Salmonellosis caused by Salmonella sp. has long been reported all over the world. Despite the availability of various diagnostic methods, easy and effective detection systems are still required. This report describes a dialysis membrane electrode interface disc with immobilized specific antibodies to capture antigenic Salmonella cells. The interaction of a specific Salmonella antigen with a mouse anti-Salmonella monoclonal antibody complexed to rabbit anti-mouse secondary antibody conjugated with HRP and the substrate o-aminophenol resulted in a response signal output current measured using two electrode systems (cadmium reference electrode and glassy carbon working electrode) and an agilent HP34401A 6.5 digital multimeter without a potentiostat or applied potential input. A maximum response signal output current was recorded for various concentrations of Salmonella viz., 3, 30, 300, 3000, 30,000 and 300,000 cells. The biosensor has a detection limit of three cells, which is very sensitive when compared with other detection sensors. Little non-specific response was observed using Streptococcus, Vibrio, and Pseudomonas sp. The maximum response signal output current for a dialysis membrane electrode interface disc was greater than that for gelatin, collagen, and agarose. The device and technique have a range of biological applications. This novel detection system has great potential for future development and application in surveillance for microbial pathogens.

Bacteriophages are bacterial cell-borne viruses that act as natural bacteria killers and they have been identified as therapeutic antibacterial agents. Bacteriophage therapy is a bacterial disease medication that is given to humans after a diagnosis of the disease to prevent and manage a number of bacterial infections. The ability of phage to invade and destroy their target bacterial host cells determines the efficacy of bacteriophage therapy. Bacteriophage therapy, which can be specific or nonspecific and can include a single phage or a cocktail of phages, is a safe treatment choice for antibiotic-resistant and recurrent bacterial infections after antibiotics have failed. A therapy is a cure for health problems, which is administered after the diagnosis of the diseases in the patient. Such non-antibiotic treatment approaches for drug-resistant bacteria are thought to be a promising new alternative to antibiotic therapy and vaccination. The occurrence, biology, morphology, infectivity, lysogenic and lytic behaviours, efficacy, and mechanisms of bacteriophages’ therapeutic potentials for control and treatment of multidrug-resistant/sensitive bacterial infections are discussed. Isolation, long-term storage and recovery of lytic bacteriophages, bioassays, in vivo and in vitro experiments, and bacteriophage therapy validation are all identified. Holins, endolysins, ectolysins, and bacteriocins are bacteriophage antibacterial enzymes that are specific. Endolysins cause the target bacterium to lyse instantly, and hence their therapeutic potential has been explored in “Endolysin therapy.” Endolysins have a high degree of biochemical variability, with certain lysins having a wider bactericidal function than antibiotics, while their bactericidal activities are far narrower. Bacteriophage recombinant lysins (chimeric streptococcal–staphylococcal constructs) have high specificity for a single bacterial species, killing only that species (lysin (CF-301) is focused to kill methicillin resistant Staphylococcus aureus (MRSA)), while other lysins have a broader lytic activity, killing several different bacterial species and hence the range of bactericidal activity. New advances in medicine, food safety, agriculture, and biotechnology demonstrate molecular engineering, such as the optimization of endolysins for particular applications. Small molecule antibiotics are replaced by lysins. The chapter discusses the occurrences of lytic phage in pathogenic bacteria in animals and humans, as well as the possible therapeutic effects of endolysins-bacteriophage therapy in vivo and in vitro, demonstrating the utility and efficacy of the therapy. Further developments in the bacteriophage assay, unique molecular-phage therapy, or a cocktail of phage for the control of a broad range of drug-resistant bacteria-host systems can promote non-antibiotic treatment methods as a viable alternative to conventional antibiotic therapy.

Day/night cycle controls neurogenesis; melatonin released from pineal gland in darkness stimulates intracellular Ca2+ dynamics thus decreasing proliferation of neural stem cells. In the daylight intracellular Ca2+ activity subsides, which stimulates neural stem cells division and increases generation of newborn neurones.

Graphical AbstractFlow chart depicting the methods, preparing, and characterizing, by histological, and scanning electron microscopy, of wet (PW-HAM) and dry (PD-HAM)of wound healing dressing, and preparation of nanoparticles (HAMP ZnO NP); and application of HAM wound dressing.

A promising therapeutic strategy for amyotrophic lateral sclerosis (ALS) treatment is stem cell therapy. Neural progenitors derived from induced pluripotent cells (NP-iPS) might rescue or replace dying motoneurons (MNs). However, the mechanisms responsible for the beneficial effect are not fully understood. The aim here was to investigate the mechanism by studying the effect of intraspinally injected NP-iPS into asymptomatic and early symptomatic superoxide dismutase (SOD)1(G93A) transgenic rats. Prior to transplantation, NP-iPS were characterized in vitro for their ability to differentiate into a neuronal phenotype. Motor functions were tested in all animals, and the tissue was analyzed by immunohistochemistry, qPCR, and Western blot. NP-iPS transplantation significantly preserved MNs, slowed disease progression, and extended the survival of all treated animals. The dysregulation of spinal chondroitin sulfate proteoglycans was observed in SOD1(G93A) rats at the terminal stage. NP-iPS application led to normalized host genes expression (versican, has-1, tenascin-R, ngf, igf-1, bdnf, bax, bcl-2, and casp-3) and the protection of perineuronal nets around the preserved MNs. In the host spinal cord, transplanted cells remained as progenitors, many in contact with MNs, but they did not differentiate. The findings suggest that NP-iPS demonstrate neuroprotective properties by regulating local gene expression and regulate plasticity by modulating the central nervous system (CNS) extracellular matrix such as perineuronal nets (PNNs).

The conceptual and experimental origins of neurosecretion are discussed. The discovery of the neurosecretory cell by the Scharrers and Bargmann and their insights that this cell type possessed the characteristics of both endocrine cells and neurons are presented. A fundamental feature of the neurosecretory cell, also referred to as a neuroendocrine cell, is the presence of many large dense core vesicles (neurosecretory vesicles) in its cytoplasm. The role of neurosecretory vesicles in the biosynthesis and secretion of the prototypic neurohormones, oxytocin and vasopressin, in the hypothalamo-neurohypophysial system is described.

The magnocellular neurons (MCNs), with their somata situated in the supraoptic and paraventricular nuclei of the hypothalamus, and their nerve terminals in the posterior pituitary (neurohypophysis), are a classical example of a neuroendocrine system. This hypothalamic-neurohypophysial system (HNS) has proven to be an important model for understanding the organization of neuronal Ca2+ homeostasis and mechanisms of neurosecretion. The MCNs synthesize, in a cell-specific manner, two neurohormones: arginine vasopressin (AVP) and oxytocin (OT), which can be released, in a Ca2+-dependent manner, both at the neurohypophysial terminal and at the somatodendritic levels. The two types of MCNs have distinct types of electrical activity leading to specific secretory patterns. OT has positive and AVP MCNs have various feedback on their own release from dendrites, but not from their axon terminals. Action potentials and the voltage-gated Ca2+ channels they open are the primary regulators of [Ca2+]i release in HNS terminals. Both HNS compartments utilize intracellular [Ca2+]i to regulate release of their peptides. However, whereas dendrites of OT neurons utilize inositol 1,4,5-trisphosphate (IP3) receptors, OT terminals utilize ryanodine receptors (RyRs) to regulate OT release. AVP release is not regulated in this way in either compartment. The somatodendritic AVP and OT release closely correlates with intracellular Ca2+ dynamics. More importantly, the Ca2+ stores in the endoplasmic reticulum (ER) play a major role in Ca2+ homeostasis in identified OT neurons. The Ca2+ homeostatic systems in the somata and dendrites differ from those active in the terminals; in the latter, it is mainly Ca2+ extrusion through the Ca2+ pump in the plasma membrane and uptake by mitochondria and neurosecretory granules (NSG) that are active. In both AVP and OT nerve terminals, no functional ER Ca2+ stores can be demonstrated experimentally. Instead, the NSG themselves store and release Ca2+. Nevertheless, trafficking of NSG appears to be the main mechanism for facilitation of peptide release in both compartments. Finally, SNARE-mediated exocytosis is different in HNS somata versus terminals. These fundamental differences in neurosecretion between somatodendrites and axon terminals highlight the importance of characterizing functional mechanisms in such compartments of neuroendocrine cells.

The extracellular matrix (ECM) consists of proteins, glycosaminoglycans and glycoproteins, that support the dynamic interactions between cells, including intercellular communication, cell attachment, cell differentiation, cell growth and migration. As such, the ECM represents an essential and very sensitive system within the tissue microenvironment that is involved in processes such as tissue regeneration and carcinogenesis. The aim of the present review is to evaluate its diversity through Ca(2+) signaling and its role in muscle cell function. Here, we discuss some methodological approaches dissecting Ca(2+) handling mechanisms in myogenic and non-myogenic cells, e.g. the importance of Ca(2+) and calpains in muscle dystrophy. We also consider the reconstruction of skeletal muscle by colonization of decellularized ECM with muscle-derived cells isolated from skeletal muscle. Therefore, it is necessary to establish new methodological procedures based on Ca(2+) signaling in skeletal muscle cells and their effect on ECM homeostasis, allowing the monitoring of skeletal muscle reconstruction and organ repair.

The aim of this study was to evaluate cell diversity by considering how Ca2+ signaling has been adapted in skeletal muscle cell function. We characterized single C2C12 myoblasts through intracellular Ca2+ signaling kinetics after exposure to specific drugs and calcium blockers using fast fluorescence microspectrofluorimetry followed by ATP effect analysis, which confirmed the expression of functional purinergic adenosine and P2 receptors. Further, we found that glutamate sensitivity of C2C12 cells was mediated by ionotropic glutamate receptors; on the other hand, most cells were responsive to cyclopiazonic acid, which inhibits the sarco-endoplasmic reticulum Ca2+-ATPase pump. These results suggest that C2C12 cells possess functional L- and P/Q-type voltage-operated Ca2+ channels, ryanodine receptors and functional sarcoplasmic reticulum Ca2+ stores (typical for muscle cells), adenosine and P2 purinergic receptors, as well as ionotropic glutamate receptors. The evaluation of intracellular Ca2+ signaling is a promising approach towards a better understanding and control of the physiopathological properties of myogenic cells that could be used as a predictive factor in the selection of optimal cells for scaffold recellularization or for tissue engineered constructs used in stem cell therapy.

Rat melanotrophs express several types of voltage-gated and ligand-gated calcium channels, although mechanisms involved in the maintenance of the resting intracellular Ca2+ concentration ([Ca2+](i)) remain unknown. We analyzed mechanisms regulating resting [Ca2+](i) in dissociated rat melanotrophs by Ca2+-imaging and patch-clamp techniques. Treatment with antagonists of L-type, but not N- or P/Q-type voltage-gated Ca2+ channels (VGCCs) as well as removal of extracellular Ca2+ resulted in a rapid and reversible decrease in [Ca2+](i), indicating constitutive Ca2+ influx through L-type VGCCs. Reduction of extracellular Na+ concentration (replacement with NMDG(+)) similarly decreased resting [Ca2+](i). When cells were champed at -80 mV, decrease in the extracellular Na+ resulted in a positive shift of the holding current. In cell-attached voltage-clamp and whole-cell current-clamp configurations, the reduction of extracellular Na+ caused hyperpolarisation. The holding current shifted in negative direction when extracellular K+ concentration was increased from 5 mM to 50 mM in the presence of K+ channel blockers, Ba2+ and TEA, indicating cation nature of persistent conductance. RT-PCR analyses of pars intermedia tissues detected mRNAs of TRPV1, TRPV4, TRPC6, and TRPM3-5. The TRPV channel blocker, ruthenium red, shifted the holding current in positive direction, and significantly decreased the resting [Ca2+](i). These results indicate operation of a constitutive cation conductance sensitive to ruthenium red, which regulates resting membrane potential and [Ca2+](i) in rat melanotrophs.

The neurohormones arginine-vasopressin (AVP) and oxytocin (OT) synthesised in supraoptic and paraventricular nuclei of neurohypophysis regulate lactation, systemic water homeostasis and nociception. Using transgenic rats expressing AVP and OT tagged with fluorescent proteins we demonstrate that both neurohormones are expressed in sensory neurones both in vitro, in primary cultures, and in situ, in the intact ganglia; this expression was further confirmed with immunocytochemistry. Both neurohormones were expressed in nociceptive neurones immunopositive to transient receptor potential vannilloid 1 (TRPV1) channel antibodies. The AVP and OT-expressing DRG neurones responded to AVP, OT, 50 mM K+ and capsaicin with [Ca2+](i) transients; responses to AVP and OT were specifically blocked by the antagonists of V-1 AVP and OT receptors. Probing the extracellular incubation saline with ELISA revealed AVP and OT secretion from isolated DRGs; this secretion was inhibited by tetanus toxin (TeNT) indicating the role for vesicular release. Expression of OT, but not AVP in DRG neurones significantly increased during lactation. Together, the results indicate novel physiological roles (possibly related to nociception and mood regulation) of AVP and OT in the sensory neurones.

Pathological remodeling, degeneration and reactivity of astrocytes are fundamental astrogliopathies contributing to all neurological diseases. In neurodegenerative disorders (including Alzheimer's disease [AD]) astroglia undergo complex changes that range from atrophy with loss of function to accumulation of reactive cells around disease-specific lesions (senile plaques in the case of AD). The cellular pathology of astroglia in the context of human AD remains enigmatic; mainly because of the severe limitations of animal models, which, although reproducing some pathological features of the disease, do not mimic its progression in full. Human-induced pluripotent stem cells technology creates a novel and potentially revolutionizing platform for studying fundamental mechanisms of the disease and for screening to identify new therapeutic compounds.

Anterior tibial muscle of C57Bl6/J mice was subjected to decellularization with hypotonic solution, detergents and DNase. Resulting acellular scaffolds were examined to characterize the content of chromatin, cell cytoplasm and extracellular matrix components incl. basal laminas, fibres and glycoproteins. Although the sarcoplasm and cell nuclei were removed, the general skeletal muscle microarchitecture with ECM of stromal components remained well preserved at light and electron microscopic levels. Moreover, basal laminas contouring honeycomb-like structures left after removal of myofibres and vascular endothelium remained intact. Immunostaining of scaffolds for collagen IV and laminin confirmed positivity of basal laminas. Histochemical staining of deparaffinised scaffold sections identified well organized fibres after staining with green trichrome, Sirius red, Weigert’s resorcin fuchsin and Gomori impregnation. Chemical analysis gave evidence of reduced dsDNA and well-preserved collagen according to high hydroxyproline content and laminin as documented by Western blotting. We cultured scaffolds seeded with murine myogenic cells in vitro and confirmed their cytocompatibility as the cells were able to adhere, grow and migrate through the ECM without affecting the scaffold structure. Myogenic cells were able to migrate in the endomysium and start to fuse. Implantation of decellularized scaffolds into an artificial cavity inside of anterior tibial muscle of mice in vivo confirmed the scaffolds were colonized soon by recipient inflammatory cells without formation of foreign body giant cells. Scaffolds were well integrated with recipient skeletal muscle and gradually resorbed within 3 weeks. Our results confirm decellularized muscle scaffold is a promising alternative for rebuilding the skeletal muscle organ as it can preserve basic chemical components and the tissue microstructure and show biocompatibility for myogenic cells as demonstrated in vitro and in vivo.

Adherent, fibroblastic cells from different tissues are thought to contain subsets of tissue-specific stem/progenitor cells (often called mesenchymal stem cells). These cells display similar cell surface characteristics based on their fibroblastic nature, but also exhibit differences in molecular phenotype, growth rate, and their ability to differentiate into various cell phenotypes. The mechanisms underlying these differences remain poorly understood. We analyzed Ca2+ signals and membrane properties in rat adipose-derived stromal cells (ADSCs) and bone marrow stromal cells (BMSCs) in basal conditions, and then following a switch into medium that contains factors known to modify their character. Modified ADSCs (mADSCs) expressed L-type Ca2+ channels whereas both L- and P/Q- channels were operational in mBMSCs. Both mADSCs and mBMSCs possessed functional endoplasmic reticulum Ca2+ stores, expressed ryanodine receptor-1 and -3, and exhibited spontaneous [Ca2+]i oscillations. The mBMSCs expressed P2X7 purinoceptors; the mADSCs expressed both P2X (but not P2X7) and P2Y (but not P2Y1) receptors. Both types of stromal cells exhibited [Ca2+]i responses to vasopressin (AVP) and expressed V1 type receptors. Functional oxytocin (OT) receptors were, in contrast, expressed only in modified ADSCs and BMSCs. AVP and OT-induced [Ca2+]i responses were dose-dependent and were blocked by their respective specific receptor antagonists. Electrophysiological data revealed that passive ion currents dominated the membrane conductance in ADSCs and BMSCs. Medium modification led to a significant shift in the reversal potential of passive currents from −40 to −50mV in cells in basal to −80mV in modified cells. Hence membrane conductance was mediated by non-selective channels in cells in basal conditions, whereas in modified medium conditions, it was associated with K+-selective channels. Our results indicate that modification of ADSCs and BMSCs by alteration in medium formulation is associated with significant changes in their Ca2+ signaling and membrane properties.

a Department of Molecular Neurophysiology, Institute of Experimental Medicine, Czech Academy of Sciences, Videnska 1083, Prague 14220, Czech Republic b Department of Neuroscience, Charles University, Second Faculty of Medicine, V Uvalu 84, Prague 15006, Czech Republic c Department of Cellular Neurophysiology, Institute of Experimental Medicine, Czech Academy of Sciences, Videnska 1083, Prague 14220, Czech Republic d Department of Neuroscience, Institute of Experimental Medicine, Czech Academy of Sciences, Videnska 1083, Prague 14220, Czech Republic e University of Manchester, School of Biological Sciences, D.4417 Michael Smith Building, Oxford Road, Manchester M13 9PT, UK f Achucarro Center for Neuroscience, IKERBASQUE, Basque Foundation for Science, 48011 Bilbao, Spain g University of Nizhny Novgorod, Nizhny Novgorod 603022, Russia h Institut National de la Santé et de la Recherche Médicale-U1198, Université Montpellier, Montpellier 34095, France i Ecole Pratique des Hautes Etudes-Sorbonne, Les Patios Saint-Jacques, 4-14 rue Ferrus, 75014 Paris, France

In eukaryotic cells, Endoplasmic Reticulum (ER) is an interconnected membranous organelle and plays important roles in protein synthesis and lipid metabolism. We have previously demonstrated that TMCO1 is an ER Ca2+ channel actively preventing ER Ca2+ overloading. Recently, we also found that TMCO1 deficiency in mouse granulosa cells (GCs) caused abnormal Ca2+ signaling, ER stress and enhanced reactive oxygen species (ROS). In this study, we further examined the roles of TMCO1 in lipid metabolism and mitochondrial functions. Intriguingly, we found that TMCO1 deletion reduced the number of lipid droplets (LDs) and the content of triglyceride (TG), which was due to ER stress associated degradation (ERAD) of the important enzyme in catalyzing TG synthesis, diacylglycerol acyltransferase 2 (DGAT2). Hypofunction in transforming non-esterification fatty acid (NEFA) to TG caused NEFA deposit, a potential risk of mitochondrial dysfunction. Furthermore, in TMCO1 deficient cells, mitochondria volume decreased and inefficient oxidative phosphorylation was detected, which underlined enhanced mitophagy and impaired mitochondrial functions. Taken these data together, we for the first time revealed the role of TMCO1 in regulating lipid-metabolism and mitochondrial function. This study may provide new insights into understanding TMCO1 defect syndrome.

The magnocellular vasopressin (AVP) and oxytocin (OT) neurones exhibit specific electrophysiological behaviour, synthesise AVP and OT peptides and secrete them into the neurohypophysial system in response to various physiological stimulations. The activity of these neurones is regulated by the very same peptides released either somato-dendritically or when applied to supraoptic nucleus (SON) preparations in vitro. The AVP and OT, secreted somato-dendritically (i.e. in the SON proper) act through specific autoreceptors, induce distinct Ca(2+) signals and regulate cellular events. Here, we demonstrate that about 70% of freshly isolated individual SON neurones from the adult non-transgenic or transgenic rats bearing AVP (AVP-eGFP) or OT (OT-mRFP1) markers, produce distinct spontaneous [Ca(2+)]i oscillations. In the neurones identified (through specific fluorescence), about 80% of AVP neurones and about 60% of OT neurones exhibited these oscillations. Exposure to AVP triggered [Ca(2+)]i oscillations in silent AVP neurones, or modified the oscillatory pattern in spontaneously active cells. Hyper- and hypo-osmotic stimuli (325 or 275 mOsmol/l) respectively intensified or inhibited spontaneous [Ca(2+)]i dynamics. In rats dehydrated for 3 or 5days almost 90% of neurones displayed spontaneous [Ca(2+)]i oscillations. More than 80% of OT-mRFP1 neurones from 3 to 6-day-lactating rats were oscillatory vs. about 44% (OT-mRFP1 neurones) in virgins. Together, these results unveil for the first time that both AVP and OT neurones maintain, via Ca(2+) signals, their remarkable intrinsic in vivo physiological properties in an isolated condition.

Isolated supraoptic neurones generate spontaneous [Ca2+]i oscillations in isolated conditions. Here we report in depth analysis of the contribution of plasmalemmal ion channels (Ca2+, Na+), Na+/Ca2+ exchanger (NCX), intracellular Ca2+ release channels (InsP3Rs and RyRs), Ca2+ storage organelles, plasma membrane Ca2+ pump and intracellular signal transduction cascades into spontaneous Ca2+ activity. While removal of extracellular Ca2+ or incubation with non-specific voltage-gated Ca2+ channel (VGCC) blocker Cd2+ suppressed the oscillations, neither Ni2+ nor TTA-P2, the T-type VGCC blockers, had an effect. Inhibitors of VGCC nicardipine, ω-conotoxin GVIA, ω-conotoxin MVIIC, ω-agatoxin IVA (for L-, N-, P and P/Q-type channels, respectively) did not affect [Ca2+]i oscillations. In contrast, a specific R-type VGCC blocker SNX-482 attenuated [Ca2+]i oscillations. Incubation with TTX had no effect, whereas removal of the extracellular Na+ or application of an inhibitor of the reverse operation mode of Na+/Ca2+ exchanger KB-R7943 blocked the oscillations. The mitochondrial uncoupler CCCP irreversibly blocked spontaneous [Ca2+]i activity. Exposure of neurones to Ca2+ mobilisers (thapsigargin, cyclopiazonic acid, caffeine and ryanodine); 4-aminopyridine (A-type K+ current blocker); phospholipase C and adenylyl cyclase pathways blockers U-73122, Rp-cAMP, SQ-22536 and H-89 had no effect. Oscillations were blocked by GABA, but not by glutamate, apamin or dynorphin. In conclusion, spontaneous oscillations in magnocellular neurones are mediated by a concerted action of R-type Ca2+ channels and the NCX fluctuating between forward and reverse modes.

Stem cells research has passed a long and exciting way from discovery to clinical applications. Every year more and more scientific reports and solid research breakthroughs are published in this fascinating field making it dif - ficult to follow new discoveries and cover the history. In the current review we overviewed the history of stem cells research starting from the discovery and ending with the current state-of-art. We discussed the obstacles and future perspectives of the cell-based therapy, with a special focus made on the protection and regeneration of the lost functions after injury/degeneration of adult central nervous system.