A cobalt-catalysed C–H carbonylation of aliphatic carboxamide derivatives is described, employing commercially available Co(ii)-salts in the presence of a silver oxidant. ii)-salts in the presence of a silver oxidant. This operationally simple process utilises an atmospheric pressure of CO and generates a range of substituted succinimide products bearing diverse functional groups that can be successfully accessed via this methodology.A cobalt-catalysed C–H carbonylation of aliphatic carboxamide derivatives is described, employing commercially available Co( Ge and co-workers described efficient intra- and intermolecular amidation reactions,9 and the Zhang group subsequently developed an alkynylation/cyclisation procedure,9 affording substituted pyrrolidinone products in excellent yields –H activation using earth abundant metals. Herein, we report the development of an oxidative Co-catalysed carbonylative cyclisation procedure of aliphatic quinolinamides to yield a range of substituted succinimide products  under a CO atmosphere.9 We observed the formation of the desired C(sp3)–H carbonylation product in only one of these cases  catalyst, C–H carbonylation did not proceed, confirming the role of the metal species in mediating this process (entry 5). Replacement of sodium benzoate base with sodium acetate, sodium carbonate or sodium phosphate proved detrimental to the efficiency of the process, with succinimide 7a being formed in diminished 23%, 3% and 2% yield, respectively (entries 6–8). Surprisingly, we found that silver salts were essential for a successful reaction; only traces of the desired product were observed upon replacing Ag(i) oxidants with Mn(OAc)3·2H2O (entry 9), and complete loss of reactivity was observed when alternative inorganic oxidants were employed (entries 10–11). Although a variety of silver salts were tolerated by the reaction, silver carbonate proved to be the superior oxidant (see ESIii) salts were found to be successful pre-catalysts for C–H carbonylation. Whilst a drop in efficiency was observed for CoBr2, a modest increase in yield of 7a to 91% was observed upon employing Co(acac)2 (entries 13–14). Lowering the catalyst concentration from 20 mol% to 10 mol% was tolerated by increasing the reaction temperature to 160 °C, allowing succinimide 7a to be observed in 94% assay yield, which could be isolated in 89% yield (entry 15). Reducing the catalyst loading to 5 mol% did not aid the reaction, and lowering the amount of the silver salt in the reaction was deleterious to the yield (entries 16–18).Next, we extensively assessed the reaction parameters of the successful process with the aim of further improving the yield as well as better understanding the role of each of the components. We found that a temperature of at least 150 °C was responsible for an effective reaction, with the yield of the process dramatically reduced upon lowering the temperature by only 15 °C –H activation at the methyl groups over traditionally more reactive sp2-hybridised C–H bonds to form 7c and 7d. Although similar selectivities were observed in a related Ni-catalysed C–H carbonylation reaction,14 this represents a rare example of competitive aliphatic over aromatic C–H activation. Spirocyclic quinolinamides comprised of a cyclohexane or substituted piperidine moiety also provided synthetically useful yields of the desired succinimide products 7f and 7g.With a set of optimal conditions in hand, we next assessed the scope of the Co-catalyzed C–H carbonylation reaction . We foun7g–7i). Succinimide 7j could also be prepared in moderate yield by the C–H carbonylation of a protected α-amino acid derivative. Selective methylene C–H activation onto a cyclopropyl group provided the bicyclic succinimide products in reasonable yields (7k–7n). Interestingly, the butyramide-derived substrate,10 displaying a partially substituted carbon atom between the carbonyl group and the site of C–H activation successfully underwent C–H carbonylation in 66% yield, to succinimide 7o; this substrate was unreactive under Ge's Co-catalysed C–H amidation procedure.9 Alanine derived 7p, containing a protected amine group, was also successfully prepared in 55% yield. We found that the Co-catalyzed carbonylation process performed well on a larger, 5 mmol scale to give an 80% yield of the succinimide product 7a.Substrates containing sensitive ester functionalities, as well as electron-withdrawing trifluoromethyl groups were all accommodated by the reaction, affording the succinimides in 51–80% yield (7c with LiAlH4 generated amino-alcohol 8 in 66% yield; hydrolysis of succinimide 7c with 3 M aqueous hydrochloric acid led to the formation of substituted succinic acid 9 in 88% yield, with release of the 8-aminoquinoline directing group; and treatment of 7c with morpholine afforded the ring-opened bis-amide 10 in 77% yield with complete regioselectivity (Finally, we demonstrated that the succinimide products were compatible with further derivatisation: reductive ring opening of ectivity .15 Moreover, to fully realise the full synthetic potential of C–H activation with earth abundant catalysts, an important challenge will be to develop processes that do not rely on precious metal additives and bespoke directing auxiliaries. On going mechanistic studies should reveal further opportunities refine these transformations and lead to the development of new efficient Co-catalyzed C–H activation reactions.In summary, we have developed a Co-catalysed carbonylative cyclisation procedure of unactivated, aliphatic C–H bonds. Central to the success of this procedure is the stabilising effect of the quinolinamide directing group. The process tolerates a range of functionalised substrates to generate substituted succinimide products. Importantly, the operationally simple reaction conditions are complemented by the ability utilise an atmospheric pressure of carbon monoxide. While the mechanism of this process remains unclear with respect to the catalytic function of the cobalt salts, an elucidation of the role of the essential stoichiometric Ag additives will also be crucial.


Misfolding and aggregation of proteins in tissues is linked to the onset of a diverse set of human neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. In these pathologies proteins usually aggregate into highly ordered and β-sheet enriched amyloid fibrils. However, the formation of these toxic structures is not restricted to a reduced set of polypeptides but rather an intrinsic property of proteins. This suggests that the number of proteins involved in conformational disorders might be much larger than previously thought. The propensity of a protein to form amyloid assemblies is imprinted in its sequence and can be read using computational approaches. Here, we exploit four of these algorithms to analyze the presence of aggregation-prone regions in the sequence and structure of the extracellular domains of several neuroreceptors, with the idea of identifying patches that can interact anomalously with other aggregation-prone molecules such as the amyloid-β peptide or promote their self-assembly. The number of amyloidogenic regions in these domains is rather low but they are significantly exposed to solvent and therefore are suitable for interactions. We find a significant overlap between aggregation-prone regions and receptors interfaces and/or ligand-binding sites, which illustrates an unavoidable competition between the formation of functional native interactions and that of dangerous amyloid-like contacts leading to disease. Protein misfolding and aggregation is associated with an increasing number of highly debilitating human diseases, including Alzheimer's disease (AD), diabetes and some types of cancer . The proAlthough any polypeptide has the potential to self-assemble into β-sheet-enriched amyloid-like conformations , the comAD is a complex neurodegenerative multifactorial pathology characterized by a progressive and irreversible decline of cognitive function. It is the most common form of dementia in individuals over 65 years of age; and the incidence of AD doubles every 5 years beyond the age of 65. According to the World Alzheimer Report 2011, it is estimated that 36 million people worldwide suffer from dementia, and patients are expected to double every 20 years to 66 million by 2030, and 115 million by 2050 . A major2+ homeostasis deregulation [A hallmark of AD is the extracellular accumulation of aggregated amyloid-β (Aβ). Aβ is a 37–43 residues peptide that results from multiple proteolytic cleavage of a large transmembrane precursor, the amyloid precursor protein . AD pathgulation . It has gulation . Such inTo address if human neuronal receptors display aggregation-prone regions susceptible of interacting with Aβ oligomeric assemblies, we have selected five different receptors for which the 3D structure were available. We have used human structures in most cases, when these were not available we used homolog proteins with a high sequential identity. In all cases we have analyzed the extracellular domains of the receptors, which include the ligand-binding sites, because it is more likely that Aβ diffusible oligomeric species will interact with them than with domains already embedded in the membrane. We have used PDBePISA   to deconWaltz  uses a pTango  is basedin vivo experiments in bacteria and on the assumption that short and specific sequence stretches modulate protein aggregation [Aggrescan  is basedregation ,30.Amylpred  is a conTo have a complete description of the aggregation properties of the selected neuronal receptors we have used four different predictors based on different concepts and calculation schemes:As a stringent criterion for identification of aggregation-prone regions we have considered as positive hits only those regions recognized simultaneously by at least three of these algorithms.Human GRM5 (P41594) is a G-protein coupled receptor for glutamate. The binding of the ligand to the receptor promotes a conformational change that initiates the signaling pathway through guanine nucleotide-binding proteins, which further modulate down-stream effectors. Signaling involves a phosphatidylinositol-calcium second messenger system, which triggers a calcium-activated chloride current. GMR5 has been shown to be a co-receptor for Alzheimer Aβ oligomers bound to cellular prion protein .GRM5 is a receptor of 1212 residues located at the cell membrane, residues 22 to 579 correspond to the extracellular region, exhibiting an α β 3-layer (aba) sandwich structure, with 32% of helical structures  and 18% of β-sheets  (PDB 3LMK).Four aggregation regions comprising residues 35–45 (IIIGALFSV), 161–166 (LLQLFN), 183–198 (TLFKYFM) and 398–410 (GFVINAIYSMAYG) are detected with the consensus of three predictors . The secl-glutamate to the receptor promotes a structural change that forces the opening of the cation channel. This action transforms the chemical input into an electrical signal, upon which the receptor desensitizes and adopts a transient inactive conformation, in which it remains bound to an agonist. This receptor is activated by the presence of Aβ oligomers at the synapsis [Human glutamate receptor 2 (AMPA 2) (P42262) plays a central role in excitatory synaptic transmission at the CNS. AMPA 2 is a receptor for glutamate that acts as ligand-gated ion channel. Binding of the excitatory neurotransmitter synapsis .AMPA 2 is a 883 residues receptor which sequence is further processed into a mature form (25–883). The crystallized extracellular region corresponds to residues 25 to 395, showing an α β 3-layer (aba) sandwich structure, with 34% helical structure  and 21% of β sheet  (PDB 2WJW) [Three hot spots of aggregation comprising residues 85–92 (VYAIFGFY), 108–113 (HVSFIT) and 136–144 (LLSLIEYYQ) are detected in the crystallized receptor . The firAfter binding to acetylcholine the mouse acetylcholine receptor (AChR) responds with an extensive change in conformation that affects all their subunits and leads to opening of an ion-conducting channel across the cellular membrane. It exhibits a pentameric structure with two α chains (P04756), and three other structurally different subunits. This receptor interacts directly with Aβ oligomers .The α chain of AChR is 457 residues long. After signal peptide processing the AChR sequence mature form comprises residues 21–457, with residues 21–230 located at the extracellular domain. Extracellular mature α AChR shows 8% helical structure  and 47% of β sheets  and is a mainly β distorted sandwich (PDB: 2QC1). Mouse α AChR shares 83.6% identity with the human AChR α receptor chain .Four regions are predicted as aggregation prone with the consensus of three predictors, comprising residues 49–62 (VQVTVGLQLIQLIN), 147–155 (YCEIIVTHF) . In thisHuman insulin receptor subunit α (P06213) is one of the subunits of the insulin receptor (IR). This transmembrane receptor is activated by insulin and belongs to the tyrosine receptors superfamily. Binding of insulin results in the phosphorylation of several intracellular substrates, which act as docking proteins for other signalling polypeptides. From a metabolic point of view, the IR is responsible for the regulation of glucose homeostasis and its involved in a wide range of pathologies, including diabetes and cancer. Aβ oligomers have been shown to reduce responsiveness to insulin in synapsis .Human IR is a 1382 amino acid residues receptor which sequence is further processed into IR subunit α (28–758). The crystallized subunit α comprises residues 25–457, which show 11% helical structure  and 29% of β-sheets  (PDB 2HR7). Two different structural domains are evident in the crystallized chain: an α β horseshoe  and a mainly β ribbon domain (192–309).Several hot spots of aggregation are detected with the consensus of three predictors, 54–69 (LIMITDYLLLFRVYGL), 87–99 (LFFNYALVIFEMV), 338–344 (GSLIINI), 502–508 (GFMLFYK), 561–571 (QYAIFVKTLVT), 628–632 (GVFSFL) . The thrHuman IGF1R (P08069) is, as IR, a member of the tyrosine kinase receptors. The activated form of this receptor is involved in cell growth and survival control. In addition, it is an important determinant of tumor transformation and the survival of malignant cells in different cancers. Binding of the ligand results in activation of the receptor kinase and receptor autophosphorylation, as well as the phosphorylation of tyrosine residues in different protein substrates, that function as signaling adapter proteins. Aβ oligomers have been shown to mediate IGF-1R activation in the hippocampus of AD brain during the early stages of disease development .IGR1R complete sequence has 1367 residues, which further processing renders the α subunit located at the extracellular environment comprising residues 31–736. The crystallized IGR1R α subunit exhibits three structural domains, (1–183) α/β horseshoe, (224–299), mainly β ribbon domain and, (300–478) another α/β horseshoe domain (PDB 1IGR).Seven aggregation prone regions are detected with the consensus of three predictors, comprising residues 27–35 (GYLHILLIS), 48–60 (LTVITEYLLLFRV), 81–92 (LFYNYALVIFEM), 416–420 (MYFAF), 490–498 (LISFTVYY), 546–551 (QYAVYV) and 695–701 (FLHNSIF) . The cryTo decipher whether the detected aggregation-prone sequences stretches might play a conserved role in different organisms we made multiple alignments of the sequences of the five analyzed receptors in vertebrates. As an example we show the results in in silico predictions, which should be further validated experimentally, our data suggest that, as previously demonstrated for other globular proteins, the extracellular domains of neuronal receptors cannot skip the presence of aggregation-prone regions in their sequences. However, the number and size of these stretches is rather small if we compare them with the average in globular proteins. This is likely a consequence of their important physiological role, since it has been shown that essential proteins are under higher selective pressure against the accumulation of aggregating sequences that nonessential ones. The detected regions are highly conserved, pointing out to an important role in the function or the structure of these receptors. Importantly, and contrary to what one could expect, many of these regions appear to be exposed to solvent in the monomeric forms. A structural inspection indicates that they are part of the interface between subunits or even overlap with the binding site for proteinic substrates. These data confirm that protein–protein interaction surfaces and regions with high aggregation propensity overlap significantly in proteins. The result is that formation of native receptor oligomers or native complexes with substrates and self-aggregation reactions or anomalous interactions with other aggregating ensembles such as Aβ oligomers are probably constantly competing in the extracellular face of neuronal cells, which might result in signaling problems. Moreover, neurons are vulnerable to perturbations in the endocytosis process in which they recycle their receptors. It is likely that Aβ oligomers might interact promiscuously with the aggregation-prone regions in conformational flexible, monomeric and unliganded receptors during this process, impeding them to arrive to their proper destinations in the neuronal membrane.Despite the work presented here is based on The formation of amyloid-like structure is an intrinsic property of most proteins and polypeptides.The presence of aggregation-prone regions can be read out directly from the primary protein sequence.Neuronal receptors exhibit aggregation-prone regions exposed to the solvent that might interact with hydrophobic diffusible amyloid-β peptide oligomers.Aggregation-prone regions in neuronal receptors often overlap with the interface between monomers or domains or coincide with the ligand-binding sites, illustrating a competition between functional and anomalous interactions.


The findings indicate that α7 nAChRs regulate cytoskeletal dynamics through local calcium signals for calpain protease activity.The α7 nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel that plays an important role in cellular calcium signaling contributing to synaptic development and plasticity, and is a key drug target for the treatment of neurodegenerative conditions such as Alzheimer’s disease. Here we show that α7 nAChR mediated calcium signals in differentiating PC12 cells activate the proteolytic enzyme calpain leading to spectrin breakdown, microtubule retraction, and attenuation in neurite growth. Imaging in growth cones confirms that α7 activation decreases EB3 comet motility in a calcium dependent manner as demonstrated by the ability of α7 nAChR, ryanodine, or IP The growth cone is a dynamic subcellular structure that guides the movement and function of growing and regenerating axons and drives synaptogenesis ,2. SignaNumerous nicotinic acetylcholine receptor (nAChR) subunits are expressed in neuronal development and contribute to cholinergic signaling important for cell proliferation, survival, and synapse formation . This clin vivo, stimulation of the α7 nAChR mediates direct structural and functional changes at the synapse in regions such as dentate gyrus and CA1 of the hippocampus . Ce. Ce345-8controls . Interes= 0.830) . The eff345-8A and repeated the spectrin assay. Western blot detection indicates a loss in the ability of choline to produce the αIIspectrin breakdown product when the α7345-8A subunit is present in the cell  = 0.101, p = 0.905)  .A number of cellular systems including differentiated PC12 cells, sympathetic, cortical, and hippocampal neuronal cultures are useful for the study of neurite development through growth cone function. Findings from such work enables an understanding of key principles that guide synaptic development and can be applied to advancing treatment for nerve regeneration –51. ResuHow calcium signaling through calpain impacts neuronal growth and function in the context of development or aging is neither entirely clear nor simple. Our findings confirm a role for endogenous calpain activity in neurite development during differentiation  as confivs. long-term neurite and cytoskeletal growth. In future studies it will be interesting to determine if α7 nAChR activation leads to the activity of calpain 1, 2, or both and if this is cell type and compartment specific. For example, high calcium levels within α7 nAChR and ER containing cellular microdmains that have been suggested in the growth cone [Studies on retinal ganglion cell degeneration suggest that calpain isoforms, which markedly differ in their calcium sensitivity and effector targets, have opposing effects on regenerative cell health with calpain 1 driving survival and calpain 2 promoting cell death . Here, wwth cone  may driv3R receptor, or uncoupling from Gαq signaling, decreases the ability of α7 nAChRs to mediate calcium transients in the growth cone [3R is sufficient to inhibit calpain function in the EB3 comet assay. In addition, metabtropic G protein signaling appears important for downstream calpain activation by the α7 nAChR as evidenced by the finding that expression of the α7345-8A mutant or application of the Gαq blocker (Sub P) impairs the ability of choline to breakdown spectrin.Morphogenic cholinergic signals are well documented in embryonic development and contribute to structure and connectivity in the CNS . A stronwth cone ,22. Basewth cone . Our finWhile a large number of α7 nAChRs are presynaptic this receptor is also expressed in somatodendritic regions and has been shown to regulate plasticity leading to long term potentiation ,58,59. SS1 File(XLSX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.


A simple and highly efficient transfer hydrogenation of alkynes and alkenes by using a two-dimensional electride, dicalcium nitride ([Ca2N]+·e–), as an electron transfer agent is disclosed. 2N]+·e–), as an electron transfer agent is disclosed. Excellent yields in the transformation are attributed to the remarkable electron transfer efficiency in the electride-mediated reactions. It is clarified that an effective discharge of electrons from the [Ca2N]+·e– electride in alcoholic solvents is achieved by the decomposition of the electride via alcoholysis and the generation of ammonia and Ca(OiPr)2. We found that the choice of solvent was crucial for enhancing the electron transfer efficiency, and a maximum efficiency of 80% was achieved by using a DMF mixed isopropanol co-solvent system. This is the highest value reported to date among single electron transfer agents in the reduction of C–C multiple bonds. The observed reactivity and efficiency establish that electrides with a high density of anionic electrons can readily participate in the reduction of organic functional groups.A simple and highly efficient transfer hydrogenation of alkynes and alkenes by using a two-dimensional electride, dicalcium nitride ([Ca The development of simple and efficient chemical transformation routes with maximal yields has been a continuously pursued challenge in synthetic chemistry. Such protocols can provide important benefits in the field of organic synthesis such as saving starting materials, reagents, and energy, thereby lowering production costs and environmental impacts.2 and TmI2), have been employed in electron transfer hydrogenation.2-mediated reactions has been achieved, the use of toxic and rare lanthanide elements, and the requirement of additives such as amines, are drawbacks that need to be resolved.For efficient electron transfer hydrogenation, it is essential to use an agent with a high reduction potential to facilitate the electron transfer. In this regard, many types of materials, such as simple metals (Mg and Yb), stabilized alkali metal systems (Na in silica-gel and in ammonia liquid), and lanthanide iodides 2·e–, was synthesized by using solvated electrons as precursors.11Electrides are ionic crystals in which loosely trapped interstitial electrons in crystallographic empty space such as cavities, channels or interlayers behave as anions.system.+1-crown-6224Al28O64]4+·4e– (C12A7:e–) mitigates the drawback of organic electrides, providing access to a new frontier in synthetic chemistry. As the inherent electrons in the electride can directly participate in chemical reactions, the use of an electride in synthetic chemical reactions would be energetically favourable compared to the electrochemical reactions, which use additional electrical energy with a low electron transfer rate, and indispensable noble metal electrodes and electrolytes. Moreover, as already demonstrated, this electride-mediated methodology is feasible for scalable reactions such as the pinacol coupling reaction using the [Ca2N]+·e– electride.In contrast, the room temperature stable inorganic electride [Ca]4+·4e– CA7:e– mit– electride was verified to be an effective electron donor in chemical reactions such as ammonia synthesis and decomposition, N2 dissociation, and CO2 splitting.2N]+·e–, which has anionic electron layers between the cationic framework layers ([Ca2N]+), was discovered.22 cm–3), similar to those of typical alkali metals. As the open layer structured [Ca2N]+·e– electride provides better accessibility to anionic electrons, when compared to the close cage structured C12A7:e– electride, we conducted direct electron transfer from a C12A7:e– electride to various organic substrates bearing C–C multiple bonds and demonstrated highly efficient electron transfer hydrogenation of alkynes and alkenes in alcoholic solvents.The C12A7:e1a , ethanol (15.9), isopropanol (17.1)], although the reaction times were prolonged. This phenomenon is ascribed to the hydrogen evolution consuming electrons by reacting with the alcohols. A relatively acidic alcohol readily reacts with electrons to liberate hydrogen gas, consuming electrons and then reducing efficiency. Thus, employing less acidic alcohols such as isopropanol is essential to achieve the high yield of 1a′ due to the enhanced electron transfer efficiency.Initially, we examined the effects of the solvent as an electride activator and proton donor, and optimized the reaction conditions . The use1a , entry 1ion rate , entry 21a′ and the electron transfer efficiency  of 1a was higher than that (–1.70 V vs. Ag/AgCl electrode) of 1j, indicating that 1a is a better electron acceptor than 1j. This result implies that the reduction potential of electrons participating in the reduction of substrates ranges between –1.54 and –1.70 V. This thermodynamic consideration indicates that an appropriate choice of electron transfer media is a critical factor to provoke the electride-mediated reactions via the control of reduction potential, since the electrons released from the [Ca2N]+·e– electride are transferred to substrates through the transfer media.It was found that no reaction occurred with the use of aliphatic substrates such as 1-dodecyne via the alcoholysis of [Ca2N]+·e–. The decomposition of the electride was confirmed by X-ray diffraction (XRD) measurements and ion-chromatography (IC) that revealed the formation of Ca(OiPr)2 and ammonia, respectively (ESIvia the single electron transfer (SET) process. The employment of various radical scavengers, such as TEMPO, 1,4-dinitrobenzene and galvinoxyl, decreased the yields of 1a′ to a level corresponding to the amount of scavengers n], indicating that this protocol is simple and efficient for the reduction of C–C multiple bonds. In comparison with other agents, the electron transfer efficiency for the [Ca2N]+·e– electride was significantly higher than that for Yb, Mg, and Na-SG, and similar to the costly compound SmI2(H2O)n. Furthermore, we estimated the electron transfer efficiency of all electride-mediated organic syntheses (ESI2N]+·e– and C12A7:e– electrides such as pinacol couplingFinally, we compared the electron transfer efficiency of the electride-mediated reactions to the efficiency of reactions using various electron transfer agents . The preheses ESI. The pre2N]+·e– electride as an electron donor in an alcoholic solvent, as a hydrogen atom donor. This methodology provides a new class of materials as a promising alternative for reducing the processes of C–C multiple bonds in terms of high electron transfer efficiency using an electride composed of naturally abundant elements.We have developed a simple and highly efficient transfer hydrogenation process utilizing a [CaSupplementary informationClick here for additional data file.


Astrocytes are implicated in modulation of neuronal excitability and synaptic function, but it remains unknown if these glial cells can directly control activities of motor circuits to influence complex behaviors in vivo. This study focused on the vital respiratory rhythm-generating circuits of the preBötzinger complex (preBötC) and determined how compromised function of local astrocytes affects breathing in conscious experimental animals (rats). Vesicular release mechanisms in astrocytes were disrupted by virally driven expression of either the dominant-negative SNARE protein or light chain of tetanus toxin. We show that blockade of vesicular release in preBötC astrocytes reduces the resting breathing rate and frequency of periodic sighs, decreases rhythm variability, impairs respiratory responses to hypoxia and hypercapnia, and dramatically reduces the exercise capacity. These findings indicate that astrocytes modulate the activity of CNS circuits generating the respiratory rhythm, critically contribute to adaptive respiratory responses in conditions of increased metabolic demand and determine the exercise capacity. Circuits of the preBötzinger complex generate rhythms needed for breathing. Here, the authors provide evidence, using a combination of chemogenetic approaches and approaches to inhibit vesicular release, that astrocytes play a role in regulating respiratory rate. Physiology of these electrically non-excitable cells of the brain is governed by intracellular Ca2+, with increases in [Ca2+]i triggering release of signaling molecules or “gliotransmitters” . Recent studies have suggested that via release of gliotransmitters astrocytes may influence activities of neural circuits controlling sleep, feeding, and chemosensing5, yet it remains unknown whether astrocytes can directly modulate motor circuits and have an impact on complex behaviors. In vitro experiments with rodent brainstem slices9 have suggested that astroglial mechanisms may play a certain role in regulating the activities of neuronal networks producing motor rhythms, including those within the preBötzinger complex (preBötC)10 in the ventrolateral medulla that generates the rhythm of breathing11. However, whether such modulation is functionally important for rhythmic motor behavior has not been determined. In this study, we accordingly focused on the preBötC that produces a fundamental, clearly defined motor output, and where local astrocytic modulation of neuronal excitability and/or synaptic transmission would directly affect respiratory motor behavior. We determined the effects of compromised preBötC astroglial vesicular release mechanisms on breathing in conscious adult rats at rest and in conditions of increased metabolic demand requiring regulatory adjustments of respiratory motor activity, including during exercise. We show that blockade of vesicular release in preBötC astrocytes reduces the resting breathing rate and frequency of periodic sighs, decreases rhythm variability, impairs respiratory responses to hypoxia and hypercapnia, and dramatically reduces the exercise capacity.Astrocytes have been proposed to modulate neuronal excitability, synaptic transmission, and plasticity12, or the dominant-negative SNARE (dnSNARE) protein13  to monitor vesicular fusion events in cultured brainstem astrocytes transduced to express dnSNARE or a control transgene (CatCh-EGFP). In dnSNARE-expressing astrocytes, the number of juxtamembrane vesicles labeled with quinacrine was reduced by 67% e 5 Fig. . The higƒR) by 11%  and by 11% , respectively 17  in preBötC astrocytes resulted in 26% higher baseline ƒR DGq Fig. . These rDGq Fig. . However Gq Fig. . Moreovetes Fig. , and factes Fig. , indicatGq expression had an opposite effect and increased respiratory variability  in rats expressing dnSNARE, by 25% (p < 0.001) in rats expressing TeLC, and by 26% (p < 0.001) in rats expressing TMPAP in the preBötC  by preBötC astrocytes modulates the variability of the respiratory rhythm and the generation of sighs.The frequency of sighs, breaths with augmented inspiration, generated periodically by the preBötC circuitsötC Fig. . Sigh fr21 and increases sigh frequency23, we next evaluated the effects of dnSNARE or TeLC expression in preBötC astrocytes on respiratory responses to systemic hypoxia (10% O2 in the inspired air) as well as the effects of dnSNARE, TeLC, TMPAP, and DREADDGq expression on sigh generation during hypoxia. Expression of dnSNARE attenuated hypoxia-induced increases in ƒR by 27% , 36% , and 44% , respectively . In conscious rats, bilateral expression of dnSNARE or TeLC in preBötC astrocytes reduced the ƒR responses to hypercapnia (6% inspired CO2) by 23%  and 20% , respectively  and 42% , respectively  neural circuits are intermingled with astroglial networks, yet the experimental evidence for astrocytes directly controlling the activities of functionally defined motor circuits to affect behavior in vivo is lacking. This study focused on the vital brainstem respiratory rhythm-generating circuits of the preBötC and determined whether astrocytes can modulate the activity of these circuits and, therefore, breathing in conscious rats. Astrocytes are well known to provide neurons with structural and metabolic support, but also have a distinct signaling function, which is mediated via the release of gliotransmitters27, blocks Ca2+-dependent vesicular fusion12, and prevents the spread of Ca2+ waves triggered by mechanical stimulation12—the effects consistent with the inhibition of vesicular ATP release28. TIRF imaging confirmed that dnSNARE expressed in astrocytes reduces the number of juxtamembrane vesicles and effectively blocks Ca2+-dependent vesicular fusion. These effects are in line with the proposed mechanisms underlying the effect of dnSNARE on exocytosis in astrocytes29.Expression of TeLC or dnSNARE protein in preBötC astrocytes reduced the resting breathing rate and frequency of periodic sighs, decreased rhythm variability, impaired respiratory responses to hypoxia and hypercapnia, and dramatically reduced the exercise capacity. TeLC is a proteolytic enzyme that cleaves SNARE proteins required for vesicular fusion. In cultured astrocytes, TeLC inhibits ATP and glutamate releaseGq involved targeting preBötC astrocytes to express this receptor followed by documenting changes in respiratory activity induced by administration of CNO. However, validation experiments of our viral vector construct revealed that in the absence of a ligand, astrocytes expressing DREADDGq exhibit a higher level of PLC activity, higher rate of spontaneous vesicular fusion, and facilitated tonic release of ATP. These results suggested that DREADDGq is constitutively active when expressed in astrocytes, an observation consistent with the properties of many hM3 receptor mutants originally described17. Since CNO appears to have low affinity for DREADDs and its effects are largely attributed to its conversion to clozapine30, which may interact with astroglial serotonin receptors31, we focused on determining the effects of the constitutive DREADDGq activity. In rats, sustained activation of PLC-mediated signaling pathways in preBötC astrocytes expressing DREADDGq was associated with higher resting breathing rate, higher frequency of periodic sighs, and increased rhythm variability. That this effect was blocked by co-expression of the potent ectonucleotidase TMPAP suggested that the stimulatory effect of DREADDGq expression in preBötC astrocytes on breathing could be mediated by direct actions of ATP and/or related purines on preBötC circuits or, alternatively, autocrine effects of ATP on Ca2+ in preBötC astrocytes leading to the release of other gliotransmitters2, neither possibility of which we rule out here.Our initial design of the gain-of-function experiment with DREADD32.The role of preBötC astrocytes in the control of breathing becomes especially important during physiological metabolic challenges, such as systemic hypoxia and hypercapnia, where enhanced respiratory effort is critical to maintain homeostasis. Although expression of dnSNARE or TeLC in preBötC astrocytes reduced resting respiratory rate, minute ventilation at normoxia/eucapnia was similar to that in animals expressing control transgene . More marked differences in ventilation between the experimental and control groups were observed during the hypoxic challenge, indicating that preBötC astrocytes are critically important for the development of the full-scale hypoxic ventilatory response. These data are consistent with the proposed role of astrocytes as CNS oxygen sensors2 and require a certain level of CO2 to operate. The preBötC has a neuronal H+/CO2-sensing mechanism33, however, our results suggest that preBötC astrocytes contribute in a significant manner to the development of the respiratory response to hypercapnia. Our data support the “distributed central chemosensitivity” hypothesis, which proposes that central respiratory sensitivity to CO2  is mediated by multiple central chemoreceptor sites (one being the preBötC), with each site providing tonic excitation in eucapnia and a fraction of the total response to systemic hypercapnia34. Previous experimental studies suggested that the contribution of the preBötC mechanism(s) to the overall respiratory response to CO2 is ~20–25%34. Our experiments showed that ventilation during eucapnia and hypercapnia is similarly reduced by ~20% in conditions when vesicular release mechanisms in preBötC astrocytes are blocked and hyperoxia is applied to reduce the drive from the peripheral chemoreceptors  in accordance with the European Commission Directive 2010/63/EU , the UK Home Office (Scientific Procedures) Act (1986), and the National Institutes of Health Guide for the Care and Use of Laboratory Animals, with project approval from the respective Institutional Animal Care and Use Committees. Animals were housed in a temperature-controlled facility with a normal light-dark cycle . Tap water and laboratory rodent chow were provided ad libitum.13 under the control of an enhanced GFAP promoter  to overexpress a potent ectonucleotidase TMPAP39. Expression of TMPAP was driven under the control of an elongation factor 1α (EF1α) promoter  to drive the expression of dnSNARE proteinq-coupled signaling pathways we generated an AVV to express DREADDGq fused with enhanced green fluorescent protein (EGFP) in astrocytes   may potentially trigger an immune response. Thus, expression of CatCh-EGFP is a much harsher control than cytoplasmic expression of EGFP, which has hardly ever been reported to cause any adverse cellular effects. Each experimental  and control (CatCh-EGFP or EGFP) animal groups were injected with the appropriate viral vector at the same time and the experimental groups were compared to their own control groups  an AVV to express calcium translocating channelrhodopsin variant (CatCh) fused with EGFP (CatCh-EGFP) under the control of the GFAP promoter (AVV-sGFAP-CatCh-EGFP); and (2) a LVV to express EGFP under the control of EF1α promoter ) and medetomidine  and placed in a stereotaxic frame. The tooth bar was adjusted so that bregma was positioned 5 mm below lambda. PreBötC areas were targeted bilaterally by advancing a pipette from the dorsal surface of the brainstem. Viral vectors . For postoperative analgesia, the animals received buprenorphine  for 3 days. No complications were observed after the surgery and the animals gained weight normally.Adult male rats (250–270 g) were anesthetized with a mixture of ketamine  as described40. An Olympus TIRF microscope was used to monitor fusion events40. Fluorescence was excited at 488 nm and collected at 500–530 nm. The imaging setup included a high-numerical-aperture (NA) oil-immersion objective , an inverted microscope , and a cooled charge-coupled-device camera (Hamamatsu). Images were analyzed using Olympus Cell^tool software (Olympus). The experiments were performed at 37 °C.In cultured brainstem astrocytes, ATP-containing vesicular compartments were visualized by quinacrine staining . The acridine derivative quinacrine is a weak base that binds ATP with high affinity and can be used to identify putative ATP-containing vesicles in living cells, including astrocytes2+]i responses in individual cultured astrocytes were visualized by recording changes in fluorescence of conventional Ca2+ indicators Fura-2 (Molecular Probes), Fluo-4, or Rhod-2 (Thermo Fisher)24. Cells were loaded with Fura-2 , Fluo-4 , or Rhod-2  with the addition of pluronic F-127 (0.005%). After the incubation with the dye, cultures were washed three times prior to the experiment. Changes in [Ca2+]i were monitored by an inverted Olympus microscope with ×20 oil-immersion objective. Excitation light provided by a Xenon arc lamp was passed sequentially through a monochromator at 340, 380, and 490 nm (Cairn Research); emitted fluorescence was measured at 515 nm (Fura-2) or 565 nm (Rhod-2). All the experiments were performed at 37 °C.The [CaGq or CatCh were incubated for 18 h in M199 medium containing 10% dialyzed fetal calf serum and 1 µCi ml−1 of [3H]-inositol (specific activity 18.5 Ci mmol−1) . Immediately prior to the assay, the incubation medium was replaced with Hanks’ balanced salt solution buffer. Lithium chloride was then added to reach a final concentration of 10 mM and cultures were incubated at 37 °C for an additional 30 min. To activate DREADDGq, CNO  was added for 20 min. Reactions were terminated by removal of the medium and the addition of 500 μl of ice-cold methanol. [3H]-Inositol phosphate ([3H]-InsP) production was determined by adding the samples to 2 ml Dowex columns pre-washed with a mixture of ammonium formate (2 M) and 0.1 M formic acid. Double-distilled water and a mixture of sodium tetraborate (5 mM) and sodium formate (60 mM) were used to elute unbound [3H]-inositol and glycosylphosphatidylinositol, respectively. Then, a mixture of ammonium formate (1 M) and formic acid (0.1 M) was added to the column to elute total [3H]-InsP into scintillation vials. The 500 µl aliquots of the eluted samples were then transferred in duplicates to liquid scintillation vials. Concentrations of [3H] in [3H]-InsP and total [3H]-inositol lipids were detected using a Beckman LS 5801 scintillation counter . The results are presented as percentages of radioactive InsP ([3H]-InsP) in the total inositol lipid pool  artificial cerebrospinal fluid  with an additional 9 mM Mg2+. The medulla was isolated and a horizontal 400 μm-thick slice was cut parallel to the ventral medullary surface42. Recordings were made in a flow chamber (3 ml min−1) at ~35 °C from the slices placed on an elevated grid to permit access of aCSF from both sides of the slice.Adult rats were transduced to express DREADD41. To control for the release of nonspecific electroactive interferents, a dual recording configuration of the ATP biosensor and control (null) biosensor was used, as described43. A “null” biosensor  current was subtracted from the current recorded by the ATP biosensor to give “net-ATP” readings, reporting release of ATP  and right (expressing CatCh) sides of the brainstem slice were swapped to determine tonic ATP release from the opposite site  were described in detail previouslyATP Fig. . Both seATP Fig. . Withoutite Fig. . Sensorsite Fig. . To conv46. Briefly, 5–7 days after the injections of viral vectors the rats were placed in a Plexiglas recording chamber (1 l) that was flushed continuously with humidified air , at a rate of 1.2 l min−1. In order to take into the account circadian variations of the physiological parameters, respiratory activity in all the animals was assessed at the same time of the day (between 1100 and 1500 hours). The animals were allowed to acclimatize to the chamber environment for ~60 min followed by 30 min recording period of resting respiratory activity. For the experiments involving hypoxic challenges, the O2 concentration in the inspired air was reduced to 10%  for 10 min. In a separate series of experiments, hypercapnia was induced by stepwise increases in CO2 concentration in the respiratory gas mixture to 3% and 6% in hyperoxic environment  to reduce the drive from the peripheral chemoreceptors. Each CO2 concentration was maintained for 5 min. Concentrations of O2 and CO2 in the plethysmography chamber were monitored online using a fast-response O2/CO2 analyzer . Data were acquired using Power1401 interface and analyzed offline using Spike2 software (CED).Whole-body plethysmography was used to record respiratory activity in unrestrained conscious adult rats−1, intraperitoneal (i.p.)) and instrumented for blood pressure recording via femoral artery cannulation. The depth of anesthesia was monitored by the stability of blood pressure and heart rate. The trachea was cannulated low in the neck with a 12-gauge cannula to maintain airway patency. Animals were then transferred to a stereotaxic frame and left to breathe spontaneously. An occipital craniotomy was performed to expose the dorsal aspect of the brainstem. Phrenic nerve activity (PNA) was recorded as an indicator of central inspiratory drive. The PNA signal was amplified (20 000×), filtered (500–1500 Hz), rectified, and smoothed (τ = 50 ms). Arterial blood gases and pH were monitored regularly using a blood gas analyzer  and maintained with the physiological ranges. Core body temperature was maintained at 37.0 ± 0.5 °C. Bombesin  was microinjected over a period of 10–15 s into the preBötC region using a single barreled micropipette (tip diameter 10–15 μm). The PNA response to bombesin was followed until returning to baseline  before a second microinjection was made into the contralateral preBötC. Data were recorded using Power1401 interface and analyzed offline using Spike2 software. At the end of the experiments, the animals were humanely killed by an overdose of pentobarbitone sodium .Adult rats were transduced to express dnSNARE and CatCh by astrocytes of the left and right preBötC regions, respectively. After 5–7 days following microinjections of viral vectors, the animals were anesthetized  and medetomidine , a laparotomy was performed, and a telemetry pressure probe  was implanted into the abdominal aorta. The abdominal muscle and skin layers were successively sutured and anesthesia was reversed . For postoperative analgesia, the animals received carprofen  for 2 days and and were allowed to recover for at least 7 days.Systemic arterial blood pressure and heart rate in exercising animals were recorded using biotelemtry. The rats were anesthetized with ketamine .Exercise capacity of experimental rats was determined using a single lane rodent treadmill (Harvard Apparatus) as described−1, i.p.), perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), and post-fixed in the same solution for 4–5 days at 4 °C. After cryoprotection in 30% sucrose, serial transverse sections (30–40 μm) of the medulla oblongata were cut using a freezing microtome. After antigen retrieval in 1% citrate buffer at 80 °C, free-floating tissue sections were incubated with chicken anti-GFP , rabbit anti-GFAP , mouse anti-NeuN , and/or goat anti-ChAT antibody  overnight at 4 °C. The sections were subsequently incubated in specific secondary antibodies conjugated to the fluorescent probes  for 1 h at room temperature. Images were obtained with a confocal microscope (Zeiss LSM 510).At the end of the experiments, the rats were given an anesthetic overdose  was measured for each respiratory cycle after the animals had habituated to the plethysmography chamber environment for at least 60 min. The average TTOT calculated for the periods of calm wakefulness and/or quiet sleep recorded in a 30-min period following acclimatization to the chamber environment was used to determine the resting respiratory frequency . The frequency distribution of the instantaneous rate of all respiratory-related events (including signing and sniffing) in the 30-min assay period was analyzed, plotted, and reported as averages for each of the experimental groups. Poincaré plots of TTOT for the nth cycle vs. TTOT for the nth+1 cycle were used to evaluate the temporal dispersion of TTOT. Variability of TTOT was determined as described previously48. Tidal volume  was determined by measuring the pressure changes in the chamber. Calculated values of minute ventilation (VE = ƒR × VT) were averaged and reported in arbitrary units. In addition to quantifying ƒR, we also determined the frequency of sighs—augmented breaths that occur on top of normal inspirations49. A sigh was defined as a high-amplitude, biphasic augmented inspiratory breath  immediately after the sigh (Supplementary Fig. Spike2 software as the frequency of augmented breaths with VT that was at least two times larger than the mean VT and a TTOT that was >50% longer than the average TTOT of the previous five cycles. In anesthetized rats, sigh was defined as a burst of phrenic nerve discharge with an amplitude of >50% higher that the preceding eupneic breath, followed by a brief period of apnea.The respiratory cycle duration (In box and whisker plots, the central black dot illustrates the mean, the central line shows the median, the edges of the box define the upper and lower quartile values, and whiskers show the minimum-maximum range of the data.U by ranks test, Wilcoxon matched-pairs signed-rank test, Kruskal–Wallis analysis of variance by ranks followed by Dunn’s post hoc test, as appropriate. Differences with p < 0.05 were considered to be significant.The data were compared using nonparametric Mann–Whitney The data that support the findings of this study are available from the corresponding authors upon request.Supplementary InformationPeer Review File


Polymorphism is a remarkable concept in chemistry, materials science, computer science, and biology. Whether it is the ability of a material to exist in two or more crystal structures, a single interface connecting to two different entities, or alternative phenotypes of an organism, polymorphism determines function and properties. In materials science, polymorphism can be found in an impressively wide range of materials, including crystalline materials, minerals, metals, alloys, and polymers. Here we report on polymorphism in a liquid crystal. A bent-core liquid crystal with a single chiral side chain forms two structurally and morphologically significantly different liquid crystal phases solely depending on the cooling rate from the isotropic liquid state. On slow cooling, the thermodynamically more stable oblique columnar phase forms, and on rapid cooling, a not heretofore reported helical microfilament phase. Since structure determines function and properties, the structural color for these phases also differs. Polymorphism is a property that allows a material to exist in two or more crystal structures. Here the authors observe thermal-induced structural polymorphism in a bent-core liquid crystal compound and show that by choosing the cooling rate, different structures with distinct structural colours are obtained. The ability of a solid material to exist in more than one form of crystal structures is called polymorphism. It can be found in many crystalline materials and has a broad relevance in fields such as pharmaceuticals and agriculture.2 and high entropy alloys3 formed at 106 to 1012 °C s−1 cooling rates4. Phase selection using controlled cooling rates in these materials is crucial to arrest (select) structures that translate into desirable macroscopic properties for various engineering applications. The vitrified amorphous structure, however, is not a polymorph; real polymorphs must have symmetries different from that of the melt they formed from on cooling.In terms of thermodynamics, there are two types of polymorphic behavior. For a “monotropic” system, the plots of the temperature-dependent free energies of the various polymorphs do not cross below all polymorphs’ melts, i.e., any transition from one polymorph to another below melting will be irreversible. For an “enantiotropic” system, the plots of the temperature-dependent free energies of the various polymorphs show a crossing point before the various melting points. Two polymorphs can be interchanged reversibly by thermal pathways or through physical contact with a lower energy polymorph. Thermal annealing or slow cooling usually lead to the formation of more stable polymorph, whereas quenching (rapid cooling) may stabilize the metastable polymorph Fig. . Apart f−1 achieved by simply removing the sample from a hotplate is frequently used to arrest a particular liquid crystalline phase as a glass circumventing crystallization5. Such vitrified structures are often utilized in freeze fracture transmission microscopy6.In liquid crystals, ‘rapid’ cooling at rates on the order of 50–100 °C s7. Recently Alaasar et al.8 reported the formation of a Dark Conglomerate (DC) phase when a bent-core liquid crystal material was cooled slowly (<2 °C min−1), and a crystalline phase when cooled at rates higher than 5 °C min−1 from the isotropic liquid phase. In this case, therefore only one of the polymorphs is liquid crystalline, the other is crystalline. In bent-core liquid crystals it is often found that, depending on the cooling rate, one of the smectic liquid crystal phases is chiral (SmCaPA), the other one is racemic (SmCsPA)10. However, apart from chirality and clinicity (anticlinic or synclinic) these two states have the same smectic structures and X-ray diffraction patterns.Examples for real polymorphism are very scarce in rod-shaped liquid crystals. One prominent example is the formation of an electric field-induced chiral smectic-C ferrielectric liquid crystal  based on a tris-biphenyl diester core with one straight 1-octyloxy side chain on the para-side and one chiral (R)-2-octyloxy side chain on the meta-side of the central biphenyl ring. The two structurally related compounds II and III shown underneath are the parent compounds with either two achiral 1-octyloxy or two chiral (R)-2-octyloxy side chains, respectively. Their corresponding phase sequences and phase transition temperatures are also shown. The achiral compound III forms two enantiotropic LC phases (both on heating and cooling), a rectangular columnar phase (Colr or B1) at elevated temperatures and a modulated helical nanofilament phase  at lower temperatures11. On further cooling, the HNFmod phase eventually forms a glassy state that persists well below room temperatures without subsequent crystallization. The homochiral compound II with two identical chiral side-chains only forms a dual modulated HNF phase (HNFmod2) with an additional interlayer electron density modulation11  and a comparison of HNF12, HNFmod13 and HNFmod2 phases), which also does not crystallize upon cooling to temperatures well below the melting point measured on heating and rather forming a glass. On the basis of the data obtained from polarized optical microscopy (POM), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), circular dichroism (CD) spectropolarimetry, atomic force microscopy (AFM), and small angle X-ray scattering (SAXS), we will show that compound 1 with only one chiral side chain forms an oblique columnar (Colob) phase exclusively upon slow cooling (with cooling rates ranging from about 0.5 to 10 °C min−1) and a new helical micro-filament phase (HμF) exclusively upon rapid cooling.Figure II and III were described earlier11. The synthesis of 1 involves the stepwise deprotection and esterification of mono-tetrahydropyranyl (THP)—mono-TBDMS (tert-butyldimethylsilyl)-protected 3,4’-dihydroxybiphenyl, which was synthesized by a Suzuki coupling15 reaction  and then immediately removed from the hotplate. Subsequent temperature-dependent POM imaging reveals grainy blue textures (both in transmission between crossed polarizers and reflection) similar to other HNF phase textures  produces entirely different thin film textures , after heating the sample to the isotropic liquid phase followed by quenching, and after heating to the isotropic liquid phase and slow cooling at a rate of 5 °C min−1. The resulting thermograms obtained from the first two processes are nearly identical in the first and second heating/cooling runs and only differ in the about 3 °C lower phase transitions recorded on cooling for the quenched sample  indicated the chirality of the twisted layers stacked as HNFs, forming a secondary twist that depends on the handedness of the individual HNFs11. CD spectra of 1 at various sample rotation angles and the sum of all individual spectra are shown in Fig. 1 after slow cooling from the isotropic liquid phase. At the same film thickness of ~10 μm, the maximum at 421 nm of the quenched sample is about 3-times larger than of the peak at 542 nm of the slowly cooled sample, however, the areas of the sum peaks are nearly equal. The exclusively positive CD signals indicate that both LC phases formed upon slow as well as rapid cooling from the isotropic liquid phase are chiral .The next set of experimental data were obtained by thin film CD spectropolarimetry Fig. 17. CD ex1 is a true reflection  color indicated by a reflection peak at 430 nm in the reflection spectrum . A summary of the obtained SAXS data are shown in Fig. 1 shows diffraction peaks labeled as qSi, where the upper index S stands for “slow cooling” and i = 1, 2… indicates the cardinal number of the peaks at increasing q values. The i = 1–9 peaks can be indexed as (11), . This Colob phase corresponds to the Bobl1Rev phase, which is one of the three main columnar phases of bent-core molecules. This phase consists of tilted polar smectic ribbons with positions shifted periodically by half molecular lengths and polarizations alternating in and out along the ribbons. This phase along with the other two main columnar phases (B1 and B1Rev) is illustrated in Supplementary Figure r or B1Rev) was observed for III, and the (11) and (02) peaks appeared at higher values (around 0.21 Å−1 and 0.3 Å−1), indicating about 30% larger lattice parameters for 1 than for III. Additionally, the FWHM values of 1 are an order of magnitude larger than for III, showing short correlated columns (correlation length ~15 nm).To characterize the structures of compound q dependences of the SAXS signal for the quenched sample are shown right after quenching (t = 0) and 3 h later (t = 180 min). Both the fresh (t = 0) and relaxed (t = 3 h) quenched samples look identical indicating long stability. They represent peak positions qQi (Q stands for “Quenched”) different from that of the slow-cooled sample. The shoulder at lower q (at 0.193 Å−1 to the left of qQ1 = 0.1609 Å−1 and marked with a blue *) is overlapping with qS1, and qQ3 = 0.2907 Å−1, qQ4 = 2qQ1 = 0.3211 Å−1 are also overlapping with qS7 and qS8. This indicates that the quenched sample is a combination of structural features inherent to the slow-cooled phase  and another phase with qQ1 = 0.1609 Å−1, qQ2 = 0.2212 Å−1, qQ3 = 0.2907 Å−1 and qQ4 = 2qQ1 = 0.3211 Å−1 peaks. The presence of features inherent to the slow-cooled phase is probably due to the insufficient speed of quenching. The fact that the peak at qQ2 becomes a minority peak compared to qQ1 and qQ4 = 2qQ1 suggests that the second phase has a modulated layer structure similar to that of the B7 phase . For example, the width of the ‘corkscrews’ is about 250–300 nm in comparison to the 40 nm width of the filaments commonly reported for non-confined HNFs12 such as compound II11. Due to the fact that the feature dimensions of these filaments far exceed the 100 nm size regime often established as the size regime for “nano”, such filaments cannot be labeled as helical nano-filaments, but rather as helical micro-filaments (HµF).We also performed SEM Fig.  and AFM II, as determined previously by TEM imaging11. The helical pitch is also much larger (about fivefold) for the HμF phase of the rapidly cooled compound 1 than for previously reported HNF phases . The diameter and pitch of the filaments are in fact in between the HNF (B4) phase and the telephone wire (and often corkscrew) structure of the B7 phase of bent-core materials22, which have modulated smectic layer structure24. On the contrary, images of the slow-cooled sample do not show discrete filaments  for both the slow cooled and the quenched sample of 1 Fig. . The datOn the contrary, the AFM images for the quenched sample Fig.  provide 1 and II, 1 and III, as well as II and III. Each contact sample was heated to a temperature where both compounds were in the isotropic liquid phase and then either cooled at a slow rate of 2 °C min−1 or rapidly cooled , composed of filaments that vastly differ in dimensions and helical pitch, meet and induce a texture with conical features , and the helical nanofilaments in both cases feature the same dimensions, the same helical pitch, and only differ in their formation of a unichiral HNF phase vs. an HNF phase conglomerate. One would assume that the homochirality of II is critical for the formation of the unichiral HNFmod2 phase, where the only additional degree of ordering is the interlayer modulation not shared by the HNFmod phase formed by III. We have previously also shown that a racemic mixture of compounds related to II with two racemic side chains , ,  and ) does not form an HNF phase, but rather short correlated crystalline ribbons with an overall columnar structure11. Astonishingly, the combination of the two molecular halves of II and III in compound 1 does not only lead to a cooling rate-dependent formation of entirely different phases, but the HNF phase formed on rapid cooling leads to differently shaped helical filaments (similar to rotini noodles or corkscrews) with much larger overall dimensions, as well as an order of magnitude larger helical pitch than any other HNF phase material  desires to form the thermodynamically more stable columnar B1 phase similar to compound III (Colob vs. Colr), whereas the left half (the meta-side) promotes the kinetically more stable filament-type B4 phase, as found in compound II. On the basis of these observations, we envision to find more examples in anisotropic bent-core molecules, where different sides of the molecule contain molecular features of symmetric bent-core molecules that have distinctly different phase sequences. Such hypothesis of course is subject to further studies. The textures observed in contact preparations at the interfaces between 1 and II, 1 and III, and between materials II and III also demonstrate the subtle differences of the free energies between the Colob (or Colr), HNF and HµF phases of these bent-core liquid crystals.Considering the phase sequence of all three compounds shown in Fig. In conclusion, the unprecedented formation of two liquid crystalline polymorphs from the isotropic liquid state under different thermal history, clearly demonstrates that these structurally different phases have almost identical free energies, so their formation can be selected by small differences in thermal history or concentrations. Astonishingly, such a small group of structurally very closely related compounds displays a very rich variety of self-assembled chiral superstructures including helical nanofilament and the heretofore never reported helical microfilament phases.Polarized optical microscopy (POM) observations were carried out under an Olympus BX-53 polarizing microscope equipped with a Linkam LTS420E heating/cooling stage.Thin film circular dichroism (CD) spectropolarimetry was performed using an OLIS spectrophotometer with quartz substrates. UV-Vis transmission and reflection spectra were measured using either a USB-4000 (Ocean Optics) or a Lambda 18 (Perkin Elmer) UV-Vis spectrophotometer.25 . The materials were filled into 1 mm diameter quartz X-ray capillary tubes, which were then mounted into a custom-built aluminum cassette that allowed X-ray detection with ± 13.5° angular range. The cassette fits into a standard hot stage (Instec model HCS402) that allowed temperature control with ± 0.1 °C precision. The stage also included two cylindrical neodymium iron boron magnetsthat supplied a magnetic induction of B = 1.5 T perpendicular to the incident X-ray beam. The analysis were proceed in Igor Pro software with Nika package26.SAXS was carried out on beamline 7.3.3 of the Advanced Light Source of Lawrence Berkeley National Laboratoryk = 5 N/m, f = 150 kHz.Scanning electron microscopy (SEM) analysis was performed using a Quanta 450 FEG SEM. Atomic force microscopy (AFM) images were taken in tapping mode at room temperature at the Molecular Foundry at the Lawrence Berkeley National Laboratory. We utilized Si probes coated with Al, The authors declare that the data supporting the findings of this study are available within the paper and its supplementary information file. Any additional information that supports the findings of this study is available from the corresponding authors upon reasonable request.Supplementary InformationPeer Review File


Thus, ligand-promoted dissolution of Cr-containing solids, a redox-independent process, must be taken into account when using sedimentary Cr isotope signatures to diagnose atmospheric oxygen levels. This work provides a step towards establishing a more robust framework for using Cr isotopes to track the evolution of the Earth’s atmosphere.The chromium (Cr) isotope system has emerged as a potential proxy for tracing the Earth’s atmospheric evolution based on a redox-dependent framework for Cr mobilization and isotope fractionation. Although studies have demonstrated that redox-independent pathways can also mobilize Cr, no quantitative constraints exist on the associated isotope fractionations. Here we survey the effects of common environmental ligands on the dissolution of Cr(III)-(oxy)hydroxide solids and associated Cr isotope fractionation. For a variety of organic acids and siderophores, δ The chromium (Cr) isotope system has emerged as a potential proxy for tracing Earth’s atmospheric evolution based on a redox-dependent framework. Here the authors show that ligand-complexation, a redox-independent process, must be considered when using Cr isotope signatures to diagnose atmospheric oxygen levels. Many of these applications are based on the assumption that Cr isotope fractionations are linked predominantly to Cr redox transformations. Consistent with this view, theoretical and experimental studies have suggested very little Cr fractionation during redox-independent processes, such as adsorption6, though recent experimental studies demonstrated large equilibrium fractionations between various Cr–Cl species7. Cr isotopes have been extensively employed in environmental geochemistry as a quantitative proxy for Cr transport and remediation efficiency (see ref. 8 and references therein). However, in order to quantitatively apply this isotope system as a tracer for Earth’s surface processes, it is critical to more precisely quantify a wider range of factors controlling the global Cr cycle and isotope budget9. Indeed, since the early proposed uses of the Cr isotope system, there have been several studies recognizing the need to account for other biogeochemical processes in addition to the redox pathway-based framework of this isotope system14.The chromium (Cr) isotope system has been exploited as a highly discriminating tracer of redox processes in the Earth’s surface environments, and is under development as a novel paleobarometer for atmospheric oxygen levels15. Thus, authigenic marine sediments capture an integrated isotopic signature reflective of the redox processes involved in Cr(III) oxidative mobilization, downstream Cr(VI) reduction, and ultimate Cr(III) burial in sediments1.The two most common oxidation states of Cr in natural environments are Cr(III) and Cr(VI). Cr(III) is typically insoluble and is the most common form of Cr in rocks and minerals at the Earth’s surface. Cr(VI) is typically soluble and is the dominant form of mobile Cr in natural environments. The standard view of the Cr isotope paleoredox proxy is grounded in the idea that oxidative weathering of Cr(III)-containing minerals by manganese (Mn) oxides, the formation of which requires free oxygen, is required for Cr redox cycling and Cr isotope fractionations. During this oxidation process, soluble Cr(VI) is released and transported to oceans, reduced to Cr(III) under reducing environments, and deposited in marine sediments16. Unweathered igneous rocks, with limited exceptions17, exhibit a narrow range of δ53Cr values of −0.124 ± 0.101‰ (defined as δ53Cr = [(53Cr/52Cr)sample/(53Cr/52Cr)standard −1] × 1000)15. Cr(III) oxidation to Cr(VI) can result in significant isotopic fractionations, both negative and positive (53εCr(VI)-Cr(III) ranging from −2.5 to +1.1‰), depending on the reaction conditions; while reduction of Cr(VI) to Cr(III) has consistently been shown to leave the remaining Cr(VI) isotopically heavy (53εCr(III)–Cr(VI) ranging from −7.6 to −0.4‰) . Because the isotopic exchange and subsequent isotopic equilibrium between Cr(III) and Cr(VI) requires three electron transfer with a significant coordination reconfiguration, the isotopic exchange rate of these species is extremely slow19. However, when dissolved Cr(VI)- and Cr(III)-containing solids are in contact for long periods, equilibrium fractionation may become relevant. The significance of such a process under actual environmental settings  can vary and is dependent on many factors, such as the relative ratio of the exchanging Cr(VI) and Cr(III) species19. Therefore, Cr isotope systematics during the Earth’s surface processes are generally assumed to be dominated by kinetic fractionations. Regardless, current experimental and empirical results suggest that both kinetic and equilibrium processes tend to lead to Cr(VI) that is isotopically heavy20.The redox transformation between Cr(III) and Cr(VI) causes a significant change in the local coordination environment of Cr. Cr(III) typically occurs as an octahedrally coordinated cation, whereas Cr(VI) primarily occurs as a tetrahedrally coordinated oxyanion. The large change in coordination environment during redox transformations leads to significant isotopic fractionation, similar to other redox-sensitive elements3 solids, one common sink phase of Cr(III), and suggest the likely complexation of Cr(III) by inorganic21 and/or organic molecules . The speciation of Cr in hydrothermal fluids, and marine settings more broadly, is also thought to be influenced by organically complexed Cr(III)26. Two classes of organic ligands, siderophores 27 and organic acids 28, have been shown to be capable of solubilizing Cr(III) from the Cr(OH)3 solid phase under environmentally relevant conditions. Both siderophores and organic acids are ubiquitous organic molecules in natural environments and are produced by a wide range of microorganisms and often co-exuded29. Siderophores are organic chelating agents with a high affinity for Fe(III) for enhancing Fe dissolution from low-solubility Fe(III)-mineral phases30. Siderophores have also been shown to have strong affinities for other trivalent metal cations such as Cr(III) due to their structural similarity  to Fe(III)31. Organic acids are known to contribute to the mobilization of metals in soils32, and a wide range of organic acids  have been shown to complex with Cr(III)35. Organic acids are likely to have been present throughout the Earth’s history through abiotic or biotic synthesis pathways  and may have played important roles in the weathering38 of poorly soluble transition metals.One generally overlooked mechanism for Cr(III) mobilization is ligand complexation. Indeed, measurements on natural waters have revealed elevated concentrations of dissolved Cr(III) that are higher than the solubility of Cr(OH)20 and observed experimentally7. Additionally, several recent studies have inferred redox-independent Cr cycling in the interpretation of relevant rock records based on the site-specific geochemistry and Cr isotope dynamics11.Despite the potential importance of Cr(III)–ligand complexes in Earth’s surface, Cr isotope fractionation during Cr(III) complexation with organic ligands has not been experimentally measured, although the potential for fractionation upon complexation with inorganic ligands has been previously suggested by theoretical studiesHere, we survey the extent of Cr isotope fractionation that occurs during ligand complexation and dissolution of Cr(III)-containing solid phases. Given the potential for analytically significant fractionations, these redox-independent processes may add complexity when using Cr isotopes to track Cr oxidative mobilization and Cr reduction during environmental remediation. We systematically characterize the dissolution of a Cr(III)-containing solid by a wide range of organic ligands, and quantified the attendant isotope effects with an eye toward better understanding the potential impact of Cr(III)–ligand complexation on global Cr cycling and interpretation of isotope signatures.In the absence of organic ligands, dissolved Cr remained <0.5 µM throughout the experiment with detectable release only occurring after 14 days Fig. . The preR2 = 0.2) , followed by oxalate (6.5 ± 0.6 × 10−14 mol m−2 s−1), and succinate and acetate (both ~ 2 × 10−14 mol m− s−1) , followed by pyoverdine (5.7 ± 0.9 × 10−13 mol m−2 s−1), and finally DFOB (4.8 ± 0.1 × 10−14 mol m−2 s−1)  relative to the absence of a ligand, for which an initial dissolution rate could not be calculated by linear regression R = 0.2 (F.2) Fig. . In the 53Cr values were observed in the solubilized Cr, from +1.23 to −0.27‰. The δ53Cr values varied based on ligand type and reaction extent. The isotope signature of dissolved Cr approaches that of the original solid with increasing reaction extent for the experiments with organic acids only. Interestingly, though the influence of DFOB on organic acid-promoted dissolution was not obvious with regard to Cr(III) release rates, a significant impact on the magnitude of isotopic fractionation was observed when comparing DFOB+organic acid experiments to organic acid-only experiments.The Cr isotopic composition of the dissolved Cr(III) was also examined throughout the experiments Fig. . A wide 39. The stability constants of Cr(III)-siderophore complexes are not well established due to the slow ligand exchange rate, weak ultraviolet–visible (UV–vis) absorbances, and low reduction potentials, all of which prevent traditional methods for the determination of stability constants (see ref. 27 and references therein). Because limited stability data are available for Cr(III)–ligand complexes for the ligands studied here, the following discussions will use the stability constants of Fe(III)–ligand complexes to estimate the potential influences of such factor on dissolution and isotope fractionation. The rationale for using Fe(III)–ligand complex stability constants as proxies is based on the similar strength of interaction between Fe(III) or Cr(III) cations and a ligand containing a hard Lewis base 40. Therefore, the ionic potential of metal cations  positively correlates with the metal–ligand stability constant40. The similarity of the ionic potential of Fe(III) (ionic radius of 0.645 Å) to Cr(III) (ionic radius of 0.615 Å)41 allows for an appropriate first estimation of Cr(III)–ligand trends. Although a more complete estimation of the Cr(III)–ligand stability constants would require the metal–ligand stability constants for a greater number of metals40, we suggest using Fe(III) stability constants as a first-order approximation of the corresponding Cr(III)–ligand complex stabilities. However, it should be noted that in some instances the measured values of Cr(III)–ligand stability, specifically for siderophores, do not necessarily follow the correlating Fe(III)–ligand stability trends, as has been observed for rhizoferrin, an α-hydroxycarboxylate-type siderophore, where the Cr(III)–rhizoferrin stability constant is 4 orders of magnitude higher than for the corresponding Fe(III)–rhizoferrin complex27. The difference in siderophore affinity has also been previously noted between Fe(III) and Mn(III) due to the identity of the binding moiety of the siderophore and the electron configuration of the metal atoms42.Organic ligands are known to promote the dissolution of metal oxides by forming inner sphere metal–ligand complexes at the solid–solution interface and weakening the metal–oxygen bond on the solid surface. The structure of the ligand influences the stability of both the surface and dissolved metal–ligand complexes and consequently the rate of dissolution. The rate-limiting step during ligand-promoted dissolution is the detachment of the metal–ligand complex from the surface; thus, increasing stability of the dissolved metal–ligand complex leads to increasing dissolution rate45. Of the organic acids utilized here, citrate, a strong chelating ligand, exhibited the greatest dissolution rate at 0.27 ± 0.01 μM d−1 , consistent with the previously reported rate of citrate-promoted Cr(OH)3 dissolution under similar experimental conditions (0.22 ± 0.09 μM d−1)28. Although the rates of organic acid-promoted dissolution of Cr(OH)3 for oxalate, succinate, and acetate have not been reported to our knowledge, inference can be made based on the trends observed with Fe oxides. For example, similar trends of dissolution rates have been observed for organic acid-promoted ferrihydrite dissolution at pH 7, which decreased in the order of citrate, oxalate, and succinate46. These trends can be described by the binding mechanism exhibited by each ligand. Citrate has been shown to adsorb to Fe oxides with each carboxyl group bound to different Fe(III) sites on the solid surface, leading to its effectiveness at dissolving metal oxides47. Oxalate has been shown to form a bidentate mononuclear complex with Fe(III) in hematite, while succinate formed a (weaker) monodentate surface complex, leading to the observed differences in dissolution rates48. Acetate is a monodentate ligand that has been shown to preferentially occupy adsorption sites on metal oxide surfaces28, though is not expected to significantly affect the dissolution rate49. In general, the trends in organic acid-promoted dissolution observed in this study are consistent with expectations.In the presence of organic acids, we find that dissolution rates generally increase with increasing stability constant of the corresponding Fe(III)–ligand complex Fig. 43–45. Of3 by siderophores has recently been studied at several pH values by Duckworth et al.27. The average surface area normalized dissolution rates at pH 7 and under similar experimental conditions (0.2 g l−1 Cr(OH)3, 100 μM siderophore, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)) were 4.3 ± 0.7 × 10−13 mol m−2 s−1 for DFOB and 3.0 ± 1.7 × 10−12 mol m−2 s−1 for protochelin (a triscatecholate-type siderophore like enterobactin)27, both of which are within a factor of 10 of rates observed in this experiment  and the difference in the number of time points considered. Unlike organic acids, the relationship between stability constant and dissolution rate of the siderophores does not appear to be correlated –siderophore stability constants. For the ligands with an available Cr-ligand stability constant and dominant species at the experimental conditions, Cr-acetate (logK = 15)44 and Cr-DFOB (logK = 33)27, general agreement between the Cr-ligand and Fe-ligand stability constant correlation with stability constant is observed. While Fe oxide dissolution rates have been shown to positively correlate to the Fe(III)-ligand stability constant50, Cr(III)–siderophore complexes have been shown to behave differently from their corresponding Fe(III)–siderophore complexes27. It is important to note that we focus here on dissolution rates rather than dissolution rate constants, which may result in some discrepancies due to siderophore adsorption affinities51, which were not the focus of this study.The dissolution of Cr(OH)45. Metal–ligand complexes with the largest stability constants (based on stability constants for the corresponding Fe(III)–ligand) resulted in less positive or more negative fractionation 44 and Cr-DFOB (logK = 33)27, Cr-acetate does not follow the predicted trend while Cr-DFOB does. A similar trend has been observed with Fe isotopes during organic acid-promoted dissolution of hornblende52. However, in that case the trend encompassed greater fractionation with increasing stability constant, opposite of the observations of this study. If the predominant control is kinetic isotope fractionation, we would expect lighter isotopes  to enrich in solution at low dissolved Cr concentrations and approach the isotopic signature of the starting solid during continued dissolution16. In this study, maximum isotopic fractionation was observed relatively soon after the experiment was initiated with isotope values eventually approaching the bulk solid isotope signature at greater dissolved Cr, similar to what has been previously observed with Fe isotopes during dissolution experiments on Fe oxides53. However, the enrichment of lighter isotopes in the solution during the early stages of dissolution was not evident in our system; instead, positive values for Δ53Cr were observed during initial dissolution  in the dissolved fraction –ligand complexes, but instead generally exhibits a negative correlation  may be caused by a fractionation during back reaction of weakly complexed liberated Cr(III). This process could be insignificant in more strongly bound Cr(III). Furthermore, if dissolution occurs layer by layer such that ligands complex and dissolve surface Cr atoms completely before the next layer is exposed and solubilized, a large fractionation may only be observed in the early stages of dissolution16. Given that Cr(OH)3 only exhibits a particle size of < 10 Å and is likely a polymer of only a few CrO6 octahedra54, rind effects are expected to be minimal for individual particles. However, since heavy aggregation is typically observed for these nanoparticles, rind effects might be more significant at an aggregate level.The stability constants of the metal–ligand complexes can also be correlated with the observed isotopic fractionation factors Fig. 43–45. Me3 solids. Therefore, despite the lower rates and extents of dissolution relative to proton-promoted dissolution27, ligand-promoted dissolution of Cr-containing phases might contribute significantly to Cr mobilization under nonacidic conditions, such as in the marine environment. For instance, isotopically fractionated Cr(III) may be leached from dusts or sourced from a benthic flux of organically complexed Cr(III). Again, the presence of ligand-bound Cr(III) in the modern oceans supports ligand complexation as an important component of the Cr cycle26. Terrestrial waters can also be near neutral or alkaline, suggesting the need to survey for significant ligand complexed Cr(III) when using Cr isotopes to monitor Cr reduction in environmental remediation studies.Overall, results from this study have important implications for research using the Cr isotope system as a paleoredox and environmental tracer. Dissolution experiments were conducted at pH 7 in order to isolate the effects of ligand-promoted dissolution on Cr(OH)57. Therefore, the results from this study bolster the case that the interpretation of the Cr isotope proxy may need to be revisited in some circumstances to account for redox-independent processes, such as the mobilization of Cr(III) by organic or inorganic complexing ligands. As previously suggested14, some sedimentary Cr isotope archives are more likely to be shaped by ligand-bound Cr and attenuating isotope fractionations associated with organic stabilization. As mentioned above, alkaline systems  may be more likely to express the effects of ligand-promoted solubilization than low pH systems, given that proton promoted dissolution scales with pH. Marine systems removed from a terrestrial source  might be expected to capture variably fractionated organically or inorganically complexed Cr(III), such as dissolved Cr(III)–carbonate complexes which have also shown to be significant during ultramafic rock weathering21. However, given the high density of organic acids in the weathering realm , soils  with evidence for intense weathering may also be susceptible to expression of Cr(III)–organic isotope fractionations . Therefore, we suggest care should be taken when interpreting negative δ53Cr values in paleosol and ancient carbonate rocks as indication of redox cycling of Cr58.The redox-independent Cr isotope fractionations observed in this study are within the range of values previously reported to reconstruct oxygenation events Fig. 1–5,9,14,53Cr values will make it difficult to directly assess the influence of Cr(III)–ligand isotope effects in deep time studies. However, we emphasize that this does not require abandonment of the basic framework for using Cr isotopes as a paleoredox proxy, where large Cr isotope variations in the sedimentary records  are a signal for oxidative Cr cycling that can be linked to atmospheric oxygen levels5. The common occurrence of authigenic but isotopically unfractionated Cr in ancient sedimentary rocks5 implies that Cr(III) moving through the Earth’s surface is not ubiquitously isotopically fractionated by ligand-promoted solubilization, though some small variability is observed in each of these studies. In this light, the possibility of some variable Cr isotope values should be expected in low oxygen systems  oxidation), and moving forward, the paleoredox Cr isotope framework must account for this possibility. Despite this added complexity, we suggest that organic complexation is unlikely to lead to a systematic shift in the mean δ53Cr value of the mobile Cr reservoir on a global scale , in contrast to extensive Cr oxidation, which can result in large 53Cr-enriched surface reservoirs as observed on the modern and Phanerozoic Earth63. Nevertheless, our results imply that some Cr isotope data from individual formations may lead to ambiguous conclusions about the presence of oxidative Cr cycling.The common occurrence of organic-bound Cr(III) in modern environments and the potential for the formation of organic acids throughout the Earth’s history indicates that the presented results must be considered when using Cr isotopes to track the Earth’s oxygenation. Further, the presence of both positive and negative ΔOur data provide a step towards accounting for Cr isotopic variability in the Earth’s rock record by pointing toward specific non-redox processes that must be considered in the calibration of the Cr isotope system as a paleoredox proxy. Our results strongly suggest that fractionations during both redox-dependent and redox-independent Cr cycling need to be assessed Fig. . In part3) was selected as a representative compound for solid-phase Cr in natural environments as it is a common Cr(III)-containing solid phase, and an endmember in the Cr(III)-Fe(III)-(oxy)hydroxide solid solution series54. A variety of organic ligands and their impacts on Cr(OH)3 dissolution and isotope fractionation were surveyed. Siderophores and organic acids were studied as they are two common classes of metal-complexing organic ligands in the natural environment as previously discussed. Siderophores are generally divided into three primary types based on the characteristic metal binding functional group, including α-hydroxycarboxylic acid (hydroxycarboxylate type), catechol (catecholate type), and hydroxamic acid (hydroxamate type)64. The most common bacterial and fungal siderophores are catecholates, and hydroxamates, respectively, though other types, including a mixed type, are also quite common and exist with a variety of chemical structures64. We chose the following types of siderophores to cover a range of binding types: DFOB (hydroxamate type), enterobactin (catecholate type), and pyoverdine (mixed type) (Table Amorphous Cr(III)-(oxy)hydroxide (hereafter referred to as Cr(OH)e) Table . A rangee) Table . Cr spec3 was synthesized following a previous procedure65. Briefly, Cr(NO3)3·9H2O (ACS grade) was dissolved in deionized water (18 MΩ cm) to obtain a total Cr concentration of 0.1 M. The solution was slowly titrated to pH 7 with 1 M NaOH. Precipitates formed from the suspension were aged for 24 h followed by dialysis to remove remaining electrolytes. The resulting wet pastes were recovered through centrifugation, freeze dried, and finely ground. Portions of the powders were dissolved in nitric acid for Cr concentration analysis by inductively coupled plasma-mass spectroscopy . The specific surface area of the synthesized solid was determined to be 95 m2 g−1 by Brunauer–Emmett–Teller (BET) gas adsorption analysis using an Autosorb-1-MP surface pore analyzer (Quantachrome Corp.).Cr(OH)3 solubilization were investigated in the presence of a single type of siderophore or organic acid, or a combination of siderophore and organic acid. The representative siderophores included DFOB , enterobactin , and pyoverdine . The representative organic acids included acetate, oxalate, succinate, and citrate .The rate and extent of Cr(OH)3 solids were suspended in a 0.1 M NaCl electrolyte solution with 10 mM HEPES buffer  at pH 7 and sonicated for 15 min to disperse the particles. HEPES buffer was previously shown to not influence the rates of siderophore-promoted dissolution of metal hydroxides . For the treatments containing a siderophore, the siderophore was added to the suspensions to achieve a final concentration of 0.1 mM. For the treatments containing an organic acid, the organic acid was added to the suspension to achieve a final concentration of 1 mM. For the treatments containing both a siderophore and an organic acid, the organic acid was added 1 min prior to the siderophore addition in order to account for its potential effect on metal hydroxide dissolution during the initial exposure67. Treatments containing organic acids were conducted in triplicate, and treatments containing siderophores were conducted in duplicate. Due to the limitations imposed by the quantities of purchased siderophores, the volumes of each replicate for the siderophores were 50, 5, and 2.4 ml for DFOB, enterobactin, and pyoverdine, respectively. All organic acid treatments were conducted at 50 ml volume. All experiments were conducted at room temperature in the dark using amber bottles, and were constantly agitated on an orbital shaker. A reaction time of ~ 1 month was chosen based on previous studies on Cr isotope exchange rates between aqueous Cr(VI) and solid Cr(III) oxyhydroxide at pH 719 and between various Cr–Cl species at low pH7, both suggesting significant to complete isotope exchange may occur within a few months.For each ligand solubilization experiment, the Cr(OH)68 on an UV–vis spectrophotometer  and were below the detection limit (0.2 μM) for all treatments. Thus, the total dissolved Cr concentration in the filtrate was considered to be Cr(III). The pH values of the reaction suspensions stayed at 7.0 ± 0.1 throughout the entire experiment for all treatments. No microbial growth was observed for the duration of the experiment. Cr release rates were determined by fitting a regression line through the linear portion of the dissolution profile for each condition, and were normalized to BET surface area for consistency of comparison with previous studies on ligand-promoted dissolution of Cr(OH)327.Throughout the experiments, 100 μl of filtered  aliquots of each sample were collected at certain time points and analyzed for total dissolved Cr concentration by ICP-MS. Cr(VI) concentrations in the filtrates were analyzed using the diphenylcarbazide assay50Cr-54Cr double spike method was used to correct for fractionation during sample purification and isotope measurement69. The double spike solution with known 50Cr/54Cr ratio (~ 1) was added as early as possible in the sample preparation process and was added in the same valence(s) as the Cr in the sample ). Proper amount of spike was added to the filtered samples to ensure an optimum spike/sample ratio of 54Crspike/52Crsample ~ 0.5. Cr was purified from matrix ions using a cation exchange resin method modified from Bonnand et al.70. Briefly, the sample–spike mixtures were gently evaporated to dryness and refluxed with 6 N HCl to allow full isotopic equilibrium between spike and sample. Samples were then dissolved in 0.5 N HCl and loaded onto Bio-Rad AG50W-X8 (100–200 mesh) cation exchange resin. Cr was eluted from the resin after loading and collected for isotope analysis. Purified Cr samples were dissolved in 0.7 N HNO3 and measured for Cr isotopic compositions on a NeptunePlus multicollector ICP-MS. Ti, V, and Fe interferences were monitored and corrected, with corrections typically < 0.1‰. A concentration matched unprocessed SRM 979 standard was analyzed before and after every sample to monitor instrument drift. Sample values were normalized to the average values of the bracketing standards. Based on standards and duplicate preparation and analysis of various types of real samples, our analytical precision was about ±0.08‰ (95% confidence level) using 200–1000 ng Cr. The starting Cr(OH)3 solid was dissolved by 0.7 N HNO3 and analyzed for Cr isotopic composition as described above and was −0.08 ± 0.04‰. The total procedural blank ranged from 0.2 to 1 ng, which is negligible compared to the sample Cr amount; therefore, blank correction was not performed.At various time points along the dissolution profile, an entire sample bottle was sacrificed for Cr isotope analysis, thus reflecting the cumulative isotope signature at that time point. The Data sets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.Supplementary Information