UCN activity show spontaneous formation of ectopic multicellular protrusions in integuments and malformed petals indicating that UCN suppresses uncontrolled growth in those tissues. In the current model UCN regulates planar growth of integuments in part by directly repressing the putative transcription factor ABERRANT TESTA SHAPE (ATS). Here we report on the identification of 3-PHOSPHOINOSITIDE-DEPENDENT PROTEIN KINASE 1 (PDK1) as a novel factor involved in UCN-mediated growth control. PDK1 constitutes a basic component of signaling mediated by AGC protein kinases throughout eukaryotes. Arabidopsis PDK1 is implied in stress responses and growth promotion. Here we show that loss-of-function mutations in PDK1 suppress aberrant growth in integuments and petals of ucn mutants. Additional genetic, in vitro, and cell biological data support the view that UCN functions by repressing PDK1. Furthermore, our data indicate that PDK1 is indirectly required for deregulated growth caused by ATS overexpression. Our findings support a model proposing that UCN suppresses ectopic growth in integuments through two independent processes: the attenuation of the protein kinase PDK1 in the cytoplasm and the repression of the transcription factor ATS in the nucleus.Tissue morphogenesis critically depends on the coordination of cellular growth patterns. In plants, many organs consist of clonally distinct cell layers, such as the epidermis, whose cells undergo divisions that are oriented along the plane of the layer. The developmental control of such planar growth is poorly understood. We have previously identified the Arabidopsis AGCVIII-class protein kinase UNICORN (UCN) as a central regulator of this process. Plants lacking  Plant organs, such as petals or roots, are composites of distinct cell layers. As a rule, cells making up a layer, for example the epidermis, the outermost layer of a tissue, divide “within the plane” of the layer. This cellular behavior results in the two-dimensional sheet-like or planar growth of the cell layer. The mechanism orchestrating such a growth pattern is poorly understood. In particular, it is unclear how uncontrolled and “out-of-plane” growth is avoided. Here we provide insight into this process. Our data indicate that higher than normal activity of a central regulator of growth and stress responses results in wavy and malformed petals and in protrusion-like aberrant outgrowths in the tissue that will develop into the seed coat. It is therefore important to keep this factor in check to allow proper formation of those tissues. We further show that a protein called UNICORN attenuates the activity of this regulator thereby ensuring the sheet-like growth of young petals or the developing seed coat. Spatial coordination of cell division patterns within a tissue layer is an essential feature of plant tissue morphogenesis. For example, the shoot apical meristem generates above-ground lateral organs, such as flowers, and is a composite of clonally distinct histogenic cell layers . Cells ounicorn (ucn) mutants exhibit spontaneous local ectopic growth revealing a defect in the regulation of planar growth [ucn mutants, one or several cells of a cell layer of the inner or outer integument divide in an oblique or periclinal, rather than anticlinal fashion. Such spatially restricted alterations in cellular behavior can be observed at various positions within integuments and at different stages of their development. Not every local occurrence results in a protrusion. As a rule, only a single protuberance originates at a proximal position in ucn mutants that may also include abnormally enlarged cells. It becomes first apparent once integument outgrowth is well underway. It is presently unclear what determines this preferential position. However, two to up to four protrusions formed at different integumentary sites can occasionally be observed as well [The genetic control of planar integument growth is poorly understood . Althougr growth . In inte as well .UCN encodes a protein kinase of the AGC VIII family [UCN mediates planar growth through the regulation of polar auxin transport. However, there is no evidence supporting this view. The available data suggest that UCN is not involved in polar auxin transport [PIN-FORMED (PIN) genes, encoding the classic regulators of intercellular polar auxin transport [I family . CertainI family –12 raisiransport ,12–14. Mransport ,16, coulransport ,18.UCN suppress ectopic growth in integuments? So far, genetic analysis has identified ABERRANT TESTA SHAPE (ATS) as an important factor involved in the UCN signaling mechanism [ATS encodes a putative transcription factor of the KANADI (KAN) family and controls integument boundary formation, integument initiation and adaxial-abaxial polarity [UCN does not control early integument development and functions independently of ARF3/ETT. Rather, the interaction between UCN and ATS is thought to be part of a later-acting surveillance mechanism that maintains planar integument outgrowth by inactivating ATS that is not in complex with ARF3/ETT [How does echanism ,14. ATS polarity –23. In tARF3/ETT ,14.ATS is a member of the KAN gene family that apparently functions specifically during integument development. Other members of the family are known to control abaxial identity during leaf formation in conjunction with ETT and ARF4 [UCN does not appear to be involved in the establishment of adaxial-abaxial polarity in integuments [UCN may regulate planar integument growth in part by maintaining the adaxial-abaxial polarity mechanism during later stages of integument outgrowth.The interaction between UCN and ATS supports the notion of a possible link between the regulation of adaxial-abaxial polarity and planar growth. Adaxial-abaxial polarity of leaves is in part regulated by antagonistic interactions between class III HD-ZIP and KAN transcription factors . ATS is and ARF4 –28. Inteand ARF4 . UCN doeeguments . Thus, U3-PHOSPHOINOSITIDE-DEPENDENT PROTEIN KINASE 1 (PDK1) represents another factor potentially involved in UCN signaling. PDK1 encodes a basal member of the AGC protein kinase family and a master regulator of downstream AGC kinases. PDK1 is well characterized in mammalian cells where it plays a central role in connecting lipid signaling to a broad range of cellular processes [PDK1 function is lethal in for example fly or mouse [rocesses ,34. Amonrocesses  and is orocesses . Completor mouse ,38. PDK1In unstimulated cells animal PDK1 is found in the cytoplasm but is largely excluded from the nucleus. Upon stimulation, however, PDK1 also accumulates in the nucleus, possibly as a means to prevent activation of cytosolic signaling pathways –41. ActiPDK1 function in tomato is lethal [PDK1 gene in Physcomitrella patens does not result in lethality but causes developmental abnormalities and late growth retardation [PDK1 genes, PDK1.1 and PDK1.2 [pdk1.1 pdk1.2 double mutants show only minor aberrations including somewhat stunted growth and reduced fertility. Moreover, they are impaired in growth promotion induced by the fungus Piriformospora indica [PDK1 gene also results in moderate dwarfism [PDK1 is also involved in growth control and that PDK1 requirement may vary from species to species.Despite impressive progress the function and regulation of PDK1 in plants is comparably less well understood –46. Resus lethal . By contardation . The Arad PDK1.2 . Loss-ofa indica . Interesdwarfism . These rPDK1 can interact with a number of AGC kinases , includiPDK1 in UCN-dependent growth control. Our data indicate that PDK1 is expressed in many different tissues and that PDK1 predominantly localizes to the cytoplasm. They further imply that PDK1 physically interacts with UCN in vitro and in plant cells. Genetic data suggest that UCN attenuates PDK1 function in vivo and that PDK1 is required for growth deregulation caused by overexpression of ATS.Here we investigated the role of Arabidopsis PDK1. The Arabidopsis genome contains two closely homologous PDK1 genes, PDK1.1 (At5g04510) and PDK1.2 (At3g10540) [PDK1-like gene in Arabidopsis  . PDK1.1 bidopsis .PDK1 transcript and the corresponding double-mutants confirmed the mild phenotype of pdk1.1 pdk1.2 double mutants reported previously [PDK1.1 and PDK1.2, showed only mild reduction in plant height and slightly reduced silique length  experiments. Nevertheless, we performed whole-mount ISH on ovules at different developmental stages. We could detect PDK1 transcripts throughout the ovule, with the exception of the nucellus   . The traionality . We coulionality . Taken tKD) by changing a conserved lysine to alanine in the catalytic domain of the two PDK1 homologs   or glutathione-S-transferase (GST:PDK1) that were produced in E. coli. In addition, we generated recombinant kinase-inactive versions of MBP:PDK1 (PDK11.2K74A) . We firs1.2K74A)  [55–57,6PDK1.1KD . The resG165S , UCNΔPIF  encompassing the predicted PIF domain), and UCNKD/ΔPIF (lacking kinase activity and PIF domain).In a next step we tested if MBP:PDK1.1 and MBP:PDK1.2 can phosphorylate GST:UCN. To this end we performed in vitro kinase assays using wild-type and various mutant forms of recombinant MBP:PDK1 and GST:UCN fusion proteins. Four different types of mutant recombinant versions of UCN were generated : UCNG165activity ), UCNKD G165S, GST:UCNKD) (ΔPIF were able to phosphorylate MBP:PDK1.1KD and MBP:PDK1.2KD (KD by MBP:PDK1.1 or MBP:PDK1.2.We observed that MBP:PDK1.1 and MBP:PDK1.2 were able to trans-phosphorylate kinase-inactive GST:UCN variants (GST:UCNT:UCNKD) . RemarkaPDK1.2KD . In bothWe further assessed if the presence of GST:UCN influenced the level of MBP:PDK1 activity. To this end titration experiments were performed in which constant levels of MBP:PDK1.1 or MBP:PDK1.2 were mixed with increasing amounts of GST:UCN or various mutant versions of GST:UCN . In bothTaken together the results indicate that in in vitro assays PDK1 and UCN phosphorylate each other and that UCN can attenuate PDK1 activity.UCN expression in the plant [pSPYNE:PDK1.1 or pSPYNE:PDK1.2 and pSPYCE:UCN constructs.  assay. We observed that UCN can interact with PDK1 in this system . Moreovehe plant  we resorhe plant . We obsestructs. . Signal structs. . The comucn-1 pdk1.1 and ucn-1 pdk1.2 double mutants. Interestingly, we found in the ucn pdk1 double mutants an essentially full restoration of the ucn ovule and flower phenotype to wild type  and transformed wild-type Ler plants with the corresponding transgenes. In both cases we investigated the phenotypes of 11 independent transgenic T2 lines homozygous for the transgene. In nine pUBQ::PDK1.1:EGFP lines we observed distorted petals and ovules with protrusions indicating that overexpression of PDK1 results in a ucn-like phenocopy   . We coulucn phenotype, such as malformed petals, were unaffected in ucn ats double mutants [The model put forward for the functional relationship between PDK1 and UCN has strong similarities to a scenario that was proposed for the UCN/ATS interaction . UCN/ATS mutants . MoreoveKD by MBP:PDK1. We next assessed if PDK1 can interact with ATS in a plant cell. We did not observe PDK1/ATS interaction in BiFC experiments indicating that the PDK1/ATS interaction observed in vitro does not occur in a plant cell  Heynh. var. Columbia (Col-0) and var. Landsberg (erecta mutant) (Ler) were used as wild-type strains. Plants were grown as described earlier [ucn-1 mutant (in Ler) was described previously [pdk-1 T-DNA lines were described before [pdk1.1 pdk1.2 mutant plants were transformed with different constructs using Agrobacterium strain GV3101/pMP90 [ATS (At5g42630), PDK1.1 (At5g04510), PDK1.2 (At3g10540), UCN (At1g51170). earlier . The ucneviously , and pdkd before . T-DNA i01/pMP90  and the 01/pMP90 . TransgeFor DNA and RNA work standard molecular biology techniques were used. PCR-fragments used for cloning were obtained using Phusion or Q5 high-fidelity DNA polymerase . All PCR-based constructs were sequenced. The Gateway-based (Invitrogen) pDONR207 was used as entry vector, and destination vectors pMDC43 and pMDC83  were useFloral tissue for quantitative real-time PCR (qPCR) was harvested from plants grown under long day conditions. With minor changes, tissue collection, RNA extraction and quality control were performed as described previously . cDNA syFor plasmid pPDK1.2::gPDK1.2:EGFP pMDC83, 4.253 kb of gPDK1.2 sequence was amplified from Col-0 genomic DNA including promoter sequence spanning genomic DNA up to the 3′ end of the adjacent gene (1.245 kb) and 3′UTR of PDK1.2 and cloned into pDONR207. pMDC83 (35S promoter was removed) was used as destination vector. For overexpression constructs, gPDK1.1 or gPDK1.2 were amplified from Col-0 genomic DNA and cloned into pDONR207. As destination vectors, pMDC43 or pMDC83 (35S promoter was replaced by either pUBQ10 or p16) were used, respectively.KD (kinase deficient version containing a K73A mutation), PDK1.2 WT and PDK1.2KD (kinase deficient version containing a K74A mutation)). For UCN, five different protein versions were expressed and purified , UCNKD (kinase deficient version containing a K55E mutation), UCNΔPIF (kinase active version lacking the PIF motif at the C-terminus), and UCNKD/ΔPIF (kinase deficient version lacking the PIF motif at the C-terminus)).UCN and PDK1 coding sequences were amplified from floral cDNA (Ler) and cloned into pGEX-6P-1  or pMal-c2x . The clones were expressed in the E. coli strain BL21 (DE3) pLysS. Expression from the pGEX vector leads to proteins fused to a N-terminal Glutathione Transferase (GST) protein, expression from pMal leads to proteins fused to a N-terminal Maltose Binding Protein (MBP). The ATS coding sequence was cloned into pET32a (Novagen) leading to an N-terminal 6xHis:Thioredoxin (Trx) fusion protein. For protein expression and purification, bacterial cultures were grown to OD 0.6–0.8 at 37°C. Then, the bacteria were induced with 0.8 mM isopropyl-beta-thio galactopyranoside (IPTG) for UCN, 1.5 mM IPTG for PDK1, and 0.5 mM IPTG for ATS, and grown at 30°C for 4 h. Subsequently, the recombinant proteins were purified from the bacteria by batch purification under native conditions using the Glutathione Sepharose 4 Fast Flow  for GST fusion proteins, amylose resin (New England Biolabs) for MBP fusion proteins, and Protino® Ni-TED packed columns for 6xHis:Trx fusion proteins according to the manufacturer’s instructions. For PDK1, four different protein versions were expressed and purified ) at RT for 1 h. Reactions were stopped by adding 4 μL 6xLaemmli buffer and boiling at 95°C for 5 min. In order to separate the proteins, 12% SDS PAGE was performed. Subsequently, the gels were stained with Coomassie Brilliant Blue G250, destained in 10% acetic acid and dried. Phosphorimager plates were exposed at RT over night and signals were detected using a Fuji BAS Phosphorimager .For kinase assays, the proteins were purified as described above and concentrations were estimated on a 12% SDS PAGE using BSA as standard protein. Assays were performed with approximately 500 ng of respective protein(s) and incubated in HMK buffer . Three independent colonies of each combination were resuspended in 500 μL ddH2O, diluted 1:100 and 10μL of the dilutions were spotted on SD-LWHA  supplemented with 5 mM 3-AT and grown at 30°C for 3 days.For yeast two-hybrid assays, the above-mentioned four PDK1 and five UCN versions were used. The coding sequences of these versions were cloned into pGBKT7 and pGADT7 vectors, respectively . Plasmids were transformed into yeast strain AH109 and transformants were selected on SDFor Bimolecular Fluorescence Complementation (BiFC), the above-mentioned four PDK1 and five UCN versions were cloned into pSPYCE-35S and pSPYNE-35S vectors, respectively . PlasmidWhole mount in situ hybridization of ovules was performed essentially as described . DigoxigS1 Table(DOCX)Click here for additional data file.S1 FigPDK1-like gene for Arabidopsis thaliana (At2g20050) [PDK1. The amino acid alignment was performed using ClustalW algorithm and BLOSUM62 matrix in Geneious 11.1.5 software (https://www.geneious.com). Amino acids are highlighted in color. Lines represent gaps. Please note that At2g20050 consists of 1,094 amino acids .A previous study had suggested a third 2g20050) . However(DOCX)Click here for additional data file.S2 Figpdk1.1–1 pdk1.2–2 (b) pdk1.1–2 pdk1.2–3. (c) Col-0. (d) pdk1.1–1 pdk1.2–2 p16::gPDK1.1:EGFP. (e) pdk1.1–1 pdk1.2–2 p16::gPDK1.2:EGFP. (f) pdk1.1–2 pdk1.2–3 pUBQ10::EGFP:gPDK1.1. (g) pdk1.1–2 pdk1.2–3 pUBQ10::EGFP:gPDK1.2. (h) pdk1.1–1 pdk1.2–2 pPDK1.2::gPDK1.2:EGFP. All constructs restore plant height. Dashed line indicates maximal plant height of the double mutants. Scale bar: 10 cm.(a) (DOCX)Click here for additional data file.S3 FigI) higher magnification of the region in (A), (B) ovule, (C) transition zone of a root, and (CI) higher magnification of the region in (C). Arrows in (AI) indicate the absence of GFP signal at the cell walls, magenta arrows in (CI) indicate most likely endoplasmic reticulum (ER), arrowheads indicate plasma membranes and asterisks indicate nuclei. Scale bars: 20 μm.Confocal micrographs are shown. (A) pUBQ10::PDK1.2:EGFP.  pUBQ10::PDK1.1:EGFP. (A) Filament, (A(DOCX)Click here for additional data file.S4 FigArabidopsis mesophyll protoplasts were used. The N-terminal part of YFP was fused to PDK1.1, PDK1.2, or UCN, respectively, and the C-terminal part of YFP was fused to different mutant versions of UCN. The variants are indicated. (A-C) Note absence of signal (n > 1500). (D) Signal is localized to the nucleus (126/425 scored protoplasts). Also compare to Fig 4 in .  N(DOCX)Click here for additional data file.S5 Fig(A) Kinase assays. Upper panel depicts autoradiogram. Lower panel shows corresponding coomassie brilliant blue (CBB)-stained gel. (upper panel) and autoradiograms (upper panel). Assays were performed for MBP:PDK1.1, MBP:PDK1.2, and GST:UCN, respectively, in combination with Trx:ATS. There is weak Trx:ATS phosphorylation signal in combination with MBP:PDK1 in comparison to GST:UCN/Trx:ATS (note the differences in the amount of Trx:ATS involved in the reactions). (B) BiFC assays of PDK1 and ATS. Note absence of signal. N = 3. Abbreviation: DIC, differential interference contrast. Scale bars: 5 μm.(DOCX)Click here for additional data file.


Moreover, Sal A increased sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) activity and expression, alleviated [Ca2+]ER depletion, and decreased ER stress-related protein expression. Sal A protects the heart from ATO-induced injury and its administration correlates with the modulation of SERCA, the recovery of Ca2+ homeostasis, and the down-regulation of ER stress-mediated apoptosis.Arsenic trioxide (ATO) has been verified as a breakthrough with respect to the management of acute promyelocytic leukemia (APL) in recent decades but associated with some serious adverse phenomena, particularly cardiac functional abnormalities. Salvianolic acid A  is a major effective component in treating ATO-induced cardiotoxicity. Therefore, the objective of our study was to assess whether Sal A had protective effects by the regulation of calcium homeostasis and endoplasmic reticulum (ER) stress. For the in vivo study, BALB/c mice were treated with ATO and/or Sal A via daily tail vein injections for two weeks. For the in vitro study, we detected the effects of ATO and/or Sal A in real time using adult rat ventricular myocytes (ARVMs) and an IonOptix MyoCam system. Our results showed that Sal A pretreatment alleviated cardiac dysfunction and Ca Arsenic-containing compounds have been used as medicines to treat multiple diseases, including cancer, syphilis and malaria, for more than 2000 years . However2+ by the release of Ca2+ from the endoplasmic reticulum/sarcoplasmic reticulum (ER/SR), resulting in calcium homeostasis imbalances /dtmax, the time to 50% peak [Ca2+]i, and the [Ca2+]i transient decay rate compared with the control group in In our subsequent experiments, Caus study , the res2+ in the intracellular by affecting SR Ca2+ intake. Intracellular Ca2+ overload and cardiac myocyte dysfunction can be regulated by reducing SERCA expression and activity i due to abnormal Ca2+ handling. Here, the results show that ATO-induced cardiac dysfunction is significantly attenuated by Sal A. Moreover, our results showed that Sal A pretreatment relieved ATO-induced decreases in the decay rates of [Ca2+]i transient, which demonstrated that Sal A increased SERCA activity. SERCA is the critical regulator of Ca2+ homeostasis and contractile activity in the cardiac SR i homeostasis. In addition, PLB, as an SR membrane protein, can regulate SERCA function, and Ser16 and Thr17 are its phosphorylation sites ) for 2 min, solution B  for 5 min, and solution B with collagenase type II for 10–15 min. After perfusion, the single cell was obtained by filtering digested heart tissues using a 300-mm nylon mesh. The cell suspension was deposited 3 times with solution A to progressively increase the extracellular Ca2+ concentration to 1.2 mM. Suspensions with a cell viability rate over 85% were used in this study, of which rod-shaped ARVMs with prominent sarcomere were contained.ARVMs were isolated using the enzymolysis method, as previously reported . In brie2+ transients, as previously reported [Our experiment used video-based sarcomere contractility and calcium recording module in a SoftEdge MyoCam System  to assess sarcomere shortening and Careported . FirstlySERCA activity of ARVMs and mice heart tissue were detected respectively. For ARVMs, the rod-shaped cardiomyocytes with prominent sarcomere were treated as followed: (a) control group: solution A for 30 min; (b) Sal A group: solution A with 1 μM Sal A for 10 min; (c) ATO group: solution A with 100 μM ATO for 20 min; and (d) ATO+Sal A group: solution A with 100 μM ATO for 20 min after solution A with 1 μM Sal A for 10 min. After all treatment, ARVMs were added to PBS buffer solution and then broken using ultrasound. Cell suspension was centrifuged for 10 min at 3500 rpm and the supernatant was used to detect the SERCA activity. For mice heart tissue, the tissue of 4 groups was added to 10 mL/g saline, and then fully homogenized. Tissue suspension was centrifuged for 10 min at 3500 rpm and the supernatant was used to detect the SERCA activity. SERCA activity was measured using a commercially available kit . The specific operation was completed according to the manufacturer’s instructions.After exposure to the group-specific solutions described in 4.1 and 4.6, the proteins of the mice heart tissue homogenates and ARVMs were obtained with the tissue or cell protein extraction reagent, and were determined by a BCA kit . Approximately 30 to 50 μg of protein was loaded onto 10% or 12% sodium dodecyl sulfate (SDS) polyacrylamide gel and then transferred to nitrocellulose membranes, which were incubated overnight at 4 °C with primary antibodies including: Santa Cruz: PLB (sc-21923), PLB-Ser16 (sc-12963), PLB-Thr17 (sc-17024), p-JNK , and JNK (sc-7345); Abcam: SERCA (ab2861), caspase-12 (ab18766), GRP78 (ab21685) and CHOP (ab11419). Then the membranes were washed and incubated with secondary antibodies  for 2h at room temperature. The immunoblots were developed using an electrochemiluminescence (ECL) kit, and then scanned; densitometric analysis was performed using Gel Pro software .p-value less than 0.05 was considered statistically significant.All data are presented as means ± standard deviation (SD) from at least 3 independent experiments. Comparisons between different groups were performed using one-way ANOVA followed by Tukey’s test with GraphPad Prism 5.0 software , and graphical presentation of data was also undertaken with GraphPad Prism. A 2+ overload and improved SERCA activity and expression, thereby alleviating ATO-induced calcium homeostasis imbalances. In addition, Sal A inhibited ATO-induced ER stress by downregulating GRP78, JNK, CHOP and caspase-12. Our findings suggest that Sal A protects against ATO-induced cardiac injury and improves myocardial performance. The cardioprotective effect seems to be due largely to the maintenance of calcium homeostasis and the attenuation of ER stress-associated apoptosis in vivo and in vitro. These results demonstrated that Sal A prevents ATO-induced cardiotoxicity, which has significant implications for clinical application. However, our study is still in its initial stages, and the exact mechanisms underlying the cardioprotective activity of Sal A require further study.Sal A treatment before ATO suppressed Ca


The European Calcium Society (ECS) is very glad to present the realization of a special issue of the International Journal of Molecular Sciences (IJMS) related to the eighth ECS workshop organized this year around the theme of “Calcium Signaling in Aging and Neurodegenerative Diseases”.This workshop took place on September 18–20, 2019 in Coimbra  and was organized by Cláudia Pereira, Cristina Rego, Luísa Cortes, and João Malva. The scientific committee consisted of Amanda Sierra, Carlos Duarte, Carlos Villalobos, Catarina Resende de Oliveira, Jan B. Parys, and Rodrigo Cunha.www.calciumsociety.eu. The ECS is especially well-known for its biennial international meetings , with workshops focusing on a single topic occurring in the intervening years [The ECS has existed to promote research into the biology of cellular calcium since 1997 and presently has a membership of 250 researchers at all career stages, from over 30 countries, both within and outside Europe. Full information about the ECS and its activities can be found via 2+ homeostasis. Moreover, the fact that these various processes are widely interconnected and influence each other does not make it any easier to unravel the underlying mechanism(s).Moreover, the topic of the workshop in Coimbra was particularly well chosen as ageing, and the related occurrence of neurodegenerative diseases, constitute an increasingly important problem in our society. The 2018 Ageing Report of the European Community . Moreove2+ signals occurring in the cell. Examples of such Ca2+-regulated processes include fertilization, cell proliferation and differentiation, metabolism, muscle contraction, vesicle secretion, gene transcription, synaptic plasticity and memory formation, autophagy, and cell death. It is therefore increasingly more appreciated that the dysregulation of Ca2+ signals leads to important malfunctions at the cellular level, and to a plethora of pathological outcomes at the organismal level [However, it is clear that most if not all cellular functions are dependent on, or at least regulated by the complex spatio-temporal Caal level ,7.2+ are directly or indirectly important for correct brain function. An important role for intracellular Ca2+ in aging and neurodegenerative diseases is therefore emerging , 13 speakers were invited, and a further 10 participants were selected to give oral communications while 19 posters were also presented. Interestingly, IJMS provided for the workshop a “Best poster award” (winner: Riccardo Filadi) and a “Best oral communication award” . Furthermore, it must be mentioned that a hands-on imaging workshop delivering practical advice on techniques for Ca2+ measurement in neuronal cells was organized by the University of Coimbra. Finally, all attendees had the opportunity to visit the ancient campus of the University of Coimbra (founded in 1290) with its famous library and science museum, declared a world heritage site by UNESCO.To obtain a better insight into the role of Ca2+ in aging and neurodegeneration were presented at the workshop. Further, whilst a number of different neurodegenerative diseases were discussed, there were some recurrent themes and clear take-home messages in the many presentations, including: the relationship between neuronal and glial cells and the Ca2+ signals arising in both cell types, Ca2+ handling at synapses, the regulation of Ca2+ sequestration and release by the endoplasmic reticulum and the role of presenilins therein, the link between Ca2+ fluxes from the endoplasmic reticulum to mitochondria and mitochondrial Ca2+ overload and neurodegeneration, the usefulness of various types of animal models in the study of neurodegeneration, the development of novel tools to measure Ca2+ at the subcellular level and to analyse the resulting Ca2+ signals, the role of Ca2+ in the regulation of cerebral blood flow, and the relation between Ca2+, lifespan, and aging.Various aspects of CaTherefore, we are very proud to present in this special issue, not only a full meeting report , but als


For an unbiased, top-down proteomic approach, select thiol- reactive reagents were used to investigate this process in release-ready native secretory vesicles. We previously characterized a biphasic effect of these reagents on Ca2+-triggered exocytosis: low doses potentiated Ca2+ sensitivity, whereas high doses inhibited Ca2+ sensitivity and extent of vesicle fusion. Capitalizing on this novel potentiating effect, we have now identified fluorescent thiol- reactive reagents producing the same effects: Lucifer yellow iodoacetamide, monobromobimane, and dibromobimane. Top-down proteomic analyses of fluorescently labeled proteins from total and cholesterol-enriched vesicle membrane fractions using two-dimensional gel electrophoresis coupled with mass spectrometry identified several candidate targets, some of which have been previously linked to the late steps of regulated exocytosis and some of which are novel. Initial validation studies indicate that Rab proteins are involved in the modulation of Ca2+ sensitivity, and thus the efficiency of membrane fusion, which may, in part, be linked to their previously identified upstream roles in vesicle docking.Regulated exocytosis enables temporal and spatial control over the secretion of biologically active compounds; however, the mechanism by which Ca This involves the coordinated actions of both lipid and protein components of the secretory vesicle membrane, particularly the later steps of Ca2+-triggered exocytosis. There have been recent advances in understanding how the lipid matrix may modulate upstream docking/priming steps free plateaus defining 0% and 100% fusion, respectively; Sr2+ data were normalized to control Ca2+ data determined in parallel, as previously described [2+]free were measured with a Ca2+ sensitive electrode  and final [Sr2+]free were calculated using MaxChelator (WINMAXC32 v2.50). Endpoint activity curves were fit using a log-normal cumulative function to determine the extent, slope and Ca2+-sensitivity of fusion free (2+-triggered membrane fusion free , 2 mM mBB (n = 6) and 500 µM dBB (n = 8) treatments  had an EC50 of 2600.6 ± 374.8 µM [Sr2+]free (n = 7), 2 mM mBB (n = 6) and 500 µM dBB (n = 7) for 20 min resulted in leftward shifts in Sr2+ sensitivity to EC50’s of 1222.2 ± 334.3 µM, 1148.7 ± 89.4 µM and 1072.8 ± 159.8 µM [Sr2+]free, respectively (n = 4), and late kinetics with 2 mM mBB (n = 4) and 500 µM dBB (n = 6) treatments, in response to low [Sr2+]free = 3110 µM  (n = 2 replicates per thiol reagent). In the eight boxed regions on the resulting 2D gels, a total of thirty-seven protein spots were observed that were labeled with LYIA, mBB and dBB  . In addireagent) . To bett and dBB . These s and dBB .n = 3 individual experiments per thiol reagent)  .2+-sensitivity of regulated exocytosis. Among the most likely candidates were cytoskeletal proteins and Rab GTPases, which have both previously been implicated in vesicular trafficking. Several Rab GTPases were not only consistently labeled by all three fluorescent thiol reagents, but were also isolated in the cholesterol-enriched membrane proteome. We, therefore, chose to test these low MW G-proteins in a series of validation trials using the well-established CV–CV fusion assays.Mascot searches of the NCBIprot database resulted in the identification of the highest abundance protein species in thirty of the thirty-seven spots from the total CV membrane proteome, yielding up to a total of twenty-seven unique proteins that were reproducibly labeled. Many of these were also identified from corresponding spots  in the cholesterol-enriched membrane proteomes, plus an additional eleven spots identified from the latter. All proteoforms identified  contain 2+ and GTP have been observed in different models of regulated secretion. While the only requirement for CV–CV fusion is an increase in [Ca2+]free [2+ sensitivity and rate of CV–CV fusion . Initial fusion kinetics were also inhibited in response to 33.7 ± 2.7 µm [Ca2+]free , but not at higher [Ca2+]free. The addition of a nonhydrolyzable analogue GTPγS did not reduce the concentration of nucleotide required to inhibit fusion (n = 4)  B. Furthe2+-triggered exocytosis, an exogenous RAB3 effector domain peptide was used free . To determine whether the effect of the RAB peptide was, in part, due to modulation of vesicle priming/docking, a modified fusion assay in which CVs were allowed to settle into contact was also carried out . As with the standard fusion assay, there was no effect on Ca2+ sensitivity . The addition of 200 µg/mL scrambled peptide had no effect on the Ca2+ sensitivity, kinetics or extent of fusion in either the standard or settle assays.To directly investigate the role of Rab-GTPases in modulating the late steps of Cawas used ; this peentified . The addentified A. Treatmried out B free . The Ca2+ sensitivity, kinetics or extent of fusion in CV pretreated with IA followed by treatment with scrambled peptide did not differ from the IA pretreatment condition.While no effect on Ca in EC50 C. Follow2+-triggered exocytosis. Second, several fluorescent thiol reagents that potentiate the Ca2+ sensitivity and kinetics of CV–CV fusion were characterized and employed as ‘functional’ tags to label CV membrane proteoforms involved. Third, prefractionation of total CV membrane or cholesterol-enriched CV membrane, followed by high-resolution 2DE, was carried out to target downstream mass spectrometric analyses on the identification of reproducibly labeled, secretory vesicle membrane proteoforms. This rigorous approach using a top-down proteomic methodology identified several viable proteoform candidates, and validation studies suggest multiple roles for Rab-GTPases in modulating the efficiency of exocytosis.Thiol-reactive agents are powerful tools in an unbiased approach to investigating proteoform functions in regulated exocytosis ,39,40,452+-triggered exocytosis [2+]free to trigger membrane fusion free can partially overcome the inhibition of fertilization envelope formation by exogenous RAB peptide in intact oocytes [To this end, we further explored the role of Rab GTPases in regulated exocytosis in the urchin CV model. An exogenous RAB peptide, containing the effector domain of Rab3 , was fouachinery . In this oocytes . Further oocytes  and neur oocytes ,77 cells2+-triggered exocytosis [2+-dependent secretion in (neuro)endocrine cells [2+ and phospholipids [2+-triggered membrane fusion increases the complexity of control over this tightly regulated process, allowing for graded responses to different external stimuli.These data highlight the dual role of specific proteoforms in exocytosis, acting as both positive and negative modulators of regulated secretion, depending on the steps of the pathway being investigated. Several Rab GTPases are associated with secretory vesicles: Rab1, Rab2, Rab3, Rab4, Rab5, Rab7, Rab10, Rab11, Rab14 and Rab27 all co-purify  during the isolation of synaptic vesicles , and we ocytosis ; howeverne cells ,79 and pne cells , respectne cells ,69,70,71ne cells ,82,83 anne cells ,86,87,88holipids ,90, whic2+-triggered exocytosis evolves, it becomes increasingly apparent that the coordinated action of a multitude of proteins and lipids are required for fast, efficient cellular secretion, and that many aspects of exocytotic pathway are very highly conserved.This does not preclude other thiol-labeled proteoforms from also contributing to efficiency of regulated exocytosis. The presence of several metabolic enzymes on secretory vesicles suggests local regulatory mechanisms to ensure optimal lipid and protein function. Along with cholesterol- and sphingomyelin-enriched membrane domains ,11,45,462+-triggered exocytosis. Thiol-reactive reagents were employed as functional tags to label candidate proteoforms that regulated the Ca2+-sensitivity and the kinetics of secretory vesicle  fusion. A targeted experimental paradigm, including subcellular fractionation and optimized 2DE resolution of labeled proteoforms, yielded a targeted list of proteins for further investigation. While a majority evidence suggests Rab GTPases have roles upstream in tethering/docking, the data here are unique in also indicating a role in later steps that modulate Ca2+-sensitivity. Furthermore, several other labeled proteoforms may have additional roles that ensure optimal function of the exocytotic machinery (both proteins and lipids) in regulated secretion.In this study, we have taken a top-down proteomic approach to studying the molecular mechanisms underlying the late steps of Ca


Being fat-soluble, PCBs are passed to newborns through maternal milk, impairing heart functionality in the adult. It is still unknown how PCBs impair cardiac contraction at cellular/molecular levels. Here, we study the molecular mechanisms by which PCBs cause the observed heart contraction defects, analysing the alterations of Ca2+ toolkit components that regulate contraction. We investigated the effect that Aroclor 1254 (Aroclor), a mixture of PCBs, has on perinatal-like cardiomyocytes derived from mouse embryonic stem cells. Cardiomyocytes, exposed to 1 or 2 µg/ml Aroclor for 24 h, were analyzed for their kinematics contractile properties and intracellular Ca2+ dynamics. We observed that Aroclor impairs cardiomyocytes contractile properties by inhibiting spontaneous Ca2+ oscillations. It disrupts intracellular Ca2+ homeostasis by reducing the sarcoplasmic reticulum Ca2+ content and by inhibiting voltage-gated Ca2+ entry. These findings contribute to the understanding of the molecular underpinnings of PCBs-induced cardiovascular alterations, which are emerging as an additional life-threatening hurdle associated to PCBs pollution. Therefore, PCBs-dependent alteration of intracellular Ca2+ dynamics is the most likely trigger of developmental cardiac functional alteration.Persistent organic pollutants are a group of chemicals that include polychlorinated biphenyls (PCBs). PCBs exposure during adult life increases incidence and severity of cardiomyopathies, whereas  However, due to their high chemical stability, PCBs are still persisting and have wide diffusion into the environment.Persistent organic pollutants are a variegated group of chemicals that include polychlorinated biphenyls (PCBs), the latter compounds containing a high number of chlorine atoms. PCBs have been widely used in electrical and electronic industries  in adult primary rat cardiomyocytes8. In guinea pig ventricular myocytes, PCB 19 exposure decreases contractile force, action potential duration and amplitude, and intracellular Ca2+ transients through inhibition of L-type voltage-gated Ca2+ channels (VGCCs)9. Also, in other adult cardiac cellular models, PCBs alter the expression and/or activity of several components of the intracellular Ca2+ signalling machinery10, such as type 2 ryanodine receptors (RyR2)11 and the Sarco-Endoplasmic-Reticulum Ca2+-ATPase (SERCA)12.Growing amounts of data link their bioaccumulation through the diet with their high toxicity; more specifically, several studies have evidenced that PCBs exposure during adult life leads to an increase in the incidence and severity of cardiomyopathies14 not attributable to inherited genetic mutations5. To this regard, exposed avian embryos display extensive cardiac dilation, thinner ventricle walls and reduced responsiveness to chronotropic stimuli15, whereas zebrafish embryos show heart defects, reduction of the heart beating rate and irregular and weak contractions16. Reduced heart size and functional deficits have been described in mouse foetuses, as well as cardiac hypertrophy in offsprings, the latter showing increased sensitivity to cardiovascular insults in adulthood5.PCBs prenatal exposure through placenta determines congenital heart defects17, increasing the myocardial wall thickness and inducing cardiac hypertrophy, thus impairing heart functionality in the adult18. Whilst the detrimental effects on the perinatal heart anatomy are evident, unknown are the PCBs effects at the cellular and molecular levels. Our study aims at dissecting the key physiological processes behind the observed heart contraction defects, by analyzing in perinatal cardiomyocytes the calcium-induced calcium-release mechanism (Ca2+ toolkit) that finely regulates contraction.Importantly, being fat-soluble, PCBs accumulate in fat tissues and are passed to newborns through maternal milkin vitro differentiation of mouse embryonic stem cells (mESCs). Functionally, these cardiomyocytes form compact beating syncytia and exhibit electrophysiological features of excitation-contraction coupling described for isolated perinatal cardiac cells21.Perinatal-like cardiomyocytes (hereafter named cardiomyocytes) were obtained from the 2, at doses in the range of environmental contamination (1 and 2 µg/ml), then their kinematics contractile properties and the intracellular Ca2+ homeostasis were evaluated.Following differentiation, cardiomyocytes were exposed for 24 h to Aroclor 1254 (Aroclor), a mixture of more than 80 PCBs isomers and congeners with high number of chlorine atoms (54%)2] and contractility, i.e. the maximum contraction velocity, [pixel/s]) and ergotropic (consumption of ATP for kinetic energy [pixel2/s2]) features were mathematically calculated from the movement of the beating syncytia22.For the evaluation of the effects of Aroclor on the contractile properties of beating syncytia, we measured their kinematics and dynamics features on AVI videos, recorded from CTR samples and after 24 h exposure to 1 or 2 µg/ml Aroclor. The chronotropic (beat frequency [Hz]), inotropic (contraction force i at both 1  reduction in basal [Ca2+]i , a selective RyR agonist, triggered a Ca2+ response in CTR samples, while it failed in the presence of Aroclor at both concentrations , known to inhibit SERCA activity thereby causing the progressive efflux of intraluminal Ca2+ through unidentified leakage channels30. This manoeuvre leads to the rapid depletion of the SR Ca2+ pool under 0Ca2+ conditions and, therefore, causes a transient increase in [Ca2+]i. The [Ca2+]i indeed returns to the original baseline due to the concerted interaction of the Na+/Ca2+ exchanger (NCX), plasma-membrane Ca2+-ATPase (PMCA) and mitochondria. CPA-induced intracellular Ca2+ mobilization is a well-known index of SR Ca2+ content and is widely employed to compare SR Ca2+ content between control and treated cells31. In our experiments, exposure to Aroclor caused a significant reduction in CPA-induced Ca2+ release under 0Ca2+ conditions  reduced in the presence of each dose of Aroclor , Ryr2 (cardiac muscle SR RyR isoform), Cacna1c (α subunit of L-type VGCCs) and Itpr2 (cardiac muscle SR InsP3R isoform). The exposure to both Aroclor concentrations did not significantly alter the quantitative expression of Atp2a2 gene (p > 0.05). Conversely, the expression of Ryr2 and Itpr2 significantly increased , whereas the expression of Cacna1c significantly decreased (0.7-fold) only in syncytia exposed to 2 µg/ml  InsP3Rs do not play a prominent role in inducing the spontaneous Ca2+ spikes, as the InsP3-sensitive SR Ca2+ pool is up-regulated by Aroclor exposure, while the amplitude and frequency of Ca2+ oscillations are reduced; and 2) InsP3Rs are, however, able to modulate RyRs-dependent Ca2+ release, as the transient depletion of the InsP3-sensitive SR Ca2+ pool leads to a short-lasting decrease in the amplitude of Ca2+ spikes in CTR cardiomyocytes. The increase in Itpr2 expression could explain the largest Ca2+ signal induced by angiotensin II in the presence of Aroclor. Consistently, 2 μg/ml Aroclor induces a dose-dependent elevation in both Itpr2 transcripts and in the peak Ca2+ response to angiotensin II.Consistent with these earlier observations, Aroclor heavily impairs spontaneous intracellular Ca2+ oscillations also in Aroclor-exposed syncytia and suggest that the signaling machinery underlying the spontaneous Ca2+ signal was not affected by the treatment. Thus, we moved our attention to single components of the Ca2+ toolkit involved in the oscillatory signal to evaluate whether and how Aroclor interferes with them. We determined that Aroclor exposure induced a significant reduction in basal [Ca2+]i and in SR Ca2+ levels in cardiomyocytes, which was reflected in a dramatic decrease in the caffeine-releasable Ca2+ pool. Additionally, Aroclor heavily inhibited voltage-dependent Ca2+ entry, as measured in response to high-KCl extracellular solution. Therefore, Aroclor affects both the components of the Ca2+ toolkit which deliver Ca2+ to the cytosol during the spiking activity. Similar results were obtained in neuronal cells, in which it was demonstrated that Aroclor 1254 induced a depletion of SR Ca2+ store through the activation of InsP3Rs37.These data indicate that RyRs and VGCCs work to shape the spontaneous Ca2+]i, suggesting that Aroclor is not able to cause a massive and rapid release of intraluminally stored Ca2+ which is the most intuitive mechanism to explain the depletion of the SR Ca2+ pool underpinning the rhythmic Ca2+ release (as discussed in more detail below). This result differs compared to that observed in rodent neurons40 and PC12 cells41, in which PCB-95 and Aroclor 1254 stimulate RyRs-dependent intracellular Ca2+ release. It has been demonstrated that PCB congeners sensitize RyRs by binding to one or more components of the RyR macromolecular complex, which could therefore act as receptors43. We speculate that in perinatal cardiomyocytes, characterized by an immature SR45, the lack of expression of components of the adult RyR macromolecular complex may explain why Aroclor fails to increase [Ca2+]i. As Aroclor did not evoke an acute Ca2+ response, we reasoned that effects observed after 24h-exposure could be explained by an interference with the Ca2+ handling machinery.The acute addition of Aroclor did not trigger any detectable increase in i49, this model would also explain the drop in resting Ca2+ levels caused by Aroclor-treated in the beating syncytia. Future investigations are required to confirm this hypothesis and to understand how Aroclor 1254 interacts with SERCA2A. Our data, however, rule out the down-regulation of Atp2a2 expression, that codes for SERCA2A, as the transcript levels of this gene are not altered after 24 h Aroclor exposure. The mechanism whereby Aroclor inhibits SERCA activity is yet to be elucidated48. However, in cardiomyocytes, SERCA activity is severely reduced by cytosolic and mitochondria-derived reactive oxygen species (ROS), which therefore lead to a dramatic reduction in SR Ca2+ concentration51. Interestingly, a recent work showed that ROS production through the mitochondrial pathway started to increase after 1 h of Aroclor exposure in the human lung cancer cell line A54952. This mechanism could explain why the inhibitory effect of Aroclor is not acute and requires a prolonged incubation also in the beating syncytia.Real Time PCR analysis demonstrated that both 1 or 2 μg/ml Aroclor induced a significant increase of the expression of 2+ levels, we found that Aroclor attenuated voltage-dependent Ca2+ entry, which is necessary to maintain the spontaneous Ca2+ oscillations over time, at both 1 and 2 μg/ml. Real Time PCR disclosed that only 2 μg/ml Aroclor 1254 decreased the expression of Cacna1c, which encodes for the α subunit of L-type VGCCs. While this mechanism is likely to play a major role in reducing voltage-dependent Ca2+ entry at the higher dose, it does not explain the inhibitory effect observed at 1 μg/ml. However, earlier reports revealed that PCBs are able to increase Ca2+ permeability across the plasma membrane in several cell types, causing a long-lasting influx of Ca2+ through unidentified Ca2+-permeable channels53 or through store-operated Ca2+ channels54. Also, Aroclor 1254 activates the Ca2+-permeable N-methyl-D-aspartate receptors55, inhibits the non-selective cation channel TRP Vanilloid 6 (TRPV6)56 and stimulates Cl–permeable γ-aminobutyric acid (GABA)A receptors57 in newborn rat neocortical neurons. All together, these findings clearly demonstrate that PCB congeners, including Aroclor 1254, have the potential to modulate plasma membrane channels, although it remains to be elucidated whether this interaction is direct or mediated by auxiliary partners. We hypothesize that, besides reducing Cacna1c expression, Aroclor 1254 inhibits L-type VGCCs through a non-yet identified mechanism in cardiomyocytes.In addition to reducing SR Ca2+ machinery. These findings contribute to the understanding of the molecular underpinnings of PCBs-induced cardiovascular alterations, which are emerging as an additional life-threatening hurdle associated to PCB pollution. Therefore, PCBs-dependent alteration of intracellular Ca2+ machinery is the most likely trigger of developmental cardiac functional alterations.In conclusion, for the first time, we demonstrate that perinatal cardiac syncytia exposed to Aroclor 1254 undergo a dramatic functional alteration of the kinematics contractile properties, due to the disruption of the intracellular Ca58. Briefly, R1 was cultivated in Knockout DMEM added with 15% ESC Qualified FBS, 2 mM L-glutamine, 1X non-essential amino acids, 0.5% penicillin/streptomycin , 0.1 mM β-mercaptoethanol (Sigma) and 500 U/ml ESGRO-LIF . The STO cell line was maintained in DMEM (Sigma) supplemented with 10% foetal bovine serum, 4 mM L-glutamine, 1X non-essential amino acids, 0.5% penicillin–streptomycin solution , 0.1 mM β-mercaptoethanol (Sigma) and 0.2 mg/mL geneticin (Sigma). ESCs were routinely passaged enzymatically every 2/3 days with trypsin/EDTA 0.05%, alternating a passage on STO feeder cells with two passages on gelatin-coated p55 dish, and maintained in an incubator at 37 °C with 5% CO2 in air.R1 mESC line  and STO cell line  were cultivated as previously describedin vitro by removing the Leukemia Inhibitory Factor (LIF) from culture medium (differentiation medium), using the hanging drop method60. For EBs formation, about seventy 20 µl droplets of differentiation medium containing 103 mESCs were plated on the lid of p55 Petri dishes. On day 3 of culture, the developing EBs were transferred on 0.1% agarose-coated tissue dishes (Corning) and from day 5, about 5–8 EBs were plated in single 1.9 cm2 well and cultivated up to 15 days.mESCs were induced to differentiate through the formation of embryoid bodies (EBs) Aroclor 1254 (Pancreac Nova Chimica) was dissolved in 100% dimethyl sulfoxide  to a concentration of 20 mg/ml. This solution was added to the differentiation medium on day 15 to a final concentration of 1 or 2 µg/ml. As control (CTR) samples, cells were exposed to 0.01% DMSO. Cells were harvested 24 h after either Aroclor or DMSO exposure. This procedure was repeated for three independent experiments.2 for video recording. For each of the three independent experiments, AVI videos of the beating syncytia were recorded from 10 randomly chosen CTR or Aroclor-exposed samples, using the Snagit software and further analyzed with the Video Spot Tracker (VST) program (http://cismm.cs.unc.edu/downloads). Then, videos were processed according to the image processing algorithm based on the Matlab programming language 61.On day 5 of differentiation, 25 EBs were plated onto 22 mm gelatin-coated Glass Bottom Dish (WillCo Wells) and cultured up to day 15. At day 15, cells were exposed to Aroclor and then transferred into the culture chamber of a Nikon BioStation IM at 37 °C and 5% CO2+ dynamics in mESCs-derived cardiomyocytes were measured by using a conventional epifluorescence Ca2+ imaging system, as shown elsewhere26. At day 5 of differentiation, EBs were plated on 12 mm coverslips in 24-multiwell plates. At day 15 beating cardiac syncytia, exposed to DMSO, 1 or 2 µg/ml Aroclor for 24 h, were analyzed for Ca2+ transients. Cells were loaded with 0.5 μM Fura-2/AM  for 20 min at 37 °C. Then, after washing with pre-warmed Ca2+-containing Tyrode’s solution , the coverslips were fixed to the bottom of a Petri dish and the cells observed by an upright epifluorescence Axiolab microscope , equipped with a Zeiss 40× Achroplan objective . Voltage-dependent Ca2+ entry was stimulated by replacing 40 mM NaCl with an equimolar amount of KCl (high-KCl solution). Intracellular Ca2+ release was monitored in the medium without Ca2+ (0 Ca2+) and supplemented with 10 mM EGTA. Cells were excited alternately at 340 and 380 nm wavelengths and the emitted light was detected at 510 nm. A first neutral density filter  reduced the overall intensity of the excitation light and a second neutral density filter  was coupled to the 380 nm filter to reach the intensity of the 340 nm wavelength. A round diaphragm was used to increase the contrast. Excitation filters were mounted on a filter wheel . Custom software, working in LINUX environment, was used to drive the camera , the filter wheel and to measure and plot on-line the fluorescence from 30–45 rectangular “regions of interest” (ROI) enclosing 30–60 areas within each of the beating syncytia. Each ROI was identified by a number. The ratio of fluorescence emitted at 340 and 380 nm was recorded as an indicator of the changes in intracellular Ca2+ concentration ([Ca2+]i). The experiments were performed at room temperature (22 °C). Ratio measurements were performed and plotted every 1.5 s for 900 s. The resting [Ca2+]i was measured by exploiting the Grynkiewicz equation, as shown in Zuccolo et al.30.CaOn day 16, following 24 h Aroclor exposure, RNA was extracted using the GenElute Mammalian Total RNA Kit according to the manufacturer’s instruction (Sigma) from about 200 EBs from each CTR and 1 or 2 µg/ml Aroclor-exposed samples. Three independent experiments were performed on a total of about 1800 EBs.2, 4 mM of each dNTP, 0.625 M oligo d(T)16, 1.875 M Random Hexamers, 20 U RNase Inhibitor, 50 U MuLV reverse transcriptase . The conditions for the reverse transcription were as follows: 25 °C for 10 min, 42 °C for 15 min, 99 °C for 5 min. One twentieth of the resulting cDNA was amplified in duplicate by Real-Time PCR in 20 µl reaction mixture with 200 nM of each specific primer  by 60 s. The amplitude of intracellular Ca2+ release in response to CPA, which is commonly used to estimate SR Ca2+ content, or caffeine and the amplitude of high-KCl-induced Ca2+ entry was measured as the difference between the ratio at the peak of intracellular Ca2+ mobilization and the mean ratio of 1 min baseline before the peak. Pooled data are given as means ± standard error (SE) and statistical significance (p < 0.05) was evaluated by the Student’s t-test for unpaired observations. Each Ca2+ trace is representative of 80–200 ROIs recorded from at least three embryoid bodies.PCR expression data are presented as means ± standard deviation (SD), while the syncytium contractile parameters are expressed as means ± 95% confidence interval for the differences between means. Data were analyzed by the one-way ANOVA and by the Supplementary InformationSupplementary Video 1Supplementary Video 2Supplementary Video 3


The transfer of Ca2+ from the endoplasmic reticulum (ER) to the mitochondria, facilitated by the presence of mitochondria-associated membranes (MAMs), is essential for several physiological mitochondrial functions such as respiration. Ca2+ transfer to mitochondria must be finely regulated because excess Ca2+ will disturb oxidative phosphorylation (OXPHOS), thereby increasing the generation of reactive oxygen species (ROS) that leads to cellular damage observed in both aging and neurodegenerative diseases. In addition, excess Ca2+ and ROS trigger the opening of the mitochondrial transition pore mPTP, leading to loss of mitochondrial function and cell death. mPTP opening probably increases with age and its activity has been associated with several neurodegenerative diseases. As Ca2+ seems to be the initiator of the mitochondrial failure that contributes to the synaptic deficit observed during aging and neurodegeneration, in this review, we aim to look at current evidence for mitochondrial dysfunction caused by Ca2+ miscommunication in neuronal models of neurodegenerative disorders related to aging, with special emphasis on AD.Age is the main risk factor for the onset of neurodegenerative diseases. A decline of mitochondrial function has been observed in several age-dependent neurodegenerative diseases and may be a major contributing factor in their progression. Recent findings have shown that mitochondrial fitness is tightly regulated by Ca Remarkably, all these processes are affected early during aging, AD pathogenesis, and other neurodegenerative conditions, suggesting a role for MAMs in the pathogenesis of these diseases . Likewise, loss of Sigma 1 receptor  has been shown to interfere with ER–mitochondria associations . Conversely, an increase in the lipidic enzymatic function of MAMs and their inter-organelle extension has been described in fibroblasts from patients with SAD, in human SAD brains, and in AD mouse models . Furthermore, MAMs are highly enriched in PS1 and PS2 proteins , a key protein in the formation of MAMs  and mitochondria couple at specific sites termed mitochondria-associated membranes (MAMs), which integrate and coordinate several cellular functions, including synthesis and exchange of phospholipid, apoptosis, mitochondrial dynamics, and Cadiseases . For exae models . Remarkae models , has recproteins  which whproteins , through of MAMs .2+ from the ER to mitochondria, resulting in either a mitochondrial Ca2+ overload, or a lack of Ca2+. If the transfer of Ca2+ is excessive, cell death occurs  Ca2+ channel by directed interaction with FAD-linked PS mutants . On the other hand, decreasing intracellular Ca2+ overload, specifically through a reduction of the InsP3R protein expression by 50%, normalizes FAD PS-associated Ca2+ signaling and rescues the biochemical, electrophysiological, and behavioral phenotypes observed in two different PS1-FAD animal models . Altogether, the above findings highlight the importance of MAMs and the transfer of Ca2+ from the ER to mitochondria in AD pathogenesis and their potential as a therapeutic target.Conditions that increase or decrease the extension of MAMs will affect the transfer of Cah occurs . If Ca2+ll death . Importand in AD . Upregulpse loss . Severaltor RyR; , or β-am sources . Another mutants , 2010 or mutants . In agre+ uptake . This in2+ transfer in long-term culture of hippocampal neurons, with correlates with an increase of the mitochondrial Ca2+ uniporter . Interestingly, a decrease in Ca2+ transfer to mitochondria and a dissociation of the MAMs have been described in cardiomyocytes from old mice suggesting that the type of modification that MAMs undergo with aging might be cell specific  takes place, which corresponds to a non-selective channel formed by a protein complex spanning the outer and inner mitochondrial membranes , and mitochondrial swelling  on neural stem cells  in AD mouse models and in post-mortem samples derived from AD human patients. The functional consequence of such an interaction is an increase in ROS production due to abnormal mitochondrial membrane permeability . Altered2+ buffer modulating cytoplasmic Ca2+ signal and hence, neurotransmission (2+ uptake capacity and increased propensity to undergo mPTP compared to non-synaptic mitochondria (2+ contribute to the impairment of mitochondria and synaptic formation remains to be explored, but given the dysregulation of Ca2+ observed during aging, AD, and other neurodegeneration, an important role is expected. Elucidating the factors that underlie early synaptic dysfunction will be key to prevent the widespread neurodegeneration associated with aging.Synapses are neuronal structures in which mitochondria are fundamental  by provismission . Synaptichondria . Likewischondria . Synaptichondria . The extchondria . Post-mochondria  and tranchondria . Along tchondria . Proteomchondria . Just rechondria . How ER 2+ is also altered in both conditions and can either energize or overload the rheostat depending on the concentration, understanding how MAM formation is regulated is important. Identifying the players that participate in the regulation to assure a proper Ca2+ transfer to mitochondria is critical in order to determine the real potential of this intracellular signaling platform as an intervention candidate to improve aging and hinder the onset of neurodegenerative disease such as AD.Aging continues to be the most relevant risk factor for AD, the most common form of dementia in the elderly, and other neurodegenerative diseases. Both aging and neurodegeneration are accompanied by a loss in the ability of the cells to adjust and rewire their metabolic networks to keep a tight balance between energy production and expenditure in an ever-changing environment. Mitochondria work as an adaptable metabolic control, a “rheostat,” that integrates inputs from the intra and extracellular environment to generate functional outputs that adjust cell behavior and energy production and consumption. Several lines of evidence suggest that mitochondrial function deteriorates with increasing age and the progression of several neurodegenerative diseases. This supports the notion that aging and the neurodegenerative diseases such as AD may share a common root, the failure of the rheostat program. Since CaMM, UA-C, and CC designed and outlined the structure and contents of the review. MM, UA-C, MS, FC, CG-B, and CC contributed to the literature review, discussion, and writing of the manuscript. All authors contributed equally to the draft revisions and final approval of the version to be published.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.


Cardiac mitochondria are essential for contractile function and regulation of cell viability, while mitochondrial dysfunction characterizes heart failure. The role of the UPRmt in the heart is unclear.The mitochondrial unfolded protein response  study the relationship among the UPRmt, mitochondrial function, and cardiac contractile function.The purpose of this study was to: 1) identify conditions that activate the UPRCultured cardiac myocytes were subjected to different stresses in vitro. Mice were subjected to chronic pressure overload. Tissues and blood biomarkers were studied in patients with aortic stenosis.mt in cultured cardiomyocytes. The UPRmt was also induced in the hearts of mice subjected to chronic hemodynamic overload. Boosting the UPRmt with nicotinamide riboside (which augments NAD+ pools) in cardiomyocytes in vitro or hearts in vivo significantly mitigated the reductions in mitochondrial oxygen consumption induced by these stresses. In mice subjected to pressure overload, nicotinamide riboside reduced cardiomyocyte death and contractile dysfunction. Myocardial tissue from patients with aortic stenosis also showed evidence of UPRmt activation, which correlated with reduced tissue cardiomyocyte death and fibrosis and lower plasma levels of biomarkers of cardiac damage (high-sensitivity troponin T) and dysfunction .Diverse neurohumoral or mitochondrial stresses transiently induced the UPRmt in the mammalian (including human) heart exposed to pathological stresses. Enhancement of the UPRmt ameliorates mitochondrial and contractile dysfunction, suggesting that it may serve an important protective role in the stressed heart.These results identify the induction of the UPR  The mammalian heart has the highest oxygen consumption rate of any organ, which may transiently increase several-fold during physiological exercise to support the increased cardiac workload. Chronic increases in cardiac workload resulting from hemodynamic overload or neurohumoral activation eventually lead to heart failure. Approximately 26 million patients worldwide are estimated to have heart failure, and the condition imposes substantial morbidity and mortality despite the use of therapies that target neurohumoral activation and cardiac rhythm disturbance Mitochondria are crucial for cardiac function through oxidative ATP generation to support muscle contraction and relaxation, the metabolism of nucleotides, amino acids and lipids, intracellular calcium buffering, and the regulation of cardiomyocyte survival. Mitochondrial dysfunction is a central feature of heart failure by contributing to energetic dysfunction, oxidative stress, calcium dysregulation, and cardiomyocyte death, and is considered a potential therapeutic target mt) is evolutionarily conserved and evoked when the mitochondrial protein folding environment is compromised and there is an accumulation of misfolded proteins mt is a mitochondrial-to-nuclear signal transduction pathway that leads to increased transcription of numerous mitochondrial protective genes, notably a repertoire of molecular chaperones, proteases, and antioxidant enzymes located primarily in the mitochondrial matrix mt serves to restore mitochondrial protein homeostasis, function, and cell survival. The UPRmt has been extensively characterized in C. elegansmt in the heart is unclear. Here, we identify the induction of the UPRmt in cardiomyocytes subjected to neurohumoral or mitochondrial stress and in the hemodynamically overloaded murine and human heart. We show that pharmacological enhancement of UPRmt with small-molecule agents ameliorates mitochondrial and contractile dysfunction in the overloaded murine heart, and in patients with aortic stenosis, elevated levels of UPRmt indexes correlate with reduced plasma biomarkers of cardiac damage and dysfunction. The UPRmt, therefore, has an important homeostatic role in the pathologically stressed mammalian heart and may be a potential therapeutic target in heart failure.The mitochondrial unfolded protein response , 2-way ANOVA, or repeated measures ANOVA as appropriate, followed by Bonferroni post-hoc testing. A p value <0.05 was considered significant.Data are presented as mean ± SEM, unless otherwise stated. Comparisons were made by Student’s mt involves the transcription factors Atf5 and CCAT-enhancer-binding protein homologous protein (CHOP), which increase the expression of mitochondrial chaperones and proteases The induction of the UPRmt induction in cardiomyocytes occurs as a process distinct from the endoplasmic reticulum unfolded protein response (ER stress) or cytosolic stress responses, we assessed the protein levels of KDEL sequence-containing markers of ER stress  and mRNA levels of the cytosolic chaperones Hsp70 and Hsp90 under these stress conditions. Protein levels of KDEL sequence-containing proteins were unaltered in cardiomyocytes treated with isoproterenol, paraquat, or Δ-OTC overexpression, whereas stimulation with tunicamycin, which induces ER stress, robustly increased the levels of these proteins  or indirectly via the pharmacological inhibition of polyADP-ribose polymerase (PARP), boosts the UPRmtPrevious studies showed that the enhancement of cellular NADmt in stressed cardiomyocytes, we compared the effects of Iso  stimulation on mitochondrial respiration with and without pre-treatment with NR or olaparib to enhance the UPRmt. Iso stimulation resulted in a significant decrease in mitochondrial maximal respiration, which was markedly improved when the UPRmt was boosted with NR , a clinical model of chronic pressure overload. The clinical and demographic characteristics of these patients are shown in mt markers among the AS group compared with control. Notably, approximately 50% of the AS subjects had substantial increases in the expression levels of these transcripts  based on their myocardial mRNA levels of UPRmt markers being higher or lower than the upper limit of “normality” (ULN). ULN was set as 2 SDs above the mean for each transcript in the control group. Subgroup A comprised AS patients with mRNA levels of UPRmt markers within the normal range, and subgroup B comprised those with mRNA levels of at least 3 UPRmt markers above the upper limit. Subjects in subgroup B in fact demonstrated significant elevation of all the UPRmt markers compared with subjects in subgroup A or control, indicating a significant activation of UPRmt in this cohort , activated the UPRproteins . We usedmt has been viewed as a cytoprotective response that promotes organelle recovery mt markers was typically transient. We therefore hypothesized that enhancing the UPRmt might elicit cytoprotective effects. Recent studies have identified agents that activate the UPRmt, thereby facilitating characterization of its functional impact under stress conditions. Notably, the augmentation of cellular NAD+ pools leads to the induction of the UPRmt, secondary to activation of NAD+-dependent deacetylases, and was shown to improve the outcome under conditions of mitochondrial stress+ pools can be achieved via dietary supplementation with NAD+ precursors such as NR, or through the inhibition of NAD+-consuming enzymes such as PARP. We confirmed that agents that augment the NAD+ pools, in particular NR, enhance the UPRmt in cardiomyocytes and that this occurs through the actions of Atf5. We found that the augmentation of the UPRmt led to significant improvement in mitochondrial dysfunction in stressed cardiomyocytes. Importantly, these effects were dependent on Atf5, indicating that they were likely to involve the UPRmt rather than other NAD+-dependent actions in the cell. To extend these data to the in vivo setting, we tested the effects of NR supplementation in mice subjected to TAC. We found that NR treatment markedly improved mitochondrial respiration, cardiomyocyte survival, and cardiac contractile function. These data provide proof-of-concept that enhancement of the UPRmt in the pathologically stressed heart may have therapeutic potential.Activation of the UPRmt and reduced indexes of ongoing myocardial damage. We found that AS patients who had an elevation of UPRmt markers had lower plasma levels of hs-TnT and N-terminal pro–B-type natriuretic peptide, both strong predictors of future adverse outcome independent of the resting LV function mt markers. These findings are consistent with the postulate from our experimental studies that increased activation of the UPRmt is beneficial by preserving mitochondrial function. In the human AS setting, a chronic improvement in mitochondrial function may manifest as a lower rate of cardiomyocyte death, with concomitant lower levels of fibrosis in the myocardium. Unlike the TAC setting, AS subjects with elevated UPRmt markers had similar cardiac function to those with low UPRmt levels, which may reflect the very slowly-progressive pathology of human AS compared with surgically induced TAC in mouse models mt in the pressure-overloaded human heart and suggest that increased activation of the UPRmt may be beneficial.To extend these findings to the human setting, we studied myocardial tissue from patients with severe AS undergoing valve replacement. A comprehensive molecular and histological analysis of myocardial tissue, clinical characteristics, and blood biomarkers revealed a striking relationship between enhanced activation of the UPRmt, may also have beneficial effects. It should be noted, however, that prolonged or dysregulated induction of the UPRmt could be detrimental, for example, contributing to an accumulation of defective mitochondria Activation of the ER stress response has been demonstrated to be an important pathophysiological mechanism in many human diseases mt activation and in different disease settings to better understand the therapeutic potential and/or toxicity of such interventions. Another limitation of our study is the fact that the human data are by necessity correlative and that the control subjects were not perfectly matched with the AS patients. However, there was a very good matching of most clinical characteristics between AS patients in the two subgroups.Future studies will need to investigate the effects of different durations of UPRmt in response to diverse in vitro and in vivo stresses in the mammalian myocardium. We also show that pharmacological boosting of the UPRmt preserves mitochondrial function and cardiomyocyte viability under pathological stress conditions relevant to heart failure. Taken together, the results suggest that the targeting of the UPRmt merits further investigation as a therapeutic strategy for heart failure.PerspectivesCOMPETENCY IN MEDICAL KNOWLEDGE: The detrimental consequences of chronic hemodynamic overload or neurohumoral stress in the heart include mitochondrial dysfunction and contractile depression. Activation of the UPRmt is able to mitigate these effects by restoring mitochondrial homeostasis.TRANSLATIONAL OUTLOOK: The UPRmt can be activated by small molecules that increase NAD+ levels. Additional studies are required to determine whether long-term enhancement of the UPRmt is beneficial in chronic hemodynamic overload.In summary, this is the first study to identify activation of the UPR


However, the underlying mechanisms are still not completely understood and due to the complexity, their elucidation remains a challenging. This review discusses the constitution of the AML niche in the bone marrow, the improvement in visualization of the complex three-dimensional niche structures and points out new therapeutic strategies to increase the overall survival of AML patients.The bone marrow is the home of hematopoiesis and is therefore a hotspot for the development of hematopoietic diseases. Complex interactions between the bone marrow microenvironment and hematopoietic stem cells must find a balance between proliferation, differentiation and homeostasis of the stem cell compartment. Changes in this tightly regulated network can provoke malignant transformation, leading to hematopoietic diseases. Here we focus on acute myeloid leukemia (AML), since this is the most frequent acute leukemia in adulthood with very poor overall survival rates and where relapse after chemotherapy continues to be a major challenge, driving demand for new therapeutic strategies. Current research is focusing on the identification of specific interactions between leukemic blasts and their niche components, which may be exploited as novel treatment targets along with induction chemotherapy. Significant progress has been gained over the last few years in the field of high-resolution imaging. Confocal  Leukemia is characterized by infiltration of the hematopoietic organs  by abnormally differentiated and nonfunctional hematopoietic blasts . High anAdult and elderly patients suffer mostly from the aggressive acute myeloid leukemia (AML) that shows an increasing incidence with age. Without therapy this disease leads to the death of the patient within a few weeks. Although the overall 5-year survival rate of AML patients improved by application of chemotherapy, it remains unsatisfactory. A positive outcome declines from 50% for those younger than age 65 years to only 23% for patients aged 65 years and higher . AlthougAML is a multi-gene disease with complex subclonal populations. After chemotherapy leukemic clones are selected to those capable of activating resistance mechanisms, leading to relapsed, and refractory disease . Therefode novo and acquired: where the latter is a sequential genetic change that finally ends in prosurvival and antiapoptotic phenotypes  and facilitates their differentiation into the manifold blood cell lineages . This stem cell-like compartment has alone long-term repopulating potential, the ability to propagate and maintain the AML phenotype, and is expected to be the main cause for AML relapse . First Lft model , 32. As n reside –35. Hencn reside . The corn reside . HoweverA number of studies have confirmed that there is a tight relationship between leukemic and endothelial cells. For instance Cogle et al. presented that vascular endothelial cells are a critical component of the AML niche and leukemic cells can integrate into the vasculature . FurtherMesenchymal stromal cells (MSCs) are known to provide microenvironmental support for HSCs but also have the potential for differentiation to multiple lineages including osteoblasts, chondrocytes and adipocytes. These processes are highly regulated and disturbances are involved in oncogenic events. Battula et al. showed that AML cells induce osteoblastic but inhibit adipogenic differentiation of MSCs by bone morphogenetic protein (BMP) signaling . This shThe role of adipocytes during leukemogenesis is also controversial. The existence of bone marrow adipocytes has been known for more than a century, but only recently has their function been deciphered. In contrast to the idea of silent fat storage, bone marrow adipocytes are highly active regulators of the bone marrow metabolism and the overall body homeostasis by secretion of adipokines . AdipocyAlthough bone marrow fibroblasts are a major component of connective tissue, knowledge about the role for AML development is very limited. It is known that fibroblasts produce extracelluar matrix fibers but also regulate the microenvironment by cytokine and chemokine secretion . FurtherThe immunological microenvironment of leukemic cells plays another fundamental role for leukemic development and chemotherapy resistance . The norIn addition to the cells of the adaptive immune system, mononuclear phagocytes contribute to the regulation of HSC populations . MacrophOne critical component for AML engraftment and progression is the adhesion to the microenvironment. Several adhesion molecules  were deciphered as playing a relevant role for AML . VLA- 4 Besides adhesion, AML cells are also regulated by soluble factors secreted by niche cells, like Notch, CCL3, TGF-β, or CXCL12 –61. We win vitro experiments, the exact mechanisms leukemia cells use for modifying their microenvironment to generate a shelter against chemotherapy still remain largely unknown.However, since most data are based on To understand how AML cells interact with their niches, it is crucial to know spatiotemporal cellular interactions and dynamics in the bone marrow. Quantitative analyses of bone marrow mononuclear cells by flow cytometry present a well-established technique and can deliver important hints of changes in cellularity but information about localization and cell-cell interactions get completely lost. Furthermore, extraction of structural cell types, such as endothelial or mesenchymal stromal cells for flow cytometry includes enzymatic tissue degradation steps, which can be inefficient and therefor significantly adulterate the data. Only imaging of native tissues enables the study of cellular interactions in the multicellular niche, without losing single components of the processed tissue.in vivo is the transgenic zebrafish (Danio rerio) (One powerful model to study the development of the hematopoietic system o rerio) . Hematopo rerio) . Howevero rerio) .In the last decades murine models became the main mammalian model system for hematopoietic research. Their convenient maintenance and handling, as well as their small body size, short reproduction cycle, and the ability for genetic manipulations are prerequisites for a biomedical research tool. The development of immunodeficient mice strains like NSG (NOD scid gamma) enables xenografting of patient-derived cells without pre-conditioning of the mouse with radiation or chemotherapy, allowing studies about interactions between human hematopoietic cells and their microenvironment . One majin situ imaging and quantitative analysis of fluorescent labeled cells within thin tissue sections  is the most high-resolution imaging technique to visualize surfaces of cells and tissues in a high-resolution manner independent of available antibodies or transgenic mouse lines. The resolution is greater than with a light microscope but information are limited to the two-dimensional surface. However, there are several SEM techniques that can obtain 3D information about a sample, enabling the reconstruction of tissue structures. Serial block face (SBF-SEM) and focused ion beam scanning electron microscopy (FIB-SEM) both allow serial sections by ablating the specimen either with a diamond knife or a beam of ions with subsequent alignment of the data to allow reconstruction of a three-dimensional volume. Within this image individual structure can be segmented and qualitative and quantitative information can be obtained . FIB-SEMin vivo antibody staining is not sufficient for direct imaging, cells of interest have to be labeled either ex vivo before transplantation or transgenic reporter mouse strains have to be generated. In particular, ex vivo treatments can influence a broad range of cellular functions like localization, division and cell-cell interactions. It is for this reason that intravital microscopy has thusfar been unable to completely replace ex vivo confocal 3D imaging and vice versa. The future will show how well-researchers will be able to combine these techniques to visualize both a detailed and dynamic picture of the bone marrow niche of hematopoietic and leukemic stem cells.In general, whole-mount imaging and computational modeling only allows visualization of snapshots of dynamic processes. The development of intravital microscopy solves this issue and has been used to study the behavior of HSCs and LICs in real time in the bone marrow. In 2005 Sipkins et al. presented two-photon microscopy data about the homing and engraftment of leukemic cells into the mouse calvarium bone marrow and described a specialized endothelial niche for acute lymphoblastic leukemia cells . This apin vivo is a sophisticated method to study leukemia-niche interactions. However, xenograft models never fully recapitulate the physiologic niche, since human cell engraftment requires the depletion of the immune system. Mice need genetic modification to deplete T-, B- and natural killer cells, and especially for AML a humanized microenvironment is necessary for the engraftment and development of the disease in vivo. But genetic engineering of mice is time and resource consuming and alternative models to study niche related pathways are required. One possibility to generate human-mouse chimeric bone marrow tissues is the usage of ossicle systems , which is covered by perivascular cells (pericytes). These pericytes are embedded in the subendothelial basement membrane and connect ECs with smooth muscle cells, which generally cover large vessels, like arteries and veins . Remarka+ and CAR cells. They form the dense and highly branched capillary network of sinusoids in the bone marrow cavity that is known to regulate the HSC compartment . Interestingly, high expression of the pro-angiogenic factor Ang2 is an independent prognostic factor for overall survival in AML . HoweverAn alternative approach may be to specifically harm the perivascular niche and the AML support by ECs with vascular disrupting agents . CombresBesides antiantiogenic approaches, other known niche components are under evaluation for targeted therapy. Developing CXCR4 inhibitors has made the most progress. The rational behind the blocking of this signaling pathway is to mobilize AML cells out of their protective niche to make them available for chemotherapy. Strong clinical evidence was shown in phase I studies for plerixafor (AMD3100), a small molecule inhibitor of CXCR4 that increased the rate of complete remissions in combination with cytarabine and daunorubicin but adverse events were frequent . Other CThe interaction between VLA-4 on AML cells and VCAM-1 on mesenchymal stromal cells represent another promising candidate for targeted therapy. Natalizumab was the first humanized VLA-4 monoclonal antibody tested in a xenograft AML model. Despite its beneficial effects on overall survival, its utility is limited since it can induce leucoencephalopathy , 132. AnLast, the therapeutic potential of the inhibition of E-selecting binding to CD44 should be noted. H90, an activating monoclonal antibody against CD44 and GMI-1271, a specific small molecule inhibitor of E-selectin, both showed a reduction of the leukemic burden in xenograft AML models , 134. ThThe immunosuppressive role of AML cells has a strong impact on chemotherapy survival and the causative mechanisms present a promising target for therapy. One approach targets the immune checkpoint molecules expressed by AML cells. Several immune checkpoints are initiated by ligand-receptor-interactions and therefore can be blocked with antibodies or blocking fragments of the receptor or ligand . UnfortuLastly, we want to mention the promising approaches with autologous or allogeneic T cells engineered with synthetic chimeric antigen receptors (CARs) . The artin vitro models that predict clinical drug responses. There is a strong demand for new agents with good tolerance in a broad range of AML patients, and increased efficacy relative to conventional chemotherapy. Any possibility to replace one or more components of treatment regimens in frail patients to reduce long-term effects on the maintenance of healthy HSCs would have significant clinical benefit. Technical developments of the past few years have yielded significant progress for understanding the physiological constitution of normal HSC niches and the malignant alterations that drive AML progression. However, the regulation of AML cells in their niche is highly complex and we have just started to understand the tremendous potential of targeting the bone marrow microenvironment to eradicate AML cells. Ongoing clinical studies and upcoming research results will point out new avenues of treatments, giving the hope that AML will 1 day become a treatable disease.There is an ever-growing number of targeted therapeutic strategies to treat acute myeloid leukemia, yet patient outcome remains poor and clinicians have an urgent need to receive new treatment strategies for patients. This result is likely due to the highly heterogeneous and highly polyclonal nature of AML and approaches that target specific mutations in AML cells may result in the eradication only of single subclones and therefor are inadequate to rid patients of the disease. Because complex interactions with the bone marrow microenvironment influence the survival and progression of AML, research is now focused on deciphering the bone marrow niche composition to identify new drugable targets. These interactions are less clone-specific and therefore represent a promising strategy to overcome the obstacles of individual cell heterogeneity. Furthermore, residual LICs are thought to drive disease relapse and targeting their sheltering niche might offer a means to improve eradication of AML. Unfortunately, blocking signaling pathways also harbors the risk of unpredictable side effects and toxicity. Most pre-clinical research is done in animal models to study drug effects on disease progression, and there are important limitations in translation of animal model results to human patients. Thus, researchers should seek to find good LB wrote the manuscript. WF and JW contributed to the writing and editing of this manuscript.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.


The ternary heterostructure was then used for the photocatalytic degradation of tetracycline in visible light, with an efficiency as high as 92% after 3 h of irradiation. Thus, the photodegradation efficiency is greatly improved compared to that of ultrathin g-C3N4, BiOBr, and black phosphorous quantum dots alone. The synthesized ternary heterostructure improves the charge separation efficiency, thus increasing the photodegradation efficiency. This work provides a new and efficient method for the degradation of antibiotics in the environment.Herein, we synthesized BiOBr/ultrathin g-C Given the increasing concern regarding environmental pollution due to antibiotics and growing awareness of anti-microbial resistance, the demand for new methods for antibiotic environmental remediation is increasingly urgent. The overuse of broad-spectrum antibiotics, such as tetracycline (TC) , has led3N4 is a lamellar, organic, non-metallic polymeric material formed from nitrogen and carbon in accordance with SP2 hybridization [3N4 one of the most suitable photodegradable antibiotic catalysts [3N4 only has high photocatalytic activity at a wavelength range above 460 nm. Therefore, there is a need to increase the photocatalytic activity range of g-C3N4 to achieve a visible light response and higher efficiency. This can be achieved through the production of new photodegradation catalysts based on g-C3N4. Several methods have been proposed to effectively enhance the activity of g-C3N4, for example, by reducing the thickness of g-C3N4 nanosheets and increasing their specific surface area or copolymerizing g-C3N4 with other semiconductors to form heterojunctions to accelerate the separation of electron (e−)–hole (h+) pairs and increase the lifetime of the photo-generated carriers [Cdization ,9. Its aatalysts ,11,12. Ucarriers ,14,15.3N4 nanosheet (UCN) by blowing dicyandiamide with ammonium chloride to increase its specific surface area [−–h+ pairs, have shown an enhanced catalytic degradation efficiency of UCN under visible light [Herein, we first synthesized an ultrathin g-Cace area ,17,18; tace area ,21,22,23le light ,25. Nevele light ,27,28.co-catalyst for visible-light-driven water splitting, and it has been demonstrated that the performance could be improved by loading BPQDs on the layered g-CN due to their unique optical and electrical properties [Recently black phosphorus has received considerable attention from the scientific community because of its excellent performance. It is a two-dimensional atomic material similar to graphene, with an energy bandgap that can be regulated by the number of layers, and with a wavelength absorbance ranging from visible light to the infrared . In addioperties .In order to further improve its photocatalytic performance, we developed a ternary heterostructure of BiOBr/UCN decorated with BPQDs for the photocatalytic degradation of TC with the required degradation efficiency.3)3·5H2O, analytical grade), potassium bromide , and TC  were purchased from Shanghai Aladdin Industrial Corporation . Anhydrous ethanol, dimethylformamide , and black phosphorus were obtained from Nanjing Materials Technology Companies .Ammonium chloride, dicyandiamide , bismuth nitrate pentahydrate (Bi(NOAmmonium chloride (20 g) and dicyandiamide (4 g) were dissolved in a suitable amount of water and stirred at 80 °C until the mixture dried out. The resulting material was then thoroughly ground and placed in a muffle furnace  and heated to 550 °C at a ramp rate of 3 °C per minute for 4 h. Finally, the product was fully ground, producing ultrathin layer UCNs ,17,18.First, 0.273 g of potassium bromide was dissolved in a suitable amount of deionized water and sonicated for 5 min; 0.3 g of UCNs were weighed, mixed into the above solution, and sonicated for 5 min. Finally, 1.113 g of bismuth nitrate pentahydrate was added to the above mixture and sonicated for 5 min. The above mixed solution was placed in a water bath at 80 °C for 3 h, and finally dried in a drying oven  to obtain the BiOBr/UCN photocatalytic system ,26,27,28Black phosphorus is a graphite-like material with excellent electron transfer properties. High-quality, low-layer black phosphorous nanosheets can be stripped by high-intensity ultrasound. Therefore, we measured 10 mg of black phosphorus and placed it in a volumetric flask  containing 10 mL of dimethylformamide, sonicated the mixture for 4 h using an ultrasonic cell crusher ; and then 1 mg/mL of BPQD dispersions were prepared.  as the positive electrode of the battery, and the sodium electrode as the negative electrode of the battery. Under the 3v voltage window, the constant current charge and discharge lasted for 2 weeks .) Next, 3N4 was observed  and 27.51° (002) were obviously weakened, indicating a change in the structural properties of UCN. The main diffraction peaks of pure BiOBr are located at 10.94°, 21.99°, 25.24°, 31.78°, 32.29°, 39.38°, 44.81°, 46.29°, 46.98°, 50.79°, 56.29°, and 57.20°, belonging to the (001), (002), (101), (102), (110), (112), (200), (004), (113), (104), (212), and (214) planes of BiOBr crystal diffraction square phase (JCPDS No. 78-0348). Following UCN and BiOBr doping at a mass ratio of 3:7, the peaks of planes (100) and (002) disappeared, the diffraction peak of BiOBr at the crystal plane (001) was weakened. Moreover, the (001) crystal phase peak observed for the BiOBr/UCN composite increased slightly in intensity following doping by BPQDs. Nevertheless, following the addition of BPQDs, the black phosphorus peaks did not appear, likely due to the minimal content [XRD was used to study the phase structure of BiOBr/UCN/BPQDs composite catalysts. The corresponding XRD patterns of g-Cobtained . Compare content ,27,35.3N4, UCN, pure BiOBr, BiOBr/UCN, and BiOBr/UCN/BPQDs were assessed by FT-IR spectroscopy  and N–H groups, respectively. The weak peak at 404.4 eV indicates that the CN atom was hybridized in the form of π bond. The O1s spectra of BiOBr and BiOBr/UCN/BPQDs  peak  peak g [27,29,−)–h+ pairs in the composites. The main emission peak was centered at 470.6 nm, with pure g-C3N4 showing the highest peak, followed by that of UCN, BiOBr/UCN, and finally BiOBr UCN/BPQDs 1/2 and photon energy hν (eV) to roughly calculate the band gap of UCN, a value of 2.4 eV was obtained, which is larger than that for pure g-C3N4 (2.2 eV). The light absorption edge of BiOBr was located near 430 nm, with a subsequent calculated band gap of 2.7 eV. Following the formation of a composite, the absorption edges of UCN and BPQDs fell between 430 and 500 nm, and increased sequentially, indicating that these composites are sensitive to and can work well under visible light [The optical properties of g-Ctroscopy . The absopy  [The catalytic activity of these five photocatalysts was measured according to the degradation of TC in water by visible light. For a typical photocatalytic measurement, 0.25 g of the photocatalyst was evenly dispersed in the solution of TC  through ultrasonication. Experiments were conducted in a 200 mL reactor, associated with a cooling water circulation system. Temperature of the system was controlled at 25 °C throughout the reaction. After reaching a state of equilibrium (30 min), the mixture was subjected to the xenon lamp  with 420 nm-cut filter. Suspension (4 mL) was sampled at given time intervals. Magnetic stirring was conducted before (the adsorption/desorption stage) and during the photocatalytic reactions. After centrifugation, the residual concentration of TC was monitored by the UV-vis spectrophotometer  Company, Shanghai, China). BiOBr/UCN/BPQDs composites showed the best photodegradation efficiency, with a degradation efficiency of up to 92% within 3 h  15,20,2,25,26. T (43.8%) ,37,38,39K is the pseudo first-order rate constant, C0 is the initial concentration of TC solution, C is the concentration of TC at times t.The kinetics of photodegradation of TC were then assessed at 30 min and at every 5 min thereafter to obtain the corresponding kinetic equation. The degradation curve of TC at 30 min a allowedK values corresponding to BiOBr/UCN/BPQDs, BiOBr/UCN, BiOBr, UCN, and pure g-C3N4 were 0.041, 0.038, 0.032, 0.025, and 0.017 min−1, respectively. Therefore, these results also indicated that had the best photocatalytic properties. The In order to assess the stability of the as-prepared BiOBr/UCN/BPQDs, we performed recycles of the photocatalytic experiments and XRD. The catalytic performance of BiOBr/UCN/BPQDs did not decrease significantly a, and th2−), hydroxyl radicals (·OH−), and e−–h+ pairs are usually involved. Therefore, t-butanol, N2, and potassium iodide were used to capture ·OH−, ·O2−, and h+, respectively. Electron paramagnetic resonance spectra indicated h+ and ·O2− were the key groups determining the catalytic degradation effect  and e––h+ pairs are the most active radicals produced during the degradation process. This highly efficient, environmentally friendly, and relatively simple to produce ternary heterostructure has potential applications in the degradation of antibiotics such as TC.The BiOBr/UCN/BPQDs tri-heterostructures in the above experiments were prepared by a sonication and hydrothermal method. Compared with BiOBr/UCN, pure g-C