Given its limited accessibility, the CA2 area has been less investigated compared to other subregions of the hippocampus. While the development of transgenic mice expressing Cre recombinase in the CA2 has revealed unique features of this area, the use of mouse lines has several limitations, such as lack of specificity. Therefore, a specific gene delivery system is required. Here, we confirmed that the AAV-PHP.eB capsid preferably infected CA2 pyramidal cells following retro-orbital injection and demonstrated that the specificity was substantially higher after injection into the lateral ventricle. In addition, a tropism for the CA2 area was observed in organotypic slice cultures. Combined injection into the lateral ventricle and stereotaxic injection into the CA2 area specifically introduced the transgene into CA2 pyramidal cells, enabling us to perform targeted patch-clamp recordings and optogenetic manipulation. These results suggest that AAV-PHP.eB is a versatile tool for specific gene transduction in CA2 pyramidal cells. Using transgenic mouse lines expressing Cre recombinase predominantly in CA2 pyramidal cells, the unique features of this region, such as processing of social memory8, dynamic regulation of the hippocampal network9, and coding of temporal information10 have been clarified. Now the CA2 Cre lines are becoming the essential tools for the study targeting the CA2 region. In the present study, we proposed an alternate approach for gene transfer to the CA2 region with adeno-associated virus (AAV) vector injections.The hippocampal CA2 region is an anatomically small area located between the CA3 and CA1 regions. Due to the restricted size, it has been less investigated than the other hippocampal areas, both molecularly and physiologically. The CA2 region was first described by Lorente de Nó based on its cytoarchitecture11. AAV-PHP.eB, a capsid variant of AAV serotype 9, was originally developed to cross the blood–brain barrier (BBB)12; intravenous infusion of AAV-PHP.eB results in efficient gene delivery to the whole brain. Yet, the transduction is not so homogenous that it preferentially infects some brain regions, including the CA2 area13.AAV vectors are among the most-favored vehicles for gene transfer to the nervous system. They have different cell affinities depending on their capsid serotypesHere, we took advantage of the tropism of AAV-PHP.eB for the CA2 region via lateral ventricle (LV) injections and developed an alternative method to label CA2 pyramidal cells using the capsid. We demonstrate the specific gene transfer to CA2 pyramidal cells by injection of this vector into the LV of mice and optogenetic manipulation of CA2 pyramidal cells. In addition, the CA2 region was preferentially infected by AAV-PHP.eB in organotypic slice cultures. Thus, the AAV vector pseudotyped with PHP.eB capsid is a versatile genetic tool for the specific labeling and manipulation of CA2 pyramidal cells.12. We infused AAV2/PHP.eB-SynTetOff-EGFP (1.4 × 1011 genome copies (gc)/mouse) by retro-orbital injection, which is a method of intravenous injection  and striatal-enriched protein tyrosine phosphatase (STEP) . Compared to that in the other hippocampal subregions, the CA2 area had a significantly larger number of GFP-expressing cells  into the bilateral LV -brain barrier was likely to be specific for AAV-PHP.eB. To verify the property, we injected a mixture of AAV2/1-SynTetOff-mRFP1 and AAV2/PHP.eB-SynTetOff-EGFP into the LV Fig. , indicat21. Although the CA2 region is known to develop in these cultures22, it is not yet fully organized at postnatal days (P)7–8, the age at which we prepared slices23. Thus, slices must be incubated for at least one week to conduct AAV infection experiments in the CA2 region. As virus vectors become less infectious in slice cultures over time, viruses should be applied within 3 days in vitro (DIV)24.We investigated whether AAV-PHP.eB had an infection preference for the CA2 region in rat hippocampal slice cultures, which preserve neuronal networks ex vivo and are suitable to study network functionsWe chose four different time points  for infection. We added a mixture of AAV2/1-SynTetOff-mRFP1 and AAV2/PHP.eB-SynTetOff-EGFP to the hippocampal slice cultures and examined both RFP and GFP signals by confocal laser scanning microscopy 48 h later Fig. a and b. 2. Based on fluorescent Nissl staining and RGS14 immunoreactivity, we determined the location of the putative CA2 region and the transverse axis along with the stratum pyramidale from CA3 to CA1  CA2 area, with injection into the LV adjacent to the intermediate hippocampus.After LV injection of the AAV2/PHP.eB vector, GFP expression was mainly observed in the dorsal CA2 region , GFP expression was observed not only in CA2 pyramidal cells but also in stratum oriens interneurons and hilar interneurons. Low dose increases the specificity for CA2 pyramidal cells, but may not be sufficient for gene expression due to the lower number of virus copies in the infected neurons. Therefore, we used the AAV-SynTetOff platform, which increases the expression level 40 times compared to that of SYN promotor15 . It is a great advantage that gene transfer to CA2 pyramidal cells can be readily accomplished by injecting the AAV vector carrying the PHP.eB capsid into the LV; in addition, an experimental model similar to that using Cre-expressing transgenic mice can be constructed just by injecting a Cre-expressing AAV vector with the PHP.eB capsid into the LV. It is known that the CA2 region is an important circuit structure in the hippocampus, responsible for social memory function8 and the occurrence of sharp-wave ripples39; therefore, there is a growing need to establish CA2-specific gene transduction methods. We believe that the proposed method, which utilizes the properties of AAV-PHP.eB capsid, is versatile for gene transfer to CA2 pyramidal cells for labeling, activity monitoring, and manipulation.In the present study, we demonstrate that the affinity of the AAV-PHP.eB capsid for CA2 pyramidal cells can be employed in a method of gene transfer into this region. This technique may contribute to the progress of unraveling the functional complexity of the CA2 region. Importantly, this method was applicable not only to the C57BL/6J but also to the BALB/c strain , juvenile C57BL/6J mice , adult BALB/c mice , and pups of Sprague–Dawley rats  were obtained from Nihon SLC. Animals were maintained under a 12-h light/dark cycle and had ad libitum access to food and water. All efforts were made to minimize animal suffering and the number of animals used. This study was carried out in compliance with the ARRIVE guidelines.All procedures involving animals were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The experiments were approved by the Committees for Animal Care and Use and those for Recombinant DNA Study at Juntendo University . Adult C57BL/6J mice 15 using LR clonase II .For construction of pAAV2-SynTetOff-mRFP1, the sequence of an mRFP1 fragment (GenBank AF506027.1)15 and inserted into the MluI/NotI sites of pBSIISK-hFLEX15, resulting in pBSIISK-hFLEX-BGHpA. The hChR2-mCherry fragment was amplified by PCR (primer set P3/P4) from pAAV-CaMKIIa-hChR2(H134R)-mCherry , and inserted into the EcoRI/SalI sites of pBSIISK-hFLEX-BGHpA, resulting in pBSIISK-hFLEX-[hChR2-mCherry]-BGHpA. The hFLEX-[hChR2-mCherry]-BGHpA fragment was inserted into the KpnI/SphI sites of pAAV2-SynTetOff-BBS41 to generate pAAV2-SynTetOff-hFLEX-[hChR2-mCherry].pAAV2-SynTetOff-hFLEX-[hChR2-mCherry] was constructed as follows: a polyadenylation signal derived from the bovine growth hormone gene  was derived from pAAV2-SynTetOff-GFPHindIII-BglII-EcoRI restriction enzyme sites and inserted into the HindIII/EcoRI sites of pAAV2-SYN-tTAad-BGHpA15 to replace the sequence of the improved version of the tetracycline-controlled transactivator (tTAad), resulting in pAAV2-SYN-HBE-BGHpA. An iCre fragment (GenBank AY056050.1) 42 was amplified by PCR (primer set P7/P8) from pBOB-CAG-iCRE-SD  and inserted into the HindIII/EcoRI sites of pAAV2-SYN-HBE-BGHpA, resulting in pAAV2-SYN-iCre-BGHpA.For construction of pAAV2-SYN-iCre-BGHpA, the oligonucleotide set (P5/P6) was annealed to form double-stranded DNA containing HindIII-iCre-F2A-SpeI-EcoRI fragment was amplified by PCR (primer set P7/P9) and inserted into pAAV2-SYN-HBE-BGHpA through the HindIII/EcoRI sites, resulting in pAAV2-SYN-iCre-F2A-SpeI-BGHpA. The EGFP sequence was then amplified by PCR (primer set P10/P11) and inserted into pAAV2-SYN-iCre-F2A-SpeI-BGHpA through the SpeI/EcoRI sites, resulting in pAAV2-SYN-iCre-F2A-EGFP-BGHpA.For construction of pAAV2-SYN-iCre-F2A-EGFP-BGHpA, a 12. The following fusion sequence was newly synthesized: (1) nucleotides 146–2202 of the wild-type AAV2 genome (GenBank AF043303.1); (2) nucleotides 1-2229 of the PHP.B capsid protein (GenBank KU056473.1); and (3) nucleotides 4411–4534 of the wild-type AAV2 genome. The fusion sequence was inserted into the XhoI/NotI sites of pBlueScript II SK (+) , resulting in pBSIISK-R2CPHP.B. We then performed overlap PCR to introduce the A587D and Q588G mutations in the PHP.B capsid protein using the primer pairs P12/P13, P14/P15, and P12/P15. The resultant PHP.eB capsid fragment (GenBank MF187357.1) was inserted into the HindIII/NotI sites of pBSIISK-R2CPHP.B to replace the PHP.B sequence, resulting in pBSIISK-R2CPHP.eB.We also prepared a helper plasmid containing the sequences of the replication protein of AAV serotype 2 (Rep2) and the capsid protein of AAV-PHP.eB43. HEK293T cells  were incubated in 10 CELLSTAR® cell culture dishes (145 mm) . Each genomic plasmid and two helper plasmids, pBSIISK-R2CPHP.eB or pBSIISK-R2C115 and pHelper  were co-transfected into HEK293T cells using polyethylenimine . Virus particles were collected from the cell lysate and supernatant and purified by ultracentrifugation with OptiPrep . The purified solution was ultrafiltrated and concentrated with Dulbecco’s phosphate-buffered saline  containing 0.001% Pluronic F-68  using an Amicon Ultra-15 . Following the ultrafiltration, each virus solution was concentrated to a volume of 100 µL. The virus titer (gc/mL) was measured using quantitative PCR (primer set P16/17) with the Fast SYBR® Green Master Mix . We measured the titer of AAV2/PHP.eB-CAG-GFP  as a reference, and the titer was approximately 10.7-fold higher at 1.07 × 1014. The following titers were corrected by dividing by 10.7; 2.5 × 1013 gc/mL for AAV2/PHP.eB-SynTetOff-EGFP; 9.3 × 1012 gc/mL for AAV2/1-SynTetOff-mRFP1; 2.6 × 1013 gc/mL for AAV2/PHP.eB-SynTetOff-FLEX-[hChR2-mCherry]; 1.6 × 1013 gc/mL for AAV2/1-SYN-iCre-F2A-EGFP-BGHpA; 6.4 × 1012 gc/mL for AAV2/1-SYN-iCre-BGHpA; and 4.1 × 1013 gc/mL for AAV2/PHP.eB-SYN-GFP-BGHpA15. The virus solutions were stored in aliquots at − 80 °C until use.Production and purification of AAV vector particles were performed as reported previously44 or the lateral tail vein14. After recovery from anesthesia, the mice were maintained under specific pathogen-free conditions with ad libitum access to food and water for 3–4 weeks.Mice were deeply anesthetized with isoflurane (Pfizer Japan). We injected 100 µL of AAV2/PHP.eB vector solutions  into the retro-orbital sinusMice were deeply anesthetized by intraperitoneal injection of a mixture of medetomidine , midazolam , and butorphanol  and placed in a stereotaxic apparatus. We injected 0.7–2.0 µL of the virus solution into the lateral ventricle  and/or 0.2 µL into the hippocampal CA2 area  by pressure through a glass micropipette attached to Picospritzer III (Parker Hannifin). After the surgery, the mice received an intraperitoneal injection of atipamezole  and recovered from anesthesia within approximately 15 min. The mice were maintained under regular health checks for one week after AAV injection.Mice were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital . Mice were then perfused transcardially with 20 mL of phosphate-buffered saline  at 20–25 °C, followed by perfusion for 3 min with the same volume of 4% paraformaldehyde  in 0.1 M phosphate buffer  at 20–25 °C. The brains were removed and post fixed overnight at 4 °C with the same fixative. After cryoprotection with 30% sucrose in 0.1 M PB, the brains were cut into 40-µm-thick parasagittal, coronal, or horizontal sections on a freezing microtome . Sections were collected in six bottles containing 0.02% sodium azide in PBS and stored at 4 °C until use for free-floating immunostaining.46. Slices of 400-μm thickness were obtained from the central region of the hippocampi using a McIlwain tissue chopper (Mickle Lab Eng.). The slices were placed on a polytetrafluoroethylene membrane filter , and the culture medium (see below for composition) was added to the bottom surface of the filter. The prepared cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO2. The slice culture medium contained 50% minimal essential medium based on Earle’s salts , 25% Hank’s balanced salt solution , and 25% heat-inactivated horse serum . The culture medium was replaced twice a week with fresh medium throughout the culture period.Whole brains were excised from anesthetized Sprague–Dawley rat pups at P6, and the hippocampi were isolated10 gc/mL with PBS, and 1 µL of the mixture was added to the culture medium. Forty-eight hours after virus application, the slices were washed with PBS for 3 min and fixed with 4% paraformaldehyde in 0.1 M PB for 4 h at 20–25 °C.For the AAV infection in the culture slices, a mixture of AAV2/1-SynTetOff-mRFP1 and AAV2/PHP.eB-SynTetOff-EGFP was diluted to 1.0 × 1047. Mice were anesthetized with isoflurane and decapitated. The brains were removed and placed in ice-cold oxygenated (95% O2/5% CO2) artificial CSF (aCSF) containing (in mM) 127 NaCl, 1.6 KCl, 1.24 KH2PO4, 1.3 MgSO4, 2.4 CaCl2, 26 NaHCO3, and 10 glucose. The brains were sliced coronally at a thickness of 400 µm using a vibratome  in ice-cold, oxygenated modified aCSF that contained (in mM) 222.1 sucrose, 27 NaHCO3, 1.4 NaH2PO4, 2.5 KCl, 1.0 CaCl2, 7.0 MgSO4, and 0.5 ascorbic acid48. Slices were maintained for 30 min at 35 °C in oxygenated aCSF and then incubated for at least 30 min at room temperature before use. All recordings were performed at 33–35 °C. Whole-cell recordings were obtained from mCherry-labeled hippocampal CA2 pyramidal cells using a MultiClamp 700B amplifier and a Digidata 1440 digitizer controlled by pCLAMP10.5 software (Molecular Devices). Glass pipettes (3–6 MΩ) were filled with a solution containing (in mM) 120 K-gluconate, 5.0 KCl, 1.0 MgCl2, 10 HEPES, 0.2 EGTA, 10 Na2-phosphocreatine, 2.0 MgATP, and 0.1 Na2GTP.Acute slices were prepared from the hippocampi of juvenile C57BL/6J miceFor optogenetic manipulation of CA2 pyramidal cells expressing hChR2(H134R)-mCherry, pulses of 465-nm light with a high-power LED illumination system  were applied for 100 ms through a 40 × objective lens . The stimulation was repeated ten times.49, or 1:1000-diluted affinity-purified guinea pig antibody against GFP51 in PBS-X containing 1% (v/v) donkey serum  and 0.12% (w/v) λ-carrageenan  (PBS-XCD). The sections and slices were incubated for 4 h with 5 μg/mL Alexa Fluor (AF) 647-conjugated donkey anti-mouse IgG , AF568-conjugated goat anti-mouse IgG , AF568-conjugated donkey anti-rabbit IgG , AF488-conjugated goat anti-guinea pig IgG , or AF488-conjugated streptavidin  in PBS-XCD, followed by incubation for 30 min with 1:200-diluted NeuroTrace 435/455 Blue Fluorescent Nissl stain  in PBS-X. The sections and slices were mounted onto aminopropyltriethoxysilane-coated glass slides , and coverslipped with 50% (v/v) glycerol and 2.5% (w/v) triethylenediaminein in 20 mM Tris–HCl (pH 7.6).All following incubations were performed at 20–25 °C and followed by rinsing twice with PBS containing 0.3% (v/v) Triton X-100 (PBS-X) for 10 min. Free-floating sections (40-µm thick) and slices (400-µm thick) were incubated for 16 h with 1:500-diluted mouse monoclonal antibody against RGS14 , 1:500-diluted mouse monoclonal antibody against STEP , 1:500-diluted rabbit polyclonal antibody against PCP4 ,1:100-diluted affinity-purified rabbit antibody against mRFP1The sections and slices were observed under a confocal laser scanning microscope  using 5× air , 10 × air , 16 × multi-immersion , and 25× water-immersion  objective lenses with a pinhole at 2–5.6 Airy disk units and a 0.8–1.2 zoom factor. NeuroTrace Blue, EGFP or AF488, AF568, mRFP1, or mCherry, and AF647 were excited using 405-, 488-, 552-, and 638-nm lasers, respectively. Their fluorescence was collected through 410–503, 495–550, 570–650, and 660–750-nm emission prism windows, respectively, and detected using the photon-counting mode of the hybrid detector .The density of GFP-expressing cells was determined by counting the cells in regions of 150 µm × 300 µm in the stratum pyramidale of the CA3, CA2, and CA1 regions, and the granule cell layer of the dentate gyrus.As shown in Fig. n are indicated in the corresponding Figure legends. For comparisons among independent groups, the Kruskal–Wallis test was used (Figs. t-test was used (Fig. P < 0.05.Data are presented as mean ± standard deviation. The exact values of ed Figs. e and 2d.Supplementary Information.


Here, we focused on adenylyl cyclase (AC) as another effector of norepinephrine stimulation. RT-PCR, immunohistochemistry, and Western blotting revealed that the abundant expression of Ca2+-stimulable AC3 was restricted to the supraventricular area, including the PVs. All the other AC isotypes hardly displayed any region-specific expressions. Immunostaining of isolated cardiomyocytes showed an enriched expression of AC3 along the t-tubules in PV myocytes. The cAMP-dependent response of L-type Ca2+ currents in the PV and LA cells is strengthened by the 0.1 mM intracellular Ca2+ condition, unlike in the ventricular cells. The norepinephrine-induced automaticity of PV cardiomyocytes was reversibly suppressed by 100 µM SQ22536, an adenine-like AC inhibitor. These findings suggest that the specific expression of AC3 along t-tubules may contribute to arrhythmogenic automaticity in rat PV cardiomyocytes.Ectopic excitability in pulmonary veins (PVs) is the major cause of atrial fibrillation. We previously reported that the inositol trisphosphate receptor in rat PV cardiomyocytes cooperates with the Na Under the [Ca2+]i-free condition with 5 mM EGTA, the β-adrenergic stimulation increased the Ca2+ currents amplitude of the PV cell by 1.50 ± 0.18 times. At [Ca2+]i of 0.1 mM, the currents were further increased by 2.59 ± 0.58 times  in the absence of [Ca2+]i . The spontaneous automaticity evoked by 10 µM norepinephrine (NE)  could nolevation . The NE-2+]i-concentrating apparatus. For example, during an action potential, the [Ca2+]i below the t-tubule is estimated to exceed 200 μM i. In the patch-clamp experiments, the cAMP-dependent response of L-type Ca2+ currents in supraventricular cells, unlike ventricular cells, is enhanced when [Ca2+]i is abundant  Highly expressed Ca2+-stimulable AC3 localizes at the t-tubule at least in PV cardiomyocytes with a well transversally organised tubular system, where it is strongly influenced by [Ca2+]i. (2) The β-adrenergic effect evaluated by the Ca2+ current is enhanced by [Ca2+]i in PV. Finally, (3) [Ca2+]i is supposedly at a high level during the PV automaticity, and the inhibitory effect of SQ22536 on the automaticity is demonstrated at an adequate concentration of 100 µM against AC3, rather than AC5, AC6, or AC9. The identity of the AC isoforms responsible for cAMP production in PV cardiomyocytes has not been previously elucidated. As a first step toward clarification of this critical issue, we employed RT-PCR, immunohistochemistry, and immunocytochemistry to determine the expression profiles in the rat heart and PVs. The gene expression profile of the AC isoforms by RT-PCR B confirmd 200 μM ,39. Beca~100 μM, . To evalnditions . This drnditions A. By connditions B,C. Acco2+-stimulable, while AC5 and AC6 are Ca2+-inhibitable i in cellular experiments i , moderately sensitive isotypes are AC1 and AC3 , and less sensitive are AC2, 4, and 7 , while AC8 and AC9 are resistant to the drug [2+-stimulable AC may be involved in the catecholamine-dependent arrhythmogenic property of PV by producing cAMP under the high-[Ca2+]i conditions. Among the nine membrane-bound AC isoforms, AC1, AC3, and AC8 are Caibitable . Residuaensitive . AC3 wasensitive  and was lmodulin . In AC3 ctograms , urine actograms . Howeverin vitro , the Ca2eriments , whereaseriments ,49. In t [Ca2+]i , Table 3-tubules ,39. As pthe drug . Accordi−/− mice have been found to have impaired responses to sympathetic stimulation as well as parasympathetic responses such as baroreceptor reflexes i conditions, rather than other Ca2+-inhibitable and -insensitive ACs. Accordingly, SQ22536 suppressed the automaticity potently and reversibly at the concentration appropriately against AC3. These findings suggest that the blockade of AC3 can suppress the ectopic excitability of PVs. Our data may provide valuable information on how ectopic excitability occurs in PVs and may contribute to the development of drugs that can pharmacologically suppress the Ca2+-dependent arrhythmogenic property of PVs because AC3-selective reagents are currently unavailable.This study identified specific AC3 expression in the rat supraventricular area, including arrhythmogenic PV cardiomyocytes. In the t-tubule membrane structure of PV cells, Ca


Ca2+ induces synaptotagmin-1 binding to its own vesicle membrane, called the cis-interaction, thus preventing the trans-interaction of synaptotagmin-1 to the plasma membrane. However, the electrostatic regulation of the cis- and trans-membrane interaction of synaptotagmin-1 was poorly understood in different Ca2+-buffering conditions. Here we provide an assay to monitor the cis- and trans-membrane interactions of synaptotagmin-1 by using native purified vesicles and the plasma membrane-mimicking liposomes (PM-liposomes). Both ATP and EGTA similarly reverse the cis-membrane interaction of synaptotagmin-1 in free [Ca2+] of 10–100 μM. High PIP2 concentrations in the PM-liposomes reduce the Hill coefficient of vesicle fusion and synaptotagmin-1 membrane binding; this observation suggests that local PIP2 concentrations control the Ca2+-cooperativity of synaptotagmin-1. Our data provide evidence that Ca2+ chelators, including EGTA and polyphosphate anions such as ATP, ADP, and AMP, electrostatically reverse the cis-interaction of synaptotagmin-1.Synaptotagmin-1 is a vesicular protein and Ca N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, which are currently considered to be the catalysts of the fusion reaction2. Neuronal SNARE proteins are selectively expressed in neurons and neuroendocrine cells, and regulate release of neurotransmitters and hormones3. Neuronal SNARE proteins consist of syntaxin-1 and SNAP-25 in the plasma membrane, and vesicle-associated membrane protein-2 (VAMP-2)  in the vesicle membrane1. Synaptotagmin-1 is a Ca2+ sensor for fast Ca2+-dependent exocytosis as an electrostatic switch4. The C2AB domain of synaptotagmin-1 coordinates Ca2+ binding, and the Ca2+-bound C2AB domain penetrates negatively-charged anionic phospholipids by electrostatic interaction2. Several different models of synaptotagmin-1 to describe the process of Ca2+-dependent vesicle fusion have been proposed, but the molecular mechanisms of synaptotagmin-1 remain controvertial5.Exocytosis is the process of vesicle fusion and neurotransmitter release regulated by soluble 5. Native vesicles contain ~ 15% anionic phospholipids including phosphatidylserine (PS) and phosphatidylinositol (PI)6, so Ca2+ induces synaptotagmin-1 binding to its own vesicle membrane, i.e., the cis-interaction8. Ca2+ fails and even slightly reduces vesicle fusion in the in-vitro reconstitution system, because synaptotagmin-1 preferentially interacts with vesicle membranes due to the physical proximity and this cis-membrane interaction prevents the trans-interaction of synaptotagmin-1 with the target membranes9. We have reported that ATP reverses this inactivating cis-interaction of synaptotagmin-1 by the electrostatic effect, and the trans-membrane interaction of synaptotagmin-1 only occurs to trigger vesicle fusion in-vivo10. This ATP effect on the cis-membrane interaction of synaptotagmin-1 has been confirmed independently: in a vesicle sedimentation assay a few hundred μM ATP electrostatically prevents a cis-configuration of synaptotagmin-111, and in a fusion assay using a colloidal probe microscopy and pore-spanning membranes ATP accelerates full fusion by preventing the cis-interaction without affecting the trans-interaction of synaptotagmin-112. However, the electrostatic regulation of the cis- and trans-membrane interaction of synaptotagmin-1 to trigger Ca2+-dependent vesicle fusion has not been described in detail.Synaptotagmin-1 is a vesicular protein and interacts with anionic phospholipids electrostatically2+ sensor for synchronous release of diverse vesicles including synaptic vesicles, large dense-core vesicles (LDCVs), and other secretory granules, the mechanism by which Ca2+-cooperativity is regulated is not clear. The Hill coefficient (n) in the Ca2+ dose–response curves for exocytosis represents Ca2+-cooperativity and the Hill coefficient varies depending on cell types from 2 to 5; e.g. calyx-of-Held synapses 15, neuromuscular junctions 16, bipolar cells 17, pituitary melanotrophs 18, and chromaffin cells 19. The Hill coefficient is the intrinsic property of each cell type and factors that regulate Ca2+-cooperativity are poorly understood.Although synaptotagmin-1 is a conserved Ca2+-binding loops of the C2 domains penetrate anionic phospholipids by reducing repulsion between anionic phospholipids and acidic residues in the C2AB domain4. The polybasic patch in the C2B domain electrostatically interacts with PIP2 in a Ca2+-independent manner20, and thereby increases the Ca2+-sensitivity of synaptotagmin-1 membrane binding21. Given that the C2AB domain has five possible Ca2+-binding sites23 and therefore may have the Hill coefficient up to 4–5, but whether local PIP2 concentrations regulate Ca2+-cooperativity is not known.Synaptotagmin-1 binds to anionic phospholipids by electrostatic interaction and the Cacis- and trans-membrane interaction of synaptotagmin-1 by using native LDCVs and the plasma membrane-mimicking liposomes (PM-liposomes). Ca2+ chelators, including EGTA and polyphosphate anions such as ATP, ADP, and AMP, electrostatically reverse the cis-interaction of synaptotagmin-1. Both ATP and EGTA, as Ca2+ chelators, have a similar effect to prevent the cis-membrane interaction of synaptotagmin-1 in free [Ca2+] of 10–100 μM, but ATP, which has a good buffering capacity in the range of 10–500 μM free [Ca2+], is an excellent Ca2+ buffer to study vesicle fusion and synaptotagmin-1 membrane binding. When the trans-membrane interaction of synaptotagmin-1 only occurs, high PIP2 concentrations in the PM-liposomes decrease the Hill coefficient of vesicle fusion and synaptotagmin-1 membrane binding to ~ 2, suggesting that local PIP2 concentrations might control Ca2+-cooperativity of synaptotagmin-1.Here we provide an assay to monitor the 24.LDCVs, also known as chromaffin granules, were purified from bovine adrenal medullae by using continuous sucrose gradient, then resuspended in a solution of 120 mM K-glutamate, 20 mM K-acetate, and 20 mM HEPES.KOH, pH 7.4, as described elsewhereE. coli strain BL21 (DE3) and purified by Ni2+-NTA affinity chromatography followed by ion-exchange chromatography as described elsewhere20. The stabilized Q-SNARE complex consists of syntaxin-1A (aa 183–288) and SNAP-25A  in a 1:1 ratio by the C-terminal VAMP-2 fragment (aa 49–96), and was purified as described earlier25. The C2AB domain of synaptotagmin-1 (aa 97–421) and soluble form of VAMP-2 lacking the transmembrane domain (VAMP-21–96) were purified using a Mono S column  as described previously26. The stabilized Q-SNARE complex was purified by Ni2+-NTA affinity chromatography followed by ion-exchange chromatography on a Mono Q column  in the presence of 50 mM n-octyl-β-d-glucoside (OG)10. The point mutated C2AB domain (S342C) was labelled with Alexa Fluor 488 C5 maleimide (C2ABA488)26.All SNARE and the C2AB domain of synaptotagmin-1 constructs based on rat sequences were expressed in l-α-phosphatidylcholine, Cat. 840055), 15% PE , 10% PS , 25% Chol , 4% PI , and 1% PIP2 . When PIP2 concentrations were changed, PI contents were adjusted accordingly. For FRET-based lipid-mixing assays, 1.5% 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- as a donor dye and 1.5% 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lissamine rhodamine B sulfonyl ammonium salt (Rhodamine-DOPE) as an acceptor dye were incorporated in the PM-liposomes (accordingly 12% unlabelled PE).All lipids were obtained from Avanti Polar lipids . Lipid composition  of the PM-liposomes that contain the Q-SNARE complex was 45% PC , then multilamellar vesicles were prepared by reverse-phase evaporation using a rotary evaporator as diethyl ether was removed. Multilamellar vesicles (0.5 mL) were extruded using polycarbonate membranes of pore size 100 nm (Avanti Polar lipids) to give uniformly-sized LUVs. After the preformed LUVs had been prepared, SNARE proteins were incorporated into them using OG, a mild non-ionic detergent, then the OG was removed by dialysis overnight in 1 L of buffer containing 150 mM KCl and 20 mM HEPES/KOH pH 7.4 together with 2 g SM-2 adsorbent beads. Proteoliposomes had protein-to-lipid molar ratio of 1:500.Incorporation of the Q-SNARE complex into large unilamellar vesicles (LUVs) was achieved by OG-mediated reconstitution, called the direct method, i.e. incorporation of proteins into preformed liposomes20. LDCV fusion reactions were performed at 37 °C in 1 mL fusion buffer containing 120 mM K-glutamate, 20 mM K-acetate, 20 mM HEPES–KOH (pH 7.4), 1 mM MgCl2, and 3 mM ATP  after addition of 0.1% Triton X-100 at the end of experiments.The PM-liposomes that contain NBD-DOPE and Rhodamine-DOPE as a donor and an acceptor dye, respectively, were incubated with LDCVs, thus leading to dequenching of donor fluorescence (NBD) as a result of lipid dilution with unlabelled vesicle membrane26. Anisotropy was measured at 37 °C in 1 mL of buffer containing 120 mM K-glutamate, 20 mM K-acetate, and 20 mM HEPES–KOH (pH 7.4), 5 mM MgCl2, 10 μM EGTA. First, 1 mM Ca2+ was applied, then ATP or EGTA was accordingly added to chelate Ca2+ and reverse the membrane binding of the C2AB domain; each time ATP or EGTA was uniformly mixed by pipetting and a magnetic stirring setup with dilution factor of 1:500 in 1 mL buffer.  was calculated using the formula r = (IVV − G × IVH)/(IVV + 2 × G × IVH), where IVV indicates the fluorescence intensity with vertically polarized excitation and vertical polarization on the detected emission and IVH denotes the fluorescence intensity when using a vertical polarizer on the excitation and horizontal polarizer on the emission. G is a grating factor used as a correction for the instrument’s differential transmission of the two orthogonal vector orientations. Lipid composition of the PM-liposomes (protein-free) was identical to those used in a fusion assay except labelled PE .The C2AB fragments  were labelled with Alexa Fluor 4882+, Ca2+, or Sr2+, thereby chelating divalent cations27. Ca2+ concentrations were calibrated with Fluo-5N, pentapotassium salt, cell impermeant, a low-affinity Ca2+ indicator with a Kd of 90 μM. Fluo-5N (500 nM) was included in buffer containing 120 mM K-glutamate, 20 mM K-acetate, 20 mM HEPES–KOH (pH 7.4), 5 mM MgCl2, and 10 μM EGTA. 5 mM ATP, ADP, or AMP  was added to chelate free Ca2+. The fluorescence signal was measured at 37 °C with wavelength of 494 nm for excitation and 516 nm for emission. The following equation was used to measure free Ca2+ concentrations:min is the fluorescence intensity in the absence of calcium with 10 mM EGTA, Fmax is the maxium fluorescence with 5 mM CaCl2, and F is the fluorescence of intermediate Fluo-5N. Fluo-5N experimental data with 5 mM ATP were correlated with the MaxChelator simulation program that calculates the free [Ca2+].ATP contains negatively charged oxygen atoms which bind to Mg50.All quantitative data are mean ± SD from ≥ 3 independent experiments. Dose–response curves were fitted using four-parameter logistic equations (4PL) (GraphPad Prism) to calculate Hill slope and EC2+ is a triggering factor of vesicle fusion and intracellular Ca2+ concentration ([Ca2+]i) is typically ~ 100 nM, but local [Ca2+]i and Ca2+ microdomains at the vesicle-release sites close to voltage-gated calcium channels increase to ~ 300 μM29. We used ATP, which is a low affinity Ca2+ buffer, to maintain ~ 10 ≤ free [Ca2+] ≤  ~ 300 μM for in-vitro assays31. ATP has a dissociation constant (Kd) ~ 230 μM [Ca2+]27, so ATP is an excellent Ca2+ buffer in the range of 10–500 μM free [Ca2+]33. We used Fluo-5N to measure free [Ca2+] in the presence of ATP to confirm the predictions of [Ca2+] and to determine how much total [Ca2+] is required to achieve a desired free [Ca2+]34, which is good for measuring around 100 μM free [Ca2+], because Kd of Ca2+ chelators should be close to the desired free [Ca2+]35. EGTA (10 μM) was included to remove contaminating Ca2+ for the calibration of free [Ca2+]. An initial total 113 μM free [Ca2+] was reduced to 26 μM in the presence of 5 mM ATP by its chelation of Ca2+  and phosphatidylinositol (PI)2+-bound synaptotagmin-1 is inserted to native vesicle membranes such as synaptic vesicles and large dense-core vesicles (LDCVs) that contain anionic phospholipids10. However, ATP electrostatically prevents the cis-interaction of synaptotagmin-1, whereas the trans-interaction of synaptotagmin-1 to the plasma membrane remains active to mediate Ca2+-dependent vesicle fusion, because PIP2 overcomes the inhibitory effect of ATP by increasing the membrane-binding affinity of the C2AB domain12.Cacis- and trans-membrane interaction of synaptotagmin-1 , which was labelled with Alexa Fluor 488 at S342C . Indeed, 5 mM ATP in the presence of 1 mM Ca2+ ; this results indicates SNARE-dependent vesicle fusion  to reverse the cis-interaction of the C2AB domain to LDCVs  millimolar range of [Ca2+] decreases the trans-interaction of synaptotagmin-1 by shielding PIP2 and (2) sub-millimolar range of [Ca2+] above 300 μM increases the cis-interaction of synaptotagmin-1 to its own vesicle membrane20. To further confirm the cis-interaction at higher [Ca2+], we performed anisotropy measurement ethane-N,N,N0,N0-tetraacetic acid (BAPTA) are well-known and reliable Ca2+ sonsor; 10–100 μM [Ca2+] exponentially induce synaptotagmin-1 binding to membrane that contain PS and PIP2 with Kd ~ 50 μM26. Therefore, ATP is an appropriate and better Ca2+ buffer than EGTA or BAPTA to study the synaptotagmin-1 activity to bind membrane and trigger vesicle fusion. Indeed, we oberserved that ATP and EGTA result in different kinetics of the Ca2+ dose–response curves of vesicle fusion and of the cis-interaction of synaptotagmin-120  in neuroendocrine cells such as pituitary melanotrophs 18 and chromaffin cells 19, but high Hill coefficient in synapses including calyx-of-Held synapses 15, neuromuscular junctions 16, and bipolar cells 17. We overserved that increasing PIP2 concentration reduces the Hill coefficient, which represents Ca2+ cooperativity  is significantly disrupted in the presence of physiological concentration of ATP/Mg2+, but this Ca2+-independent interaction remains strong when the PM-liposomes contain high PIP2 (10% PS/5% PIP2), suggesting that high PIP2 concentrations are required for Ca2+-independent binding of the C2AB domain in physiological ionic strength20. Here, we have used 10% PS/1% PIP2 in the PM-liposomes to selectively examine the Ca2+-dependent membrane interaction and binding of the C2AB domain. However, in the pre-fusion state for vesicle docking and priming, the C2AB domain of synaptotagmin-1 is most likely bound to the plasma membrane through the PIP2-interacting polybasic region of the C2B domain20 or the SNARE complex49 in a Ca2+-independent manner. Ca2+ can induce a re-orientation of the C2AB domain on the plasma membrane by changing the binding mode with the SNARE complex49 or PIP245. This change in orientation may act as a switch to trigger synaptotagmin-1-dependent vesicle fusion in neurons and neuroendocrine cells. Our results do not rule out the possibility for Ca2+-independent interactions of synaptotagmin-1 with the SNARE complex despite extremely weak interaction49 and it remains a topic of further study to include Ca2+-independent interactions of synaptotagmin-1 in our system for physiological relevance.In this study, we investigate the electrostatic regulation of C2AB binding to vesicle membrane and the PM-liposomes. We have previously observed that Ca


KMT2A-SEPT9, KMT2A-ELL, NUP98-NSD1, and RUNX1-USP42 and 81 somatic mutations. Overall, all patients had genetic aberrations comprising of not only cytogenetic changes, but also gene fusions and mutations. We also demonstrated several characteristic genetic mutations according to the AML-MRC subgroup. TP53 was the most commonly mutated gene  and all were found in the AML-MRC subgroup with myelodysplastic syndrome-defining cytogenetic abnormalities (AML-MRC-C) (p = 0.002). These patients showed extremely poor overall survival not only in AML-MRC, but also within the AML-MRC-C subgroup. The ASXL1  and SRSF2  mutations were associated with the AML-MRC subgroup with >50% dysplasia in at least two lineages (AML-MRC-M) and were frequently co-mutated . Both mutations could be used as surrogate markers to diagnose AML-MRC, especially when the assessment of multilineage dysplasia was difficult. IDH1/IDH2  were most commonly mutated in AML-MRC, followed by CEBPA , PTPN11 , FLT3 , IDH1 , and RUNX1 . These mutations were not limited in any AML-MRC subgroup and could have more significance as a risk factor or susceptibility marker for target therapy in not only AML-MRC, but also other AML categories.Acute myeloid leukemia with myelodysplasia-related changes (AML-MRC) includes heterogeneous conditions such as previous history and specific cytogenetic and morphological properties. In this study, we analyze genetic aberrations using an RNA-based next-generation sequencing (NGS) panel assay in 45 patients with AML-MRC and detect 4 gene fusions of  NPM1 and double CEBPA mutations. Nonetheless, some AML categories are diagnosed based on bone marrow (BM) morphology and other associated findings. One of the aforementioned categories is AML with myelodysplasia-related changes (AML-MRC), which is diagnosed in patients who have previous history or specific cytogenetic or morphological properties. The former two conditions are more obvious than the latter. Studies have questioned the independent predictive value of myelodysplasia in the absence of high-risk cytogenetic abnormalities in AML [The current diagnosis of acute myeloid leukemia (AML) is largely dependent on genetic aberrations . In the s in AML ,3 and has in AML . HoweverIn this study, we analyzed the genetic aberrations in AML-MRC using an RNA-based NGS panel assay and detected gene fusions, mutations, and expressions. We compared the genetic profile among AML-MRC subgroups and endeavored to determine characteristic genetic mutations according to subgroup and to elucidate their clinical significance.We evaluated all consecutive patients who were diagnosed with AML-MRC at Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, from 2013 to 2018. Patients’ medical records, including history of myelodysplastic syndrome (MDS), myelodysplastic syndrome/myeloproliferative disorder (MDS/MPN), or chemotherapy; laboratory data, BM morphology, and immunophenotypes, were reviewed. Cytogenetic abnormalities were classified according to the 2020 International System for Human Cytogenetic Nomenclature guidelines . Patient® Myeloid assay for Illumina  according to the manufacturer’s instructions. Briefly, reverse transcription using random primers was performed for synthesis of cDNA, followed by end repair and adenylation steps. Cleanup of cDNA using Agencourt® AMPure® XP beads and ligation of molecular barcode (MBC) adapters and universal primer sites was performed. The MBC adapter-attached cDNA was amplified by the GSP1 primer pool and primer complementary to universal primer site, and a second PCR using the GSP2 primer pool was performed. The libraries were quantitated using a KAPA Universal Library Quantification Kit , normalized, and loaded into NextSeq (Illumina). Data were analyzed by Archer® Analysis version 5.1.7 (ArcherDX). For AMP-based NGS analysis, variants were selected and annotated using analytics algorithms and public databases [Anchored multiplex PCR-based enrichment RNA-sequencing libraries were generated from 250 ng of RNA using the ArcherDx FusionPlexTP53 and FLT3-internal tandem duplication mutations were separately analyzed using Sanger sequencing and fragment analysis according to methods used in previous studies, respectively [ectively ,9. Recurectively .p-value <0.10 in the univariate analysis were entered into a Cox proportional hazards model or proportional hazards model for a subdistribution of competing risk factors. All statistical analyses were performed using SPSS, version 13.0  and R software .Categorical variables were compared using the Chi-square test or Fisher’s exact test, while continuous variables were analyzed with the Mann–Whitney U test and Kruskal–Wallis H test. Overall survival (OS) curves were plotted using the Kaplan–Meier method and were analyzed with the log-rank test. Results were expressed as the hazard ratio with a 95% confidence interval (95% CI). For multivariate analysis, variables with a n = 45) are summarized in FLT3-ITD, NPM1, RUNX1, ASXL1, and TP53 mutations [p = 0.002). Most of the AML-MRC-C were included in the adverse group , while 56% of AML-MRC-M (10/18) and 33% of AML-MRC-H (1/3) were in the adverse group.Patient characteristics , 43 (96%), and 36 (80%) patients, respectively. Monocytic markers CD11c and CD64 were positive in 32 (71%) and 12 (27%) patients, respectively. The aberrant expression of CD7 and CD56 was observed in 13 (29%) and 12 (27%) patients, respectively.KMT2A-SEPT9, KMT2A-ELL, NUP98-NSD1, and RUNX1-USP42, and 82 somatic mutations in 20 genes. The median number of somatic mutations was one per patient , and 44% of patients (n = 20) had more than one mutation. When combined with cytogenetics, all AML-MRC cases had at least one genetic aberration. The most frequent mutation was present in TP53 , followed by ASXL1 , IDH2 , SRSF2 , CEBPA , PTPN11 , FLT3 , IDH1 , and RUNX1 . All TP53 mutations were observed in AML-MRC-C (p = 0.002), specifically in patients with complex karyotype (p < 0.001) and monosomy 5 or del(5q) (p = 0.001). On the other hand, ASXL1 and SRSF2 mutations were more commonly detected in AML-MRC-M compared to AML-MRC-C  and were frequently co-mutated . Patients with ASXL1 mutation were older, with a median age of 69 years (46–87), compared to those without the mutation . They showed a lower expression of CD274 and WT1 genes . Patients with SRSF2 were older, with a median age of 73 years , and showed a lower expression of CD274, IRF8, MECOM, and PDCD1 genes  compared to those without the mutation. Patients with the ASXL1/SRSF2 co-mutation were older, with a median age of 73 years (69–87), compared to those without the co-mutation (p = 0.002). IDH1 and IDH2  were commonly mutated in AML-MRC and were mutually exclusive. Patients with the IDH1/IDH2 mutation showed higher proportions of blasts in peripheral blood  and BM . In terms of immunophenotypes, the TP53 mutation was associated with HLA-DR negativity (p = 0.012) and the ASXL1 mutation was associated with CD33 negativity (p = 0.004). CEBPA and PTPN11 mutations were associated with cy-MPO negativity . PTPN11 was associated with aberrant CD7 expression (p = 0.007)  and a lower expression of IRF8, MECOM, and MYH11  compared to those without the mutation  . In addimutation .p = 0.004), respectively, after the median follow-up period of 84.8 months for survivors (p = 0.010) or only those who had undergone intensive chemotherapy (p = 0.016) , but also in those receiving any treatment (p = 0.010) in the univariate analysis for OS (p = 0.003) (Excluding 6 patients (13.3%) without treatment, 31 (68.9%) and 8 patients (17.8%) received intensive chemotherapy and a low-intensity treatment, respectively . In detaurvivors a. This s= 0.016) b,c. Whens for OS . In the = 0.003) .KMT2A rearrangement. The other two fusions were NUP98-NSD1 and RUNX1-USP42, which could be missed on a cytogenetic analysis. NUP98-NSD1 was reported at a relatively low frequency in AML and MDS and had an impact on poor prognosis [RUNX1-USP42 fusion is a rare RUNX1 rearrangement in AML and further emphasizes the need for the collection of additional cases [AML-MRC has been estimated to represent 24–35% of all AML cases and is more commonly seen in older AML patients . In thisrognosis ,13. The al cases . The genTP53 mutation was most common in AML-MRC and highly associated with AML-MRC-C. All cases with the TP53 mutation had complex karyotypes, while a subset of cases with complex karyotype did not have the TP53 mutation, indicating that complex karyotypic changes generally precede the mutation [TP53 mutations were considered adverse prognostic markers, as replicated in this study. Moreover, the TP53 mutation itself was an adverse prognostic marker within AML-MRC-C. These results collectively indicated that TP53 mutations are associated with an extremely poor overall survival not only in AML-MRC, but also in the AML-MRC-C subgroup [First, the mutation ,16. Bothsubgroup .ASXL1 and SRSF2 mutations were associated with AML-MRC-M, and co-mutations were frequent. Results from this and a previous studies showed that ASXL1/SRSF2 co-mutated AML was associated with old age, AML-MRC, and monocytic differentiation [ASXL1 mutation was defined as an adverse prognostic marker in AML [ASXL1 or the ASXL1/SRSF2 co-mutation. We carefully postulated that ASXL1 mutated AML consists of heterogeneous cases with morphological signs of dysplasia [SRSF2 as well as ASXL1 could have a potential role as a surrogate marker in the AML-MRC-M subgroup, especially when the morphological assessment of multilineage dysplasia is difficult [Second, ntiation . The ASXr in AML , but we ysplasia , which eifficult .IDH1 and IDH2 mutations were detected in all subgroups of AML-MRC without prognostic significance. IDH mutations were also common genetic alterations in AML and MDS. Among AML-MRC, IDH1 and IDH2 mutations have been reported at frequencies of approximately 4% and 21%, respectively [IDH1 mutation was associated with myeloid dysplasia in mice, which exhibited anemia, ineffective erythropoiesis, and increased immature progenitors and erythroblasts [IDH1/IDH2 mutation categorized into groups other than AML-MRC in cases without definable multilineage dysplasia. The IDH1/IDH2 mutation has been scrutinized more as a susceptible genetic marker for current target therapies [IDH1/IDH2 mutations is informative for therapeutic planning rather than AML categorization [Third, ectively . A recenherapies ,23. Takerization .CEBPA, PTPN11, FLT3, and RUNX1 mutations. These mutations were detected in all AML-MRC subgroups. Previous studies demonstrated that dysplasia was not relevant for AML with CEBPA and RUNX1 mutations, and RAS pathway mutations and FLT3-ITD were significantly more frequent in cases without evaluable erythroid cells [The last group included id cells ,26. Accoid cells , and theid cells ,29.For treatment decisions, the diagnosis of AML-MRC by itself may have potential clinical implications. In a previous study by Seymour et al., authors revealed that the use of azacitidine improves clinical outcomes in older patients with AML-MRC as compared to conventional care regimens such as low-dose cytarabine . FurtherAlthough we clearly demonstrated the characteristic genetic changes in AML-MRC, several limitations were noted. We did not fully evaluate MDS-associated genetic mutations because we used an RNA-based NGS panel assay . We did TP53 mutation was closely associated with AML-MRC-C and the extremely poor outcome in AML-MRC. ASXL1 and SRSF2 mutations were associated with AML-MRC-M and could be used as surrogate markers to diagnose AML-MRC. Mutations in IDH1/IDH2, CEBPA, PTPN11, FLT3, and RUNX1 were not limited to any AML-MRC subgroup and could be more significant as risk factors or susceptible markers for target therapy in not only AML-MRC, but also other AML categories.In summary, AML-MRC is composed of heterogenous cases with different risk categories and genetic characteristics. Most AML-MRC patients had genetic aberrations, including gene fusions and mutations, as well as cytogenetic changes. In terms of genetic mutations, AML-MRC showed characteristics according to subgroup, and each had its own significance. The


Herpesvirus sylvilagus”. Thereafter, he moved to Colorado and later accepted a faculty position in the Department of Neurology at the University of Colorado School of Medicine. As a member of the Don Gilden laboratory, he devoted his career toward the study of transcription, latency and pathogenesis of varicella-zoster virus. In mid-career, he developed an interest in organizing scientific meetings. Based on knowledge acquired from that responsibility, he established CALS in 2011, specifically to bring together what he called a disjointed assemblage of virologists with shared interests in viral latency. CALS was a success. In this Festschrift, his friends and colleagues have submitted their contributions. The authors come from virology research centers in several countries, including the United States, the United Kingdom, the Netherlands, Germany, France, Switzerland, Israel, India, China and Japan. The viruses include, besides varicella-zoster virus and herpes simplex virus, cytomegalovirus, dengue virus, and coronavirusRandall Cohrs established the Colorado Alphaherpesvirus Latency Society (CALS) in 2011. The main function of CALS was to support an annual meeting of investigators interested in herpesvirus latency and reactivation. Randall Cohrs died on 30 July 2021. In memory of Randy, we have assembled a Festschrift of articles written by virologists who attended CALS, as well as the Rocky Mountain Virology Association and the International Herpesvirus Workshop. Randy received his PhD degree from Southern Illinois University; the title of his doctoral thesis in the department of microbiology was “The molecular biology of Randall Cohrs was the president of the Colorado Alphaherpesvirus Latency Society (CALS). The main purpose of CALS was to share scientific insights and foster collaborative research efforts among the gathered virologists. After his unexpected death in 2021, we have assembled a Festschrift in memory of Randy and his numerous achievements. Scientists who attended CALS, as well as Randy’s friends and colleagues from other virology meetings, were invited to contribute an article.Randy was a child of the American tall grass prairie. This massive ecosystem encompasses most of the states of North Dakota, South Dakota, Nebraska, Kansas, Iowa as well as sections of Illinois west and south of Chicago. The prairies formed 10,000 years ago and were once home to millions of bison. Long after the bison disappeared, Randy was raised in Illinois. The land is dotted with small villages and medium-sized towns, interspersed with thousands of acres of farmland. In turn, the farmland is crisscrossed by thousands of miles of train tracks. Even as a child, Randy always wanted to know more about how things worked. Randy enjoyed high school, where he enrolled in all the science classes; but he also won a letter for high achievement in cross-country running. Like all teenagers, he also had a variety of summer and after-school jobs, such as a short-order cook, a groundskeeper and even a cemetery caretaker. However, he never passed up a chance to go fishing. Herpesvirus sylvilagus”. Experimental infection with Herpesvirus sylvilagus produces clinical and histopathologic changes in its natural host, the cottontail rabbit (Sylvilagus genus), that are similar to those observed in humans acutely infected with Epstein–Barr virus (infectious mononucleosis). Randy’s most important Ph.D. research publication was a paper in the Journal of Virology, entitled Herpesvirus sylvilagus: Polypeptides of virions and nucleocapsids [After high school, he spent both his undergraduate and microbiology graduate college years at Southern Illinois University (SIU) in Carbondale, Illinois. He always described these years as fulfilling. He obtained his Ph.D. degree under the mentorship of Professor Hassan Rouhandeh on 20 March 1985. The title of his doctoral thesis was “The molecular biology of Asymptomatic reactivation and shed of infectious varicella zoster virus in astronauts [Comparison of virus transcription during lytic infection of the Oka parental and vaccine strains of varicella-zoster virus determined that viral IE62 (ICP4 homolog) was not the sole determinant of attenuation in the live varicella vaccine strain Oka [Randy moved to Colorado soon after graduation in order to be closer to his family who had moved there while Randy was in college. He accepted a post-doctoral position at the AMC Cancer Research Center in Lakewood, Colorado, with Professor Opendra Sharma, to study the role of vitamin E and the stability of RNA in cancer cells . Soon aftronauts . Anotherrain Oka . Randy wrain Oka ,8,9.At the seventh annual meeting of the Rocky Mountain Virology Club in 2007, Kathryn Holmes, from the University of Colorado, summoned Randy, Tony Schountz and Joel Rovnak from across the room to the business meeting, already in progress, and conferred upon them the administrative duties of procuring snacks and beverages for the next meeting. Two years later, Randy and Joel volunteered to run the meeting and Tony set up the website. At the same time, they established the Rocky Mountain Virology Association (RMVA), a 501(c)3 corporation. The RMVA could accept tax-free donations, but more importantly, RMVA could apply for support from the National Institutes of Health (NIH). From 2010 forward, the annual RMVA meeting held at the scenic Colorado State University Mountain Campus has been partially funded by the National Institute of Allergy and Infectious Diseases. With this NIH support, the organizers invited distinguished speakers from around the country. This change expanded the RMVA from a small regional meeting to one with national status. During these meetings, nothing excited Randy as much as making connections between investigators across a broad range of fields .For a brief moment in this commentary, we introduce some pop-psychology to explain Randy’s enthusiasm for organizing virology meetings. There has been considerable research into the traits acquired by children in a family, based on their birth order. Almost everyone agrees that the first-born child is typically a high achiever. Randy was the middle child, having both an older and a younger brother. Randy had the archetypical persona of a middle child, including being a people-pleaser, thriving on friendships, having a large social circle, and playing the role of peacemaker. We believe that these characteristics gave him the ability, along with the valuable experience acquired from supervising the RMVA meeting, to establish the Colorado Alphaherpesvirus Latency Society (CALS) in 2011 (together with Don Gilden). We consider CALS to be one of the crowning achievements of Randy’s career. For nine consecutive years (2011–2019) until halted by the COVID-19 epidemic, CALS was held annually in mid-May at the Christiania Hotel in Vail, Colorado. CALS began as a meeting of 40 scientists interested in latency of alphaherpesviruses, but expanded to 70 when Don and Randy were able to secure rooms in the nearby Tivoli Lodge for additional participants. The rules were simple: submit an abstract, and give a 10 min talk followed by a 10 min discussion. The sessions over 2 days were divided into themes relevant to latency. Each CALS session had one guest speaker; the speaker at the 2019 CALS was the Nobel laureate Stanley Prusiner [Each author will present below an anecdote about Randy, which provides more insight into his persona both inside and outside the world of science.Both Randy and I lived similar childhoods. We talked about trains, not model toy trains but real trains. Both of us were raised on the prairie, where trainspotting was a popular game for children. When a train was first seen or when a train whistle was heard miles away, he walked to the tracks to be only a few feet away when the 200-ton locomotive rumbled past him. The engineers always waved. As his first scientific experiment, he placed penny coins on the tracks and waited to see which became the widest after being crushed by the locomotive. Randy’s favorite railroad was the Soo line because of its weird name, a phonetic abbreviation of the Minneapolis, Saint Paul and Sault Sainte Marie railroad (French word Sault = Soo in English). When Randy moved to Colorado, he switched his allegiance to the scenic Denver and Rio Grande Railroad. Joel later told me that Randy liked Joel’s office because Joel could hear sounds of passing trains from his office.In the late 1970s, both Randy and I were graduate students at SIU in microbiology and chemistry, respectively. Later, we were colleagues working together on varicella-zoster virus latency and reactivation in the same department at the University of Colorado School of Medicine. Having had the privilege of interacting closely with Randy for the past 4 decades, I noticed that the five words that Randy used most often was “how can I help you“. Whether it was a person that he met in the street for the first time or a longtime acquaintance, Randy was always finding ways to help them when there was a need. I can never forget how Randy carried my hand carriage with the broken wheel up the hill to my room in Regensburg, Germany, and when he rolled my 3-year-old son in his stroller throughout the museums in Washington, DC. Additionally, Randy had a knack of making friends independent of language and cultural barriers. I remember him spending more than an hour with a total stranger (who did not know English), whom he bumped into during his visit to India. Randy’s passion for life and curiosity to learn will be missed.Randy and I spent hours brain-storming transcription regulation and working out chromatin immune precipitation protocols. He was an early proponent of next-generation sequencing and did everything he could to promote the core sequencing capacity at the University of Colorado School of Medicine and to support investigators with the skills to analyze sequence data. To that end, we spent a lot of time selecting guest speakers from the sequencing and transcription fields for our virology meetings, more than one of whom lamented that they were not virologists and questioned why were we inviting them to a virology meeting. They came to the meetings and came back again and again. One of Randy’s greatest joys was introducing people who would never meet otherwise. He knew that they needed to meet and just had not realized it yet.VIRUSES has given his friends and colleagues an opportunity to assemble a Festschrift by which to recognize and remember the legacy of Randy Cohrs.On Friday, 30 July 2021, Randy was working late in the laboratory as usual. Just before he left, he arranged meetings the next day with students and colleagues to practice talks for a meeting on Monday. While driving home he must have known something was amiss with his health, because he took care to pull over to the side of the highway out of traffic, one last act of consideration for others. The impact of his death extends far and wide. Randy is buried in Golden, Colorado, at the historic Golden Cemetery, Section 238. Graciously, the journal


However, the Se–TAX nanocomplex appeared to be the most effective, displaying a lower working concentration range and negligible pro-oxidant effect compared with pure SeNPs. The mechanism of Se–TAX beneficial effects involved the activation of some antioxidant enzymes and the suppression of ROS-generating systems during OGD/reoxygenation, while TAX and “naked” SeNPs were less effective in regulating the cellular redox status. Naked SeNPs inhibited a global increase in Ca2+ ions in cytosol, but not OGD-induced hyperexcitation of the neuroglial network, while Se–TAX suppressed both [Ca2+]i rise and hyperexcitation. The effect of TAX at similar doses appeared exclusively in inhibiting OGD-induced hyperexcitation. Analysis of necrosis and apoptosis after OGD/reoxygenation revealed the highest efficiency of the Se–TAX nanocomplex as well. Se–TAX suppressed the expression of proinflammatory and proapoptotic proteins with simultaneous activation of protective genes. We conclude that the Se–TAX nanocomplex combines the antioxidative features taxifolin and the antiapoptotic effect of nanoselenium, involving the regulation of Ca2+ dynamics. The neuroprotective effect of the natural antioxidant taxifolin (TAX) is well known for ischemic pathologies. However, the limitations of taxifolin application are described—poor solubility, low ability to penetrate the blood–brain barrier, and side effects from high doses for stroke therapy. We proposed the problem of targeted delivery of taxifolin and achievement effective concentrations could be solved by developing a nanocomplex of selenium nanoparticles (SeNPs) with taxifolin (Se–TAX). In this study, we developed a selenium–taxifolin nanocomplex based on selenium nanoparticles with a 100 nm size. It was shown that TAX, SeNPs, and Se–TAX were all able to suppress the production of ROS in neurons and astrocytes under exposure to exogenous H Stroke therapies are significantly limited due to rapid brain metabolism and poor transport of most neuroprotectors across the blood–brain barrier . The casAll these processes lead to excitotoxicity, hyperexcitation of neuronal networks, necrosis, and apoptosis activation ,3. The sTaxifolin  is an important bioflavonoid polyphenolic antioxidant, and it can be found in fairly large amounts in fruits, vegetable oils, red wine, tea, Siberian larch, and so on ,5. TaxifThe trace element selenium (Se) has a pleiotropic effect and a high therapeutic potential for the treatment of various diseases. It attracts a lot of attention for the use in therapy and nanomedicine. According to the World Health Organization (WHO), the Se consumption rate is: for adult women, 55 µg/day, for adult men, 70 µg/day. The upper permissible level of Se consumption ranges from 300 to 600 μg/day. The toxic dose is considered to be 900 μg/day ,13,14. T2+ homeostasis and promote cell survival in the penumbra zone i, cell cultures were loaded with Fura-2 . The cells were stained with the probe dissolved in Hank’s balanced salt solution (HBSS) composed of (mM): 156 NaCl, 3 KCl, 2 MgSO4, 1.25 KH2PO4, 2 CaCl2, 10 glucose, and 10 HEPES, pH 7.4. To measure [Ca2+]i, we used the system based on an inverted motorized microscope, Leica DMI6000B, with a high-speed monochrome CCD-camera HAMAMATSU C9100. For excitation and registration of Fura-2 fluorescence, we used an FU-2 filter set  with the excitation filters BP340/30 and BP387/15, beam splitter FT-410, and emission filter BP510/84. Illuminator Leica EL6000 with a high-pressure mercury lamp was used as a source of excitation light. To distinguish neurons and astrocytes, we used short-term applications of 35 mM KCl before the main experiments. This method is described in detail in our previous work i. The application of “naked” SeNPs or the Se–TAX nanocomplex caused dose-dependent generation of Ca2+ signals in astrocytes, including an increase in the base concentration of [Ca2+]i, Ca2+ oscillations, and other modes of behavior of the Ca2+ signaling system. Note that SeNPs and Se–TAX showed similar EC50 values. This indicates the effect of nanoselenium on the Ca2+ signaling system of astrocytes, but not the taxifolin adsorbed on the nanoparticles.Therefore, taxifolin at the studied concentrations did not cause the generation of high-amplitude Ca2O2-induced ROS production at a concentration of 3 μg/mL. Additionally, EC50 for activating the Ca2+ signaling system of astrocytes was about 1 μg/mL. This result implies the use of a higher concentration of nanoparticles in experiments for more efficient activation of Ca2+ responses. Therefore, we used SeNPs and Se–TAX at a concentration of 3 μg/mL in further experiments. During OGD, we revealed two phases of an increase in ROS production. The first phase occurs 15–20 min after the OGD induction, and the second can be observed as the increase in ROS production after 30–35 min of DCF fluorescence registration  during ischemia and had almost no effect on the Ca2+ dynamics. As a result, it was significantly less effective in suppressing necrosis.Therefore under ischemia-like conditions (OGD), the selenium–taxifolin nanocomplex showed the highest efficiency in suppressing ROS production, inhibiting the global increase in i. It has been shown that short-term or chronic consumption of taxifolin enhances the antioxidant status of hippocampal cells through increased expression of antioxidant enzymes Sod1 and catalase and suppression of the increase in Ca2+ ions in the cytosol [2+]i can lead to cell death when such an increase is irreversible [2+]i in hippocampal cells during OGD [2+]i during OGD in cortical cells. However, they are able to inhibit reversible Ca2+ signals, which is a sign of neuronal hyperexcitation. It is known that ROS scavengers, and especially taxifolin, enhance the GABAergic component of neurotransmission during hypoxia and anoxia, as well as upon the addition of exogenous H2O2 [2+]i. However, the nanocomplex of selenium with taxifolin (Se–TAX) exhibits the beneficial features of both components, suppressing both the total increase in [Ca2+]i and ischemic hyperexcitation of the neuronal network.The processes of ROS production, mitochondrial damage, and cell death are closely connected to the dynamics of i and necrotic death through the activation of reactive astrogliosis and the expression of genes encoding cytoprotective proteins [A cascade of damaging intracellular events triggered with ischemia/reoxygenation in brain cells ultimately leads to the induction of necrosis and apoptosis. Previously, we showed that prolonged incubation of cerebral cortex cells with SeNPs leads to the inhibition of the OGD-induced global increase of [Caproteins . In thisproteins . After Oproteins . Interesproteins , suppresproteins ,68,69, a2+ homeostasis and promote cell survival in the penumbra zone [The anti-inflammatory effect of taxifolin has also been shown: its administration (30 mg/kg/day) leads to a decrease in the expression of TNFα, NOS, IL-1β, Caspase-3, and Bax, which occurs simultaneously with an increase in the level of Bcl-2 in the hippocampus and cerebral cortex of LPS-treated mice . A similbra zone ,71. Othe2+ cell dynamics and the expression of genes encoding proteins responsible for cell survival. Note that there are only few studies on the cytoprotective mechanisms of action of taxifolin in the form of nanocomplexes with selenium, especially in complex neuroglial networks during ischemia and reoxygenation. The taxifolin nanocomplex with nanoselenium—P80-Que@Se nanocomposites (P80-Que@Se NCs)—was shown to suppress the aggregation of Aβ 1–42, to attenuate oxidative stress, and to hold promise for the treatment of Alzheimer’s disease . It is s2O2. The nanocomplex has high antioxidant properties and antiapoptotic and anti-inflammatory effects, increasing the level of expression of protective proteins and suppressing the expression of damaging ones. In general, the effectiveness of the studied agents in ischemia/reoxygenation can be arranged in descending order when using similar concentrations: Se–TAX > SeNPs > TAX. It should be said that the use of a nanocomplex based on selenium nanoparticles doped with taxifolin is a promising neuroprotective approach for brain ischemia due to the low toxicity of both components of the nanocomplex. At the same time, the prospect of using the nanocomplex is provided by the effect of the selective delivery of effective doses of taxifolin with the help of taxifolin. The limitation of this nanocomplex should consider the need for additional in vivo experiments.The developed selenium–taxifolin nanocomplex, created on the basis of selenium nanoparticles with a size of 100 nm, showed high cytoprotective efficiency in the cortical cells during ischemia/reoxygenation and the action of exogenous H


Export of one Fe2+ by Fpn is coupled to import of two H+ to maintain charge balance. Here, we show that human Fpn (HsFpn) binds to and mediates Ca2+ transport. We determine the structure of Ca2+-bound HsFpn and identify a single Ca2+ binding site distinct from the Fe2+ binding sites. Further studies validate the Ca2+ binding site and show that Ca2+ transport is not coupled to transport of another ion. In addition, Ca2+ transport is significantly inhibited in the presence of Fe2+ but not vice versa. Function of Fpn as a Ca2+ uniporter may allow regulation of iron homeostasis by Ca2+.Ferroportin (Fpn) is a transporter that releases ferrous ion (Fe SLC40A1 gene, is the only known Fe2+ exporter in human (Ferroportin (Fpn), encoded by the in human . Fpn is in human . Activitin human . Mutatioin human .2+/2H+ exchanger, that is, the export of one Fe2+ is accompanied by the import of two H+  that binds to HsFpn with nanomolar affinity (2+ transport. As the fragment of antigen-binding (Fab) of 11F9 is known to target the intracellular side of HsFpn , first in the absence of a preset membrane potential and then in the presence of –120 mV membrane potential , we added valinomycin to clamp the membrane potential to –120 mV and we observed a large increase of Ca2+ influx. The rate of Ca2+ uptake in the second phase is faster than that in the first phase  µM  (2+ uptake in HEK cells expressing HsFpn and obtained a KM of 85.9 (36.6–193.0) µM  . We also93.0) µM .2+ by cryo-electron microscopy (cryo-EM) to an overall resolution of 3.0 Å  and a triple alanine mutation (Gln99_Asn100_Glu219 to Ala) to the Ca2+ binding site show significantly reduced Ca2+ transport activity in the proteoliposome assay , which is one helical turn away from Asp39 at Ca2+ binding site, maintains high Ca2+ transport activity . We found that Ca2+ binding is an endothermic process with a dissociation constant (DK) of ~32 µM   either  . We then binding . As a cotype WT; . In addi) either . Combine2+ in the presence of Co2+, and vice versa. In the presence of 2 mM Co2+, Ca2+ binding affinity is reduced by ~fourfold as shown in DK from fitting of the ITC data. We interpret the reduced Ca2+ binding in the presence of Co2+ as the loss of Asp39 to the coordination of Co2+ at S1 (2+ binding affinity (DK) is not affected in the presence of 2 mM Ca2+, as shown in 2+. This result is similar to the effect of the S1 mutation on Co2+ binding   . We inte2+ transport in the presence of Fe2+ or Co2+, and Fe2+ or Co2+ transport in the presence of Ca2+. We found that Fe2+ or Co2+ transport is not affected significantly in the presence of either 1.25 mM or 0.5 mM Ca2+ , a bacterial homolog of human Fpn  was cloned into a pFastBac dual vector. A Tobacco Etch Virus (TEV) protease site and an octa-histidine (8×His) tag was appended to the C-terminus of the protein. HsFpn was expressed in Sf9 (Spodoptera frugiperda) cells using the Bac-to-Bac method (Invitrogen). Purification of HsFpn follows the same protocol reported for Tarsius syrichta Fpn (TsFpn) (Codon-optimized cDNA of HsFpn (UniProt ID:  (TsFpn) . Purifie2 for 30 min on ice before cryo-EM grid preparation.Membrane scaffold protein (MSP) 1D1 was expressed and purified following an established protocol . For lip-/Å2 for each stack. The stacks were motion corrected with MotionCor2 (The cryo-EM grids were prepared using Thermo Fisher Vitrobot Mark IV. The Quantifoil R1.2/1.3 Cu grids were glow-discharged with air for 15 s at 10 mA using Plasma Cleaner (PELCO EasiGlow). Aliquots of 3.5 µl nanodisc sample were applied to the glow-discharged grids. After blotted with filter paper  for 4.0 s, the grids were plunged into liquid ethane cooled with liquid nitrogen. A total of 4498 micrograph stacks were collected on a Titan Krios at 300 kV equipped with a K3 direct electron detector (Gatan) and a Quantum energy filter (Gatan) at a nominal magnification of 81,000×and defocus values from –2.5 µm to –0.8 µm. Each stack was exposed in the super-resolution mode with an exposing time of 0.0875 s per frame for a total of 40 frames per stack. The total dose was about 50 etionCor2  and binntionCor2 . The deftionCor2 .ab initio reconstruction with limited particles from the best 2D classes. Multiple rounds of heterogeneous refinement were performed with particles selected from the 2D classification and four initial reference models until no more than 5% of input particles were classified into bad classes. A final of 437,959 particles after heterogeneous refinement were subjected to non-uniform (NU) refinement with an adaptive solvent mask. After handedness correction, local refinement and CTF refinement were performed with a soft mask around the Fpn and the Fv region of the Fab. Resolutions were estimated with the gold-standard Fourier shell correlation 0.143 criterion  low-pass filtered to 20 Å in RELION 3.1 . Particlriterion . Local rriterion .The structure of apo HsFpn (from PDB ID 6W4S) and 11F9 Fab (from PDB ID 6VYH) were individually docked into density maps in Chimera . The doc2 or 5 mM CoCl2 was prepared in the same FPLC buffer. For competition binding, either 2 mM CoCl2 or CaCl2 was added to protein samples prior to ligand titration. The ITC measurements were performed in Auto-iTC200  at 25 °C. A total of 25 injections were administered (1.01 μL for injections 1 and 2.02 μL for injections 2–25) with a 150 s interval between injections. Background-subtracted data were fitted with binding models in the Origin 8 software package  to extract DK, ΔH, and entropy change (ΔS).The WT and mutant HsFpn proteins were purified as described above and concentrated to 50–75 μM (3–4.5 mg/mL) in the FPLC buffer. The buffer was degassed, and all the protein samples were centrifuged at 18,000×g for 20 min to remove aggregates. The injectant of 2 mM CaCl2 for 2 days, cells were harvested and solubilized in the lysis buffer  plus 1% LMNG and Protease Inhibitor Cocktail (Roche) for 1 hr at 4 °C. Insoluble fractions were pelleted by centrifugation and supernatants were run in SDS-PAGE. Proteins were visualized by western blotting with mouse anti-Strep (Invitrogen/Thermo Fisher) and IRDye-800CW anti-mouse IgG (Licor). Images were taken on an Odyssey infrared scanner (Licor).The cDNAs of WT and mutant HsFpn were subcloned into a modified pEG BacMam vector with a C-terminal Strep-tag. The resulting plasmids with Fpn or the empty plasmid were transfected into HEK 293 F cells on six-well plates with 293fectin transfection reagent (Invitrogen/Thermo Fisher) per the manufacturer’s protocol. After incubation at 37 °C with 8% CO2, and 5 mM D-glucose. The loading of Fluo-4  for Ca2+ uptake assays or pHrodo Red  for H+ transport assays was performed following manufacturer’s protocols. After the loading finished, free dyes were washed away, and cells in each well were maintained in 90 µL LCIS. Both the Ca2+ uptake and H+ transport assays were performed in the FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices) at 37 °C. Fluorescence changes were recorded at an excitation and emission wavelength of 485 nm and 538 nm for Ca2+ uptake assays, and 544 nm and 590 nm for H+ transport assays with 5 s intervals. Transport was triggered by the addition of 10 µL ligand stock solution (CaCl2 or CoCl2) to achieve the desired concentration of extracellular Ca2+ or Co2+. The H+ transport was assayed with 500 µM Co2+ or Ca2+. To test the effect of extracellular pH on Ca2+ uptake, the extracellular buffer was changed to pre-warmed LCIS with adjusted pH soon before the addition of 500 µM Ca2+. All the mutants were assayed with 500 µM Ca2+. For Ca2+ uptake assays, the initial rate is defined by slope of a linear fit to the first 25 s of data points. For H+ transport assays, relative fluorescence changes at the equilibrium stage were averaged to represent intracellular pH changes.The pEG BacMam plasmids with HsFpn or the empty plasmid were transfected into HEK 293 F cells on black wall 96-well microplates coated with poly-D-lysine (Invitrogen/Thermo Fisher). After 2 days, cells were washed in the live cell imaging solution (LCIS) containing 20 mM HEPES (pH 7.4), 140 mM NaCl, 2.5 mM KCl, 1.0 mM MgClhttp://n2t.net/addgene:104463; RRID:Addgene_104463)  domain. Ca2+ binding to CaM triggers conformational changes that results in increased GFP fluorescence. Ca2+ uptake in the presence of Fe2+ or Co2+ were performed in FlexStation 3 as described above except that the excitation and emission wavelength were set at 485 nm and 513 nm. Fe2+ (NH4Fe(SO4)2) or Co2+(CoCl2) ions were loaded into cells by incubation for ~10 min at 37 °C, during which time fluorescence readings were recorded. For Fe2+ loading, 1 mM sodium ascorbate was used to protect Fe2+ ions from oxidation. To start the export of Fe2+ or Co2+, the extracellular buffer was exchanged to pre-warmed LCIS ~30 s before the addition of Ca2+. LCIS with adjusted pH was used when testing the effect of extracellular pH. The initial rate is defined as the slope of a linear fit to the first 25 s of transport data.The WT and mutant HsFpn were expressed in HEK 293 F cells as described above except that the plasmid for mammalian expression of jGCaMP7s (pGP-CMV-jGCaMP7s) was co-transfected. The pGP-CMV-jGCaMP7s was a gift from Douglas Kim & GENIE Project  . GCaMP iPOPE and POPG lipid (Avanti Polar Lipids) were mixed at a 3:1 molar ratio, dried under Argon, and vacuumed overnight to remove chloroform. The dried lipid was resuspended in the reconstitution buffer  to a final concentration of 10 mg/mL. After hydration for 2 hr, the liposome sample was sonicated to transparency and incubated with 40 mM n-decyl-β-D-maltoside  for 2 hr at room temperature under gentle agitation. Then HsFpn protein was added at a 1:100  ratio. For the empty control, the same volume of blank buffer was added. Detergent was removed by dialysis at 4 °C against the reconstitution buffer. Dialysis buffer was changed every day for 4 days. The proteoliposome or empty liposome sample was aliquoted and frozen with liquid nitrogen, and was stored at –80 °C for future use.2+ influx assays, or with 250 μM calcein (Invitrogen/Thermo Fisher) for Fe2+/Co2+ influx assays. The dye was incorporated during three cycles of freeze-thaw. Liposomes were extruded to homogeneity with a 400 nm filter . Removal of free dyes outside liposomes and exchange of outside buffer was achieved by passing samples through a desalting column  equilibrated with buffer required for a desired transport condition. Liposome samples were transferred to a quartz cuvette for fluorescence recording in a FluoroMax-4 spectrofluorometer (HORIBA). Fluorescence changes were recorded at an excitation and emission wavelength of 494 nm and 513 nm with 10 s intervals at 37 °C.Proteoliposomes with HsFpn or empty liposomes were thawed and mixed with 100 μM Fluo-4  for Ca2+ influx, transport was initiated by the addition of CaCl2 to the desired concentration. When testing the inhibition by hepcidin or 11F9 Fab (i)/(o), 20 µM human hepcidin (Sigma) or purified 11F9 Fab (2. For the Fab (o) condition, 20 µM Fab was added to samples collected from desalting column. When testing the effect of different pHs, an outside buffer has the same components as the inside buffer but was adjusted to a desired pH. When testing the effect of different Na+, K+, or Cl-, the inside and outside of liposomes have symmetrical buffers with 100 mM NaCl, KCl, or K-Gluconate. For measurements with –120 mV membrane potential shown in 2. For the –120 mV group, the outside buffer contained 20 mM HEPES (pH 7.5), 1 mM KCl, and 99 mM NaCl while the inside buffer has 20 mM HEPES (pH 7.5) and 100 mM KCl. For the 0 mV group, the outside buffer was the same as the inside buffer with 20 mM HEPES (pH 7.5) and 100 mM KCl. For the +120 mV group, the inside buffer has 20 mM HEPES (pH 7.5), 1 mM KCl, and 99 mM NaCl while the outside buffer contained 20 mM HEPES (pH 7.5) and 100 mM KCl.To monitor Ca11F9 Fab  was adde2+/Co2+ influx assays, symmetrical buffer  was used and transport was initiated by dilution (10×) of 30 µL of liposome sample into 270 µL of outside buffer containing 50 µM of NH4Fe(SO4)2 or CoCl2. In the case of Fe2+ influx, 1 mM sodium ascorbate was added. To load liposomes with Ca2+, 1.25 mM or 0.5 mM CaCl2 was added prior to freeze-thaw cycles. Buffer with 1.25 mM or 0.5 mM CaCl2 was used during the desalting step. To create a 10-fold Ca2+ gradient opposite to the Fe2+ or Co2+gradient, samples were diluted into the outside buffer without Ca2+.For Fe +-driven transporter for Fe2+-efflux is also capable of passive Ca2+ influx. The evidence supporting the conclusions is convincing, but the physiological relevance of Ca2+ entry by Fpn is yet to be established. The work will be of broad interest to cell biologists and biochemists.This study makes an important contribution to the function of the iron transporter Ferroportin (Fpn). By using a combination of proteoliposome assays, mutagenesis and structural studies by cryo EM, the authors are able to demonstrate that the H public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:2+ transport by ferroportin" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including David Drew as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Kenton Swartz as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Aashish Manglik (Reviewer #3).Thank you for submitting your article "Mechanism of CaThe reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Major concerns:2+ binding. What is unclear is if Ca2+ is actually transported to a significant level? Is the Fpn protein just capable of one turnover in proteoliposomes? We highly recommend kinetics using 45CaCl2 (eg. PNAS E5354-E5362) to calculate actual Ca2+ turnover (kcat).You have been able to robustly demonstrate CaReviewer #1 (Recommendations for the authors):2+ binding. What is unclear to me is how much Ca2+ is actually transported? Is the Fpn protein just capable of one turnover in proteoliposomes? The "cleanest" experiment would be to measure Ca2+ uptake using 45CaCl2 (eg. PNAS E5354-E5362) so one can calculate Vmax and kcat. From my understanding, metalloproteins do not necessarily use the optimum metal for catalysis, but the metal available in the appropriate concentration in where they function. I wonder if the Ca2+ site is just not selected against, because its not needed too, i.e., Ca2+ doesn't compete for Fe2+ uptake and the actual "turnover" for Ca2+ is very low?The authors have been able to robustly demonstrate CaReviewer #2 (Recommendations for the authors):2+, although it does not appear to function as an Fe2+-Ca2+ antiporter. What is less clear is whether Fpn will transport calcium bi-directionally. There are technical issues that would make this a bit hard to study, but if the authors think the answer is clear from their data, they should be explicit in explaining this. A further question that needs explaining is why bind and transport calcium? Cells have a high capacity for calcium flux independent of Fpn. Is there a physiological importance to this activity?The data reported here appear to be of high quality and are convincing; the experimental design is excellent and the necessary controls are appropriately employed. A couple of issues need clarification in the text. FPN is clearly an iron efflux pump and these studies make clear that Fpn can also import Ca Major concerns:2+ binding. What is unclear is if Ca2+ is actually transported to a significant level? Is the Fpn protein just capable of one turnover in proteoliposomes? We highly recommend kinetics using 45CaCl2 (eg. PNAS E5354-E5362) to calculate actual Ca2+ turnover (kcat).You have been able to robustly demonstrate Ca2+ is measured by changes of Fluo-4 fluorescence. The fluorescence intensity increases progressively as we increase the concentrations of the Ca2+ and reaches up to five-fold higher than the initial value. As shown in Author response 1, a five-fold increase of fluorescence intensity requires >20 µM of Ca2+. If we assume that a lipid headgroup has an area of ~60 Å2 and that liposomes have an average diameter of ~400 nm, we arrive at a rough estimate of >100 turnovers for each Fpn under 500 µM external Ca2+ as shown in Figure 1—figure supplement 4a.Uptake of CaReviewer #1 (Recommendations for the authors):2+ binding. What is unclear to me is how much Ca2+ is actually transported? Is the Fpn protein just capable of one turnover in proteoliposomes? The "cleanest" experiment would be to measure Ca2+ uptake using 45CaCl2 (eg. PNAS E5354-E5362) so one can calculate Vmax and kcat. From my understanding, metalloproteins do not necessarily use the optimum metal for catalysis, but the metal available in the appropriate concentration in where they function. I wonder if the Ca2+ site is just not selected against, because its not needed too, i.e., Ca2+ doesn't compete for Fe2+ uptake and the actual "turnover" for Ca2+ is very low?The authors have been able to robustly demonstrate CaSee response to Major concerns.Reviewer #2 (Recommendations for the authors):2+, although it does not appear to function as an Fe2+-Ca2+ antiporter. What is less clear is whether Fpn will transport calcium bi-directionally. There are technical issues that would make this a bit hard to study, but if the authors think the answer is clear from their data, they should be explicit in explaining this. A further question that needs explaining is why bind and transport calcium? Cells have a high capacity for calcium flux independent of Fpn. Is there a physiological importance to this activity?The data reported here appear to be of high quality and are convincing; the experimental design is excellent and the necessary controls are appropriately employed. A couple of issues need clarification in the text. FPN is clearly an iron efflux pump and these studies make clear that Fpn can also import Ca2+ transport when the Fab was added only to the external side, and we found that the rate of transport is higher than the rate when the Fab was added to both sides and lower than the rate when no Fab was added . We take this result as further indication that Fpn is reconstituted in both orientations.We added an explanation that the Fpn in liposomes is randomly oriented and made a schematic drawing  to illustrate this point. We also measured Ca2+ uptake by Fpn. We speculate that loss of cellular Fe2+ will lead to Ca2+ influx through Fpn, which may trigger further cellular responses.We are also intrigued by the calcium transport activity of Fpn, and more study is required to address the questions on biology Ca2+ transport, both studies share common grounds on the calcium binding site and transport of Fe2+ in the presence of Ca2+. We made a comment on this in Discussion.Although our study arrived at a different conclusion from that of Deshpande et al. 2018 with regard to Ca