Since the discovery of lysosomes more than 70 years ago, much has been learned about the functions of these organelles. Lysosomes were regarded as exclusively degradative organelles, but more recent research has shown that they play essential roles in several other cellular functions, such as nutrient sensing, intracellular signalling and metabolism. Methodological advances played a key part in generating our current knowledge about the biology of this multifaceted organelle. In this review, we cover current methods used to analyze lysosome morphology, positioning, motility and function. We highlight the principles behind these methods, the methodological strategies and their advantages and limitations. To extract accurate information and avoid misinterpretations, we discuss the best strategies to identify lysosomes and assess their characteristics and functions. With this review, we aim to stimulate an increase in the quantity and quality of research on lysosomes and further ground‐breaking discoveries on an organelle that continues to surprise and excite cell biologists. Lysosomes play essential roles beyond their well‐known degradative function, including nutrient sensing, intracellular signalling and metabolism. To better understand these functions, it is essential to employ the best methodological approaches and know their advantages and limitations. Here, we review current methods used to analyze lysosome morphology, positioning, motility and function. The proper use of these methods will allow new discoveries to be made about this fascinating organelle. Feeding leads to the activation of the metabolic sensor kinase mammalian target of rapamycin (mTOR) at the surface of lysosomes to aid in the recycling of the digested macromolecules. Conversely, cellular starvation leads to inhibition of mTOR and the activation of the master transcription factor EB (TFEB), responsible for the expression of a set of genes leading to the biogenesis of new lysosomes.Recent advances in lysosome biology have been comprehensively described in excellent recent reviews.2trans‐Golgi network (TGN) to endosomes via the cation‐dependent or the cation‐independent MPR, where they dissociate from the receptor in the acidic endosome lumen. MPRs recycle to the Golgi while the lysosomal enzymes are retained within the endosome lumen for subsequent lysosomal delivery. The low pH of late endosomes, their accumulation of internal membranes and their content of newly synthesized ‘lysosomal’ proteins, raises the question of how lysosomes can be distinguished from endosomes. Lysosomes are, however, distinct entities to which soluble cargo and intraluminal vesicles are delivered via full fusion or kiss‐and‐run  between late endosomes and pre‐existing lysosomes.Progression along the endocytic pathway from early to late endosomes is characterized by a gradual lowering of pH, removal of recycling proteins and accumulation of intraluminal vesicles that form by inward invagination of the endosomal limiting membrane. Endosomes also have an important role in lysosomal maintenance by delivering newly synthesized lysosomal membrane proteins and enzymes. Many lysosomal enzymes are delivered from the Lysosomes are heterogeneous in terms of morphology, composition, pH and intracellular distribution.e.g., LAMP2 is involved in chaperone‐mediated autophagyThe number and identity of lysosomal membrane proteins depends on the cell type, the method of isolation and how a lysosomal membrane protein is defined. As an example, more than 700 proteins were found enriched in liver lysosomal membranes, of which more than 200 were known or predicted endolysosomal proteins.+)‐ATPase (v‐ATPase), which maintains the acidic lumen of the lysosome, and multiple ion channels  and transporters . Components of the trafficking machinery that regulate the interaction/fusion of lysosomes with other organelles are also used as lysosomal markers. These include small guanosine triphosphatases , the homotypic fusion and vacuolar protein sorting (HOPS) complex proteins, some soluble N‐ethylmaleimide sensitive factor attachment protein receptors (SNARE) proteins, including vesicle associated membrane protein 7 (VAMP7) and syntaxins 7 and 8, as well as the Ca2+ binding protein synaptotagmin VII.Degradative enzymes, of which there are over 70, are also used as lysosomal markers, although the relative abundance of different enzymes can vary between cell types. As described above, many lysosomal membrane proteins and enzymes, including LAMPs and cathepsins, respectively, are delivered to lysosomes via the endocytic pathway and so will not be entirely restricted to lysosomes. Indeed, the degree to which a ‘lysosomal marker’ extends along the endocytic pathway can be highly cell type‐dependent. One way around this issue is to compare the distribution of the ‘lysosomal’ marker with that of MPRs, which can be found throughout the endocytic pathway, but not in lysosomes. Additional potential lysosomal markers include subunits of the vacuolar  were originally described as cell‐type specific specialized secretory organelles, mainly found in cells of the haemopoietic lineage, which share characteristics with lysosomes. LROs are formed using machinery that was first identified in studies of genetic disorders such as Chediak‐Higashi syndrome, Griscelli syndrome and Hermansky‐Pudlak syndrome While LRO‐specialized contents and their morphology are very diverse, partly reflecting different contents, at least some of the machinery underlying their biogenesis and secretion is shared, including the small GTPase Rab27, which is usually found on the membrane of LROs, while absent from conventional lysosomes.i.e., Weibel‐Palade bodies) initially form in an AP‐1‐dependent process at the trans‐Golgi network (TGN),Conventionally, LROs arise from an endosomal compartment modified by receiving specialized cargo delivered by an adaptor protein complex (AP)‐3‐dependent route. However, this is not the only model; endothelial LROs . We44.1Microscopic visualization is one of the first steps in the analysis of lysosomes. The discovery of new lysosomal proteins along with the development of new antibodies and dyes to selectively label lysosomes have provided an invaluable set of tools for the study of this subcellular compartment.The visualization of lysosomes by light microscopy, mainly using fluorescence, can be achieved through different approaches: (a) immunolabelling of lysosomal proteins, either membrane‐bound proteins or soluble proteins such as enzymes; (b) use of cargoes internalized by endocytosis; (c) use of freely diffusible dyes that emit fluorescence only in the lysosomal acidic environment or in the presence of active lysosomal hydrolases. Immunolabelling of lysosomes can be performed by using antibodies against lysosomal membrane proteins, such as LAMP1 and LAMP2.e.g., Magic Red—see Section e.g., LysoTracker—see Section Labelling of luminal lysosomal hydrolases can also be used in this context, for which different approaches are available. One is immunolabelling with antibodies against cathepsins such as cathepsin B  or cathepsin D . These antibodies allow the visualization of lysosomes either by immunofluorescence or immunohistochemistry. Other suitable methodologies rely on the use of fluorescently labelled inhibitors of lysosomal hydrolases such as Bodipy FL‐Pepstatin A , a cathepsin D inhibitor that targets it within lysosomes of living and fixed cells, or fluorescently labelled small inhibitors that specifically recognize active lysosomal hydrolases such as the glucocerebrosidase (GBA).i.e., with Triton X‐100), this can interfere with the localization of LAMPs. In this case, a two‐step immunofluorescence labelling is required. For this, cells are fixed and permeabilized with saponin or digitonin, blocked with BSA or species‐matched serum and incubated with anti‐LAMP antibody and subsequently with a fluorescently‐labelled secondary antibody. Cells are then post‐fixed with paraformaldehyde (PFA) and then permeabilized with Triton X‐100, without perturbing LAMP localization. As noted above, it is essential to keep in mind that LAMPs do not label lysosomes specifically, as these molecules are present also in late endosomes, at the plasma membrane and on LAMP carrier vesiclesVisualization of lysosomes by fluorescence microscopy is also a fast and reliable way to infer possible pathological alterations in lysosomes by studying the distribution, size and presence of accumulated luminal material. However, fixation and permeabilization conditions may require optimization, especially in the case of co‐labelling of lysosomes and other markers. If the labelling of a marker of interest requires harsher permeabilization  and in vivo  processes. Thus, depending on the applications, it is available in different molecular weights, with a neutral or anionic net charge. Dextrans are biologically inert because they are composed of a poly‐ linkage that cannot be degraded by lysosomal hydrolases. Dextrans are available linked to different fluorophores such as Alexa Fluor, Oregon Green and Lucifer Yellow, the latter being useful also to monitor water flux in intracellular organelles and lysosomes given its sensitivity to deuterium (D2O) concentration.2O‐containing medium according to the extent of D2O influx in lysosomes. Dextrans are stable within lysosomes, and, for this reason, represent a bona fide marker of lysosomal terminal compartments. The conditions to allow dextran to be used as a reliable lysosomal marker are cell‐specific and careful optimization of dextran concentration and pulse/chase times must be carried out for each cell type. For instance, in kidney epithelial cells, dextran is administered in complete medium at concentrations ranging from 50 to 500 μg/mL, for 2 hours (pulse).Dextran is a hydrophilic polysaccharide with low immunogenicity. It can be conjugated with different dyes to evaluate a variety of uterium DO concente.g., HAP‐1, Figure This assay is a powerful tool to monitor endocytosis4.1.2DQ‐BSA is a fluorescent probe composed of a BSA derivative conjugated to the self‐quenched red‐BODIPY dye. After DQ‐BSA internalization by fluid‐phase endocytosis, this probe is degraded by proteolysis in lysosomes. This results in the release of bright fluorescent fragments Figure , allowin4.2e.g., in LSDs) or even within a single population of cells, the following approaches can be taken to facilitate lysosomal recognition, as well as providing additional information  as electron‐dense organelles enclosed by a single membrane, typically ranging from 200 to 600 nm in diameter. The formation of electron‐dense multilamellar whorls  is a useful distinguishing feature of lysosomal ultrastructure4.2.1e.g., EGF)Coupled with morphological analyses, pulse/chase experiments with fluid phase or receptor‐targeted probes can also provide information on efficiency of lysosomal delivery. Fluid‐phase probes such as BSA or ligands/antibodies to the extracellular domains of proteins targeted for lysosomal degradation can be coupled to electron‐dense gold particles or to horseradish peroxidase (HRP) to aid lysosome visualization. HRP  is relatively resistant to lysosomal degradation and can be readily visualized by the presence of electron‐dense 3,3′‐diaminobenzidine  reaction product.4.2.2in situ to electron‐dense particles visible by EM.N‐{3‐[amino]propyl}‐N‐(3‐aminopropyl)methylamine dihydrochloride , which selectively accumulates in acidic organelles and, as well as emitting a fluorescent signal, can also be detected by antibodies targeting dinitrophenol  projections produced from three‐dimensional (3D) samples. Aligned 2D images obtained from thicker sections, tilting the sample in a series of different angles, can be digitally combined to generate 3D reconstructions in a technique known as electron tomography. The increased information provided by 3D tomographic reconstructions is especially useful where the membrane may dip in and out of a 2D projection, for example in characterizing tubular lysosomes4.2.4Lysosomes are highly dynamic organelles and while EM can capture lysosomal positioning, live cell light microscopy of fluorescent probes or ectopically expressed/gene‐edited fluorescent lysosomal proteins is ideally suited for studying lysosome dynamics. By combining information from fluorescence and EM in correlative light EM (CLEM), dynamic cellular events and localization of fluorescent proteins can be visualized in the context of the cellular ultrastructure, at resolutions that can only be achieved by EM. However, this can be a challenging technique. The focal plane needs to be considered for accurate correlation of ultrathin TEM sections with confocal images and light microscopy can be coupled with SBF‐SEM or FIB‐SEM.4.32+ signalling,2+ dynamics,There is increasing evidence that lysosome biogenesis and function are strongly influenced by connections with other organelles at MCS. A surge in interest in MCS that form between the ER and late endocytic organelles has led to major advances in our understanding of their regulation and function over the last decade.MCS are generally defined as regions where the membranes of neighbouring organelles are 5 to 30 nm apart. As the only means to accurately resolve this distance, EM remains the gold‐standard for MCS analysis, even allowing visualization of the tethers that bridge the interface of the two organelles to stabilize the MCS. However, not all labs have access to EM and while MCS are dynamic, often transient, events, EM is limited to fixed samples. Therefore, several fluorescence microscopy techniques have been developed to study the dynamics of inter‐organelle interactions.4.3.1in situ detection of protein interactions.e.g., LAMP1 for lysosomes). Oligonucleotide‐labelled secondary antibodies  allows 4.3.2Advances in imaging and post‐image analysis of organelle‐targeted fluorescent proteins has transformed our ability to study the dynamics of inter‐organelle associations. Live cell confocal and lattice light sheet spectral imaging has been coupled with imaging informatics to map dynamic MCS between multiple organelles in living cells.Biochemical approaches have been developed that can be coupled with standard confocal microscopy imaging for studying MCS. These include complementation assay approaches that depend on the generation of a fluorescent signal when two proteins or fragments that are targeted to different organelles are in close proximity. They include fluorescence resonance energy transfer (FRET),4.3.3e.g., LAMP1) and cloned upstream of a self‐cleaving viral 2A peptide. The complementing GFP1‐10 is targeted to the lysosome‐associated organelle of interest  and cloned downstream, to ensure an equimolar amount of the two transgenes. The construct is under the control of a bidirectional UAS promoter, together with a cytosolic DsRed for identification of expressing cells, to allow GAL4‐driven expression of the UAS promoter.A splitGFP contact site sensor (SPLICS) has been developed as a one‐step approach to dynamically detect inter‐organelle juxtaposition. This approach has been successfully used to visualize ER:mitochondria MCS in zebrafish neurons,4.4A critical aspect of lysosomal function is its acidic pH. Lysosomal acidification is achieved by the activity of the v‐ATPase, a multisubunit protein complex that uses the energy derived from ATP hydrolysis to transport protons across the lysosomal membrane into the lumen of this organelle.4.4.1LysoTracker dyes are fluorescent acidotropic probes that label acidic organelles in live cells and are available in a variety of colours and versions such as the Lysotracker Red DND‐99 , which retains good fluorescence after aldehyde fixation, but not after fixation with other reagents like glyoxal.4.4.2The assessment of the lysosomal luminal pH by ratiometric fluorescence is indicative of relative differences in pH. Moreover, a calibration curve of the signal can be used to convert the fluorescence ratio in absolute pH values. LysoSensor yellow/blue DND‐160  exhibits a pH‐dependent increase in fluorescence intensity upon acidification, in contrast with LysoTracker probes.e.g., fluorescein isothiocyanate [FITC] and pHrodo dyes—Invitrogen, D1820 and Invitrogen, P35368, respectively) and a second, pH‐insensitive fluorophore .Ratiometric assessment of lysosomal pH can also be performed using pulse‐chased fluid phase markers, like dextrans labelled with Oregon Green , a fluorinated analog of fluorescein that is intrinsically ratiometric or with both a pH‐sensitive fluorophore  vinyl]‐3H‐benzo[e]indole—BiDLFinally, to overcome dye loading, leakage, optical imprecisions or photobleaching, efforts have been done do develop new ratiometric probes to quantify lysosome pH  a tandem fusion of pHluorin‐mCherry linked to the luminal domain of LAMP14.52+ in the hundred‐micromolar range in their lumen. Furthermore, in contrast to organelles such as the ER, lysosomes and LROs display a membrane potential dependent on K+ channels, such as the Ca2+‐activated potassium channel (BK) and TMEM175, of around −20 mV (convention states that the positive lumen is considered the same as the extracellular space),2+ storage organelle allows them to buffer and release Ca2+, and thus are now recognized as playing an important role in Ca2+ signalling and homeostasis. Because Ca2+ signals are highly localized due to cellular buffering mechanisms, release from small mobile or positioned organelles can deliver Ca2+ to a specific cellular target. Lysosomal Ca2+ is important for membrane fusion events and trafficking, possibly luminal Ca2+ for enzyme activity, and Ca2+ release for both distinct and more general roles in Ca2+ signalling and thus needs to be measured and manipulated.Lysosomes contain free Ca2+ is taken up into the endolysosomal system from the extracellular milieu by endocytosis.2+ filling/storage is pH‐dependent. However, membrane contact sites between the ER and lysosomes have also been proposed as sites of Ca2+ transfer between the two organelles, and this may be bidirectional.2+ release from lysosomes may trigger a much larger Ca2+‐induced Ca2+ release (CICR) from the ER, which will greatly overestimate their Ca2+ content. Hence, determination of lysosomal Ca2+ content should be done under conditions of ER Ca2+ depletion (in Ca2+‐free media to avoid capacitative Ca2+ entry) or block of the major ER Ca2+ release channels inositol 1,4,5‐trisphosphate receptors (IP3Rs) and the ryanodine receptors (RyRs).Ca2+ permeable channels have been demonstrated to be present in lysosomal membranes, which can mediate Ca2+ release from lysosomes in response to various stimuli, including metabolic factors. These include two‐pore channel subtype 2 (TPC2), P2X4 and the Trp channels, TRPML1, transient receptor potential melastatin‐related 2 (TRPM2) and transient receptor potential ankyrin 1 receptor (TRPA1).2+ selective and pass other cations such as Na+ and H+.2+ release from lysosomes can be directly measured by expressing channel‐genetically encoded Ca2+ indicator (GECI) chimeras containing a lysosomal targeting sequence. This has been done for the luminescent Ca2+ reporter protein aequorin, which is less subject to pH influence.2+ but reports Ca2+ fluxes specifically from lysosomes. Controls should be performed comparing Ca2+ signals from other sources . Surprisingly, using this approach, it has been found that Ca2+ fluxes are highly compartmentalized, with flux through one class of channel not detected by another channel type bearing the probe.A number of Ca2+ can be performed indirectly by emptying the organelle of Ca2+ with lysosomotropic agents such as glycyl‐l‐phenylalanine 2‐naphthylamide . Dye‐esters do not appear to load into lysosomes sufficiently perhaps surprisingly because of lack of luminal esterases.2+ probes to the extracellular medium and allowing them to be endocytosed and trafficked to the terminal lysosome compartment.2+‐insensitive. This ratiometric approach allows for a quantification of free luminal Ca2+. Because the acidic pH of the lysosomal lumen decreases the apparent affinity of Ca2+ probes, luminal pH must be simultaneously measured and corrected for. For resting lysosomal luminal Ca2+ measurements, the acidic pH can be advantageous, as it decreases the in situ affinity of probes for Ca2+, because low affinity probes are required because the resting levels of lysosomal luminal Ca2+ are around 500 μM, which would normally saturate higher affinity indicators. Another recent development is the use of DNA‐based lysosomal fluorescent Ca2+ probes such as CalipHluor, which is also pH correctable.2+ environment and the perilysosomal space can also be manipulated to dampen or remove lysosomal Ca2+ in a more selective way than the pharmacological approaches above. Dextran‐linked 1,2‐bis(o‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA) based probes such as Cal520‐dextran  can traffic to the lysosomal lumen, similar to the Ca2+ probes mentioned above, to buffer luminal Ca2+. Furthermore, a rapalog system has been used to target a Ca2+ chelating protein like calbindin, specifically to the lysosomal surface, to chelate Ca2+ signals originating from the lysosome.Luminal measurement of lysosomal free Ca4.6i.e., nutrient starvation) or genetic  cues may boost the induction of this cellular process.Lysosome biogenesis is the process by which new lysosomes are generated and is essential to guarantee that enough lysosomes are available to support cellular metabolism, in particular catabolic pathways such as autophagy, while generating new building blocks for anabolism. Intracellular trafficking of lysosomal membrane proteins and soluble hydrolases is fundamental for the generation of new functional lysosomes.Regulation of lysosome biogenesis is operated at both transcriptional and translational levels. The existence of transcriptional regulation of lysosome biogenesis was demonstrated by the identification of TFEB as a master regulator of several genes encoding lysosomal membrane proteins and hydrolases.GLA), generated by CRISPR/Cas9. As expected, GLA knockout (KO) cells display lysosomal globotriaosylceramide (Gb3) storage in almost all LAMP1‐positive vesicles, visualized using fluorescently‐labelled subunit B of Shiga toxin, which specifically binds Gb3. GLA KO cell starvation triggers lysosome biogenesis, which does not induce clearance of Gb3 storage from older lysosomes but leads to a marked increase in small LAMP1‐positive vesicles free of Gb3 storage. The appearance of storage‐free lysosomes is indicative of lysosome biogenesis, because in cells in which this process is blocked, there are no storage‐free lysosomes.In addition to the evaluation of lysosome number as a proxy of lysosome biogenesis, it was recently demonstrated that cell models of LSDs represent valuable tools to analyze lysosomes that have been formed through stimulus‐induced lysosome biogenesis. An assay has been developed in a cell model of Fabry disease, an LSD caused by the loss of function of α‐galactosidase gene ‐TFEB signalling axis is a major hub that allows the cell to adjust lysosome number, activity and function, based on the cellular nutritional status.4.7.1TFEB activity is strictly regulated through post‐translational modifications and spatial organization.e.g., nutrient fluctuation) may alter TFEB activity.TFEB localization can be monitored by immunofluorescence analysis using TFEB antibodies, from Cell Signaling (4240) for human cells or from Bethyl Laboratories (A303‐673A), recommended for mouse cells. The active (nuclear) TFEB is dephosphorylated in several serine residues and hence TFEB lower molecular weight can be confirmed by WB using the same antibodies mentioned above. Alternatively, the phosphorylation status of specific serine residues can be monitored by WB using phosphoantibodies, such as phospho‐TFEB (Ser211) from Cell Signaling (37681) or phospho‐TFEB (Ser142) from Millipore (ABE1971). However, because of the low abundance of TFEB in most cell lines, the use of these antibodies usually requires constitutive expression of exogenous TFEB. Furthermore, when assessing TFEB nuclear translocation, cell culture conditions should be carefully controlled because alterations in several environmental conditions  or localize more peripherally and close to the plasma membrane. Juxtanuclear lysosomes are more acidic, immotile and prone to fuse with endosomes or autophagosomes, while peripheral lysosomes are more motile and can undergo exocytosisThe positioning of lysosomes, labelled as described (see Section D(x))/N(x)] * sqrt[N(x)/A]), where D(x) represents the spatial distance to the next neighbour of pixels belonging to the lysosome region; N(x) the number of pixels belonging to the lysosome region; and A the number of pixels of the cell object. This requires lysosome automatic object segmentation .https://quorumtechnologies.com/index.php/component/content/category/31-volocity-software).https://github.com/PTschaikner/RadialIntensityProfile).Different strategies have been used to assess lysosome positioning in a more unbiased way. Automated measurement of lysosome clustering can be done with the Clark aggregation index (CAI)In addition, the morphometric analysis of lysosomes and their distribution in normal or pathological conditions can be assessed by HCI using Opera  a phenotypic image‐based ‘machine learning’ script that calculates the percentage of cells showing a clustered lysosomal distribution vs. cells showing lysosomes correctly distributed throughout the cell Figure .4.94.9.1Lysosome transport along microtubules to the periphery (anterograde movement) is dependent on kinesin motors, while the transport toward the juxtanuclear region (retrograde movement) is dependent on dynein.Lysosomes should be labelled by combining LysoTracker or dextran , a plugin developed for the study of mitochondria movement can be used.Kymographs (plots of line profiles over time) are especially useful for visualization and quantification of movement along dendrites or axons and can be generated semi‐automatically. For this, the selection and tracing of axonal or dendritic segments is done manually in ‘straightened’ neurites or using a segmented line (Fiji/ImageJ). Then, kymographs are automatically plotted using the ‘reslice’ function (Fiji/ImageJ). If there are multiple segments, the multiple kymograph plugin can be used, enabling a quick visualization of moving lysosomes, as well as stationary ones. Moreover, the direction of movement can be easily identified. For example, if the cell body is positioned at the left, the retrograde moving vesicles are toward the left and the anterograde moving ones to the right in the kymograph. Importantly, the manual analysis of moving lysosomes may introduce biases. Therefore, the Trackmate Fiji plugin can be used to analyze lysosome movement, and parameters such as velocity and distance can be also easily obtained.4.9.3in vivo studies , the best examples arise from model systems such as Drosophila melanogaster and zebrafish (Danio rerio). Drosophila is a tractable model system with a sophisticated genetic toolkit, ideal for in vivo studies, where the labelling of organelles or other vesicles, including live visualization of lysosomes, is rather accessible.in vivo analysis of lysosome morphology and motility. In addition to labelling lysosomes, it is also possible to co‐label other markers of interest .e.g., kymographs, see Section ex vivo tissues is a powerful method to study live organelle dynamics. However, some studies require prolonged imaging or imaging during different developmental timepoints. For this, methods for prolonged imaging of Drosophila larvae or adult flies have been developed, and it is now also possible to image intact animals during extended periods.Drosophila larvae, Kakanj and colleagues developed a method that allows the visualization of tissues, including epidermis, fat body, gut, imaginal discs, neurons, tracheae, muscles and hemocytes, for up to eight consecutive hours.in vivo imaging of lysosomes in Drosophila can be performed in every life cycle stage, and be coupled to other manipulations, including genetic, pharmacological or others. The development of super‐resolution microscopy techniques is also fuelling the quality of in vivo analysis of intracellular trafficking.For in vivo studies of endosome biology in this model organism.in vivo lysosomal function are now possible, for example, the quantitative monitoring of H2S. H2S is a physiological mediator of inflammation and is generated in lysosomes of inflamed cells, although its role may be context specific. This gasotransmitter can be monitored using a luminescent probe based on a Ruthenium(II) complex, namely Ru‐MDB methyl 2‐thio)benzoate), which exhibits high specificity and sensitivity for H2S and low cytotoxicity.2S generation in live cells under drug stimulation. Therefore, this method can lead to a better understanding of the physiological and pathological functions of H2S in live organisms. Another useful tool is the generation of fusion proteins of apolipoprotein A‐1 (APOA‐I‐mCherry) to perform cholesterol trafficking studies in vivo. This is achieved by feeding TopFluor‐cholesterol , a fluorescent cholesterol analogue, to zebrafish. In this way, quantitative microscopy analysis of transgenic zebrafish expressing fluorescently tagged protein markers of late endosomes/lysosomes provides live imaging of cholesterol transport in the intestinal endosomal‐lysosomal system.In zebrafish, valuable tools have been generated such as Rab‐based transgenic lines for Rab5, Rab7 and Rab11 to use in 4.10The modern ‘omics’ technologies have opened the possibility of characterizing the full molecular inventory of an organelle. This specialized area of science is now referred to as organellar omics and relies on the constant optimization of purification methodologies. Organellar omics allows researchers to gain detailed insight, understand the dynamic nature of organelles and identify not only ‘permanent resident’ proteins but also temporarily associated proteins (interactors) that carry out specialized functions and transitory molecules such as cargo to be processed.https://www.addgene.org/102930/). TMEM192‐3xHA expression can be evaluated by WB using HA antibody and its lysosomal localization confirmed by monitoring HA and LAMP1 co‐localization by immunofluorescence.Different protocols have been developed to efficiently isolate lysosomes from other cellular components. Lysosome‐enriched fractions can be obtained by methods based on density gradient media Applications of LysoIP include the characterization of lysosome proteome composition, investigation of lysosomal cargo proteins and metabolites, monitoring of dynamic changes of the lysosome proteome/metabolome upon treatments and unravelling of post‐translational modifications of lysosomal proteins. Overall, this is a rapid and highly reproducible technique. However, several factors ought to be considered before performing this technique. Firstly, the type of cells employed to isolate lysosomes. For example, rat chondrosarcoma (RCS) cells are resistant to mechanical and physical stress and, for this reason, the lysis procedure requires a cell homogenizer. For most cell lines, that is, HeLa  or ARPE‐19 , a micro hand‐operated Potter‐Elvehjam homogenizer with a PTFE pestle or 10 passages through a 27G syringe needle are enough to guarantee proper cell lysis and release of the intracellular components. Another factor that should be considered, especially when comparing different experimental settings, is the quantity of lysosomes that can be immunoprecipitated. Different cell lines have different amounts of lysosomes and therefore, the choice of cell line is essential for the final result. Consequently, the total amount of cell lysate used should be optimized considering the average abundance of lysosomes. This calculation becomes critical when the experimental setting employs a single cell line but different treatments that influence lysosome biogenesis or turnover. Another issue is the cross‐contamination with other organelles. This may not be linked to the technical quality of the LysoIP but rather to an increased amount of contact sites between lysosomes and other cytosolic organelles. To evaluate LysoIP efficiency, one can load the flowthrough, also referred to as unbound fraction on sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and perform a WB for lysosomal markers, comparing with the input and immunoprecipitated fractions. The greatest advantage of LysoIP is that it does not require sophisticated equipment and it can, therefore, be performed in any cell biology laboratory. Indeed, the available commercial kits for lysosome enrichment are based on density gradient separation, which often requires an ultracentrifugation step. Moreover, LysoIP has a higher yield in terms of lysosome concentration and a lower amount of starting material is required. Noteworthy, a new methodology was recently developed to systematically map the autophagosome content, combining proximity labelling and organelle enrichment with quantitative proteomics.4.11l‐Leucyl‐l‐Leucine methyl ester hydrochloride  is significantly increased, compared to untreated cells.Lysosomal insults or dysfunction often result in LMP, which causes the release of the lysosomal contents, leading to cell dysfunction or death.The integrity of the lysosomal membrane can also be inferred by assessing the acidification of this organelle using the lysosomotropic fluorescent probe Acridine Orange , which emits red light in acidic compartments and green light in alkaline compartments. Thus, the quantification of the red fluorescence can be used to count acidic lysosomes, whereas the reduction in red fluorescence and the increase in green fluorescence can be used to measure lysosomal alkalinization or LMP Figure . For thi4.12Lysosomes contain over 70 different acid hydrolases that can catalyze the breakdown of proteins, lipids, polysaccharides, DNA and RNA.Historically, the measurement of lysosomal hydrolase activity has been the gold‐standard for LSD diagnosis,4.12.1e.g., galactocerebrosidase, acid sphingomyelinase and lysosomal acid lipase).in vitro assay in which the enzymes are not in direct contact with these proteins. In the case of GBA, the activation can be achieved by using taurocholate in the assay buffer.Lysosomal hydrolase activity can be easily assayed in the blood (serum/plasma or leukocytes) and other biological samples when the enzyme is sufficiently abundant.In 4‐MU‐based assays, when optimal conditions are defined, the enzyme will hydrolyze a fraction of the substrate containing the 4‐methylumbelliferyl moiety, resulting in the release of the 4‐MU group, which is highly fluorogenic at basic pH (pH 10‐11). Alkaline buffers are thus routinely used to quench and stop the enzymatic reaction, improving the fluorescence yield. The enzymatic activity can then be calculated as a direct proportion of the fluorescence intensity.in vivo enzymatic activity may thus be distinct from that assayed in cell or tissue preparations. Indeed, these assays are usually unable to fully mimic the local lysosomal environment regarding elements such as co‐factors, chaperones and activators, which are essential for enzyme stability, processing and structure.The downsides of using artificial substrates include the fact that these assays do not fully reflect the physiological conditions that exist in cells. The 4.12.2in vitro activity assays discussed above have triggered the development of more accurate and sensitive tools to monitor lysosomal hydrolases in their natural environment. Substrate‐based probes are broadly available tools that can penetrate the cellular membranes and allow tracking of the activity of lysosomal enzymes in situThe limitations associated with in vivo and in situ, ABPs must be able to efficiently reach lysosomes and fluoresce within the acidic environment of the lysosome lumen.e.g., BODIPY, DAMP, Cy5.5, etc.) have optimal fluorescence in the low pH of the lysosome and have been commonly used for labelling lysosomal hydrolases.Whenever the catalytic mechanism of an enzyme is known, it is possible to develop chemical probes that covalently bind to the enzyme's active site and allow the detection of the enzyme via a reporter moiety grafted to the probe.A broad range of fluorogenic substrates to monitor lysosomal hydrolase activity is now commercially available, making them accessible and easy‐to‐use tools. Magic Red substrates, in particular, have recently been adapted for use in HCI analysis protocols to monitor lysosome function in LSD models by using Opera and Operetta systems (PerkinElmer).4.132+‐regulated process that occurs in almost all cell types. Therefore, Ca2+ ionophores like ionomycin 2+ channel present in the lysosome membrane —TRPML1— such as ML‐SA1  and MK6‐83 , are commonly used to induce lysosome exocytosis.Although lysosomes are generally regarded as terminal degradative organelles, they can also undergo exocytosis, releasing their luminal contents.e.g., anti‐LAMP1 H4A3) provides a good readout of lysosome exocytosis.One of the most commonly used methods to monitor lysosome exocytosis is the detection of lysosomal membrane proteins, such as LAMP1, at the plasma membrane. When lysosomes fuse with the plasma membrane, the luminal domain of LAMP1 is exposed. Thus, detection of LAMP1 with a monoclonal antibody for a luminal epitope of the protein .The detection of released lysosomal enzymes in the extracellular milieu is also often used to evaluate lysosome exocytosis. The unprocessed and processed (mature) forms of lysosomal enzymes can be detected in the extracellular media, by WB, using specific antibodies that are commercially available  microscopy. For this purpose, it is necessary to establish cell lines expressing, preferably stably, lysosomal markers conjugated with pH‐sensitive probes such as pHluorin. The fluorescence signal from pHluorin is quenched in acidic environment. As lysosomes undergo fusion with the plasma membrane, pHluorin contacts the extracellular medium neutral pH and the fluorescence signal can be detected as a flash of light. Therefore, the pHluorin signal is a good readout of lysosome exocytic events.EM is also a powerful tool for the analysis of lysosome exocytosis. This approach must be combined with tannic acid treatment, which allows exocytic structures to be arrested at the stage of their docking/fusion with the plasma membrane.5Even though lysosomes were discovered more than 70 years ago, essential concepts regarding their function and life cycle were only uncovered in recent years. This was possible because of methodological advances such as imaging techniques that allow the visualization of the dynamics of this organelle in live cells. We now have a better understanding of the heterogeneity of different lysosome populations and their characteristics. However, we are still starting to understand their functions in cell physiology. New insights will arise from future technological developments as the resolution of imaging techniques, both in time and space, further increases.i.e., ‘lysosome rejuvenation’) might represent a promising anti‐ageing strategy. In addition, a deeper understanding of lysosome biology can provide novel therapeutic strategies for LSDs and other lysosomal disorders, as well as for age‐related degenerative diseases. In order to achieve these goals, lysosome research must continue, using cutting edge methodology. It seems inevitable that the lysosome will continue to surprise us for many years to come.The outdated view of lysosomes as simply degradative organelles has been changed by the finding that they play a central role in several other cellular functions, such as nutrient sensing, intracellular signalling and metabolism. Indeed, lysosomes are essential for many aspects of cell physiology and failure in their functions can result in cellular ageing and age‐associated phenotypes such as cell senescence. Therefore, the reestablishment of lysosome functions (The authors declare no potential conflict of interest.6https://publons.com/publon/10.1111/tra.12839.The peer review history for this article is available at Data S1 Supporting Information.Click here for additional data file.


Ucp1) and peroxisome proliferator-activated receptor-gamma coactivator-1alpha (Ppargc1a) (2+) is fundamental to a wide variety of cellular processes including, but not limited to, muscle contraction, gene transcription, exocytosis, and cellular metabolism in virtually all eukaryotic cells  have centered on the cyclic adenosine monophosphate (cAMP)-related signaling . Acting pargc1a) . While tic cells , whether2+-ATPase (SERCA)–intracellular Ca2+ axis in adipocytes  serves as the main Ca2+ store that determines intracellular Ca2+ homeostasis. Among the ER membrane-localized Ca2+ channels, SERCA is the solely Ca2+ influx pump that has been shown to fine-tune intercellular Ca2+ dynamics. By performing immunoprecipitation and mass spectrometry analysis, we identified that SERCA2 is able to interact and precipitate with FNIP1, an adaptor protein originally identified through its interaction with FLCN and AMP-activated protein kinase (AMPK)  adipocytes, the reuptake of cytosolic Ca2+ into the ER lumen mediated by SERCA is reduced, resulting in elevated Ca2+ levels and an increased duration of Ca2+ transients in the cytosol upon external stimuli. More importantly, we showed that, functionally, loss of adipocyte FNIP1 in mice results in enhanced thermogenic remodeling in WAT. This improves systemic glucose homeostasis and prevents hepatic steatosis when mice are fed a high-fat diet (HFD).In this study, we set to investigate the intracellular Cae (AMPK) . We then+ beige adipocyte formation and mitochondrial biogenesis in the absence of cold exposure or β-adrenergic stimulation. Therefore, FNIP1 acts as an endogenous ‘brake’ to inhibit adipocyte beiging. Intriguingly, our results also indicate that FNIP1 suppression of thermogenic programs occurs in a depot-selective manner, the reason for which is not clear. This could reflect the intrinsic differences between beige adipocytes and brown adipocytes that affect the adaptive range of thermogenic remodeling.We observed that FNIP1 deficiency in adipocytes triggers a robust UCP1While we have demonstrated previously that FNIP1 acts through AMPK but not mammalian target of rapamycin (mTOR) complex 1 signaling to regulate mitochondrial function in the skeletal muscle , interesPreviously published studies have revealed that FNIP1 could actually act as a co-chaperone that regulates the chaperone function of Hsp90 . We foun2+ cycling in beige fat via the SERCA2–RyR pathway can promote a UCP1-independent thermogenesis  What are the mechanisms by which the intracellular Ca2+ signals activate the thermogenic program? (ii) How is the interaction between FNIP1 and SERCA regulated under the physiological setting? (iii) Does this FNIP1–SERCA–intracellular Ca2+ pathway represent a broader metabolic regulatory network in other organs? (iv) Will it be possible to exploit such FNIP1–intracellular Ca2+ signals as treatment strategies?In summary, our recent work suggests that FNIP1 acts as a negative regulator of beige adipocyte thermogenesis to control systemic glucose homeostasis through modulating SERCA . Linking


In this review, we examined the role of different endothelial TRP channel isoforms in tumor vessel formation, a process that is essential in tumor growth and metastasis.Tumor diseases are unfortunately quick spreading, even though numerous studies are under way to improve early diagnosis and targeted treatments that take into account both the different characteristics associated with the various tumor types and the conditions of individual patients. In recent years, studies have focused on the role of ion channels in tumor development, as these proteins are involved in several cellular processes relevant to neoplastic transformation. Among all ion channels, many studies have focused on the superfamily of Transient Receptor Potential (TRP) channels, which are non-selective cation channels mediating extracellular Ca These systems either extrude Ca2+ across the plasma membrane, such as the plasma membrane Ca2+-ATPase and the Na+/Ca2+ exchanger (NCX), or sequester cytosolic Ca2+ in the endoplasmic reticulum (ER), the largest intracellular Ca2+ reservoir (Sarco-Endoplasmic Reticulum Ca2+-ATPase (SERCA)) [2+ concentration ([Ca2+]i) in both vascular endothelial cells i also stimulates proliferation, migration, tube formation and neovessel formation in circulating ECFCs i can recruit a number of downstream Ca2+-dependent pro-angiogenic decoders, such as the transcription factors nuclear factor of activated T-cells (NFAT), nuclear factor-kappaB (NF-kB), cAMP responsive element binding protein (CREB), myosin light chain kinase (MLCK), myosin 2, endothelial nitric oxide synthase (eNOS), extracellular signal-regulated kinases 1/2 (ERK 1/2), and serine/threonine kinase (Akt) [An increase in endothelial [Case (Akt) .2+ response to pro-angiogenic signals can occur as recurrent Ca2+ spikes or a transitory or biphasic increase in [Ca2+]i. Phospholipase Cγ (PLCγ), which cleaves phosphatidylinositol-4,5-bisphosphate (PIP2) to generate the second messengers inositol-1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG), is brought about by the binding of VEGF to its specific receptor, VEGFR-2, on the plasma membrane. The latter triggers the proto-oncogene, serine/threonine kinase (RAF1)-MEK-ERK1/2 cascade by activating the DAG-sensitive protein-kinase C (PKC). InsP3 then activates the InsP3R on the ER membrane, thereby causing Ca2+ to be released from the ER. The following decrease in ER Ca2+ levels induces stromal interaction molecule 1 (Stim1), an ER Ca2+ sensor, to oligomerize and translocate from perinuclear to peripheral ER cisternae, where it interacts with the Ca2+-permeable channel, Orai1, at ER-plasma membrane junctions known as puncta (10–25 nm). The influx of Ca2+ through Orai1 channels has been termed store-operated Ca2+ entry (SOCE) and maintains the Ca2+ response to VEGF in both vascular endothelial cells i in response to Vm ≥ −60 mV. The remaining [Ca2+]i response depends on TRPC1,3,4,5,6/TRPV1,3/TRPA1 i, whereas Na+ influx causes membrane depolarization, activating voltage-gated Ca2+ channels in excitable cells and altering the driving-force for Ca2+ entry in non-excitable cells i globally [2+ entry in vascular ECs subjected to a variety of acetates, hormones, and mechanical stimuli, such as pulsatile stretch and laminar shear stress [M by conducting a depolarizing inward current carried by Na+ and Ca2+, resulting in a positive shift in VM that may be amplified by the Ca2+-dependent recruitment of TRPM4  downstream of VEGFR, TRPC3 is activated. This causes Naogenesis . In contppressed . In EPCsferation . Silencin HUVECs . Specifin HUVECs . TRPC6 an HUVECs . Similarn HUVECs . In EPCsn HUVECs . The Fign HUVECs .2+ influx, which induces activation of CaMKII signaling and enhances TRPV1-eNOS complex formation, leading to NO production and angiogenic tube formation in vitro [According to a recent study, TRPV4 has long been recognized to control angiogenesis and neovascularization by promoting EC migration and proliferation . TRPV4 iin vitro .The 2+ entry through TRPM2 activation. Furthermore, matrigel plugs supplemented with VEGF injected subcutaneously into TRPM2 knockout mice show significantly reduced blood vessel formation compared to wild type mice [2+ deficit on the development and migration of microvascular ECs, suggesting that magnesium and TRPM7 are regulators of angiogenesis [It has also been discovered that TRPM2, TRPM4, and TRPM7 are involved in angiogenesis . VEGF haype mice . In respype mice . An earlogenesis . Figure 2+-permeable channels or whether they come together to form a supermolecular complex that may also include TRPC4. SOCE can be recruited by SDF-1α, to increase ECFC migration in vitro and neovessel development in vivo [2+-sensitive transcription factor NF-kB, SOCE activates the ERK1/2 and PI3K/Akt signaling pathways to promote SDF-1α-induced ECFC motility [2+ oscillations generated by VEGF [2+ entry causes the dynamic interaction between InsP3Rs and SOCE to alter the spiking Ca2+ response to VEGF, increasing the proliferation of UCB-ECFC [2+ oscillations in UCB-ECFCs, which is related to their higher proliferative capability, has been postulated to be caused by TRPC3 participation [A preliminary report showed that only TRPC1 and TRPC4 were expressed by human ECFCs generated from peripheral blood (PB-ECFCs), although TRPC3 was also present in ECFCs produced from umbilical cord blood . Followi in vivo , as well in vivo ,112. Spemotility ,52. STIMmotility , just asmotility . TRPC3 i by VEGF . GeneticUCB-ECFC . The inccipation . This ficipation .2+ entry [The two most significant endothelial TRPV isoforms involved in angiogenesis and arteriogenesis are TRPV1 and TRPV4. Multiple independent studies revealed that human ECFCs also express TRPM7 , TRPV1 , and TRP2+ entry . Subsequ2+ entry  found th2+ entry . A recen2+ entry .2+ mechanism is essential for neovascularization and resistance to anti-angiogenic and chemotherapeutic treatments [In a growing number of cancers, recent data have suggested that deregulation of the endothelial Caeatments ,60,61. A2+ entry in migrating B-TECs compared to non-migrating cells. Furthermore, AA has been shown to stimulate extracellular Ca2+ entry in B-TECs during the first stages of the tubulogenic process, but not when a capillary-like network was already developed. These findings clearly suggest that the early stages of breast cancer angiogenesis are characterized by the over-expression of endothelial TRPV4 channels [TRPV4 has been the first endothelial TRP channel directly linked to malignant angiogenesis. In line with this, TRPV4 expression was noticeably increased in breast-tumor-derived endothelial cells (B-TECs), and TRPV4 activation with AA or 4αPDD stimulated migration in B-TECs, but not in the control human microvascular endothelial cells. On the other hand, when cells were transfected with a short hairpin RNA that selectively targets TRPV4 (shTRPV4), AA-induced B-TEC migration was stopped . Notablychannels   involves a complex remodeling of endothelial TRP channels. TRPA1, TRPV2, and TRPC3 were shown to be up-regulated in three different endothelial cell lines established from PCa patients as well as in endothelial cells lining tumor capillaries in vivo . In PCa-isoforms .2+ influx blocked the ERK1/2 pathway, reducing the rate of A-TEC proliferation , thereby reducing their mechanosensitivity to extracellular matrix (ECM) rigidity. This, in turn, favored A-TEC motility. The tumor vasculature in TRPV4 KO mice was characterized by an increased proportion of hyperpermeable, pericyte-free and dilated microvessels, which are known to moderate the therapeutic effect of anticancer treatments . A subseferation . These r2+ signaling machinery; in accord, primary tumor samples express more Orai1 and TRPC6 channel proteins than normal renal tissues [2+ concentration and InsP3R expression, while they displayed a remarkable up-regulation of Stim1, Orai1, and TRPC1. These changes result in a remarkable rewiring of their Ca2+ toolkit, which render RCC-ECFCs less responsive to VEGF [The most common form of kidney cancer in adults is renal cellular carcinoma (RCC) and there is substantial evidence that ECFCs may play a primary role in RCC neovascularization ,130,131. tissues ,133. In  tissues . These c tissues , the los tissues . In addi to VEGF . However to VEGF  (Figure 2+ entry pathway in ECs, EPCs, and ECFCs. However, they can also block angiogenesis, as reported for TRPM4 in HUVECs and TRPM7 in cerebral microvascular endothelial cells. An important aspect of endothelial TRP signaling that deserves much attention is its involvement in the abnormal vascularization that characterizes cancer. A crucial contribution to the angiogenic switch that transforms a dormant lesion into a growing tumor is provided by the recruitment of myeloid angiogenic cells and ECFCs, which are released from vascular stem cell niches, respectively. The abnormal expression and activity of TRP channels in T-ECs and T-ECFCs, may promote tumor neovascularization, however, several aspects are still not entirely clear, e.g., which endothelial TRP isoforms are dysregulated, and therefore further studies are needed.Endothelial TRP channels play a crucial role in vascular remodeling, regulating angiogenesis, arteriogenesis and vasculogenesis. These channels are highly heterogeneous in their gating mechanisms, with the propensity of some isoforms to form heteromeric complexes, making them the most versatile Ca


Trichophyton, Microsporum, and Epidermophyton. Dermatophyte infection is not only a threat to the health of small animals, but also an important zoonotic and public health issue because of the potential transmission from animals to humans. Rabbit dermatophytosis is often clinically identified; however, limited information was found in Asia. The aims of this study are to investigate the prevalence and to evaluate the risk factors of dermatophytosis in pet rabbits in Northern Taiwan. Between March 2016 and October 2018, dander samples of pet rabbits were collected for fungal infection examination by Wood’s lamp, microscopic examination (KOH preparation), fungal culture, and PCR assay (molecular identification). Z test and Fisher’s exact test were performed to evaluate the potential risk factors, and logistic regression analysis was then performed to build the model of risk factors related to dermatophyte infection. Of the collected 250 dander samples of pet rabbits, 29 (11.6%) samples were positive for dermatophytes by molecular identification. In those samples, 28 samples were identified as the T. mentagrophytes complex and 1 sample was identified as M. canis. Based on the results of the Firth’s bias reduction logistic analyses, animal source (rabbits purchased from pet shops) and number of rearing rabbits (three rabbits or more) were shown as the main risks for dermatophyte infection in the pet rabbits in Taiwan. The results of the present study elucidate the prevalence of rabbit dermatophyte infection, pathogens, and risk factors in Taiwan, and provide an important reference for the prevention and control of rabbit dermatophytosis.Dermatophytes are the group of keratinophilic fungi that cause superficial cutaneous infection, which traditionally belong to the genera  Epidermophyton, Trichophyton, and Microsporum spp. [Trichophyton spp. and Microsporum spp. cause skin diseases in animals, such as T. mentagrophytes, T. verrucosum, and M. canis, which are known as zoophilic dermatophytes. The transmission of these zoophilic dermatophytes occurs via infective arthrospores coming from the hair coat of infected animals or the environment [Dermatophytes, referred to as the ringworm fungi, are traditionally divided into three closely related genera, including rum spp. ,2. Trichironment ,4. Infecironment ,4,5,6.T. mentagrophytes complex and M. canis species [Dermatophytoses are common fungal infections in rabbit farm, laboratory, and pet rabbits ,12,13,14 species ,16,17. T species ,19,20. S species ,22,23. T species .M. ferrugineum and T. violaceum are the main causative agents of human dermatophytoses in Northern and Southern Taiwan, respectively [In Taiwan, various epidemiological surveys of human dermatophytoses have been reported ,29,30,31ectively ,29. In 2ectively . Some caFrom March 2016 to October 2018, a total of 250 pet rabbits were examined for dermatophyte infection at Guting animal hospital in Taipei City, Rabbit Saving Association, and Taiwan Stray Bunny Protection Association . The pet rabbits’ data, including breed, weight, age, gender, neuter status, source, location, living space, number of rearing rabbits, rearing with other animals, history of ectoparasite infestation, and season of sample collection, were collected for analysis of potential risk factors of dermatophyte infection.For each pet rabbit, Wood’s lamp was firstly used to detect the fluorescent reaction of the dermatophytes ,32. SomeAll dander samples were cultured on Mycosel agar plates  and incubated at 25 °C for up to 4 weeks . The funMacroscopic morphology was observed by the size, texture, and color of colonies on agar plates . The micAll DNA samples were extracted from hyphae of the suspected dermatophyte isolates using an MX-16 automatic nucleic acid extractor  with a Maxwell 16 Tissue DNA Purification kit  following the manufacturer’s instructions. All DNA samples were stored at −20 °C until PCR processing for dermatophyte detection.ITS) region of Trichophyton spp. (603 bp.) and Microsporum spp. (632 bp.) [2 , 0.125 μL of Taq DNA polymerase , and adjusted to a final volume of 25 μL with sterile distilled water. The PCR condition was described by White et al. [Dermatophyte DNA was detected by using PCR assay . The pri632 bp.) . PCR mixe et al. . The expThe dermatophyte PCR-positive products from hyphae samples were purified using a Plus DNA Clean/Extraction Kit  and sent for nucleotide sequencing . Sequence data were compared with known sequences deposited in the GenBank database using the NCBI nucleotide BLAST tool. For genetic analysis, validated sequences were aligned and analyzed by using MegAlign .p < 0.05. Logistic regression analysis was then performed to build the model of risk factors related to dermatophyte infection [R statistical software  was used in this study to perform descriptive statistical analyses and to assess the association between dermatophyte infection and potential risk factors. The descriptive statistics were presented as mean and standard deviation for measurement data, and medians and interquartile ranges for categorical data. Z test was used for the continuous variable (weight), and Fisher’s exact test was used for the categorical variables , with significance at nfection .The Kappa value coefficient was used to interpret the consistency of the results among the detection methods of Wood’s lamp, microscopic examination, and culture/molecular identification, and Excel software was used for calculation and analysis. According to Fleiss’s Kappa value, five levels of agreement were considered as follows: 0.0–0.20 (extremely low), 0.21–0.40 (fair), 0.41–0.60 (moderate), 0.61–0.80 , and 0.81–1  [From March 2016 to October 2018, a total of 250 pet rabbits were examined at Guting animal hospital, Taiwan Rabbit Saving Association, and Taiwan Stray Bunny Protection Association. The average weight of the recruited rabbits was 1.64 ± 0.76 kg, and the average age was 34.88 ± 30.60 months. Among the animals, 135 (54%) rabbits were from districts of New Taipei City, while 115 (46%) rabbits were from districts of Taipei City . The petTrichophyton spp.  pet rabbits presented fluorescent reaction on the bodies examined by Wood’s lamp a. All ofton spp. c, and 1 rum spp. d, by colTrichophyton and 1 Microsporum PCR-positive samples. From Trichophyton PCR-positive products, 11 samples were randomly selected and sent for sequencing. Ten sequences showed 100% similarity with T. interdigitale isolates , and one sequence showed 100% similarity with the T. benhamiae isolate (KY827233) listed in the GenBank database. Based on the phylogeny, T. interdigitale and T. benhamiae were assumed as the T. mentagrophytes complex. The sequence from the Microsporum PCR-positive sample showed 100% similarity with the M. canis (MN808763) listed in the GenBank database.The PCR results revealed that all 29 suspected colonies harbored dermatophyte DNA fragments, which were 28 In the comparison of positive results among different detection methods, Wood’s lamp examination, microscopic examination (KOH preparation), and molecular identification was shown as follows: (1) In the 250 recruited pet rabbits, 29 rabbits were detected positive for dermatophytes by molecular identification, while 8 rabbits were positive by Wood’s lamp examination. Four rabbits were detected positive by both methods; (2) In the 250 recruited pet rabbits, 29 rabbits were positive for dermatophytes by molecular identification, while 9 rabbits were positive by microscopic examination (KOH preparation). Five rabbits were detected positive by both methods; (3) In the 250 recruited pet rabbits, 8 rabbits were positive for dermatophytes by Wood’s lamp examination, while 9 rabbits were positive by microscopic examination (KOH preparation). Four rabbits were detected positive by both methods.Comparative analysis of the methods for dermatophyte detection was performed by using the Kappa value coefficient. The Kappa value of the dermatophyte detection between Wood’s lamp examination and molecular identification was 0.17, indicating that the detection results of the two methods were extremely low consistency. The Kappa value of the dermatophyte detection between microscopic examination (KOH preparation) and molecular identification was 0.22, indicating that the detection results of the two methods were in very low consistency. In addition, the Kappa value of the dermatophyte detection between Wood’s lamp and microscopic examination (KOH preparation) was 0.42, indicating that the detection results of the two methods were in moderate consistency.p < 0.05, and the prevalence of dermatophyte infection in each categorical variable is shown in p value = 0.4297). In Taipei City, eleven samples from Daan District , Neihu District , and Zhongzheng District  were detected as dermatophyte positive, but those from the rest of the districts were all negative     . The categorical variables were analyzed using Fisher’s exact test with significance at negative a. In Newnegative b. Regard × 10−5) . The pre= 0.007) . In addi= 0.017) . The rab= 0.012) .T. mentagrophytes complex isolates were mainly from mixed breed rabbits bought from pet shops. Among those 28 rabbits, 15 rabbits were reared in the cages, while 11 rabbits were reared indoors and the other 2 were reared both indoors and in cages. In addition, six rabbits that detected positive for the T. mentagrophytes complex were infested by fur mites . The regression model isan three .The interpretation of the variables obtained from the Firth’s bias reduction logistic regression model is mentioned as follows: the risk of dermatophyte infection in pet rabbits purchased from pet stores was 15.6 and 16.6 times higher than the rabbits from adoption and personal breeding, respectively; the risk of dermatophyte infection in pet rabbits reared with two rabbits was 1.247 times higher than the rabbits that were reared alone; the risk of dermatophyte infection in pet rabbits reared with three rabbits or more was 11.416 times higher than the rabbits that were reared alone.This is the first study on the prevalence of dermatophyte infection in pet rabbits in Taiwan and also the first report on the related risk factors in Asia. The prevalence of dermatophyte infection in pet rabbits (13.9%) in the present study was higher than those detected from veterinary clinics, pet shops, and pet cafés reported in Southern Italy (3.29%), the Netherlands (3.80%), Chile 7.14%), and Thailand 12.1%) ,42,43,44.14%, and.1% ,42,4T. mentagrophytes is the most common dermatophyte species to infect rabbits from laboratories, veterinary clinics, pet shops, and rabbit farms [T. mentagrophytes, the rabbits reared in the farms were also reported to be infected with M. canis [T. mentagrophyte complex is also the predominant dermatophyte (96.5%) that infects pet rabbits in Northern Taiwan. After fungal infection was identified in pet rabbits, the treatment of Itraconazole (5 mg/kg) was given for at least 6 weeks. After 6 weeks of oral medication, dermatophyte culture from dander samples was performed. After the negative results were confirmed, Itraconazole (5 mg/kg) was applied for another 2 weeks.it farms ,43,44,45M. canis ,9,17. BaIn the present study, the risk of dermatophyte infection in pet rabbits purchased from pet stores  was 15.6 and 16.6 times higher than the rabbits from adoption (1.1%) and personal breeding (0%), respectively. The rabbits in pet shops were mainly brought from different commercial farms and reared until sold. Because dermatophyte spores can remain viable for many years, the contaminated cages or environment in pet shops can facilitate mutual infection among the rabbits ,46. In aT. mentagrophytes spores can not only be isolated from infected rabbits and asymptomatic carriers, but also from rabbit nests, cages, and the surrounding environment [Another risk factor of rabbit dermatophytosis in this study is the number of rearing rabbits. It was found that the more rabbits were reared together, the higher possibility of dermatophyte infection. ironment ,4,49. A ironment . M. canis had been predominantly reported on dogs and cats and has also been found on pet rabbits [M. canis from rabbit samples could be due to the presence of dogs and cats in the surrounding environment, which can be an asymptomatic carrier for this dermatophyte species [M. canis was isolated from the rabbit reared with dogs. On the other hand, T. mentagrophytes spores were detected from a dog’s basket, which showed the potentiality of dermatophyte infection in humans and other animals by direct contact with a dog or its living area [ rabbits ,51,52. W species ,51. Thising area . Cheyletiella parasitovorax is a non-burrowing fur mite, which is commonly found on rabbits and is transmitted among rabbits by direct contact. The obligate parasite causes a disruption of the stratum corneum, which may facilitate a dermatophyte infection during an infestation of rabbits [In the present study, fur mite infestation was found to be associated with the dermatophyte infection.  rabbits . Another rabbits . Autumn was the period with the highest risk for dermatophyte infection in pets (including rabbits) in Italy . HoweverT. mentagrophytes, was associated with dermatophytosis in rabbits which were reared in human living and working areas [T. benhamiae and had a contact history with her two pet rabbits [T. benhamiae infection had a history of contact with rabbits [The risk of human dermatophyte infection, especially ng areas ,9,14. Du rabbits . Further rabbits . The comRabbits infected with dermatophytes in pet shops, farms, or houses are undesired. This is not only because of zoonotic exposure for humans, but also for the risk of spreading the infection to other animals. Pet shops are the main source of pet rabbits in Taiwan, and the rabbits from there have a higher risk for dermatophyte infection; therefore, preventive measures in pet shops and their source, rabbit farms, should be taken. The rabbits arriving in pet shops should be quarantined in a clean environment, and fungal culturing is an option to achieve dermatophyte-free animals . In addi


Lännergren et al. (2001) using Rhod2 reported calcium accumulation in mitochondria during prolonged contractile activity in Xenopus fibers but not in mouse fast flexor digitorum brevis (FDB) fibers. 2 yr later, matrix calcium in both muscles. The absence of mitochondrial calcium uptake during tetanic stimulation was further confirmed on FDB fibers loaded with Rhod2 . In contrast, cameleon probes targeted to mitochondria produced clear-cut evidence of transient increases of mitochondrial Ca2+ concentration in electrically stimulated murine muscles with consistent results in tibialis anterior fibers in situ  and in isolated FDB fibers . The matrix-free Ca2+ concentration follows closely cytosolic-free Ca2+ variations, even during a single twitch, although with a somewhat different kinetics. A fast rise phase was followed by a slow decrease, and this allowed the integration of the signals coming from repeated stimulations/action potentials. Importantly, the time scale of the metabolic activation triggered by calcium is much faster compared to the activation induced by ADP and creatine which implies energy depletion. Thus, calcium-activation could be interpreted as a feed-forward regulation, while ADP activation appeared as a feed-back control.In the first decade of the new century, the free CaThe third paragraph of the Mitochondria in skeletal muscle fibers section has been corrected, and it appears below with the corrections underlined. The errors appear only in PDF versions downloaded on or before September 12, 2022.


A variety of oncologic therapies and some candidate drugs induce their antitumor effects (in part or in whole) through the modulation of [Ca2+]i. Cervical cancer is one of most common cancers among women worldwide. Recently, major research advances relating to the Ca2+ signals in cervical cancer are emerging. In this review, we comprehensively describe the current progress concerning the roles of Ca2+ signals in the occurrence, development, and prognosis of cervical cancer. It will enhance our understanding of the causative mechanism of Ca2+ signals in cervical cancer and thus provide new sights for identifying potential therapeutic targets for drug discovery.Intracellular calcium (Ca For example, intracellular Ca2+ overloading promotes apoptosis of myoblasts, and increasing intracellular Ca2+ can cause an escalation of reactive oxidative species [2+]i depends on various calcium channels, calcium pumps, and interacting proteins and ligands in the body to ensure its level within an appropriate range and give full play to its biological effects. Therefore, as a messenger, the concentration of [Ca2+]i is kept within a strict range i concentrations  in the ER and transmits this signal to Orai1 channels in the cell surface membrane to promote Ca2+ influx. In cancer cells, SOCE plays an important role in the cell cycle process, proliferation, migration, metastasis, and evasion of apoptosis i or protein signaling and ER releases Ca2+ through these channels [Intracellular Cachannels ,68.2+ signaling pathway is an extremely important part of maintaining body homeostasis. The signaling pathway has direct and indirect effects on the action of cells, including control of cell metabolism, secretion, fertilization, proliferation, and smooth muscle contraction [2+ signaling are important factors in the development of a large number of human diseases [2+ signaling system are responsible for altered Ca2+ signaling in many cancer cells, such as lung cancer and cholangiocarcinoma clear cell renal cell carcinoma, and are closely related to the occurrence, development, and prognosis of cancer [2+ signaling pathway, and its genetic polymorphism is associated with an increased risk of cervical squamous cell carcinoma [The IP3R/Catraction ,70. Chandiseases . Increasf cancer ,73,74. Tf cancer . The ITParcinoma ,75.2+ from intracellular stores during excitation–contraction coupling in cardiac and skeletal muscle [2+ release, thus leading to the generation of a quick, transient increase in cytosolic Ca2+ levels [2+ signaling. Such studies cannot conclusively provide ideas for obtaining corresponding therapeutic targets.RyR channels are the largest known ion channels that are located in the membrane of the ER and are expressed in a restricted subset of cell subtypes, such as cardiac and skeletal muscle cells . One of l muscle . RyRs ha+ levels . RyRs ar+ levels . RyRs ca+ levels ,80,81. S+ levels . Abdul e+ levels . Law et + levels . However2+ homeostasis by releasing Ca2+, SERCA-dependent Ca2+ transport is the only Ca2+ uptake mechanism in this organelle [2+ homeostasis according to cell type and its differentiation state. Regulation of SERCA activity can affect cell differentiation and survival [2+ homeostasis and inducing ER stress-mediated cancer cell death. The study also found that SBF-1 inhibited the growth and migration of HeLa cells depending on the activity and level of SERCA2 [Calcium is actively accumulated in the ER by sarcoendoplasmic reticulum (SR) calcium transport ATPase (SERCA enzymes). Unlike the above-mentioned channels that regulate ER Carganelle . Therefosurvival . Three Ssurvival ,84. Studsurvival . SBF-1, survival . In humaf SERCA2 . In addif SERCA2 .Escherichia coli efflux-multidrug resistance E), and the Ca2+ sensitive inhibitory regulatory subunits MICU1 and MICU2 [2+ across the inner mitochondrial membrane to the mitochondrial matrix. Due to this central role and ability to influence cell behavior and fate, several research groups are investigating the role of the MCU complex in different cancers and cancer-related diseases [2+ uptake. Furthermore, in HeLa cervical cancer cells, overexpression of MCU or knockdown of MICU1 resulted in constitutive mitochondrial Ca2+ influx [MCU is the pore-forming subunit of the MCU complex, which consists of MCU, the scaffold protein EMRE  functions are strictly dependent on Caeostasis ,96,97. I+ stores . In addiansition ,100. Relansition ,102,103.ansition . The finansition . When usgression . It furtgression .2+ signaling and cervical cancer, we can easily find that Ca2+ signaling plays an important role in cervical cancer growth, proliferation, and metastasis. Changes in various Ca2+ channels, pumps, exchangers, sensors, and Ca2+ activation effectors related to the regulation of Ca2+ homeostasis can affect the concentration of Ca2+ inside and outside the cell, thereby affecting cervical cell metabolism, proliferation, and apoptosis, and eventually leading to the development of cervical cancer  and SPCA2 genes are significantly increased in breast cancer [2+-ATPase (PMCA) is ubiquitously expressed, which is critical for maintaining low resting cytosolic Ca2+ in cells. Studies spanning many years have revealed that some cancers are associated with a remodeling of PMCA expression [2+-dependent signaling cascades associated with tumor cell migration by promoting local Ca2+ influx [2+ channel [In addition to the Cat cancer ,105. Thepression . The sodpression . Piezo1,+ influx . Piezo1 + influx ,109. In  channel .2+ homeostasis and signal transduction are related to the occurrence, development, and prognosis of cervical cancer. Research findings in other tumors may provide new ideas for cervical cancer research. Existing studies on Ca2+ signaling related to cervical cancer treatment are still few, and the current research has not included Ca2+ signaling targets as the focus of cervical cancer treatment. Increasing expression or silencing of some Ca2+ channel proteins related to tumor cell apoptosis has been confirmed in the treatment of other tumors, which provides a new idea for the targeted therapy of cervical cancer. Admittedly, little is known about the effect of Ca2+ signaling in cervical cancer; there is, thus, a need for additional research in this field.However, there is no relevant research to show whether these channels or protein- regulated Ca


Our study showed that the endogenous H2S content and the expression of cystathionine gamma-lyase (CSE), which is the key enzyme of H2S production, were decreased in arterial SMCs of diabetic mice. The expression of PCNA and Cyclin D1 was increased, and the expression of p21 was decreased in the diabetic state. After administration of dopamine 1-like receptors (DR1) agonist SKF38393 and exogenous H2S donor NaHS, the expression of CSE was increased and the change in proliferation-related proteins caused by diabetes was reversed. It was further verified by cell experiments that SKF38393 activated calmodulin (CaM) by increasing the intracellular calcium ([Ca2+]i) concentration, which activated the CSE/H2S pathway, enhancing the H2S content in vivo. We also found that SKF38393 and NaHS inhibited insulin-like growth factor-1 (IGF-1)/IGF-1R and heparin-binding EGF-like growth factor (HB-EGF)/EGFR, as well as their downstream PI3K/Akt, JAK2/STAT3 and ERK1/2 pathways. Taken together, our results suggest that DR1 activation up-regulates the CSE/H2S system by increasing Ca2+-CaM binding, which inhibits the IGF-1/IGF-1R and HB-EGF/EGFR pathways, thereby decreasing their downstream PI3K/Akt, JAK2/STAT3 and ERK1/2 pathways to achieve the effect of inhibiting HG-induced VSMCs proliferation.Tissue ischemia and hypoxia caused by the abnormal proliferation of smooth muscle cells (SMCs) in the diabetic state is an important pathological basis for diabetic microangiopathy. Studies in recent years have shown that the chronic complications of diabetes are related to the decrease of endogenous hydrogen sulfide (H Therefore, preventing the abnormal proliferation of vascular smooth muscle in the diabetic state is a key factor for improving the microvascular complications of diabetic patients.Diabetes mellitus is a multifactorial metabolic disorder whose main cause is an absolute or relative lack of insulin, or the peripheral tissues are not sensitive to insulin . Hypergl2+]i). In contrast, DR2 are coupled to Gαi/o and negatively regulate the production of cAMP and the inhibition of PKA, as well as decrease [Ca2+]i [The physiological actions of dopamine are mediated by five different but closely related G protein-coupled receptors (GPCRs) that are divided into two major groups: D1-like receptors and D2-like receptors . D1-like [Ca2+]i . Dopamin [Ca2+]i , kidney  [Ca2+]i , and vas [Ca2+]i  tissue.  [Ca2+]i . It has  [Ca2+]i  and redu [Ca2+]i . In the 2S) has been proven to be a gasotransmitter like carbon monoxide (CO) and nitric oxide (NO). It is endogenously generated and manifests significant effects at physiologically relevant concentrations i in heart tissue and cardiomyocytes i and activating CaM, thereby increasing the endogenous H2S content and leading to the inhibition of the abnormal proliferation of VSMCs in the diabetic state. Overall, this study opens new paths to the prevention and treatment of diabetes and its complications.Calmodulin (CaM) is a protein that has physiological functions after being activated by binding to calcium ions. It is widely expressed in eukaryotic cells and has a variety of biological meanings . Yang et2S . Interesmyocytes . HoweverC57BL/6J mice were obtained from the Animal Research Institute of Harbin Medical University (Harbin. China). Animals were housed in a climate-and temperature-controlled room on a 12:12-h light-dark cycle. The mice were maintained on a standard diet and water ad libitum. All animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals published by the China National Institutes of Health and approved by the Animal Care Committees of Harbin Medical University .3) and AG1478 (an inhibitor of EGFR) were purchased from Sigma Chemical Co. . The primary antibodies for CSE, Cyclin D1, PCNA, P21 and β-actin were from Proteintech . The 7-azido-4-methyl-coumarin (H2S probe), CaM, t-ERK1/2, p-ERK1/2, t-PI3K, p-PI3K, t-Akt, p-Akt, t-JAK2, p-JAK2, t-STAT3, p-STAT3, HB-EGF, IGF-1, t-EGFR, p-EGFR, t-IGF-1R and p-IGF-1R antibodies were obtained from Cell Signaling Technology . DR1 antibody was purchased from GENE TEX . The protein A/G Magnetic Beads was obtained Selleckchem, . The Cell Counting Kit-8 (CCK-8) was obtained from Boster Bio-engineering Limited Company . The EdU was obtained from RiboBio Company . Control vector plasmid and over-DR1 plasmid, control siRNA, CSE siRNA, IGF-1 siRNA, HB-EGF siRNA and calcein AM were purchased from Santa Cruz . All other chemicals were from Sigma or Santa Cruz.SKF38393 , PPG , streptozocin (STZ), Sodium hydrogen sulfide (NaHS), AG1024 (an inhibitor of IGF-1), AG490 (an inhibitor of JAK2), PD98059 (an inhibitor of ERK1/2), LY294002 (an inhibitor of PI3K), Tetracaine (a RyR inhibitor), 2-APB  mice were made diabetes by a single injection of streptozotocin . The STZ was dissolved in 100 μM citrate buffer (citric acid: sodium citrate=1:1.32). After 3 days, the mice with glucose levels above 16.67 mmol/L were considered hyperglycemic (diabetes) . SimultaAorta tissue samples were fixed in 10% neutral buffered formaldehyde and dehydrated through a serial alcohol gradient and embedded in paraffin wax blocks. 4-um-thick aorta tissue sections were dewaxed in xylene, rehydrated through decreasing concentrations of ethanol, and washed. And then stained with H&E for general histopathological evaluation and Masson for show the fibers in the tissue.2O2 solution for 10 minutes. The sections were blocked for 1 hour with 5% goat serum and incubated with α-SMA primary antibodies overnight at 4 °C. Next, the slides were incubated with the secondary antibody for 30 minutes at 37? following the instruction. For signal detection, the slides were incubated with DAB solution for 3 minutes. Finally, slides were counterstained with hematoxylin. All images were acquired by microscope with 10× or 60× objectives.IHC was conducted as described previously . The aorThe mouse aortic SMCs line (MOVAS)were purchased from the Shanghai Institute of Biochemistry and Cell Biology  and were cultured in growth medium DMEM containing 10% foetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin. The experiments were performed when the cells reached 70-80% confluence by trypsinization according to the standard procedures and the medium was changed every 48 h.The MOVAS were randomly divided into the following 7 groups and each group included 6 samples (n=6): (1) Control group (Control): The MOVAS were cultured 2% FBS-DMEM containing 5.5 mM glucose; (2) High glucose group (HG): The MOVAS were cultured with 2% FBS-DMEM containing HG (40.0 mM glucose); (3) HG+SKF38393 group: 5 μM SKF38393 was added to the medium containing HG at the same time; (4) HG+NaHS group: 100 μM NaHS was added to the medium containing HG at the same time; (5) HG+PPG+SKF38393 group: 5 mM PPG was added to the medium for 1h before HG and SKF38393 incubation, the other procedure was similar to that for group 3; (6) HG+2-APB (or W-7 or Tetracaine)+SKF38393 group: The procedure was similar to that for group 5, except that PPG was replaced by 3 μM 2-APB (or 10 μM W-7 or 100 μM Tetracaine); (7) HG+AG1024  group: 5 μM AG1024  was added to the medium for 1h before HG incubation. Then all the MOVAS were incubated for 48 h.3 cells/well). After the cells were serum-starved for 12 h, exposed to each treatment for 48 h. The cell counting kit-8 (CCK-8) was added to each well according to the manufacturer's introductions. Optical density was measured by a microplate spectrophotometer at a wavelength of 570 nm (A570).Cells were seeded in 96-well plates , which has been proved to selectively respond to H2S as described previously i) was measured by Ca2+ indicator Fluo 4-AM as described previously i, the expression of CaM and the interaction of DR1 and CSE were detected in HG-induced MOVAS. The [Ca2+]i of the HG group was higher than the control group. After SKF38393 was given, the [Ca2+]i was further up-regulated. The addition of W-7, an inhibitor of CaM, did not affect the up-regulation effect of HG and SKF38393 on the [Ca2+]i. 2-APB (an IP3 inhibitor) and tetracaine (a RyR inhibitor) closed the channel for internal calcium release and reduced the increase in intracellular calcium caused by SKF38393  is one of the most common metabolic disorders and is estimated to affect 4.4% of the global population in the next 20 years . The vasnous H2S .2S, we supplemented with a DR1 agonist SKF38393, an exogenous H2S donor NaHS and a CSE inhibitor PPG in vivo and in vitro and found that SKF38393 significantly increased DR1 and CSE expression and endogenous H2S production, while NaHS only increased CSE expression and endogenous H2S production, but had no effect on DR1 expression. The CSE inhibitor PPG abolished the effect of SKF38393  is a pleiotropic hormone that regulates VSMCs migration, proliferation, apoptosis, and differentiation. These actions are mediated by IGF-1R . Rui Wantic SMCs . Heparintic SMCs , 46. Thetic SMCs . IGF-1R tic SMCs . EGFR intic SMCs . The JAKtic SMCs . Phosphotic SMCs . Our res2S production in T1MD mice and HG-induced VSMCs. How does the activation of DR1 increase the production of endogenous H2S? To answer this question, we tested [Ca2+]i, the expression of CaM and the interaction of CaM and CSE. In the case of HG, the level of [Ca2+]i is increased  type 3 and 6 and TRPM4 channels, and voltage-dependent calcium channels, thereby up-regulating Ca2+ regulated myogenic tone i [2+]i. SKF38393 further increased [Ca2+]i. 2-APB (an IP3R inhibitor) and tetracaine (a RyR receptor inhibitor) blocked the effect of SKF38393 (2S production by enhancing Ca2+-CaM pathway in HG-induced VSMCs.The above experimental results confirmed that DR1 activation inhibits VSMCs proliferation by increasing endogenous Hncreased . This is stimuli , 52. Theuli [2+]i, 54. In nic tone . Ca2+ is biology . A direc earlier . Yang etal cells . Other r pathway . Our preay [2+]i . Our datSKF38393 . In addiSKF38393 . These r2S system are decreased, IGF-1/IGF-1R and HB-EGF/EGFR as well as their downstream PI3K/Akt, JAK2/STAT3 and ERK1/2 pathways are activated, VSMCs are in a state of abnormal proliferation. Administration of DR1 agonists SKF38393 can increase [Ca2+]i and activate CaM, thereby activating the CSE/H2S system, releasing endogenous H2S, and then down-regulating IGF-1/IGF-1R and HB-EGF/EGFR as well as their downstream PI3K/Akt, JAK2/STAT3 and ERK1/2 pathways, finally, inhibiting VSMCs proliferation. A potential target was found for the up-regulation of H2S, so as to achieve the protective effect of H2S on diabetes mellitus and its complications.In summary , in the The data set used and/or analyzed in the current research can be obtained from the corresponding author upon reasonable request.www.aginganddisease.org/EN/10.14336/AD.2021.1104.The Supplementary data can be found online at:


Pasteurella multocida (P. multocida) infection can cause a series of diseases in different animals and cause huge economic losses to the breeding industry. P. multocida is considered to be one of the most significant pathogens in rabbits. In order to elucidate the pathogenic mechanism and innate immune response of P. multocida, an infection experiment was carried out in this study.P. multocida can rapidly replicate in a variety of tissues, and the colonization in most of the tested tissues reached the maximum at 2 dpi and then decreased at 3 dpi. The number of P. multocida in lung and thymus remained high level at 3 dpi. Toll-like receptors 2 and 4 signaling pathways were activated after P. multocida infection. The expression of Il1β, Il6, Il8, and Tnf-α was significantly increased. The expression of most proinflammatory cytokines peaked at 2 dpi and decreased at 3 dpi, and the expression trend of cytokines was consistent with the colonization of P. multocida in rabbit tissues.Our results showed that the clinical symptoms of rabbits were severe dyspnoea and serous nasal fluid. During the course of the disease, the deaths peaked at 2 days post infection (dpi) and mortality rate was 60%. The pathological changes of the lung, trachea, and thymus were observed. In particular, consolidation and abscesses appeared in lung. Histopathologic changes in rabbits showed edema, hemorrhage, and neutrophil infiltration in the lung. P. multocida can rapidly replicate in various tissues of rabbit and cause bacteremia after infection. TLRs signaling pathways were activated after P. multocida infection, significantly inducing the expression of proinflammatory cytokines, which is might the main cause of respiratory inflammation and septicemia.The  Pasteurella multocida (P. multocida) is responsible for different diseases in a variety of animals, including farm animals , wild mammals, reptiles, and laboratory animals [P. multocida-related diseases of huge economic losses to the breeding industry mainly include fowl cholera in avian, haemorrhagic septicemia and bovine respiratory disease in ruminant, porcine progressive atrophic rhinitis and swine plague, and complex respiratory diseases in rabbits [P. multocida has been serologically divided into five serotypes  based on capsular antigens [P. multocida are host specific, and can cause different diseases and pathological types among different animal species. Serotypes associated with fowl cholera are generally A:1, A:3, and A:4 [P. multocida serogroup F was confirmed to be highly pathogenic in rabbits [ animals . P. mult rabbits . It can  rabbits . Using Cantigens , 5. On t and A:4 ; haemorr and A:4 ; bovine  and A:4 ; and por and A:4 . Pasteur rabbits . In Ital rabbits .Pasteurella species, P. multocida is considered to be part of the normal flora of the oropharyngeal. Rabbits are often colonized with P. multocida for long periods without clinical symptoms. It is also a primary or opportunistic pathogen of the respiratory tract. Diseases develop when animals are subjected to certain stresses, such as transportation, overcrowding, changes in ambient temperature or humidity. Stresses are the important factor, which seems to be conducive to promote the proliferation and toxicity of microorganisms, and its mechanism is not fully understood [P. multocida-related diseases is very difficult. Similar to P. multocida in various livestock and poultry varieties under intensive production conditions, P. multocida also causes high economic losses in rabbits. Although P. multocida has been discovered for decades, there are few studies on the pathogenesis of the disease caused by P. multocida in rabbits.In rabbits, pasteurellosis is considered one of the most common diseases, and the prevalence rate ranges from 4.3 to 100% , 13. Likderstood . TherefoP. multocida infection in rabbits mainly causes a series of inflammatory diseases and septicemia. Innate immune system is the first line of defense against the invasion of pathogenic microorganisms. Pattern recognition receptors detect pathogen-associated molecular patterns and damage-associated molecular patterns [P. multocida has been reported in rabbits, study on the role of host innate immune response against P. multocida infection are scarce. In this study, we investigated the colonization of P. multocida, pathological changes, clinical symptoms, and innate immune responses in rabbits after P. multocida infection.patterns . Among apatterns . TLR2 anpatterns . In the patterns . AlthougP. multocida C51-17 strain (serovar A) was cultured on sheep blood agar plates supplemented with 5% sheep red blood cells at 37 °C for 24–36 h. The P. multocida colony was grown in trypticase soybean broth supplemented with 5% fetal bovine serum at 37 °C shaker for 12 h. According to the results of viable count, the bacterial culture was diluted in PBS to 106 colony forming unit per mL (CFU/mL) for the challenge experiment.The P. multocida infection, nasal and conjunctival swabs were collected from all rabbits before challenge. The swabs were plated onto sheep blood agar plates at 37 °C for 24–36 h for bacterial examination. The swab samples were also detected by 16S rRNA gene which is species-specific for P. multocida . Rabbits of the control group were injection with 1 mL of PBS.A total of 50 healthy weaned New Zealand White rabbits (35 days old) used in this study were purchased from a commercial rabbitry and housed individually in stainless steel rabbit cages, which were placed in isolated rooms with independent ventilation. The rabbits were randomly separated into two groups and free access to water and feed. Each group was housed in a separate room. In order to confirm the absence of Ten rabbits were selected to calculate the mortality and clinical symptoms scores independently by assigning random numbers . The attitude, temperature, and chest murmur, incidence, and deaths were observed and counted daily. Clinical symptoms were scored separately from 0 to 3 Table . The surThe thymus, lung, and trachea of 5 rabbits in each group were collected at 1, 2, and 3 dpi to observe histopathological changes. The tissues were fixed with 4% paraformaldehyde solution at room temperature. The fixed tissue samples were embedded in paraffin and then sectioned at 5 μm. Next, the sections were stained with hematoxylin and eosin (H&E). Histopathological changes were observed using a Leica DM2000 LED light microscope .P. multocida at 1, 2, and 3 dpi. DNA was extracted with the Bacteria Genomic DNA kit  and stored at − 20 °C until further use. Quantitative real-time PCR (qRT–PCR) reaction system was carry out in a total volume of 20 μL with TransStartR Tip Green qPCR SuperMix (+Dye II) . The primers qPm-F and qPm-R were designed for the detection of P. multocida . RNA (1 μg) was reverse transcribed with TransScriptR one-step gDNA Removal and cDNA Synthesis SuperMix . The synthesized cDNA was stored at − 20 °C. For qRT–PCR, the primers for Tlr2, Tlr4, Il1β, Il6, Il8, Tnf-α, and Gapdh were based on the previous studies (Table TransStartR Tip Green qPCR SuperMix (+Dye II) . qRT–PCR was performed with the LineGene 9600 Plus real-time PCR System (Bioer Technology). The qRT–PCR program involved 1 cycle of 94 °C for 30 s, followed by 40 cycles of 94 °C for 5 s and 60 °C for 34 s and finally followed by dissociation curves. The housekeeping gene Gapdh was used as the internal control to normalize the target genes. The relative mRNA transcriptional levels of target genes were calculated using the 2−ΔΔCt method. The results were expressed as the fold change of target gene expression in infected rabbits (n = 5) vs. that of the control group (n = 5). Make three replicates for each sample.The lung and thymus of 5 rabbits in each group were collected to investigate the transcriptional levels of innate immunity-related genes at 1, 2, and 3 dpit-test. A value of p < 0.05 was set as statistical significance.The statistical analysis was performed by SPSS 23.0 software . The difference of survival rate of rabbits was analyzed using the Kaplan–Meier method. Other data were analyzed with the Student’s P. multocida was detected in the nasal and conjunctival swabs, indicating that all rabbits were free of P. multocida infection. No clinical symptoms and deaths were observed in control group during the experiment. Depression, fever, loss of appetite, shortness of breath, and serous nasal discharge occurred at 1 dpi, of which 2/10 rabbits showed moderate diarrhea. As shown in Fig. Before challenge, no No gross lesions of the disease were observed in rabbits of control group. The pathological changes were mainly focus on the lung, with slight bleeding and consolidation at 1 and 2 dpi. The lungs developed typical consolidation at 3 dpi, with abscesses and fibrous exudates. Hemorrhage occurred in the tracheal ring after challenge, most severe at 3 dpi. Similarly, the thymus exhibited obvious bleeding spots, which are the most severe at 2 dpi . The expression of Tlr2 reached the peak at 2 dpi in thymus, significantly up-regulated by 12.53-fold (p < 0.05). The expression trend of Tlr4 in thymus was similar to that of Tlr2, significantly up-regulated by 4.30-fold at 2 dpi (p < 0.05) and only a 1.72-fold increase at 3 dpi. However, the expression of Tlr4 was suppressed in the lung, especially by a significant 0.42-fold at 2 dpi . Similarly, the expression of Il6 was the highest at 2 dpi, up-regulated 4.26-fold in the lung and 70.81-fold in the thymus (p < 0.05) . In lung, the expression of Il8 significantly up-regulated 12.46-fold at 1 dpi, then gradually decreased, and up-regulated 5.41-fold at 3 dpi (p < 0.05)  increased in lung [P. multocida infections. After activation of TLRs, NF-κB signaling was stimulated to resist bacterial infection by regulating production of cytokines [P. multocida is partially dependent on TLR4 in macrophages. The expression of pro-IL-1β was significantly up-regulated after P. multocida infection, but partially decreased in TLR4 knockout macrophages [Il1β was significantly increased in lung and thymus. IL-6 and IL-8 are important cytokines in the inflammatory response, and cooperate with other cytokines to regulate the inflammatory response process. TNF-α is an endogenous pyrogen that induces fever and stimulates endothelial cells and leukocytes to release various inflammatory mediators that promote neutrophil phagocytosis. It has been reported that P. multocida highly induced the secretion of proinflammatory cytokines  in mouse [Il6, Il8, and Tnf-α was significantly increased, indicating that the TLRs pathway was activated after P. multocida challenge. The expression of most cytokines peaked at 2 dpi and decreased at 3 dpi. This trend was consistent with the colonization of P. multocida in rabbit tissues. We speculated that the high expression of proinflammatory cytokines  in lung might be the main cause of respiratory inflammation and septicemia.In mice and cattle, the inflammatory mediators were highly expressed in lung after  in lung . These iytokines , 33. Recrophages . In currin mouse . SimilarP. multocida infection in rabbit were investigated. The clinical symptoms were mainly respiratory symptoms, with severe dyspnoea and serous nasal fluid. The pathological changes are directly related to colonization of P. multocida in rabbit tissues. TLRs signaling pathways were activated after P. multocida infection, the overexpression of inflammatory factors aggravates tissue damage and mortality, which is also the main cause of respiratory inflammation and septicemia. Altogether, this study has pioneered the systematic exploration of the colonization, innate immune responses, and etiology in P. multocida-infected rabbits. This study also provide new insights into the relationship between P. multocida pathogenicity and defense response in rabbits.In conclusion, the whole disease process of