Mechanical loading promotes bone formation and osteocytes are a major mechanosensory cell in the bone. Both Piezo1 channels and connexin 43 hemichannels (Cx43 HCs) in osteocytes are important players in mechanotransduction and anabolic function by mechanical loading. However, the mechanism underlying mechanotransduction involving Piezo1 channels and Cx43 HCs in osteocytes and bone remains unknown.2+ signaling and activate Cx43 HCs, while Yoda1 antagonist Dooku1 inhibited Ca2+ and Cx43 HC activation induced by both mechanical loading and Yoda1. Moreover, the intracellular Ca2+ signal activated by Yoda1 was reduced by the inhibition of Cx43 HCs and pannexin1 (Panx1) channels, as well as ATP-P2X receptor signaling. Piezo1 and Cx43 HCs were co-localized on the osteocyte cell surface, and Yoda1-activated PI3K-Akt signaling regulated the opening of Cx43 HCs. Furthermore, Cx43 HCs opening by mechanical loading on tibias was ablated by inhibition of Piezo1 activation in vivo.We showed that, like mechanical loading, Piezo1 specific agonist Yoda1 was able to increase intracellular Ca2+ activated by Piezo1 regulates the opening of HCs through PI3K-Akt and opened Cx43 HCs, along with Panx1 channels, and ATP-P2X signaling sustain the intracellular Ca2+ signal, leading to bone anabolic function.We demonstrated that upon mechanical stress, increased intracellular CaThe online version contains supplementary material available at 10.1186/s13578-022-00929-w. Osteocyide (NO) . Ca2+ reeostasis . Connexi and K+) . Connexi and K+) , 7. OsteCx43 HCs , 9, and olecules , 11. Ext loading , 13, whi loading , 15. Mor loading , 17.During the last decade, Piezo proteins were identified as mechanosensitive ion channel subunits, and the mechanosensitive function and mechanisms of Piezo ion channels, Piezo1/2, have been unveiled , 19. Pie2+ channels and Cx43 HCs in osteocytes. Both in vivo and in vitro studies showed that the opening of Cx43 HCs is regulated by Piezo1 activation upon mechanical loading or Yoda1. In addition, intracellular Ca2+ increase by mechano-activated Piezo1 is involved in the regulation of Cx43 HCs and pannexin 1 channels, and HC opening is regulated by activated PI3K signaling. Moreover, Piezo1 and Cx43 HCs are co-localized on the cell surface of osteocytes, and FFSS enhances the co-localization.In the present study, we unveiled the functional and structural relationship between mechanosensitive Piezo1 Ca3−) supplemented with 2.5% fetal bovine serum and 2.5% bovine calf serum and incubated in a 5% CO2 incubator at 37 °C as described previously [Osteocytic MLO-Y4 cells derived from murine long bones were provided by Dr. Lynda Bonewald at Indiana University School of Medicine. Lentiviral-mediated CRISPR/Cas9 genome editing technology using lentiCRISPRv2:Cx43-sgRNA or ROSA26-sgRNA was applied to generate Cx43 KD and Rosa26 control MLO-Y4 cells as previously described [eviously .2 were generated, and a cell surface area of 5 cm2 was used for dye uptake assay. MLO-Y4 cells were pre-incubated with Cx43(E2) antibody, generated and affinity-purified as previously described [10Panx , Dooku1 , BAPTA-AM , apyrase , and LY294002 . The cells were then subjected to FFSS , followed by dye uptake assay. In dye uptake assay, cells were incubated with 10 μM ethidium bromide (EtBr) (Mw: 394 Da) and FITC-Dextran (Mw: 10 kDa) at room temperature (RT) for 5 min and FITC-Dextran was used to detect the apoptotic and unhealthy cells. The FITC-Dextran-positive cells were excluded for quantification of dye uptake level. The treatment with Yoda1  was performed in the presence of the dyes. Cells were rinsed with PBS and fixed with 2% paraformaldehyde, and the mean intensity of the nuclear region was determined using NIH ImageJ software  [FFSS experiments were conducted as previously described . Brieflyescribed , 10Panx 5 cells on a collagen-coated 35 mm culture dish with an inner 25-mm diameter circle glass slide 48 h prior to live Ca2+ imaging. Before imaging capturing, cells were incubated with Cx43(E2) antibody, 10Panx, Dooku1, BAPTA-AM, oATP , and apyrase  at RT for 30 min, followed with Fluo-4-AM , a Ca2+ indicator, at 37 ℃ for 40 min. The culture medium was then rinsed and replaced with recording medium , and the glass slide was placed in the Coverslip Cell Chamber . The chamber was placed on the objective stage of the microscope and set still before capturing images. Ca2+ signaling (488 nm) of the MLO-Y4 cells was imaged using a Nikon sweptfield laser scanning confocal microscope  with a 40X objective. The entire imaging process lasted 600 s, and each image was taken with a 2-s interval, and Yoda1 was loaded at 240 s. For sequential inhibition of Cx43 HCs or Panx1 Channel, Cx43(E2) antibody or 10Panx was added at 360 s. The BAPTA-AM was added at 360 s. For the recording of Ca2+ signal in mechanically loaded MLO-Y4 cells, the cells cultured on coverslips were then placed in the FFSS chamber and connected with the fluid circulation system, and live calcium images were captured at 900 s with FFSS (8 dynes/cm2) stimulation applied at 300 s and lasted for 300 s. The entire process lasted for 900 s with 3 phases (300 s each) rest, stimulation, and recovery.MLO-Y4 cells were cultured at a total of 2.5 × 102) for 1 h or Yoda1 for 30 min and rinsed 3 times with cold (4 ℃) DPBS. The live cells on the slides were incubated with rabbit polyclonal anti-Cx43(E2) antibody (1:100 dilution) in recording medium supplemented with 2.5% fetal bovine serum and 2.5% bovine calf serum at 4 ℃ for 90 min. After rinsing 3 times with cold DPBS supplemented with 1 mM Ca2+, cells were fixed with 2% paraformaldehyde for 10 min, followed by blocking with donkey blocking buffer . The cells were then incubated with 1:100 dilution mouse monoclonal anti-Piezo1 antibody  at 4 ℃, overnight. The labeling by primary antibodies was performed by sequential incubation of secondary antibodies first with donkey anti-mouse Alexa Fluor 488-conjugated  for 1 h and then with donkey anti-rabbit Alexa 594  for 1 h. Slides were mounted and sealed for microscopic analysis. Confocal sectional images were taken with an optical microscope  using the appropriate filters.MLO-Y4 cells cultured on collagen-coated glass slides were subjected to FFSS  and proteinase inhibitors) and then ruptured by pipetting through a 20-gauge needle. Cell lysates with Cx43 KD or Rosa26 control were ultracentrifuged at 45,000 g for 45 min at 4 ℃ to prepare crude membrane extracts. Protein concentration of protein lysates was determined by Micro BCA Protein Kit . 30 µg proteins were boiled in SDS sample loading buffer  glycerol, 10 mM DTT, 0.05% (w/v) bromophenol blue), subjected to 10% SDS–polyacrylamide gel electrophoresis, and electroblotted onto a nitrocellulose membrane. Membranes were blocked with donkey serum blocking buffer at RT for 1 h, then incubated with a 1:1000 dilution of rabbit polyclonal anti-Akt antibody  and rabbit polyclonal anti- phosphorylated-AKT Ser473 antibody  (1:300 dilution) for LY-294002/Yoda1 treated MLO-Y4 cell samples or incubated with 1:500 dilution of rabbit polyclonal anti-Cx43(CT) antibody or 1:150 of mouse monoclonal anti-Piezo1 antibody  for Cx43 KD and Rosa26 samples. Primary antibodies were detected with goat anti-rabbit IgG conjugated IRDye® 800CW and goat anti-mouse IgG conjugated IRDye® 680RD (1:15 000 dilutions) using a Licor Odyssey Infrared Imager . The band intensity was quantified by densitometry using NIH ImageJ software.MLO-Y4 cell lysates were resuspended in immunoprecipitation (IP) buffer . Lysates were precleared with protein A/G plus-agarose beads  and pelleted by centrifugation at 7000 rpm at 4 ℃ for 5 min. Supernatants (precleared lysates) were then incubated with rabbit polyclonal anti-Piezo1 antibody  overnight at 4 ℃, followed by precipitation of the complexes after incubation with protein A/G plus-agarose beads at 4 ℃. Immunoprecipitants were separated on 10% SDS–polyacrylamide gel and immunoblotted with mouse monoclonal anti-Cx43  (1:200 dilution) or mouse monoclonal anti-Piezo1 antibody  (1:500 dilution).We subjected 15-week-old male C57Bl/6 mice to mechanical loading through tibial compression using a loading setup established in our laboratory , 30, 31.p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.GraphPad Prism8 statistics software  was used for statistical analysis. Student-Newman Keul’s test and One-way ANOVA were used for two or multiple comparison analyses. Two-way ANOVA with post hoc Tukey test was used to assess the difference between multiple groups with two variables. Data were shown as mean ± SEM, and asterisks indicate the degree of significant differences compared with the controls, *Both Cx43 HCs and Piezo1 channels expressed in osteocytes are mechanosensitive channels; however, the functional relationship of these two channels has not been assessed. MLO-Y4 cells were treated with different concentrations  of Yoda1, and time-lapse images with 5 min intervals of EtBr fluorescence were taken. The cell uptake of EtBr indicated an increase in the membrane permeability. The apoptotic, unhealthy cells were assessed with FITC-Dextran, a dye with Mw of 10 kDa which is impermeable to HCs (< 1.2 kDa) but can be taken up by apoptotic, dying cells. We observed that FITC-Dextran-positive cells were overall very low as shown in Additional file 2+ indicator, were subjected to 5-min FFSS (8 dynes/cm2), and live-cell images were captured. The representative images, which are the first images of each phase in the pretreated Dooku1 and control groups, showed dramatically increased levels of intracellular Ca2+ after FFSS stimulation. The intracellular Ca2+ rise was blocked significantly with Dooku1 in the recording media  -induced dye uptake  antibody, pre-treatment of Dooku1 and Apy did not inhibit HCs uptake induced by ionomycin  at the 120 s time point in the recording media. As expected, the second dose of BAPTA-AM suppressed the Ca2+ signal to a relatively low and steady level  and latent phase (up to 360 s) , a potent inhibitor of ATP-activated P2X purinergic receptors  antibody and Cx43 knockdown. MLO-Y4 cells were pre-incubated with Cx43(E2) antibody (2 μg/ml), then live Ca2+ images were captured, and Yoda1 was applied during the imaging. Compared with the control, the intracellular Ca2+ signal (first peak) decreased significantly, while exhibiting a parallel declining curve for the rest of the recording time periods  antibody-treated group  antibody.Since Cx43 HCs can mediate extracellular Ca release –41, we dods Fig. A. By quaoup Fig. B. Similaods Fig. C and AUCods Fig. D. Intere10Panx (150 μM), an inhibitory mimetic peptide for Panx1 channels, also significantly reduced Yoda1-induced dye uptake , 180 s, and 360 s showed that both Cx43(E2) antibody and 10Panx could significantly reduce Ca2+ signals at both acute phase and latent phase  antibody and 10Panx in sequential order by first preincubating cells with Cx43(E2) antibody  antibody showed a lower level of Ca2+ signal , there was an increased colocalization of Piezo1 and Cx43  antibody, a mouse monoclonal antibody that binds to the extracellular domine of Cx43 and inhibits HCs [2, a critical anabolic factor in response to mechanical stress [2+ levels, leading to the opening of Cx43 HCs and Panx1 channels. Moreover, Piezo1 activation leads to the activation of the PI3K-Akt signaling pathway required for the opening of Cx43 HCs. We also observed the colocalization of Piezo1 and Cx43 HCs, suggesting the proximity of the two proteins, and this co-localization was enhanced by mechanical stimulation. Furthermore, we found that the activity of Cx43 HCs along with Panx1 channels and the activation of P2X receptors was necessary to further elevate intracellular Ca2+ levels initiated by Piezo1 channels. The role of Piezo1 in regulating Cx43 HC opening by mechanical loading was also confirmed in osteocytes of tibial bone in situ. Together, as illustrated in the diagram  antibody; however, a greater level of reduction was not observed by combined treatment, probably due to the presence of other pathways that uptake the dye and are sensitive to 10Panx. Interestingly, the action of Cx43 HCs and Panx1 channels was not additive, but interconnected, requiring each other to be active. Several studies have shown that P2X receptor activation is unaffected by genetic deletion or pharmacological inhibition of Panx1 [10Panx on Panx1 channels may not affect P2X receptors [We confirmed the involvement of Panx1 channels using activity . In addiactivity . Howeveractivity . Panx1 creceptor . The intreceptor . We showof Panx1 , which ieceptors .2+, which is required for the following ATP release [2+ concentration. Previous studies proved that the opening of Cx43 HCs is triggered by moderate intracellular Ca2+ concentration rises (≤ 500 nM), and HCs opening allows the influx of Ca2+ [2+ imaging results, opening of Cx43 HCs is believed to occur at a later phase, while Piezo1 channel activation is responsible for the initial intracellular Ca2+ peak. Moreover, we also observed that the opening of Cx43 HCs allows the release of molecules like ATP that could activate P2X channels expressed on the cell surface, suggesting an indirect Ca2+ signaling pathway for the sustained influx of Ca2+ mediated by Cx43 HCs  are also involved in the regulation of cytoplasmic Ca2+ (Ca2+cyt). The Ca2+cyt and Ca2+ER oscillation was subtly synchronized, and the increased Ca2+cyt was proven to refill Ca2+ER, and the T-Type VSCC facilitates this behavior [2+ signaling under mechanical stimulation remained largely elusive. We investigated intracellular Ca2+ signals by live imaging upon mechanical loading and activated Piezo1. Unlike the Ca2+ oscillation pattern on a single cell, in this study, the averaged Ca2+ signal of 18 to 36 cells from 3 to 6 independent experiments showed a relatively consistent pattern. The difference might be due to the delayed synchronization between cells, as well as the calculated Ca2+ signal derived from combined Ca2+cyt and Ca2+ER. Interestingly, the inhibition of Cx43 HCs and Panx1 both decrease intracellular Ca2+ signals at both acute and latent phases.The acute event that takes place within 1 min of mechanical stimulation is the increase of intracellular Ca release . Our dat of Ca2+ . Based o release , 56. ATP release , 44, 57. loading . Endoplabehavior . However2+ signal, which may indicate that Cx43 HCs engage more in sustaining later phase intracellular Ca2+. Regardless of the sequential order of the inhibitors being applied, a similar, although slightly greater, inhibition of intracellular Ca2+ activated by Piezo1, was shown by inhibiting Panx1 channels. It has been known that elevated intracellular Ca2+ activates both types of channels [2+ is likely to be a result of the opening of both Cx43 HCs and Panx1 channels that subsequently mediate Ca2+ increase. The slightly greater reduction by first applying the Panx1 inhibitor may indicate that the Panx1 channel contributes slightly more to the overall intracellular Ca2+ level. A limitation of the study is the time resolution since 2 s time intervals could be relatively too long to resolve the temporal difference attributed by Cx43 HCs and Panx1 channels upon Piezo1 activation. The dye uptake data suggest a functional dependency between Cx43 HCs and Panx1 channels. Similar to the inhibitory effect by Cx43 HCs and Panx1 channels, oATP and apyrase reduced intracellular Ca2+ at both acute and latent phases, which indicates the involvement of ATP-activated P2X receptor signaling in regulating intracellular Ca2+ dynamics triggered by Piezo1. Moreover, we also observed that apyrase also inhibited HCs dye uptake. These results suggest that a high local concentration of extracellular ATP released, possibly by osteocytic HCs formed by Cx43 and Panx1 channels, activates purinergic receptors, leading to further opening of HCs and Ca2+ influx at the latent phase.To explore the temporal relationship of Cx43 HCs and Panx1 channels, we inhibited these two channels in a sequential manner. Despite the similar reduction of the first peak, the application of Cx43(E2) antibody at the later phase showed a lower Cachannels . These d2+ regulation and the PI3K-Akt signaling pathway. Ca2+ influx induces the Ca2+ release from the endoplasmic reticulum via IP3R activation [2+ activates PI3K-Akt, which modulates GSK3β activity and therefore regulates the Wnt/β-catenin pathway [We showed that Yoda1 increased Akt activation, and inhibition of PI3K-Akt signaling inhibited HCs induced by Yoda1. Previous studies reported a close relationship between Cativation . The cyt pathway . Moreove pathway . We rece pathway . Here, w2+ and open HCs. In summary, it is likely that the interplay of Piezo1 and integrin activation upon mechanical loading activates the PI3K-Akt signaling pathway that induces the opening of Cx43 HCs [Before the discovery of Piezo1 as a mechanosensitive ion channel, Piezo1 was reported to be involved in integrin activation by recruiting the small GTPase R-Ras to the ER . The PieCx43 HCs , 45, 66,It is well accepted that fluid flow through the bone lacuna-canalicular system generates shear stress, and therefore in vitro FFSS models have been developed to mimic in vivo shear stress. However, under in vivo situations, the number of dendrites per cell, the changes in lacunar and canalicular number with age, and the configuration of osteocytes vary significantly . Here, w2+-dependent contractions and enhances the production and release of extracellular vesicles (EVs) containing bone regulatory proteins [2+-related mechanisms such as T-Type VSCC, Ca2+ER, and EVs under mechanical loading conditions and to investigate the structural relationship between Piezo1, integrin, and Cx43 HCs, as well as the underlying mechanism, intracellular Ca2+ influx, and activated PI3K-Akt pathway. Together, our findings established a critical relationship between major players in the anabolic function of mechanical loading on bone, Piezo1, Cx43, and Panx HCs.Recent studies also revealed that mechanical stimulation of osteocytes activates Caproteins . FurtherAdditional file 1: Figure S1. (A) Western blot analysis of Cx43 and Piezo1 protein expression of Rosa26 and Cx43 KD in MLO-Y4 cell lysate. (B and C) Western blot quantification of p-Akt and Akt protein expression of different duration  of Yoda1 treatment in lysates of MLO-Y4 cells, n=3 from three independent experiments. Data are shown with mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001. Statistical analysis was performed using One-way ANOVA for multiple comparison analysis. (D) Representative EtBr/FITC-dextran dye uptake images of MLO-Y4 cells pre-incubated with LY294002, followed with Yoda1 treatment from three independent experiments, scale bar=20 μm; (E) Cell lysates and immunoprecipitants with Piezo1 antibody were immunoblotted with Cx43 or Piezo1 antibody.


These Ca2+ transients are primarily sequestered in subcellular domains, including primary branches, branchlets and leaflets, and endfeet. In previous studies, it suggests that aging causes functional defects in astrocytes. Until now, it was unclear whether and how aging affects astrocytic Ca2+ transients at subcellular domains. In this study, we combined a genetically encoded Ca2+ sensor (GCaMP6f) and in vivo two-photon Ca2+ imaging to determine changes in Ca2+ transients within astrocytic subcellular domains during brain aging. We showed that aging increased Ca2+ transients in astrocytic primary branches, higher-order branchlets, and terminal leaflets. However, Ca2+ transients decreased within astrocytic endfeet during brain aging, which could be caused by the decreased expressions of Aquaporin-4 (AQP4). In addition, aging-induced changes of Ca2+ transient types were heterogeneous within astrocytic subcellular domains. These results demonstrate that the astrocytic Ca2+ transients within subcellular domains are affected by aging differently. This finding contributes to a better understanding of the physiological role of astrocytes in aging-induced neural circuit degeneration.Astrocytic Ca We found that astrocytic Ca2+ transients diversely change during aging within the astrocytic subcellular domains. This finding provides us with a more precise understanding of how astrocytes contribute to brain function and dysfunction during the aging process.To determine the aging-induced Male C57BL/6J mice were obtained from the GemPharmatech Company in Nanjing. The mice were divided into three groups according to their ages: the 2–3-month-old, 7–10-month-old, and 13–16-month-old groups. 6–9 mice were used per group, and 23 mice were used in total. Mice were maintained in a 12-h light-dark cycle at 22–25°C and 50–60% relative humidity with freely available food and water. All animal experiments were performed according to the Institutional Animal Care and Use Committee of the Third Military Medical University, China.2 and placed in stereotaxic frames. A small vertical incision was made in the skin. The part of the skull directly above the injection site was thinned using a small bone drill to allow penetration by a glass micropipette containing pAAV. To detect the characteristics of Ca2+ transients of astrocytes, the AAV construct  was injected with a glass micropipette at a 45° forward tilt into the somatosensory cortex [anteroposterior (AP): + 0.5 mm; mediolateral (ML): −1.56 mm, 12 vg/mL) were applied at dorsoventral (DV): −0.7 and −0.5 mm. After the injection, the pipette was held in place for 15 min before being slowly retracted from the brain. The scalp incision was closed with tissue adhesive ; postinjection analgesics were administered to aid recovery.The mice were anesthetized with 1–1.5% isoflurane in pure Oin vivo two-photon Ca2+ imaging was performed as previously described . A craniotomy (approximately 3 mm diameter) centered on the previous virus injection site was made by a cranial drill. During the two-photon Ca2+ imaging, the concentration of isoflurane decreased to 0.5–1% (breathing rates of mice were kept at 100–120 times per minute). The recording chamber was perfused with warm artificial cerebrospinal fluid (ACSF), as previously described  and perfused with 4% paraformaldehyde (PFA). Free-floating coronal brain slices (40 μm thick) were obtained and stained as previously described . In brie2+ imaging was completed according to our previously reported methods (2+ transients were expressed as relative fluorescence changes (Δf/f), corresponding to the mean fluorescence from all pixels within specified regions of interest (ROIs), as reported by our previous studies /f0, where the fluorescence f0 was estimated as the 25th percentile of the entire fluorescence recording , multipeak (MP), and plateau (PL) according to a previous study (2+ imaging data were removed according to the rule of 1.5 × interquartile range (IQR) before statistics. Normality was tested by the D’Agostino–Pearson test since the data didn’t fit the normal distribution. As such, multiple group comparisons were performed using a Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Fractions of active/inactive ROIs  and types of Ca2+ transients were compared with χ2-test or Fisher’s exact test followed by Bonferroni correction. Data were expressed as the mean ± SEM. P < 0.05 was considered statistically significant.Data analysis of two-photon Ca methods  using La methods . Microdo methods  using ou studies , 2021. Tecording . Ca2+ siecording . Therefous study . SP meanus study . All sta2+ elevations in the peripheral districts of astrocytic processes like branchlets and leaflets could generate propagating Ca2+ signals in the primary branches through the opening of endoplasmic reticulum (ER) Ca2+ release channels  at approximately 12–20 μm from the soma perimeter were applied to represent Ca2+ transients within the branchlets and leaflets  is the predominant water channel localized in the astrocytic endfeet and mediates water transport into the brain parenchyma . A previ endfeet . We also endfeet . Similar endfeet , the cur endfeet , which c endfeet .2+ transients within the somata of astrocytes were assessed during brain aging. Data indicated that aging did not affect the characteristics  and proportions of Ca2+ transient types within astrocytic somata  primary branches emanating from soma; (ii) branchlets and leaflets that occupy most of the astrocyte territory also known as an anatomic domain; and (iii) endfeet that are specialized extensions of astrocytic branches contacting and plastering blood vessel . Most of domains , heterog domains , and are signals . These lstrocyte ; in bransmission ; in endfcoupling . Therefo+ buffering and glutamate clearance  in response to stimulation of metabotropic receptors  sets the threshold for the generation of spreading Ca2+ events and determines the probability of Ca2+ transient initiation , ionotropic receptors or channels in leaflets   in the aged brain  . Additioed brain . It has trocytes .2+ transients within the endfeet are involved in the modulation of neurovascular coupling (2+ transients are partly triggered by AQP4-mediated water influx, which promotes ATP release and activation of P2 purinergic receptors (2+ transients within the endfeet (2+ transients within the endfeet are also mediated by mitochondria (2+ uptake (2+ transients observed within endfeet. In addition, according to previous studies, IP3R2-mediated Ca2+ transients are present within branches and branchlets (2+ transients within these subcellular domains.Previous studies have found that Cacoupling . These Ceceptors . In a noeceptors  and our eceptors  both ind endfeet  could bechondria  and GABAchondria . Therefo+ uptake  and decr+ uptake  can alsoanchlets , but absanchlets , which c2+ transients at subcellular domains during brain aging. Previous studies indicated that aging reduces cellular surface area and morphological complexity of astrocytes (2+ transients in old astrocytes (2+ and more frequent Ca2+ transients in astrocytic network (Apart from that, both the age-dependent morphological changes and astrocytic network alterations may induce the aberrant astrocytic Catrocytes . These atrocytes . In addi network .2+ transient types based on their shapes: singlepeaks, multipeaks, and plateaus (2+ transient types indicate diverse mechanisms of signaling (2+ transient types changed with aging in primary branches (2+ signaling in astrocyte subcellular domains. Specifically, it suggested that the IP3-mediated release of ER calcium stores contributed to singlepeaks and multipeaks (2+ transient changes during aging within primary branches (There are three different Caplateaus . Differeignaling . In thisbranches  and branbranches , but rembranches . This sultipeaks . As suchltipeaks  could bebranches , branchlbranches .2+ transients within astrocytic somata during brain aging (2+ transients remained stable (in vitro methods and chemical calcium indicators used in these experiments.Our study indicated that there was no change of Cain aging , suggestd stable  or declid stable  during ain vivo results demonstrate that aging alters the characteristics of Ca2+ transients within astrocyte subcellular domains. However, these changes in Ca2+ transients are heterogeneous in the subcellular domains, which indicates that the aging-induced degenerative mechanisms differ at the subcellular level in astrocytes. This finding provides us a better understanding of how astrocytes contribute to brain dysfunction during aging and other neural degenerative diseases, like Alzheimer’s disease and Parkinson’s disease.Altogether, our The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of the Third Military Medical University.2+ image. FD, ZY, JLi, CY, YW, and MG performed the immunohistochemistry. FD, SL, QD, JLy, ZZ, HJ, and XL performed the data analysis. XC, XL, LF, and KZ wrote the manuscript with input from all co-authors. All authors contributed to the article and approved the submitted version.LF and KZ designed the work. FD, SL, RL, ZY, YH, SY, QD, JZ, JLy, ZZ, MH, HW, JLi, CY, YW, MG, SC, and HJ performed the main experiments. FD, ZY, MH, and HW completed the virus injections. FD, RL, YH, and JZ performed the two-photon Ca


Malignant central airway stenosis is a life-threatening condition. However, treatment of malignant airway stenosis remains challenging. There is currently a severe lack of an excellent animal model of malignant airway stenosis to facilitate treatment approaches. This is the first study to establish a rabbit model of malignant airway stenosis for bronchoscopic interventional studies.New Zealand White rabbits were used in this study, randomly divided into group A (18 rabbits) and group B (6 rabbits). A VX2 fragment suspension was injected into the submucosal layer of rabbits’ airway by bronchoscopy. Bronchoscopic examinations were performed once a week after VX2 tumor implantation to observe tumor growth and the degree of airway stenosis. Randomly, three rabbits were generally dissected after a weekly bronchoscopic examination in group A. The rabbits that reached grade III airway stenosis underwent stent implantation in group B.A total of 24 rabbits were successfully implanted with the VX2 fragment suspension in the airway without significant adverse events, and the success rate of the tumor growth was 100%. The degree of airway stenosis reaching grade III took 2 to 3 weeks after implantation of the VX2 tumor. The median survival time of rabbit models without stent implantation and rabbits with stent implantation was 32.5 and 32.0 days, respectively.The implanting method is safe and effective for the establishment of a rabbit model of malignant airway stenosis. When the tumor grows to 2 to 3 weeks, the rabbit model is available for stent implantation. We recommend the models for more preclinical animal studies on bronchoscopic interventional treatments. Malignant central airway stenosis is a life-threatening condition caused by malignant tumors, most commonly occurring in locally advanced lung cancer , 2. For Although many of these technical changes can be evaluated directly in the patient, clinical investigation often needs preliminary support from animal studies. However, there is currently a severe lack of an excellent animal model of malignant airway stenosis to facilitate treatment-related studies.The VX2 tumor, a squamous cell cancer model, which was firstly proposed by Shope and Hurst  in 1933,This is the first study that aims to develop a rabbit model of malignant airway stenosis in which we describe the use of submucosal VX2 fragments in the tracheal wall and to investigate the safety and efficiency of the method, as well as the characteristics of the airway stenosis model. Furthermore, we observed the feasibility of stent implantation in the novel model.Male or female New Zealand White rabbits  weighing between 2.5 and 3.5 kg were used in this study. The VX2 tumor previously stored at -80°C was obtained from the Surgery Laboratory in the First Affiliated Hospital of Wenzhou Medical University. All experimental procedures were approved by the Laboratory Animal Center of Wenzhou Medical University (ID: wydw2021-0289) and were performed in accordance with the National Institute of Health’s Guide for the Care and Use of Laboratory Animals.Four rabbits with hind-limb tumors were used to propagate and maintain VX2 tumors. Approximately 0.2 ml of saline with VX2 tumor tissue fragments, which could pass through a 1-ml syringe connecting with an 18-gauge needle, was injected deeply into the hind limb of gluteal muscles of rabbits. After 2~3 weeks, the animals were executed and the hind-limb tumors were processed. All VX2 tumors were cleaned from the surrounding normal tissue, and necrotic portions of the tumors were removed.The collected tumors were cut into small pieces approximately 0.5 mm in diameter, which could pass through the 20-gauge needle, and preserved in saline for fragment implantation. Approximately 0.6 ml tumor fragment suspension was placed in a 1-ml syringe connecting with a 20-gauge transfusion needle  was used to observe the characteristics of the airway stenosis model, and group B (6 rabbits) was implanted with a metal stent when malignant stenosis occurred in the rabbit airway.via the ear vein and then secured in supine position. Its anterior neck was exposed, and then preoperative hair removal was prepared. A medical tooth pad was fixed in the oral cavity of the rabbit. An ordinary bronchoscope with an outer diameter of 4.9 mm  was inserted into the airway from the tooth pad, and 1 ml of lidocaine (2%) was injected into the vocal area to reduce the intense cough reaction of the rabbit.Each rabbit was firstly anesthetized by injection with 3% pentobarbital sodium (1 ml/kg) Then the anterior neck between two tracheal cartilages was punctured with the 20-gauge transfusion needle in the guidance of a light source of the bronchoscope -stained histopathology is the gold standard for the diagnosis of carcinoma.The rabbits were injected with 3% pentobarbital sodium (1 ml/kg) In group B, a self-expandable metal stent (8 mm in diameter and 30 mm in length) was implanted when the degree of airway stenosis in the rabbit reached grade III. Firstly, the distance from the distal end of the airway tumor to medical teeth was measured by bronchoscopy and a guidewire was inserted across the stenosis into the main bronchus. Then the delivery device was gently passed over the guidewire and the stent was released according to the measured distance before. After withdrawing the delivery device, ideal positioning of the stent was assessed and adjusted endoscopically. Finally, bronchoscopic examinations were performed every week to observe the stent-related complications and airway restenosis.All 24 rabbits tolerated the bronchoscopy procedure without clinically significant adverse events. When implanting VX2 fragment suspension, no apnea or massive hemorrhage, or even death, occurred Table 1.In the first week after tumor implantation, small nodules attached to the outer wall of the trachea in all living rabbits. All rabbits were confirmed to have tumor growth in their airway by dissection and then histopathology. The success rate of tumor growth in the rabbit airway was 100% Table 1.In group A, the macroscopic external and internal views of the tracheal tumor are as shown in In histopathological examination, the tumor cells grew vigorously in the first 3 weeks. Moreover, some tumors  showed slightly necrotic portions in the second and third weeks. However, the tumors showed obviously necrotic portions in the fourth week , 39% (7/18), and 5% (1/18) rabbits in the first week, respectively.In the second week, the percentages of non-appreciable stenosis, grade I stenosis, grade II stenosis, and grade III stenosis were 13% (2/15), 34% (5/15), 40% (6/15), and 13% (2/15), respectively. Moreover, in the third week, the percentages of grade II stenosis, grade III stenosis, and grade IV stenosis were 42% (5/12), 42% (5/12), and 16% (2/12), respectively. In the fourth weekly bronchoscopic examination, three rabbits (33%) showed grade III airway stenosis and six rabbits (67%) showed grade IV stenosis. The data are summarized in The gross classical appearance of the tumors varied from small submucosal microbulge to larger mass in bronchoscopy. In rabbits with malignant airway stenosis in the study, different structural stenosis types including extrinsic compression of the airway, tumor invasion of the airway wall or intraluminal tumor, and a combination growth are shown in The airway tumor did not metastasize to other organs of rabbits in the first and second weekly necropsy examinations. In the third week, one rabbit was observed to have diffuse pulmonary metastases. In the fourth week, three rabbits were verified with diffuse pulmonary metastases while one of them was attacked by metastatic esophageal carcinoma. The remaining rabbits with their survival time of more than 28 days all showed pulmonary metastases; one of them also had esophageal metastasis, and four of them presented with multiple metastatic organs including lung, esophagus, liver, and kidney. The manifestations of metastatic lesions and corresponding histopathology are demonstrated in The remaining six rabbits in group A suffered from respiratory failure. Before they received humane sacrifice, the bronchoscopic examination presented grade IV stenosis on the airway. The survival time of rabbits varied from 31 to 34 days, and the median survival time was 32.5 days after VX2 tumor implantation.In group B, the time of airway showing grade III stenosis was 3 weeks in four rabbits and 2 weeks in other rabbits. The metal stents were successfully implanted into the narrow airway of six rabbits. Moreover, the airway of all six rabbits recovered patency at once after stent implantation Figure 5In the current study, the bronchoscopic procedure was minimally invasive with a high success rate of implantation and establishment of a malignant airway stenosis model. The adequate follow-up data showed a degree of airway stenosis in different periods, different airway stenosis types, survival time, tumor growth, and metastases of rabbits after VX2 implantation, which indicated that the animal models were reliable for mimicking the disease of human malignant central airway stenosis. When the tumor grows to 2 to 3 weeks, the rabbit model is available for stent implantation.125I seeds, due to the current lack of animal models of malignant airway stenosis  of VX2 tissue suspension. Future studies are needed to observe the relationship between stenosis types and the amount of VX2 suspension.In conclusion, the implanting method described above is safe and effective for the establishment of a rabbit model of malignant airway stenosis to mimic the progression of this human disease. When the tumor grows at 2 to 3 weeks, the rabbit model is available for stent implantation. We recommend the model for more preclinical animal studies on bronchoscopic interventional treatments.The original contributions presented in the study are included in the article/The animal study was reviewed and approved by Laboratory Animal Center of Wenzhou Medical University (ID: wydw2021-0289).XL was a major contributor in writing the manuscript. CC and YL designed the study. MY and XJ analyzed the data. LZ and WZ performed the study. All authors contributed to the manuscript revision and read and approved the submitted version.The study was supported by grants from the Key Laboratory of Interventional Pulmonology of Zhejiang Province (2019E10014) and the Zhejiang Provincial Key Research and Development Program (2020C03067).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Myeloid sarcomas (MS) are rare extramedullary (EM) hematological tumors that generally arise during the natural course of acute myeloid leukemia (AML), occurring concomitantly with the onset of systemic leukemia; it can also occur following onset but rarely before. Common sites of EM involvement include the lymph nodes, skin, soft tissue, bone and peritoneum. Herein, we report the case of a 63-year-old man who presented EM AML upon initial diagnosis involving the bone marrow, lymph nodes and skin (leukemia cutis). A diagnosis was made based on immunohistochemistry (IHC). This case presents a diagnostic dilemma due to its atypical presentation and the sites involved. It also highlights the importance of IHC in the diagnosis of EM AML. The potential role of hypomethylating agents and Venetoclax in cases not eligible for hematopoietic stem cell transplant are also discussed. Hematological malignancies may manifest as extramedullary soft tissue masses that can occur at sites other than the bone marrow. Extramedullary (EM) manifestations of acute myelogenous leukemia (AML) are described in the literature as occurring in 2–9% of newly diagnosed AML ,2. MyeloA 63-year-old man was admitted to the emergency department after having suffered from night sweats and weight loss for 3 weeks. His previous medical history included lumbar disc pathology and unilateral leg pain; he was also a heavy smoker (55 pack-years). Upon admission, the patient was hemodynamically stable and afebrile, presenting ECOG 1. A general examination revealed pallor, bilateral enlarged lymph nodes  and palpable hepatomegaly. The cutaneous examination showed purple/reddish patches on the upper body. Laboratory tests showed pancytopenia with severe neutropenia and increased lactate dehydrogenase .A peripheral blood smear revealed the apparent presence of blasts. The patient was admitted with suspected hematological malignancy. Empirical antibiotic therapy with amoxicillin and clavulanic acid was administered along with folic acid. Viral serology and blood cultures were negative. A computed tomography scan (CT) confirmed hepatomegaly and multiple mediastinal, axillary, celiac, hepatic, inguinal and mesenteric lymphadenopathy with hepatomegaly but no splenomegaly A,B. A thBone marrow evaluation and excisional axillary adenopathy biopsy were performed. The flow cytometry was inconclusive, showing a lack of lineage specificity with CD11c+, CD33+, CD64+ (low), myeloperoxidase (MPO)+ (dubious) and HLA-DR. Bone marrow morphology showed massive infiltration but no clear conclusions due to the extensive immaturity and the aberrant cell morphology C. The paThe spectrum of AML clinical presentation is variable and highly dependent on the location or the organ involved with leukemia . Very raThe majority of extramedullary blastic tumors can be classified as pre-B, pre-T or MS using a panel of monoclonal antibodies including myeloid , together with B and T lineage markers. Staining for MPO was conventionally used to help differentiate lesions from lymphoma. MS is a rare entity, which may occur as the presenting feature before, or concurrent with, the onset of overt Leukemia, resulting in frequent misdiagnosis. The expression of blast cell CD56  has been associated with both MS and LC . The rarEM involvement in AML is a relatively rare but clinically significant occurrence that often causes therapeutic and diagnostic dilemmas for both hematologists and pathologists. A history of AML will point towards this as a diagnosis, but prior knowledge of the condition as well as a high degree of suspicion are essential. Our case demonstrates how challenging the diagnosis of AML may be when presenting with atypical extramedullary disease and highlights how important it is to obtain an early and accurate diagnosis. To the best of our knowledge, there are no case reports in the literature combining EM AML with concurrent bone marrow, lymph node and skin involvement, thus establishing it as a rare entity with poor prognosis. The current therapeutic recommendations are based on extrapolation from medullary AML but further studies targeting EM AML are warranted.


Panicum virgatum L.), a perennial C4 biofuel crop, is considered an ideal plant for phytoremediation due to its high efficiency in removing Cd and other heavy metals from contaminated soil. The key to understanding the mechanisms of switchgrass Cd tolerance is to identify the genes involved in Cd transport. Heavy-metal ATPases (HMAs) play pivotal roles in heavy metal transport, including Cd, in Arabidopsis thaliana and Oryza sativa, but little is known about the functions of their orthologs in switchgrass. Therefore, we identified 22 HMAs in switchgrass, which were distributed on 12 chromosomes and divided into 4 groups using a phylogenetic analysis. Then, we focused on PvHMA2.1, which is one of the orthologs of the rice Cd transporter OsHMA2. We found that PvHMA2.1 was widely expressed in roots, internodes, leaves, spikelets, and inflorescences, and was significantly induced in the shoots of switchgrass under Cd treatment. Moreover, PvHMA2.1 was found to have seven transmembrane domains and localized at the cell plasma membrane, indicating that it is a potential transporter. The ectopic expression of PvHMA2.1 alleviated the reduction in primary root length and the loss of fresh weight of Arabidopsis seedlings under Cd treatment, suggesting that PvHMA2.1 enhanced Cd tolerance in Arabidopsis. The higher levels of relative water content and chlorophyll content of the transgenic lines under Cd treatment reflected that PvHMA2.1 maintained water retention capacity and alleviated photosynthesis inhibition under Cd stress in Arabidopsis. The roots of the PvHMA2.1 ectopically expressed lines accumulated less Cd compared to the WT, while no significant differences were found in the Cd contents of the shoots between the transgenic lines and the WT under Cd treatment, suggesting that PvHMA2.1 reduced Cd absorption from the environment through the roots in Arabidopsis. Taken together, our results showed that PvHMA2.1 enhanced Cd tolerance in Arabidopsis, providing a promising target that could be engineered in switchgrass to repair Cd-contaminated soil.Cadmium (Cd) in soil inhibits plant growth and development and even harms human health through food chain transmission. Switchgrass ( Cadmium (Cd) pollution in soil severely inhibits plant growth and development. Cd is absorbed via plant root cells and transferred to stems and leaves, which causes chlorophyll decomposition and leaf yellowing, resulting in plant growth restriction and even death. Cd-contaminated plants go into the food chain and are consumed by herbivorous animals and even human, leading to serious health problems ,2,3. Man1B-type ATPase subfamilies, which function as transporters to hydrolyze ATP for the transport of metal ions [Arabidopsis thaliana named as AtHMA1-AtHMA8 [Oryza sativa named as OsHMA1-OsHMA9 [Arabidopsis HMA members are functionally characterized as participating in heavy metal ion transport and translocation. AtHMA1-AtHMA4 are classified into the zinc/cadmium/cobalt/lead group, while AtHMA5-AtHMA8 are categorized into the copper/silver group [Arabidopsis AtHMA3, overexpression of OsHMA3 in rice enhances Cd tolerance, which is mainly attributed to the sequestration of Cd into root vacuoles, thus preventing Cd from moving to the shoots [OsHMA3 also promotes the expression of ZIP genes, which are involved in zinc transport, and increases the absorption of zinc [Arabidopsis AtHMA5 and rice OsHMA5 both promote the root-to-shoot copper translocation and improve growth and development [HMAs are members of the Ptal ions . Various1-AtHMA8  and nine1-OsHMA9 . HMAs ar1-OsHMA9 . Arabidoer group . AtHMA1 er group . AtHMA3 er group . AtHMA3 er group . AtHMA2 er group ,17,18. Aer group ,20, whiler group ,22. OsHMer group , and OsHer group . Similare shoots . Overexp of zinc ,26. OsHM of zinc . Arabidoelopment . OsHMA6 elopment ,29. Panicum virgatum L.) HMAs in the plant’s response to Cd stress is less clear at present. Switchgrass is an important perennial C4 bioenergy grass with a high biomass, strong resistance, wide adaptability [HMA3 was upregulated in switchgrass inoculated with plant-growth-promoting bacteria (PGPB) under Cd stress [HMA genes involved in the plant’s response to Cd stress is significant as it contributes to understanding the mechanisms of Cd transport and provides a potential way for the remediation of Cd-contaminated soil.HMAs play important roles in the response to Cd stress in distinct species, but the function of switchgrass  and Pavir.4NG313000.1 (PvHMA2.2) as the orthologs of rice OsHMA2, which amino acid sequences, transmembrane domains, and gene expression patterns in response to Cd treatment were highly similar. In this study, we found that PvHMA2.1 is a plasma membrane-localized protein. The ectopic expression of PvHMA2.1 mitigated the inhibitions in primary root length, fresh weight of both shoot and root, and chlorophyll content, and maintained a high level of relative water content in Arabidopsis under Cd stress. Lower Cd contents in the roots of the transgenic lines indicated that the ectopic expression of PvHMA2.1 reduced Cd absorption by roots and might promote root-to-shoot Cd translocation, leading to an alleviation of Cd toxicity in Arabidopsis. This study is the first to identify the function of PvHMA2.1 in Arabidopsis, highlighting a novel regulation mechanism of switchgrass HMAs in response to Cd stress.In this study, we identified 22 HMAs from switchgrass and conducted a phylogenetic analysis with 8 Arabidopsis HMAs (AtHMA1-AtHMA8) were retrieved from TAIR. Using a local BLASTP process, we identified 25 potential HMA members in switchgrass. Three of the HMA members were excluded because they are short in length, being only about a quarter or one-fifth of the other potential PvHMAs. To reveal the evolutionary relationship of the HMA family of switchgrass, Arabidopsis, and rice, the protein sequences of an additional nine rice HMAs (OsHMA1-OsHMA9) were retrieved from the Rice Genome Annotation Project. A neighbor-joining tree was conducted using the MEGA 11, based on the 39 protein sequences aligned using the online MAFFT. All HMA proteins were clustered into four groups   . AtHMA1 HMAs are distributed on 12 chromosomes, and 1-3 of the HMA genes are unevenly located on each chromosome , and Pavir.4KG357400 are located on Chr04K, while Pavir.4NG281800, Pavir.4NG313000 (PvHMA2.2), and Pavir.4NG274400 are located on the corresponding parts of Chr04N. Each two genes are present on chromosome Chr01 , Chr02 , and Chr07 , and only one gene is present on Chr06  and Chr09 . The HMA genes exist in pair on ChrK and ChrN in terms of location, together with their evolutionary relationship  and Pavir.4NG313000 (PvHMA2.2) were cloned to study their responses to Cd stress.The phylogenetic analysis showed a similar classification of the 22 PvHMA proteins compared to the general classification in and rice B. The coand rice . These rPvHMA2.1 and PvHMA2.2 are 3306 bp and 3309 bp, respectively, and each of them encodes 1101 and 1102 amino acid residues, respectively, with a similarity of 92.70%  lengths of f 92.70% . Next, tf 92.70% A,B. TherPvHMA2.1 and PvHMA2.2 in P. virgatum Alamo. PvHMA2.1 is expressed in various tissues, such as roots, internodes, leaves, spikelets, and inflorescences  treatment of 50 µM of Cd, the transcript of PvHMA2.1 obviously increased in shoots, and the significant increase happened quickly after a 1-day treatment and reached a peak level after a 3-day treatment -2, -3, -5, -6, -10, -13, and -19, were obtained from the T1-generation plants of PvHMA2.1 ectopically expressed Arabidopsis  or with 50 µM of Cd for phenotyping. After 8-day cultivation, there was no obvious difference in seedling growth between the WT and the PvHMA2.1 ectopically expressed lines under the control condition. Under 50 µM of Cd treatment, the root growth of the WT was extremely inhibited, but that of the PvHMA2.1 ectopically expressed lines was less restricted  were chosen to study PvHMA2.1 effect under Cd treatment A. Specifstricted B. Moreovreatment B. To furectively C. These Arabidopsis seedlings under Cd treatment, we measured the fresh weights of seedling roots and shoots. There were no significant differences in the fresh weights of the roots or the shoots between the WT and the PvHMA2.1 ectopically expressed lines under the control condition. Under the Cd treatment, the fresh weights of both the roots and the shoots of three PvHMA2.1 ectopically expressed lines were higher than those of the WT, especially the fresh weights of the OM5-2 and OM19-13 lines were significantly higher compared to the WT  had similar relative water contents under the control condition, while the relative water contents of the three transgenic lines were significantly higher than those of the WT during the exposure to Cd D, indica1B-type ATPase members, have been proven to play pivotal roles in Cd detoxification in many plants [Arabidopsis.Cd is an unessential trace element for plants, which is inadvertently taken up through the absorption pathways of some essential divalent metal ions and affects many aspects of plant physiology and biochemistry . The dety plants ,38,39. HArabidopsis, rice, and switchgrass, respectively) were classified into four groups according to the phylogenetic analysis  were downloaded from TAIR . Using them as the queries, a local BLASTP against the annotated protein sequences of switchgrass genome of P. virgatum v5.1 from phytozome 13  was conducted, with an e-value cutoff of 0.05 and identity of 50%. In total, 25 potential PvHMAs were found in switchgrass, while 3  out of 25 candidate PvHMAs were excluded from further analyses due to their shortness in sequence length.The protein sequences of eight Arabidopsis HMAs, and 9 rice HMAs (OsHMA1-OsHMA9), which were retrieved from the Rice Genome Annotation Project , using MAFFT  with default parameters, and a phylogenetic tree was constructed using the MEGA 11, using the neighbor-joining method with 1000 bootstrap replicates. The chromosomal location of the 22 PvHMA genes was visualized using Gene Location Visualize from the GTF/GFF module of TBtools [https://meme-suite.org/meme/tools/meme, accessed on 9 November 2022) with 10 motifs and the Conserved Domain Search from NCBI  with the initial parameters, respectively. The TBtools was used to visualize the conserved motifs and domains [https://services.healthtech.dtu.dk/service.php?TMHMM-2.0, accessed on 10 November 2022) with the initial parameters, respectively. The sequence numbers of HMAs in P. virgatum, A. thaliana, and O. sativa are listed in the To further investigate the evolutionary relationship of the remaining 22 PvHMAs, multiple sequence alignment was performed with the protein sequences of the 22 switchgrass HMAs, 8  TBtools . The con domains . Amino aP. virgatum cultivar Alamo was used in this study. Switchgrass wild-type plants were cultivated in a greenhouse with natural light and night-day temperature ranging from 25–35 °C. Roots, internodes, leaves, and spikelets at reproductive stage 1 (R1), and inflorescences at R3 were harvested for RNA extraction and determination of tissue-specific expression patterns of PvHMA2.1 and PvHMA2.2 [PvHMA2.2 .2 (0 µM CdCl2 as the control condition) in a growth chamber under 25 °C (16 h light/8 h dark cycles). During 7-day time-course  Cd treatment, the seedlings of switchgrass were quickly rinsed with deionized water for three times, absorbed surface moisture with filter papers, placed in centrifuge tubes, frozen in liquid nitrogen, and stored at −80 °C for RNA extraction.Surface-sterilized seeds of wild-type switchgrass were placed in the dark for 3 days at 4 °C, and then transferred to the wet filter paper to germinate and grow for 7 days. The seedlings were transferred to 1/4 Hoagland’s nutrient solution for another 7 days, and then treated under 50 µM CdClPvUbiquitin (GenBank: FL955474.1) and AtActin2 (AT3G18780) were used as the references in switchgrass and Arabidopsis, respectively. The primers for qPCR are listed in −ΔΔCT [Total RNA was extracted using the Quick General Plant RNA Extraction Kit  and then reverse-transcribed to cDNA using the PrimeScript™ RT reagent Kit . Real-time fluorescent quantitative PCR (qPCR) was performed using the TB Green™ Premix Ex Taq™ II Kit . in −ΔΔCT .PvHMA2.1 CDS was amplified using PCR, which was cloned into the vector of pMDC83-GFP driven by the 35S promoter, using the specific primers listed in 35S:PvHMA2.1-GFP, the control of 35S:GFP, p19, the cell plasma membrane marker CBL-RFP, and the nucleus marker Athook-RFP were introduced into agrobacterium strain GV2260, respectively. The mixtures of 35S:PvHMA2.1-GFP, p19, and CBL-RFP/Athook-RFP were transiently expressed in the leaves of 3-week-old N. benthamiana, and the mixtures of 35S:GFP, p19 and CBL-RFP/Athook-RFP were used as the controls. The subcellular localization of PvHMA2.1 was visualized using a confocal microscope after 2–3 days of infiltration.PvHMA2.1 CDS was cloned into the plant binary vector pUbi1301 [BamHI and KpnI, using the specific primers listed in pUbi1301-PvHMA2.1 was transformed into agrobacterium strain GV3101, which was subsequently introduced to wild-type Arabidopsis Columbia by the floral-dipping method [Arabidopsis transgenic plants was performed on a ½ MS medium with 50 mg/L of hygromycin and 100 mg/L of ampicillin and further confirmed using PCR and qPCR. Three independent transgenic lines with elevated expression levels of PvHMA2.1 were used for the investigation of PvHMA2.1 in response to Cd stress.pUbi1301  via the g method ,60. The Arabidopsis T4 transgenic lines  and the WT seeds were sterilized with 6% NaClO for 10–15 min and placed in the dark for 3 days at 4 °C. The surface-sterilized seeds were grown on a 1/2 MS medium for 3 days and transferred to a 1/2 MS medium without Cd or with 50 µM of CdCl2 for another 8 days. The primary root lengths of all seedlings were measured individually using IMAGEJ 1.52 after the photographs were taken. The shoots and roots of the seedlings were sampled, with 10 plants in each replicate, and their fresh weights were measured after removing surface moisture by filter papers. Four samples were repeated. The Arabidopsis seedlings, with 10 plants in each replicate, were measured according to the methods mentioned in [Arabidopsis seedlings were tested as described previously [To quantify the effect of Cd toxicity, the relative conductivity and the malondialdehyde content of the ioned in . The proeviously . Four biArabidopsis seedlings were grown on a 1/2 MS medium without Cd or with 50 µM of CdCl2 for 8 days. The shoots and roots of the Arabidopsis seedlings, with 10 plants in each replicate, were rinsed by deionized water three times, and the fresh weights of the shoots and roots were recorded. Next, the fresh samples were dehydrated for 6 h at 80 °C and digested in HNO3 [ in HNO3 . Cd contp < 0.05, p < 0.01, and p < 0.001, respectively.Statistical analyses were performed using Excel and GraphPad Prism 9. The values are represented as mean ± SD. Significance of differences was determined using Student’s t-test, as indicated in figures. One, two, and three asterisks indicate PvHMA2.1 in Arabidopsis reduces Cd absorption in roots and may promote Cd transport from roots to shoots, thereby enhancing Cd tolerance in Arabidopsis. At the physiological level, the enhancement of Cd tolerance in the PvHMA2.1 ectopically expressed lines is due to the maintenance of water holding capacity and the photosynthesis stability of Arabidopsis seedlings by PvHMA2.1 under Cd stress. PvHMA2.1 enhances Cd tolerance and may promote Cd transport from roots to shoots in Arabidopsis, while its function in switchgrass is still unknown. In future research, we will continue to investigate the function of PvHMA2.1 in switchgrass, which may be of great significance for the use of switchgrass as a resource to repair Cd-contaminated soil.Taken together, the ectopic expression of


Ising machines based on analog systems have the potential to accelerate the solution of ubiquitous combinatorial optimization problems. Although some artificial spins to support large-scale Ising machines have been reported, e.g., superconducting qubits in quantum annealers and short optical pulses in coherent Ising machines, the spin stability is fragile due to the ultra-low equivalent temperature or optical phase sensitivity. In this paper, we propose to use short microwave pulses generated from an optoelectronic parametric oscillator as the spins to implement a large-scale Ising machine with high stability. The proposed machine supports 25,600 spins and can operate continuously and stably for hours. Moreover, the proposed Ising machine is highly compatible with high-speed electronic devices for programmability, paving a low-cost, accurate, and easy-to-implement way toward solving real-world optimization problems. Many combinatorial optimization problems are classified as the non-deterministic polynomial-time (NP)-hard or NP-complete complexity classes; these are difficult to be solved on standard digital computers because the number of combinations grows exponentially or factorially as the problem size N increases. Although some software algorithms, such as simulated annealing and other approximate algorithms9, have been developed to accelerate the computation, using a Von Neumann architecture to solve these problems remains time-consuming due to the step-by-step computation mode and limited operating frequency10. Interestingly, the solution to these problems can be effectively accelerated by mapping them onto an analog Ising machine. The Hamiltonian of an N-spin Ising machine without an external field is given by Ji,j is the spin interaction between the i-th and j-th spins, and σi and σj respectively denote the z projection of the spins with eigenvalues of either 1 or −1. Finding the optimal answer is equivalent to searching the ground state of an Ising machine11, and computational acceleration is achieved by utilizing the intrinsic convergence property of the system, as convergence to the ground state often occurs at high speed.Combinatorial optimization problems can be found everywhere in modern society, such as drug discovery5. Many Ising machines, e.g., those based on trapped ions13, superconducting circuits17, molecules19, optical33, optoelectronic34, and electrical systems38, have been reported. The optimization principle of most types of these Ising machines is based on the idea of minimum power dissipation39. Among them, coherent Ising machines (CIMs) using degenerate optical parametric oscillators (DOPOs) have drawn a substantial amount of attention due to their attractive advantages, such as large scale23 and flexible connectivity33. It has also been reported that DOPO-based Ising machines are superior to quantum annealers32 and digital computers33 in some specific optimization problems. However, as the artificial spins are represented by the phases of the short optical pulses and stored in a long fiber cavity, the stability is weak because the optical phase is very sensitive to the cavity delay variation24. According to Large-scale, stable, and programable Ising machines are highly desired to solve real-world optimization problems40. In the proposed CIM, the artificial Ising spins are represented by the phases of the short microwave pulses generated in an optoelectronic cavity. The number of generated spins is as large as 25,600, and the machine stably operates over 12 h. One-dimensional (1D) and two-dimensional (2D) Ising model simulations were conducted on the proposed CIM, and the experimental results suggest the machine has promising potential in finding the low-energy state. In addition, the machine was used to solve max-cut problems, which are well-known NP-hard problems, and a high success rate of finding the maximum cut was achieved.Here, we propose a large-scale, stable Ising machine based on an optoelectronic parametric oscillator (OEPO)40. In the proposed machine, the oscillations in the OEPO cavity are discrete pulses with half the frequency of a local oscillation and locked with a relative 0 or π phase via an electric mixer . The function of the electric mixer is similar to that of the nonlinear crystal in the DOPO. The binary-phase oscillation can then be used to simulate an artificial Ising spin, e.g., the 0-phase/π-phase state represents up/down spin.Figure 22 or a measurement-feedback circuit33. The delay-line scheme, implemented by a wavelength-division multiplexing (WDM) system and tunable optical delay lines (ODLs), is adopted in our demonstration. The oscillating microwave signal is distributed to different optical channels using the WDM as a beam splitter. Each channel propagates through a specific delay line (with an n-bit delay). These channels are combined using another WDM as a beam adder and launched into the photodetector (PD); thus, the i-th spin and the (i + n)-th spin interact. In this scheme, the coupling strength is controlled by the corresponding channel’s laser power, and the coupling sign can be reversed by tuning the optical delay line.With the help of time-division multiplexing, large-scale, discrete microwave pulse oscillations can be obtained in a single long fiber cavity. In such an oscillation network, the spin-spin interaction can be implemented by delay linesJ, would change the global loss of the OEPO network and result in the corresponding phase configuration . Phase configuration with minimum loss has the maximum possibility to be selected and oscillate stably20. The OEPO network can operate around the minimum-loss state and outputs the corresponding microwave signal whose phase configuration corresponds to the optimal solution of the given Ising problem.A specific spin-spin interaction, defined by matrix J is a unit matrix) artificial spin network. The results are exhibited in Fig. 9 to 106, the artificial spins in the OEPO are much less sensitive to temperature fluctuation and ambient vibration, yielding much higher stability compared to the DOPO counterpart.The large scale and high stability of the proposed system were verified in a non-interaction  from the (i-1)-th to the i-th spin for i ≤ 25,600 and from the 25,600-th to the first spin.To implement a 1D Ising simulation, a second channel with an additional 1-bit delay was used to interact with the neighboring spins. The formed 1D Ising model was a closed loop, and the coupling was unidirectional . Due to the additional constraints imposed by the vertical coupling, the machine took much less time to evolve from the noise state to the low-energy state compared to the 1D Ising simulation, as shown in Fig. Gs = {1.01, 1.05, 1.1} are shown in Fig. As with the 1D simulation, the possibility and the time taken to reach lower energy were highly related to the cavity gain. To reveal the evolution process, some snapshots at specific roundtrips at different small-signal gains 42. A similar freeze-out effect was also observed for the proposed Ising machine, which prevented the machine from reaching the ground state 43. Fortunately, the error detection and the feedback control technique used in the DOPO-based Ising machine can also be established in our scheme to improve the machine performance45. Depending on the comparison between the current energy and the previously visited, one can increase or decrease the cavity gain or the mutual injection strength to help the machine escape from the local energy minima and find a better one. The small signal gain is determined by the power of the pump laser in the OEPO, which can be precisely and independently tuned by an additional intensity modulator.Another possible reason is that weaker spin-amplitude nonuniformity may be obtained in the OEPO-based machine, leading to a lower Ising energy since it damages the machine performanceIt should also be noted that the lower Ising energy is obtained at the expense of a slower repetition rate and a longer feedback loop as compared with those of DOPO-based CIMs, which means that a longer time is required to implement a single roundtrip. Fortunately, fewer roundtrips are required to achieve the same low Ising energy in the proposed Ising machine. Moreover, it is worthwhile to obtain lower Ising energy at the expense of a predictable time when solving certain combinatorial optimization problems.27, the spatial multiplexing of the spins on integrated chips26, and analog electronic systems38. Detailed comparisons between different Ising machines are available in section 9 in Supplementary Materials.Some platforms have also been proposed to implement Ising machines that can avoid the stability issue of the fiber-based feedback loop of DOPO-based CIMs, e.g., via recurrent feedback using free-space spatial light modulators (SLMs)24. Taking advantage of its arbitrary programmability, the multi-body interaction and the nonlinear interaction can also be realized. Moreover, recent booming development in photonics matrix multiplication may provide a new way for arbitrary spin interaction46. Overall, the microwave photonic Ising machine is expected to be a large-scale, highly-stable, and programable optimizer, thus paving the way for solving real-world combinatorial optimization problems.Currently, the proposed machine still suffers from connectivity issues. Fortunately, flexible programmability can be easily realized using measurement-feedback schemes, as the microwave spin can be easily measured and controlled with high-speed electrical devicesπ phase. When the oscillation is degenerate, the transmission function of the mixer can be described as:β is the frequency conversion coefficient from the input port, and γ is the leakage coefficient of the radio frequency (RF) signal . Assuming the slow-varying envelope applied to the second MZM in Fig. τ can be given as follows47:t is the slow time scale of the order of the cavity delay (~100 μs), GEA is the power gain of the electrical amplifier (EA), RPD is the responsivity of the PD, IPD is the power launched into the PD, ZPD is the PD impedance, J1[·] is the first-order Bessel function of the first kind, and Vπ is the half-wave voltage. Near the steady state, s(t) varies on a slow time scale. The following approximation can be used: s0 | , ϕ0) of Eq.  = 0:The mixer is the key device required to lock the phase of the oscillating signal with 0 or s0 | satisfy Eq. \documentN and Γ can be equivalent to the Ising Hamiltonian. Equation  or fall  by |Ji,j | . Since all the couplings from other spins have an integrated contribution i-th spin is given by J, the interactions between the spins are firstly programmed into the oscillation network, either in the analog or in the digital domain. And then, the cavity gain is increased gradually to search for the minimum-loss state. Meanwhile, the amplitudes of spins increase, and their phases are adjusted towards the lowest configuration. When the oscillation network runs stably, one can measure the corresponding phase configuration, from which the best answer to the given problems can be solved.As one can find that the second term in Eq.  has the Equation  conformsd as Eq. . To solv49. Fortunately, limiting the amplitude of spins at the feedback and optimizing the nonlinear function could reduce the amplitude inhomogeneity to improve the success probability44. Details about how the nonuniformity impedes the machine from finding the best answer and how to relieve such negative impact are under investigation.However, the assumption that all the spins have the same amplitude is not always satisfied. When solving complex problems, the spins experience different interactions along the optoelectronic cavity, which would change the spin gain and ultimately results in amplitude non-uniformity. This non-uniformity may cause inconsistency between the minimal-loss configuration and the optimal solution to the corresponding problem. The negative impact of nonuniformity also can be found in the DOPO-based Ising machineSupplementary MaterialsA typical frezee-out evolutionThe construction of max-cut solvers


Additionally, we compared the Ca2+ activation properties of isolated slow- and fast-twitch myofibers from 3-month-old WT and BLAJ male mice. Differences were observed for some Ca2+ handling and glucose/glycogen metabolism-related protein levels between BLAJ soleus and EDL muscles (compared with WT) that may contribute to the previously reported differences in function in these BLAJ muscles. Dysferlin deficiency did not impact glycogen content of whole muscles nor Ca2+ activation of the myofilaments, although soleus muscle from 10-month-old BLAJ mice had more glycogen than EDL muscles. These results demonstrate a further impact of dysferlin deficiency on proteins associated with excitation-contraction coupling and glycogen metabolism in skeletal muscles, potentially contributing to altered contractile function in dysferlinopathy.Dysferlinopathies are a clinically heterogeneous group of muscular dystrophies caused by a genetic deficiency of the membrane-associated protein dysferlin, which usually manifest post-growth in young adults. The disease is characterized by progressive skeletal muscle wasting in the limb-girdle and limbs, inflammation, accumulation of lipid droplets in slow-twitch myofibers and, in later stages, replacement of muscles by adipose tissue. Previously we reported myofiber-type specific differences in muscle contractile function of 10-month-old dysferlin-deficient BLAJ mice that could not be fully accounted for by altered myofiber-type composition. In order to further investigate these findings, we examined the impact of dysferlin deficiency on the abundance of calcium (Ca Contrary to this, however, we reported slowed relaxation in the BLAJ EDL ) > 8 to 4.5, prepared by mixing varying ratios of ‘relaxing solution’ and ‘maximal Ca2+-activating solution’. The relaxing solution was composed (in mM) of 126 K+, 36 Na+, 10.3 Mg2+, 50 EGTA, 90 HEPES, 8 ATP, 10 creatine phosphate, and 1 NaN3, with a pH of 7.10 ± 0.01. The maximal Ca2+-activating solution was comprised (in mM) similarly, but with 47.5 Ca2+ and 8.1 Mg2+. The solutions were prepared according to established methods [2+ concentrations were calculated using a previously determined apparent affinity constant for EGTA of 4.78 × 106 [The Ca methods . Free Ca78 × 106 .2+ solutions, to identify each myofiber as either slow- (type 1) or fast-twitch (type 2), myofibers were exposed to two strontium (Sr2+)-EGTA-buffered solutions of known Sr2+ concentration, prepared by mixing different proportions of maximal Sr2+-activating solution with the relaxing solution A to achieve a power of Sr2+ of 5.5 (‘low’) and 4.5 (‘high’). Maximal Sr2+-activating solution was composed similarly to maximal Ca2+-activating solution but with 40 mM Sr2+ instead of Ca2+. Myofiber-type classification was determined by the ratio of the force responses to ‘low’ and ‘high’ pSr solutions, where slow-twitch myofibers (which are more sensitive to Sr2+ than fast-twitch myofibers) had a much higher ratio (~75–85%) compared with fast (~0–5%) [Following exposure to the Ca (~0–5%) .2) was calculated as maximum Ca2+-activated force (mN) normalized to myofiber CSA (mm2) estimated from myofiber diameter measurements taken from a minimum of three sites along the myofiber. Myofiber CSA was assumed to be ellipsoidal and was approximated by the formula: CSA = (π × a × b)/4, where a is the major axis of the ellipse (taken as the largest diameter measurement), and b is the minor axis of the ellipse .Specific force , and the Ca2+-sensitivity properties: Hillslope coefficient , and pCaX (the amount of Ca2+ needed to produce ‘X’ amount of force) were derived.Cat-tests, or non-parametric Mann-Whitney U tests where appropriate. Post hoc comparisons with Holm-Bonferroni corrections were conducted for each statistical test where appropriate, with statistical significance taken as p < 0.05. Statistical analyses were performed using Jamovi (Version 1.6).All data are presented as mean ± standard error of mean (SEM). Data were examined using separate one-way ANOVAs, two-way ANOVAs, Student’s 2+ handling and metabolism in the context of muscle contraction is of interest. Moreover, our findings related to altered EC coupling and glucose metabolism (as a fuel to provide energy for contraction), strongly indicate distinct intrinsic myofiber-type specific effects of dysferlin deficiency with important implications for a more precise understanding of the molecular basis of the disease. The marked myofiber-type differences between BLAJ slow soleus and fast EDL muscles, further emphasize the potential importance of myofiber-type composition in different muscles for the manifestation of dysferlinopathy.This study, together with the results of Lloyd et al.  showing


It has been suggestive of the Ca2+ signaling-dependent cellular processes to be tightly regulated by the numerous types of Ca2+ channels, pumps, exchangers and sensing receptors. Consequently, dysregulated Ca2+ homeostasis leads to a series of events connected to elevated malignant phenotypes including uncontrolled proliferation, migration, invasion and metastasis, all of which are frequently observed in advanced stage lung cancer cells. The incidence of bone metastasis in patients with advanced stage lung cancer is estimated in a range of 30% to 40%, bringing about a significant negative impact on both morbidity and survival. This review dissects and summarizes the important roles of Ca2+ signaling transduction in contributing to lung cancer progression, and address the question: if and how Ca2+ signaling might have been engaged in metastatic lung cancer with bone metastasis, thereby potentially providing the multifaceted and promising solutions for therapeutic intervention.Intracellular Ca In unexcited states, (Ca2+)i ions are maintained at a very low level (in a range of 50–150 nM) nonetheless, a transient elevation of (Ca2+)i, obtained through either (Ca2+)i efflux from intracellular organelles into cytosol or through (Ca2+)i influx into cytosol from extracellular milieu, could mediate activation of various downstream signaling cascades  levels  pathway keeps a major role in regulating a variety of cellular processes such as proliferation, differentiation and surivial. The abnormal expression of MAPKs is frequently observed in NSCLC -4-phenylpiperazine) (KN-62), a specific CaMKII antagonist, Williams, et al.  as well as cell-cell attachment. Cells are connected to the ECM at focal adhesion points by structural complexes linking membrane spanning integrins to the cytoskeleton. Therefore, migratory capacity of cancer cells are principally assessed by the rate of focal adhesion assembly and disassembly. Noticeably, Carequired . The atte front) , 86. Fole front) , which pe front) . Further calpain , and reg calpain , 91, and lamella . For retchannels . Ca2+-deactility , promotiactility , 96.2+ pulses” in the front of migrating cells are released from ER via IP3-induced activation of IP3Rs [2+ release by the axis of RTK-PLC-IP3R would be major source of Ca2+ pulses in the front of migrating cells. Indeed, EGFR, also known as HER1, belonging to the ErbB family of structurally related RTKs that comprises four isoforms: ErbB2 (HER2), ErbB3 (HER3) and ErbB4/HER4 [2+ pulses in the cell front.It is well-characterized that “local Caof IP3Rs , which ibB4/HER4 , is overbB4/HER4 . It is cbB4/HER4 ; furtherbB4/HER4  reported2+ pulses in the cell front via activating PLC, which hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIP2) to release IP3, which binds to IP3R to trigger transient Ca2+ release [2+ sensitizes SOC channels located at PM to infux (Ca2+)e across PM [2+-activated potassium (KCa) channel family, regulated the constitutive (Ca2+)e entry through Orai1 localization within the lipid raft, which affected the migratory ability of breast cancer cells. Disruption of interaction between SK3 and Orai1 from lipid rafts weakened SK3-mediated Ca2+ entry, migration and bone metastasis [2+)i level. STIM1, the ER Ca2+ sensor, and Orai3, constituent a native SOC entry essential for NSCLC progression [2+ pulses in the front of migrating cells [2+ pulses near its leading edge cause depletion of Ca2+ in the front ER, resulting in activation of STIM1 at the cell front [In addition to RTK, G-protein coupled receptors (GPCRs) on local Ca release , 103. Incross PM . Chantômcross PM  once reptastasis . Moreovegression , 50. Incng cells . In migrll front . More spll front .2+ permeable channels, PMCA and NCX, by the specific blockers, which are vanadate (V5+) and KB-R7943, respectively, led to a decrease in migratory capacity of MDCK-F cells, suggesting these channels were of significant importance for cell migration [2+)i into the ER lumen, triggering a leak of the ER lumical Ca2+ into cytosol [2+)i concentration caused MLCK saturation and myosin contractility [2+, thereby disabling further Ca2+ signaling through IP3Rs, suggesting a physiological importance of SERCA on lung cancer progression, invasion and metastasis.In addition, inhibition of the activity of Caigration . Further cytosol . The higactility . Indeed,actility  once rep2+ signaling network preferentially towards bone metastasis is essential for development of the efficacious drug program.Bone is one of the most common metastatic sites for lung cancer, in which 36% of patients with bone lesions, and a range of 20%–60% with bone marrow micrometastasis , 110. Me3K and MAPK pathways. Nonetheless, the detailed mechanisms of how CXCR4 /CXCL12 interaction stimulates metastasis and/or tumor growth and their complete implications on metastatic lung cancer in bone are unknown.Bone is a dynamic organ included a variety of embryo-derived cells such as hematopoietic, stromal, endothelial, adipocytes, OCs, OBs and osteocytes . Two impBone homeostasis is maintained by two major types of bone cells, consisting of OBs and OCs, which are responsible for bone formation and bone resorption, respectively . Of thes2 deficiency) and high level of (Ca2+)e, enabling tumor cells to establish an acidic microenvironment via production of a large amount of lactic acid, which then creates the local areas inside bone, thereby accelerating tumor cell dormancy and promotes osteolysis [2+)e triggers activation of Ca2+-sensing receptor (CaR), a G protein-coupled receptor, on PM of tumor cells and OBs [2+-sensing receptors enhances the secretion of parathyroid hormone-related peptide (PTHrP), which subsequently binds to its receptor, PTHR1, to increase receptor activator of nuclear factor kappa-B (RANK) ligand (RANKL) expression in bone marrow stromal cells, thereby promoting osteolysis [3R-induced local Ca2+ release, inducing activation of one of the master transcription factors of osteoclastogenesis, the NFATc-1 [2+)e might have been responsible for activating the Ca2+ channels, pumps and exchangers to promote differentiation and growth of metastatic lung cancer cells (MLCCs) in bone.Bone has several particular characteristics such as acidified milieu, hypoxia  migration, (2) adhesion and invasion to bone, and (3) proliferation, growth and metastasis in bone. However, it is unclear whether aberrant changes in Ca2+ signals are one of the primary causes of initiation of lung cancer progression.In this article, I have reviewed the role of Ca2+-regulated processes? Accumulating preclinical and clinical evidence has elucidated the relationship between aberrant Ca2+ signaling and tumor progression. Using the specific blockers of Ca2+ channels, pumps and/or exchangers has demonstrated the significant antitumor effects on lung cancer progression (2+ signaling would be a promising target for novel lung cancer treatments. However, before contemplating such efficacious therapeutic interventions based on pharmacological modulation of Ca2+-regulators, it is crucial to design more potent and specific, but less off-target drugs targeting Ca2+-regulators, including Ca2+ channels, pumps and/or exchangers. Therefore, further studies are required to verify the toxicity and pharmacokinetic of such modulators prior to the clinical tests.To what extent can therapeutic strategies exploit these Cagression , indicat


In addition, dysregulation of Ca2+ can cause morphological and functional changes in MSNs and CPNs. Therefore, Ca2+ imaging techniques have the potential of visualizing changes in Ca2+ dynamics and neuronal activity in HD animal models. This minireview focuses on studies using diverse Ca2+ imaging techniques, including two-photon microscopy, fiber photometry, and miniscopes, in combination of Ca2+ indicators to monitor activity of neurons in HD models as the disease progresses. We then discuss the future applications of Ca2+ imaging to visualize disease mechanisms and alterations associated with HD, as well as studies showing how, as a proof-of-concept, Ca2+imaging using miniscopes in freely-behaving animals can help elucidate the differential role of direct and indirect pathway MSNs in HD symptoms.Huntington’s disease (HD) is a fatal, hereditary neurodegenerative disorder that causes chorea, cognitive deficits, and psychiatric symptoms. It is characterized by accumulation of mutant Htt protein, which primarily impacts striatal medium-sized spiny neurons (MSNs), as well as cortical pyramidal neurons (CPNs), causing synapse loss and eventually cell death. Perturbed Ca HD is ctin gene . While ttin gene .Htt gene, often displaying neuropathology that is highly consistent with human HD  receptor-mediated excitotoxicity  receptor 1, making it more sensitive to InsP3 upon its release when glutamate binds to its receptors. InsP3R1, when activated by InsP3, causes Ca2+ release from the endoplasmic reticulum , probably as a compensatory mechanism. SOCs are membrane-bound Ca2+-conducting channels that activate in response to depletion of Ca2+ stores . In the last section, we discuss future applications of Ca2+ imaging to visualize HD disease mechanisms, as well as some of our own studies showing how, as a proof-of-concept, Ca2+ imaging using miniscopes in freely-behaving animals can help elucidate the role of CPNs and direct and indirect pathway MSNs in HD symptoms. We apologize for any omissions of the already vast literature on Ca2+ imaging in HD.The focus of this minireview is not to describe in detail diverse Cavailable . Insteadvailable . ComparePast investigations into circuits and pathways involved in HD development have been limited by difficulty recording from large neuron populations and constraints in temporal and spatial resolution. While a leading tool for investigating neuronal dynamics has been electrophysiological recordings of neuron electrical activity in the form of action potential firing (both extracellularly and intracellularly) due to high temporal resolution, these methods can be limited in spatial resolution, specificity of neuron types, or are typically able to record from only a few neurons. This presents challenges in investigating altered circuits in HD due to the relatively isolated neuronal recordings.2+ imaging, fluorescent genetically encoded Ca2+ indicators (GECIs) bind to intracellular Ca2+ and emit fluorescent signals upon excitation, which can be acquired through the microscope. Since Ca2+ influx is associated with action potentials, it provides an indirect indication of neuronal firing activity. Cell type-specific expression of the Ca2+ indicator can be used to limit fluorescence to neurons of interest. In brain slices, 2-PLSM allows for recording from 50 to 100 cells simultaneously and extracting spike estimates from Ca2+ signals (2+ activity in HD and wildtype (WT) mice. Fernandez-Garcia et al., for example, used fluorescence imaging of striatal and cortical cell cultures from R6/1 and WT mice to examine Ca2+ dynamics. Notably, spontaneously active neurons and coordinated ensemble activity were reduced in striatal but not in cortical cultures, suggesting reduced ability to integrate excitatory inputs  has opened the door to neuron recordings in deeper tissue, using laser excitation methods that are able to reduce background fluorescence noise and discern individual neurons . Pioneer signals , openingy inputs . Howevercontrols . This re neurons .2+ buffering capacity may be associated with mitochondrial dysfunction. Indeed, mitochondrial Ca2+ levels also are dysregulated in HD. Using Ca2+ probes that do not permeate the mitochondrial membrane as indicators of extramitochondrial Ca2+, it is possible to indirectly monitor influxes of Ca2+ into the mitochondria, as well as total Ca2+ retained in the mitochondria upon opening of permeability transition pore (PTP) and Ca2+ release into the cytosol  and GECIs  can be made to specifically target organelles such as the mitochondria, directly providing insights into mitochondrial Ca2+ transport  lens is surgically implanted into the mouse brain expanding imaging to deep tissues inaccessible by traditional two-photon benchtop microscopes, and allowing use in freely-moving animals . Challen2+ indicators, whose fluorescence is then carried back through the fibers , and allows for recordings from multiple brain regions simultaneously, as well as in deep tissue , which has much lower spatial resolution and does not allow for visualization of specific neuronal interactions. It has also been observed that fiber photometry is more correlated with non-somatic Caactivity . Despiteysiology , allowin2+ indicators of different colors  as well as direct and indirect pathway MSNs (in D1- and A2A-Cre mice).Currently, our lab is using the UCLA miniscope (v3) to record Cag 2-PLSM , we foun6/2 mice . However2+ imaging regarding low spatial resolution and limitations on behavior and movement. The remaining limitations with miniscopes, but also present with other imaging techniques, include general obstacles with Ca2+ imaging regarding the duration of the transient signal. Ca2+ imaging is in general slower to respond than voltage-based imaging due to delay in binding of Ca2+ indicators. Also, due to slow release of Ca2+ indicators, cell fluorescence can remain after the firing of action potentials. The extended decay time (in the order of seconds) of the signal limits visualization/deconvolution on the duration of Ca2+ responses, and only reliably represents the beginning of the signal. For some proposed mechanisms for disease pathology of HD  miniature 2-photon microscopes are being developed (Miniscopes represent an improvement on challenges faced by other forms of Cagy of HD  that sugeveloped , 2022, aThough still in development, these systems enable monitoring with two-photon imaging over weeks, with minimal restrictions on behavior. However, similar to other methods using implanted lenses, the GRIN lenses used in miniscope imaging can cause some brain tissue displacement or damage.2+ activity from multiple neuron populations (Future research with miniscopes is focused on imaging more cell types in a circuit, potentially using multiple GECIs with wavelengths that can be distinguished, thus visualizing Caulations . Additioulations  expand tulations . Behavioulations . Currentulations  also con2+ imaging modalities and their applications to investigate the development and disease progression of HD in mouse models. These methods have been used to compare alterations in Ca2+ dynamics, which are likely key in HD manifestation. Current advances in Ca2+ imaging will make it possible to use Ca2+ imaging in conjunction with other methods to corroborate and formulate hypotheses about specific neuronal networks, dynamic alterations, and circuits involved with HD. For example, use of miniscopes in different Cre mouse lines may be able to elucidate the potentially differential role of striatal direct and indirect pathway neurons during the onset of motor symptoms, as well as the role of different types of interneurons in striatum and cerebral cortex.This minireview has presented several different CaAll authors wrote the first draft of the manuscript, worked on revision, read, and approved the submitted version of the manuscript.


The renal angiomyolipoma (AML) is a benign tumor characteristically composed of fat, smooth muscle tissue, and vessels. We collected AMLs from our nephrectomy database, reclassified them according to their histological appearance, recorded the demographic, clinical, and pathological parameters, and compared them with oncocytoma (RO) and renal cell carcinoma (RCC). Immunohistochemistry was ordered in 41 cases. In 2224 nephrectomies, we found 52 AMLs with a 53 mm median size. The mean age was 52.76. Forty-eight tumors were sporadic, while four were hereditary. The revision resulted in 31 classic, 13 leiomyoma-like, five lipoma-like, two epithelioid, and one AML with epithelial cysts. SMA was diffusely positive, except for the epithelioid type, while MelanA harbored stronger expression than HMB45. AML was more frequent in females and appeared ten and 7 years earlier than RO and RCC, respectively. The follow-up time was 7.42 years, and neither tumor-related death nor relapse occurred. AML is rare in nephrectomies and develops primarily in females in their 50s with an average size of 50–60 mm at the surgery. The histological appearance in order of frequency is classic, leiomyoma-like, lipoma-like, epithelioid, and cystic. The MelanA, HMB45, and SMA immunohistochemistry can support the light-microscopic findings. TSC1 or TSC2 genes [via the mTOR pathway [TSC1 and TSC2 are necessary [The angiomyolipoma (AML) is a benign tumor occurring mainly in the kidney and belongs to the perivascular epithelioid cell tumors (PEComa) , 2. GeneC2 genes , 9, whicC2 genes , 10. The pathway . AML occ pathway . Most of pathway , 13. In  pathway , 15, and pathway . Surgica pathway . If appl pathway . For cla pathway , but tum pathway  and leio pathway  morpholoecessary . Peripheecessary . We collAML cases were collected from the archive of the Department of Pathology, Albert Szent-Györgyi Medical School, University of Szeged. Two pathologists (AJ and LK) reviewed all hematoxylin eosin-stained slides and available immunohistochemical staining. They reclassified the cases according to the current classification scheme, and the subtypes were as follows: classic AML, AML with epithelial cysts (AMLEC), lipoma-like AML, leiomyoma-like AML, oncocytic AML, and epithelioid AML (eAML) . In thisAll immunohistochemical stains available in 22 tumors were reviewed. By using tissue microarray technique, another 19 AMLs were stained by MelanA , HMB45 , and SMA . Two 2-mm-thick tissue cores represented the tumors. The reactions were evaluated in a semiquantitative fashion . The FFPE blocks were unavailable in eleven cases; hence, no immunohistochemistry was performed for these tumors.p < .05.For parametric and non-parametric tests, the SPSS software package was applied, and the differences were deemed significant if Fifty-two AML cases were diagnosed from 2224 nephrectomy specimens. The mean age of all patients was 52.76 years (range 27–76 years). In males, 7 tumors, while, in females, 45 AMLs occurred . Forty-eight tumors were sporadic in our data set, and four were linked to TSC. The mean ages of sporadic and TSC cases were 54.04 and 37.75, respectively. One tumor developed in polycystic kidney disease, and another one evolved in a graft kidney. Tumor rupture and hemorrhagic complications occurred in eight patients shown in , and in The median size of all tumors was 53 mm (range: 4–300 mm), but in cases with tuberous sclerosis, it was found to be 260 mm. The revision of the cases resulted in thirty-one classic, thirteen leiomyoma-like, five lipoma-like, two epithelioid, and one AMLEC. No oncocytic AML was seen. Synchronous tumors were observed in four cases . Seven AClassic AML was the most common subtype (59.61%). Two tumors occurred in males (case #31 and #33). In case #35, the tumor developed in damaged kidney parenchyma (polycystic kidney), while, in case #33, the patient used finasteride to treat benign prostatic hyperplasia. All TSC-linked cases showed classic morphology. This morphology was seen in 25% of the AMLs investigated. These tumors contained only a small amount of fat tissue; therefore, macroscopically, they caused no impression of AML shown in .We registered a lipoma-like appearance in five cases, and case #35 evolved in a transplanted kidney. We diagnosed two tumors as eAML. This subtype required several immunohistochemical staining at the original histological diagnosis. Invasion, mitotic activity, and tumor cell necrosis were exclusively seen in these tumors shown in .This tumor was discovered in a 46-years-old male. The relatively small lesion was accidentally noticed during a urological investigation. Histologically, the tumor shared some characteristics with metanephric stromal tumor, and the tumor cells were estrogen and progesterone receptor-positive. We have no data on any hormonal treatment. p < .001; AML vs. RCC p < .001). Concerning the age among AML, onocytoma and RCC patients, the AML occurs 10 years and 7 years prior to the two aforementioned tumors, respectively . There is no difference in terms of the tumor size among the three tumors .We compared the gender, age and tumor size of AML patients with those, who were operated with oncocytoma (RO) and renal cell carcinoma (RCC). The AML is more common in females (AML vs. RO; TSC1 or TSC2, which impairs the regulation of the mTOR signaling pathway and leads to increased cell proliferation [TP53 mutation has also been noted in eAML, possibly contributing to the malignant clinical course [Oncocytoma and AML are the most common benign tumors of the kidney . The forferation . A TP53 l course . Geneticl course . Expressl course . Focal Sl course . The trel course . A closel course . Surgical course . The tuml course . The coml course . To mainl course . In doubl course , 62. Sysl course , 64.AML is rare in nephrectomy specimens, and it is either sporadic or associated with TSC. It typically develops in women around 50 years, and the average size of surgically treated cases is about 50–60 mm. Histologically, the tumor may be classic, leiomyoma-like, lipoma-like, epithelioid, or cystic, in order of frequency. The eAML may be malignant, so such tumors should be treated like RCC and closely monitored. The pathological diagnosis is usually problem-free, and the light-microscopic findings may be supplemented by MelanA, HMB45, and SMA immunostaining.