Ionic Milieu Research Articles

Ionic release from bioactive SiO2-CaOCME/poly(tetrahydrofuran)/poly(caprolactone) hybrids drives human-bone marrow stromal cell osteogenic differentiation

This study demonstrates that dissolution products of inorganic/organic SiO2-CaOCME/PTHF/PCL-diCOOH hybrid (70S30CCME-CL) drive human bone marrow stromal cells (h-BMSCs) down an osteogenic pathway with the production of mineralised matrix. We investigated osteogenesis through combined analyses of mRNA dynamics for key markers and targeted staining of mineralised matrix. We demonstrate that h-BMSCs undergo accelerated differentiation in vitro in response to the 70S30CCME-CL ionic milieu, as compared to incubation with osteogenic media. Extracts from 70S30CCME-CL promote osteogenesis by inducing changes in cellular metabolic activity, promoting changes in cell morphology consistent with the osteogenic lineage, and by enhancing mineralisation of hydroxyapatite in the extracellular matrix. Additionally, our results show that 70S30CCME-CL hybrids prove sustained functional resilience by maintaining osteostimulatory effects despite cumulated dissolution cycles. In co-differentiation medium, 70S30CCME-CL ionic release can modulate signalling pathways associated with non-osteogenic functions, further supporting their potential for bone regeneration applications. Overall, our study provides compelling experimental evidence that the 70S30CCME-CL hybrid is a promising biomaterial for bone tissue regeneration applications.

Ca2+-dependent thermal sensitivity of bacterial MreB assemblies

AbstractIn the bacterial cells, the actin homolog MreB manages the cellular motions and morphology. MreB polymers are important for cell-wall growing and cell shape determination. Fluorescence microscopy studies investigated that in bacterial cells the MreB polymer forms ribbon-like structures that likely helical nearby of the cell wall at the periphery of the cell. As we presented earlier, the thermal motion of the ribbon-like MreB polymers was slowed down by the addition of millimolar Ca2+. The rapid Ca2+ depletion, via EGTA treatment, reordered the polymers into extensive sheets in the presence of magnesium, and further treatment with calcium led to fissured monolayer sheets and the dissociation of filaments into web-like structures which attached to the glass surface. The heat denaturation of MreB assemblies, under varying Ca2+ concentrations, was investigated by DSC, and the Ca2+-dependent MreB polymer rearrangement rates were assessed by isoperibol calorimetry. Here, we measured Ca2+-dependent thermodynamics of prokaryotic MreB assemblies. Under high ionic strength, the MreB polymers show multiple thermal components around 60 °C and 82 °C, generated by less and more stable structures. MreB polymers with a relatively slow exothermic kinetics turned to be more stable due to adding millimolar Ca2+. However, changing the calcium concentration from micromolar to nanomolar and subsequently recovered it to micromolar initialized endothermic remodeling of MreB assemblies and the majority of them showed higher stability than before the treatment. Presumably, the final cell shape depends on the assembling of MreB polymers and the ionic milieu. Calcium concentration-induced changes of MreB structure makes sense in membrane remodeling during prokaryotic cell division or osmotic adaptation.

Signaling Roleplay between Ion Channels during Mammalian Sperm Capacitation.

In order to accomplish their primary goal, mammalian spermatozoa must undergo a series of physiological, biochemical, and functional changes crucial for the acquisition of fertilization ability. Spermatozoa are highly polarized cells, which must swiftly respond to ionic changes on their passage through the female reproductive tract, and which are necessary for male gametes to acquire their functional competence. This review summarizes the current knowledge about specific ion channels and transporters located in the mammalian sperm plasma membrane, which are intricately involved in the initiation of changes within the ionic milieu of the sperm cell, leading to variations in the sperm membrane potential, membrane depolarization and hyperpolarization, changes in sperm motility and capacitation to further lead to the acrosome reaction and sperm-egg fusion. We also discuss the functionality of selected ion channels in male reproductive health and/or disease since these may become promising targets for clinical management of infertility in the future.

Astrocyte Calcium Signaling Shifts the Polarity of Presynaptic Plasticity

Astrocytes have been increasingly acknowledged to play active roles in regulating synaptic transmission and plasticity. Through a variety of metabotropic and ionotropic receptors expressed on their surface, astrocytes detect extracellular neurotransmitters, and in turn, release gliotransmitters to modify synaptic strength, while they can also alter neuronal membrane excitability by modulating extracellular ionic milieu. Given the seemingly large repertoire of synaptic modulation, when, where and how astrocytes interact with synapses remain to be fully understood. Previously, we have identified a role for astrocyte NMDA receptor and L-VGCCs signaling in heterosynaptic presynaptic plasticity and promoting the heterogeneity of presynaptic strengths at hippocampal synapses. Here, we have sought to further clarify the mode by which astrocytes regulate presynaptic plasticity by exploiting a reduced culture system to globally evoke NMDA receptor-dependent presynaptic plasticity. Recording from a postsynaptic neuron intracellularly loaded with BAPTA, briefly bath applying NMDA and glycine induces a stable decrease in the rate of spontaneous glutamate release, which requires the presence of astrocytes and the activation of A1 adenosine receptors. Upon preventing astrocyte calcium signaling or blocking L-VGCCs, NMDA + glycine application triggers an increase, rather than a decrease, in the rate of spontaneous glutamate release, thereby shifting the presynaptic plasticity to promote an increase in strength. Our findings point to a crucial and surprising role of astrocytes in controlling the polarity of NMDA receptor and adenosine-dependent presynaptic plasticity. Such a pivotal mechanism unveils the power of astrocytes in regulating computations performed by neural circuits and is expected to profoundly impact cognitive processes.

WNK kinases sense molecular crowding and rescue cell volume via phase separation

When challenged by hypertonicity, dehydrated cells must recover their volume to survive. This process requires the phosphorylation-dependent regulation of SLC12 cation chloride transporters by WNK kinases, but how these kinases are activated by cell shrinkage remains unknown. Within seconds of cell exposure to hypertonicity, WNK1 concentrates into membraneless condensates, initiating a phosphorylation-dependent signal that drives net ion influx via the SLC12 cotransporters to restore cell volume. WNK1 condensate formation is driven by its intrinsically disordered C terminus, whose evolutionarily conserved signatures are necessary for efficient phase separation and volume recovery. This disorder-encoded phase behavior occurs within physiological constraints and is activated invivo by molecular crowding rather than changes in cell size. This allows kinase activity despite an inhibitory ionic milieu and permits cell volume recovery through condensate-mediated signal amplification. Thus, WNK kinases are physiological crowding sensors that phase separate to coordinate a cell volume rescue response.

The ins and outs of virus trafficking through acidic Ca2+ stores

Many viruses exploit host-cell Ca2+ signaling processes throughout their life cycle. This is especially relevant for viruses that translocate through the endolysosomal system, where cellular infection is keyed to the microenvironment of these acidic Ca2+ stores and Ca2+-dependent trafficking pathways. As regulators of the endolysosomal ionic milieu and trafficking dynamics, two families of endolysosomal Ca2+-permeable cation channels – two pore channels (TPCs) and transient receptor potential mucolipins (TRPMLs) – have emerged as important host-cell factors in viral entry. Here, we review: (i) current evidence implicating Ca2+ signaling in viral translocation through the endolysosomal system, (ii) the roles of these ion channels in supporting cellular infection by different viruses, and (iii) areas for future research that will help define the potential of TPC and TRPML ligands as progressible antiviral agents.

Charging Up the Periphery: Glial Ionic Regulation in Sensory Perception.

The peripheral nervous system (PNS) receives diverse sensory stimuli from the environment and transmits this information to the central nervous system (CNS) for subsequent processing. Thus, proper functions of cells in peripheral sense organs are a critical gate-keeper to generating appropriate animal sensory behaviors, and indeed their dysfunction tracks sensory deficits, sensorineural disorders, and aging. Like the CNS, the PNS comprises two major cell types, neurons (or sensory cells) and glia (or glia-like supporting neuroepithelial cells). One classic function of PNS glia is to modulate the ionic concentration around associated sensory cells. Here, we review current knowledge of how non-myelinating support cell glia of the PNS regulate the ionic milieu around sensory cell endings across species and systems. Molecular studies reviewed here suggest that, rather than being a passive homeostatic response, glial ionic regulation may in fact actively modulate sensory perception, implying that PNS glia may be active contributors to sensorineural information processing. This is reminiscent of emerging studies suggesting analogous roles for CNS glia in modulating neural circuit processing. We therefore suggest that deeper molecular mechanistic investigations into critical PNS glial functions like ionic regulation are essential to comprehensively understand sensorineural health, disease, and aging.

Modeling of sustained spontaneous network oscillations of a sexually dimorphic brainstem nucleus: the role of potassium equilibrium potential.

Intrinsic oscillators in the central nervous system play a preeminent role in the neural control of rhythmic behaviors, yet little is known about how the ionic milieu regulates their output patterns. A powerful system to address this question is the pacemaker nucleus of the weakly electric fish Apteronotus leptorhynchus. A neural network comprised of an average of 87 pacemaker cells and 20 relay cells produces tonic oscillations, with higher frequencies in males compared to females. Previous empirical studies have suggested that this sexual dimorphism develops and is maintained through modulation of buffering of extracellular K+ by a massive meshwork of astrocytes enveloping the pacemaker and relay cells. Here, we constructed a model of this neural network that can generate sustained spontaneous oscillations. Sensitivity analysis revealed the potassium equilibrium potential, EK (as a proxy of extracellular K+ concentration), and corresponding somatic channel conductances as critical determinants of oscillation frequency and amplitude. In models of both the pacemaker nucleus network and isolated pacemaker and relay cells, the frequency increased almost linearly with EK, whereas the amplitude decreased nonlinearly with increasing EK. Our simulations predict that this frequency increase is largely caused by a shift in the minimum K+ conductance over one oscillation period. This minimum is close to zero at more negative EK, converging to the corresponding maximum at less negative EK. This brings the resting membrane potential closer to the threshold potential at which voltage-gated Na+ channels become active, increasing the excitability, and thus the frequency, of pacemaker and relay cells.

Label‐free subcellular mapping of iron‐bound proteins in breast cancer cells

Dysregulation of iron transport, storage and utilization is at the basis of a variety of human diseases, since free iron can participate in redox reactions that are deleterious to lipid membranes and signaling pathways. As intracellular iron transport occurs in a pH-dependent manner, changes in endosomal pH, often associated with cancer, could lead to alterations in iron homeostasis. Essential to iron transport is transferrin (Tf), which binds iron at a neutral pH for safe transport throughout the body and releases it upon uptake and delivery to a mildly acidified endosome. Excess iron can be stored in cytoplasmic protein ferritin (Ft) or delivered into mitochondria to be incorporated into proteins with heme or iron-sulfur structures. Recently, we have developed a novel label-free Raman hyperspectral microscopy methodology to identify the subcellular distributions and relative quantities of iron-bound Tf, Ft and heme proteins within heterogenous cell populations. Previously, we have shown that Raman hyperspectral imaging could identify endocytic distributions of iron-bound Tf using a specific Raman peak at 1300 cm-1 in intact breast cancer cells. Moreover, we have shown that alterations in the subcellular localization of iron-bound Tf populations correlated with altered endocytic regulation of iron-bound Tf in two distinct breast cancer cell lines. Based on these results, we propose that breast cancer cells display a disrupted iron homeostasis due to iron release delays caused by alterations in the pH or ionic milieu of the early endosomes. Currently, we are testing a putative correlation between pH gradient alterations and iron release delays in different breast cancer cells. To further investigate cellular iron regulation, Ft cell-specific Raman signature was determined via evaluation of cells incubated in the presence or absence of iron-loaded Ft. Since cells may internalize Ft through an endocytic pathway, this approach allowed for identification of Ft Raman signature. However, the subcellular distribution of internalized iron-loaded Ft may not be reflective of normal cytoplasmic Ft in cells. Internalization of iron-loaded Ft results in multiple significantly different Raman spectra peaks at several wavelengths. Importantly, we have used multivariate analysis to show a significant separation of cell Raman spectra from that of cells incubated with Ft. Further we can map a previously known heme protein peak at 1372 cm-1, which is specifically associated with the ferric iron heme. In the future, following further validation of Ft peaks the mapping of Tf, Ft and heme can be evaluated across multiple cell lines and tissue samples. The application of this research has far reaching potential in understanding how dysregulation of endocytic pH may lead to alterations in intracellular iron transport and storage in breast cancer cells.

Acid-induced gelation of enzymatically cross-linked caseinates: Small and large deformation rheology in relation to water holding capacity and micro-rheological properties

Caseinate dissolved in different ionic milieus was used as model substrate for cross-linking by microbial transglutaminase and subsequent acid-induced gelation. The study follows up on previous research, where the molecular properties of cross-linked casein nanoparticles were linked to certain rheological characteristics of acid gels (Raak et al., 2019, Food Hydrocoll 86:43–49), and presents additional insight into physical gel properties. The frequency dependency of the storage modulus followed a power law relationship, with the exponent being linearly related to the phase angle. The frequency dependency of the loss factor could be fitted with a second-order polynomial function, the minimum of which was shifted to lower frequency at increasing cross-linking extent. Strain sweep experiments showed a less pronounced strain hardening behaviour and reduced fracture strain with increasing cross-linking extent, indicating lower flexibility of cross-linked casein nanoparticles and a lower number of non-covalent bonds, respectively. The water holding capacities of the gels were in a good relationship with the fracture properties, suggesting that the serum drainage during centrifugation was also affected by the strain hardening behaviour. Additionally, diffusing wave spectroscopy showed that the casein particles interacted already prior to the gelation onset, and that the presence of Ca2+ ions increases the particle size in both the caseinate solutions and gels

Abundant Monovalent Ions as Environmental Signposts for Pathogens during Host Colonization.

Host colonization by a pathogen requires proper sensing and response to local environmental cues, to ensure adaptation and continued survival within the host. The ionic milieu represents a critical potential source of environmental cues, and indeed, there has been extensive study of the interplay between host and pathogen in the context of metals such as iron, zinc, and manganese, vital ions that are actively sequestered by the host. The inherent non-uniformity of the ionic milieu also extends, however, to "abundant" ions such as chloride and potassium, whose concentrations vary greatly between tissue and cellular locations, and with the immune response. Despite this, the concept of abundant ions as environmental cues and key players in host-pathogen interactions is only just emerging. Focusing on chloride and potassium, this review brings together studies across multiple bacterial and parasitic species that have begun to define both how these abundant ions are exploited as cues during host infection, and how they can be actively manipulated by pathogens during host colonization. The close links between ion homeostasis and sensing/response to different ionic signals, and the importance of studying pathogen response to cues in combination, are also discussed, while considering the fundamental insight still to be uncovered from further studies in this nascent area of inquiry.

Size Modulation of Enzymatically Cross-Linked Sodium Caseinate Nanoparticles via Ionic Strength Variation Affects the Properties of Acid-Induced Gels

Enzymatic cross-linking by microbial transglutaminase is a prominent approach to modify the structure and techno-functional properties of food proteins such as casein. However, some of the factors that influence structure-function-interrelations are still unknown. In this study, the size of cross-linked sodium caseinate nanoparticles was modulated by varying the ionic milieu during incubation with the enzyme. As was revealed by size exclusion chromatography, cross-linking at higher ionic strength resulted in larger casein particles. These formed acid-induced gels with higher stiffness and lower susceptibility to forced syneresis compared to those where the same number of ions was added after the cross-linking process. The results show that variations of the ionic milieu during enzymatic cross-linking of casein can be helpful to obtain specific modifications of its molecular structure and certain techno-functional properties. Such knowledge is crucial for the design of protein ingredients with targeted structure and techno-functionality.

Left Atrial Remodeling Mechanisms Associated with Atrial Fibrillation.

Heart disease has always been one of the important diseases that endanger health and cause death. Therefore, it is particularly important to understand left atrium reconstruction and atrial fibrillation before heart image processing. The purpose of this paper is to provide an important review of the mechanisms of left atrial remodeling (LAR) associated with atrial fibrillation (AF). LAR refers to the spectrum of pathophysiological changes in (i) atrial structure and physiological function, and (ii) electric, ionic, and molecular milieu of the LA, in response to stresses imposed by conditions such as hypertension, myocardial ischemia, autonomic denervation and congestive heart failure. The main mechanisms of LAR include electrical remodeling, structural remodeling, metabolic remodeling, autonomic remodeling, neurohormones and inflammation, and other influencing factors. LAR is not only the basic mechanism of AF and heart failure, but also the pathophysiological basis of its progression. In clinical practice, AF is the most common persistent arrhythmia, and is believed to be the result of a combination of mechanisms that have triggers and maintenance mechanisms, including spontaneous ectopic pacing and multiple wavelet reentry. While LA electrophysiological, structural, and ultra-structural changes trigger AF, in turn, AF alters the LA electrical and structural properties that promote its maintenance and recurrence. Chronic AF leads to extensive changes in atrial cellular substructures, including loss of myofibrils, accumulation of glycogen, changes in mitochondrial shape and size, fragmentation of sarcoplasmic reticulum, and dispersion of nuclear chromatin. Electrical remodeling and structural remodeling of the atria during AF, involving structural changes and functional impairment of the left atrium, can lead to serious decline in left ventricular function and severe heart failure. Therefore, LAR and AF are inter-activating phenomena, and the resulting complications can cause serious disabling and fatal events. In this paper, we present (i) the mechanisms of LAR, in the form of structural, electrical, metabolic, and neurohormonal changes, and (ii) their interactive roles in initiating and maintaining AF. These in-depth understanding of the atrial remodeling mechanisms can in turn provide useful insights into the treatment of AF and heart failure.

A Neurotoxic Ménage-à-trois: Glutamate, Calcium, and Zinc in the Excitotoxic Cascade.

Fifty years ago, the seminal work by John Olney provided the first evidence of the neurotoxic properties of the excitatory neurotransmitter glutamate. A process hereafter termed excitotoxicity. Since then, glutamate-driven neuronal death has been linked to several acute and chronic neurological conditions, like stroke, traumatic brain injury, Alzheimer’s, Parkinson’s, and Huntington’s diseases, and Amyotrophic Lateral Sclerosis. Mechanisms linked to the overactivation of glutamatergic receptors involve an aberrant cation influx, which produces the failure of the ionic neuronal milieu. In this context, zinc, the second most abundant metal ion in the brain, is a key but still somehow underappreciated player of the excitotoxic cascade. Zinc is an essential element for neuronal functioning, but when dysregulated acts as a potent neurotoxin. In this review, we discuss the ionic changes and downstream effects involved in the glutamate-driven neuronal loss, with a focus on the role exerted by zinc. Finally, we summarize our work on the fascinating distinct properties of NADPH-diaphorase neurons. This neuronal subpopulation is spared from excitotoxic insults and represents a powerful tool to understand mechanisms of resilience against excitotoxic processes.

Quantitative label-free imaging of iron-bound transferrin in breast cancer cells and tumors

Transferrin (Tf) is an essential serum protein which delivers iron throughout the body via transferrin-receptor (TfR)-mediated uptake and iron release in early endosomes. Currently, there is no robust method to assay the population of iron-bound Tf in intact cells and tissues. Raman hyperspectral imaging detected spectral peaks that correlated with iron-bound Tf in intact cells and tumor xenografts sections (~1270-1300 cm−1). Iron-bound (holo) and iron-free (apo) human Tf forms were endocytosed by MDAMB231 and T47D human breast cancer cells. The Raman iron-bound Tf peak was identified in cells treated with holo-Tf, but not in cells incubated with apo-Tf. A reduction in the Raman peak intensity between 5 and 30 min of Tf internalization was observed in T47D, but not in MDAMB231, suggesting that T47D can release iron from Tf more efficiently than MDAMB231. MDAMB231 may display a disrupted iron homeostasis due to iron release delays caused by alterations in the pH or ionic milieu of the early endosomes. In summary, we have demonstrated that Raman hyperspectral imaging can be used to identify iron-bound Tf in cell cultures and tumor xenografts and detect iron release behavior of Tf in breast cancer cells.

Phase Diagram of Purified CNS Myelin Reveals Continuous Transformation between Expanded and Compacted Lamellar States.

Purified myelin membranes (PMMs) are the starting material for biochemical studies, from individual components up to the isolation of detergent-resistant membrane (DRM) fractions or detergent-insoluble glycosphingolipid (DIG) fractions, which are commonly believed to resemble physiological lipid rafts. The normal DIG isolation protocol involves the extraction of lipids under moderate cooling. The isolation of PMMs also involves the cooling of myelin as well as exposure to low ionic strength (IS). Here, we addressed the combined influence of cooling and IS on the structure of PMMs. The phase behaviour was investigated by small angle X-ray diffraction. Analysis of the diffraction peaks revealed the lamellar periodicity (), the number of periodically correlated bilayers (), and the relatives fractions of each phase. Departure from physiological conditions induced a phase separation in myelin. The effect of monovalent and divalent ions was also compared at equivalent IS, showing a differential effect, and phase diagrams for both ion types were established—Ca2+ induced the well-known over-compacted phase, but additionally we also found an expanded phase at low IS. Na+ promoted phase separation, and also induced over-compaction at sufficiently high IS. Finally, exploring the whole phase diagram, we found evidence for the direct isothermal transformation from the expanded to the compacted phase, suggesting that both phases could in fact originate from the identical primary lateral phase separation, whereas the apparent difference lies in the inter-bilayer interaction that is modulated by the ionic milieu.

Cannabinoids, TRPV and nitric oxide: the three ring circus of neuronal excitability.

Endocannabinoid system is considered a relevant player in the regulation of neuronal excitability, since it contributes to maintaining the balance of the synaptic ionic milieu. Perturbations to bioelectric conductances have been implicated in the pathophysiological processes leading to hyperexcitability and epileptic seizures. Cannabinoid influence on neurosignalling is exerted on classic receptor-mediated mechanisms or on further molecular targets. Among these, transient receptor potential vanilloid (TRPV) are ionic channels modulated by cannabinoids that are involved in the transduction of a plethora of stimuli and trigger fundamental downstream pathways in the post-synaptic site. In this review, we aim at providing a brief summary of the most recent data about the cross-talk between cannabinoid system and TRPV channels, drawing attention on their role on neuronal hyperexcitability. Then, we aim to unveil a plausible point of interaction between these neural signalling systems taking into consideration nitric oxide, a gaseous molecule inducing profound modifications to neural performances. From this novel perspective, we struggle to propose innovative cellular mechanisms in the regulation of hyperexcitability phenomena, with the goal of exploring plausible CB-related mechanisms underpinning epileptic seizures.

Potassium response and homeostasis in Mycobacterium tuberculosis modulates environmental adaptation and is important for host colonization.

Successful host colonization by bacteria requires sensing and response to the local ionic milieu, and coordination of responses with the maintenance of ionic homeostasis in the face of changing conditions. We previously discovered that Mycobacterium tuberculosis (Mtb) responds synergistically to chloride (Cl-) and pH, as cues to the immune status of its host. This raised the intriguing concept of abundant ions as important environmental signals, and we have now uncovered potassium (K+) as an ion that can significantly impact colonization by Mtb. The bacterium has a unique transcriptional response to changes in environmental K+ levels, with both distinct and shared regulatory mechanisms controlling Mtb response to the ionic signals of K+, Cl-, and pH. We demonstrate that intraphagosomal K+ levels increase during macrophage phagosome maturation, and find using a novel fluorescent K+-responsive reporter Mtb strain that K+ is not limiting during macrophage infection. Disruption of Mtb K+ homeostasis by deletion of the Trk K+ uptake system results in dampening of the bacterial response to pH and Cl-, and attenuation in host colonization, both in primary murine bone marrow-derived macrophages and in vivo in a murine model of Mtb infection. Our study reveals how bacterial ionic homeostasis can impact environmental ionic responses, and highlights the important role that abundant ions can play during host colonization by Mtb.

Acid-induced gelation of enzymatically cross-linked caseinate in different ionic milieus

Acid casein powder was used to prepare caseinate solutions (27 g/kg) with different ionic milieus: sodium caseinate (NaCN, I ∼0.015 mol/L) and calcium caseinate (CaCN, I ∼0.03 mol/L) were obtained by neutralisation with NaOH and Ca(OH)2, respectively, and dissolving in phosphate buffer resulted in a high ionic strength caseinate solution (CN-PB, I ∼0.16 mol/L). Treatment with microbial transglutaminase (mTGase) for defined incubation times lead to different extents of casein cross-linking, which were characterised by size exclusion chromatography and the N-ε-(γ-glutamyl)-lysine isopeptide content (IC). Maximum polymer size was reached at ∼90% casein polymerisation, and increased in the order NaCN < CN-PB < CaCN. Further enzyme treatment, however, increased the IC, pointing to cross-links within existing polymers. We suggest that the maximum polymer size is determined by the size of casein particles resulting from self-assembly in solution and that mTGase preferably acts on molecules within the same particle. Enhanced association at higher ionic strength (CN-PB) or in the presence of bivalent cations (CaCN) may therefore result in larger covalently cross-linked casein polymers. Furthermore, oscillation rheometry revealed that the relationship between casein cross-linking and stiffness of gels acidified with glucono-δ-lactone depends on the ionic milieu. While for NaCN G'MAX increased with the time of cross-linking, the presence of ions resulted in the highest G'MAX at moderate cross-linking intensities. This was also observed when NaCl was added to cross-linked NaCN. The results suggest that electrostatic attraction during gel formation are interfered by ions and cannot be compensated by rearrangements in case of extensively cross-linked casein particles.

Introducing biomimetic shear and ion gradients to microfluidic spinning improves silk fiber strength

Silkworm silk is an attractive biopolymer for biomedical applications due to its high mechanical strength and biocompatibility; as a result, there is increasing interest in scalable devices to spin silk and recombinant silk so as to improve and customize their properties for diverse biomedical purposes (Vepari and Kaplan 2007 Prog. Polym. Sci. 32). While artificial spinning of regenerated silk fibroins adds tunability to properties such as degradation rate and surface functionalization, the resulting fibers do not yet approach the mechanical strength of native silkworm silk. These drawbacks reduce the applicability and attractiveness of artificial silk (Kinahan et al 2011 Biomacromolecules 12). Here, we used computational fluid dynamic simulations to incorporate shear in tandem with biomimetic ion gradients by coupling a modular novel glass microfluidic device to our previous co-axial flow device. Fibers spun with this combined apparatus demonstrated a significant increase in mechanical strength compared to fibers spun with the basic apparatus alone, with a three-fold increase in Young’s modulus and extensibility and a twelve-fold increase in toughness. These results thus demonstrate the critical importance of ionic milieu and shear stress in spinning strong fibers from solubilized silk fibroin.