Design of neural organoids engineered by mechanical forces.

Neural organoids consist of three-dimensional tissue derived from pluripotent stem cells that could recapitulate key features of the human brain. During the past decade, organoid technology has evolved in the field of human brain science by increasing the quality and applicability of its products. Among them, a novel approach involving the design of neural organoids engineered by mechanical forces has emerged. This review describes previous approaches for the generation of neural organoids, the engineering of neural organoids by mechanical forces, and future challenges for the application of mechanical forces in the design of neural organoids.

Large-scale cultivation of human iPS cells in bioreactor with reciprocal mixing.

A substantial number of human iPS cells (hiPSCs) is needed for cell therapy to be successful against various diseases. We previously reported on a bioreactor with reciprocal mixing that produces specific physical properties that differ from those of conventional bioreactors with rotary paddle stirring. Moreover, such reactors not only provide a homogeneous environment but also allow the control of spheroid size by changing the mixing speed. In this study, we applied this bioreactor to the large-scale cultivation of hiPSCs. Approximately 10 billion hiPSCs were obtained from 2.0 L of culture, and the high expression of pluripotency markers was maintained. Our findings indicate that a bioreactor with reciprocal mixing can be used for large-scale hiPSC cultivation.

Tree-rings stable isotope (δ18O and δ2H) based 368 years long term precipitation reconstruction of South Eastern Kashmir Himalaya.

The hydroclimatic variability in Kashmir Himalaya is influenced by the western disturbances and the Indian Summer Monsoon. To investigate long-term hydroclimatic variability, 368 years tree-ring oxygen and hydrogen isotope ratios (δ18O and δ2H) extending from 1648 to 2015 CE were analysed. These isotopic ratios are calculated using five core samples of Himalayan silver fir (Abies pindrow) collected from the south-eastern region of Kashmir valley. The relationship between the long and short periodicity components of δ18O and δ2H suggested that physiological processes had a minimum effect on the tree-ring stable isotopes in Kashmir Himalaya. The δ18O chronology was developed based on the average of five-individual tree-ring δ18O time series covering the time span of 1648-2015 CE. The climate response analysis revealed the strongest and most significant negative correlation between tree ring δ18O and precipitation amount from the previous year's December to current year's August (D2Apre). The reconstructed D2Apre (D2Arec) explains precipitation variability from 1671 to 2015 CE and is supported by historical and other proxy-based hydroclimatic records. The reconstruction has two distinguishing features: first, it is characterized by stable wet conditions during the last phase of Little Ice Age (LIA) i.e., from 1682 to 1841 CE; and second, the southeast Kashmir Himalaya had experienced drier conditions as compared to recent and historical period with intense pluvial events since 1850. The present reconstruction shows, there have been more extreme dry events than extreme wet events since 1921. A tele-connection is observed between D2Arec and Sea Surface Temperature (SST) of the Westerly region.

Improved recombinant protein production in Aspergillus oryzae lacking both α-1,3-glucan and galactosaminogalactan in batch culture with a lab-scale bioreactor.

Filamentous fungi are used as production hosts for various commercially valuable enzymes and chemicals including organic acids and secondary metabolites. We previously revealed that α-1,3-glucan and galactosaminogalactan (GAG) contribute to hyphal aggregation in the industrial fungus Aspergillus oryzae, and that production of recombinant protein in shake-flask culture is higher in a mutant lacking both α-1,3-glucan and GAG (AGΔ-GAGΔ) than in the parental strain. Here, we compared the productivity of the wild type, AGΔ-GAGΔ, and mutants lacking α-1,3-glucan (AGΔ) or GAG (GAGΔ) in batch culture with intermittent addition of glucose in a 5-L lab-scale bioreactor. The hyphae of the wild type and all mutants were dispersed by agitation, although the wild type and AGΔ formed small amounts of aggregates. Although mycelial weight was similar among the strains, the concentration of a secreted recombinant protein (CutL1) was the highest in AGΔ-GAGΔ. Evaluation of fluid properties revealed that the apparent viscosities of mycelial cultures of the wild type and AGΔ-GAGΔ decreased as the agitation speed was increased. The apparent viscosity of the AGΔ-GAGΔ culture tended to be lower than that of the wild-type strain at each agitation speed, and was significantly lower at 600 rpm. Overall, the lack of α-1,3-glucan and GAG in the hyphae improved culture rheology, resulting in an increase in recombinant protein production in AGΔ-GAGΔ. This is the first report of flow behavior improvement by a cell-surface component defect in a filamentous fungus.

Induction of inverted morphology in brain organoids by vertical-mixing bioreactors.

Organoid technology provides an opportunity to generate brain-like structures by recapitulating developmental steps in the manner of self-organization. Here we examined the vertical-mixing effect on brain organoid structures using bioreactors and established inverted brain organoids. The organoids generated by vertical mixing showed neurons that migrated from the outer periphery to the inner core of organoids, in contrast to orbital mixing. Computational analysis of flow dynamics clarified that, by comparison with orbital mixing, vertical mixing maintained the high turbulent energy around organoids, and continuously kept inter-organoid distances by dispersing and adding uniform rheological force on organoids. To uncover the mechanisms of the inverted structure, we investigated the direction of primary cilia, a cellular mechanosensor. Primary cilia of neural progenitors by vertical mixing were aligned in a multidirectional manner, and those by orbital mixing in a bidirectional manner. Single-cell RNA sequencing revealed that neurons of inverted brain organoids presented a GABAergic character of the ventral forebrain. These results suggest that controlling fluid dynamics by biomechanical engineering can direct stem cell differentiation of brain organoids, and that inverted brain organoids will be applicable for studying human brain development and disorders in the future.

Improved cultivation of Chinese hamster ovary cells in bioreactor with reciprocal mixing.

We have constructed a new bioreactor with reciprocal mixing that is better suited for the cultivation of delicate animal cells. In-silico simulation (computational fluid dynamics) suggested both maximum and average shear stresses in the bioreactor with reciprocal mixing to be remarkably lower than in a conventional bioreactor with rotary mixing. Although we could not find any difference in growth speed and cell density between the bioreactors with reciprocal and rotary mixing, we did find cell viability in the reciprocal-mixing bioreactor to be retained longer than in the rotary-paddle bioreactor. This implied that cell culture in a bioreactor with reciprocal mixing could be prolonged for the production of target proteins. Leakage of lactate dehydrogenase activity into the culture medium was suppressed much more in the reciprocal-mixing bioreactor than in the rotary-paddle one. Production of human tissue plasminogen activator in the former system was also observed to be much higher than in the latter. Therefore, a bioreactor with reciprocal mixing was concluded to be better suited for the cultivation of animal cells and efficient production of proteins, such as antibody drugs and various growth factors.

Comparative Effects of Etelcalcetide and Maxacalcitol on Serum Calcification Propensity in Secondary Hyperparathyroidism: A Randomized Clinical Trial.

BACKGROUND AND OBJECTIVES: Vitamin D receptor activators and calcimimetics (calcium-sensing receptor agonists) are two major options for medical treatment of secondary hyperparathyroidism. A higher serum calcification propensity (a shorter T50 value) is a novel surrogate marker of calcification stress and mortality in patients with CKD. We tested a hypothesis that a calcimimetic agent etelcalcetide is more effective in increasing T50 value than a vitamin D receptor activator maxacalcitol. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: A randomized, multicenter, open-label, blinded end point trial with active control was conducted in patients with secondary hyperparathyroidism undergoing hemodialysis in Japan. Patients were randomly assigned to receive intravenous etelcalcetide 5 mg thrice weekly (etelcalcetide group) or intravenous maxacalcitol 5 or 10 µg thrice weekly (maxacalcitol group). The primary, secondary, and tertiary outcomes were changes in T50 value, handgrip strength, and score of the Dementia Assessment Sheet for Community-Based Integrated Care System from baseline to 12 months, respectively. RESULTS: In total, 425 patients from 23 dialysis centers were screened for eligibility, 326 patients were randomized (etelcalcetide, n=167; control, n=159), and 321 were included in the intention-to-treat analysis (median age, 66 years; 113 women [35%]). The median (interquartile range) of T50 value was changed from 116 minutes (interquartile range, 90-151) to 131 minutes (interquartile range, 102-176) in the maxacalcitol group, whereas it was changed from 123 minutes (interquartile range, 98-174) to 166 minutes (interquartile range, 127-218) in the etelcalcetide group. The increase in T50 value was significantly greater in the etelcalcetide group (difference in change, 20 minutes; 95% confidence interval, 7 to 34 minutes; P=0.004). No significant between-group difference was found in the change in handgrip strength or in the Dementia Assessment Sheet for Community-Based Integrated Care System score. CONCLUSIONS: Etelcalcetide was more effective in increasing T50 value than maxacalcitol among patients on hemodialysis with secondary hyperparathyroidism. There was no difference in handgrip strength or cognition between the two drugs. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: VICTORY; UMIN000030636 and jRCTs051180156.

Intradialytic exercise in the treatment of social frailty: a single-center prospective study-preliminary results during the unexpected COVID-19 pandemic.

BACKGROUND: Social frailty-the lack of a connection to society and infrequent social activities-has been reported to be associated with future declines in physical function in elderly individuals. This study aimed to evaluate both the association of social frailty with the physical function and the efficacy of intradialytic exercise as a therapy for social frailty among hemodialysis patients. METHODS: All 16 outpatient hemodialysis patients in the hemodialysis department of a single medical center were enrolled in this single-center prospective single-arm interventional study. Patients received five questions which asked about going out infrequently, lack of visiting friends, feeling unhelpful to friends or family, living alone, and lack of talking with someone. Those to whom two or more of the above were applicable were categorized as socially frail. All patients were placed into exercise therapy to be performed during their thrice-weekly hemodialysis visits. Participants' physical function (walking speed), muscle strength (grip strength), muscle mass (appendicular skeletal muscle mass index), and social frailty were evaluated at baseline and after 3 months of therapy. RESULTS: Four (25%) of the 16 participants (median age 71.5 years, 8 women) were categorized as being socially frail. In comparison to the non-socially frail group (non-SF), the socially frail group (SF) had a significantly lower walking speed (0.70 ± 0.12 m/s vs 1.15 ± 0.26 m/s, p = 0.005) and significantly worse performance on the Short Physical Performance Battery. Three months of intradialytic exercise therapy significantly improved their walking speed, from 1.04 ± 0.30 m/s to 1.16 ± 0.29 m/s (p = 0.003). intradialytic exercise therapy significantly improved walking speed in both the SF group and the non-SF group. The 2019 coronavirus disease pandemic unexpectedly occurred in the middle of the intervention period of this study, and although it was not statistically significant, the number of socially frail individuals among our participants increased to seven (43.8%, p = 0.248). CONCLUSIONS: Social frailty was associated with reduced physical function among hemodialysis patients. Intradialytic exercise therapy improved physical function regardless of the presence of social frailty. TRIAL REGISTRATION: UMIN-CTR, UMIN-CTR000038313. Registered November 1, 2019, https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000043639.

Magnesium isotopes reveal bedrock impacts on stream organisms.

Magnesium is an essential element to aquatic organisms and understanding the origin of Mg is important for understanding their growth. Ultimately, Mg in streams is derived from the chemical weathering of bedrock in the catchment. In this study, we used Mg stable isotope ratios (δ26Mg) to test whether stream organisms reflect lithological sources in stream catchments. In November 2017 and May 2018, we sampled aquatic insects and small gobies from six temperate streams in the Lake Biwa area (central Japan). Three of these streams had up to 38% limestone in their catchment (limestone streams), and three streams lacked limestone (non-limestone streams). We hypothesised that stream organisms from limestone streams had significantly lower δ26Mg values compared to those of the same organisms from non-limestone streams. Aquatic insects from limestone streams had an average of 0.78‰ lower δ26Mg values than those of the same organisms from non-limestone streams, thereby indicating a lithological control on the δ26Mg of aquatic insects. Aquatic insects often showed an offset to higher δ26Mg values compared to those of stream water, thereby pointing to a 26Mg-enriched diet as an additional Mg source to water and/or Mg isotope fractionation during Mg accumulation. Instead, stream water was the main Mg source for small gobies, as their bones reflected the δ26Mg of water. We concluded that δ26Mg could trace Mg sources of aquatic organisms, and the same methodology can be applied to other metals.

Turbulence Activates Platelet Biogenesis to Enable Clinical Scale Ex Vivo Production.

The ex vivo generation of platelets from human-induced pluripotent cells (hiPSCs) is expected to compensate donor-dependent transfusion systems. However, manufacturing the clinically required number of platelets remains unachieved due to the low platelet release from hiPSC-derived megakaryocytes (hiPSC-MKs). Here, we report turbulence as a physical regulator in thrombopoiesis in vivo and its application to turbulence-controllable bioreactors. The identification of turbulent energy as a determinant parameter allowed scale-up to 8 L for the generation of 100 billion-order platelets from hiPSC-MKs, which satisfies clinical requirements. Turbulent flow promoted the release from megakaryocytes of IGFBP2, MIF, and Nardilysin to facilitate platelet shedding. hiPSC-platelets showed properties of bona fide human platelets, including circulation and hemostasis capacities upon transfusion in two animal models. This study provides a concept in which a coordinated physico-chemical mechanism promotes platelet biogenesis and an innovative strategy for ex vivo platelet manufacturing.

Using food network unfolding to evaluate food-web complexity in terms of biodiversity: theory and applications.

Food-web complexity often hinders disentangling functionally relevant aspects of food-web structure and its relationships to biodiversity. Here, we present a theoretical framework to evaluate food-web complexity in terms of biodiversity. Food network unfolding is a theoretical method to transform a complex food web into a linear food chain based on ecosystem processes. Based on this method, we can define three biodiversity indices, horizontal diversity (DH ), vertical diversity (DV ) and range diversity (DR ), which are associated with the species diversity within each trophic level, diversity of trophic levels, and diversity in resource use, respectively. These indices are related to Shannon's diversity index (H'), where H' = DH  + DV  - DR . Application of the framework to three riverine macroinvertebrate communities revealed that D indices, calculated from biomass and stable isotope features, captured well the anthropogenic, seasonal, or other within-site changes in food-web structures that could not be captured with H' alone.

A comparison of freeze-drying and oven-drying preparation methods for bulk and compound-specific carbon stable isotope analyses: examples using the benthic macroinvertebrates Stenopsyche marmorata and Epeorus latifolium.

RATIONALE: Carbon stable isotope analysis of bulk samples and fatty acids is an established method for tracing carbon flow pathways and reconstructing trophic interactions, but there is no consensus on which sample drying method should be used for sample preparation. The aim of this study was to determine if freeze-drying and oven-drying treatments used to prepare samples of the benthic macroinvertebrates Stenopsyche marmorata and Epeorus latifolium for bulk and fatty-acid-specific carbon stable isotope analysis yield different isotopic ratio values. METHODS: Five individuals each from two species were split in half; one half was freeze-dried and the other half was oven-dried. The samples were ground and the δ(13)C values of the bulk samples and eight fatty acids were measured following combustion using an isotope ratio mass spectrometer coupled to an elemental analyzer or gas chromatography system. RESULTS: The mean difference in the bulk and fatty acid δ(13)C values between freeze-dried and oven-dried samples was small (≤0.1‰ in both cases), although relatively large variations were observed in individual fatty-acid-specific δ(13)C values (maximum of ≤0.9 ‰). There were no significant differences in either bulk sample or fatty-acid-specific δ(13)C values between freeze-dried or oven-dried samples of the same species. CONCLUSIONS: Freeze-drying and oven-drying are equally acceptable methods for preparing freshly caught S. marmorata and E. latifolium samples for bulk and fatty-acid-specific carbon stable isotope analyses.

Stable nitrogen isotopic composition of amino acids reveals food web structure in stream ecosystems.

The stable N isotopic composition of individual amino acids (SIAA) has recently been used to estimate trophic positions (TPs) of animals in several simple food chain systems. However, it is unknown whether the SIAA is applicable to more complex food web analysis. In this study we measured the SIAA of stream macroinvertebrates, fishes, and their potential food sources (periphyton and leaf litter of terrestrial C3 plants) collected from upper and lower sites in two streams having contrasting riparian landscapes. The stable N isotope ratios of glutamic acid and phenylalanine confirmed that for primary producers (periphyton and C3 litter) the TP was 1, and for primary consumers (e.g., mayfly and caddisfly larvae) it was 2. We built a two-source mixing model to estimate the relative contributions of aquatic and terrestrial sources to secondary and higher consumers (e.g., stonefly larva and fishes) prior to the TP calculation. The estimated TPs (2.3-3.5) roughly corresponded to their omnivorous and carnivorous feeding habits, respectively. We found that the SIAA method offers substantial advantages over traditional bulk method for food web analysis because it defines the food web structure based on the metabolic pathway of amino groups, and can be used to estimate food web structure under conditions where the bulk method cannot be used. Our result provides evidence that the SIAA method is applicable to the analysis of complex food webs, where heterogeneous resources are mixed.