The relapse rate for children with acute myeloid leukemia (AML) remains high despite advancements in risk classification, multi-agent chemotherapy intensification, stem cell transplantation, and supportive care guidelines. Prognosis for this subgroup of children with relapsed/refractory AML remains poor. It is well known that the ceiling of chemotherapy intensification has been reached, limited by acute and chronic toxicity, necessitating alternative treatment approaches. In the last several years, our improved understanding of disease biology and critical molecular pathways in AML has yielded a variety of new drugs to target these specific pathways. This review provides a summary of antibody drug conjugates (ADCs), small molecule inhibitors, and tyrosine kinase inhibitors with an emphasis on those that are currently under clinical evaluation or soon to open in early phase trials for children with relapsed/refractory AML. Acute myeloid leukemia (AML), a hematopoietic stem cell disorder characterized by clonal expansion of abnormally differentiated myeloid lineage blasts, accounts for 20% of childhood leukemia . AML conAntibody-based therapies have emerged as an effective and well-tolerated approach to cancer care, whereby attaching a cytotoxic drug to an antibody can lead to increased dose intensity with reduced toxicity  Figure , Table 1The cell surface antigen, CD33, is present in more than 80% of patients with AML but is absent from hematopoietic stem cells, rendering it an ideal immunoconjugate target . From ea2/dose. This landmark study showed that 3-year event free survival (EFS) was significantly improved with the addition of GO  by a significant reduction in 3-year relapse risk (RR) , particularly in the low and intermediate risk groups. Furthermore, the study did not demonstrate an increased risk of sinusoidal obstructive syndrome (SOS) in the GO group as had been the case in early studies using higher doses of GO [The most promising data for targeting CD33 in both children and adults have come from studies with Gemtuzumab ozogamicin , a humanized IgG4 anti-CD33 antibody conjugated to calicheamicin, a DNA-cleaving cytotoxic agent, leading to its FDA approval in 2000. A subsequent randomized controlled trial in adults, Southwest Oncology Group (SWOG) S0106, failed to meet its primary end points of improved remission induction and safety, resulting in withdrawal of approval . Howeveres of GO . This suMesothelin is a cell-surface tumor differentiation antigen expressed on mesothelial cells of serosal lining. It has been associated with malignant transformation, cellular proliferation, and tumor aggressiveness in a variety of solid tumors, including lung, pancreatic, and ovarian origin. Mesothelin was recognized as an attractive candidate for targeted cancer therapy due to its limited expression in normal tissue and high expression in cancer tissue ,18. AnetCD123, the alpha subunit of the IL-3 receptor, is overexpressed in multiple hematologic malignancies, including AML, ALL, and blastic plasmacytoid dendritic cell neoplasm (BPDCN). Because of its high expression on leukemic blasts as compared with normal hematopoietic stem cells, CD123 has emerged as an attractive candidate for molecularly targeted therapeutics . Tagraxon = 66) and 43% (n = 7) of BPDCN patients had an objective response (complete remission (CR), complete remission with incomplete hematologic recovery (CRi), and partial remission (PR)) to IMGN632 [IMGN632 is comprised of a novel humanized anti-CD123 antibody, G4723A, linked to a unique DNA-alkylating payload of the recently developed IGN (indolinobenzodiazepine pseudodimer) class of cytotoxic compounds ,29. Kovt IMGN632 . These e IMGN632 . ThroughWith the discovery of multiple pathways critical to the proliferation and survival of AML cells, small molecule inhibitors have emerged as a promising therapeutic strategy in AML , Table 1Binding of E-selectin adhesion molecules to AML blasts enhances the adherence of AML cells within the vascular bone marrow niche via activation of the Wnt signaling pathway. Protection within the vascular marrow niche supports leukemia cell quiescence, limits their exposure to cell-cycle dependent chemotherapy, and prolongs their survival ,34,35. TUproleselan (Glycomimetics (GMI)-1271) is a small molecule inhibitor designed to prevent the binding of E-selectin to ligands on leukemia blasts. Preclinical work demonstrated that GMI-1271 can overcome adhesion-mediated chemotherapy resistance in vitro and reduce leukemia burden in mice engrafted with primary AML cells ,37. UproKMT2A (formerly known as MLL or mixed lineage leukemia)-rearranged acute leukemia, involving fusions of 11q23, is a particularly aggressive leukemia affecting approximately 15–20% of childhood AML. KMT2A rearrangements are found in several groups of leukemia patients: Infantile ALL, AML, and acute megakaryoblastic leukemia (AMKL), a rare subtype of AML that occurs mainly in children with Down syndrome [syndrome ,41,42. Osyndrome ,41. CleaDOT1L (disruptor of telomeric silencing 1-like) is a histone methyltransferase at H3K79 that in its normal form transcriptionally modifies chromatin of downstream target genes to help maintain the integrity of chromosomes and genes. However, the aberrant recruitment of DOT1L by a majority of KMT2A-fusion partners results in an inappropriate hypermethylation and overexpression of downstream, targeted genes that promote leukemia. The oncogenic drive of KMT2A-rearranged leukemia is dependent on the activation of DOT1L ,44. TherAnother area of therapeutic interest has been to inhibit the binding between MLL and menin, an essential co-factor of the oncogenic component of the KMT2A complex that also acts as a histone methyltransferase to transcriptionally regulate targeted genes that are critical to leukemogenesis. Several small molecule menin inhibitors have been developed to date ,49. One Finally, another menin-KMT2A inhibitor, Kura Oncology (KO)-539, has been shown to inhibit leukemia growth and prolong survival in KMT2A-rearranged cell lines and in vivo models, earning the FDA Orphan Drug Designation . This coTargeting more than one component of the KMT2A complex has great potential for targeted treatment. Although a DOT1L inhibitor by itself has not shown promising results in Phase I trials, Dafflon et al. showed that the combination of a DOT1L inhibitor and a menin inhibitor demonstrated robust synergy in its anti-leukemic growth inhibition effect in a mouse model injected with KMT2A-AF9 leukemic cells, compared to either drug alone ,52. FurtKRAS, NRAS, fms-like tyrosine kinase 3 (FLT3) or PTPN11 mutations [Mitogen-activated protein kinase (MAPK) cascade is a signaling pathway crucial for the regulation of cell differentiation, proliferation, and survival. The cascade is activated by ligand binding to a receptor tyrosine kinase (RTK) to activate Ras then Raf, followed by Mitogen-activated protein kinase , finally resulting in activation of the last component of the pathway: Extracellular signal-regulated kinase (ERK). Activated ERK can then activate a variety of downstream substrates, and so the suppression of MEK/ERK activation can have profound effects on controlling cell growth ,54,55,56utations , and norutations . The mosThe preclinical efficacy of MEK inhibitors in reducing leukemia burden and prolonging survival in cell lines, primary patient cells, and mouse xenograft models harboring RAS-mutated acute leukemia cells has been demonstrated in a variety of studies ,60. DataTP53) mutation is an infrequent event in AML [Although Tumor Protein p53 -246, a methylated, small-molecule derivative of PRIMA-1 (p53 re-activation and induction of massive apoptosis), which has been shown to bind and reactivate mutant and inactivated p53 by restoring its wild-type transcriptional activity to induce cell apoptosis ,81. An oFLT3 internal tandem duplications (FLT3-ITDs), the most common class of FLT3 gene mutations found in AML, lead to constitutive activation of the kinase, promoting cell growth, survival, and anti-apoptotic signaling. FLT3-ITDs are associated with a poor prognosis due to a high relapse rate [FLT3 (fms-like tyrosine kinase 3), a cytokine receptor (CD135) belonging to the receptor tyrosine kinase class III, is expressed mainly on hematopoietic cells and plays a pivotal role in myeloid and lymphoid cell proliferation and survival. pse rate . There hp = 0.078) and following induction II . Furthermore, 3-year EFS was improved to 57.5% from the historical control of 34.3%, p = 0.007, and the RR was reduced with sorafenib treatment , although 3-year OS was not significantly different  [First generation FLT3 inhibitors including sorafenib and midostaurin are multi-kinase inhibitors that show activity against a host of growth factors, including FLT3, KIT, platelet-derived growth factor receptor (PDGFR), and vascular endothelial growth factor (VEGF), leading to more off-target toxicity. Sorafenib is FDA-approved for treatment of renal cell carcinoma. There have been mixed results in early phase clinical trials of Sorafenib in combination with chemotherapy for adults with de novo FLT3-ITD+ AML ,84. Howe= 0.375) . SorafenMidostaurin, another first-generation FLT3 inhibitor, is the first tyrosine kinase inhibitor to be FDA approved for AML therapy in the first line. Approval largely resulted from the Alliance 10603 trial, in which adults with newly diagnosed, FLT3-mutated AML, who received midostaurin in combination with standard chemotherapy had superior EFS and OS as compared to standard chemotherapy alone . In chilSecond-generation FLT3 inhibitors are much more highly selective in targeting FLT3, thereby showing more limited off-target toxicity . QuizartFinally, gilteritinib is another potent and highly selective second generation FLT3 inhibitor that exerts dual activity against FLT3 and AXL, an additional receptor tyrosine kinase that promotes proliferation and survival of AML cells . Due to In summary, the last several years have been pivotal for AML clinicians, researchers, and patients, representing a period of robust discovery and development of targeted drugs for AML. Many of these drugs, as described in this review, have advanced from bench to bedside and are being evaluated in adults with either ongoing or emerging studies in children. The impact of these ADCs, small molecule inhibitors, and FLT3 inhibitors on improving survival in children with leukemia and other cancers remains to be seen as pediatric clinical trials become increasingly available. While the evaluation of these novel drugs as monotherapy is a critical first step, it will be equally critical to test their safety and efficacy in strategic combination with cytotoxic chemotherapy and/or other novel therapies. The standard of care for upfront AML therapy in children remains a risky, stratified, multi-agent chemotherapy regimen, but as evidenced by the last several landmark COG AML trials, incorporation of targeted therapies, including Gemtuzumab ozogamicin and tyrosine kinase inhibitors targeting FLT3-ITD mutations, are improving outcomes. The AML molecular landscape has evolved along with our understanding of the biology and genetic makeup of AML, and the potential of ongoing research may dramatically inform future treatment options.


APP), presenilin-1 (PSEN1), and presenilin-2 (PSEN2) genes. AD mutations contribute to the generation of toxic amyloid β (Aβ) peptides and the formation of cerebral plaques, leading to the formulation of the amyloid cascade hypothesis for AD pathogenesis. Many drugs have been developed to inhibit this pathway but all these approaches currently failed, raising the need to find additional pathogenic mechanisms. Alterations in cellular calcium (Ca2+) signaling have also been reported as causative of neurodegeneration. Interestingly, Aβ peptides, mutated presenilin-1 (PS1), and presenilin-2 (PS2) variously lead to modifications in Ca2+ homeostasis. In this contribution, we focus on PS2, summarizing how AD-linked PS2 mutants alter multiple Ca2+ pathways and the functional consequences of this Ca2+ dysregulation in AD pathogenesis.Alzheimer’s disease (AD) is the most common form of dementia. Even though most AD cases are sporadic, a small percentage is familial due to autosomal dominant mutations in amyloid precursor protein ( Alzheimer’s disease (AD) is an age-related neurodegenerative disorder, considered the most common cause of dementia. According to the original 1984 diagnostic criteria, and the revised clinical criteria evaluated by the National Institute on Aging-Alzheimer’s Association (NIA-AA) in 2011 ,4. Even APOE gene polymorphisms are the best characterized. APOE encodes an apolipoprotein that has a prominent role in cholesterol homeostasis. It exists in different polymorphic forms, and the Epsilon 4 (ε4) form correlates with a major risk of AD, both in homozygosis and heterozygosis ). 2+ homeostasis and contribute to the cardiac pathology ) that is ensured by a highly complex molecular Ca2+ toolkit composed of cell surface receptors, channels, pumps, antiporters, Ca2+ buffers, and Ca2+ sensors. They have specific distributions and roles within the cell, cooperating in the maintenance of cell Ca2+ homeostasis [2+] gradient between the cytosol ([Ca2+]c ~ 100 nM) and the extracellular medium ([Ca2+]e ~ 1.2–2 mM), in resting conditions, and to increase the [Ca2+]c up to 1–3 μM, upon cell stimulation. Indeed, Ca2+ can cross the PM and/or can be released from intracellular stores  and refilling of empty stores [2+] in the ER lumen  reachable in the cytosol upon cell stimulation (2–3 µM). This implies that mitochondria can take up Ca2+ very rapidly only when microdomains of high [Ca2+] occur close to their surface. These Ca2+ microdomains are typically formed near the mouth of channels and receptors permeable to Ca2+. Thus, the mitochondria that are able to rapidly take up Ca2+, during cytosolic Ca2+ rises, are those in close proximity to PM Ca2+ channels or ER Ca2+ releasing channels [2+ levels occur for any rise of cytosolic [Ca2+], but these increases are slow and small for organelles distant from the microdomains. In particular, sites of proximity between ER and mitochondria constitute specific subcellular regions, called MAM  andmed NCLX , and thetiporter ; Figure  to MCUC ,115, Ca2changers . It is wchannels . It needview see ; Figure iewed by ).2+ homeostasis could be responsible for AD neurodegeneration  caused by mutated PS2, augmenting the number of Ca2+ microdomains on organelle surface, thus increasing the efficiency of ER to mitochondria Ca2+ transfer , by different mechanisms .2+ homeostasis c   experiments in cell populations  in real-time within specific organelles, restricting the analysis only to co-transfected cells expressing also the protein under investigation. They can be divided into two main groups: single fluorophore-based GECIs  and a peptide sequence from the myosin light chain kinase (M13)  C. These , GCaMP6 , has bee created , allowin created ,199. How created ,201 impo2+ responsive element [2+ dynamics in living cells. They are based on cyan (CFP) and yellow (YFP) variants of GFP and they exploit M13 and CaM for sensing Ca2+. Briefly, Ca2+ ions bind to CaM causing a conformational change that allows M13 to wrap around CaM, forcing the two FPs closed by. These changes in the molecule’s structure allow FRET to occur between the two fluorophores  and human FAD APP mutated genes  [Numerous tg mouse lines expressing wt or mutated human APP and/or PS have been generated to provide animal models of AD. The expression of human FAD mutant of PS1 or PS2 in mice does not cause the characteristic histological hallmarks found in AD patients as amyloid plaques or neurofibrillary tangles are absent. This is because the mouse APP is not-amyloidogenic. Generally, and 671) ,208,209.and 671) .APP knock-in strategy and present overproduction of Aβ42 without APP overexpression. Like the first-generation mouse models, also these mice present limitations, i.e., they do not exhibit tau pathology or neurodegeneration  compared to PS2APP mice, and approximately 30-fold higher levels of PSEN2 mRNA. They also have about 2-fold higher Aβ levels than PS2APP mice [Focusing on PS2 and APP, the homozygous tg lines PS2-N141I (PS2.30H) and PS2APP are commonly used. The PS2-N141I (PS2.30H) mouse line expresses the human promoter . This mopromoter . The othpromoter . In thispromoter , but onlAPP mice . The B6.APP mice , in whicAPP transcript at 12 months [In B6.152H mice, amyloid plaques are absent at 3 months of age. The overt Aβ deposition in the brain appears only at approximately 6 months, with heavy plaque load in the hippocampus, frontal cortex, and subiculum at 10 months. Aβ deposits in blood vessels were observed sporadically and mainly in large vessels. The amount of cerebral amyloid deposits correlated with levels of the human 2 months ,215. Dec2 months . Further2 months . FinallyIn general, despite several AD mouse models have been generated, none of them fully recapitulates the human pathology ,218, str


Acute Myeloid Leukemia is an aggressive disease with poor outcomes. New targeted therapies that can boost the effects of currently used chemotherapy medications without added toxicity are needed. Targeting an overactive kinase, called the protein Kinase CK2 in AML, helps leukemia cells undergo cell death and helps certain chemotherapy drugs work better. Here, we present evidence that CX-4945, a CK2 inhibitor drug, effectively kills leukemia cells in mouse models and shows the mechanism of action responsible for these effects. Leukemia cells are more sensitive to a decrease in CK2 kinase levels than normal cells. Our results show that inhibiting CK2 kinase makes AML cells more susceptible to anthracycline-induced cell death. Anthracyclines like daunorubicin and doxorubicin are widely used to treat leukemia in children and adults. A rational combination of protein kinase CK2 inhibitors with the standard of care chemotherapy may help treat AML more effectively.Protein Kinase CK2 (Casein Kinase 2 or CK2) is a constitutively active serine-threonine kinase overactive in human malignancies. Increased expression and activity of CK2 in Acute Myeloid Leukemia (AML) is associated with a poor outcome. CK2 promotes AML cell survival by impinging on multiple oncogenic signaling pathways. The selective small-molecule CK2 inhibitor CX-4945 has shown in vitro cytotoxicity in AML. Here, we report that CX-4945 has a strong in vivo therapeutic effect in preclinical models of AML. The analysis of genome-wide DNA-binding and gene expression in CX-4945 treated AML cells shows that one mechanism, by which CK2 inhibition exerts a therapeutic effect in AML, involves the revival of IKAROS tumor suppressor function. CK2 phosphorylates IKAROS and disrupts IKAROS’ transcriptional activity by impairing DNA-binding and association with chromatin modifiers. Here, we demonstrate that CK2 inhibition decreases IKAROS phosphorylation and restores IKAROS binding to DNA. Further functional experiments show that IKAROS negatively regulates the transcription of anti-apoptotic genes, including BCL-XL . CX-4945 restitutes the IKAROS-mediated repression of BCL-XL in vivo and sensitizes AML cells to apoptosis. Using CX-4945, alongside the cytotoxic chemotherapeutic drug daunorubicin, augments BCL-XL suppression and AML cell apoptosis. Overall, these results establish the in vivo therapeutic efficacy of CX-4945 in AML preclinical models and determine the role of CK2 and IKAROS in regulating apoptosis in AML. Furthermore, our study provides functional and mechanistic bases for the addition of CK2 inhibitors to AML therapy. Acute Myeloid Leukemia (AML) is a heterogeneous hematological malignancy with poor prognosis, despite aggressive therapy, in children and adults alike . CytotoxProtein Kinase CK2, formerly known as Casein Kinase 2 (CK2), is a pleiotropic, constitutively active, serine-threonine kinase essential for cell survival and development . CK2 exiAlthough previous studies suggested several pathways through which CK2 inhibitors might operate, the in vivo therapeutic action mechanism for CK2 inhibition in AML is not well understood ,14,21. IBCL2L1). Furthermore, we demonstrate that the CK2 overexpression impairs IKAROS’ ability to repress BCL-XL. CX-4945 restores the IKAROS-mediated repression of BCL-XL and sensitizes AML cells to apoptosis. Finally, CX-4945 showed anti-tumor activity and prolonged survival in an AML patient-derived xenograft model. We show that in AML, the central mechanism of BCL-XL repression following the CK2 inhibition is due to the restored IKAROS transcription factor activity. These results provide a mechanistic basis to justify the clinical development of CK2 inhibitors combined with cytotoxic therapy to treat AML.Here, we report that IKAROS is hyperphosphorylated in AML cells with CK2 overexpression. The molecular or pharmacological inhibition of CK2 decreases IKAROS phosphorylation and increases IKAROS binding to target gene promoter regions. Genome-wide DNA-binding and gene expression analyses in CX-4945-treated AML cells revealed a strong IKAROS binding to the BCL-2 family genes. We performed loss of function and gain of function experiments showing that IKAROS represses the transcription of B cell Lymphoma Like–extra-large (BCL-XL) encoded by B cell Lymphoma Like 2–like 1  and the German Collection of Microorganisms and Cell Cultures (DSMZ). De-identified patient samples were provided by Loma Linda University  and collaborators at the Penn State Cancer Institute in compliance with institutional review board regulations. Cells were cultured with RPMI 1640 plus  with a 10% heat-inactivated Fetal bovine serum (FBS)  and 1% penicillin-streptomycin. HEK-293T cells were cultured in DMEM (CellGro) supplemented with 10% FBS. Human AML primary cells (AML-1), previously expanded in mice, were cultured in StemSpan SFEM , supplemented with a recombinant human stem cell factor , IL3 (20 ng/mL), FMS-like tyrosine kinase ligand , G-CSF (20 ng/mL), and GM-CSF , as well as 1% penicillin-streptomycin.The CK2 inhibitor CX-4945 sodium salt was a gift from Senhwa Biosciences. CX-4945 and daunorubicin hydrochloride were purchased from MedChem Express .U937-GFP-luc cells were transplanted into NRG-S 1Eav/J) mice via tail vein injection at a dose of 25,000 cells per mouse. The treatment was initiated when a bioluminescence intensity (BLI) signal of 100,000 photons/sec was detected. Mice were randomized based on the average BLI signal and treated with CX-4945 100 mg/kg or a vehicle, given via an oral gavage twice daily for 7 days. Bioluminescence imaging ) was used to track the leukemia progression. AML patient samples were selected based on their increased CK2 protein expression (mRNA and protein). Irradiated NRG-S mice were injected with 2 million cells per mouse via the tail vein. Engraftment was defined as 2–5% AML cells  in peripheral blood (PB). We started the CX-4945 treatment of 100 mg/kg twice daily after confirming the engraftment, and continued for a total of 21 days. After the treatment period, mice were sacrificed, and mononuclear cells were isolated from the bone marrow, spleen, and peripheral blood. The cell count and flow cytometry analysis were performed  using AML antibodies against human CD45+, CD13+, and CD33+ cells. One cohort was monitored for moribund signs, and the survival time was noted.Apoptosis assays were performed following the manufacturer’s instructions using the Annexin V-7AAD apoptosis detection kit  and analyzed on the Muse Cell Analyzer . Briefly, 100 μL of cells were added to 100 μL of reagent, incubated for 20 min at room temperature, and analyzed.The colorimetric WST-1 cell proliferation assay  was performed in 96-well white clear-bottom plates  in quadruplicate experiments, according to the manufacturer’s instructions. Absorbance at 440 nm (reflects the number of viable cells) was measured using a BioTek Synergy Mx plate reader.2, 1% Triton X-100, 100 mM NaCl, 10 mM NaF, 1 mM Na3VO4, and a protease inhibitor cocktail. The protein was quantified using the Bradford assay and used for Western blot analysis and immunoblotting. The antibodies used for the Western blot assay are listed in Cells were treated with 5 and 10 µM of CX-4945 or the vehicle control for 48 h and the whole-cell lysate was collected. Briefly, samples were prepared on ice using lysis buffer containing 10 mM Tris-HCl (pH 7.4), 5 mM MgClCells were treated with different doses (5 or 10 μM) of CX-4945 for 48 h. Cells were washed twice with phosphate-free RPMI 1640 and incubated with 0.5 mCi/ml [32P] orthophosphate  in a phosphate-free RPMI 1640 medium for 6 h. Cells were collected and washed twice with cold PBS and the nuclear protein was extracted using the NE-PER Nuclear and Cytoplasmic extraction reagent kit . The lysis buffer was supplemented with a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Then, IKAROS was immuno-precipitated using the Dynabead Protein G Immunoprecipitation Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Briefly, lysates were incubated with the Dynabeads Protein G and IKAROS antibody  complex for 2 h on a rotator-mixer. IKAROS was eluted, separated by SDS-PAGE, transferred to a PVDF membrane, and imaged by radiography.2. Colonies were counted under an inverted light microscope. Colonies that contained around 50 cells or more were counted for analysis.Cells were pretreated with CX-4945 alone or combined with daunorubicin, or the vehicle control for 48 h. Cells were washed with PBS and plated in triplicate in a 6-well plate containing the 1.1 ml MethoCult H4100 medium  supplemented with RPMI-1640 and 10% FBS for U937 cells, as well as a MethoCult H4034 Optimum (STEMCELL Technologies) medium for AML-1 cells. Colonies were propagated for 7–14 days in an incubator at 37 °C and 5% COTotal RNA (ribonucleic acid) was isolated from cells using the QIAshredder and RNeasy Mini Kit . Complementary DNA (cDNA) was generated from 1 μg of total RNA using the Superscript First-Strand Synthesis System (Invitrogen). The qRT-PCR was performed using a StepOne Plus real-time PCR machine (Applied Biosystems) with PerfeCTa SYBR Green FastMix (Quanta Biosciences). Values were normalized to 18 s RNA and the relative expression values were determined by the 2-ΔΔCt method. Primers used for target gene studies include:BCL2L1-5′-TTGGATGGCCACTTACCTGAAT-3′; BCL2L1-Rev 5′-CCGCCGTTCTCCTGGAT-3′; CK2α-5′-AGCGATGGGAACGCTTTG-3′; CK2α-Rev 5′-AAGGCCTCAGGGCTGACAA-3’; 18s-For 5’-GTAACCCGTTGAACCCCATT-3′ 18s-Rev5′-CCATCCAATCGGTAGTAGCG-3′.The luciferase assay was performed using the LightSwitch Luciferase Assay System (SwitchGear Genomics). Human embryonic kidney (HEK)-293T cells were seeded into 24-well plates. HEK-293T cells do not have endogenous IKAROS. After 24 h, cells were transiently transfected with 0.15 μg of indicated promoter-reporter constructs or pROM vector and 0.15 μg of pcDNA3.1-IKAROS or pcDNA3.1 vector in triplicate experiments for each group using lipofectamine 2000  according to manufacturer’s instructions. According to the manufacturer’s instructions, 24 h after transfection, cells were lysed in 100 μL of a LightSwitch Assay Solution  and rocked at RT for 30 min. Lysates were measured by a luminometer (Promega GloMax 20/20 Luminometer). Luciferase activities were calculated as a fold change relative to the vector-only cells and normalized to pcDNA3.1 vector readings. All transfection and reporter assays were performed in triplicate experiments.IKAROS qChIP assays were performed as described previously. Primersp > 0.05 (ns); p ≤ 0.05 (*); p < 0.01 (**); p < 0.001 (***); p < 0.0001 (****). Statistical significance in Figure 3B,E was performed using the one-way analysis of variance (ANOVA). Statistical analysis for all other column graphs used multiple two-tailed t-tests by the Holm-Sidak method, with alpha = 0.05. Each row (representing a cell line or drug concentration) was analyzed individually, without assuming a consistent Standard Deviation (SD). The number of t-tests per analysis was dependent on the number of conditions. The qChIP values where the signal was more than 2-fold greater than the background anti-Immunoglobulin G (anti-IgG) level were analyzed. The Kaplan-Meier method and the log-rank test was used to perform the survival analysis and compare survival differences .Graphed data are represented as mean values with bars representing the standard deviation (mean ± SD) of three technical replicates and at least two independent experiments. Statistical analysis was performed using GraphPad Prism 9.0.0. P-value summaries are as follows: In line with the previously published data ,16,36, wIn B-ALL, IKAROS is hyperphosphorylated by CK2 ,38. We tWe measured the CK2 activity indirectly by using an antibody against the phospho-CK2 substrate (motif pS/pTDXE) to measure the amount of phosphorylated CK2 substrates ,41,42,43We studied the effect of CK2α downregulation and pharmacological inhibition of CK2α on BCL-XL expression. We achieved a pharmacological inhibition of CK2 using a specific inhibitor, CX-4945. The treatment of U937 and AML-1 with CX-4945 showed dose-dependent decreases in BCL-XL mRNA A and proThese results demonstrate that the CK2α downregulation and pharmacological inhibition of CK2 results in decreased IKAROS phosphorylation and BCL-XL repression in AML cells. The overexpression of CK2α promotes cell viability in vitro, and accelerates leukemia progression in vivo likely by increasing the expression of anti-apoptotic BCL-XL. We developed a patient-derived xenograft (PDX) model by transplanting previously expanded human primary AML cells (AML-1) into irradiated NRG-S mice via tail vein injection at a dose of 2 million cells per mouse. These mice were treated with CX-4945 orally at 100 mg/kg twice daily for 21 days A. After We developed a cell line derived xenograft mouse model by transplanting luciferase labeled U937 and THP-1 cells (U937-GFP-luc and THP-1-GFP-luc) into NRG-S mice via tail vein injection. Engraftment was confirmed by bioluminescence imaging. Mice were treated with CX-4945 orally at 100 mg/kg twice daily for 7 days. The CX-4945 treated mice showed decreased BLI D. After IKZF1 loss of function mutations is rare, hyperphosphorylation by CK2 can impair IKAROS’ DNA-binding ability and subsequent regulatory functions. Using chromatin immunoprecipitation followed by highly parellel DNA sequencing (ChIP-seq), we did a global analysis of changes in the genome-wide DNA-binding of IKAROS in AML cells following the treatment with CX-4945. U937 cells were treated with 10 µM of CX-4945 for 72 h to assess IKAROS mediated epigenomic changes. A link to access the ChIP-seq data in the UCSC browser is provided in the supplemental material. Here, we show that the binding of IKAROS to the promoter region of the BCL2L1 (BCL-XL) gene increases after the CX-4945 treatment (BCL-XL promoter region using a quantitative chromatin immunoprecipitation assay (qChIP). The CX-4945 treatment showed increased binding of IKAROS to the BCL-XL promoter in U937 . The modification of “histone mark” influences gene expression by changing how accessible the chromatin is to the transcription. The histone mark is a specific modification (acetylation or methylation) of a specific histone protein. Acetylation of lysine 9(K9) of the H3 histone protein is noted as H3K9Ac. Methylation of lysine 27 (K27) of H3 histone is noted as H3K27me3. Enrichment of H3K9 acetylation (H3K9Ac) is an indicator of open and active chromatin. Enrichment of H3K27 tri-methylation (H3K27me3) is a marker of closed and repressive chromatin. Repressive chromatin signature is noted as an enrichment of Histone (H) 3 lysine (K) 27 tri-methylation (H3K27me3) and loss of H3K9 acetylation (H3K9ac) and H3K4 tri-methylation (H3K4me3) [BCL-XL promoter region. Here, we investigated the mechanism by which IKAROS represses its target gene, H3K4me3) . We perfBCL-XL promoter in U937 cells with forced IKAROS overexpression  is one of the most commonly used cytotoxic therapies for AML that induces DNA damage and apoptosis. AML cells often overexpress BCL-XL and other anti-apoptotic genes as one mechanism of chemoresistance. As shown in The addition of CX-4945 potentiates DNR induced apoptosis A,B. DownProtein kinase CK2 is essential for embryonic development, cell survival, growth, and maintenance ,47. The IKZF1 gene) is a master regulator of lymphoid hematopoiesis and a tumor suppressor in leukemia [IKZF1 genetic alterations, less than 10% of patients in AML have IKZF1 genetic mutations [In lymphoblastic leukemia, the tumor suppressor IKAROS is one of the important substrates of CK2 . IKAROS leukemia . Unlike utations . The funutations . BCL-XL (BCL2L1) gene. Further validation by the quantitative ChIP experiment showed that treating U937 and AML primary patient cells (AML-1) with CX-4945 enhances IKAROS binding to the BCL-XL gene. Functional experiments using the loss of function or gain of the IKAROS function have shown that IKAROS negatively regulates BCL-XL transcription via chromatin remodeling (formation of repressive chromatin). IKAROS is hyperphosphorylated in AML. The CK2 inhibition can reverse this process and restore the IKAROS regulation of BCL-XL. Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) was performed to assess IKAROS binding to DNA in U937 AML cells following the treatment with CX-4945. Results showed enhanced IKAROS binding peaks at the promoter region of several genes. IKAROS binding peaks were significantly increased at the promoter region of the Here, we show that the forced overexpression of CK2 in AML cells led to increased cell proliferation, decreased apoptosis, and resistance to DNR. More importantly, CK2 overexpressing AML cells engrafted rapidly and decreased the survival time of mice. Downregulation and pharmacological inhibition of CK2 decreased the BCL-XL expression and sensitized the AML cells to DNR-induced apoptosis. Anthracyclines (DNR and mitoxantrone) are used extensively in the treatment of AML. These results provide a mechanistic basis for the use of CX-4945 in combination with DNR. BCL-XL. Other anti-apoptotic genes regulated by CK2 and IKAROS include BCL2A1 and MCL-1. Here, we focused on the transcriptional regulation of the anti-apoptotic gene BCL-XL, as this is one of the important genes regulating leukemia cell resistance to apoptosis [This study shows that, in AML, IKAROS and CK2 play an essential role in the transcriptional regulation of the anti-apoptotic gene poptosis . The clipoptosis . Here, wBCL-XL gene (In summary, we report for the first time on the in vivo therapeutic efficacy of CX-4945 in AML patient-derived xenograft models. These results establish that one of the central mechanisms by which CX-4945 exerts an anti-leukemia effect in vivo is via the revival of IKAROS-mediated transcriptional repression of the -XL gene . The fun


The pursuit of relating the location of neural damage to the pattern of acquired language and general cognitive deficits post-stroke stems back to the 19th century behavioural neurology. While spatial specificity has improved dramatically over time, from the large areas of damage specified by post-mortem investigation to the millimetre precision of modern MRI, there is an underlying issue that is rarely addressed, which relates to the fact that damage to a given area of the brain is not random but constrained by the brain’s vasculature. Accordingly, the aim of this study was to uncover the statistical structure underlying the lesion profile in chronic aphasia post-stroke. By applying varimax-rotated principal component analysis to the lesions of 70 patients with chronic post-stroke aphasia, we identified 17 interpretable clusters, largely reflecting the vascular supply of middle cerebral artery sub-branches and other sources of individual variation in vascular supply as shown in classical angiography studies. This vascular parcellation produced smaller displacement error in simulated lesion–symptom analysis compared with individual voxels and Brodmann regions. A second principal component analysis of the patients’ detailed neuropsychological data revealed a four-factor solution reflecting phonological, semantic, executive-demand and speech fluency abilities. As a preliminary exploration, stepwise regression was used to relate behavioural factor scores to the lesion principal components. Phonological ability was related to two components, which covered the posterior temporal region including the posterior segment of the arcuate fasciculus, and the inferior frontal gyrus. Three components were linked to semantic ability and were located in the white matter underlying the anterior temporal lobe, the supramarginal gyrus and angular gyrus. Executive-demand related to two components covering the dorsal edge of the middle cerebral artery territory, while speech fluency was linked to two components that were located in the middle frontal gyrus, precentral gyrus and subcortical regions . Future studies can explore in formal terms the utility of these principal component analysis-derived lesion components for relating post-stroke lesions and symptoms. et al. applied principal component analysis to post-stroke lesions to construct a parcellation of the middle cerebral artery cortical supply. This parcellation was used in lesion–symptom mapping, and mis-localization analyses showed lower error compared to Brodmann areas.Brain damage post-stroke is not random but is constrained by the neurovascular system. Zhao   This allowed researchers to infer the location of damage and determine the effects on behaviour in greater detail  (3) and novel acquisition protocols allow imaging of different tissue properties . This engineering technology has been combined with advances in analytical techniques   and MRI AT scan) . MRI scaLesion mapping methodologies have been used widely in the stroke aphasia literature e.g. . For theThe key aim of this study, therefore, was to use a data-driven approach to uncover the underlying patterns in the lesions of 70 chronic aphasic post-stroke cases. We achieved this aim by using principal component analysis (PCA) with varimax rotation to investigate the patients’ lesion structure. One previous study on coma used PCA on brain images to compare with a probabilistic frequency map  was used in the previous studies (This study included 70 post-stroke patients with chronic aphasia (either ischaemic or haemorrhagic) with damage restricted to the left hemisphere  using an eight-element SENSE head coil. A T1-weighted inversion recovery sequence with three-dimensional acquisition was employed, with the following parameters: repetition time = 9.0 ms, echo time = 3.93 ms, flip angle = 8°, 150 contiguous slices, slice thickness = 1 mm, acquired voxel size 1.0 × 1.0 × 1.0 mm3, matrix size 256 × 256, FOV = 256 mm, inversion time = 1150 ms, SENSE acceleration factor 2.5 and total scan acquisition time = 575 s.High-resolution structural T1 images using a brain extraction tool optimized for patient brains (1 image (not brain extracted) and use the transformation matrix to warp the mask into Montreal Neurological Institute space. The analysis used Statistical Parametric Mapping software  and a modified unified segmentation–normalization procedure  and correlated to the lesion profile based on Thttp://icatb.sourceforge.net/) to estimate the number of components within the data that explained the most variance with a penalty for increasing the number of components. We used the ‘princomp’ function to extract the components and then applied a varimax rotation to help with the interpretation of the resulting components (using the ‘rotatefactors’ function). We projected the PCA factor scores back into brain space and converted the data into z-scores and used |z| > 3.29 (P < 0.001 two-tail) as the cut-off value. Clusters ˃500 voxels (volume: 4 cm3) were reported using the Harvard–Oxford  and natbrainlab (white matter) atlases. Having obtained the structure of the underlying lesions, we explored which lesion component(s) were related to the behavioural variations. First, we conducted Pearson correlation analysis between patients’ 20 lesion component scores and 4 behaviour component scores (Bonferroni corrected at P < 0.05). To test which of the lesion components were the most important predictors, we built a stepwise regression model where the lesion component scores were used to predict the behavioural component scores. The lesion component(s) that significantly predicted behavioural factors were then mapped onto the brain and converted into z-scores [|z| > 3.29 (P < 0.001 two-tail) as the cut-off value]. Clusters ˃200 voxels (volume: 1.6 cm3) were reported using the Harvard–Oxford  and natbrainlab (white matter) atlases.We conducted the PCA of the lesion data in MATLAB (R2012a) and used the minimum description length criteria and Kolmogorov information criterion   corrected for multiple comparisons. We calculated the centre of mass of the significant cluster and determined the Euclidean distance between this point and ground truth voxel. This process was repeated for 57 632 voxels, for which more than three patients had damage. Next, we simulated the ground truth using BAs, where for each area the sample was split into intact/damaged based on at least 20% of the area being damaged and a case with damage had a 90% chance of exhibiting a behavioural deficit , and the resulting cluster was used to calculate the displacement error based on the centre of gravity. This process was repeated for 29 areas, which had at least three patients with damage. Finally, the same process was repeated using the lesion principal components (the vascular territories). A Welch t-test was used to statistically compare the displacement values across the three lesion models.We conducted two analyses to help determine how well the lesion territory parcellation fared compared to a single-voxel or Brodmann atlas model. First, we determined the displacement error induced by different lesion models by changing the ground truth . Similar analyses in the literature have used different parcellations, which contain brain regions of differing sizes ; The conditions of our ethical approval do not permit the public archiving of anonymized study data. The anonymized data necessary for reproducing the results in this article can be requested from the corresponding author.A summary of the patients’ scores is provided in The factor analysis on the behavioural data revealed four orthogonal dimensions see , which rN = 40 was 17, while for N = 60 it was 19. For a sample size N = 65 and 70, the median value was 20 components. This result suggests that there are likely to be 20 components and, accordingly, this is the value we used in the study.Before reporting the results, it should be noted that the sign (positive or negative) of the coefficients is arbitrary, since variance does not depend on sign; however, the order of the components is important, with the first component accounting for the most variance and the last component the least. The minimum description length criteria  suggesteThe components could be grouped based on location within the fronto-parietal lobes or temporal lobe. Most components belonged to the MCA territory and were located in the fronto-parietal lobes, where Component 1 was subcortical, Components 3, 6, 7, 8, 12, 16, and 17 were frontal and Component 14 was parietal. There were three components in the temporal lobe . Two or three components were located along the watershed between territories (Components 9 and 11 and possibly 16). Component 13 fell within the medial frontal region and was likely to belong to the anterior cerebral artery territory and Component 9 in the occipital lobe was likely to belong to the posterior cerebral artery territory. The four other components were either scattered or included regions in the right hemisphere and thus hard to interpret  and precentral gryus. Component 6 was located at the central opercular cortex, extending to insular and parietal operculum cortex. Components 7, 3, 17 and 12 corresponded to the orbitofrontal, precentral, central and postcentral branches, respectively. These four are the territories of the frontal cortical branches, originating from the M4 segment. Component 7 came after the subcortical branch, located at the orbital frontal cortex, extending to frontal pole and inferior frontal gyrus (pars triangularis). Following it was Component 3, located at the middle frontal gyrus, extending to the precentral gyrus and inferior frontal gyrus. Component 17 was lateral to Component 3, lying on the central sulcus, occupying both precentral and postcentral gyri. Component 12 was posterior to Component 3 and was located at the postcentral gyrus, but occupying a large section of the superior parietal lobule. Component 16 was located at the middle and superior frontal gryus and was superior to the other frontal components. It is possible that this either reflected the most dorsal frontal branch or belonged to the watershed with the anterior cerebral artery. Component 14 was completely within the parietal lobe, including supramarginal gyrus and angular gyrus, and was linked to the posterior parietal branch.Moving inferiorly into the temporal lobe territories, we found Component 2, which was in the lateral temporal lobe, extending from the anterior superior and middle temporal gyrus to the angular gyrus and supramarginal gyrus. As the peak was posterior, we suggest that this reflects the posterior temporal branch. The medial temporal lobe structures made up Component 4, including the inferior longitudinal fasciculus and posterior segment of the arcuate fasciculus, therefore was likely to reflect the posterior temporal–occipital branch. We note that this component covered the ventricle and possibly reflected atrophic changes. The anterior inferior temporal lobe made up of Component 15, including the anterior portion of the inferior longitudinal fasciculus. The one component in the occipital lobe (Component 5) was located over the inferior division of lateral occipital cortex, occipital fusiform gyrus and occipital pole and, therefore, was more likely to belong to the posterior cerebral artery.We labelled Components 9 and 11 as watershed components as they overlapped with the territory between cerebral arteries (P < 0.01 with voxel P < 0.001). There were no significant clusters for the executive factor at this statistical threshold. The cluster related to phonology was found in the dorsal auditory language pathway, including middle and superior temporal gyrus, extending posterior to supramarginal gyrus and medial whiter matter substrates, e.g. inferior longitudinal fasciculus and arcuate posterior segment . Semantic ability was related to two clusters. The first one covered the lateral and medial anterior temporal lobe and extending to medial temporal lobe inferior longitudinal fasciculus . The second cluster was at the posterior end of the inferior longitudinal fasciculus and extended into the superior division of lateral occipital cortex . Finally, the fluency factor correlated with a large cluster in the frontal lobe, covering the precentral and postcentral gyrus, thalamus, central opercular cortex and also white matter including the frontal aslant tract, cortico-spinal, corpus callosum and cingulum .The neural correlates identified using voxel-based correlational methodology (VBCM) for phonology, semantics and fluency are shown in r = −0.409) and 4 (r = 0.419) correlated with phonology components score, Component 15 (r = −0.495) correlated with semantics, Component 9 (r = 0.477) correlated with executive function and, lastly, Components 3 (r = 0.436) and 6 (r = −0.407) correlated with fluency .The Pearson correlation analysis  = 10.288, P < 0.001), which included Component 4 (beta = 0.429) and Component 8 (beta = 0.244), with an adjusted r square of 0.212. We mapped the z-transformed coefficients back into brain space and found two significant clusters. The largest cluster was located over the left inferior longitudinal fasciculus and the posterior segment of the arcuate fasciculus, while the second cluster was located in the left pars opercularis and frontal operculum cortex. A significant model predicted semantic ability , which included Component 15 (beta = −0.418), Component 14 (beta = −0.336) and Component 12 (beta = 0.256), with an adjusted r square of 0.316. We found three significant clusters on the brain, of which the largest cluster was located within the left temporal lobe and the inferior longitudinal fasciculus. The second cluster was identified in the left supramarginal gyrus, and the third cluster was in the left optic radiations. We observed a significant model for executive ability , which included Component 9 (beta = 0.443) and Component 10 (beta = −0.266), with an adjusted r square of 0.276. Neural regions related to the executive factor were located in the watershed regions. The largest cluster overlapped with inferior temporal lobe running posterior to the occipital lobe, while the second cluster was located within the left middle frontal gyrus, the frontal pole and the inferior frontal gyrus. Finally, a significant model was identified for speech fluency ability , which included Component 3 (beta = 0.352) and Component 1 (beta = −0.274), with an adjusted r square of 0.236. The neural results revealed two significant clusters, the largest of which was located in the left middle frontal gyrus and precentral gyrus. The smaller cluster was found in subcortical regions such as the cortico-spinal tract, putamen and thalamus.We used stepwise regression to determine if the lesion components can be used to predict behavioural factors. The results are shown in t-test, t = 2.14, P = 0.0493, two-tailed) and the Brodmann parcellation simulation [Welch’s t-test, t = 2.66, P = 0.0117, two-tailed; see We conducted two analyses to test how useful the PCA territories were, compared with a single-voxel model and another whole brain atlas (Brodmann map). In the first instance, we simulated different ‘ground truths’ using a single voxel, BA or vascular territories. We found that the mean displacement error was largest for the Brodmann parcellation , followed by the single-voxel model  and then the vascular territories model . The displacement of the vascular territories was significantly smaller than the single-voxel simulation  when using a specific set of ground truth ROIs based on the Broadmann map [BA 37 and 38 alone, BA 37 plus 38 and BA 39 plus 44; replicating the models reported by in vivo lesion mapping. Many previous studies using lesion–symptom mapping have not taken into account the non-random distribution of brain damage after stroke, which is constrained by the vasculature. In the present study, we showed that PCA of the lesions in post-stroke aphasia can reveal the underlying components of damage across the MCA territory, which are striking similarity to the vascular structure and forms of vascular individual variation identified by seminal angiography studies  plus individual differences in vascular supply. In contrast, there are numerous variations in cytoarchitecture or cortical functions and, thus, it seems inevitable that there will be many more regions in those resultant parcellations of the brain. Future studies that have access to much larger lesion databases can assess that the spatial clusters identified in this study are efficient in capturing the post-stroke damage, by evaluating predictive modelling of out-of-sample data.We note that, inescapably, the spatial maps of principal components depend on the number of the components extracted. When a different optimization criterion was applied, a larger estimated number of components were derived yet the maps relating to these components were much harder to interpret and did not relate obviously to the known cortical vascular supplies see . The numWe provided two sets of results (i.e. manipulating the ground truth and manipulating the inference method) to help understand the effect on lesion–symptom mapping. Our results showed that, when we altered the ground truth, the vascular territories parcellation produced smaller displacement to the simulated ground truth compared to a single-voxel or Brodmann map model. The displacement values for the single-voxel model and BAs derived in the current study are very similar to recent studies i.e. . By projIn the second set of analyses, we did not observe clear differences between the mass univariate or multivariate methods. This might appear contradictory to recent reports [e.g. support vector machine classifier in By the way of a preliminary exploration, we compared voxel-based analyses (VBCM) with the results from the lesion component to behavioural mapping. The results for phonological ability showed overlap between both methods in the posterior temporal region including the underlying posterior segment of the arcuate fasciculus, which has been shown to be involved in the dorsal auditory language pathway . AlthougVBCM and the behaviour-lesion components regression both revealed clusters in the white matter of anterior temporal lobe when mapping semantic ability. This finding converges with evidence that the anterior temporal lobe is important for successful semantic representation  and restSpeech fluency is usually the first domain on which aphasia patients are categorized . In our Finally, VBCM analysis failed to identify any neural correlates for the executive factor  . FurtherStudies of cognitive and language impairments in stroke patients have provided valuable insights into understanding the organization of the brain and provide implications for clinical application. In the present study, we have demonstrated that the underlying structure of the lesion reflects the brain’s vasculature that can be utilized in lesion–symptom mapping to reduce mis-location error in stroke patients. In the future, one would need to formally test and validate the use of these PCA-derived lesion components in lesion–symptom mapping, prediction modelling and related neuropsychological approaches that aim to relate brain to behaviour.fcaa062_Supplementary_DataClick here for additional data file.


On the other hand, Ca2+ confined at z = 19 Å crosslink charged saccharides and prevent the incorporation of cationic surfactants. We found that the addition of nonlethal aromatic alcohols facilitate the incorporation of cationic surfactants by the significant roughening of the chain/saccharide interface. Combination of precise localisation of ions and molecular-level structural analysis quantitatively demonstrated the synegtestic interplay of ingredients to achieve a high antibacterial activity.Antimicrobial resistance is a major threat to public health. Although many commercial sanitisers contain a combination of cationic surfactants and aromatic alcohols, the physical mechanisms where these two substances bind to or how they disturb bacterial membranes are still largely unknown. In this study, we designed a well-defined model of Gram-negative bacteria surfaces based on the monolayer of lipopolysaccharides with uniform saccharide head groups. Since commonly used X-ray reflectivity is sensitive to changes in the thickness, roughness and electron density but is not sensitive to elements, we employed grazing incidence X-ray fluorescence. In the absence of Ca This is due to the overuse of therapeutic agents by over-prescribing antibiotics in the clinical treatment of human patients as well as livestock in the farming industry5. Although the development of antimicrobial agents is necessary for the sustainable society, it is often overlooked that a proper use of sanitisers already enables us to achieve a sufficient hygiene level as well as to reduce the number of antibiotic treatments6. Bacteria, including pathogenic and non-pathogenic species, form colonies, often called biofilms, in households, industry sectors, and hospitals. A recent multicentre study reported patients’ bath basins as potential bacterial reservoirs, which may become a source of transmission of nosocomial infections7.As stated in a report issued by the World Health Organization in 2014, antimicrobial resistance is a major threat to public health8. For example, benzalkonium chloride  the electrostatic binding of positively charged quaternary nitrogen to negatively charged lipids on bacterial membrane surfaces, (2) the integration of hydrocarbon chains of QACs into the hydrophobic core of bacterial membranes, which results in (3) the disruption of cell membranes and subsequent loss of cytoplasm11. However, although QACs have been extensively used since the 1930s, some bacterial strains have gained resistance. For example, Methylobacterium mesophilicum KMC10, which form biofilms in bathrooms, are tolerant against BAC treatment up to a concentration of 5 vol%12, suggesting that one of the above mentioned steps is hindered. Intriguingly, some alcohols, such as benzyl alcohol , bisbiguanides (chlorhexidine), and polymeric biguanides, are among the most commonly used disinfectantsMethylobacterium exposed to either [BAC] = 10 µM or [BzA] = 100 mM alone exhibited no major differences from the control, suggesting that these conditions are nonlethal for bacteria. However, they found that all bacteria died within 3 min of exposure to a BAC and BzA mixture, indicating the synergistic potential of this combination11. Using fluorescent probes, they found that the membrane fluidity increased in the presence of nonlethal BzA (100 mM). In line with previous accounts, they interpreted that BzA destabilised the membrane structures13 and enhanced BAC permeation. However, there has been no quantitative study on the influence of BAC and BzA on the stratified membrane structure and ion distribution near the bacterial surface.Current understanding on the physical mechanism how this combination treatment promotes the antimicrobial function. Yano et al. previously reported that the colony forming unit (CFU) of + ions bound to the saccharide head groups of LPS Ra are displaced by divalent Ca2+ ions, resulting in the maintenance of stratified membrane structures even in the presence of cationic antimicrobial peptides, ranging from fish protamines used for food preservation to peptide-based, antiseptic drug candidates17. Coarse-grained Monte Carlo simulations demonstrated the accumulation of Ca2+ ions, which enables the bacterial LPSs to form an electrostatic potential barrier to protect them from the intrusion of cationic antimicrobial peptides18. Moreover, interfacial shear rheology data indicated that LPS Ra molecules forms a two-dimensional gel in the presence of Ca2+ at the air/water interface, while the same membrane behaves as a sol in the absence of Ca2+ ions19. Based on these data, we hypothesized that the addition of nonlethal aromatic alcohol is necessarry for QAC (BAC) to overcome the barrier made out of negatively charged core saccharides crosslinked by Ca2+.Previously, we demonstrated that the monovalent KSalmonella enterica (serovar Minnesota) rough mutant R60 (LPS Ra) As shown in Fig. 20. The combination of specular X-ray reflectivity (XRR) and grazing incidence X-ray fluorescence (GIXF)21 enables us to quantify the detailed structures of LPS membranes in the direction perpendicular to the membrane within sub-Å resolution, as well as the precise position of K+ and Ca2+ within a Δz = ± 3 Å resolution17. First, we assessed bacterial killing by measuring the CFU of Salmonella enterica sv. Minnesota Ra in the presence of a commercial sanitiser and mixtures of BAC and BzA. Then, we measured XRR and GIXF of the LPS Ra monolayer in the presence of BAC, BzA, and BAC plus BzA  have any protective functions, all experiments were performed in Ca2+-free and Ca2+-loaded buffers.In the present study, we fabricated a model of bacterial outermost surfaces by depositing a monolayer of lipopolysaccharides (LPSs) at the air/water interface, and quantitatively determined how these two compounds influence the structural integrity and specific distribution of each element/ion near the interface. As the most realistic and well-defined building blocks, we chose LPSs purified from Salmonella enterica (serovar Minnesota) rough mutant R60. As shown in Fig. 6 bacteria was deposited on Lysogeny broth (LB) agar, the surface was fully covered with bacterial colonies after an overnight incubation at 37 °C. On the other hand, we found that only 2 bacteria survived when 10 times more bacteria were exposed to a 0.1% (v/v) sanitiser for 5 min before seeding  to confirm its antimicrobial activity to Salmonella enterica after the exposure to solutions of either aromatic alcohol (BzA) or the cationic surfactant (BAC) at different concentrations, in order to understand how each ingredient individually contributes to the killing of bacteria. Figure Salmonella enterica after 5 min exposure to BzA solutions of various concentrations in the absence and presence of Ca2+ ions. Compared to the control containing intact bacteria CFU/mLCa-free ≈ 1012, the CFU/mL levels decreased but remained high even at high BzA concentrations. For example, CFU/mLBzA(Ca-free) ≈ 108 and CFU/mLBzA(Ca-loaded) ≈ 1010 at [BzA] = 100 mM, suggesting that the aromatic alcohol (BzA) alone does not promote the killing of bacteria. Moreover, the CFU/mL levels in Ca2+-loaded buffer (solid symbols) were higher than those in Ca2+-free buffer (open symbols) at all BzA concentrations, indicating the protective function of Ca2+ ions against BzA. All bacteria in Ca2+-free buffer died at [BzA] = 200 mM, while those in Ca2+-loaded buffer still survived, CFU/mLBzA(Ca-loaded) ≈ 107. Figure Salmonella enterica after a 5 min exposure to solutions with different BAC concentrations in the absence and presence of Ca2+ ions. We found that all bacteria were already killed at [BAC] = 1 µM in Ca2+-free buffer (open symbols), while Salmonella enterica could withstand BzA even at 105 times higher dose in the same buffer , the CFU/mL still remained high, CFU/mLBAC(Ca-loaded) ≈ 1010. In the presence of Ca2+ ions, the BAC concentration necessary for killing all bacteria ([BAC] = 20 µM) was more than an order of magnitude higher. The obtained results suggest the critical role of cationic surfactant (BAC) in bacteria killing and the protective function of Ca2+ ions.After confirming the killing capability of the commercial sanitiser containing both BAC and BzA, we measured the normalised colony-forming unit CFU/mL of fer Fig. A. Conver2+-loaded buffer  rough mutant at the air/water interface . Note that π = 0 mN/m of the green curve was defined as the surface pressure of the subphase containing 100 mM BzA (πBzA ≈ 15 mN/m). The data including this offset level are presented in Supporting Information Figure σ = 3.9 Å to 5.6 Å by the presence of BzA, suggesting that BzA molecules adsorb to the interface between hydrocarbon chains and carbohydrate head groups and roughen this interface. However, the roughness of the other interfaces showed no remarkable changes. This seems to explain our data showing that 100 mM BzA caused a decrease in CFU/mL in Ca2+-free buffer but is not sufficient to kill all bacteria . As presented in Fig. 2+. In fact, the electron density profiles reconstructed from the XRR data are almost identical  rough mutant R60.The pressure-area isotherms of the LPS Ra monolayer in the presence of Cafer Fig. A, the incal Fig. F. The pr2+-free buffer. The surface pressure in the presence of BAC was also corrected to the pressure with respect to the subphase containing BAC (πBAC ≈ 7 mN/m). The data including this offset level are presented in Supporting Information Figure π = 20 mN/m increased by ΔA ≈ 90 Å2 in the presence of BAC, suggesting the incorporation of the hydrocarbon chains of BAC into the LPS membrane6. XRR data in the presence of BAC  modulated the layered structure of bacterial membranes with aid of XRR. Figure BAC Fig. B lost peBAC Fig. C clearlyBAC Fig. . This seBAC Fig. B.Figure 2+-loaded buffer , the increase in the mean molecular area caused by BAC, ΔA ≈ 30 Å2, was less than one half compared to the data on Ca2+-free subphase  at [BAC] = 5 µM. The fact that 5 µM BAC alone is not able to disturb the stratified LPS Ra membrane structure in the presence of Ca2+ coincides with the resistance of bacteria against QAC.Intriguingly, the influence of 5 µM BAC on the isotherms seems less pronounced on Ca2+-free buffer, which coincides with the composition in a sanitiser. Note that the surface pressure was corrected by that of the subphase containing 100 mM BzA and 5 µM BAC (πBzA+BAC ≈ 20 mN/m). The data including the offset level are presented in Supporting Information Figure π = 20 mN/m, denoting that the LPS membrane was significantly disturbed  and cationic surfactant (BAC) promotes the disturbance of bacterial outer membranes. Figure N/m Fig. B is simi2+-loaded buffer. Remarkably, the global shape of the XRR curve  are from K+ near the interface, while the data from the angle beyond the critical angle reflect K+ in bulk. The higher counts at qz ≤ qc suggests the accumulation of K+ near the interface. The solid lines are the best-fit results calculated by Eq.  = 4.6 M at zmax(K) = 17 Å from the air/chain interface. The maximum concentration is 46 times higher than the bulk level (100 mM), whereas the peak position corresponds to the position of inner core saccharides consisting of two phosphorylated glucosamine units and two 2-keto-3-deoxyoctonoic acid units. The integration of the concentration along z-axis yields the lateral density of K+ ions, cL(K) = 2.6 × 1014 ions/cm2. From the area per LPS Ra molecule at π = 20 mN/m, A ≈ 210 Å2, the number of K+ ions associated with one LPS Ra molecule can be determined to be N = 5.5. The obtained value seems reasonable from the net charge per core saccharide, 6 e−14. As suggested by XRR  is within the spatial resolution, ± 3 Å18. In fact, the calculated lateral ion density (cL(K) = 2.9 × 1014 ions/cm2) and the number of associating K+ ions (N = 5.7) suggest that the charge neutrality and thus the electrostatic barrier were not disturbed by 100 mM BzA. Although a clear decrease in the counts indicates a distinct decrease in K+ ions near the interface, we could not achieve reasonable fitting results for the GIXF data measured in the presence of 5 µM BAC and 5 µM BAC and 100 mM BzA  = 19 Å. From the lateral density of Ca2+ ions, cL(Ca) = 2.5 × 1014 ions/cm2, we calculated the number of Ca2+ ions associated with one LPS Ra molecule, N = 3.9, which is in good agreement with our previous reports indicating that the binding of Ca2+ to core saccharides resulted in the displacement of K+17. It is notable that the presence of 100 mM BzA , and, more remarkably, the lateral density of Ca2+ ions decreased to cL(Ca) = 2.4 × 1014 ions/cm2. As a consequence, the number of Ca2+ ions associated with one LPS Ra molecule decreased to N = 3.8, which allows the binding of cationic BAC. When the LPS Ra monolayer is in contact with both 5 µM BAC and 100 mM BzA  rough mutant Ra (LPS Ra). The combination of structure analysis (XRR) and element-specific localisation (GIXF) enabled us to demonstrate how the main ingredients of sanitisers, a cationic surfactant (BAC) and an aromatic alcohol (BzA), modulate the structures and electrostatics of LPS Ra membranes in a quantitative manner. As it has been established that the presence of divalent cations, such as Ca2+ and Mg2+, facilitates the survival of bacteria, we used Ca2+-free and Ca2+-loaded buffers to discern the protective role of Ca2+. As a control, we performed CFU counting assays  and BzA (100 mM) in sanitisers are not at lethal levels when only one of them is used. Thus, to clearly discriminate the differential roles of the two compounds in the presence and absence of Caane Fig. . The sim µM Fig. B. In con2+-loaded subphase  or the number of ions associated with one LPS molecule. On Ca2+-free subphase, the presence of BAC alone disabled the calculation of K+ density profile, because the layered structure was already disturbed. On Ca2+-loaded subphase, the binding of BAC to LPS, suggested by XRR, caused a slight shift in the peak position and a decrease in the number of Ca2+ associated with one LPS molecule. However, the “layer” of Ca2+ was not disturbed, because the incorporation into the membrane core was blocked by crosslinked core saccharides. In contrast, the ion concentration profiles near the interface could not be calculated in the coexistence of BAC and BzA due to the loss of structural integrity. Interestingly, the concentration of BzA seems to have a significant influence on the synergestic effect; when we exposed the LPS Ra monolayer to 5 µM BAC and 50 mM BzA, we found that the monolayer remained intact in the presence of Ca2+  and the Ministry of Economy, Trade, and Industry (METI) of Japan published the list of surfactants that help the removal of severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2), which includes QAC (https://www.meti.go.jp/english/press/2020/0529_001.html). Simultaneous XRR and GIXF can be used to monitor the initial phase of interactions between virus particles and host cell membranes. Since RNA in coronavirus is surrounded by lipid membranes, the simultaneous XRR and GIXF experiments of membranes with the same composition would help us optimize the molecular structure and dose for the effective virus removal and/or inactivation.The localisation of various elements within Å resolution opens a large potential to determine the density profiles of not only ions but also molecules. For example, the position and density of proteins bound to the membrane surface can be determined by monitoring S 1S signals from S-containing amino acids2+ protects the bacterial outermembrane, we hypothesized that the addition of nonlethal (100 mM) BzA facilitates either the binding or incorporation of BAC. In the absence of Ca2+, all bacteria  died at [BAC] ≥ 1 µM. In fact, the stratified structures of the model of bacterial outermembranes and the layer of K+ ions bound to negatively charged core saccharides (z = 17 Å) were significantly disturbed by the exposure to the subphase containing only [BAC] = 5 µM, which corresponds to the actual concentration of BAC in sanitisers. On the other hand, BAC alone were not able to disturb the layered structure. A slight shift of the Ca2+ peak and a decrease in the number of associated Ca2+ in the presence of BAC indicate that BAC bind to LPS but the membrane incorporation was blocked by negatively charged core saccharides crosslinked by Ca2+ ions. Remarkably, the addition of nonlethal BzA (100 mM) disturbed the structural integrity of bacterial outermembranes by the roughening of the chain/saccharide interface. Our XRR data indicated the additional BzA caused a significant increase in the interfacial roughness between hydrocarbon chains and saccharide head groups, which facilitates the incorporation of cationic surfactants.We unraveled the physical mechanism how cationic surfactants (BAC) and aromatic alcohol (BzA), the main ingredients of commercial sanitisers, synergestically overcome the resistance of bacteria against BAC. Specular X-ray reflectivity (XRR) and grazing incidence X-ray fluorescence (GIXF) were combined to gain the molecular-level structures and ion-specific distributions near the bacterial surface simultaneously. As our previous studies suggested that the crosslinking of charged core saccharides by CaThe function of reagents with mutiple ingredients is often explained by the simple summation of the function of each ingredient. Although commonly used reflectivity-based techniques, such as XRR, are powerful tools to detect subtle changes in the thickness, density and roughness on the molecular level, they are not sensitive to the element specific density profiles in the vicinity of interfaces. Our data demonstrated that the simultaneous XRR and GIXF measurements provide more “element-specific” information, which helps us gain insight into physical mechanisms of reactions at complex, biological interfaces, such as binding of virus particles to the host cell membranes and inactivation of virus by surfactants.Salmonella enterica (serovar Minnesota) Ra R6029 and lyophilised as previously reported31. The lyophilised powder of LPS Ra was dissolved in a mixture of liquid phenol, chloroform, and petroleum ether at a volume ratio of 2:5:8. Benzalkonium chloride  and benzyl alcohol (BzA) were purchased from Sigma-Aldrich . Lysogeny broth (LB) and LB agar were purchased from Sigma-Aldrich . Both Ca2+-free buffer  and Ca2+-loaded buffer  were adjusted to pH 7.4 by titrating with KOH and Ca(OH)2, respectively Throughout this study, double deionised water  with a specific resistance of ρ > 18 MΩ cm was used. Unless stated otherwise, other chemicals were purchased from Sigma-Aldrich  and used with no further purification.LPS Ra was purified from the bacterial rough strains of 11. In brief, 10 µL of Salmonella enterica (serovar Minnesota) rough mutant suspension containing 7 × 1010 bacteria/mL was incubated with 1 mL of 0.1% (v/v) bathroom cleaner/sanitiser . To highlight the impact of the two main ingredients, a cationic surfactant (BAC) and an aromatic alcohol (BzA), we also mixed the same bacterial suspensions with 1 mL of BzA and BAC individually and in combination, dissolved in Ca2+-free/loaded buffers. The mixture was always incubated for 5 min at 20 °C in a rotator  to ensure homogenous mixing. The mixture was serially diluted in LB medium, and each fraction was deposited on an LB agar plate. After an overnight incubation at 37 °C, the colony forming units (CFU) were normalised by volume of bacterial suspension  and grazing incidence X-ray fluorescence (GIXF) experiments were carried out at beamline ID10 of the European Synchrotron Radiation Facility . The samples were irradiated with a monochromatic synchrotron beam with a photon energy of either 8 keV or 10 keV . The film balance was kept in a sealed He gas atmosphere to minimise the scattering of the fluorescence emission by air, and the stability of monolayer was confirmed by monitoring the surface pressure throughout the XRR/GIXF measurement, which takes about 40 min  the maximum concentration cmax, and (2) the peak position of concentration zmax.Supplementary information.


Here, we synthesize the current understanding of astrocyte morphology, coupling local synaptic activity to astrocytic Ca2+ in perisynaptic astrocytic processes and morphology-defined mechanisms of Ca2+ regulation in a distributed model. To this end, we build simplified realistic data-driven spatial network templates and compile model equations as defined by local cell morphology. The input to the model is spatially uncorrelated stochastic synaptic activity. The proposed modeling approach is validated by statistics of simulated Ca2+ transients at a single cell level. In multicellular templates we observe regular sequences of cell entrainment in Ca2+ waves, as a result of interplay between stochastic input and morphology variability between individual astrocytes. Our approach adds spatial dimension to the existing astrocyte models by employment of realistic morphology while retaining enough flexibility and scalability to be embedded in multiscale heterocellular models of neural tissue. We conclude that the proposed approach provides a useful description of neuron-driven Ca2+-activity in the astrocyte syncytium.Neuronal firing and neuron-to-neuron synaptic wiring are currently widely described as orchestrated by astrocytes—elaborately ramified glial cells tiling the cortical and hippocampal space into non-overlapping domains, each covering hundreds of individual dendrites and hundreds thousands synapses. A key component to astrocytic signaling is the dynamics of cytosolic Ca Astrocytes of the cortical and hippocampal gray matter are important actors in a number of information processing processes, including synaptic plasticity, long-term potentiation, and synchronization of neuronal firing Haydon,  as well 2+ dynamics on data-driven irregular structures, both for an individual cell and for a network; (iii) to reveal how realistic morphological features are manifested in the spatiotemporal patterns of the calcium dynamics.In summary, this frames a new mindset for understanding the function of astrocytes and at the same time poses a challenge for modeling studies. Namely, the morphological features should now be considered as a specific control parameter that significantly contribute to the both single-cell dynamics and network activity patterns. This problem breaks down into three specific tasks: (i) to provide tractable, but still biologically reasonable mathematical account for contribution of subcellular morphological features to intracellular calcium dynamics; (ii) to further develop approaches to modeling of CaWe address these tasks in more detail in the rest of the Introduction.2+-activity at multiple spatial and temporal scales  due to their proximity to synaptic connections between neurons. The transients located in the leaflets primarily rely on influx of Ca2+ through plasma membrane, in part because of the high surface-to-volume ratio in this region and in part because the leaflets are often devoid of organelles including ER  production by phospholipase C, it can trigger Ca2+-induced release of Ca2+ from intracellular stores via IP3 receptors (IP3Rs) of the ER.The coupling from synaptic activity to local CaVolterra , early wVolterra , from miVolterra , etc. OnVolterra : neurotr3 receptors  postsynaptic neurons at distant synaptic terminals, although most Ca2+ events are confined to a single astrocyte spatial domain. Propagating calcium waves can travel distances of more than 100 μm with speed from 7 to 27 μm/s in culture and brain slices  and store-dependent mechanisms scale with astrocytic process morphology, as defined by surface to volume ratio, cytoplasm volume fraction and the physical presence of ER in the process. This has been studied in point-models by Oschmann et al.  contains only astrocyte, while in extreme class II regions (leaflets), Ca2+ dynamics is dominated by exchange with plasma membrane and each modeled pixel contains a mesh of extremely thin astrocyte processes tangled with neuropil. We thus define a mapping of each pixel in the spatial model template to either class III (no astrocyte) or to a continuous variable between the extreme cases of class I and II with implications in local calcium dynamics and diffusion.In this work we aim to conceptualize our current understanding of spatial organization of the astrocytic Ca2+-related mechanism extremely challenging. Not surprisingly, there is a substantial body of published models that aim to account for the essential features of calcium dynamics in astrocytes, which do not completely agree with each other  with intensity p Hz.We assume that astrocytic CaAstrocytes, like neurons, have complex morphology. Ideally, an algorithm to create spatial templates should provide means to “grow” realistic branching 3D shapes of astrocytes from a set of randomly placed “seed” locations. Indeed, there are many experimental and modeling studies of the branching patterns for various types of neurons  . An example of the described mapping from image intensity to AVF is shown in min (class II) to 1 (class I). Thus, one can set up computation for a specific spatial template by simply drawing it algorithmically or with an indexed palette using a graphical editor.Our simulations are based on compiling an encoded raster image representation (a template) of the model space to region-specific equations. For the sake of computational simplicity, we use two-dimensional spatial layout—each pixel of the spatial template can be interpreted as a thin slab, occupied either exclusively  or partly by astrocyte cytosol; or as belonging to extracellular space. As follows from this approach, each pixel in the model space has to be assigned to either astrocyte-free space (class III) or astrocyte-occupied space, ranging from class I, astrocyte soma and thick branches, to class II, i.e., elements of volume containing a tangle of thin astrocyte processes and unresolved neuronal structures, e.g., synaptic boutons. We account for a graded transition from thick branches to thin processes to leaflets by introducing a local astrocyte volume fraction (AVF) parameter, which defines the landscape of how much of each pixel volume is occupied by astrocyte in the 3D prototype. AVF here is defined as a ratio between local fluorescence intensity of the template and the intensity at the soma AVF = max extracellular glutamate concentration [Glu].The principal variables of the model are (i) the cytosolic calcium concentration r ∈ ]). SVR scales relative inputs of Ca2+ exchange through plasma membrane and with ER as described below, while AVF scales effective diffusion coefficients for Ca2+ and IP3.To account for the morphology-based spatial heterogeneity of astrocytes, we introduce a parameter The equation set for the principal variables reads:JER is the total flow of calcium ions in exchange between the cytosol and endoplasmic reticulum; Jpm is the total flow of calcium ions through the plasma membrane in exchange between the cytosol and extracellular space; IGlu and ICa stand for glutamate- and calcium-dependent inositol trisphosphate production mechanisms, mediated by phospholipases β and δ; Ieq is a simplified first-order equilibration of inositol trisphosphate concentration to the basal level [IP3]0; [Glu]amb is the ambient concentration of extracellular glutamate, and τGlu is the timescale of its clearance and return to the baseline level; ξp(t) is stochastic source of glutamate from nearby located neuronal synapses triggered by Poisson spike trains in each pixel. Additionally, Jdiff, Idiff, and Gdiff describe the finite-element approximation of diffusion of cytosolic Ca2+, IP3 and extracellular glutamate, respectively and are described below.where s accounts for the stratification of intracellular dynamics according to r = rmin ≪ 1 and s ≈ 1, while for deep cytosol locations r = 1 and s ≈ 0. With this we (i) describe that input from all plasma membrane calcium currents is maximal in the leaflets and (ii) assume that endoplasmic reticulum does not invade leaflets much, thus ER exchange is large only in thicker branches and soma. We also assume that all IP3 is produced in the plasma membrane by means of G-protein coupled phospholipase β or Ca2+-dependent phospholipase δ.The weighting coefficient 3R-mediated current JIP3, leak of Ca2+ from endoplasmic reticulum Jleak, and contribution of ER membrane Ca2+ pump Jpump:Total calcium flow across the endoplasmic membrane is composed of IP2+ current via IP3 receptors JIP3 is modeled in the same way as in Ullah et al.  in Ullah et al. ; JGlu = γ[Glu] describes the direct effect of extracellular glutamate on additional calcium influx.where 3 concentration from Equation (3) has the following terms: first, we use a lumped first-order description of [IP3] equilibration to a resting level [3]0IP:The dynamics for IP2+-stimulated IP3 production in the same way as in Ullah et al.  in each pixel is modeled as quantal release triggered by a spike train drawn from a homogeneous Poisson process with intensity p, which agrees with statistics of neuronal firing  term in Equation (4) is given byStochastic glutamate source ξA is the instantaneous increase in glutamate release rate associated with each presynaptic event and tk are times of presynaptic spikes in the given pixel following Poisson process with intensity psyn.where 2+ can remain confined to the spatial domain of a single astrocyte, but can also spread to the neighboring astrocytes  diffusion of intracellular IP3 and Ca2+ between contacting astrocytic leaflets, via gap junctions. Relative input of the two mechanisms differs across brain regions and for the cortical astrocytes the one mediated by the gap junctions has been reported to prevail  than Ca2+ (≈ 10 μm2/s) due to Ca2+ buffering and often disregard Ca2+ diffusion altogether. However, the effective rate of IP3 diffusion can occur on a much slower timescale , e.g., for Ca2+ scaled by spatial grid step δx and local AVF value r: i enumerates the N nearest neighboring astrocyte-containing units. Here, we regard diffusion in porous media and assume that larger AVF is associated with larger cross-sectional area open for diffusion, as the astrocyte process diameter increases, and simultaneously with less tortuous paths taken up by diffusing molecules as the processes become less entangled. This leads to approximately quadratic scaling of D* with r. The diffusive term for IP3 is defined in a similar way.where Finally, neighboring pixels with different AVF values obviously contain unequal volumes of astrocyte cytoplasm; hence, small concentration changes in areas with high AVF should cause larger diffusion-mediated concentration changes in the neighboring pixels with low AVF. This was accounted for by scaling the concentration rates of change due to diffusive exchange by the ratio of the AVF values of the two neighboring pixels.2+ is taken as a lower-bound estimate in Allbritton et al.  implemented in AGEOM–CUDA software  of areas and lifetimes of individual Ca2+ transients in all single-cell spatial templates to avoid selection bias. Ca2+ transients were thresholded at 25% change from the local baseline level. The resulting contiguous TXY volumes of suprathreshold Ca2+ concentration were treated as discrete events. CCDF X is defined as the probability P that X is greater than some x value: To quantify spatiotemporal properties of the simulated CaThe proposed model, including the modifications to the local calcium dynamics and spatial mapping, was tested in a number of simulation experiments with different parameter settings and different spatial templates . To test for agreement between model behavior and the experimentally observed dynamics, first, we looked at the effect of the level of mean neuronal firing rate  on spatio-temporal dynamics of astrocytic calcium, and second, we tested whether the artificial spatial templates could provide for realistic intercellular calcium waves or other collective variants of astrocytic calcium dynamics.psyn = 0.005 Hz) most Ca2+ events were spatially confined and tended to start at a small number of sites, as shown with max-span contours and red labeling in psyn = 0.01 Hz) many Ca2+ events spread to occupy the whole cell domain and again tended to initiate at the same sites. Synaptic signaling events were integrated into a spatial glutamate profile as shown in psyn = 0.01 Hz. There were no obvious differences between these cells and all the others in overall morphology or AVF statistics. All cells did generate whole-cell transients at a higher excitation of psyn = 0.02 Hz. Most cells demonstrated a decline in the number of small events, covering less than 25% of the cell area with excitation, as a larger proportion of events was enabled to spread over larger areas, while the rate of event initiation could remain stable. The area of large (> 25%) events increased for all cells which generate large events under low drive conditions except the three, which did not generate large events at all, apart from other cells. The average area of the small (< 25%) events increased with excitation strength for all cells.The tendency, exemplified by a single template in 2+ transients and slower IP3 production. Both integrate over time to steady-state levels of the model variables. Because the relative input of plasma membrane transport is defined by AVF in our model, we expected that the steady state levels and the probability of Ca2+ event initiation should depend on AVF as well. Steady-state values of [Ca2+] and [IP3] decreased with growing AVF . Due to the large range of steady-state [IP3]i concentrations, the event is unclear in absolute [IP3]i values (middle row), but is obvious in the relative scale (bottom row).An example of a single large Ca2+ activity in most cells is self-organized in a repeated pattern of Ca2+ transient initiation and spreading . The left column of 2 were likely to expand further and cover the whole cell.We next describe statistics of areas and lifetimes of individual Ca2+ transients in 2D spatial templates, which we use to generate astrocytic network in the next section to reduce computational load. We note however that the presented modeling approach is extendable to 3D with minimal modifications. An example of simulation for a 3D single-cell spatial template is presented in 2+ transients at different spatial and temporal scales, waves that expand toward soma and vice versa, there are visible repeated patterns of activation.We so far examined Ca2+ activity was not uniform across the spatial template: there were active “hot” brims in the periphery of most of the cell domains and some cells were less active than others, as shown in 2+ transients originating in some astrocytes and expanding to their neighbors in a wavelike manner, an example of such a wave is shown in 2+]i, separated by 1 s.After testing model behavior at microdomain and single-cell level, we turned to ensembles of connected cells. Collective dynamics of astrocytes was simulated using spatial templates containing about 40 cells as shown in 2+]i and [IP3]i in 3, which reflects individual properties of each cell. IP3 concentration peaks are wider and occur with a small delay in comparison to Ca2+ peaks, which reflects the slower kinetics of IP3 production timescale and a lag due to diffusion of IP3 from the periphery to the somatic region.To simplify the description of emerging spatiotemporal patterns, each cell can be considered as an element that “fires,” i.e., producing a global whole-cell spike, or remains silent. This cell-wise activity is shown for i and created spike-triggered 50 s-long averages from the raster of Ca2+ signals in all patches. Percentage of all points with [Ca2+]i > 0.2 μM in such spike-triggered windows was used as the repeatability score for a given patch. The patches were then projected back onto the spatial template, with averaging of the values in overlapping areas between patches. This resulted in an automated mapping of areas leading to repeated downstream activation at the network level, revealing cells, serving as hubs in spreading multicellular events  an algorithm for data-based generation of 2D spatial templates matching realistic astrocyte morphology, and (ii) morphology-dependent spatially non-uniform parameter landscape for the calcium dynamics. To this end, we introduce the AVF parameter, which sets locally the relative input of the plasma membrane and ER based pathways and scales effective intracellular diffusion coefficients. The central idea underlying this separation is that astrocytes “sense” synaptic activity with fine processes, and it is where Ca2+ transients are relying on extracellular Ca2+ rather than intracellular stores, and where the bulk of IP3 can be produced, while thicker branches and somata provide the positive feedback gain mechanism for IP3-mediated Ca2+-induced Ca2+ release from ER. This mechanism separation is directly mapped to cell morphology in our approach.We proposed a spatially detailed model of astrocytic calcium activity, which reflects current understanding of the two distinct mechanisms of Ca2+ dynamics driven by synaptic activity, represented by spatially uncorrelated point-sources of glutamate release coupled to focal Ca2+ entry, triggered by independent stochastic Poisson spike trains. At the single-cell level, the statistics of Ca2+ event durations and expansion areas turned out to have a power-law distribution resembling experimental data  is confined to small beads, whereas the rest chemical components of the reaction can diffuse freely, which leads to complex dynamical regimes and interaction between neighboring beads with immobilized catalyst. Another similar concept is that of volume transmission and intercellular communication via diffusing signaling molecules secreted by excitable cells  systems in coupled reactor volumes, e.g., microemulsions .The model code and data, used for network spatial template generation and simulations are available for download at DV: computer simulations, analysis of simulation results, figure preparation. AV: computer simulations, analysis of simulation results, figure preparation, writing the manuscript. DP: program design and architecture for simulations, writing the manuscript. AB: conceptualization of the model, network generation algorithm, figure preparation, writing the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


TRPC3-mediated Ca2+ influx has been implicated in a variety of signaling processes in both excitable and non-excitable cells. Kidneys play a commanding role in maintaining whole-body homeostasis and setting blood pressure. TRPC3 is expressed abundantly in the renal vasculature and in epithelial cells, where it is well positioned to mediate signaling and transport functions in response to GPCR-dependent endocrine stimuli. In addition, TRPC3 could be activated by mechanical forces resulting from dynamic changes in the renal tubule fluid flow and osmolarity. This review critically analyzes the available published evidence of the physiological roles of TRPC3 in different parts of the kidney and describes the pathophysiological ramifications of TRPC3 ablation. We also speculate how this evidence could be further translated into the clinic.TRPC3 is a Ca Located in the perirenal space of the retroperitoneum, kidneys perform numerous vital functions ranging from removal of waste products and harmful substances, maintaining the homeostasis of water and solutes, supervising acid–base balance, and control of blood pressure to the synthesis of hormones and biologically essential products. Genetic or acquired renal diseases are among the leading factors to mortality and are recognized as both major health problem and economic burden –3. Moreo+, K+, Cs+) and divalent  cations, it is thought that TRP-dependent Ca2+ influx and subsequent [Ca2+]i elevations are responsible for the majority of TRP channel physiological roles i overload leading to effacement and ultimately podocyte loss. The decrease in podocyte population is further exacerbated due to their limited capacity for regeneration. It was also noted that the magnitude of TRPC6 potentiation correlates with the severity of disease progression i elevations positively correlates with TRPC3 expression in afferent arterioles of postnatal and 20 days old piglets i elevations were dependent upon the stimulation of purinergic P2Y2 receptors and downstream PLC cascade i in the CD cells by activation of the ryanodine – but not IP3-dependent stores followed by store-operated Ca2+ entry (SOCE) i signaling i to initiate proper cellular response(s) i elevations in native CD cells regardless of the status of intracellular Ca2+ stores [2+]i elevations in CD cells. However, it is not known whether TRPC3 could be directly activated by a mechanical stretch of the plasma membrane due to unique structural features of the channel, such as extra-long S3 transmembrane segment i elevations in the CNT/CD cells in response to increased tubular flow i responses to hypotonicity in freshly isolated split-opened CDs i signaling and AQP2-water transport in the CD to regulate renal water handling and systemic water balance.It also appears that TRPC3-dependent Ca2+-permeable channel, which is known to be activated in response to GPCR signaling as well as mechanical distortions of the plasma membrane. It is broadly expressed in the kidney where it is well suited to participate in a variety of signaling and transport processes (summarized in 2+ influx contributes to maintaining the contractility tone of afferent arterioles and glomerular mesangial cells, which could be instrumental in regulating the rate of filtration. Understanding why the upregulation of TRPC3 expression is not as detrimental for podocyte function as increased TRPC6 might be of use in designing new strategies to fight diabetic nephropathy and glomerulonephritis in clinic. Inhibition of TRPC3-dependent Ca2+ influx in the interstitial fibroblasts has been shown to reduce fibrosis and thus could be beneficial during ischemia-reperfusion kidney injury and acute kidney failure. Strong evidence also implicates TRPC3 in mediating trans-cellular Ca2+ reabsorption in the proximal tubule and collecting duct. Stimulation of this mechanism might reduce the formation of renal stones. Finally, TRPC3 functions as an osmosensor in the collecting duct to regulate AQP2-dependent water reabsorption and subsequently urine volume/osmolarity. It is conceivable to speculate that TRPC3 stimulation might facilitate AQP2 trafficking to ameliorate the copious urine production during congenital or lithium-induced diabetes insipidus.TRPC3 is a Ca