Isotonic Medium Research Articles

Effects of a DC offset on an electrothermal microparticle trap assembled with an AC electric field

Micro and nano-scale colloidal particles can be rapidly assembled at electrode-electrolyte interfaces in highly organized structures. These particles aggregate through various forces which are, among others, chemical, electrical, and thermal in nature. Patterning biological cells in such structures has high impact applications but it remains a challenge due to their limited viability under said manipulation forces. Rapid electrokinetic patterning (REP) uses AC electrothermal micro-vortices to aggregate both synthetic particles and biological cells. In this work, we explore the effects of a DC offset on a REP trap performance in bio-relevant isotonic medium. REP traps were characterized by measuring the inter-particle distance under different DC offsets, using a Delaunay triangulation. The inter-particle distance was measured in a steady-state trap followed by the disassembly of the aggregate as the REP vortex was turned off. DC offset enhanced the trapping performance for micro-particles suspended in a sugar-based isotonic medium. It was observed that an increasingly negative DC offset increased the steady-state inter-particle distance and reduced the rate of disassembly. However, no such trend was found with positive DC offsets. Changing the offset in small steps (<500 mV) affected only the inter-particle distance whereas, larger steps significantly affected the trap stability and size.

Optimization of Zwitterionic Polymers for Cell Cryopreservation.

Cryopreservation techniques are valuable for the preservation of genetic properties in cells, and the development of this technology contributes to various fields. In a previous study, an isotonic freezing medium composed of poly(zwitterion) (polyZI) has been reported, which alleviates osmotic shock, unlike typical hypertonic freezing media. In this study, the primitive freezing medium composed of emerging polyZI is optimized. Imidazolium/carboxylate-type polyZI (VimC3C) is the optimal chemical structure. The molecular weight and degree of ion substitution (DSion) are not significant factors. There is an impediment with the primitive polyZI freezing media. While the polyZI forms a matrix around the cell membrane to protect cells, the matrix is difficult to remove after thawing, resulting in low cell proliferation. Unexpectedly, increasing the poly(VimC3C) concentration from 10% to 20% (w/v) improves cell proliferation. The optimized freezing medium, 20% (w/v) poly(VimC3C)_DSion(100%)/1% (w/v) NaCl aqueous solution, exhibited a better cryoprotective effect.

Modulation of prostaglandin transport activity of SLCO2A1 by annexin A2 and S100A10.

Solute carrier organic anion transporter family member 2A1 (SLCO2A1) is a prostaglandin (PG) transporter and serves as the osmosensitive ATP-permeable maxi-anion channel (Maxi-Cl). Since a heterotetrameric complex of annexin A2 (ANXA2) and S100A10 is obligatory for the channel activity, the present study aimed to determine if they regulate SLCO2A1-mediated PG transport. This study examined PGE2 uptake and ATP release in Anxa2 and/or S100a10 knockout (KO) murine breast C127 cells. Deletion of Slco2a1 decreased PGE2-d4 uptake by wild-type (WT) cells in an isotonic medium (290 mosmol/kgH2O). Decreased osmolarity (135 mosmol/kgH2O) stimulated ATP release but did not affect PGE2 uptake kinetics, showing Km (1,280 nM) and Vmax (10.38 pmol/15 s/mg protein) similar to those in isotonic medium (1,227 nM and 10.65 pmol/15 s/mg protein), respectively, in WT cells. Deletion of Anxa2 associated with loss of S100a10 diminished SLCO2A1-mediated ATP release and uncompetitively inhibited PGE2 uptake with lowered Km (376 nM) and Vmax (2.59 pmol/15 s/mg protein). Moreover, the immunoprecipitation assay confirmed the physical interaction of ANXA2 with SLCO2A1 in WT cells. Enforcement of ANXA2 expression to Anxa2 KO cells partially restored PGE2 uptake and increased Km (744.3 nM) and Vmax (9.07 pmol/15 s/mg protein), whereas the uptake clearance (Vmax/Km) did not change much regardless of ANXA2 expression. These results suggest that an ANXA2/S100A10 complex modulates PG transport activity but osmolality has little effect on it; therefore, the bound form of SLCO2A1, which functions as a PG transporter and Maxi-Cl, may exist regardless of changes in the cell volume.NEW & NOTEWORTHY A previous study indicated that the ANXA2/S100A10 complex represents the regulatory component of SLCO2A1-mediated Maxi-Cl channel activity. The present study showed that apparent PGE2 uptake by C127 cells was osmoinsensitive and uncompetitively inhibited by loss of ANXA2 expression, demonstrating that ANXA2 is a regulatory factor of SLCO2A1-mediated PG transport activity.

Dielectrophoretic profiling of erythrocytes to study the impacts of metabolic stress, temperature, and storage duration utilizing a point-and-planar microdevice

Dielectrophoresis (DEP) is widely utilized for trapping and sorting various types of cells, including live and dead cells and healthy and infected cells. This article focuses on the dielectric characterization of erythrocytes (red blood cells or RBCs) by quantifying DEP crossover frequency using a novel point-and-planar microwell device platform. Numerical simulations using COMSOL Multiphysics software demonstrate that the distribution of the DEP force is influenced by factors such as the shape of the point electrode, spacing between the point and planar electrodes, and the type of bioparticle being investigated. The dependency on electrode spacing is experimentally evaluated by analyzing the DEP crossover response of erythrocytes. Furthermore, the results are validated against the traditional electrical characterization technique called electrorotation, which typically requires laborious fabrication and operation using quadrupole electrodes. Other significant factors, including erythrocyte storage age and the changes in cell properties over time since collection, osmolarity, and temperature, are also assessed to determine the optimal conditions for erythrocyte characterization. The findings indicate a significant difference between fresh and stored erythrocyte samples (up to 4 days), highlighting the importance of maintaining an isotonic medium for cell storage.

Suppression of neutrophils by sodium exacerbates oxidative stress and arthritis.

Typical Western diet, rich in salt, contributes to autoimmune disease development. However, conflicting reports exist about the effect of salt on neutrophil effector functions, also in the context of arthritis. We investigated the effect of sodium chloride (NaCl) on neutrophil viability and functions in vitro, and in vivo employing the murine K/BxN-serum transfer arthritis (STA) model. The effects of NaCl and external reactive oxygen species (H2O2) were further examined on osteoclasts in vitro. Hypertonic sodium-rich media caused primary/secondary cell necrosis, altered the nuclear morphology, inhibited phagocytosis, degranulation, myeloperoxidase (MPO) peroxidation activity and neutrophil extracellular trap (NET) formation, while increasing total ROS production, mitochondrial ROS production, and neutrophil elastase (NE) activity. High salt diet (HSD) aggravated arthritis by increasing inflammation, bone erosion, and osteoclast differentiation, accompanied by increased NE expression and activity. Osteoclast differentiation was decreased with 25 mM NaCl or 100 nM H2O2 addition to isotonic media. In contrast to NaCl, external H2O2 had pro-resorptive effects in vitro. We postulate that in arthritis under HSD, increased bone erosion can be attributed to an enhanced oxidative milieu maintained by infiltrating neutrophils, rather than a direct effect of NaCl.

Formation of nuclear CPSF6/CPSF5 biomolecular condensates upon HIV-1 entry into the nucleus is important for productive infection

The early events of HIV-1 infection involve the transport of the viral core into the nucleus. This event triggers the translocation of CPSF6 from paraspeckles into nuclear speckles forming puncta-like structures. Our investigations revealed that neither HIV-1 integration nor reverse transcription is required for the formation of puncta-like structures. Moreover, HIV-1 viruses without viral genome are competent for the induction of CPSF6 puncta-like structures. In agreement with the notion that HIV-1 induced CPSF6 puncta-like structures are biomolecular condensates, we showed that osmotic stress and 1,6-hexanediol induced the disassembly of CPSF6 condensates. Interestingly, replacing the osmotic stress by isotonic media re-assemble CPSF6 condensates in the cytoplasm of the cell. To test whether CPSF6 condensates were important for infection we utilized hypertonic stress, which prevents formation of CPSF6 condensates, during infection. Remarkably, preventing the formation of CPSF6 condensates inhibits the infection of wild type HIV-1 but not of HIV-1 viruses bearing the capsid changes N74D and A77V, which do not form CPSF6 condensates during infection1,2. We also investigated whether the functional partners of CPSF6 are recruited to the condensates upon infection. Our experiments revealed that CPSF5, but not CPSF7, co-localized with CPSF6 upon HIV-1 infection. We found condensates containing CPSF6/CPSF5 in human T cells and human primary macrophages upon HIV-1 infection. Additionally, we observed that the integration cofactor LEDGF/p75 changes distribution upon HIV-1 infection and surrounds the CPSF6/CPSF5 condensates. Overall, our work demonstrated that CPSF6 and CPSF5 are forming biomolecular condensates that are important for infection of wild type HIV-1 viruses.

Optically induced electrothermal microfluidic tweezers in bio-relevant media

Non-contact micro-manipulation tools have enabled invasion-free studies of fragile synthetic particles and biological cells. Rapid electrokinetic patterning (REP) traps target particles/cells, suspended in an electrolyte, on an electrode surface. This entrapment is electrokinetic in nature and thus depends strongly on the suspension medium’s properties. REP has been well characterized for manipulating synthetic particles suspended in low concentration salt solutions (~ 2 mS/m). However, it is not studied as extensively for manipulating biological cells, which introduces an additional level of complexity due to their limited viability in hypotonic media. In this work, we discuss challenges posed by isotonic electrolytes and suggest solutions to enable REP manipulation in bio-relevant media. Various formulations of isotonic media (salt and sugar-based) are tested for their compatibility with REP. REP manipulation is observed in low concentration salt-based media such as 0.1× phosphate buffered saline (PBS) when the device electrodes are passivated with a dielectric layer. We also show manipulation of murine pancreatic cancer cells suspended in a sugar-based (8.5% w/v sucrose and 0.3% w/v dextrose) isotonic medium. The ability to trap mammalian cells and deposit them in custom patterns enables high-impact applications such as determining their biomechanical properties and 3D bioprinting for tissue scaffolding.

Neuroprotection in ophthalmology. Targets in the most frequent diseases. A clinician's point of view

AbstractObjectives: To briefly review the most frequent ocular diseases in which a neuroprotective treatment would be needed and to present the results of a translational research based on the neuroprotective capacities of allogeneic bone marrow‐derived mesenchymal stem cells (MSCs) in an ocular disease that currently lacks treatment, the non‐arteritic acute anterior ischemic optic neuropathy. (NA‐AION) considered as an infarct of the optic nerve head.Methods: After extensive preclinical evaluation of safety in rabbits, a prospective, non‐randomized, phase II clinical trial (Eudra CT number 2016–003029‐40 and ClinicalTrials.gov NCT03173638) was approved. Five patients with acute unilateral NA‐AION diagnosed within the first 2 weeks after symptom onset were included. After informed consent, patients received an intravitreal injection of 0.05 ml containing 150 μl of isotonic medium composed of Ringer's lactate, 0.5% human albumin, and 5 mM glucose, with a cell concentration of 1.5 × 106 cells/ml. The injection was administered using a 25‐gauge needle via the pars plana after topical anaesthesia. Patients were followed on days 1, 7, 30, 90, 180 and 1 year after injection. Safety was defined as the absence of an inflammatory reaction. The best‐corrected visual acuity, visual evoked potentials (VEP) and retinal nerve fibre layer (RNFL) and ganglion cell layer (GCL) thicknesses were measured.Results: No patients had signs of intraocular inflammation at any time. An epiretinal membrane (ERM) developed in one patient. One phakic patient had a retrolental aggregate that did not progress to cataract. Four patients had visual improvement and three had increased amplitudes on the VEP recordings after treatment. The RNFL and GCL thicknesses decreased significantly in parallel with the visible papillary atrophy that developed during the follow‐up period.Conclusions: Despite development of an ERM in one patient and a possible cellular deposit in the retrolental space, the functional results are encouraging. A larger multicentre study of treated patients that includes a control group is currently running. The paracrine capacities of allergenic MSCs should be more extensively investigated in the clinic, taking advantage of their non‐immunogenicity, their ease of obtaining and their apparent safety.Financial support: Funded by Strategic Action in Health of the Institute of Health Carlos III (PIC18/00018), the Department of Regional Health of the Castilla y Léon Government (GRS 1928/A/19), and the Department of Education of the Castilla y León Regional Government (Grant VA077P17).

Rapid Reversible Osmoregulation of Cytoplasmic Biomolecular Condensates of Human Interferon-α-Induced Antiviral MxA GTPase.

We previously discovered that exogenously expressed GFP-tagged cytoplasmic human myxovirus resistance protein (MxA), a major antiviral effector of Type I and III interferons (IFNs) against several RNA- and DNA-containing viruses, existed in the cytoplasm in phase-separated membraneless biomolecular condensates of varying sizes and shapes with osmotically regulated disassembly and reassembly. In this study we investigated whether cytoplasmic IFN-α-induced endogenous human MxA structures were also biomolecular condensates, displayed hypotonic osmoregulation and the mechanisms involved. Both IFN-α-induced endogenous MxA and exogenously expressed GFP-MxA formed cytoplasmic condensates in A549 lung and Huh7 hepatoma cells which rapidly disassembled within 1-2 min when cells were exposed to 1,6-hexanediol or to hypotonic buffer (~40-50 mOsm). Both reassembled into new structures within 1-2 min of shifting cells to isotonic culture medium (~330 mOsm). Strikingly, MxA condensates in cells continuously exposed to culture medium of moderate hypotonicity (in the range one-fourth, one-third or one-half isotonicity; range 90-175 mOsm) first rapidly disassembled within 1-3 min, and then, in most cells, spontaneously reassembled 7-15 min later into new structures. This spontaneous reassembly was inhibited by 2-deoxyglucose (thus, was ATP-dependent) and by dynasore (thus, required membrane internalization). Indeed, condensate reassembly was preceded by crowding of the cytosolic space by large vacuole-like dilations (VLDs) derived from internalized plasma membrane. Remarkably, the antiviral activity of GFP-MxA against vesicular stomatitis virus survived hypoosmolar disassembly and subsequent reassembly. The data highlight the exquisite osmosensitivity of MxA condensates, and the preservation of antiviral activity in the face of hypotonic stress.

Water transport regulates nucleus volume, cell density, Young’s modulus, and E-cadherin expression in tumor spheroids

Cell volume is maintained by the balance of water and solutes across the cell membrane and plays an important role in mechanics and biochemical signaling in cells. Here, we assess the relationship between cell volume, mechanical properties, and E-cadherin expression in three-dimensional cultures for ovarian cancer. To determine the effect of water transport in multi-cellular tumors, ovarian cancer spheroids were subjected to hypotonic and hypertonic shock using water and sucrose mixtures, respectively. Increased osmolality resulted in decreased nucleus volume, increased Young’s modulus, and increased tumor cell density in ovarian cancer spheroids. Next, we looked at the reversibility of mechanics and morphology after 5 min of osmotic shock and found that spheroids had a robust ability to return to their original state. Finally, we quantified the size of E-cadherin clusters at cell-cell junctions and observed a significant increase in aggregate size following 30 min of hypertonic and hypotonic osmotic shocks. Yet, these effects were not apparent after 5 min of osmotic shock, illustrating a temporal difference between E-cadherin regulation and the immediate mechanical and morphology changes. Still, the osmotically induced E-cadherin aggregates which formed at the 30-minute timepoint was reversible when spheroids were replenished with isotonic medium. Altogether, this work demonstrated an important role of osmolality in transforming mechanical, morphology, and molecular states.

Osmosensing by cytoplasmic biomolecular condensates of antiviral MxA GTPase

Cellular and tissue‐level edema is a common feature of acute viral infections, including covid‐19. However, there is little understanding of the effects of tissue edema on antiviral effector mechanisms. In as much as defects in IFN‐α function (such as due to presence of autoantibodies) are now implicated in enhanced severity of covid‐19 disease, we investigated how hypoosmolarity and cellular edema might affect the antiviral activity of IFN‐α‐induced human MxA GTPase, a major antiviral effector against several RNA and DNA viruses. We had previously discovered that cytoplasmic human MxA existed in the cytoplasm in phase‐separated membraneless biomolecular condensates with a gel‐like internal consistency. The vesicular stomatitis virus (VSV) nucleocapsid (N) protein associated with such MxA condensates in cells exhibiting an antiviral phenotype. Unexpectedly, MxA condensates displayed rapid and reversible osmosensing properties. Condensates of both IFN‐α‐induced endogenous MxA and of exogenous GFP‐MxA expressed in human A549 lung and Huh7 hepatoma cells rapidly disassembled within 1‐2 min when cells were exposed to hypotonic buffer (approx. 50 mOsm), and rapidly reassembled into new structures within 1‐2 min of shifting of cells to isotonic culture medium (approx. 330 mOsm). GFP‐MxA condensates in cells continuously exposed to culture medium in the range one‐fourth, one‐third or one‐half isotonicity first rapidly disassembled within 1‐2 min, and then, in most cells. spontaneously reassembled 7‐15 min later into new structures. The data are consistent with cytoplasmic “crowding” as the mechanism driving MxA condensate formation. In terms of antiviral function, one 5 min cycle of disassembly and reassembly of GFP‐MxA condensates at 1 or 2 hr after VSV infection did not affect MxA antiviral activity. Additionally, continuous exposure of GFP‐MxA expressing A549 lung cells to culture medium at one‐fourth or one‐third tonicity (thus triggering a cycle of disassembly and slower spontaneous reassembly in the continued presence of hypotonic culture medium) together with a VSV challenge, left antiviral activity largely unaffected. Such cultures showed an increase in number of cells with VSV N colocalized with the reassembled GFP‐MxA condensates. The data reveal a novel property of MxA biomolecular condensates ‐ rapid and reversible osmosensing. Importantly, antiviral activity was largely unaffected by tonicity‐driven disassembly/reassembly mimicking conditions of cellular and tissue‐level edema.

A case of multipyramidal kidneys with smooth surface in a New Zealand white rabbit

The focus of the present study was to present a case of multilobar kidneys with smooth surface in one New Zealand white rabbit. It is well known that the kidneys of the rabbits are unipyramidal. During dissection it was found that there was an exception in one female animal which was clinically healthy and sexually matured, aged 8 months and with weight from 2.5 kg to 3.2 kg. After evisceration of both kidneys, and incision in the lateral borer of the fresh organs, it was found that the cortex and medulla were constructed by pyramidal shaped lobes. The apex of the lobes formed papillae and got up into calices into the real sinus. The renal pelvis was a concave structure. We conducted an imaging anatomical study. The anatomical preparations were studied in liquid isotonic medium, using ultrasound device with linear transducer. Thus we confirmed the results with these of the organs’ morphological features. The cortex with the fibrous capsule were hyperechoic, compared to the relatively hypoechoic image of the pyramidal lobes. The papillae forming the apex were outlined by the hyperechoic calices. The renal pelvis and hilus were hypoechoic findings. After fixation the kidneys in 10 % water solution of formalin the pyramidal shaped lobes were preserved and with well distinguished papillae. The calices protruded into the sinus. In all methods we found seven number of well-defined pyramidal shaped lobes.

Ram epididymal sperm frozen in an extender containing ethylene glycol have higher post-thaw longevity and in vitro fertility.

Establishing an efficient, simple and inexpensive method for freezing ram epididymal sperm so that the quality and fertility of spermatozoa could be maintained for a longer period after thawing is of great practical value. To optimize freezing and thawing protocol for ram epididymal sperm using either ethylene glycol (EG) or glycerol (GLY) as cryoprotectants (CPAs). Then, to evaluate the post-thaw longevity and in vitro fertility of spermatozoa that were frozen and thawed according to the optimized protocol. At first, an optimum protocol for freezing and thawing sperm using EG or GLY were investigated, and the next experiments were performed using the spermatozoa that had been frozen and thawed according to the optimized protocol for each CPA. In the next experiments, frozen-thawed and fresh sperm were diluted in an isotonic culture medium and subsequently incubated at 39°C for 4h. The motility characteristics and functional membrane integrity (FMI) of spermatozoa were evaluated after thawing, after dilution (t0 ), and after incubation (t4 ). The in vitro fertility of the spermatozoa was assessed at t0 and t4 . For both CPAs, the highest motility parameters and FMI was found for spermatozoa frozen at 3cm above LN2 and thawed at 50 and 65°C (P<0.05). In comparison to the spermatozoa of GLY group, the spermatozoa of the EG group had higher total and progressive motility at t0 , as well as higher FMI, total and progressive motility, and linearity at t4 (P<0.05). Fertility of frozen-thawed sperm was higher than that of fresh sperm at t0 (P<0.05). Incubation treatment increased the fertility of fresh sperm while decreased the fertility of frozen-thawed sperm, and this decline was more severe in GLY than in the EG group. Based on the findings, EG can be a more suitable CPA for freezing ram epididymal sperm.

Black Sea turbot sperm motility depending on the dilution of seawater

The sperm motility of fish reflects their fertilizing ability. Sperm activation in fish with external fertilization occurs in an isotonic medium (sea or freshwater). The duration of sperm motility is a compromise between the level of energy reserves possessed by the sperm and the process of osmotic damage it experiences in the activating medium. Under natural conditions, various biotic and abiotic factors can affect the characteristics of motility. At the same time, when assessing the reproductive potential of males in laboratory conditions, the dilution and methods of its activation have a significant effect on the characteristics of sperm quality (namely, speed, the proportion of motile spermatozoa, and the time of their activity). In our work, we determined the main characteristics of the motility of the Black Sea turbot sperm at a dilution of 1:10 and 1: 100 with seawater. We have shown that, the average speed and percentage of motile sperm significantly decrease with higher dilution of seawater.

Relationship of Motility Activation to Lipid Composition, Protein Profile, and Swelling Rate of Burbot Lota lota Spermatozoon Following Change of Temperature and Osmolality

Despite available information on the roles of osmotic pressure, potassium (K+), and calcium (Ca2+) in activation of Eurasian burbot spermatozoon motility, the changes in milt biochemical composition and mechanisms underlying their activation at temperatures above optimal spawning temperature is still unclear. We explored spermatozoon swelling, lipid composition and proteome in relation to osmolality and temperature of swimming medium. The result revealed that temperature increment from 4 to 30°C increases burbot spermatozoa vulnerability to osmotic pressure, decreasing motility in extremely hypotonic media, and the presence of Ca2+decreases swelling of spermatozoa. No spermatozoon swelling was observed in non-ionic isotonic media at all studied temperatures. A role of swelling in activation of spermatozoa motility at 30°C was rejected. No differences were found in protein profile and lipid composition with respect to activation of burbot spermatozoa at 30°C. Burbot spermatozoon activation occurs at 30°C without modification of the spermatozoon membrane. Elucidation of the intrinsic signaling pathway of burbot spermatozoon spontaneous activation requires further study.

Formation of Non-Nucleoplasmic Proteasome Foci during the Late Stage of Hyperosmotic Stress.

Cellular stress induces the formation of membraneless protein condensates in both the nucleus and cytoplasm. The nucleocytoplasmic transport of proteins mainly occurs through nuclear pore complexes (NPCs), whose efficiency is affected by various stress conditions. Here, we report that hyperosmotic stress compartmentalizes nuclear 26S proteasomes into dense nuclear foci, independent of signaling cascades. Most of the proteasome foci were detected between the condensed chromatin mass and inner nuclear membrane. The proteasome-positive puncta were not colocalized with other types of nuclear bodies and were reversibly dispersed when cells were returned to the isotonic medium. The structural integrity of 26S proteasomes in the nucleus was slightly affected under the hyperosmotic condition. We also found that these insulator-body-like proteasome foci were possibly formed through disrupted nucleus-to-cytosol transport, which was mediated by the sequestration of NPC components into osmostress-responding stress granules. These data suggest that phase separation in both the nucleus and cytosol may be a major cell survival mechanism during hyperosmotic stress conditions.

The effects of incubation media on the assessment of the shape of human erythrocytes by flow cytometry: a contribution to mathematical data interpretation to enable wider application of the method.

Flow cytometry (FC) analysis of erythrocyte shape and related biomechanical properties, such as osmotic fragility, have not moved from a research tool to regular clinical testing. The main reason is existing evidence that various pre-analytical factors influence the mathematical interpretation of the data obtained. With an aim to contribute to the standardization and broaden the use of FC for human erythrocyte shape assessment, freshly prepared peripheral blood erythrocytes isolated from healthy donors were incubated in iso and hypo-osmotic solutions (pure saline, saline with potassium and calcium, and phosphate buffered saline) and examined by FC using values of forward scatter (FSC) and side scatter (SSC). Kurtosis, skewness, Pearson's second skewness coefficient of dissymmetry (PCD), and spherical index, calculated from FSC distributions, were used for the erythrocyte shape evaluation. In all isotonic media FSC distribution and FSC-based morphology parameters showed huge inter-individual and inter-medium variation. With decreasing osmolality, in all media and samples, the size of the erythrocytes increased, and swelling index and kurtosis decreased. However, changes in skewness and PCD were influenced by the medium used and the sample tested. Compared to FSC, SSC signal in isotonic and its change in hypotonic media showed lower inter-individual variation and was not influenced by the type of medium. We propose a spherical index and kurtosis as FSC-based indicators of erythrocyte shape. As more resistant to the influence of the preanalytical treatment, SSC data appeared to be unfairly neglected for the assessment of erythrocyte shape, in comparison to the usually employed FSC data.

Mechanism of CuSO4 cytotoxicity in goat erythrocytes after high-level in vitro exposure to isotonic media

Copper (Cu) is a common environmental pollutant in nature. Cu-poisoning can cause liver damage and erythrocytes hemolysis. To evaluate the effect of CuSO4 poisoning on the morphological and functional characteristics of goat red blood cells. Five 10–14-month-old goats were selected for jugular vein blood sampling to obtain erythrocytes, and then the erythrocytes were processed with different concentrations (0, 10, 20, 30, 40 and 50 μmol/L) of CuSO4 for 48 h, and 40 μmol/L doses CuSO4 incubated for different time (12, 24, 36, 48 and 60 h) to process erythrocytes. We observed the changes in erythrocyte morphology through scanning electron microscopy, and detected the antioxidant function and activities of three ATPases. Additionally, biological properties were examined from the perspectives of phospholipids and membrane protein components, permeability fragility, and fluidity in erythrocytes. We found that after CuSO4 treatment, the antioxidant capacity of erythrocytes decreased, which was manifested as increased MDA content and decreased CuZn-SOD and GSH-Px activities (p < 0.05). In addition, we also found that erythrocyte fluidity decreased, osmotic fragility increased, membrane phospholipid percentage and protein composition changes abnormally, and Na+/K+-ATPase, Mg2+-ATPase and Ca2+-ATPase activities decreased (p < 0.05). From the results, it can be concluded that CuSO4 exposure causes hemolysis of goat erythrocytes through oxidative stress to the structure and function of erythrocytes, showing a dose-time effect.

A method for the preparation of electrocompetent cells to transform unicellular green algae, Coccomyxa (Trebouxiophyceae, Chlorophyta) strains Obi and KJ

Unicellular green algae, Coccomyxa spp. strains Obi and KJ are capable of growing at broad pH ranges, and accumulate lipids at levels higher than 50% of the dry cell weight under nitrogen-depleted conditions. An efficient genetic transformation method applicable to the two strains is required for their genetic improvement to facilitate their application in commercial production of biofuels. After an extended trial and error period, we fortuitously determined conditions required for the transformation of the strains through electroporation. First, the growth of the strains in isotonic medium e.g., minimal salt medium containing 0.6 M sorbitol or mannitol was required. Second, the growth under a light/dark cycle was critical. In the dark period in the cycle, cells contained two or four endospores surrounded by a mother-cell wall. Within half an hour after the beginning of the light period, the mother-cell wall was cleaved, and unicellular autospores that were electrocompetent were released. The application of a single electroporation pulse at an electric-field strength of 10 kV cm−1 to the autospores yielded transformants at a frequency of >10−6 per input cell for strain KJ and >10−7 per input cell for strain Obi. The transformation method is relatively simple, and provided several to several dozen higher transformation frequencies than those applying the biolistic bombardment method that we previously developed. The present study also provides insights that could facilitate the development of genetic transformation systems for microalgae, which reproduce by endospore formation.

A novel waterjet technology for transurethral cystoscopic injection of viable cells in the urethral sphincter complex.

In a recent preclinical study, we noticed that injection of cells in the urethral sphincter by needle through a cystoscope under visual control frequently yielded in misplacement or loss of cells. We, therefore, investigated if a needle-free waterjet device delivers viable cells under defined settings, including injection volume and pressure, fluid velocity and transportation media, precisely through the urothelium and connective tissue close to the sphincter muscle without full penetration of the sphincter apparatus. Mesenchymal stromal cells (MSCs) were prepared for needle-free waterjet injections. Upon injections into liquids cell viability and yield were investigated by trypan blue dye exclusion. Upon injection into cadaveric urethral tissue samples, cells were isolated from the urethrae and expanded to prove that this novel method delivered viable cells into the tissue. MSC injections by William's needle served as controls. Waterjet injections of MSCs into isotonic cell culture medium resulted in equal or better yields of viable cells when compared with needle injections. Upon injection in urethral tissue samples, the waterjet technology facilitated fast and precise injections of viable cells through urothelial, mucosal and submucosal layers to reach the sphincter muscle. By controlling the injection pressure, loss of cells due to insufficient thrust or unintended full penetration was avoided. Needle-free waterjet injections deliver cells in the urethra faster and more precisely when compared with needle injections without compromising their viability. This is the first proof-of-concept study providing evidence that a waterjet transports viable cells precisely into the targeted tissue.