Due to an improvement in life expectancy, the incidence of age-related neurodegenerative diseases has significantly increased. However, there is no effective protective treatment or therapy available but rather only very limited palliative treatment. Therefore, there is an urgent need for the development of preventive strategies and disease-modifying therapies to treat AD/PD. Because dysregulated Ca2+ metabolism drives oxidative damage and neuropathology in these diseases, the identification or development of compounds capable of restoring Ca2+ homeostasis and signaling may provide a neuroprotective avenue for the treatment of neurodegenerative diseases. In addition, a set of strategies to control mitochondrial Ca2+ homeostasis and signaling has been reported, including decreased Ca2+ uptake through voltage-operated Ca2+ channels (VOCCs). In this article, we review the modulatory effects of several heterocyclic compounds on Ca2+ homeostasis and trafficking, as well as their ability to regulate compromised mitochondrial function and associated free-radical production during the onset and progression of AD or PD. This comprehensive review also describes the chemical synthesis of the heterocycles and summarizes the clinical trial outcomes. Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the two most common neurodegenerative diseases in the elderly. The key histopathological features of these diseases are the presence of abnormal protein aggregates and the progressive and irreversible loss of neurons in specific brain regions. The exact mechanisms underlying the etiopathogenesis of AD or PD remain unknown, but there is extensive evidence indicating that excessive generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), along with a depleted antioxidant system, mitochondrial dysfunction, and intracellular Ca An impaired antioxidant system or aberrant and sustained free-radical formation can result in redox balance modifications and concomitant alteration in redox-sensitive signaling pathways. At the physiological level, brain reactive oxygen species (ROS) and reactive nitrogen species (RNS) are second messengers involved in intracellular signaling, but increased levels of free radicals cause harmful effects to biological macromolecules that contribute to the aging process and the pathogenesis of neurodegenerative diseases. Lipid peroxidation (LPO) consists of the abstraction of allylic hydrogen atoms from side-chains of polyunsaturated fatty acids (PUFAs) by ROS and RNS. A major deleterious outcome of LPO is the production of a variety of reactive aldehyde species, such as malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE). The reaction between O2•− and NO• leads to the formation of peroxynitrite, which targets tyrosine residues in proteins via free-radical addition to generate 3-nitrotyrosine (3-NT). Protein carbonylation is an oxidative stress-driven nonenzymatic and irreversible post-translational modification (PTM). Oxidative deamination of alkaline amino acids such as arginine, lysine, and histidine leads to protein carbonylation. Advanced glycation end products (AGEs) are generated in a nonenzymatic reaction involving lipids, proteins, or nucleic acids and reducing sugars. The interaction of AGEs with their receptors (RAGEs) causes oxidative stress. ROS/RNS may also interact with nucleobases of the DNA  to form 8-hydroxy-2-deoxyguanosine (8-OHdG) while oxidative RNA damage produces 8-hydroxyguanosine (8-OHG).The brain is particularly prone to oxidative insult due to a complex interconnected myriad of factors, such as high metabolic activity, neurotransmitter autoxidation, elevated content of redox active transition metals, modest antioxidant defense, glutamate excitotoxicity, and altered calcium  to form adenosine triphosphate (ATP). Moreover, PTMs involve enzyme-mediated covalent addition of specific functional groups  to proteins after their synthesis. Redox-related PTMs can modulate the activity of proteins implicated in a variety of cellular signaling pathways, such as protein folding and degradation, transcription factor expression and activity, and energy metabolism by regulating the tricarboxylic acid cycle (TCA) and glycolytic enzymes, fatty-acid metabolism, and protein cysteine thiol nitrosation, sulfenylation, or glutathionylation. Mitochondria along with the ER play an important role in controlling intracellular Ca2+ homeostasis, which regulates several neuronal processes, including synaptic plasticity, metabolic regulation, proliferation, gene expression, and apoptosis.Compelling evidence has demonstrated that the mitochondrial electron transport chain (ETC) is the major endogenous source of ROS/RNS generation, although the endoplasmic reticulum (ER) and peroxisomes can also be an important cause of free-radical production . Specifi2+ can behave as a pathological effector in these neurological disorders. Cytosolic and organelle Ca2+ overload promotes its mitochondrial accumulation, which triggers the opening of the mitochondrial permeability transition pore (mPTP) and the disruption of the mitochondrial membrane potential (ΔΨm). Sustained mPTP opening leads to Ca2+ release, mitochondrial depolarization, OXPHOS disruption, compromised structural and functional integrity of the inner mitochondrial membrane, and release of cytochrome c (CYT C) and other apoptotic proteins from the outer mitochondrial membrane -fluro-2-deoxyglucose demonstrated a progressive decline in cerebral glucose metabolism in AD patients cyt). This process is limited by endogenous Ca2+ buffers, particularly Ca2+-binding proteins  and Ca2+ extrusion systems, such as plasma membrane Ca2+ ATPases (PMCAs), sarcoplasmic and ER Ca2+ ATPases (SERCAs), and plasma membrane and Na+/Ca2+ exchangers (NCXs) that remove Ca2+ from cytosol to the extracellular space or intracellular organelles (mainly ER). Mitochondria also influence Ca2+ levels and signaling. Cytosolic Ca2+ enters the mitochondrial matrix through the mitochondrial Ca2+ uniporter (MCU) complex, a process driven by the ΔΨm. Importantly, subtle modifications in the ΔΨm may considerably modify mitochondrial Ca2+ uptake ability cyt, as well as Ca2+ store and release from the ER, simultaneously with an increased Ca2+ trafficking from the ER into mitochondria. Aged neurons exhibited a significant decrease in the store-operated Ca2+ entry (SOCE), a pathway related to dendritic spine stability and memory storage. Ca2+ homeostasis disruptions found in aging neurons may promote energy production but with the potential risk of increased susceptibility to mitochondrial Ca2+ overload, reduced spine stability, and neuronal loss. There is a correlation between these functional changes and altered expression of the IP3, NMDA, and TLR4 receptors, the mitochondrial MCU, and the plasma membrane molecular players involved in SOCE cyt oscillations were abolished by different antagonists of synaptic transmission (NMDAR) and antagonists of action potential propagation (tetrodotoxin). Our results suggest the involvement of both NMDAR and amyloid channels in the primary response to Aβ oligomers, further enhanced by networking activity. In support of this notion, we also demonstrated that non-neuronal cells expressing NMDAR exhibit Ca2+ responses to Aβ oligomers, in contrast to cells lacking NMDA. Expression of NMDAR subunits NR1/NR2A and NR1/NR2B in these cells restored Ca2+ responses to NMDA but not to Aβ oligomers, indicating that Aβ oligomers may promote Ca2+ entry via amyloid channels and NMDAR. Aβ oligomers may also activate distant neurons intertwisted by synaptic connections, spreading excitation by recruiting additional NMDAR and specific VOCCs, and leading to excessive Ca2+ entry, excitotoxicity, and neuron degeneration in AD -fluro-2-deoxyglucose PET imaging, glucose hypometabolism was detected in the cerebral cortex of individuals with early-stage PD and the hippocampus and in the temporo-parietal and occipital regions of PD dementia (PDD) patients cyt oscillations that can promote mitochondrial OXPHOS activity, which may render them vulnerable to stressor-mediated damage, including genetic mutations, mitochondrial toxins, and defective aging  the maintenance of a massive axonal tree that requires anterograde/retrograde transport of cytosolic metabolites and organelles, (ii) the maintenance and propagation of action potentials (APs) and the restoring of ionic gradients, and (iii) the large number of synaptic vesicles cycling. DA neurons display large undeca-7,9,11-trien-4-yl]ethanoate)  in 1990 . The fir in 1990 . Schwartlubility . Enantio% yield) .18 in the synthesis of 3  indole) 4 was originally synthesized using the Fischer indole reaction. Nevertheless, a more recent synthesis reported by Zheng et al. used p-toluidine 23 and 2-methyl-5-vinylpyridine 24 as commercial starting materials and achieved the desired product 4 in four reaction steps, with a ~16% overall reaction yield [p-tolyl)hydrazinyl)ethyl)pyridine 26 and 1-methylpiperidin-4-one 27 was performed at reflux temperature with 80% HAc ethyl]-1on yield . Cycliza 80% HAc .4 was also produced employing an efficient ruthenium(III) catalysis [32 from ortho-substituted aryl azide 31 catalyzed by RuCl3·nH2O. The total synthesis comprised six steps with an ~47% overall yield  [2+]cyt concentration in cortical neurons of 3xTg-AD or APPsw mice [2+ trafficking. Nevertheless, Ca2+ current was limited by nifedipine [2+ influx, negatively modulating L-type VOCCs. STIM1-KO cells displayed an elevated Ca2+ entry in response to membrane depolarization, which was nifedipine-sensitive. Hippocampal neurons from 5xFAD mice exposed to nifedipine inhibit abnormal VOCC and store-operated Ca2+ (SOC) entry, a process regulated by STIM1. Nifedipine inhibited mutant tau-induced intracellular Ca2+ responses to membrane depolarization [2+ influx, generation of ROS, and overall toxicity.Nifedipine (Procardia) is a first-generation dihydropyridine Catoxicity . Up to dg., PS1) . Treatmeg., PS1) . Treatmeg., PS1) . NifedipPsw mice . An age-fedipine . The strrization . Further2+ entry through the high-threshold potential nifedipine-sensitive L-type VOCCs [2+ that interacts with nifedipine, resulting in Ca2+-mediated CREB phosphorylation and c-fos gene expression [2+ spike (HTS) and the slow oscillatory potential (SOP) are diverse sources of Ca2+ conductance that play an important role in the generation of action potentials in SN DA neurons. While nifedipine showed a slight inhibitory effect on HTS, the molecule was able to block SOP. Moreover, nifedipine steadily abolished the spontaneous firing pattern [2+ homeostasis and signaling, synaptic function, oxidative insult, and apoptotic cell death. Oral administration of nifedipine had a safety profile at doses of up to 50 mg/kg in rats over a period of 13 weeks. In dogs, no damage was detected up to 100 mg/kg dosage for a period of 4 weeks. Rats tolerated daily intravenous administration of 2.5 mg/kg nifedipine over a period of 3 weeks while dogs tolerated up to 0.1 mg/kg nifedipine for 6 days. An overdose of nifedipine can induce severe hypotension, systemic vasodilation, and reflex tachycardia. Data derived from a single available clinical study found no evidence of correlation between nifedipine and PD prevalence. However,pe VOCCs . Althougpression . Subcutapression . Nifedippression . The hig pattern . Nifedip pattern . In summ5 -1,4-dihydropyridine-3,5-dicarboxylate) was reported by Singh et al.[35 as the β-keto ester, 2-nitro-benzaldehyde 36 as the aldehyde, and ammonium acetate as the nitrogen donor [The first synthesis of nifedipine gh et al.using an en donor   at 0.167 mL·min−1 to obtain nifedipine 5 in 71% yield  and CO2 in aqueous solution  is a Car’s type . A syster’s type . Long-teproteins . Nimodipproteins . The ageg memory . Nimodipg memory . Nimodipg memory . Exposurmulation . In addiu levels . Tau ovemodipine . Further2+ content worsened Aβ-mediated behavioral impairment and defective chemotaxis, but exposure to nimodipine extended lifespan and rescued motor lesions, synaptic deficiencies, and DAergic degeneration [+-induced increase in cytosolic Ca2+ content, mitochondrial depolarization and fragmentation, and neuronal death [• and LPS-activated microglia [•−). In summary, the L-type Ca2+ channel blocker antagonist nimodipine shows beneficial effects on AD and PD. The molecule ameliorates behavioral outcome, increases synaptic transmission and neuron survival, improves mitochondrial function, and attenuates oxidative stress and inflammation. Nimodipine is, in general, well tolerated although sensations of warmth and skin reddening can occur. High concentrations of the nimodipine can result in reduced blood pressure, headache, nausea, muscle weakness, and gastrointestinal issues [To date, there have been no clinical trials with nimodipine in individuals with PD. Elevated Caneration . Subcutaneration . Exposurneration . Moreoveneration . Nimodipal death . In addial death . Nimodipal death . Nimodipal death . Exposural death . Nimodipicroglia . Nimodipl issues . Isolate6 and other bioactive dihydropyridines [N-tethered enamino component 42 with the corresponding 2-benzylidene α-keto ester 43 provided the conjugated enamine 44. This intermediate was treated with trifluoroacetic acid to obtain the free enamino ester, which spontaneously cyclizes in solution to obtain the desired product 6  5-propan-2-yl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate) yridines . Condensroduct 6 .48 and 2-methoxyethyl-2-(3-nitrobenzyl-idene)acetoacetate 49. Cyclocondensation of both compounds employing iPrOH as the solvent, in the presence of hydrochloric acid, afforded the final product 6 in high yield and purity (More recently, due to a considerable demand of nimodipine in the Russian market, Pharm. Sintez Co. developed a new methodology to produce the drug in pilot batches . The appd purity .2+-induced toxicity, which are important factors in the neurodegenerative process described in AD and PD. According to the combination of preclinical and clinical data, there is a general consensus that development of therapeutic interventions targeting mitochondrial Ca2+ signaling may slow or stop the progression of neurodegenerative diseases, including AD and PD. A set of strategies to normalize mitochondrial Ca2+ homeostasis and signaling has been described. One potential approach involves Ca2+ uptake blockade through the L-type VOCCs, which inhibits Ca2+ transient at either presynaptic or postsynaptic sites and attenuates glutamate-mediated excitotoxicity. Selective antagonists of Cav1.2–1.3 L-type VOCCs also exhibit beneficial properties since prolonged opening of L-type channels results in augmented Ca2+ release and influx, as well as its accumulation in DA neurons. The capability of heterocyclic agents to modulate mitochondrial Ca2+ content and signaling, and their ability to preserve mitochondrial biogenesis and function provide a plausible biological basis for their neuroprotective effect (Multiple candidate drugs for treating AD or PD have failed over the last years. Further research is still needed to determine the therapeutic time window and the stage of the disease that should be used in clinical trials. Neuropathological and biochemical assessments support the notion that mitochondrial dysfunction causes an increase in free radicals and Cae effect .


Drug resistance and poor treatment response are major obstacles to the effective treatment of acute myeloid leukemia (AML). A deeper understanding of the mechanisms regulating drug resistance and response genes in AML is therefore urgently needed. Our previous research has highlighted the important role of nuclear factor E2-related factor 2 (NRF2) in AML, where it plays a critical role in detoxifying reactive oxygen species and influencing sensitivity to chemotherapy. In this study, we identify a core set of direct NRF2 targets that are involved in ferroptosis, a novel form of cell death. Of particular interest, we find that glutathione peroxidase 4 (GPX4) is a key ferroptosis gene that is consistently upregulated in AML, and high expression of GPX4 is associated with poor prognosis for AML patients. Importantly, simultaneous inhibition of NRF2 with ML385 and GPX4 with FIN56 or RSL3 synergistically targets AML cells, triggering ferroptosis. Treatment with ML385 + FIN56/RSL3 resulted in a marked reduction in NRF2 and GPX4 expression. Furthermore, NRF2 knockdown enhanced the sensitivity of AML cells to the ferroptosis inducers. Taken together, our results suggest that combination therapy targeting both NRF2 and GPX4 may represent a promising approach for the treatment of AML.The online version contains supplementary material available at 10.1186/s40164-023-00411-4. Acute myeloid leukemia (AML) is the most common type of leukemia in adults, characterized by the uncontrolled proliferation of myeloid cells in the blood, bone marrow, and other tissues –4. DespiNuclear factor E2-related factor 2 (NRF2) is a crucial regulator in the anti-oxidative stress pathway . HoweverFerroptosis is a newly identified form of non-apoptotic cell death that is characterized by iron-dependent activation of lipoxygenase and subsequent lipid peroxidation (LPO), and it has emerged as a critical factor in various types of cancers –20, inclIn this study, we identified a novel mechanism by which NRF2 activation leads to overexpression of GPX4 in AML, resulting in chemoresistance and evasion of ferroptosis. By using various ferroptosis inducers and inhibitors, they have shown that NRF2-deficient AML cells are particularly sensitive to ferroptosis inducers and that NRF2-induced GPX4 supports AML cell survival against drug treatment. The study also demonstrates that combined inhibition of NRF2 and GPX4 can significantly reduce the expression of both genes, thereby facilitating ferroptosis. These findings provide important insights into the mechanism of chemoresistance in AML and suggest that targeting NRF2 and GPX4 may be a promising therapeutic strategy for the treatment of this disease.Bone marrow (BM) samples were obtained from sixteen patients with newly diagnosed AML .AML cell lines HL60, MV4;11, KG1a, U937, HEL, Molm13, NB4, KG1 and Kasumi-1 were cultured in RPMI 1640 (Invitrogen) containing 10% fetal bovine serum (FBS). HEK293T cells were cultured in DMEM (Invitrogen) medium containing 10% FBS. All these cell lines were cultured in a humidified atmosphere containing 5% CO5 MV4;11 cells were co-cultured with virus supernatant plus 8 µg/mL polybrene . Then 5 µg/mL puromycin was applied to select positively infected cells for at least 5 days. The shRNA sequences are listed in Supplemental Material Specific oligonucleotides directed against human NRF2 were designed and cloned into the pLKO.1-puro plasmid according to the protocol recommended by Addgene, and empty pLKO.1-puro plasmid served as control. Lentiviruses carrying pLKO.1-puro were prepared by the transfection of standard packaging vectors into HEK293T cells using lipofectamine 3000 . Viral supernatant was harvested 24 and 48 h after transfection. A total of 2 × 10For analysis of cell death, cells were harvested, and the Annexin-V-APC/PI Detection Kit  was used following the manufacturer’s protocols and performed as previously described , 26. ForTotal RNA from cells were extracted using TRIzol reagent  following the manufacturer’s instructions and was reversed transcribed into cDNA using High-Capacity cDNA Reverse Transcription Kits . Quantitative real-time PCR (qRT-PCR) was performed with SYBR Green  in a total volume of 20 µl using the following program: 95°C for 5 min followed by 40 cycles of 95℃ for 30s, 60℃ for 30s, and 72℃ for 30s. Relative target gene expression levels were normalized to the mRNA level of β-actin. The primers for qRT-PCR are shown in Supplemental Material Cells were collected and lysed in 1х SDS loading buffer containing protease inhibitors. Protein lysates were separated using 12% SDS-PAGE and subsequently transferred to polyvinylidene fluoride membrane  following by blocking with QuickBlock™ blocking buffer  and incubation with antibodies directed against GPX4  or NRF2  overnight at 4℃. Anti-β-actin antibody  served as control. After incubation with a secondary antibody, ECL  was used to amplify the binding antibody signal, and images were obtained with a UVITEC photo documenter. The full gel images in the paper figures are displayed in Figure 5 cells were suspended in a volume of 10 µl with 100 pmol oligonucleotides and transfected three times in each 12-well plate. After electroporation, cells were cultured in RPMI 1640 medium supplemented with 10% FBS at 37 °C and 5% CO2 for 24 h, and subsequently divided into two groups with or without drug treatment. The cells were then collected for protein level detection and evaluation of cell death. Supplemental Material The Neon® Transfection System (Invitrogen) was used to transfect 100 pmol oligonucleotides into MV4;11 cells in a total volume of 10 µl , 28. In 2 in a cell incubator away from light for 60 min. The cells were then washed twice with PBS, resuspended in 200 µL of fresh PBS, and analyzed using flow cytometry.To detect LPO, C11-BODIPY 581/591  was employed following the manufacturer’s instructions. In brief, cells were labeled with C11-BODIPY (2.5 µM) in 400 µL RPMI 1640 medium and incubated at 37 °C and 5% CORNA from LV-NC and LV-NRF2 MV4;11 cells was extracted using TRIzol reagent. Amplified cDNA was sequentially converted into short-read sequencing libraries using the VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina® Hisat2 (version: 2.0.4). Sequencing was performed by Novogene Co. Ltd., Tianjin Biotechnology Corporation. The expression level of each gene was calculated as fragments per kilobase of exon model per million mapped reads (FPKM).p value < 0.05 was considered significant. *, p < 0.05, **, p < 0.01, ***, p < 0.001.Unless otherwise stated in the figure legends, all data are presented as the mean ± SD of three independent experiments. Statistical analysis was conducted using SPSS 22.0 software on data from independent biological replicates. Student’s t-test was used to determine the significance of differences between two groups. One-way ANOVA followed by either Bonferroni or Dunnett’s post hoc test was applied to compare three or more groups, as specified in the figure legends. A Our recent studies have demonstrated that NRF2 promotes the expression of key molecules involved in AML development and drug sensitivity. To identify potential therapeutic targets for AML, we performed a broad screening of direct NRF2 target genes Fig. a. Our hyUsing qRT-PCR analysis, we validated that several of these genes are indeed transcriptional targets of NRF2 in AML cells Fig. f-g. AmonTo confirm the regulatory role of NRF2 on GPX4 in AML, we used western blotting to detect changes in GPX4 expression after manipulating NRF2 expression. As shown in Fig. It has been reported that RSL3 and FIN56 can specifically inhibit GPX4 to induce the ferroptosis of tumor cells , 29, 30.Further studies revealed that treatment with ML385 and a GPX4 inhibitor significantly increases the cell death of MV4;11 and Kasumi-1 cells Fig. b. FurthePrevious studies have shown that GPX4 plays a crucial role in ferroptosis and chemoresistance in human cancers. Activation of the NRF2 pathway plays a dominant role in protecting human cancer cells against cell death. To investigate potential synergistic effects of ML385 and GPX4 inhibitors on ferroptosis, we assessed the combined impact of these agents on lipid peroxidation levels using the C11-BODIPY probe. As depicted in Fig. To further investigate the synergistic effects of NRF2 and GPX4 inhibition, MV4;11 and Kasumi-1 cells were treated with ML385 or GPX4 inhibitor alone or ML385 combined with GPX4 inhibitor, and the expression of NRF2 and GPX4 was detected by western blotting. As shown in Fig. In this study, we made three significant observations. First, the NRF2 protein induced high constitutive GPX4 levels in AML. Second, GPX4 is one of the most important NRF2 target genes in AML. Third, NRF2/GPX4 attenuates ferroptosis in AML.Previously, we established that NRF2 plays a vital role in the detoxification of ROS and the resistance to chemotherapy in AML . As suchElevated GPX4 levels are frequently observed in many types of cancer, implying that it may be protumorigenic. Our study has found that GPX4 is highly expressed in AML cells, and elevated GPX4 levels correlate with poor prognosis in AML patients. This suggests that GPX4 may play a critical role in the development and treatment of AML. GPX4 functions as a central defensive enzyme against ferroptosis, and previous studies have indicated that NRF2 may regulate GPX4 expression and be related to ferroptosis in solid cancers , 33. SimChemotherapy-induced cell death is often associated with the generation of ROS –36. HighOur investigation has revealed that the expression of GPX4 in AML is likely attributed to the activity of NRF2 in AML cells. Furthermore, we have demonstrated that the NRF2/GPX4 axis plays a crucial part in ferroptosis and the antileukemia response. Based on these findings, our study provides supporting evidence and a basis for the targeting of NRF2/GPX4 as a strategy for developing therapeutics to improve chemo-sensitivity in AML.Below is the link to the electronic supplementary material.Supplementary Material 1. Clinical information and primer sequences.Supplementary Material 2. The KEGG enrichment of 349 NRF2-Chip intersection genes.Supplementary Material 3. The information of 70 candidate genes.Supplementary Material 4. The top 20 KEGG enrichments of the 70 candidate genes.Supplementary Material 5. Supplementary figures.


Even if it seems to be an important anatomical parameter for tissue regeneration, few studies in literature evaluate the “mean measure” of root divergence. Most of them are linear measurements, which hardly describe the dental furcation conformation in its entirety. It is left to the subjectivity of the operator deciding whether a furcation is convergent or divergent. The goal of this study is to create a visual evaluation method using specific measurements applied on endo-oral X-rays to overcome these problems, giving a conformation of the entire interradicular space and its divergence.A user-friendly software  was used to take three different measurements on endo-oral radiographs of upper and lower molars. Three blind operators measured 20 radiographs, to analyze the intra- and inter-operator reproducibility of the measurements. Then, the technique was repeated on 250 radiographic images to identify an average value and define a main conformation of the interradicular space. The ratio of these three measurements allowed to develop a new visual evaluation method of the interradicular space.Intra and inter-operator reproducibility was statistically confirmed on a sample of 20 anonymous endo-oral radiographs measured by 3 blind operators, indicating that the measurement technique was not operator dependent. Measurement made on 250 X-rays obtained with this technique permitted to subdivide in five groups the conformation of the interradicular space and define a mean value of the interradicular space.A new anatomical evaluation of the interradicular space in its entirety, which could help the clinicians in diagnostic and decisional phase in the therapy of furcated molars, can be obtained.A pre-operative evaluation of interradicular space conformation could affect therapy treatment choice. Molar teeth with furcation defects are considered one of the main challenges in periodontal therapy. While Class I furcations are generally maintainable with non-surgical therapy alone , Class ITherefore, the aim of the present study is to propose a method for describing roots conformation and to test its intra- and inter-rater reliability.This is an intra-rater repeatability and inter-rater test–retest reliability study. Three blind operators measured 20 intraoral radiographs of upper and lower molars to evaluate the reproducibility of the measurement technique. A pre-calibrated operator measured 250 intraoral radiographs of lower molars for the definition of medium radicular space in molars vestibular furcation. The X-rays were collected using the DBSWIN software  in a progressive numbering archive.Periapical radiographs of upper and lower molars were retrieved. On upper molars, only the vestibular furcation was evaluated.Radiographs made with paralleling techniqueGood image definition (clearly distinguishable roots margins)Complete view of the interradicular spaceComplete view of root apicesRadiographs were included in the present study if they met the following inclusion criteria:Radiographs with extreme roots overlapping, incomplete view of interradicular space, or any other reason which made the images unreadable were excluded.D)Furcation roof width (T)Interradicular furcation width (L)Interradicular vertical septum length , a vertical line was drawn from the roof of the furcation to the midpoint of an imaginary line passing through both root tips. This line reflected the vertical length of the interradicular septum. A second horizontal line was drawn within two thirds coronal of the interradicular septum, where the interradicular septum had the maximum width. Lastly, an “osculating circle” was traced along the curvature of the furcation roof to extrapolate the width of the roof itself. The osculating circle  and the length of the line from the roof of the furcation to the midpoint of an imaginary line passing through the tips of the roots (L) and between the line drawn in the maximum width of the interradicular septum (T) and the line from the roof of the furcation to an imaginary line passing through the tips of the roots (L) . Ratios are created as follows: between the diameter of the circle ((L) Fig. .Fig. 2A D/L ratio reflects please consider replacing “express” with “reflects” the roof furcation width (RFW), and the T/L reflects same as before the interradicular furcation width (IFW).The D, T, and L and calculated their ratios at time 0 (T0). After 30 days (T1), the operators repeated the same evaluation on the same 20 cases. All the measurements were made independently each operator from the other.Three dentists , without previous knowledge of the selected cases, blindly assessed furcation divergence with the proposed evaluation system on a sample of 20 periapical X-rays. The 3 operators carried out the measurements of D, T, and L and the ratios D/L (RFW) and T/L (IFW). The ratios were then statistically analyzed and an average measure of the reports, reflecting the interradicular space, was created, based on the statistical distribution of the measurements collected.A sample of 249 periapical radiographs was collected by a pre-calibrated operator (A.C.), who measured The reliability of the novel evaluation of furcation divergence was explored among the same examiner at different timepoints (intra-reliability) and between different examiners (inter-reliability) on 20 periapical X-rays. Bland–Altman plots were constructed to investigate the intra-rater and inter-rater operator reliability. A generalized linear model was used to evaluate the repeatability of the two measurements in relation to the operators (fixed effects) and the measurements carried on the same objects (random effects).R-square) of 83.4% and that the investigator seems to have a significant role in determining the measurements (p < 0.01). Examiner 1 was found to give statistically significantly different measurements compared to operator 2; however, the difference between the two operators, and overall, among each operator, are < 5%.The linear generalized model for RFW as dependent variable showed a variability (R-square) of 94.8%, with the operator having a significant role in the model (p < 0.05). A statistically significant difference between examiner 1 and 3 was observed (p < 0.05); however, this difference was within 5%.The linear generalized model for IFW as dependent variable showed a variability (p > 0.05). Although a statistically significant different was found between the measurements of two examiners for RFW and IFW, these differences were within 4 hundredths of a millimeter  and can therefore be considered not clinically relevant and negligible   does notSubdivision in quintiles of the range of measurements of RFW and IFW Table , showed Among the limitations of the present study, the lack of standardization of the periapical X-rays utilized to conduct the reliability study and the determination of the “mean furcation configuration” must be mentioned. Also, it is not possible to apply this method to the palatal furcation of upper molars due to overlapping of the mesial and distal roots. In addition, the sample size is relatively limited and represent a specific population only and therefore further studies are needed to explore the generalizability of this method in other populations. Lastly, although this preliminary study provides the basis for utilizing this evaluation system in everyday case scenarios, clinical studies are needed to evaluate whether the root/furcation anatomy, described with the proposed method and ratios, affects the outcomes of periodontal therapies.The present study introduced a new anatomical evaluation of the interradicular space, which could help clinicians and researchers when searching for an objective quantitative description of the furcation configuration/anatomy. This visual evaluation method has proven to be reliable and consistent among the same operators and different examiners. Subdivision in quintiles of the range of measurements could establish a classification of the interradicular area based on the value of more representative interradicular space. The ability to measure these values and to classify them objectively may allow to weigh their influence on bone regeneration and improve the way bone defects are managed. Further studies are needed to establish a possible positive correlation with the regenerative potential.


Until a few years ago, the onset of acute myeloid leukemia (AML) was entirely ascribed to genetic lesions in hematopoietic stem cells. These mutations generate leukemic stem cells, which are known to be the main ones responsible for chemoresistance and relapse. However, in the last years, increasing evidence demonstrated that dynamic interplay between leukemic cells and bone marrow (BM) niche is of paramount relevance in the pathogenesis of myeloid malignancies, including AML. Specifically, BM stromal niche components, such as mesenchymal stromal cells (MSCs) and their osteoblastic cell derivatives, play a key role not only in supporting normal hematopoiesis but also in the manifestation and progression of myeloid malignancies. Here, we reviewed recent clinical and experimental findings about how genetic and functional alterations in MSCs and osteolineage progeny can contribute to leukemogenesis and how leukemic cells in turn generate a corrupted niche able to support myeloid neoplasms. Moreover, we discussed how the newest single-cell technologies may help dissect the interactions between BM stromal cells and malignant hematopoiesis. The deep comprehension of the tangled relationship between stroma and AML blasts and their modulation during disease progression may have a valuable impact on the development of new microenvironment-directed therapeutic strategies, potentially useful for a wide cohort of patients. In recent years, there have been major advances in our understanding of AML, including new knowledge about the molecular pathogenesis, leading to an update of the disease classification,3 technological progress in genomic diagnostics and assessment of minimal residual disease,5 and the successful development of new therapeutic agents.6 Disease-specific chemotherapy regimens and consolidation with hematopoietic cell transplant (HCT) have enhanced AML outcomes, reaching an overall survival of ~60% at 5 years across different studies,8 and chemo-free regimens have opened new possibilities for elderly patients not eligible for chemotherapy treatment.9 Despite that, AML patients still face a risk of treatment-resistance and relapse that severely affects the outcome. Furthermore, the application of new targeted therapies into clinical practice remains challenging due to the high complexity of this disease, including multiple driving mutations and the coexistence of several competing tumorigenic clones. These persistent difficulties necessitate the identification of innovative therapeutic approaches that are effective for a larger cohort of AML patients.Acute myeloid leukemia (AML) is a hematopoietic malignancy originating from hematopoietic stem cells (HSCs) and progenitor cells. The annual incidence is around 2.4 cases per 100,000 individuals and it increases progressively with age, to a peak of 12.6 per 100,000 in adults of 65 years of age or older.10 LSCs are characterized by quiescence and self-renewal capability, which make them able to induce AML onset when transplanted in secondary and preferentially tertiary recipient mice and to sustain cell survival in optimized ex vivo co-culture systems.11 LSCs arise from the accumulation of somatic DNA mutations in HSCs in genes involved in quiescence, cell cycle, and cell proliferation , self-renewal potential, differentiation capability (NPM1 and CEBPA), and epigenetic regulation such as DNA-methylation-related genes .12 Advances in cancer genomics have revealed that most AML genomes display a small number of mutations, which are acquired in a stepwise manner (named the two-hit theory).13 LSC therapeutic targeting and elimination are mandatory to improve the dismal AML outcome. However, LSCs not only share surface markers with HSCs and AML blasts, but also present a large heterogeneity within and among patients, and thus the identification of therapeutic LSC-specific targets is challenging.14The main cause of relapse in AML patients is the presence of chemoresistant leukemic stem cells (LSCs) nested in the bone marrow (BM) niche.15 Indeed, malignant cells reside within the niche and extensively communicate with its components. These interactions contribute to the initiation of malignancy, support of disease progression, resistance to chemotherapy, and loss of normal hematopoiesis.The BM niche is a complex network of molecular and cellular entities composed of mesenchymal stromal cells (MSCs), endothelial cells, osteoblastic cells, nerves from the sympathetic nervous system, and accessory cells . It plays a role not only in BM homeostasis but also in hematological malignancies, such as myeloid diseases.This review describes the latest findings regarding the interplay between myeloid malignancies, such as AML, MDS and MPN, and the BM stromal niche, focusing on clinical and experimental aspects that support the pivotal role of the MSCs and their osteoblastic progeny in supporting disease initiation and progression. Unraveling the molecular basis of these interactions, by using new sophisticated imaging and sequencing technologies, is a prerequisite for identifying new targetable cell-extrinsic factors in the BM niche that, in addition to the classical targeting of leukemic cell-intrinsic mechanisms, can improve the therapeutic benefit for these patients.17 Regarding myeloid diseases, the most striking evidence of BM niche alteration in patients is the BM fibrosis that occurs during the development of MPN, especially in myelofibrosis (MF). Although intrinsic MSC alterations have been observed in MF patients,19 pathological HSCs play a pivotal role in subverting the BM niche.20 This concept is reinforced by data from patients that recovered after HCT where a complete response is defined by the complete regression of BM fibrosis.21The BM niche can become altered during hematopoietic malignancies, as it has been demonstrated in multiple murine models of leukemia and has been very well-described in patients with multiple myeloma.22, the authors analyzed the size of marrow adipocytes in BM sections from 70 patients with primary AML, observing a correlation between increased amounts of small BM adipocytes and a poorer prognosis. Another study showed that BM biopsies of AML patients present a significant increase of CD271+ MSCs, which in turn enhance the production of reticular fibers,23 commonly associated with therapy failure and decreased overall survival.24 Several studies reported an increased number of vessels and other vascular abnormalities in AML patient-derived BM biopsies, raising the interest in antiangiogenic therapies also in AML.25 However, whether increased angiogenesis constitutes a prognostic factor for AML treatment response remains unclear. Furthermore, a decrease of osteocalcin (OCN), a noncollagenous protein strictly correlated with bone turnover, was found in BM and peripheral blood serum of AML patients, especially those with adverse-risk disease.26 Notably, a reduced OCN level correlates to inferior overall survival, poor response to chemotherapy, and lower relapse-free survival in AML patients. Moreover, chemotherapeutic treatments can cause a restoration of OCN levels as a consequence of an increase in osteoblast (OB) number in the BM.27In AML patients, some BM morphological features that correlate with the outcome have been described. In the study of Lu and colleagues28 but also by specific signaling pathways triggered by the altered BM components, especially MSCs. Indeed, it has been extensively described how MSCs endow AML cells with resistance to several therapeutic agents, including standard chemotherapy and new molecular targeted agents such as Fms-like tyrosine kinase (FLT3) inhibitors . Co-cultures of AML cells harboring internal tandem duplication of FLT3 (FLT3-ITD) together with MSCs protect blasts from FLT3 inhibitors through the alteration of cytokines, chemokines, RAS/MAPK, and ERK signaling pathways.29 However, the precise population of cells and molecular mechanisms involved in chemoresistance have only been partially elucidated.The BM microenvironment is implicated in drug resistance not only by changing the pharmacokinetics of drugs by the alteration of BM vascular permeability,31 In particular, Uy and colleagues31 demonstrated an increased overall survival and relapse-free survival in AML patients treated with plerixafor in combination with chemotherapy. A similar strategy was adopted in combination with hypomethylating agents such as decitabine or azacytidine32 or in combination with targeted drugs such as sorafenib.33 Treatment with sorafenib, G-CSF, and plerixafor significantly increases the mobilization of blasts and CD34+/CD38− stem/progenitor cells, and the overall response rate as well.An important demonstration of the role of the BM niche in protecting leukemic cells from chemotherapy comes from the clinical use of the CXCL12/CXCR4 axis inhibitors. Indeed, different groups have tried to boost chemotherapy activity by stripping the LSCs from their niche, upon administration of granulocyte colony-stimulating factor (G-CSF) or plerixafor in combination with antileukemic treatments, with promising results.34 Within the BM niche, HSCs are found predominantly located in perivascular regions, including sinusoidal and arteriolar vessels.36 Only a minor fraction of HSCs resides near trabecular and cortical bone surfaces.37 However, HSCs are motile within the niche and cannot be strictly localized within a specific area.38 Multiple cell types, including stromal and endothelial cells, populate perivascular regions contributing to the maintenance of HSCs. The stromal fraction includes MSCs, and cells belonging to different mesodermal lineages differentiated from them, in particular OBs and adipocytes. MSCs differ between the distinct BM regions, where periarteriolar MSCs display a propensity to undergo osteogenesis, whereas sinusoidal MSCs show an enhanced adipogenic profile.41In the last years, our understanding of HSCs and their niche has increased exponentially, thanks to technological advancements, including the use of reporter mice, refinement of HSC markers, single-cell RNA sequencing (scRNA-seq), CyTOF, full bone sections, and live animal imaging.42 Multiple MSC subsets with distinct impacts on HSC behavior have been identified both in mice and in human.43BM MSCs colocalize closely with HSCs and regulate their homeostasis by direct interaction through the production of soluble factors, including CXCL12, angiopoietin (ANGPT), stem cell factor (SCF), osteopontin (OPN), and interleukin 7. Moreover, MSCs protect HSCs from stress signals, such as oxidative stress and inflammation.44 Osteolineage cells produce many HSC-supporting molecules, including OPN, CXCL12, SCF, thrombopoietin, and ANGPT. However, recent evidence suggests that they are not the predominant source of these factors, because other populations are the main producers.47Osteolineage cells, including pre-OBs and OBs, were among the first components of the BM niche to be associated with hematopoiesis regulation, yet the entire mechanism is still controversial. They affect HSC renewal, expansion and homing, and, depending on their stage of differentiation, their maturation along different lineages. Although the most immature uncommitted progenitor subset influences HSC maintenance and proliferation, committed progenitors or mature OBs regulate HSC differentiation along the lymphoid, myeloid, and erythroid lineages.48 Adipocytes secrete fatty acids, hormones, adipokines, and cytokines, which have profound effects on the function of other neighboring cell populations in the BM microenvironment. Although previous studies identified BM adipocytes as negative regulators of hematopoiesis,49 recent works have revealed that they positively regulate HSC regeneration under stress conditions.50The role of BM adipocytes in hematopoiesis regulation is complex and still under investigation.51 The permeability of blood vessels regulates reactive oxygen species (ROS) in adjacent HSCs and niche populations, and less permeable arterioles maintain HSCs in a quiescent state with low ROS levels, whereas leaky sinusoids increase ROS levels in HSCs, thus leading to their activation and mobilization into the circulation. Endothelial cells provide soluble factors that promote HSC self-renewal and hematopoietic regeneration after injury.BM endothelial cells, lining the surface of blood vessels, control vascular integrity, which in turn affects HSC trafficking and functions.52 Moreover, terminally differentiated hematopoietic cells, including megakaryocytes, macrophages, neutrophils, and regulatory T cells, play a key role in the BM niche.44The sympathetic nervous system, which innervates both the bone and the BM, also regulates HSC functions.AML is characterized not only by the blocking of HSC differentiation and the accumulation of blasts but also by alterations in the nonhematopoietic compartment of the BM microenvironment, which sustain leukemogenesis while suppressing normal hematopoiesis. MSCs and their progeny can contribute to leukemogenesis at least in 2 different ways: they can acquire mutations or functional alterations, caused by aging and chronic inflammation, that predispose for malignancy development, or they can be corrupted by transformed hematopoietic cells, facilitating disease manifestation and/or progression. Both these contributions can coexist during leukemia initiation and expansion.RAR-γ) microenvironment transplanted with wild-type (WT) BM rapidly developed MPN.53 Similarly, the inactivation of the Retinoblastoma (Rb) gene, a central regulator of the cell cycle, in both HSCs and BM microenvironment established a myeloproliferative disorder.54 MPN was also developed after transplanting normal HSCs into mice carrying inactivation of Mind bomb 1 (Mib1), the primary regulator of endocytosis of Notch ligands, in BM stromal cells.55Pioneering studies on reciprocal BM transplantation using mouse models carrying mutations in nonhematopoietic or hematopoietic cells demonstrated the potential of dysregulated stroma to drive the initiation of myeloid diseases, including MPN, MDS, and AML Figure . For insDicer1, an RNAse III endonuclease, in osterix-expressing osteoprogenitors, which caused MDS-like disease with increased propensity to develop AML.56Dicer1 depletion in mature OCN-expressing OBs did not result in a hematopoietic phenotype.The first evidence that alterations of a specific BM stromal population represented by osteolineage cells can drive myeloid malignancies arose from the deletion of Ptpn11 gene, encoding for the protein tyrosine phosphatase SHP2, in MSCs and osteoprogenitors but not in mature osteolineage cells showed the MPN development. These mice are characterized by the generation of a proinflammatory environment. Specifically, overproduction of the chemokine CCL3 by Ptpn11-mutated stromal cells can cause the recruitment of monocytes, which secreted inflammatory cytokines such as IL-1β and hyperactivated HSCs, driving the disease progression.57Mice presenting activating mutations in the 58 The increased nuclear β-catenin interacts with Forkhead box protein O1 (FoxO1), inducing the activation of Notch signaling and consequently increasing the expression of the ligand Jagged1 on OBs.59 The subsequent aberrant activation of Notch signaling in HSCs induces their leukemic transformation and ultimately leads to AML development. Finally, it has been demonstrated that HSCs from β-catenin mutated mice acquire intrinsic alterations and, once they are transplanted in WT mice, they provoke AML onset.58Furthermore, constitutive activation of β-catenin in OBs leads to AML development in mice.61 Genetic depletion of healthy mature OBs in an AML murine model causes an increase in circulating blasts and in tumor engraftment in the BM and spleen, an increase of myelopoiesis associated with a compromised B lymphopoiesis and erythropoiesis, and shortened survival.27 On the contrary, maintenance of the OB pool treating mice with an inhibitor of the duodenal serotonin synthesis increased mice survival, reducing circulating blasts and restoring the normal marrow function.27 Similar results were obtained by Bowers and colleagues62, which demonstrated that OB ablation significantly accelerates leukemia development and reduces the survival of transgenic BCR-ABL mice.More recent studies provided evidence that nongenetic changes, such as BM niche remodeling caused by premature or physiologic aging, can also foster myeloid cell expansion.63 Deletion of signal-induced proliferation-associated gene 1 (Sipa1) in mesenchymal and endothelial cells also results in MPN.64Osteolineage cells are not the only BM cell type involved in leukemogenesis. Loss of canonical Notch signaling in endothelial cells leads to MPN-like disease, through constitutive activation of mir-155 and NFKB signaling.Despite all these studies demonstrating that BM populations, particularly osteolineage cells, can lead to a preleukemic myeloid disease when mutated or dysfunctional in mice, similar evidence is still missing in patients.67 Although the phenotypic profile of cell surface markers defining stromal MSCs in the BM niche does not change between healthy subjects and myeloid malignancy patients, specific subpopulation proportions, functions, or molecular profiles appear to be altered. Cytogenetic abnormalities, such as hypodiploidy, chromosomal translocations, duplications, and deletions, have been observed in AML-MSCs. Interestingly, these mutations are different from those detected in the leukemic blasts of the same individual.68 These alterations have also been confirmed by Kim and colleagues69, who described the presence of genomic instability and a global hypomethylation in AML-MSCs compared with the healthy ones. The functional consequences of these MSC alterations are still debated. Several studies carried out on MSCs from patients with AML have noted normal proliferative capacity, colony-forming unit-fibroblast capability, survival, and ability to support ex vivo hematopoiesis. In contrast, other studies showed defective hematopoietic supportive capacity, reduced expression of adhesion molecules (VCAM1 and CD49F/ITGA6), increased apoptosis and senescence, and augmented production of inflammatory cytokines in MSCs derived from patients with myeloid neoplasm.70 In stromal cells from MDS and AML patients, the expression of molecules involved in the interaction with HSCs is decreased, whereas the population of CD271+ MSCs, which favors blast expansion through upregulation of CXCL12, is increased.26 MSCs sustain the survival and chemoresistance of AML blasts in several ways, such as supplying prosurvival factors, providing metabolic substrates alternative to glucose, and rewiring their metabolism.73A possibility to approach the modifications of the human BM microenvironment in AML is to isolate and study the MSC populations derived from AML patients (AML-MSCs). Leukemic cells reprogram MSCs through cell-to-cell contact, secreted factors, and exosomes.67Recent bioengineering advances were made in recreating BM stroma through organ-on-a-chip devices that would allow the investigation of MSC-mediated chemoresistance mechanisms. Biomimetic scaffolds capable of mimicking bone extracellular matrix were used to study the effects of MDS/AML blasts on MSCs, reprogramming their transcriptome toward novel prooncogenic activities.76 Battula and colleagues77 showed that AML-MSCs present increased expression of tissue nonspecific alkaline phosphatase and a switch from adipogenic to osteogenic differentiation, driven by a BMP-dependent mechanism. Similarly, AML cells upregulated the expression of connective tissue growth factor in normal MSCs and activated Smad1/5 signaling, inducing MSCs to differentiate into committed osteoprogenitors, but not mature OBs. Our group recently demonstrated that AML-MSCs, even when removed from their pathological environment, retain an intrinsically abnormal differentiation pattern with altered osteogenesis. Using an in vivo system specific to assess their osteogenic potential, AML-MSCs exhibit a reduced capacity to form mature bone and develop an osteoprogenitor-rich niche with the presence of Osterix+/OCN− pre-OBs and OCN+/Dentin matrix acid phosphoprotein 1− immature osteocytes.78The capacity of MSCs derived from patients with AML to differentiate into osteolineage cells is controversial. Several studies demonstrated that AML-MSCs present an impairment in the differentiation capability into the three mesodermal lineages, and in particular in the osteogenic differentiation.These data indicate that AML cells educated MSCs to overproduce functionally altered osteoprogenitors, although the reasons for this differentiation impairment remain to be elucidated.79 The ability of AML cells to alter BM vasculature is further supported by the extensive vascular remodeling observed in PDX models and BM biopsies from patients.28 Interestingly, the rescue of endosteal vessels preserves HSCs and improves chemotherapy efficacy.79 In addition, Xiao et al demonstrated that AML MLL-AF9 cell infiltration in the BM causes an expansion of Early B-cell factor 2+ (Ebf2+) MSCs with reduced CXCL12 expression, which favors AML establishment.80The BM niche becomes dramatically altered during AML progression, as clearly demonstrated in many murine models of leukemia Figure . The end82 increasing the number of functionally altered osteoprogenitor cells and immature OBs. Blocking the terminal differentiation of MSCs into mature OBs seems to contribute to AML progression. In an MLL-AF9 AML model, the leukemia development causes local neuropathy, reducing the number of NG2+ periarteriolar cells, which are involved in the maintenance of HSC quiescence, and leading to the osteogenic differentiation of Nestin+ MSCs. Despite this commitment to osteoblastic lineage, these mice show a lack of terminally differentiated OBs and deficient bone mineralization.83 Similar results were reported also for a JAK2V617F-MPN mouse model, in which sympathetic neuropathy causes a reduction of Nestin+ MSCs, which promotes the expansion of mutant HSCs and facilitates disease progression by loss of HSC-retention factor expression, including CXCL12, SCF, ANGPT1, and VCAM1.84 Frisch and colleagues81, using an immunocompetent murine model of leukemia, demonstrated that AML blasts alter directly normal OB functions. Engrafted animals presented decreased OPN-positive OBs, loss of trabecular structures, and lower serum levels of the bone formation marker OCN. Osteoblastic inhibition by leukemia seems to be mediated by chemokine CCL3, which is increased in malignant BM cells, both in leukemic mice and patients.81 Furthermore, FLT3-ITD-positive mice showed alterations in bone morphology suggesting a possible impact of aberrant oncogenic FLT3 signaling on the process controlling bone homeostasis.86Furthermore, leukemic myeloid cells alter the fine control of the osteogenic niche,66 noted that AML patients present an increase in exosome secretion, which correlates with the reduction of OCN plasma levels. Moreover, transplantation of AML-derived exosomes induces in the murine BM microenvironment alterations similar to those described for mice engrafted with AML cells . Interestingly, AML-derived exosomes transfer to the BM stroma the negative regulator of osteogenesis Dickkopf-1 (DKK1), which causes suppression of OB differentiation and supports a more rapid leukemia progression. Treatment of transplanted mice with DKK1 inhibitors significantly delays AML progression and increases their overall survival. In addition, AML-derived exosomes induce downregulation of HSC-supporting factors  in BM stromal cells, reducing their capacity to support normal hematopoiesis.66 Doron and colleagues87 demonstrated that AML cells can use extracellular vesicles to transmit endoplasmic reticulum stress and to promote the unfolded protein response in stromal cells, inducing their osteogenic commitment partially through the transfer of BMP2.Kumar and colleagues88 identified kynurenine (Kyn), a tryptophan catabolite, as a new promoter of the BM niche remodeling in AML. AML-secreted Kyn binds to the serotonin receptor 1B (HTR1B) on OBs, inducing a proinflammatory state characterized by increased expression of acute-phase protein serum amyloid A (SAA). SAA acts with a mechanism of positive feedback on leukemia cells by increasing the expression of indoleamine 2,3-dioxygenase-1 (IDO1), the rate-limiting enzyme for Kyn synthesis, thereby enabling AML progression. Genetic and pharmacological inhibition of kyneurin-HTR1B interaction between leukemia cells and OBs hampers AML proliferation.Recently, Galan-Diez and colleagues90 Battula and colleagues77 developed a human AML niche model, which consists of subcutaneous implantation of human bone fragments freshly collected from healthy subjects into the flanks of NSG mice transplanted with human AML cell lines. The authors demonstrated that mice transplanted with human AML cell lines presented a strong upregulation of early-stage OB markers Runx2 and Osterix in both the endosteal surface and medullary cavity of implants, compared with those harvested from control mice, confirming an increased but incomplete osteogenic activity also in humanized leukemic BM.As the study of the BM niche contribution to leukemia development is hindered in humans, reliable models of the human leukemic niche are fundamental for the confirmation of these findings and their translational application. In this regard, humanized BM niche models offer a valuable alternative. Over the years, several models using different scaffold materials and implant techniques have been proposed to recreate a humanized BM microenvironment that allows studying the ability of human stromal cells to sustain the normal and/or leukemic engraftment and to evaluate the interactions between hematopoietic and nonhematopoietic compartments.91 very little is known about the nonhematopoietic niche in human BM at the molecular level.Comprehensive mapping of the interactions occurring within the BM niche during AML pathogenesis and treatment would have a paramount impact on developing new therapeutic approaches targeting the leukemic microenvironment, aimed at improving the patient outcome. Although the impact of AML on transcriptomic changes in the immune microenvironment has recently been addressed in patients,92 and spatially resolved transcriptomics41 have recently allowed to unravel the complexity of heterogeneous stromal progenitors in murine BM. Under homeostatic conditions, all these studies found considerable overlap between the identified niche populations. To examine how nonhematopoietic BM cells are altered during AML development, Baryawno and colleagues39 compared scRNA-seq profiles from healthy and MLL-AF9-knock-in leukemic mice. They identified 17 distinct BM populations at steady state, including 3 endothelial , 1 pericyte, 2 osteolineage (OLC-1 and OLC-2), 5 fibroblastic, 1 mesenchymal (LepR-MSCs), and 4 chondrocyte clusters. The MLL-AF9 mouse model showed significant alterations in the cellular composition of the BM microenvironment, with a marked decrease in osteolineage-differentiated LepR-MSCs and a concurrent increase in pre-OBs. This suggests an impairment in osteogenic differentiation due to the induction of genes inhibiting bone formation and calcification and is consistent with the decrease of bone formation and OB number reported in murine models of AML.83 Even the adipogenic differentiation of LepR-MSCs was impaired as revealed by the downregulation of several adipocytic-related genes. This is in line with a previous study reporting defective adipogenesis in AML PDX models.93 In addition, the presence of leukemic cells reduced the expression of regulatory molecules necessary for normal hematopoiesis in specific stromal subsets, including endothelial cells.scRNA-seq92 examined the changes affecting BM stromal cells during AML development using an AML PDX model.92 They showed that major niche components, including CD31+ cells, Col1a1+ cells, and Nestin+ cells, which overlapped with the signature of endothelial, skeletal, and smooth muscle cells, respectively, underwent gene expression changes upon AML engraftment. Integrating BM transcriptome and secretome, they described a deregulated proteomic signature similar to the one reported for AML patients.94 Interestingly, several signaling mediators of the complement and coagulation cascade result downregulated, in agreement with clinical evidence of bleeding disorders and prothrombotic state in AML patients. Moreover, mediators associated with neutrophil degranulation are reduced, while chemokines involved in monocyte chemotaxis results increased, suggesting alterations in the regulation of innate immunity.With a similar approach, Passaro and colleagues95Similarly, a scRNA-seq study on BM stroma of mice with MPN reported a functional reprogramming of 2 specific MSC subsets into collagen-producing myofibroblasts, responsible for extracellular matrix deposition and fibrosis. Inflammation plays a critical role, as increased expression of alarmin heterodimer S100A8/S100A9 is related to stroma reprogramming from hematopoiesis support toward fibrotic transformation.97 Similarly, the analysis of the stromal compartment in BM of multiple myeloma patients led to the identification of a myeloma-specific inflammatory MSC subset, which spatially colocalized with tumor and immune cells and showed the expression of genes involved in tumor survival and immune modulation.98 These studies represent the first steps to unraveling alterations of BM stroma components during the development of hematological malignancies in humans.Very recently, a scRNA-seq approach has been applied to deconvolute the heterogeneity of MSCs in healthy human BM, leading to similar findings compared with the murine experiments, revealing adipo-, osteo-, and chondrogenic clusters.94 performed proteomic profiling of the noncellular soluble compartment of the BM microenvironment in patients with AML using the SOMAscan assay. They found 91 upregulated and 77 downregulated proteins in the leukemic BM. The proteomic signature of the AML BM niche revealed perturbation of signaling networks, particularly those associated with chemokine and cytokine signaling. Specifically, levels of proteins involved in bone homeostasis such as BMP10, CCL3, CX3CL1, OPN, endoglin, parathyroid hormone-like hormone, the secreted regulators of BMP signaling pathway, chordin-like protein 1, and matrix metalloproteinase 7 displayed altered values.Although RNA-seq studies are useful, proteome-based studies would be essential to investigate molecules that are regulated by posttranscriptional mechanisms. Çelik and colleaguesAlthough more effort is necessary to unravel the BM stromal cell changes in different AML stages, all transcriptomic and proteomic data indicate a shift toward an accumulation of pre-OBs that might be instrumental in disease evolution and should be further explored to identify novel therapeutic targets.The studies reviewed herein, largely performed in animal models, highlight that leukemic myeloid cells and the BM stroma interact extensively, having mutual effects on each other. However, despite a growing set of evidence in patients sustains that leukemic cells can alter the stromal compartment by producing factors that corrupt the BM niche to their advantage, the role of somatic mutations in BM stromal cells in leukemia initiation has not yet been confirmed in patients.3 The ultimate goal of research in the field of hematologic malignant niches is the development of therapies targeting the deregulated microenvironment, which, alone or in combination with the existing cell-intrinsic therapies, may increase leukemia susceptibility to treatment, leading to the complete eradication of all malignant clones. This could be accomplished by targeting the interactions between blasts and their niche, or blast-intrinsic pathways that influence the niche or directly the niche components. Because several mechanisms involved in leukemia progression often overlap across multiple leukemic subtypes, niche-oriented therapeutic targets would be effective for a wide cohort of patients. Recent evidence about the main role of pre-OBs on AML progression led to considering the crosstalk taking place between blasts and osteolineage cells as a potential target for therapy.The elucidation of mechanisms behind the interaction between AML cells and BM stroma can represent a turning point in AML treatment. Indeed, despite the improvements in the management of this disease, AML patients still face a risk of chemoresistance and relapse that severely affect the outcome.Our knowledge about cellular populations and molecular mechanisms of the BM niche involved in interactions with healthy and malignant hematopoiesis is still in its infancy. For this reason, the application of new imaging and sequencing technologies for the analysis of BM samples from patients represents a valuable option not only to shed light on the pathogenesis of myeloid neoplasms but also to identify possible novel therapeutic targets. Furthermore, monitoring the specific signature of the leukemic niche at diagnosis and during therapy could be exploited as a potential tool to predict response in clinical trial settings.The authors thank Fondazione Matilde Tettamanti and Comitato Maria Letizia Verga for their generous and constant support.CT, BR, and AP wrote the article. AB and MS revised and edited the article. All authors have read and agreed to the final version of the article.The authors have no conflicts of interest to disclose.This work was partially supported by AIRC 5x1000 “Immunity in Cancer Spreading and Metastasis (ISM)” (grant 2018-21147) to AB, AIRC IG 2022 (grant 2022-27507) to MS, PRIN 2021-NAZ-0033 to AB, and by Associazione SKO Arianna Amore ONLUS (research fellowship to CT).


Pseudomonas chlororaphis produces various benzobactins, whose biosynthesis is highlighted by a synergistic effect of two unclustered genes encoding enzymes on boosting benzobactin production; the formation of non‐proteinogenic 2‐hydroxymethylserine by a serine hydroxymethyltransferase; and the types I and II NRPS architecture for structural diversity. Our findings reveal the biosynthetic potential of a widespread benzobactin gene cluster.Benzoxazolinate is a rare bis‐heterocyclic moiety that interacts with proteins and DNA and confers extraordinary bioactivities on natural products, such as C‐1027. However, the biosynthetic gene responsible for the key cyclization step of benzoxazolinate remains unclear. Herein, we show a putative acyl AMP‐ligase responsible for the last cyclization step. We used the enzyme as a probe for genome mining and discovered that the orphan benzobactin gene cluster in entomopathogenic bacteria prevails across Proteobacteria and Firmicutes. It turns out that  Benzoxazolinate is a rare bis‐heterocyclic moiety that interacts with proteins and DNA. It was found that a putative acyl AMP‐ligase mediates the last cyclization step to afford benzoxazolinate. The enzyme was then used as a probe for genome mining, which led to the discovery of a new class of benzoxazolinate‐containing compounds in diverse bacteria. However, the enzyme catalyzing intramolecular attack of the NH2 on the carboxylate of the enolpyruvate to afford a 1,4‐oxazolinate ring remains undefined.Benzoxazolinate  moiety iVibrio.2) is the product of the xvb pathway in the entomopathogenic bacterium Xenorhabdus vietnamensis,xvb BGC coding for types I and II non‐ribosomal peptide synthetase (NRPS) hybridXenorhabdus and Photorhabdus BGCs to the MIBiG database,2 is yet to be characterized. To date, benzoxazolinate‐containing natural products were only accessible with these four bacterial producers. Here, we present that genome mining enabled by gene functional characterization of benzoxazolinate biosynthesis provides a prolific producer for a class of benzoxazolinate derivatives, whose BGC is widely distributed across Gram‐negative and Gram‐positive strains.AuromomycinXenorhabdus and Photorhabdus, we used the protein sequence of SgcG as a probe to search for putative BGCs. We found that in addition to XvbC encoded by the known xvb BGC in X. vietnamensis,X. szentirmaii DSM 16338 and X. szentirmaii US are located in two highly similar gene clusters, of which the BGC in X. szentirmaii DSM 16338 is designated as xsb. The xsb BGC . The xsb BGC lacks an SgcD homolog, and only xsbA (encoding a dehydrogenase) and xsbC (encoding a phenylacetate‐CoA ligase family enzyme) contain a counterpart in the sgc BGC. The remaining xsb BGC is made up of xsbB which encodes an NRPS with an adenylation (A) and a thiolation (T) domain, xsbD that encodes a 3‐oxoacyl‐ACP synthase, and xsbE that encodes an isochorismate synthase.To explore potential alternative benzoxazolinate‐producing hosts among xsb BGC, we inserted an arabinose‐inducible PBAD promoterxsbA and found that the induced PBADxsbA mutant yielded a single peak  and NMR spectroscopy . However, in contrast to the homologous expression of xsb BGC, the heterologous expression of xsbABCDE in Escherichia coli BL21(DE3) did not produce 1 , which has a high expression level in the wild‐type strain.xsbABCDE with the ADIC synthase encoding gene xpzC , consistent with previously described.xsze_03053 and xsze_03255, were found when using SgcD as a probe to search for homologs in the genome of X. szentirmaii DSM 16338. We were concerned that Xsze_03053 and Xsze_03255 could also synthesize ADIC and such genetic redundancy within X. szentirmaii DSM 16338 could functionally complement XpzC. Therefore, we deleted xpzC and the obtained PBADxsbA ΔxpzC mutant lost production of 1  with XpzS2 on the carboxylate without forming a CoA‐bound thioester intermediate, as observed during the biosyntheses of coumermycin A1,xsbB, xsbC, and xsbD. While the PBADxsbA ΔxsbB and PBADxsbA ΔxsbD mutants did not affect the product profile  harboring xsbA, xpzC, and xsbC produced 1, while no production of 1 was observed in E. coli expressing xsbA and xpzC . Most importantly, a C‐terminal extension with Lys429 in NatL2 is also present in XsbC. Lys429 from the other monomer forms a salt bridge with the AMP , and thus C‐terminal extension crossing to the other monomer is proposed to lock the functional homodimer in the “close” conformation, which prevents a CoA from entering the binding pocket.To support our hypothesis that XsbC belonging to the phenylacetate‐CoA ligase family mediates OPA cyclization via an ATP‐dependent adenylating manner Figure a, we purTaking all these data into account, we propose a three‐gene cassette consisting of an anthranilate synthase component I homolog (e.g. SgcD) or ADIC synthase (e.g. XpzC), an FMN‐dependent and [Fe−S]‐containing dehydrogenase (e.g. SgcG and XsbA), and an acyl‐AMP ligase (e.g. XsbC) being responsible for benzoxazolinate biosynthesis.xvb BGC responsible for benzobactin biosynthesis in entomopathogenic bacteria Xenorhabdus and PhotorhabdusPseudomonas, and is prevalent across two phyla of bacteria  and Firmicutes . Such extensive similarities to the xvb BGC suggest that benzoxazolinate‐containing natural products are more widespread in diverse bacteria—from ocean to land—than we thought, and that Pseudomonas being the most abundant genus among all these strains might be prolific producers for these rare natural products.To expand the biosynthetic repertoire of benzoxazolinate‐containing natural products, we carried out a survey of all bacterial genomes available in the NCBI database using the protein sequence of XsbC as a query. Besides C‐1027Pseudomonas strains for homologously expressing the target BGC by a promoter‐exchange strategy.pbz, from Pseudomonas chlororaphis subsp. piscium DSM 21509 was successfully activated as indicated by the significant distinction of HPLC profiles between induced and non‐induced mutants  as determined by untargeted analysis, and therefore we attempted to isolate them for structure elucidation. The compound with a mass of 305, designated as benzobactin B (3) confirmed by NMR spectroscopy , has one 2‐hydroxymethylserine residue less than 2. Compounds with larger masses  were not isolated due to their low production levels and instability. Among them, the structure of a compound with a mass of 609, designated as benzobactin C (4), was tentatively assigned to be a dimer of 3 by tandem MS/MS and isotope labeling experiments . Based on the sum formula (Table S5), we proposed that benzobactins‐628 and 1012 are a dimer and tetramer of 3, respectively, while their exact chemical structures cannot be formulated at the current stage.We selected different pbz BGC contains a typical three‐gene benzoxazolinate cassette pbzCEF. An ADIC synthase (PhzE) in a phenazine biosynthetic operon that is 2.6 megabase pairs away from the pbz BGC is also present in the genome  revealed that PbzI and its homologs do not have any histidine or aspartic acid residues in the second and third positions . PbzI as a free‐standing enzyme with an atypical core motif C3 might be a new example to understand the catalytic mechanism of condensation domains/enzymes.From a functional point of view, PbzI appears to be a canonical condensation enzyme catalyzing amide‐bond formation and regioselectively uses the α‐tertiary amine with steric hindrance as a nucleophile, which differs from the PbzD‐Cxsb) in the entomopathogenic bacterium X. szentirmaii DSM 16338 ; an FMN‐dependent and [Fe−S]‐containing dehydrogenase  is assumed to convert ADIC into OPA as previously described;1 is formed by intramolecular cyclization of OPA under the mediation of a putative acyl AMP‐ligase (XsbC). Given the structural uniqueness and the fascinating biological function of benzoxazolinate and benzoxazolinate‐containing natural products, we then performed a targeted BGC‐based genome mining by using the protein sequence of XsbC, and discovered that the orphan benzobactin pathway (xvb) in entomopathogenic bacteria Xenorhabdus and Photorhabduspbz BGC in P. chlororaphis subsp. piscium DSM 21509 , as well as benzobactins‐628 and 1012 that are yet to be characterized. Our recent whole‐genome‐sequencing of X. vietnamensis DSM 22392 revealed that the xvb cluster resides on a 51‐kb plasmid and is flanked with transposase and integrases, while the pbz cluster resides on the chromosome. This suggests that the benzobactin BGC is likely to spread horizontally in bacteria at a great evolutionary distance.We first characterized the biosynthesis of benzoxazolinate (2–4), we observe that the anthranilate synthase component I (PbzF in the benzobactin pathway) and the ADIC synthase (PhzE in the phenazine pathway), which share low protein sequence similarity, have a synergistic positive effect on the production level of benzobactins  catalyzes mono‐ and bis‐hydroxymethylation to afford 2‐hydroxymethylserine which is an unusual building block in natural products  should be addressed to the authors.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.


Calcium-evoked release of neurotransmitters from synaptic vesicles (SVs) is catalysed by SNARE proteins. The predominant view is that, at rest, complete assembly of SNARE complexes is inhibited (‘clamped’) by synaptotagmin and complexin molecules. Calcium binding by synaptotagmins releases this fusion clamp and triggers fast SV exocytosis. However, this model has not been quantitatively tested over physiological timescales. Here we describe an experimentally constrained computational modelling framework to quantitatively assess how the molecular architecture of the fusion clamp affects SV exocytosis. Our results argue that the ‘release-of-inhibition’ model can indeed account for fast calcium-activated SV fusion, and that dual binding of synaptotagmin-1 and synaptotagmin-7 to the same SNARE complex enables synergistic regulation of the kinetics and plasticity of neurotransmitter release. The developed framework provides a powerful and adaptable tool to link the molecular biochemistry of presynaptic proteins to physiological data and efficiently test the plausibility of calcium-activated neurotransmitter release models. A computational modelling framework demonstrates how synaptotagmin-1 and −7 can jointly regulate the kinetics and plasticity of neurotransmitter release and provides a powerful tool to test molecular models of calcium-triggered release in silico. In addition to synapses, similar SNARE proteins mediate the fusion of virtually all membranous organelles in living cells4.Synaptic release of neurotransmitters forms the basis of information transfer in the brain. It is well established that synaptic vesicle (SV) fusion with the plasma membrane is mediated by SNARE  proteins, namely VAMP2 on the SV (v-SNARE) and syntaxin1 and SNAP25 on the plasma membrane (t-SNAREs) in most central synapses2+ channels (VGCCs) located in the AZ, resulting in the formation of local Ca2+ nano/microdomains near RRP vesicles , which triggers SV exocytosis and synchronous neurotransmitter release on a millisecond timescale5. VGCCs close within several milliseconds after an AP, resulting in the collapse of Ca2+ nano/microdomains. However, the presynaptic Ca2+ level remains elevated in the low micromolar range for tens to hundreds of milliseconds. This long-lasting increase in residual 7. Asynchronous release and synaptic facilitation are, in large part, mediated by the presynaptic membrane-associated high-affinity Ca2+ release sensor synaptotagmin 7 (Syt7), which can be activated by 11. Thus, it is emerging that the synaptic release of neurotransmitters is synergistically regulated by low- and high-affinity synaptotagmins acting on the same pool of vesicles or even on the same SNARE complex. How this occurs in molecular terms remains poorly understood.A distinct property of SV exocytosis is that it is tightly coupled to neuronal activity and controlled by action potential (AP)-evoked increases in presynaptic 2+-activation of synaptotagmin molecules and act cooperatively to drive rapid SV exocytosis leading to neurotransmitter release14. However, it has not been quantitatively tested whether this ‘release of inhibition’ model can adequately describe the millisecond kinetics of synchronous neurotransmitter release or if additional mechanisms, such as membrane bending and/or membrane bridging by Syt121, are also critical.The current prevailing view is that each RRP vesicle contains several partially assembled SNARE complexes (‘SNAREpins’) that are arrested (‘clamped’) in this state by synaptotagmins and the soluble presynaptic protein complexin. The SNAREpins are thought to be released by Ca25. For example, it has been shown that a given SNAREpin can simultaneously bind two Syt1 molecules, one at the ‘primary’ interface, independently of complexin, and another at the ‘tripartite’ interface, in conjunction with complexin , whilst the tripartite site appears to be universally accessible to all synaptotagmin isoforms, including Syt721. Dual synaptotagmin binding at primary and tripartite interfaces has the potential to explain, in molecular terms, how different synaptotagmin isoforms cooperatively regulate neurotransmitter release and short-term plasticity13. However, this hypothesis remains to be tested.Recently, considerable progress has been made in understanding the structural and functional organisation of the SNARE pre-fusion complexxin Fig. 21. Inter2+ at the AZ is challenging due to its spatial scale (200–600 nm) and the inherently low signal-to-noise ratio of fluorescent Ca2+ indicators when imaging with millisecond resolution. Another obstacle is that, at present, it is not possible to directly track the molecular states of different synaptotagmin isoforms on RRP vesicles. Data-constrained realistic computational models of presynaptic terminals are, therefore, essential tools that can bypass the limitations of experimental approaches. Indeed, we have previously created a set of computational tools to model presynaptic Ca2+ dynamics at different types of synapses during physiological patterns of activity30.One difficulty in addressing these questions is that measuring the spatio-temporal dynamics of Ca2+ transients that occur at the presynaptic AZ and to test how their activation triggers SV exocytosis for different molecular models of the fusion clamp. We find that release of inhibition is sufficient to explain the millisecond kinetics of AP-evoked SV exocytosis. Our results indicate that, irrespective of the triggering Ca2+ signal’s shape, or the nature of the fusion clamp, the majority of synchronous vesicular fusion occurs when 3 SNAREpins are simultaneously free from inhibition. Furthermore, our simulations reveal that the Syt1/SNARE interaction at the primary site alone can account for the millisecond kinetics of AP-evoked synchronous release and that binding of Syt1 or Syt7 to SNARE complexes at the tripartite interface provides an additional level of regulation of vesicular fusion. In particular, dual Syt1/Syt7 binding to the same SNAREpin can explain the role of Syt7 in the regulation of short-term synaptic plasticity and the kinetics of vesicular release.Here we describe an experimentally constrained computational modelling framework that allows us to model the activation of Syt1 and Syt7 by physiologically relevant Ca21. It has also been shown that Syt1 can simultaneously interact with the lipid bilayer via PIP2 interaction and with SNAREs via the primary interface24. It is thus likely that Syt1 binding at the primary interface occurs at an early stage of vesicle docking, preceding the SNARE assembly process. In contrast, the tripartite interface is generated only when SNAREs are partially zippered and can bind complexin. Furthermore, the tripartite interface binding motif is present in both Syt1 and Syt721. Based on these data, we considered three distinct synaptotagmin fusion clamp architectures. In all cases, the primary interface was occupied by Syt1, whilst the tripartite interface was either unoccupied  or occupied  either by Syt1 (Syt1P/Syt1T) or by Syt7 (Syt1P/Syt7T)  Fig. . To mode2+ synchronised neurotransmitter release33. Furthermore, recent cryo-electron tomography analysis in cultured hippocampal synapses demonstrated a circular symmetric arrangement of six protein densities at the interface between docked SVs and the presynaptic membrane, each possibly corresponding to a single SNARE-associated exocytic module34. Therefore, as a default setting, we assumed that each RRP vesicle was associated with six SNAREpins.Biochemical and physiological analyses have shown that a small number of SNAREs are sufficient to achieve fast, Ca2+ binding leads to rapid insertion of the aliphatic loops of synaptotagmin C2 domains into the membrane and that this step is critical for triggering neurotransmitter release36. Indeed, structural and biochemical analyses have indicated that a Ca2+-triggered reorientation of Syt1 C2 domains displaces Syt1 from the primary SNARE interface37. Therefore, we assumed that Ca2+ binding and subsequent membrane loop insertion of synaptotagmin C2 domains induces the removal of the fusion clamp, i.e. ‘release of inhibition’ . The fusion dynamics of RRP vesicles in response to diverse 49, as detailed in the Methods section.Finally, we assumed that the repulsive forces between an SV and the plasma membrane constitute a potential energy barrier, which is lowered by the independent energetic contributions of individual assembled SNARE complexes. SV fusion was triggered when the barrier was overcome by thermal fluctuations at a rate given by the Arrhenius equation Fig. 42,43. Th2+-activation of SV fusion has been quantitatively described in the calyx of Held—a giant synapse in the auditory brainstem. In these experiments, flash photolysis was used to generate spatially uniform, step-like increases of 2+-evoked vesicular release was monitored using post-synaptic patch-clamp recordings51. The measured kinetics and the [Ca2+] dependency of glutamate release at this synapse have been described by several empirical mathematical models53. Among these, the six-state allosteric model proposed by Lou et al.  required at least three uninhibited SNAREpins. This value is consistent with previous experimental and modelling estimates for the number of SNAREs required to mediate synchronous release of neurotransmitters43. Indeed, the apparent rate of synchronous vesicle fusion in our modelling framework is limited by the time taken for three SNAREpins to be released from the fusion clamp, which depends on the −1 . This implies that once three out of six SNAREpins are simultaneously unclamped, vesicle fusion is practically inevitable. In line with this prediction, the peak release rate was directly proportional to the fraction of vesicles with three uninhibited SNAREpins, irrespective of the synaptotagmin clamp architecture  . Adding either a second Syt1 or Syt7 clamp at the tripartite interface reduced the vesicular release by similar amounts. The amplitude of AP-evoked [Ca2+] transients decreased with increasing coupling distance, which resulted in a corresponding reduction of both the peak release rate and the overall release probability  and coupling distances (30–80 nm). We next simulated vesicular release for different synaptotagmin clamp architectures in response to the obtained Ca2+ dynamics and calculated the paired-pulse ratio Short-term plasticity of synaptic neurotransmitter release is commonly assessed by measuring vesicular release in response to pairs of APs. We therefore tested how the molecular architecture of the synaptotagmin clamp shapes vesicular release in response to paired-pulse stimulation Fig. . Using t2+] transients at vesicular release sites were comparable for the 1st and 2nd AP , we observed depression of vesicular release in response to the 2nd AP, which was most prominent at short coupling distances where [Ca2+]local transients had the greatest amplitude. The depression can be explained by the depletion of the RRP vesicles due to the absence of vesicle replenishment in these simulations and the relatively high release probability at the 1st AP  the release probability at both the 1st and the 2nd APs was low, and the PPR was close to 1 for the whole range of coupling distances and inter-stimulus intervals tested. Strikingly, the inclusion of Syt7 at the tripartite interface (Syt1P/Syt7T) led to a facilitation of vesicular release at the 2nd AP . This means that at the time of arrival of the 2nd AP these unfused vesicles are expected to have up to 40% of Syt7 clamps already released  progressive increase of peak [Ca2+]local at vesicular release sites due to Ca2+ buffer saturation and (ii) activation of Syt7 due to increase of [Ca2+]residual28.We next assessed whether the release of inhibition model could explain the complex patterns of vesicular release observed in response to bursts of neuronal activity. To test this, we chose the mossy fibre bouton (MFB)—CA3 pyramidal neuron synapse in the hippocampus as a model environment. This synapse, which is also called a ‘detonator synapse’, has a very low initial release probability . Therefore, we used the release efficacy, We previously estimated Cadel Fig. . In this28. Indeed, by its design, the allosteric model accounts for facilitation triggered by increases in peak [Ca2+]local at vesicular release sites  the initial release efficacy was several-fold higher than in the case of the benchmark allosteric model, P/Syt1T and 0.0015 for Syt1P/Syt7T. Consequently, both dual clamp models showed very strong facilitation, P/Syt1T and 100 for Syt1P/Syt7T  and dual Syt1 and/or Syt7 clamps at primary and tripartite interfaces (Syt1P/Syt1T and Syt1P/Syt7T). Our analysis shows that the release of a single or dual synaptotagmin clamp can account for sub-millisecond kinetics of Ca2+-triggered neurotransmitter exocytosis. Furthermore, the dual binding Syt1P/Syt7T arrangement also reproduced facilitation of SV exocytosis in response to pairs or burst of APs. This result shows that the release of inhibition model also provides a mechanism by which Syt7 can regulate short-term plasticity.Based on the available structural data59. In contrast, the relevance of the tripartite interface remains unclear because the interaction of SNAREs/synaptotagmins/complexin at this site cannot be measured biochemically and is thus expected to be very weak61. However, considering the high local concentration of the vesicular release machinery components at RRP vesicles, it is reasonable to expect that the tripartite interface can be at least partially occupied under physiological conditions. In fact, our simulations argue that the weak interaction at this site may play an important functional role and the dynamic occupancy of the tripartite interface by either Syt1 or Syt7 could provide direct control of the kinetics and plasticity of neurotransmitter release by changing the strength and Ca2+-activation properties of the SV fusion clamp. Indeed, we find that a mixed model combining both single and dual clamp architectures can closely describe the release kinetics and short-term plasticity in response to trains of APs in hippocampal MFB terminals.The functional importance of the primary interface has been well established, both in live synapses and under reconstitution conditions2+-activation of synaptotagmin and removal of the fusion clamp, it has several simplifications. We assumed that release of the fusion clamp occurs simultaneously with Ca2+-triggered membrane insertion of the C2 domains for both Syt1 and Syt7. This is likely the case for Syt1 interaction at the primary interface24, but the kinetics of synaptotagmin/complexin/SNARE interactions at the tripartite interface are not currently known. Similarly, we assumed that restoration of the clamp occurs instantaneously after reversal of synaptotagmin membrane insertion. Thus, the current model provides an upper estimate for the magnitude of synchronous release and lower estimates for asynchronous release and short-term facilitation. We also note that in many synapses the degree of short-term facilitation is determined not only by the activation of Syt7 but also by an increase in the amplitude of [Ca2+] transients at the vesicular release sites during repetitive stimulation. This increase can be attributed to various phenomena, including buffer saturation62, action potential broadening63, Ca2+ channel facilitation64 and Ca2+ release from the intracellular stores66.While our parsimonious model describes the key molecular elements of Ca67, inclusion of the second C2 domains in the models will increase the overall Ca2+/membrane affinity of synaptotagmin molecules and the Ca2+ cooperativity. At present, the binding modalities of Syt7 to the SNARE complex remain unknown; thus, we modelled Syt7 binding to SNAREs via the tripartite interface based on structural homology21. However, it is worth noting that the output for the Syt1/Syt7 dual clamp model will be the same even if Syt7 binds at a different site. Furthermore, in addition to the synaptotagmins and canonical SNAREs considered in our model, different modes of Ca2+-evoked release might utilise vesicles with distinct compositions of SNAREs and Ca2+ sensors .For simplicity, we only modelled the activation of Syt1 C2B and Syt7 C2A domains. Considering that the C2A and C2B domains act synergistically43 or via formation of supramolecular complexes, e.g. Syt1 oligomerisation72. In the framework of the mechanical coupling model, the partially assembled SNAREpins interact with each other via long-range mechanical forces, mediated by the surrounding scaffolding membranes. Therefore, each clamped SNAREpin acts as an additional mechanical obstacle for the fusing membranes, thereby generating a negative feedback loop. Given that the exact energetic cost of a clamped SNAREpin remains undefined, we explored the potential influence of this mechanism by introducing an extra energy barrier of 2 kBT for each SNAREpin  Syt1 and Syt7 C2 domains can bind two Ca2+ ions independently, with similar intrinsic affinities 45. It has been shown that the rate of Ca2+ binding by synaptotagmin C2 domains is limited by diffusion (~0.1–10 µM−1 ms−1)48. Therefore, we assumed −1 ms−1. Because of the symmetry between the free and fully Ca2+-bound states  Fig. . We esti2+](t) traces were generated by Monte Carlo estimation from multiple stochastic simulations. Specifically, we used the direct Gillespie algorithm49 which proceeds by iteratively generating a randomised time at which the system next changes its state and then randomly selecting the identity of the new state. The Ca2+ binding and SV fusion dynamics of the allosteric model were described by a six-state kinetic scheme with a single occupied state which is updated at each step of the algorithm. The release of inhibition models consisted of either six (in the case of Syt1P) or twelve (in the cases of Syt1P/Syt1T and Syt1P/Syt7T) four-state kinetic schemes , corresponding to an average error of less than 1%, making this the expected upper limit of prediction errors due to stochastic variation.In order to assess the accuracy of these release estimates generated using the Monte Carlo approach, we compared the 2+ influx, buffering, diffusion and extrusion was performed in the Virtual Cell (VCell) simulation environment (vcell.org) as described in detail in our previous studies. Specifically, [Ca2+](t) transients approximating Ca2+ dynamics at the AZ of small excitatory boutons (Figs. 29 and ref. 28 respectively. The properties of endogenous Ca2+ buffers used in the model were previously estimated in refs. 80. and are shown in Supplementary Table Three-dimensional modelling of AP-evoked presynaptic Cans Figs.  and 4 anns Figs.  and 6 weFurther information on research design is available in the Peer Review FileSupplementary InformationDescription of Supplementary MaterialsCustom MatLab codeReporting Summary