Native Physiological Environment Research Articles

NaV1.8/NaV1.9 double deletion mildly affects acute pain responses in mice.

The 2 tetrodotoxin-resistant (TTXr) voltage-gated sodium channel subtypes NaV1.8 and NaV1.9 are important for peripheral pain signaling. As determinants of sensory neuron excitability, they are essential for the initial transduction of sensory stimuli, the electrogenesis of the action potential, and the release of neurotransmitters from sensory neuron terminals. NaV1.8 and NaV1.9, which are encoded by SCN10A and SCN11A, respectively, are predominantly expressed in pain-sensitive (nociceptive) neurons localized in the dorsal root ganglia (DRG) along the spinal cord and in the trigeminal ganglia. Mutations in these genes cause various pain disorders in humans. Gain-of-function missense variants in SCN10A result in small fiber neuropathy, while distinct SCN11A mutations cause, i. a., congenital insensitivity to pain, episodic pain, painful neuropathy, and cold-induced pain. To determine the impact of loss-of-function of both channels, we generated NaV1.8/NaV1.9 double knockout (DKO) mice using clustered regularly interspaced short palindromic repeats/Cas-mediated gene editing to achieve simultaneous gene disruption. Successful knockout of both channels was verified by whole-cell recordings demonstrating the absence of NaV1.8- and NaV1.9-mediated Na+ currents in NaV1.8/NaV1.9 DKO DRG neurons. Global RNA sequencing identified significant deregulation of C-LTMR marker genes as well as of pain-modulating neuropeptides in NaV1.8/NaV1.9 DKO DRG neurons, which fits to the overall only moderately impaired acute pain behavior observed in DKO mice. Besides addressing the function of both sodium channels in pain perception, we further demonstrate that the null-background is a very valuable tool for investigations on the functional properties of individual human disease-causing variants in NaV1.8 or NaV1.9 in their native physiological environment.

Engineering a Biomimetic Glomerular Filtration Barrier: Coculturing Endothelial Podocytes on Kidney ECM-Bacterial Cellulose Membrane Hybrid.

A novel avenue for advancing our understanding of kidney disease mechanisms and developing targeted therapeutics lies in overcoming the limitations of the existing in vitro models. Traditional animal models, while useful, do not fully capture the intricacies of human kidney physiology and pathophysiology. Tissue engineering offers a promising solution, yet current models often fall short in replicating the complex microarchitecture and biochemical milieu of the kidney. To address this challenge, we propose the development of a sophisticated in vitro glomerular filtration barrier (GFB) utilizing advanced biomaterials and a kidney decellularized extracellular matrix (kdECM). In our approach, we employ a bacterial cellulose membrane (BC) as a scaffold, providing a robust framework for cell growth and interaction. Coating this scaffold with kdECM hydrogel derived from caprine kidney tissue via a detergent-free decellularization method ensures the preservation of vital extracellular matrix proteins crucial for cellular compatibility and signaling. Our engineered GFB not only supports the growth of endothelial and podocyte cells but also exhibits the presence of key markers such as CD31 and nephrin, indicating successful cellular integration. Furthermore, the expression of collagen IV, an essential extracellular matrix (ECM) protein, validates the fidelity of our model in simulating cellular interactions within a kdECM matrix. Additionally, we assessed the filtration efficiency of the developed GFB model using albumin, a standard protein, to evaluate its performance under conditions that closely mimic the native physiological environment. This innovative approach, which faithfully recapitulates the native microenvironment of the glomerulus, holds immense promise for elucidating kidney disease mechanisms, conducting permeability studies, and advancing personalized therapeutic strategies. By leveraging cutting-edge biomaterials and tissue-specific coculture technology, this study can be further extended to develop GFB for the treatment of renal diseases, ultimately improving patient outcomes and quality of life.

Chemically cross-linked hydrogels from repetitive protein arrays

Biomaterials for tissue regeneration must mimic the biophysical properties of the native physiological environment. A protein engineering approach allows the generation of protein hydrogels with specific and customised biophysical properties designed to suit a particular physiological environment. Herein, repetitive engineered proteins were successfully designed to form covalent molecular networks with defined physical characteristics able to sustain cell phenotype. Our hydrogel design was made possible by the incorporation of the SpyTag (ST) peptide and multiple repetitive units of the SpyCatcher (SC) protein that spontaneously formed covalent crosslinks upon mixing. Changing the ratios of the protein building blocks (ST:SC), allowed the viscoelastic properties and gelation speeds of the hydrogels to be altered and controlled. The physical properties of the hydrogels could readily be altered further to suit different environments by tuning the key features in the repetitive protein sequence. The resulting hydrogels were designed with a view to allow cell attachment and encapsulation of liver derived cells. Biocompatibility of the hydrogels was assayed using a HepG2 cell line constitutively expressing GFP. The cells remained viable and continued to express GFP whilst attached or encapsulated within the hydrogel. Our results demonstrate how this genetically encoded approach using repetitive proteins could be applied to bridge engineering biology with nanotechnology creating a level of biomaterial customisation previously inaccessible.

Rubisco Function, Evolution, and Engineering.

Carbon fixation is the process by which CO2 is converted from a gas into biomass. The Calvin-Benson-Bassham cycle (CBB) is the dominant carbon-consuming pathway on Earth, driving >99.5% of the ∼120 billion tons of carbon that are converted to sugar by plants, algae, and cyanobacteria. The carboxylase enzyme in the CBB, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco), fixes one CO2 molecule per turn of the cycle into bioavailable sugars. Despite being critical to the assimilation of carbon, rubisco's kinetic rate is not very fast, limiting flux through the pathway. This bottleneck presents a paradox: Why has rubisco not evolved to be a better catalyst? Many hypothesize that the catalytic mechanism of rubisco is subject to one or more trade-offs and that rubisco variants have been optimized for their native physiological environment. Here, we review the evolution and biochemistry of rubisco through the lens of structure and mechanism in order to understand what trade-offs limit its improvement. We also review the many attempts to improve rubisco itself and thereby promote plant growth.

Getting to the heart of it - Small GTPase activity probed at an atomic level in cells.

Ras homology protein (RhoA), a small G protein (SmG), plays a crucial pathophysiological role and is implicated in cardiovascular diseases such as diabetes, heart failure, hypertension, and stroke. RhoA regulates cytoskeleton remodeling through actin fibrillation and aids in overall cell survival. Like all SmGs that function as regulatory switches, the amount of RhoA in the heart is delicately controlled, with large changes resulting in faulty signaling that can lead to cardiac disease. ∼2-fold overexpression protects myocytes from ischemic injury and decreases fibrosis during the stress response, while a large excess (∼20-fold) is associated with cardiac hypertrophy, fibrosis, and cardiomyopathy. A myriad of studies have been conducted on the structure-function relationship of SmGs, with most structural biology and biophysical investigations employing a reductionist in vitro strategy in which the native physiological context is ignored. In order to assess how proteins work in vivo, there is a growing emphasis to investigate systems in a native physiological environment. In this presentation, I will report on my investigation of conformational dynamics and enzymatic activity of the RhoA protein in a crowded environment. I will discuss RhoA behavior using 2D 15N-1H NMR, 19F NMR and fluorescence spectroscopy and will present my findings on both the enzyme activity and conformations of GDP- and GTP- bound RhoA under the influence of macromolecular crowders, small molecule metabolite crowding, and the role of cardio-specific metabolites. In particular, I will use 19F nuclei as robust cellular NMR tracers and reporters on live in-cell using19F labeled RhoA protein

Generating intravital super-resolution movies with conventional microscopy reveals actin dynamics that construct pioneer axons

ABSTRACTSuper-resolution microscopy is broadening our in-depth understanding of cellular structure. However, super-resolution approaches are limited, for numerous reasons, from utilization in longer-term intravital imaging. We devised a combinatorial imaging technique that combines deconvolution with stepwise optical saturation microscopy (DeSOS) to circumvent this issue and image cells in their native physiological environment. Other than a traditional confocal or two-photon microscope, this approach requires no additional hardware. Here, we provide an open-access application to obtain DeSOS images from conventional microscope images obtained at low excitation powers. We show that DeSOS can be used in time-lapse imaging to generate super-resolution movies in zebrafish. DeSOS was also validated in live mice. These movies uncover that actin structures dynamically remodel to produce a single pioneer axon in a ‘top-down’ scaffolding event. Further, we identify an F-actin population – stable base clusters – that orchestrate that scaffolding event. We then identify that activation of Rac1 in pioneer axons destabilizes stable base clusters and disrupts pioneer axon formation. The ease of acquisition and processing with this approach provides a universal technique for biologists to answer questions in living animals.

Growth- and Stress-Induced PASTA Kinase Phosphorylation in Enterococcus faecalis.

Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes Such PASTA kinases regulate critical processes, including antibiotic resistance, cell division, toxin production, and virulence, and are essential for viability in certain organisms. Based on in vitro studies with purified extracellular and intracellular fragments of PASTA kinases, a model for signaling has been proposed, in which the extracellular PASTA domains bind currently undefined ligands (typically thought to be peptidoglycan, or fragments thereof) to drive kinase dimerization, which leads to enhanced kinase autophosphorylation and enhanced phosphorylation of substrates. However, this model has not been rigorously tested in vivoEnterococcus faecalis is a Gram-positive intestinal commensal and major antibiotic-resistant opportunistic pathogen. In E. faecalis, the PASTA kinase IreK drives intrinsic resistance to cell wall-active antimicrobials, suggesting that such antimicrobials may trigger IreK signaling. Here we show that IreK responds to cell wall stress in vivo by enhancing its phosphorylation and that of a downstream substrate. This response requires both the extracellular PASTA domains and specific phosphorylatable residues in the kinase domain. Thus, our results provide in vivo evidence, with an intact full-length PASTA kinase in its native physiological environment, that supports the prevailing model of PASTA kinase signaling. In addition, we show that IreK responds to a signal associated with growth and/or cell division, in the absence of cell wall-active antimicrobials. Surprisingly, the ability of IreK to respond to growth and/or division does not require the extracellular PASTA domains, suggesting that IreK monitors multiple parameters for sensory input in vivoIMPORTANCE Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes and regulate critical processes. The prevailing model for signaling by PASTA kinases proposes that the extracellular PASTA domains bind ligands to drive kinase dimerization, enhanced autophosphorylation, and enhanced phosphorylation of substrates. However, this model has not been rigorously tested in vivo We show that the PASTA kinase IreK of Enterococcus faecalis responds to cell wall stress in vivo by enhancing its phosphorylation and that of a downstream substrate. This response requires the PASTA domains and phosphorylatable residues in the kinase domain. Thus, our results provide in vivo evidence, with an intact full-length PASTA kinase in its native physiological environment, that supports the prevailing model of PASTA kinase signaling.

Convergence of Highly Resolved and Rapid Screening Platforms with Dynamically Engineered, Cell Phenotype-Prescriptive Biomaterials.

Biophysical and biochemical cues from the cellular microenvironment initiate intracellular signaling through cellular membrane receptors and trigger specific cell developmental programs. Extracellular substrates and matrix scaffolds engineered to mimic cell's native physiological environment must incorporate the multifactorial parameters (composition, micro and nanoscale organization and topography) of the extracellular matrix as well as the dynamic nature of the matrix. The design of such engineered biomaterials is challenged by the inherent complexity and dynamic nature of the cell-extracellular matrix reciprocity, while the validation of robust microenvironments requires a deeper, higher content phenotypic resolution of cell-matrix interactions alongside a rapid screening capability. To this end, high-throughput platforms are integral to facilitating the screening and optimization of complex engineered microenvironments for directing desired cell developmental pathway. This review highlights the recent advances in biomaterial platforms that present dynamic cues and enable high throughput screening of cell's response to a combination of micro-environmental factors. We also address some newer techniques involving high content image informatics to elucidate emergent cellular behaviors with a focus on stem cell regenerative endpoints.

Labeling Monomeric Insulin with Renal-Clearable Luminescent Gold Nanoparticles.

In the native physiological environment, inorganic nanoparticles (NPs) often induce nonspecific protein adsorption, which could significantly alter the function of the proteins they labeled. As a result, small fluorescent dyes are still widely used in the imaging of proteins in animals due to their minimal interference with protein function. Here, we used monomeric insulin as a model and compared its bioactivity before and after labeling with renal-clearable near-infrared-emitting gold NPs. These NPs were chosen because they have high resistance to serum protein adsorption and low nonspecific accumulation. We have found that a 1:1 insulin-NP ratio can be achieved, where the insulin-NPs show minimal serum protein binding with fully retained bioactivity comparable to that of unlabeled insulin. These results show a proof of concept that renal-clearable NPs can behave like small molecules in protein labeling without changing the individual protein's function, laying down a foundation for in vivo tracking of proteins with multimodality imaging techniques.

Zigzag Generalized Lévy Walk: the In Vivo Search Strategy of Immunocytes.

Immune responses are based on the coordinated searching behaviors of immunocytes that are aimed at tracking down specific targets. The search efficiency of immunocytes significantly affects the speed of initiation and development of immune responses. Previous studies have shown that not only the intermittent walk but also the zigzag turning preference of immunocytes contributes to the search efficiency. However, among existing models describing immunocytes' search strategy, none has captured both features. Here we propose a zigzag generalized Lévy walk model to describe the search strategy of immunocytes more accurately and comprehensively by considering both the intermittent and the zigzag-turning walk features. Based on the analysis of the searching behaviors of typical immune cell types, dendritic cells and leukocytes, in their native physiological environment, we demonstrate that the model can describe the in vivo search strategy of immunocytes well. Furthermore, by analyzing the search efficiency, we find that this type of search strategy enables immunocytes to capture rare targets in approximately half the time than the previously proposed generalized Lévy walk. This study sheds new light on the fundamental mechanisms that drive the efficient initiation and development of immune responses and in turn may lead to the development of novel therapeutic approaches for diseases ranging from infection to cancer.

Nonlinear optical microscopy for immunoimaging: a custom optimized system of high-speed, large-area, multicolor imaging.

The nonlinear optical microscopy has become the current state-of-the-art for intravital imaging. Due to its advantages of high resolution, superior tissue penetration, lower photodamage and photobleaching, as well as intrinsic z-sectioning ability, this technology has been widely applied in immunoimaging for a decade. However, in terms of monitoring immune events in native physiological environment, the conventional nonlinear optical microscope system has to be optimized for live animal imaging. Generally speaking, three crucial capabilities are desired, including high-speed, large-area and multicolor imaging. Among numerous high-speed scanning mechanisms used in nonlinear optical imaging, polygon scanning is not only linearly but also dispersion-freely with high stability and tunable rotation speed, which can overcome disadvantages of multifocal scanning, resonant scanner and acousto-optical deflector (AOD). However, low frame rate, lacking large-area or multicolor imaging ability make current polygonbased nonlinear optical microscopes unable to meet the requirements of immune event monitoring. We built up a polygon-based nonlinear optical microscope system which was custom optimized for immunoimaging with high-speed, large-are and multicolor imaging abilities. Firstly, we validated the imaging performance of the system by standard methods. Then, to demonstrate the ability to monitor immune events, migration of immunocytes observed by the system based on typical immunological models such as lymph node, footpad and dorsal skinfold chamber are shown. Finally, we take an outlook for the possible advance of related technologies such as sample stabilization and optical clearing for more stable and deeper intravital immunoimaging. This study will be helpful for optimizing nonlinear optical microscope to obtain more comprehensive and accurate information of immune events.

Imaging Fast Calcium Currents beyond the Limitations of Electrode Techniques

The current understanding of Ca2+ channel function is derived from the use of the patch-clamp technique. In particular, the measurement of fast cellular Ca2+ currents is routinely achieved using whole-cell voltage-clamp recordings. However, this experimental approach is not applicable to the study of local native Ca2+ channels during physiological changes of membrane potential in complex cells, since the voltage-clamp configuration constrains the membrane potential to a given value. Here, we report for the first time to our knowledge that Ca2+ currents from individual cells can be quantitatively measured beyond the limitations of the voltage-clamp approach using fast Ca2+ imaging with low-affinity indicators. The optical measurement of the Ca2+ current was correlated with the membrane potential, simultaneously measured with a voltage-sensitive dye to investigate the activation of Ca2+ channels along the apical dendrite of the CA1 hippocampal pyramidal neuron during the back-propagation of an action potential. To validate the method, we analyzed the voltage dependence of high- and low-voltage-gated Ca2+ channels. In particular, we measured the Ca2+ current component mediated by T-type channels, and we investigated the mechanisms of recovery from inactivation of these channels. This method is expected to become a reference approach to investigate Ca2+ channels in their native physiological environment.

Long-term culture of bovine nucleus pulposus explants in a native environment

Background contextChronic low back pain is a disease with tremendous financial and social implications, and it is often caused by intervertebral disc degeneration. Regenerative therapies for disc repair are promising treatments, but they need to be tested in physiological models. PurposeTo develop a physiological in vitro explant model that incorporates the native environment of the intervertebral disc, for example, hypoxia, low glucose, and high tissue osmolarity. Study designBovine nucleus pulposus (NP) explants were cultured for 42 days in conditions mimicking the native physiological environment. Two different approaches were used to balance the swelling pressure of the NP: raised medium osmolarity or an artificial annulus. MethodsBovine NP explants were either cultured in media with osmolarity balanced at isotonic and hypertonic levels compared with the native tissue or cultured inside a fiber jacket used as an artificial annulus. Oxygen and glucose levels were set at either standard (21% O2 and 4.5 g/L glucose) or physiological (5% O2 and 1 g/L glucose) levels. Samples were analyzed at Day 0, 3, and 42 for tissue composition (water, sulfated glycosaminoglycans, DNA, and hydroxyproline contents and fixed charge density), tissue histology, cell viability, and cellular behavior with messenger RNA (mRNA) expression. ResultsBoth the hypertonic culture and the artificial annulus approach maintained the tissue matrix composition for 42 days. At Day 3, mRNA expressions of aggrecan, collagen Type I, and collagen Type II in both hypertonic and artificial annulus cultures were not different from Day 0; however, at Day 42, the artificial annulus preserved the mRNA expression closer to Day 0. Gene expressions of matrix metalloprotease 13, tissue inhibitor of matrix metalloprotease 1, and tissue inhibitor of matrix metalloprotease 2 were downregulated under physiological O2 and glucose levels, whereas the other parameters analyzed were not affected. ConclusionsAlthough the hypertonic culture and the artificial annulus approach are both promising models to test regenerative therapies, the artificial annulus was better able to maintain a cellular behavior closer to the native tissue in longer term cultures.

A Little Engine That Could: ATP-Powered Electrical Battery and Heater Inside Cells

A Little Engine That Could: ATP-Powered Electrical Battery and Heater Inside Cells

High Throughput Screening for Small Molecule Therapy for Gaucher Disease Using Patient Tissue as the Source of Mutant Glucocerebrosidase

Gaucher disease (GD), the most common lysosomal storage disorder, results from the inherited deficiency of the lysosomal enzyme glucocerebrosidase (GCase). Previously, wildtype GCase was used for high throughput screening (HTS) of large collections of compounds to identify small molecule chaperones that could be developed as new therapies for GD. However, the compounds identified from HTS usually showed reduced potency later in confirmatory cell-based assays. An alternate strategy is to perform HTS on mutant enzyme to identify different lead compounds, including those enhancing mutant enzyme activities. We developed a new screening assay using enzyme extract prepared from the spleen of a patient with Gaucher disease with genotype N370S/N370S. In tissue extracts, GCase is in a more native physiological environment, and is present with the native activator saposin C and other potential cofactors. Using this assay, we screened a library of 250,000 compounds and identified novel modulators of mutant GCase including 14 new lead inhibitors and 30 lead activators. The activities of some of the primary hits were confirmed in subsequent cell-based assays using patient-derived fibroblasts. These results suggest that primary screening assays using enzyme extracted from tissues is an alternative approach to identify high quality, physiologically relevant lead compounds for drug development.

In Situ Imaging of Metals in Cells and Tissues

In Situ Imaging of Metals in Cells and Tissues

Frequency modulation atomic force microscopy in ambient environments utilizing robust feedback tuning

Frequency modulation atomic force microscopy (FM-AFM) is rapidly evolving as the technique of choice in the pursuit of high resolution imaging of biological samples in ambient environments. The enhanced stability afforded by this dynamic AFM mode combined with quantitative analysis enables the study of complex biological systems, at the nanoscale, in their native physiological environment. The operational bandwidth and accuracy of constant amplitude FM-AFM in low Q environments is heavily dependent on the cantilever dynamics and the performance of the demodulation and feedback loops employed to oscillate the cantilever at its resonant frequency with a constant amplitude. Often researchers use ad hoc feedback gains or instrument default values that can result in an inability to quantify experimental data. Poor choice of gains or exceeding the operational bandwidth can result in imaging artifacts and damage to the tip and/or sample. To alleviate this situation we present here a methodology to determine feedback gains for the amplitude and frequency loops that are specific to the cantilever and its environment, which can serve as a reasonable "first guess," thus making quantitative FM-AFM in low Q environments more accessible to the nonexpert. This technique is successfully demonstrated for the low Q systems of air (Q approximately 40) and water (Q approximately 1). In addition, we present FM-AFM images of MC3T3-E1 preosteoblast cells acquired using the gains calculated by this methodology demonstrating the effectiveness of this technique.

Mirk/Dyrk1B: A Multifunctional Dual-Specificity Kinase Involved in Growth Arrest, Differentiation, and Cell Survival

Minibrain-related kinase (Mirk)/dual-specificity tyrosine-regulated kinase (Dyrk)1B is one of the best functionally characterized members of the Dyrk/Minibrain family of dual-specificity kinases. Dyrk family kinases are highly conserved mediators of growth control and differentiation. Mirk is expressed at high levels in skeletal muscle; thus, most of the recent studies of Mirk have used myogenesis as a model system to explore the function of Mirk in a native physiological environment. These studies have revealed that Mirk is a multifunctional Ser/Thr kinase that plays a critical role in muscle differentiation by regulatory effects on motility, transcription, cell cycle progression, and cell survival. Mirk also is found at elevated levels in various solid tumors, where it seems to act as a tumor survival factor. This review summarizes the known regulators and functions of Mirk kinase and outlines opportunities for future studies of Mirk in the fields of muscle and tumor biology.