Altered Ca2+ Homeostasis in Red Blood Cells of Polycythemia Vera Patients Following Disturbed Organelle Sorting during Terminal Erythropoiesis

Over 95% of Polycythemia Vera (PV) patients carry the V617F mutation in the tyrosine kinase Janus kinase 2 (JAK2), resulting in uncontrolled erythroid proliferation and a high risk of thrombosis. Using mass spectrometry, we analyzed the RBC membrane proteome and showed elevated levels of multiple Ca2+ binding proteins as well as endoplasmic-reticulum-residing proteins in PV RBC membranes compared with RBC membranes from healthy individuals. In this study, we investigated the impact of JAK2V617F on (1) calcium homeostasis and RBC ion channel activity and (2) protein expression and sorting during terminal erythroid differentiation. Our data from automated patch-clamp show modified calcium homeostasis in PV RBCs and cell lines expressing JAK2V617F, with a functional impact on the activity of the Gárdos channel that could contribute to cellular dehydration. We show that JAK2V617F could play a role in organelle retention during the enucleation step of erythroid differentiation, resulting in modified whole cell proteome in reticulocytes and RBCs in PV patients. Given the central role that calcium plays in the regulation of signaling pathways, our study opens new perspectives to exploring the relationship between JAK2V617F, calcium homeostasis, and cellular abnormalities in myeloproliferative neoplasms, including cellular interactions in the bloodstream in relation to thrombotic events.

The transfer temperature from slow cooling to cryogenic storage is critical for optimal recovery of cryopreserved mammalian cells

Cryopreservation is a key step for the effective delivery of many cell therapies and for the maintenance of biological materials for research. The preservation process must be carefully controlled to ensure maximum, post-thaw recovery using cooling rates slow enough to allow time for cells to cryodehydrate sufficiently to avoid lethal intracellular ice. This study focuses on determining the temperature necessary at the end of controlled slow cooling before transfer to cryogenic storage which ensures optimal recovery of the processed cell samples. Using nucleated, mammalian cell lines derived from liver (HepG2), ovary (CHO) and bone tissue (MG63) this study has shown that cooling must be controlled to -40°C before transfer to long term storage to ensure optimal cell recovery. No further advantage was seen by controlling cooling to lower temperatures. These results are consistent with collected differential scanning calorimetry data, that indicated the cells underwent an intracellular, colloidal glass transition between -49 and -59°C (Tg’i) in the presence of the cryoprotective agent dimethyl sulfoxide (DMSO). The glass forms at the point of maximum cryodehydration and no further cellular dehydration is possible. At this point the risk of lethal intracellular ice forming on transfer to ultra-low temperature storage is eliminated. In practice it may not be necessary to continue slow cooling to below this temperature as optimal recovery at -40°C indicates that the cells have become sufficiently dehydrated to avoid further, significant damage when transferred into ultra-low temperature storage.

Effects of Low Temperature on Shrimp and Crab Physiology, Behavior, and Growth: A Review

As important aquaculture species worldwide, shrimps and crabs are thermophilic animals with a feeble thermoregulation ability. Changes in environmental factors are the main reason for the decrease in the immunity and disease resistance ability of cultured organisms. Water temperature is one of the most common abiotic stress factors for aquatic ectotherms. It influences nearly all biochemical and physiological processes in crustaceans, resulting in an imbalance in ion and water homeostasis, neuromuscular function loss, cellular dehydration, and altered metabolic pathways. The present review summarizes the current knowledge on the effects of low temperature on the physiological response, and the behavior, development, and growth of shrimp and crab. We suggest a deeper research to understand the physiological processes involved in thermoregulation; this knowledge could be used to reduce the adverse effects in the shrimps and crabs during the culture.

Physiological and Proteomic Analyses of Different Ecotypes of Reed (Phragmites communis) in Adaption to Natural Drought and Salinity

Drought and salinity are the two major abiotic stresses constraining the crop yield worldwide. Both of them trigger cellular dehydration and cause osmotic stress which leads to cytosolic and vacuolar volume reduction. However, whether plants share a similar tolerance mechanism in response to these two stresses under natural conditions has seldom been comparatively reported. There are three different ecotypes of reed within a 5 km2 region in the Badanjilin desert of Northwest China. Taking the typical swamp reed (SR) as a control, we performed a comparative study on the adaption mechanisms of the two terrestrial ecotypes: dune reed (DR) and heavy salt meadow reed (HSMR) by physiological and proteomic approaches coupled with bioinformatic analysis. The results showed that HSMR and DR have evolved C4-like photosynthetic and anatomical characteristics, such as the increased bundle sheath cells (BSCs) and chloroplasts in BSCs, higher density of veins, and lower density and aperture of stomata. In addition, the thylakoid membrane fluidity also plays an important role in their higher drought and salinity tolerance capability. The proteomic results further demonstrated that HSMR and DR facilitated the regulation of proteins associated with photosynthesis and energy metabolism, lipid metabolism, transcription and translation, and stress responses to well-adapt to the drought and salinity conditions. Overall, our results demonstrated that HSMR and DR shaped a similar adaption strategy from the structural and physiological levels to the molecular scale to ensure functionality in a harsh environment.

Cellular dehydration acutely degrades mood mainly in women: a counterbalanced, crossover trial

It is unclear if mild-to-moderate dehydration independently affects mood without confounders like heat exposure or exercise. This study examined the acute effect of cellular dehydration on mood. Forty-nine adults (55 % female, age 39 (sd 8) years) were assigned to counterbalanced, crossover trials. Intracellular dehydration was induced with 2-h (0·1 ml/kg per min) 3 % hypertonic saline (HYPER) infusion or 0·9 % isotonic saline (ISO) as a control. Plasma osmolality increased in HYPER (pre 285 (sd 3), post 305 (sd 4) mmol/kg; P < 0·05) but remained unchanged in ISO (pre 285 (sd 3), post 288 (sd 3) mmol/kg; P > 0·05). Mood was assessed with the short version of the Profile of Mood States Questionnaire (POMS). The POMS sub-scale (confusion-bewilderment, depression-dejection, fatigue-inertia) increased in HYPER compared with ISO (P < 0·05). Total mood disturbance score (TMD) assessed by POMS increased from 10·3 (sd 0·9) to 16·6 (sd 1·7) in HYPER (P < 0·01), but not in ISO (P > 0·05). When TMD was stratified by sex, the increase in the HYPER trial was significant in females (P < 0·01) but not in males (P > 0·05). Following infusion, thirst and copeptin (surrogate for vasopressin) were also higher in females than in males (21·3 (sd 2·0), 14·1 (sd 1·4) pmol/l; P < 0·01) during HYPER. In conclusion, cellular dehydration acutely degraded specific aspects of mood mainly in women. The mechanisms underlying sex differences may be related to elevated thirst and vasopressin.

Identifying in-silico how microstructural changes in cellular fruit affect the drying kinetics

Convective drying of fruits leads to microstructural changes within the material as a result of moisture removal. In this study, an upscaling approach is developed to understand and identify the relation between the drying kinetics and the resulting microstructural changes of apple fruit, including shrinkage of cells without membrane breakage (free shrinkage) and with membrane breakage (lysis). First, the effective permeability is computed from a microscale model as a function of the water potential. Both temperature dependency and microstructural changes during drying are modeled. The microscale simulation shows that lysis, which can be induced using various pretreatment processes, enhances the tissue permeability up to four times compared to the free shrinkage of the cells. Second, via upscaling, macroscale modeling is used to quantify the impact of these microstructural changes in the fruit drying kinetics. We identify the formation of a barrier layer for water transport during drying, with much lower permeability, at the tissue surface. The permeability of this layer strongly depends on the dehydration mechanism. We also quantified how inducing lysis or modifying the drying conditions, such as airspeed and relative humidity, can accelerate the drying rate. We found that inducing lysis is more effective in reducing the drying rate (up to 26%) than increasing the airspeed from 1 to 5 m/s or decreasing the relative humidity from 30% to 10%. This study quantified the need for including cellular dehydration mechanisms in understanding fruit drying processes and provided insight at a spatial resolution that experiments almost cannot reach.

Similar Yet Different–Structural and Functional Diversity among Arabidopsis thaliana LEA_4 Proteins

The importance of intrinsically disordered late embryogenesis abundant (LEA) proteins in the tolerance to abiotic stresses involving cellular dehydration is undisputed. While structural transitions of LEA proteins in response to changes in water availability are commonly observed and several molecular functions have been suggested, a systematic, comprehensive and comparative study of possible underlying sequence-structure-function relationships is still lacking. We performed molecular dynamics (MD) simulations as well as spectroscopic and light scattering experiments to characterize six members of two distinct, lowly homologous clades of LEA_4 family proteins from Arabidopsis thaliana. We compared structural and functional characteristics to elucidate to what degree structure and function are encoded in LEA protein sequences and complemented these findings with physicochemical properties identified in a systematic bioinformatics study of the entire Arabidopsis thaliana LEA_4 family. Our results demonstrate that although the six experimentally characterized LEA_4 proteins have similar structural and functional characteristics, differences concerning their folding propensity and membrane stabilization capacity during a freeze/thaw cycle are obvious. These differences cannot be easily attributed to sequence conservation, simple physicochemical characteristics or the abundance of sequence motifs. Moreover, the folding propensity does not appear to be correlated with membrane stabilization capacity. Therefore, the refinement of LEA_4 structural and functional properties is likely encoded in specific patterns of their physicochemical characteristics.