printing , and combustion in micro- operations in space. Additionally, a native (fully un-fibrillized gravity. Use of select interfacial flow regimes such as steady, monomeric, or dimeric at 1.6 pH) 2 mg/mL sample was also pulsatile, or oscillatory flows, allows fluid devices to mimic the prepared, ultimately used in a serendipitous seeding trial. After npj Microgravity (2022) 41 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA P. McMackin et al. Fig. 6 Images of gelation front progression in pre-sheared insulin cases. If present, southward-moving gelation fronts are indicated by yellow dashed lines. Experimental trials Experimental trials began after crew members installed the 12 mL sample syringe and test cell (test cells containing the RSD’s pinning rings and deployment tube) into the experimental hardware and sealed the MSG. Drop deployment followed installation by 0.5 days to dissipate any static charge accumulated during installation and to place deployment during crew sleep, avoiding deleterious accelerations. Drops were deployed at a rate of 10 mL/min in two stages to the total volume of 8.58 mL (volume of a 2.54 cm diameter sphere). Steady shear commenced after deployment with the lower ring rotating at 30 rpm, corresponding to a Reynolds number of 180 (where Re = Ωa /ν, where Ω is ring rotation rate, a is ring radius, and ν is the kinematic viscosity of water). Wide-field image data (example in Fig. 1a) of steadily- Fig. 7 Gelation front polar position, θ , vs time for pre-sheared sheared drops was collected every 0.5 days, with pre-sheared trials insulin cases. This data was extracted using the videos from which shearing for 3.5 days and the native trial shearing for 9.5 days with Fig. 6 was generated. a midpoint seeding injection of 0.4 mL of insulin fibrils after 5.6 days. LED light modules used for illumination were deactivated outside of sampling to maintain ambient conditions within the test cell. preparation, all samples were degassed under a 710 mmHg vacuum for 0.5 days to minimize air bubbles and subsequently frozen at −20 C before transportation and launch to the ISS. On Protein amyloidogenesis orbit, sample syringes were thawed 1 day at ambient temperature Protein amyloidogenesis was quantified using the measured before installation in the RSD hardware. After each experimental intensity of light from a drop’s bulk fluid in both the native trial, each protein solution was withdrawn back from the drop into seeding trial and the pre-sheared fibrillization trials. As fibrillization the syringe and placed in cold stowage (4 C) until returning to proceeded the size of particles within a solution increased leading Earth. Each sample syringe had two controls, a flight control which to increased scattering of light and visible increases in turbidity. accompanied samples to the ISS and a ground control that While such optical methods of measurement lag behind spectro- remained on Earth. Both controls underwent no shear experi- scopic measurements of monomer extinction (as sufficiently mentation and were identically degassed, frozen, thawed and small fibrils and pre-fibril aggregates will not scatter light), trends subsequently refrigerated. in fibrillization remain observable. Measured intensity was defined Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 41 P. McMackin et al. as the average of normalized red, green, and blue intensity values 6. Jenkinson, I. R., Seuront, L., Ding, H. & Elias, F. Biological modification of mechanical properties of the sea surface microlayer, influencing waves, ripples, within an interrogation area centered on the drop, calculated foam and air-sea fluxes. Elem. Sci. Anth. 6,1–32 (2018). using a MATLAB script summing over the 120 frames (30 fps) 7. Li, J. et al. Interfacial stress in the development of biologics: Fundamental recorded at each time point. Measurement uncertainty was understanding, current practice, and future perspective. AAPS J. 21,1–17 (2019). defined as the standard deviation of these intensity values within 8. Thomas, J. H. Fluid dynamics of cerebrospinal cluid flow in perivascular spaces. J. the interrogation window over each of the 120 frames of the video R. Soc. Interface 16,1–11 (2019). camera. This average intensity was then normalized to produce 9. Adamson, A. W. & Gast, A. P. Physical Chemistry of Surfaces. 6th edn. (John Wiley the measured intensity, I, which ranged between 0 and 1 and Sons Inc, NY, USA, 1997). corresponding to no detected light and a fully saturated camera 10. Otzen, D. E. Amyloid Fibrils ad Prefibrillar Aggregates. (Wiley-VCH, Weinheim, sensor. Furthermore, optical measurement was also utilized for the Germany, 2013). quantification of the final stage of amyloidogenesis, protein 11. Morrison, F. A. Understanding Rheology. (Oxford University Press, NY, USA, 2001). 12. Doi, M. Soft Matter Physics. (Oxford University Press, Oxford, United Kingdom, gelation. As a protein solution transitioned from a liquid 2013). suspension of fibers to a linked network of fibers, a noticeable 13. Snell, E. H. & Helliwell, J. R. Macromolecular crystallization in microgravity. Rep. change occurred in the optical properties of regions undergoing Prog. Phys. 68, 799–853 (2005). this phase transition. Differences in turbidity, highly turbid 14. Timofeev, V. I. et al. X-ray investigation of gene-engineered human insulin unmoving regions indicative of gel, allowed for the measurement crystallized from a solution containing polysialic acid. Acta Cryst. 66, 259–263 of gelation progression using optical tracking of gelation fronts. (2010). 15. Snell, E. H. & Helliwell, J. R. Microgravity as an environment for macromolecular crystallization - an outlook in the era of space stations and commercial space Model fit flight. Crystallogr. Rev. 10, 1080 (2021). A three-parameter sigmoidal function was utilized to obtain I (t), 16. Tobin, B. W., Uchakin, P. N. & Leeper-Woodford, S. K. Insulin secretion and representing a solution’s intensity based on a specific fibril sensitivity in space flight: Diabetogenic effects. Nutrition 18, 842–848 (2002). content as a function of time: 17. Bergouignan, A. et al. Towards human exploration of space: The theseus review series on nutrition and metabolism research priorities. npj Microgravity 2,1–8 1 1 (2016). I ðtÞ¼ I  : (1) f 0 kðt tÞ kt h h 1 þ e 1 þ e 18. Hughson, R. L. et al. Increased postflight carotid artery stiffness and inflight insulin resistance resulting from 6-mo spaceflight in male and female astro- This three-parameter function originated from protein fibrillization nauts. Am. J. Physiol. Heart Circ. Physiol. 310, 628–638 (2016). theory and contains constants that are of relevance to biophysical 19. D’Souza, A., Theis, J. D., Vrana, J. A. & Dogan, A. Pharmaceutical amyloidosis 10,25,29 properties . I is the initial intensity describing a fully monomeric associated with subcutaneous insulin and enfuvirtide administration. Amyloid 21,71–75 (2014). protein solution, t is the time (days) required to reach a half fibrillized 20. Zapadka, K. L., Becher, F. J., dos Santos, A. L. G. & Jackson, S. E. Factors affecting solution, and k is the intrinsic rate coefficient(1/days)thatdepends on the physical stability (aggregation) of peptide thereputics. Interface Focus 7, both the processes of nucleation and fibril elongation. Measured 1–18 (2017). intensity values of the pre-sheared trials were fittoEq. (1)using a 21. Szymczak, P. & Cieplak, M. Hydrodynamic effects in proteins. J. Phys. Condens. nonlinear least-squares MATLAB algorithm to obtain these biophysical Matter 23,1–14 (2010). parameters as functions of protein concentration. 22. Bekard, I. B., Asimakis, P., Bertolini, J. & Dunstan, D. E. The effects of shear flow on protein structure and function. Biopolymers 95, 733–745 (2011). 23. McBride, S. A., Tilger, C. F., Sanford, S. P., Tessier, P. M. & Hirsa, A. H. Comparison Reporting summary of human and bovine insulin amyloidogenesis under uniform shear. J. Phys. Further information on research design is available in the Nature Chem. B 119, 10426–10433 (2015). Research Reporting Summary linked to this article. 24. Pandey, L. M. et al. Surface chemistry at the nanometer scale influences insulin aggregation. Colloids Surf. B 100,69–76 (2012). 25. McBride, S. A., Sanford, S. P., Lopez, J. M. & Hirsa, A. H. Shear-induced amyloid DATA AVAILABILITY fibrillization: The role of inertia. Soft Matter 12, 3461–3467 (2016). 26. Balaraj, V. S. et al. Surface shear viscosity as a macroscopic probe of amyloid The data collected during this study is available from the corresponding authors fibril formation at a fluid interface. Soft Matter 13, 1780–1787 (2017). upon reasonable request. 27. Grigolato, F. & Arosio, P. The role of surfaces on amyloid formation. Biophys. Chem. 270, 106533–106546 (2021). 28. Adam, J. A., Middlestead, H. R., Debono, N. E. & Hirsa, A. H. Effects of shear rate CODE AVAILABILITY and protein concentration on amyloidogenesis via interfacial shear. J. Phys. The code written during this study is available from the corresponding authors upon Chem. B 125, 10355–10363 (2021). reasonable request. 29. Nielsen, L. et al. Affect of environmental factors on the kinetics of insulin fibril formation: Elucidation of the molecular mechanism. Biochemistry 40, 6036–6046 Received: 23 May 2022; Accepted: 23 August 2022; (2001). 30. Krebs, M. R. H. et al. The formation of spherulites by amyloid fibrils of bovine insulin. Proc. Natl Acad. Sci. Usa. 101, 14420–14424 (2004). 31. Pasternack, R. F. et al. Formation kinetics of insulin-based amyloid gels and the effect of added metalloporphyrins. Biophysical J. 90, 1033–1042 (2006). 32. Schleeger, M. et al. Amyloids: From molecular structure to mechanical proper- REFERENCES ties. Polymer 54, 2473–2488 (2013). 1. Clement, G. & Slenzka, K. Fundamentals of Space Biology. (Springer Science 33. Surmacz-Chwedoruk, W., Babenko, V., Dec, R., Szymczak, P. & Dzwolak, W. The +Business Media LLC, NY, USA, 2006). emergence of superstructural order in insulin amyloid fibrils upon multiple 2. Council, N. R. Recapturing a Future for Space Exploration: Life and Physical Sci- rounds of self-seeding. Sci. Rep. 6,1–12 (2016). ences Research for a New Era. (The National Academic Press, Washington DC, 34. Iadanza, M. G. et al. A new era forunderstanding amyloid structures and disease. USA, 2011). Nat. Rev. Mol. Cell Biol. 19, 755–773 (2018). 3. Barzegari, A. & Saei, A. A. An update to space biomedical research: Tissue 35. Fowler, D. M., Koulov, A. V., Balch, W. E. & Kelly, J. W. Functional amyloid - from engineering in microgravity bioreactors. BioImpacts 2,23–32 (2012). bacteria to humans. Trends Biochem. Science 32, 217–224 (2007). 4. Blue, R. S. et al. Supplying a pharmacy for nasa exploration spaceflight: Chal- 36. Otzen, D. & Riek, R. Functional amyloids. Cold Spring Harb. Perspect. Biol. 11, lenges and current understanding. npj Microgravity 5,1–12 (2019). 1–29 (2019). 5. Smith, S. M., Zwart, S. R., Douglas, G. L. & Heer, M. Human Adaptation to 37. Balistreri, A., Goetzler, E. & Chapman, M. Functional amyloids are the rule rather Spaceflight: The Role of Food and Nutrition. 2nd edn (National Aeronautics and than the exception in cellular biology. Microorganisms 8,1–13 (2020). Space Administration, TX, USA, 2021). npj Microgravity (2022) 41 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA P. McMackin et al. 38. Hortschansky, P., Schroeckh, V., Christopeit, T., Zandomeneghi, G. & Fandrich, M. 69. Iliff, J. J. et al. The emerging relationship between interstitial fluid-cerebrospinal The aggregation kinetics of alzheimer’s β-amyloid peptide is controlled by fluid exchange, amyloid β and sleep. Biol. Psychiatry 83, 328–336 (2018). stochastic nucleation. Protein Sci. 14, 1753–1759 (2005). 70. Rasmussen, M. K., Mestre, H. & Nedergaard, M. The glymphatic pathway in 39. Murphy, M. P. & LeVine(III), H. Alzheimer’s disease and the β-amyloid peptide. J. neurological disorders. Lancet Neural 17, 1016–1024 (2018). Alzheimers Dis. 19, 311–323 (2010). 71. Frankel, R. et al. Autocatalytic amplification of alzheimer-associated aβ42 pep- 40. Chen, G. et al. Amyloid beta: Structure, biology and structure-based therapeutic tide aggregation in human cerebrospinal fluid. Commun. Biol. 2,1–11 (2019). development. Acta Pharmacologica Sin. 38, 1205–1235 (2017). fang. 72. Kylkilahti, T. M. et al. Achieving brain clearance and preventing neurodegen- 41. Yagi-Utsumi, M. et al. Characterization of amyloid β fibril formation under erative diseases - a glymphatic perspective. J. Cereb. Blood Flow. Metab. 0,1–13 microgravity conditions. npj Microgravity 6,1–6 (2020). (2021). 42. Nizynski, B., Dzwolak, W. & Nieznanski, K. Amyloidogenesis of tau protein. Pro- 73. Roberts, D. R. et al. Effects of spaceflight on astronaut brain structure as indi- tein Sci. 26, 2126–2150 (2017). cated on mri. N. Engl. J. Med. 377, 1746–1753 (2017). 43. Joie, R. L. et al. Rospective longitudinal atrophy in alzheimer’s disease correlates 74. Zhang, L.-F. & Hargens, A. R. Spaceflight-induced intracranial hypertension and with the intensity and topography of baseline tau-pet. Sci. Transl. Med. 12,1–13 visual impairment: Pathophysiology and countermeasures. Physiol. Rev. 98, (2020). 59–87 (2018). 44. Araki, K. et al. Parkinson’s disease is a type of amyloidosis featuring accumula- 75. Kramer, L. A. et al. Intracranial effects of microgravity: A prospective longitudinal tion of amyloid fibrils of α-synuclein. Proc. Natl Acad. Sci. U. S. A. 116, mri study. Radiology 295, 640–648 (2020). 17963–17969 (2019). 76. ichi Iwasaki, K. et al. Long-duration spaceflight alters estimated intracranial 45. de Oliveira, G. A. P. & Silva, J. L. Alpha-synuclein stepwise aggregation reveals pressure and cerebral blood velocity. J. Physiol. 599, 1067–1081 (2021). features of an early onset mutation in parkinson’s disease. Commun. Biol. 2,1–13 77. Roy-O’Reilly, M., Mulavara, A. & Williams, T. A review of alterations to the brain (2019). during spaceflight and the potential relevance to crew in long-duration space 46. Ghetti, B. et al. Prion protein amyloidosis. Brain Pathol. 6, 127–145 (1996). exploration. npj Microgravity 7,1–9 (2021). 47. Choi, J.-K. et al. Amyloid fibrils from the n-terminal prion protein fragment are 78. Barisano, G. et al. The effect of prolonged spaceflight on cerebrospinal fluid and infectious. Proc. Natl Acad. Sci. U. S. A. 113, 13851–13856 (2016). perivascular spaces of astronauts and cosmonauts. Proc. Natl Acad. Sci. U. S. A. 48. Marzban, L., Park, K. & Verchere, C. B. Islet amyloid polypeptide and type 1 119,1–3 (2022). diabetes. Exp. Gerontol. 38, 347–351 (2003). 79. Gulati, S., Raghunandan, A., Rasheed, F., McBride, S. A. & Hirsa, A. H. Ring-sheared 49. Hull, R. L., Westermarl, G. T., Westermark, P. & Kahn, S. E. Islet amyloid: A critical drop (rsd): Microgravity module for containerless flow studies. Microgravity Sci. entity in the pathogenesis of type 2 diabetes. J. Clin. Endocrinol. Metab. 89, Technol. 29,81–89 (2017). 3629–3643 (2004). 80. Gulati, S., Riley, F. P., Lopez, J. M. & Hirsa, A. H. Mixing within drops via surface 50. Abedini, A. & Schmidt, A. M. Mechanisms of islet amyloidosis toxicity in type 2 shear viscosity. Int. J. Heat. Mass Trans. 125, 559–568 (2018). diabetes. FEBS Lett. 587, 1119–1127 (2013). 81. Gulati, S., Riley, F. P., Hirsa, A. H. & Lopez, J. M. Flow in a containerless liquid 51. ISS safety requirements document. Tech. Rep. SSP 51721, National Aeronautics system: Ring-sheared drop with finite surface shear viscosity. Phys. Rev. Fluids 4, and Space Administration, Houston, Texas (2019). 1–9 (2019). 52. Amin, S., Barnett, G. V., Pathak, J. A., Roberts, C. J. & Sarangapani, P. Protein 82. McMackin, P. M. et al. Simulated microgravity in the ring-sheared drop. npj aggregation,particle formation, characterization and rheology. Curr. Opin. Colloid Microgravity 6,1–7 (2020). Interface Sci. 19, 438–449 (2014). 83. Riley, F. P., McMackin, P. M., Lopez, J. M. & Hirsa, A. H. Flow in a ring-sheared 53. Gong, Z., You, R., Chang, R. C.-C. & Lin, Y. Viscoelastic response of neural cells drop: Drop deformation. Phys. Fluids 33,1–12 (2021). governed by the deposition of amyloid-β peptides (aβ). J. Appl. Phys. 119,1–7 84. Lopez, J. M. & Hirsa, A. H. Coupling of the interfacial and bulk flow in kinfe-edge (2016). viscometers. Phys. Fluids 27,1–13 (2015). 54. Mattana, S., Caponi, S., Tamagnini, F., Fioretto, D. & Palombo, F. Viscoelas- 85. A Pojman, J., Bessonov, N., Volpert, V. & Paley, M. S. Miscible fluids in micogravity ticity of amyloid plaques in transgenic mouse brain studied by brillouin (mfmg): A zero-upmass investigation on the international space station. microspectroscopy and correlative raman analysis. J. Innov. Opt. Health Sci. Microgravity Sci. Technol. XIX-1,33–41 (2007). 10,1–24 (2017). 86. Derkach, S. R., Kragel, J. & Miller, R. Methods of measuring rheological properties 55. Wang, R., Yang, X., Cui, L., Yin, H. & Xu, S. Gels of amyloid fibers. Biomolecules 9, of interfacial layers (experimental methods of 2d rheology). Colloid J. 71,1–17 1–12 (2019). (2009). 56. Woodard, D. et al. Gel formation in protein amyloid aggregation: A physical 87. Tamim, S. I. & Bostwick, J. B. Oscillations of a soft viscoelastic drop. npj Micro- mechanism for cytotoxicity. PLoS ONE 9,1–8 (2014). gravity 7,1–8 (2021). 57. Jean, L., Lee, C. F., Hodder, P., Hawkins, N. & Vaux, D. J. Dynamics of the for- 88. Guo, X., Chen, X., Zhou, W. & Wei, J. Effect of polymer drag reducer on rheo- mation of a hydrogel by a pathogenic amyloid peptide: Islet amyloid poly- logical properties of rocket kerosene solutions. Materials 15,1–14 (2022). peptide. Sci. Rep. 6,1–10 (2016). 89. Correia, A. C. M., Boue, G., Laskar, J. & Rodriguez, A. Deformation and tidal 58. Courchesne, N.-M. D., Duraj-Thatte, A., Tay, P. K. R., Nguyen, P. Q. & Joshi, N. S. evolution of close-in planets and satellites using a maxwell viscoelastic rheol- Scalable production of genetically engineered nanofibrous macroscopic mate- ogy. Astron. Astrophys. 571,1–16 (2014). rials via filtration. ACS Biomater. Sci. Eng. 3, 733–741 (2017). 90. Samuel, H., Lognonne, P., Panning, M. & Lainey, V. The rheology and thermal 59. Reynolds, N. P. Amyloid-like peptide nanofibrils as scaffolds for tissue engi- histroy of mars revealed by the orbital evolution of phobos. Nature 569, neering: Progress and challenges (review). Biointerphases 14,1–8 (2019). 523–527 (2019). 60. Hill, E. K., Krebs, B., Goodall, D. G., Howlett, G. J. & Dunstan, D. E. Shear flow 91. Suresh, R. & Simranjeet, S. Exoplanets and their structure, rheology and induces amyloid fibril formation. Biomacromolecules 7,10–13 (2006). dynamics. Int. Res. J. Eng. Technol. 7,44–49 (2020). 61. Dunstan, D. E., Hamilton-Brown, P., Asimakis, P., Ducker, W. & Bertolini, J. Shear 92. Trinh, E. H. & Depew, J. Solid surface wetting and the deployment of drops in flow promotes amyloid-β fibrilization. Protein Eng. Des. Sel. 22, 741–746 (2009). microgravity. Microgravity Sci. Technol. 7, 299–306 (1993). 62. Trumbore, C. N. Progress in Molecular Biology and Translational Science. (Elsevier 93. Brutin, D. et al. Sessile drop in microgravity: Creation, contact angle and inter- Inc, Amsterdam Netherlands, 2019). face. Microgravity Sci. Technol. 21, S67–S76 (2009). 63. Schladitz, C., Vieira, E. P., Hermel, H. & Mohwald, H. Amyloid-β-sheet formation at 94. Xu, S.-H., Wang, C.-X., Sun, Z.-W. & Hu, W.-R. The influence of contact line velocity the air-water interface. Biophys. J. 77, 3305–3310 (1999). and acceleration on the dynamic contact angle: An experimental study in 64. Jean, L., Lee, C. F. & Vaux, D. J. Enrichment of amyloidogenesis at an air-water microgravity. Int. J. Heat. Mass Trans. 54, 2222–2225 (2011). interface. Biophys. J. 102, 1154–1162 (2012). 95. Rizzardi, L. F. et al. Evaluation of techniques for performing cellular isolation and 65. Campioni, S. et al. The presence of an air-water interface affects formation and preservation during microgravity conditions. npj Microgravity 2,1–10 (2016). elongation of alpha-synuclein fibrils. J. Am. Chem. Soc. 136, 2866–2875 (2014). 96. Baba, P., Toth, A. & Horvath, D. Surface-tension-driven dynamic contact line in 66. Duerkop, M., Berger, E., Durauer, A. & Jungbauer, A. Impact of cavitation, high microgravity. Langmuir 35, 406–412 (2019). shear stress and air/liquid interfaces on protein aggregation. Biotechnol. J. 13, 97. Amberg, G. Detailed modelling of contact line motion in oscillatory wetting. npj 1–9 (2018). Microgravity 8,1–8 (2022). 67. Zhou, J. et al. Effects of sedimentation, microgravity, hydrodynamic mixing and 98. Ludwicki, J. M. et al. Is contact-line mobility a material parameter? npj Micro- air-water interface on α-synuclein amyloid formation. Chem. Sci. 11, 3687–3693 gravity 8,1–8 (2022). (2020). 99. Torres, L. J. & Weislogel, M. M. The ejection of large non-oscillating droplets from 68. Padayachee, E. R. et al. Cerebrospinal fluid-induced retardation of amyloid β a hydrophobic wedge in microgravity. npj Microgravity 7,1–10 (2021). aggregation correlates with alzheimer’s disease and the apoe ϵ4 allele. Brain Res 100. Weislogel, M. M. et al. How advances in low-g plumbing enable space 1651,11–16 (2016). exploration. npj Microgravity 8,1–11 (2022). Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 41 P. McMackin et al. 101. Reitz, B. et al. Additive manufacturing under lunar gravity and microgravity. 126. Edwards, D. A., Brenner, H. & Wasan, D. T. Interfacial Transport Processes and Microgravity Sci. Technol. 33,1–12 (2021). Rheology. (Butterwortj-Heinemann, MA, USA, 1991). 102. Dietrich, D. L. et al. Droplet combustion experiments aboard the international space station. Microgravity Sci. Technol. 26,65–76 (2014). 103. Meyer, F. et al. Oxygen droplet combustion in hydrogen under microgravity ACKNOWLEDGEMENTS conditions. Combust. Flame 241,1–11 (2022). The authors would like to thank Louise Littles, Sridhar Gorti, Hong Q. Yang, Kevin 104. Amidon, G. L., DeBrincat, G. A. & Najib, N. Effects of gravity on gastric emptying, Depew, Michael Hall, James McClellan, Heidi Parris, Shawn Reagan, Ryan Reeves, intestinal transit, and drug absorption. J. Clin. Pharmacol. 31, 968–973 (1991). Shawn Stephens, Paul Galloway, Ben Murphy, and Fran Chiramonte for their 105. Yang, J.-Q. et al. The effects of microgravity on the digestive system and the continued support of both the RSD project and the operations team at Rensselaer new insights it brings to the life sciences. Life Sci. Space Res 27,74–82 (2020). Polytechnic Institute. The authors also thank astronauts Raja Chari, Shane Kimbrough, 106. Bureau, L. et al. Blood flow and microgravity. C. R. Mecanique 345,78–85 (2017). Christina Koch, Akihiko Hoshide, Megan McArthur, Luca Parmitano, Thomas Pasquet, 107. Navasiolava, N. et al. Vacsular and microvascular dysfunction induced by and Mark Vande Hei for their excellence and flexibility during real-time space microgravity and its analogs in humans: Mechanisms and countermeasures. operations. The authors are also grateful for the support to this study given by NASA Front. Phys. 11, 420–428 (2020). BPS, NASA MSFC, NASA JSC, NSF-CASIS, and Teledyne-Brown Engineering. This work 108. Galvin, I., Drummond, G. B. & Nirmalan, M. Distribution of blood flow and ventilation was supported by NASA Grant 80NSSC20K1726 and NSF Grant 1929134. in the lung: gravity is not the only factor. Br. J. Anaesth. 98,420–428 (2007). 109. Prisk, G. K. Microgravity and the respiratory system. Eur. Respir. J. 43, 1459–1471 (2014). AUTHOR CONTRIBUTIONS 110. Janmaleki, M., Pachenari, M., Seyedpour, S. M., Shahghadami, R. & Sanati-Nez- P.M., J.A., and A.H. conceptualized and designed the experiment. J.A. prepared insulin had, A. Impact of simulated microgravity on ctoskeleton and viscoelastic solutions. P.M. and J.A. performed the remote ISS operations and experimental properties of endothelial cell. Sci. Rep. 6,1–11 (2016). measurements. P.M. performed the analysis of experimental measurements. S.G. 111. Bradbury, P. et al. Modeling microgravity at the cellular level: Implications for performed supporting computational fluid dynamics simulations. P.M. produced all human disease. Front. Cell Dev. Biol. 8,1–11 (2020). the figures and tables. P.M., J.A., and A.H. wrote the manuscript. P.M., J.A., S.G., and 112. Kennedy, M. J. & Volz, P. A. The effect of space flight irradiation on sacchar- A.H. critically evaluated data and results, including data interpretation, figure omyces cerevisiae growth and respiration. FEMS Microbiol. Lett. 19, 125–128 development, and manuscript editing. (1983). 113. Bouloc, P. & D’Ari, R. Escherichia coli metabolism in space. J. Gen. Microbiol. 137, 2839–2843 (1991). COMPETING INTERESTS 114. Kobayashi, Y., Kikuchi, M., Nagaoka, S. & Watanabe, H. Recovery of deinococcus radiodurans from radiation damage was enhanced under microgravity. Biol. Sci. The authors declare no competing interests. Space 10,97–101 (1996). 115. Walther, I., Bechler, B., Muller, O., Hunzinger, E. & Cogoli, A. Cultivation of sac- charomyces cerevisiae in a bioreactor in microgravity. J. Biotechnol. 47, 113–127 ADDITIONAL INFORMATION (1996). Supplementary information The online version contains supplementary material 116. Saffary, R. et al. Microbial survival of space vacuum and extreme ultraviolet available at https://doi.org/10.1038/s41526-022-00227-2. irradiation: strain isolation and analysis during a rocket flight. FEMS Microbiol. Lett. 215, 163–168 (2002). Correspondence and requests for materials should be addressed to Amir Hirsa. 117. Horneck, G., Klaus, D. M. & Mancinelli, R. L. Space microbiology. Microbiol. Mol. Biol. Rev. 74, 121–156 (2010). Reprints and permission information is available at http://www.nature.com/ 118. Nislow, C. et al. Genes required for survival in microgravity revealed by genome- reprints wide yeast deletion collections cultured during spaceflight. BioMed. Res. Intern. 10,1–10 (2015). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims 119. Ott, E. et al. Molecular repertoire of deinococcus radiodurans after 1 year of in published maps and institutional affiliations. exposure outside the international space station within the tanpopo mission. Microbiome 8,1–16 (2020). 120. Padgen, M. R. et al. Ecamsat spaceflight measurements of the role of σsin antibiotic resistance of stationary phase escherichia coli in microgravity. Life Sci. Open Access This article is licensed under a Creative Commons Space Res 24,18–24 (2020). Attribution 4.0 International License, which permits use, sharing, 121. McMackin, P. M. et al. Effects of microorganisms on drop formation in micro- adaptation, distribution and reproduction in any medium or format, as long as you give gravity during a parabolic flight with residual gravity and jitter. Microgravity Sci. appropriate credit to the original author(s) and the source, provide a link to the Creative Technol. 34,1–9 (2022). Commons license, and indicate if changes were made. The images or other third party 122. Walther, I. Space bioreactors and their applications. Adv. Space Biol. Med. 8, material in this article are included in the article’s Creative Commons license, unless 197–213 (2002). indicated otherwise in a credit line to the material. If material is not included in the 123. Menezes, A. A., Cumbers, J., Hogan, J. A. & Arkin, A. P. Towards synthetic bio- article’s Creative Commons license and your intended use is not permitted by statutory logical approaches to resource utilization on space missions. J. R. Soc. Interface regulation or exceeds the permitted use, you will need to obtain permission directly 12,1–20 (2015). from the copyright holder. To view a copy of this license, visit http:// 124. Painter, N. A. & Sisson, E. An overview of concentrated insulin products. Diabetes creativecommons.org/licenses/by/4.0/. Spectr. 29, 136–140 (2016). 125. Knopp, J. L., Holder-Pearson, L. & Chase, J. G. Insulin units and conversion factors: A story of truth, boots, and faster half-truths. J. Diabetes Tech. Soc. 13, © The Author(s) 2022 597–600 (2019). npj Microgravity (2022) 41 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA

Journal

npj MicrogravitySpringer Journals

Published: Sep 20, 2022

There are no references for this article.