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The escalating contamination caused by lead ions (Pb 2 ⁺) and its harmful effects on all life forms has raised global concerns. Certain microalgae thrive in metal mining sites characterized by low pH and high concentrations of Pb 2 ⁺, which are usually prohibitive for many microorganisms. Little is known about the mechanisms underlying the adaptation of such microalgae to these hostile conditions. In this study, we elucidated the adaptive strategies of the green microalga Micractinium belenophorum strain AUMW, isolated from a lead mining site, and its application for the removal of Pb +2 . Results revealed that strain AUMW can efficiently tolerate up to 200 ppm of Pb +2 in an F/2 medium. Further experimental variables were optimized through response surface methodology (RSM), and 99.6 % removal of Pb 2 ⁺ was achieved. Novel adaptive responses of strain AUMW to high levels of Pb 2 ⁺ include: (i) activation of metal-protective response by modulation of quantum yield ( F v /F m ) and non-photochemical quenching (NPQ) of photosystem II; (ii) extracellular silicification encapsulated cells of strain AUMW and altered cell morphology from oval to hexagonal; (iii) silicification prevented intracellular translocation of Pb +2 ; (iv) silicification boosted adsorption of Pb +2 , thus enhanced its removal. This study offers new insights into the protective role of silicification in green microalgae and its potential for the removal of metals from metal-polluted sites, waste from energy storage battery industries, and spent batteries. It also provides a solid base to explore the genetic and metabolic pathways involved in the adaptation of strain AUMW to elevated levels of Pb +2 ., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Cells evolved some 4 billion years ago, and since then the integrity of the structural and functional continuity of cellular life has been maintained via highly conserved and ancient processes of cell reproduction and division. The plasma membrane as well as all the cytoplasmic structures are reproduced and inherited uninterruptedly by each of the two daughter cells resulting from every cell division. Although our understanding of the evolutionary emergence of the very first cells is obscured by the extremely long timeline since that revolutionary event, the generally accepted position is that the de novo formation of cells is not possible; all present cells are products of other prior cells. This essential biological principle was first discovered by Robert Remak and then effectively coined as Omnis Cellula e Cellula (every cell of the cell) by Rudolf Virchow: all currently living cells have direct structural and functional connections to the very first cells. Based on our previous theoretical analysis, all cells are endowed with individual sentient cognition that guides their individual agency, behaviour and evolution. There is a vital consequence of this new sentient and cognitive view of cells: when cells assemble as functional tissue ecologies and organs within multicellular organisms, including plants, animals and humans, these cellular aggregates display derivative versions of aggregate tissue- and organ-specific sentience and consciousness. This innovative view of the evolution and physiology of all currently living organisms supports a singular principle: all organismal physiology is based on cellular physiology that extends from unicellular roots., (© 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Aquaporin-1 (Aqp1), a water channel, has garnered significant interest for cell-based medicine and in vivo synthetic biology due to its ability to be genetically encoded to produce magnetic resonance signals by increasing the rate of water diffusion in cells. However, concerns regarding the effects of Aqp1 overexpression and increased membrane diffusivity on cell physiology have limited its widespread use as a deep-tissue reporter. In this study, we present evidence that Aqp1 generates strong diffusion-based magnetic resonance signals without adversely affecting cell viability or morphology in diverse cell lines derived from mice and humans. Our findings indicate that Aqp1 overexpression does not induce ER stress, which is frequently associated with heterologous expression of membrane proteins. Furthermore, we observed that Aqp1 expression had no detrimental effects on native biological activities, such as phagocytosis, immune response, insulin secretion, and tumor cell migration in the analyzed cell lines. These findings should serve to alleviate any lingering safety concerns regarding the utilization of Aqp1 as a genetic reporter and should foster its broader application as a noninvasive reporter for in vivo studies., (© 2024. The Author(s).)

The PHB2 gene is located on chromosome 12p13 and encodes prohibitin 2, a highly conserved protein of 37 kDa. PHB2 is a dimer with antiparallel coils, possessing a unique negatively charged region crucial for its mitochondrial molecular chaperone functions. Thus, PHB2 plays a significant role in cell life activities such as mitosis, mitochondrial autophagy, signal transduction, and cell death. This review discusses how PHB2 inhibits transcription factors or nuclear receptors to maintain normal cell functions; how PHB2 in the cytoplasm or membrane ensures normal cell mitosis and regulates cell differentiation; how PHB2 affects mitochondrial structure, function, and cell apoptosis through mitochondrial intimal integrity and mitochondrial autophagy; how PHB2 affects mitochondrial stress and inhibits cell apoptosis by regulating cytochrome c migration and other pathways; how PHB2 affects cell growth, proliferation, and metastasis through a mitochondrial independent mechanism; and how PHB2 could be applied in disease treatment. We provide a theoretical basis and an innovative perspective for a comprehensive understanding of the role and mechanism of PHB2 in cell function regulation., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Carbon-based quantum dots (CQDs) have been shown to have promising application value in tumor diagnosis. Their use, however, is severely hindered by the complicated nature of the nanostructures in the CQDs. Furthermore, it seems impossible to formulate the mechanisms involved using the inadequate theoretical frameworks that are currently available for CQDs. In this review, we re-consider the structure-property relationships of CQDs and summarize the current state of development of CQDs-based tumor diagnosis based on biological theories that are fully developed. The advantages and deficiencies of recent research on CQDs-based tumor diagnosis are thus explained in terms of the manifestation of nine essential changes in cell physiology. This review makes significant progress in addressing related problems encountered with other nanomaterials., (© 2023 The Authors.)

In the microbial world, cell size and shape impact physiology, but students struggle to visualize spatial relationships between cells and macromolecules. In prokaryotic cells, cell size is limited by reliance on diffusion for nutrient uptake and the transport of nutrients within the cell. Cells must also meet a minimum size threshold to accommodate essential cellular components such as ribosomes and DNA. Using 3D printing allows for the creation of custom models that can be influential teaching tools in the biology classroom. This lesson uses 3D cell models to teach students enrolled in an introductory microbiology course about bacterial cell size and the biological importance of surface-area-to-volume ratio. During the lesson, students interact with 3D cell models and discuss a series of questions in small groups. Student learning was assessed using quantitative and qualitative student response data collected pre- and post-lesson. Student achievement of learning objectives, and their confidence in their knowledge of these concepts, improved post-lesson, and these gains were statistically significant. Our findings suggest that interacting with 3D-printed cell models improves student understanding about bacterial cell size and diffusion., Competing Interests: The authors declare no conflict of interest., (Copyright © 2023 Wollmuth et al.)

Kidney tubuloids are cell models that are derived from human or mouse renal epithelial cells and show high similarities with their in vivo counterparts. Tubuloids grow polarized in 3D, allow for long-term expansion, and represent multiple segments of the nephron, as shown by their gene expression pattern. In addition, human tubuloids form tight, functional barriers and have been succesfully used for drug testing. Our knowledge of mouse tubuloids, on the other hand, is only minimal. In this study, we further characterized mouse tubuloids and differentiated them towards the collecting duct, which led to a significant upregulation of collecting duct-specific mRNAs of genes and protein expression, including the water channel AQP2 and the sodium channel ENaC. Differentiation resulted in polarized expression of collecting duct water channels AQP2 and AQP3. Also, a physiological response to desmopressin and forskolin stimulation by translocation of AQP2 to the apical membrane was demonstrated. Furthermore, amiloride-sensitive ENaC-mediated sodium uptake was shown in differentiated tubuloids using radioactive tracer sodium. This study demonstrates that mouse tubuloids can be differentiated towards the collecting duct and exhibit collecting duct-specific function. This illustrates the potential use of mouse kidney tubuloids as novel in vitro models to study (patho)physiology of kidney diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Olde Hanhof, Dilmen, Yousef Yengej, Latta, Ammerlaan, Schreurs, Hooijmaijers, Jansen, Rookmaaker, Orhon, Verhaar and Hoenderop.)

Acute oxygen (O 2 ) sensing and adaptation to hypoxia are essential for physiological homeostasis. The prototypical acute O 2 sensing organ is the carotid body, which contains chemosensory glomus cells expressing O 2 -sensitive K + channels. Inhibition of these channels during hypoxia leads to cell depolarization, transmitter release, and activation of afferent sensory fibers terminating in the brain stem respiratory and autonomic centers. Focusing on recent data, here we discuss the special sensitivity of glomus cell mitochondria to changes in O 2 tension due to Hif2α-dependent expression of several atypical mitochondrial electron transport chain subunits and enzymes. These are responsible for an accelerated oxidative metabolism and the strict dependence of mitochondrial complex IV activity on O 2 availability. We report that ablation of Epas1 (the gene coding Hif2α) causes a selective downregulation of the atypical mitochondrial genes and a strong inhibition of glomus cell acute responsiveness to hypoxia. Our observations indicate that Hif2α expression is required for the characteristic metabolic profile of glomus cells and provide a mechanistic explanation for the acute O 2 regulation of breathing., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Soil salinity is a major environmental stress that has been negatively affecting the growth and productivity of rice. However, various salt-resistant plant growth-promoting rhizobacteria (PGPR) have been known to promote plant growth and alleviate the damaging effects of salt stress via mitigating physio-biochemical and molecular characteristics. This study was conducted to examine the salt stress potential of Bacillus strains identified from harsh environments of the Qinghai-Tibetan plateau region of China. The Bacillus strains NMTD17, GBSW22, and FZB42 were screened for their response under different salt stress conditions (1, 4, 7, 9, 11, 13, and 16%). The screening analysis revealed strains NMTD17, GBSW22, and FZB42 to be high-salt tolerant, moderate-salt tolerant, and salt-sensitive, respectively. The NMTD17 strain produced a strong biofilm, followed by GBSW22 and FZB42. The expression of salt stress-related genes in selected strains was also analyzed through qPCR in various salt concentrations. Further, the Bacillus strains were used in pot experiments to study their growth-promoting ability and antioxidant activities at various concentrations (0, 100, 150, and 200 mmol). The analysis of growth-promoting traits in rice exhibited that NMTD17 had a highly significant effect and GSBW22 had a moderately significant effect in comparison with FZB42. The highly resistant strain NMTD17 that stably promoted rice plant growth was further examined for its function in the composition of rhizobacterial communities. The inoculation of NMTD17 increased the relative abundance and richness of rhizobacterial species. These outcomes propose that NMTD17 possesses the potential of PGPR traits, antioxidants enzyme activities, and reshaping the rhizobacterial community that together mitigate the harmful effects of salinity in rice plants., Competing Interests: GM was employed by Shenzhen Batian Ecotypic Engineering Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ali, Ayaz, Mu, Hussain, Yuanyuan, Yu, Xu, Manghwar, Gu, Wu and Gao.)

Background: Transferrin receptor 1 (TfR1), encoded by TFRC, is a key regulator of iron homeostasis and plays important roles in many diseases, including cancers., Objective: To decipher the underlying molecular functions of TfR1 based on its influence on transcriptome profile in cancer cells., Methods: In this study, we first identified the expression pattern and prognostic influence of TFRC in cervical cancer patients from TCGA database. To explore the regulatory outcomes of TfR1 from the view of whole transcriptome profile, we generated TFRC knockdown (TFRC-KD) HeLa cells and negative control (NC) cells using short hairpin RNA (shRNA) method. Unbiased transcriptome sequencing (RNA-seq) experiment was used to analyze the global expression level and alternative splicing (AS) changes between TFRC-KD and NC cells., Results: We found TFRC was consistently elevated in cervical cancer samples and tightly associated with prognosis of patients. Differential expression analysis revealed that 629 differentially expressed genes (DEGs) were identified between TFRC-KD and NC. Functional enrichment analysis of these DEGs revealed that TFRC-KD extensively disturbed cell physiology related pathways, including immunity, cell metabolism and gene expression. Moreover, dysregulated AS profile also indicated that TfR1 has important roles in the AS regulation. Hundreds of TfR1-regulated AS genes were involved in DNA repair, cell death, transcription and viral reproduction pathways, which were tightly associated with cancer cell progression., Conclusions: In summary, we for the first time explored the molecular functions of TfR1 at transcriptional and post-transcriptional levels. These results demonstrate TfR1 participates in the progression of cervical cancer by affecting the expression and AS levels of genes in cancer associated pathways, which greatly extends our understanding of TfR1 functions besides iron homeostasis and provide novel options in cancer treatment by targeting TfR1., (© 2021. The Author(s) under exclusive licence to The Genetics Society of Korea.)

Oxygen (O 2 ) is essential for life and therefore the supply of sufficient O 2 to the tissues is a major physiological challenge. In mammals, a deficit of O 2 (hypoxia) triggers rapid cardiorespiratory reflexes (e.g. hyperventilation and increased heart output) that within a few seconds increase the uptake of O 2 by the lungs and its distribution throughout the body. The prototypical acute O 2 -sensing organ is the carotid body (CB), which contains sensory glomus cells expressing O 2 -regulated ion channels. In response to hypoxia, glomus cells depolarize and release transmitters which activate afferent fibers terminating at the brainstem respiratory and autonomic centers. In this review, we summarize the basic properties of CB chemoreceptor cells and the essential role played by their specialized mitochondria in acute O 2 sensing and signaling. We focus on recent data supporting a "mitochondria-to-membrane signaling" model of CB chemosensory transduction. The possibility that the differential expression of specific subunit isoforms and enzymes could allow mitochondria to play a generalized adaptive O 2 -sensing and signaling role in a wide variety of cells is also discussed.

Real-time estimation of physiological properties of the cell during recombinant protein production would ensure enhanced process monitoring. In this study, we explored the application of dielectric spectroscopy to track the fed-batch phase of recombinant Escherichia coli cultivation for estimating the physiological properties, namely, cell diameter and viable cell concentration (VCC). The scanning capacitance data from the dielectric spectroscopy were pre-processed using moving average. Later, it was modeled through a nonlinear theoretical Cole-Cole model and further solved using a global evolutionary genetic algorithm (GA). The parameters obtained from the GA were further applied for the estimation of the aforementioned physiological properties. The offline cell diameter and cell viability data were obtained from particle size analyzer and flow cytometry measurements to validate the Cole-Cole model. The offline VCC was calculated from the cell viability % from flow cytometry data and dry cell weight concentration. The Cole-Cole model predicted the cell diameter and VCC with an error of 1.03% and 7.72%, respectively. The proposed approach can enable the operator to take real-time process decisions to achieve desired productivity and product quality., (© 2021 Wiley Periodicals LLC.)

Cell adhesion plays a critical role in cell communication, cell migration, cell proliferation, and integration of medical implants with tissues. Focal adhesions physically link the cell cytoskeleton to the extracellular matrix, but it remains challenging to image single focal adhesions directly. Here, we show that plasmonic scattering microscopy (PSM) can directly image the single focal adhesions in a label-free, real-time, and non-invasive manner with sub-micrometer spatial resolution. PSM is developed based on surface plasmon resonance (SPR) microscopy, and the evanescent illumination makes it immune to the interference of intracellular structures. Unlike the conventional SPR microscopy, PSM can provide a high signal-to-noise ratio and sub-micrometer spatial resolution for imaging the analytes with size down to a single-molecule level, thus allowing both the super-resolution lateral localization for measuring the nanoscale displacement and precise tracking of vertical distances between the analyte centroid and the sensor surface for analysis of free-energy profiles. PSM imaging of the RBL-2H3 cell with temporal resolution down to microseconds shows that the focal adhesions have random diffusion behaviors in addition to their directional movements during the antibody-mediated activation process. The free-energy mapping also shows a similar movement tendency, indicating that the cell may change its morphology upon varying the binding conditions of adhesive structures. PSM provides insights into the individual focal adhesion activities and can also serve as a promising tool for investigating the cell/surface interactions, such as cell capture and detection and tissue adhesive materials screening.

Otopetrins comprise a family of proton-selective channels that are critically important for the mineralization of otoliths and statoconia in vertebrates but whose underlying cellular mechanisms remain largely unknown. Here, we demonstrate that otopetrins are critically involved in the calcification process by providing an exit route for protons liberated by the formation of CaCO 3 Using the sea urchin larva, we examined the otopetrin ortholog otop2l , which is exclusively expressed in the calcifying primary mesenchymal cells (PMCs) that generate the calcitic larval skeleton. otop2l expression is stimulated during skeletogenesis, and knockdown of otop2l impairs spicule formation. Intracellular pH measurements demonstrated Zn 2+ -sensitive H + fluxes in PMCs that regulate intracellular pH in a Na + /HCO 3 - -independent manner, while Otop2l knockdown reduced membrane proton permeability. Furthermore, Otop2l displays unique features, including strong activation by high extracellular pH (>8.0) and check-valve-like outwardly rectifying H + flux properties, making it into a cellular proton extrusion machine adapted to oceanic living conditions. Our results provide evidence that otopetrin family proton channels are a central component of the cellular pH regulatory machinery in biomineralizing cells. Their ubiquitous occurrence in calcifying systems across the animal kingdom suggest a conserved physiological function by mediating pH at the site of mineralization. This important role of otopetrin family proton channels has strong implications for our view on the cellular mechanisms of biomineralization and their response to changes in oceanic pH., Competing Interests: The authors declare no competing interest.

The meninges shield the nervous system from diverse, rather harmful stimuli and pathogens from the periphery. This tissue is composed of brain endothelial cells (BECs) that express diverse ion channels and chemical-transmitter receptors also expressed by neurons and glial cells to communicate with each other. However, information about the effects of ATP and angiotensin II on BECs is scarce, despite their essential roles in blood physiology. This work investigated in vitro if BECs from the meninges from rat forebrain respond to ATP, angiotensin II and high extracellular potassium, with intracellular calcium mobilizations and its second messenger-associated pathways. We found that in primary BEC cultures, both ATP and angiotensin II produced intracellular calcium responses linked to the activation of inositol trisphosphate receptors and ryanodine receptors, which led to calcium release from intracellular stores. We also used RT-PCR to explore what potassium channel subunits are expressed by primary BEC cultures and freshly isolated meningeal tissue, and which might be linked to the observed effects. We found that BECs mainly expressed the inward rectifier potassium channel subunits Kir1.1, Kir3.3, Kir 4.1 and Kir6.2. This study contributes to the understanding of the functions elicited by ATP and angiotensin II in BECs from rat meninges. SIGNIFICANCE OF THE STUDY: Brain endothelial cells (BECs) express diverse ion channels and membrane receptors, which they might use to communicate with neurons and glia. This work investigated in vitro, if BECs from the rat forebrain respond to angiotensin II and ATP with intracellular calcium mobilizations. We found that these cells did respond to said substances with intracellular calcium mobilizations linked to inositol trisphosphate and ryanodine receptor activation, which led to calcium release from intracellular stores. These findings are important because they might uncover routes of active communication between brain cells and endothelial cells., (© 2021 John Wiley & Sons Ltd.)

In Science Signaling, Gualdani and colleagues provide evidence that the ion channel TRPV4 functions as a mechanosensor in the renal proximal tubules and show that TRPV4 activity modulates protein reabsorption., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Current cell-based biosensors have progressed substantially from mere alternatives to molecular bioreceptors into enabling tools for interfacing molecular machineries and gene circuits with microelectronics and for developing groundbreaking sensing and theragnostic platforms. The recent literature concerning whole-cell biosensors is reviewed with an emphasis on mammalian cells, and the challenges and breakthroughs brought along in biomedical analyses through novel biosensing concepts and the synthetic biology toolbox. These recent innovations allow development of cell-based biosensing platforms having tailored performances and capable to reach the levels of sensitivity, dynamic range, and stability suitable for high analytic/medical relevance. They also pave the way for the construction of flexible biosensing platforms with utility across biological research and clinical applications. The work is intended to stimulate interest in generation of cell-based biosensors and improve their acceptance and exploitation.

We study the dynamics of a model of membrane vesicle transport into dendritic spines, which are bulbous intracellular compartments in neurons driven by molecular motors. We reduce the lubrication model proposed in Fai et al. (Phys Rev Fluids 2:113601, 2017) to a fast-slow system, yielding an analytically and numerically tractable equation equivalent to the original model in the overdamped limit. The model's key parameters include: (1) the ratio of motors that prefer to push toward the head of the dendritic spine to the motors that prefer to push in the opposite direction, and (2) the viscous drag exerted on the vesicle by the spine constriction. We perform a numerical bifurcation analysis in these parameters and find that steady-state vesicle velocities appear and disappear through several saddle-node bifurcations. This process allows us to identify the region of parameter space in which multiple stable velocities exist. We show by direct calculations that there can only be unidirectional motion for sufficiently close vesicle-to-spine diameter ratios. Our analysis predicts the critical vesicle-to-spine diameter ratio, at which there is a transition from unidirectional to bidirectional motion, consistent with experimental observations of vesicle trajectories in the literature.

Microbial physiology is an essential characteristic to be considered in the research and industrial use of microorganisms. Conventionally, the study of microbial physiology has been limited to carrying out qualitative and quantitative analysis of the role of individual components in global cell behaviour at a specific time and under certain growth conditions. In this framework, groups of observable cell physiological variables that remain over time define the physiological states. Recently, with advances in omics techniques, it has been possible to demonstrate that microbial physiology is a dynamic process and that, even with low variations in environmental culture conditions, physiological changes in the cell are provoked. However, the changes cannot be detected at a macroscopic level, and it is not possible to observe these changes in real time. As an alternative to solve this inconvenience, dielectric spectroscopy has been used as a complementary technique to monitor on-line cell physiology variations to avoid long waiting times during measurements. In this review, we discuss the state-of-the-art application of dielectric spectroscopy to unravel the physiological state of microorganisms, its current state, prospects and limitations during fermentation processes. Key points • Summary of the state of the art of several issues of dielectric spectroscopy. • Discussion of correlation among dielectric properties and cell physiological states. • View of the potential use of dielectric spectroscopy in monitoring bioprocesses.

The cortical collecting duct (CCD) of the mammalian kidney plays a major role in the maintenance of total body electrolyte, acid/base, and fluid homeostasis by tubular reabsorption and excretion. The mammalian CCD is heterogeneous, composed of Na + -absorbing principal cells (PCs) and acid-base-transporting intercalated cells (ICs). Perturbations in luminal flow rate alter hydrodynamic forces to which these cells in the cylindrical tubules are exposed. However, most studies of tubular ion transport have been performed in cell monolayers grown on or epithelial sheets affixed to a flat support, since analysis of transepithelial transport in native tubules by in vitro microperfusion requires considerable expertise. Here, we report on the generation and characterization of an in vitro, perfusable three-dimensional kidney CCD model (3D CCD), in which immortalized mouse PC-like mpkCCD cells are seeded within a cylindrical channel embedded within an engineered extracellular matrix and subjected to luminal fluid flow. We find that a tight epithelial barrier composed of differentiated and polarized PCs forms within 1 wk. Immunofluorescence microscopy reveals the apical epithelial Na + channel ENaC and basolateral Na + /K + -ATPase. On cessation of luminal flow, benzamil-inhibitable cell doming is observed within these 3D CCDs consistent with the presence of ENaC-mediated Na + absorption. Our 3D CCD provides a geometrically and microphysiologically relevant platform for studying the development and physiology of renal tubule segments.

Cell manipulation using optically induced dielectrophoresis (ODEP) in microfluidic systems has attracted the interest of scientists due to its simplicity. Although this technique has been successfully demonstrated for various applications, one fundamental issue has to be addressed-Whether, the ODEP field affects the native properties of cells. To address this issue, we explored the effect of ODEP electrical conditions on cellular properties. Within the experimental conditions tested, the ODEP-based cell manipulation with the largest velocity occurred at 10 Vpp and 1 MHz, for the two cancer cell types explored. Under this operating condition, however, the cell viability of cancer cells was significantly affected (e.g., 70.5 ± 10.0% and 50.6 ± 9.2% reduction for the PC-3 and SK-BR-3 cancer cells, respectively). Conversely, the exposure of cancer cells to the ODEP electrical conditions of 7-10 Vpp and 3-5 MHz did not significantly alter the cell viability, cell metabolic activity, and the EpCAM , VIM , and ABCC1 gene expression of cancer cells. Overall, this study fundamentally investigated the effect of ODEP electrical conditions on the cellular properties of cancer cells. The information obtained is crucially important for the utilization of ODEP-based cell manipulation in a microscale system for various applications.

Sarcoids are the most common skin neoplasm in the Equidae family. Sarcoids are benign, but may cause severe damage in affected animals. Due to the high risk of post-treatment recurrence and the lack of an effective method of treatment, it is reasonable to perform studies on the molecular aspects of this neoplasm. Therefore, the present studies analyzed five genes (cell cycle control binding protein alpha, coronin 1b, metalloproteinase 2, tissue inhibitor of metalloproteinases 3 and vimentin) related to cell mobility and invasion traits. Primary healthy fibroblasts and sarcoid cells were obtained from skin biopsies. Cell lines were cultured in two different medium types with different concentrations of foetal bovine serum (10% and 0.5% FBS) to study its influence on the analyzed genes. Gene expression was measured using the real-time PCR method. The results showed significant differences in two genes (coronin and vimentin) depending on culture conditions. In conclusion, the results enabled finding two new genes, related to cell motility and invasion traits, in which gene expression is deregulated. Results of the study may put new knowledge into the complexity of the genetic background of this disease and show the importance of further analysis on this subject.

The last five decades of molecular and systems biology research have provided unprecedented insights into the molecular and genetic basis of many cellular processes. Despite these insights, however, it is arguable that there is still only limited predictive understanding of cell behaviours. In particular, the basis of heterogeneity in single-cell behaviour and the initiation of many different metabolic, transcriptional or mechanical responses to environmental stimuli remain largely unexplained. To go beyond the status quo , the understanding of cell behaviours emerging from molecular genetics must be complemented with physical and physiological ones, focusing on the intracellular and extracellular conditions within and around cells. Here, we argue that such a combination of genetics, physics and physiology can be grounded on a bioelectrical conceptualization of cells. We motivate the reasoning behind such a proposal and describe examples where a bioelectrical view has been shown to, or can, provide predictive biological understanding. In addition, we discuss how this view opens up novel ways to control cell behaviours by electrical and electrochemical means, setting the stage for the emergence of bioelectrical engineering.

Glucose-induced (physiological) insulin secretion from the islet β-cell involves interplay between cationic (i.e., changes in intracellular calcium) and metabolic (i.e., generation of hydrophobic and hydrophilic second messengers) events. A large body of evidence affirms support for novel regulation, by G proteins, of specific intracellular signaling events, including actin cytoskeletal remodeling, transport of insulin-containing granules to the plasma membrane for fusion, and secretion of insulin into the circulation. This article highlights the following aspects of GPCR-G protein biology of the islet. First, it overviews our current understanding of the identity of a wide variety of G protein regulators and their modulatory roles in GPCR-G protein-effector coupling, which is requisite for optimal β-cell function under physiological conditions. Second, it describes evidence in support of novel, noncanonical, GPCR-independent mechanisms of activation of G proteins in the islet. Third, it highlights the evidence indicating that abnormalities in G protein function lead to islet β-cell dysregulation and demise under the duress of metabolic stress and diabetes. Fourth, it summarizes observations of potential beneficial effects of GPCR agonists in preventing/halting metabolic defects in the islet β-cell under various pathological conditions (e.g., metabolic stress and inflammation). Lastly, it identifies knowledge gaps and potential avenues for future research in this evolving field of translational islet biology. Published 2020. Compr Physiol 10:453-490, 2020., (This article is a U.S. Government work and is in the public domain in the U.S.A.)

Nitric oxide, studied to evaluate its role in cardiovascular physiology, has cardioprotective and therapeutic effects in cellular signaling, mitochondrial function, and in regulating inflammatory processes. Heme oxygenase (major role in catabolism of heme into biliverdin, carbon monoxide (CO), and iron) has similar effects as well. CO has been suggested as the molecule that is responsible for many of the above mentioned cytoprotective and therapeutic pathways as CO is a signaling molecule in the control of physiological functions. This is counterintuitive as toxic effects are related to its binding to hemoglobin. However, CO is normally produced in the body. Experimental evidence indicates that this toxic gas, CO, exerts cytoprotective properties related to cellular stress including the heart and is being assessed for its cytoprotective and cytotherapeutic properties. While survival of adult cardiomyocytes depends on oxidative phosphorylation (survival and resulting cardiac function is impaired by mitochondrial damage), mitochondrial biogenesis is modified by the heme oxygenase-1/CO system and can result in promotion of mitochondrial biogenesis by associating mitochondrial redox status to the redox-active transcription factors. It has been suggested that the heme oxygenase-1/CO system is important in differentiation of embryonic stem cells and maturation of cardiomyocytes which is thought to mitigate progression of degenerative cardiovascular diseases. Effects on other cardiac cells are being studied. Acute exposure to air pollution (and, therefore, CO) is associated with cardiovascular mortality, myocardial infarction, and heart failure, but changes in the endogenous heme oxygenase-1 system (and, thereby, CO) positively affect cardiovascular health. We will review the effect of CO on heart health and function in this article.

We develop a mathematical model to study the immediate effect of low-dose radiation on the G2 checkpoint and the G2/M transition of the cell cycle via a radiation pathway (the ATM-Chk2 pathway) of an individual mammalian cell. The model consists of a system of nonlinear differential equations describing the dynamics of a network of regulatory proteins that play key roles in the G2/M transition, cell cycle oscillations, and the radiation pathway. We simulate the application of a single pulse of low-dose radiation at different intensities ([Formula: see text] 0-0.4 Gy) and times during the latter part of the G2-phase. We use bifurcation analysis to characterize the effect of radiation on the G2/M transition via the ATM-Chk2 pathway. We show that radiation between 0.1 and 0.3 Gy can delay the G2/M transition, and radiation higher than 0.3 Gy can fully activate the G2 checkpoint. Also, our results show that radiation can be low enough to neither delay the G2/M transition nor activate the G2 checkpoint ([Formula: see text] 0.1 Gy). Our model supports the idea that the cell response to radiation during G2-phase explains hyper-radiosensitivity and increased radioresistance (HRS/IRR) observed at low dose.

Phosphatidylinositol (PI) lipids have a predominance of a single molecular species present through the organism. In healthy mammals this molecular species is 1-stearoyl-2-arachidonoyl (18:0/20:4) PI. Although the importance of PI lipids for cell physiology has long been appreciated, less is known about the biological role of enriching PI lipids with 18:0/20:4 acyl chains. In conditions with dysfunctional lipid metabolism, the predominance of 18:0/20:4 acyl chains is lost. Recently, molecular mechanisms underpinning the enrichment or alteration of these acyl chains in PI lipids have begun to emerge. In the majority of the cases a common feature is the presence of enzymes bearing substrate acyl chain specificity. However, in cancer cells, it has been shown that one (not the only) of the mechanisms responsible for the loss in this acyl chain enrichment is mutation on the transcription factor p53 gene, which is one of the most highly mutated genes in cancers. There is a compelling need for a global picture of the specificity of the acyl chain composition of PIs. This can be possible once high-resolution spatio-temporal information is gathered in a cellular context; which can ultimately lead to potential novel targets to combat conditions with altered PI acyl chain profiles., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A glucose-glycerol mixed carbon source (MCS) can substantially reduce batch fermentation time and improve ε-poly-L-lysine (ε-PL) productivity, which was of great significance in industrial microbial fermentation. This study aims to disclose the physiological mechanism by transcriptome analyses. In the MCS, the enhancements of gene transcription mainly emerged in central carbon metabolism, L-lysine synthesis as well as cell respiration, and these results were subsequently proved by quantitative real-time PCR assay. Intracellular L-lysine determination and exhaust gas analysis further confirmed the huge precursor L-lysine pool and active cell respiration in the MCS. Interestingly, in the MCS, pls was remarkably up-regulated than those in single carbon sources without transcriptional improvement of HrdD, which indicated that the improved ε-PL productivity was supported by other regulators rather than hrdD. This study exposed the physiological basis of the improved ε-PL productivity in the MCS, which provided references for studies on other biochemicals production using multiple substrates.

The unique microenvironment found within the liver in vivo plays a key role in the induction of functional maturation in the developing hepatocyte. During organogenesis, hepatocytes acquire a polar phenotype that allows them to perform their functions of bile production and transport, protein synthesis, metabolism, and detoxification simultaneously, independently, and efficiently. It is thought that the induction of polarity and functional maturation in hepatocytes is dependent on the complex interplay of cell-cell and cell-extracellular matrix (ECM) interactions. While this process is highly efficient in the human liver, it has been shown that hepatocytes rapidly lose their functions when placed in cell culture. This poses a challenge for the development of a bioartificial liver (BAL) support system, which utilizes a live cellular source to perform hepatic functions in the event of acute liver failure or primary nonfunction. However, once the molecular mechanisms underlying the induction of hepatocyte polarity are fully identified, it will be possible to develop highly functional hepatic cells from human pluripotent stem cells (hPSCs). This new cell line would be an ideal cellular source for a BAL system, as it would have both the functionality and longevity to support a patient through the entire clinical course of treatment. In this review, we explore the literature that has examined the potential mechanisms that induce polarity in the developing hepatocyte and discuss the future implications of this knowledge in a clinical setting from a bioengineering perspective., (Copyright © 2019 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Interaction of Staphylococcus aureus alpha-toxin (hemolysin A, Hla) with eukaryotic cell membranes is mediated by proteinaceous receptors and certain lipid domains in host cell plasma membranes. Hla is secreted as a 33 kDa monomer that forms heptameric transmembrane pores whose action compromises maintenance of cell shape and epithelial tightness. It is not exactly known whether certain membrane lipid domains of host cells facilitate adhesion of Ha monomers, oligomerization, or pore formation. We used sphingomyelinase (hemolysin B, Hlb) expressed by some strains of staphylococci to pre-treat airway epithelial model cells in order to specifically decrease the sphingomyelin (SM) abundance in their plasma membranes. Such a pre-incubation exclusively removed SM from the plasma membrane lipid fraction. It abrogated the formation of heptamers and prevented the formation of functional transmembrane pores. Hla exposure of rHlb pre-treated cells did not result in increases in [Ca 2+ ] i , did not induce any microscopically visible changes in cell shape or formation of paracellular gaps, and did not induce hypo-phosphorylation of the actin depolymerizing factor cofilin as usual. Removal of sphingomyelin from the plasma membranes of human airway epithelial cells completely abrogates the deleterious actions of Staphylococcus aureus alpha-toxin.

The term 'mitochondrial dynamics' is commonly used to refer to ongoing fusion and fission of mitochondrial structures within a living cell. A growing number of diseases, from Charcot Marie Tooth Type 2a neuropathies to cancer, is known to be associated with the dysregulation of mitochondrial dynamics, leading to irregularities of mitochondrial network morphology that are associated with aberrant metabolism and cellular dysfunction. Studying these phenomena, and potential pharmacological interventions to correct them, in cultured cells is a powerful approach to developing treatments or cures. Appropriately designed experiments and quantitative approaches for characterizing mitochondrial morphology and function are essential for furthering our understanding. In this chapter, we discuss the importance of cell incubation conditions, choices around imaging modalities, and data analysis tools with respect to experimental outcomes and the interpretation of results from studies of mitochondrial dynamics. We focus primarily on the quantitative analysis of mitochondrial morphology, providing an overview of the available tools and approaches currently being used and discussing some of the strengths and weaknesses associated with each. Finally, we discuss how the ongoing development of imaging and analysis tools continues to improve our ability to study normal and aberrant mitochondrial physiology in vitro and in vivo.

Many aspects of cell physiology, including migration, membrane function, and cell division, are best understood by observing live cell dynamics over time using video microscopy. To probe these phenomena in colon epithelial cells using simple components with a limited budget, we have constructed an inexpensive (<$410) self-contained apparatus, consisting of a closed-loop, feedback-controlled system regulated by a PID (proportional-integrative-derivative) controller contained within a 0.077 m 3 insulated acrylic box. Temperature, humidity, pH, and proliferative capacity of colon epithelial cells in this system mimic those in a standard tissue culture incubator for over four days. Our system offers significant advantages over existing cost-prohibitive commercially available and custom-made devices because of its very low cost, use of PID temperature control, lack of reliance on constant infusion of external humidified, heated air or carbon dioxide, ability to directly measure cell culture medium temperature, and combination of exquisite cellular detail with minimal focus drift under physiological conditions for extended periods of time. Using this apparatus, coupled with an inverted microscope equipped with phase contrast optics and a programmable digital camera, we have observed many events in colon epithelial cells not visible by static imaging, including kinetics of normal and abnormal mitoses, dynamic membrane structures, intracellular vesicle movements, and cell migration. © 2018 International Society for Advancement of Cytometry., (© 2018 International Society for Advancement of Cytometry.)

Coccolithophores belong to the most abundant calcium carbonate mineralizing organisms. Coccolithophore biomineralization is a complex and highly regulated process, resulting in a product that strongly differs in its intricate morphology from the abiogenically produced mineral equivalent. Moreover, unlike extracellularly formed biological carbonate hard tissues, coccolith calcite is neither a hybrid composite, nor is it distinguished by a hierarchical microstructure. This is remarkable as the key to optimizing crystalline biomaterials for mechanical strength and toughness lies in the composite nature of the biological hard tissue and the utilization of specific microstructures. To obtain insight into the pathway of biomineralization of Emiliania huxleyi coccoliths, we examine intracrystalline nanostructural features of the coccolith calcite in combination with cell ultrastructural observations related to the formation of the calcite in the coccolith vesicle within the cell. With TEM diffraction and annular dark-field imaging, we prove the presence of planar imperfections in the calcite crystals such as planar mosaic block boundaries. As only minor misorientations occur, we attribute them to dislocation networks creating small-angle boundaries. Intracrystalline occluded biopolymers are not observed. Hence, in E. huxleyi calcite mosaicity is not caused by occluded biopolymers, as it is the case in extracellularly formed hard tissues of marine invertebrates, but by planar defects and dislocations which are typical for crystals formed by classical ion-by-ion growth mechanisms. Using cryo-preparation techniques for SEM and TEM, we found that the membrane of the coccolith vesicle and the outer membrane of the nuclear envelope are in tight proximity, with a well-controlled constant gap of ~4 nm between them. We describe this conspicuous connection as a not yet described interorganelle junction, the "nuclear envelope junction". The narrow gap of this junction likely facilitates transport of Ca 2+ ions from the nuclear envelope to the coccolith vesicle. On the basis of our observations, we propose that formation of the coccolith utilizes the nuclear envelope-endoplasmic reticulum Ca 2+ -store of the cell for the transport of Ca 2+ ions from the external medium to the coccolith vesicle and that E. huxleyi calcite forms by ion-by-ion growth rather than by a nanoparticle accretion mechanism., (© 2017 Phycological Society of America.)

Cells need to act upon the elastic extracellular matrix and against steric constraints when proliferating in a confined environment, leading to the build-up, at the population level, of a compressive, growth-induced, mechanical stress. Compressive mechanical stresses are ubiquitous to any cell population growing in a spatially-constrained environment, such as microbes or most solid tumors. They remain understudied, in particular in microbial populations, due to the lack of tools available to researchers. Here, we present various mechano-chemostats: microfluidic devices developed to study microbes under pressure. A mechano-chemostat permits researchers to control the intensity of growth-induced pressure through the control of cell confinement, while keeping cells in a defined chemical environment. These versatile devices enable the interrogation of physiological parameters influenced by mechanical compression at the single cell level and set a standard for the study of growth-induced compressive stress., (© 2018 Elsevier Inc. All rights reserved.)

Several factors affect protein expression in Pichia pastoris, one among them is the carbon source. In this work, we studied the effect of this factor on the expression level of rabies virus glycoprotein (RABV-G) in two recombinant clones harboring seven copies of the gene of interest. The expression was driven either by the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter or the inducible alcohol oxidase1 (AOX1) promoter. Clones were compared in terms of cell physiology and carbon source metabolism. The transcription levels of 16 key genes involved in the central metabolic pathway, the methanol catabolism, and the oxidative stress were investigated in both clones. Cell size, as a parameter reflecting cell physiological changes, was also monitored. Our results showed that when glucose was used as the sole carbon source, large cells were obtained. Transcript levels of the genes of the central metabolic pathway were also upregulated, whereas antioxidative gene transcript levels were low. By contrast, the use of methanol as a carbon source generated small cells and a shift in carbon metabolism toward the dissimilatory pathway by the upregulation of formaldehyde dehydrogenase gene and the downregulation of those of the central metabolic. These observations are in favor of the use of glucose to enhance the expression of RABV-G in P. pastoris., (© 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

The world's oceans today have become a place for the disposal of toxic waste, which leads to the degradation of marine mammal habitats and populations. Marine mammal cell cultures have proven to be a multifunctional tool for studying the peculiarities of the cell physiology and biochemistry of these animals as well as the destructive effects of anthropogenic and natural toxicants. This review describes the sources of marine mammal live tissues and the methods required for establishing cell cultures, their use, and long-term storage. Approaches to conserving rare animal species by applying cell biology methodologies are also discussed., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Constitutively expressed small heat shock protein HspB1 regulates many fundamental cellular processes and plays major roles in many human pathological diseases. In that regard, this chaperone has a huge number of apparently unrelated functions that appear linked to its ability to recognize many client polypeptides that are subsequently modified in their activity and/or half-life. A major parameter to understand how HspB1 is dedicated to interact with particular clients in defined cellular conditions relates to its complex oligomerization and phosphorylation properties. Indeed, HspB1 structural organization displays dynamic and complex rearrangements in response to changes in the cellular environment or when the cell physiology is modified. These structural modifications probably reflect the formation of structural platforms aimed at recognizing specific client polypeptides. Here, I have reviewed data from the literature and re-analyzed my own studies to describe and discuss these fascinating changes in HspB1 structural organization.

Synthetic biology is an emerging interdisciplinary research field. By designing and constructing new or re-designing the existing natural systems, it confers them novel functions, which do not exist in nature. Owing to the predictability and controllability, synthetic biology attracts more and more interest from biologists, physicists, and engineers. Synthetic biology approaches not only can be widely used for biotechnological applications but also can be used to study complex biological systems to address fundamental questions. Here, we reviewed the recent studies following the concept of "build-to-understand", particularly, the studies to understand intracellular network structure, cell physiology, the behavior of multicellular populations, and ecosystems.

Rabies is a fatal disease that can be prevented by vaccination. Different approaches were investigated to develop novel human rabies vaccines with improved features compared to the current available vaccines, among them is the use of heterologous gene expression technology. Here, we describe the expression of the surface rabies virus glycoprotein (RABV-G), which is the major antigen responsible for the induction of protective immunity, in Pichia pastoris. Six transformants were selected according to their gene copy number as determined by real time qPCR. Upon induction by methanol, low level of RABV-G was secreted into the culture medium, around 60 ng/mL. To understand the effect of foreign gene dosage on cellular physiology of P. pastoris, transcriptional analysis of key genes involved in unfolded protein response (UPR) and endoplasmic reticulum associated degradation (ERAD) pathway was performed. Results showed that these pathways were highly activated; misfolded RABV-G was degraded in the cytosol via the ERAD mechanism. To study the functionality of the secreted RABV-G, in vitro competitive neutralizing assay was conducted. Data showed the secreted recombinant RABV-G had enabled a reduction of the neutralizing activity of human immune rabies serum, indicating that the secreted recombinant protein had reached its correct conformational form., (© 2015 International Union of Biochemistry and Molecular Biology, Inc.)

Studies within the last decade have localized the functional expression of olfactory receptors (ORs) to cells outside of the olfactory epithelium. In human hepatocarcinoma and prostate cancer cells, the activation of ORs by odors modulates elementary physiological processes and leads to an inhibitory effect on proliferation. Cells of the respiratory tract are in direct contact with the surrounding air, in which a myriad of volatile molecules, especially odors, are present. Non-small-cell lung cancer (NSCLC) has a high prevalence, a high mortality rate and is difficult to treat. NSCLC cells are nearly resistant to common chemotherapeutic approaches, and surgical resection provides the only possible chance of a cure for most patients. New approaches for the treatment of NSCLC are the focus of many current studies. Thus, it is of interest to characterize the functional expression of ORs in cancer cells of the lung and to investigate the impact of ORs on pathophysiological processes. In the present study, we demonstrate that the expression of OR2J3 and cytosolic Ca 2+ increase via the activation of the agonist helional in the NSCLC cell line A549. We further investigated the underlying pathway. Helional triggers phoshoinositol-3 kinase (PI3K), signaling the release of intracellular Ca 2+ and phosphorylation of ERK. We observed that OR2J3 activation induces apoptosis and inhibits cell proliferation and migration in long-term stimulus experiments with helional. Our study provides the first evidence of the functional expression of an OR in NSCLC cells and its putative therapeutic impact., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Understanding the impact of gene knockouts on cellular physiology, and metabolism in particular, is centrally important to quantitative systems biology and metabolic engineering. Here, we present a comprehensive physiological characterization of wild-type Escherichia coli and 22 knockouts of enzymes in the upper part of central carbon metabolism, including the PTS system, glycolysis, pentose phosphate pathway and Entner-Doudoroff pathway. Our results reveal significant metabolic changes that are affected by specific gene knockouts. Analysis of collective trends and correlations in the data using principal component analysis (PCA) provide new, and sometimes surprising, insights into E. coli physiology. Additionally, by comparing the data-to-model predictions from constraint-based approaches such as FBA, MOMA and RELATCH we demonstrate the important role of less well-understood kinetic and regulatory effects in central carbon metabolism., (Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Recently, we reported the cloning and characterization of short isoform of the icilin-activated cold receptor TRPM8 channel in keratinocytes, dubbed eTRPM8. We demonstrated that eTRPM8 via fine tuning of the endoplasmic reticulum (ER) - mitochondria Ca(2+) shuttling regulates mitochondrial ATP and superoxide (O2(•-)) production and, thereby, mediates control of epidermal homeostasis by mild cold. Here, we provide additional information explaining why eTRPM8 suppression and TRPM8 stimulation both inhibit keratinocyte growth. We also demonstrate that stimulation of eTRPM8 with icilin may give rise to sustained oscillatory responses. Furthermore, we show that ATP-induced cytosolic and mitochondrial Ca(2+) responses are attenuated by eTRPM8 suppression. This suggests positive interplay between eTRPM8 and purinergic signaling pathways, what may serve to facilitate the ER-mitochondria Ca(2+) shuttling. Finally, we demonstrate that cold (25°C) induces eTRPM8-dependent superoxide-mediated necrosis of keratinocytes. Altogether, these results are in line with our model of eTRPM8-mediated cold-dependent balance between keratinocyte proliferation and differentiation.

This study combines novel multibeam electron microscopy with a geographical information system approach to create a first, seamless, navigable anatomic map of the human hip and its cellular inhabitants. Using spatial information acquired by localizing relevant map landmarks (e.g. cells, blood vessels), network modeling will enable disease epidemiology studies in populations of cells inhabiting tissues and organs., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Renal ischemia and reperfusion injury causes loss of renal epithelial cell polarity and perturbations in tubular solute and fluid transport. Na(+),K(+)-ATPase, which is normally found at the basolateral plasma membrane of renal epithelial cells, is internalized and accumulates in intracellular compartments after renal ischemic injury. We previously reported that the subcellular distribution of Na(+),K(+)-ATPase is modulated by direct binding to Akt substrate of 160 kD (AS160), a Rab GTPase-activating protein that regulates the trafficking of glucose transporter 4 in response to insulin and muscle contraction. Here, we investigated the effect of AS160 on Na(+),K(+)-ATPase trafficking in response to energy depletion. We found that AS160 is required for the intracellular accumulation of Na(+),K(+)-ATPase that occurs in response to energy depletion in cultured epithelial cells. Energy depletion led to dephosphorylation of AS160 at S588, which was required for the energy depletion-induced accumulation of Na,K-ATPase in intracellular compartments. In AS160-knockout mice, the effects of renal ischemia on the distribution of Na(+),K(+)-ATPase were substantially reduced in the epithelial cells of distal segments of the renal tubules. These data demonstrate that AS160 has a direct role in linking the trafficking of Na(+),K(+)-ATPase to the energy state of renal epithelial cells., (Copyright © 2015 by the American Society of Nephrology.)

Raman micro-spectroscopy provides a convenient non-destructive and location-specific means of probing cellular physiology and tissue physiology at sub-micron length scales. By probing the vibrational signature of molecules and molecular groups, the distribution and metabolic products of small molecules that cannot be labeled with fluorescent dyes can be analyzed. This method works well for molecular concentrations in the micro-molar range and has been demonstrated as a valuable tool for monitoring drug-cell interactions. If the small molecule of interest does not contain groups that would allow for a discrimination against cytoplasmic background signals, "labeling" of the molecule by isotope substitution or by incorporating other unique small groups, e.g. alkynes provides a stable signal even for time-lapse imaging such compounds in living cells. In this review we highlight recent progress in assessing the physiology of cells and tissue by Raman spectroscopy and imaging., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Upon activation by with-no-lysine kinases, STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) phosphorylates and activates SLC12A transporters such as the Na(+)-Cl(-) cotransporter (NCC) and Na(+)-K(+)-2Cl(-) cotransporter type 1 (NKCC1) and type 2 (NKCC2); these transporters have important roles in regulating BP through NaCl reabsorption and vasoconstriction. SPAK knockout mice are viable and display hypotension with decreased activity (phosphorylation) of NCC and NKCC1 in the kidneys and aorta, respectively. Therefore, agents that inhibit SPAK activity could be a new class of antihypertensive drugs with dual actions (i.e., NaCl diuresis and vasodilation). In this study, we developed a new ELISA-based screening system to find novel SPAK inhibitors and screened >20,000 small-molecule compounds. Furthermore, we used a drug repositioning strategy to identify existing drugs that inhibit SPAK activity. As a result, we discovered one small-molecule compound (Stock 1S-14279) and an antiparasitic agent (Closantel) that inhibited SPAK-regulated phosphorylation and activation of NCC and NKCC1 in vitro and in mice. Notably, these compounds had structural similarity and inhibited SPAK in an ATP-insensitive manner. We propose that the two compounds found in this study may have great potential as novel antihypertensive drugs., (Copyright © 2015 by the American Society of Nephrology.)

Small fragments circulating in the blood were formally identified by the end of the nineteenth century, and it was suggested that they assisted coagulation via interactions with vessel endothelia. Wright, at the beginning of the twentieth century, identified their bone-marrow origin. For long, platelets have been considered sticky assistants of hemostasis and pollutants of blood or tissue samples; they were just cell fragments. As such, however, they were acknowledged as immunizing (to specific HPA and HLA markers): the platelet's dark face. The enlightened face showed that besides hemostasis, platelets contained factors involved in healing. As early as 1930s, platelets entered the arsenal of medicines were transfused, and were soon manipulated to become a kind of glue to repair damaged tissues. Some gladly categorized platelets as cells but they were certainly not fully licensed as such for cell physiologists. Actually, platelets possess almost every characteristic of cells, apart from being capable of organizing their genes: they have neither a nucleus nor genes. This view prevailed until it became evident that platelets play a role in homeostasis and interact with cells other than with vascular endothelial cells; then began the era of physiological and also pathological inflammation. Platelets have now entered the field of immunity as inflammatory cells. Does assistance to immune cells itself suffice to license a cell as an "immune cell"? Platelets prove capable of sensing different types of signals and organizing an appropriate response. Many cells can do that. However, platelets can use a complete signalosome (apart from the last transcription step, though it is likely that this step can be circumvented by retrotranscribing RNA messages). The question has also arisen as to whether platelets can present antigen via their abundantly expressed MHC class I molecules. In combination, these properties argue in favor of allowing platelets the title of immune cells.

Accumulation of abnormal protein inclusions is implicated in motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Autophagy, an intracellular process targeting misfolded proteins and damaged organelles for lysosomal degradation, plays crucial roles in survival and diseased conditions. Efforts were made to understand the role of autophagy in motor neuron degeneration and to target autophagy in motor neuron for ALS treatment. However, results were quite contradictory. Possible autophagy defects in other cell types may also complicate the results. Here, we examined autophagy activity in skeletal muscle of an ALS mouse model G93A. Through overexpression of a fluorescent protein LC3-RFP, we found a basal increase in autophagosome formation in G93A muscle during disease progression when the mice were on a regular diet. As expected, an autophagy induction procedure (starvation plus colchicine) enhanced autophagy flux in skeletal muscle of normal mice. However, in response to the same autophagy induction procedure, G93A muscle showed significant reduction in the autophagy flux. Immunoblot analysis revealed that increased cleaved caspase-3 associated with apoptosis was linked to the cleavage of several key proteins involved in autophagy, including Beclin-1, which is an essential molecule connecting autophagy and apoptosis pathways. Taking together, we provide the evidence that the cytoprotective autophagy pathway is suppressed in G93A skeletal muscle and this suppression may link to the enhanced apoptosis during ALS progression. The abnormal autophagy activity in skeletal muscle likely contributes muscle degeneration and disease progression in ALS., (© 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)