However, previous attempts to model carbon–phosphorus-oxygen feedbacks have neglected key parameters that could shape the global P cycle. Here we present new diagenetic models to fully parameterize marine P burial. We have also coupled this diagenetic framework to a global carbon cycle model. We find that seawater calcium concentration, by strongly influencing carbonate fluorapatite (CFA) formation, is a key factor controlling global phosphorus cycling, and therefore plays a critical role in shaping the global oxygen cycle. A compilation of Cenozoic deep-sea sedimentary phosphorus speciation data provides empirical support for the idea that CFA formation is strongly influenced by marine Ca concentrations. Therefore, we propose a previously overlooked coupling between Phanerozoic tectonic cycles, the major-element composition of seawater, the marine phosphorus cycle, and atmospheric pO2.The marine phosphorus cycle plays a critical role in controlling the extent of global primary productivity and thus atmospheric  Previous work suggests that marine oxygen levels and bioturbation are important factors that shape phosphorus burial and the size of the marine biosphere. Here the authors show that seawater calcium concentration is a key factor in controlling marine P burial, and thus the global oxygen cycle. However, our understanding of the factors that govern the retention or regeneration of P during burial and subsequent chemical alteration of marine sediments (diagenesis) arguably remains limited. Traditionally, bottom-water oxygen concentrations have been assumed to exert the largest influence on P burial4. More recently, it has been suggested that the rise of bioturbation in the early Paleozoic significantly increased marine P burial8. However, although these factors may influence the burial of organic and iron-bound P phases, the dominant controls on the formation of carbonate fluorapatite (CFA)—the largest modern authigenic P burial flux9—are relatively poorly understood. As CFA accounts for >50% of marine P burial9, this translates into considerable uncertainty regarding the chief factors responsible for global P cycling and, by extension, the processes regulating global organic carbon burial and atmospheric pO2 on geologic time scales.Although there are few constraints on the how the size of the biosphere has changed through Earth’s history, phosphorus (P) is commonly considered to be the major limiting nutrient for primary production on geologic time scales. It is also generally accepted that P has a critical role in regulating the extent of organic carbon (C) burial and thus atmospheric oxygen levels (10), and the roles played by other key CFA components such as Ca2+, CO32−, and F− are largely overlooked. In particular, calcium (Ca) is the most abundant ionic species in CFA (Ca9.54Na0.33Mg0.13(PO4)4.8(CO3)1.2F2.48, ref. 11) and seawater dissolved Ca is the single most important Ca source for CFA formation in the upper marine sediment pile. Intuitively, the large swings in seawater Ca concentration during the Phanerozoic14 could have significantly influenced the saturation state and thus precipitation rate of CFA. Here, using models and empirical data, we make a case that seawater Ca concentrations are a key factor shaping marine P burial. Our model results also suggest a significant role for seawater Ca on the evolution of Earth’s redox budget.The factors regulating CFA formation and burial in marine sediments and the relative importance of environmental and biological boundary conditions in influencing this process have been underexplored. For example, the kinetics of CFA formation are typically simplified for use in diagenetic models; commonly, CFA formation is parameterized as a linear relationship with dissolved phosphate . In contrast to the simplified rate law used in previous studies10, this model includes a rate law that describes the formation rate of CFA as a function of its saturation state, which is consistent with experimental observations16. This exercise indicates that an increase in seawater Ca concentrations will drive a significant increase in CFA formation . We calibrated our CFA rate law in our fully parameterized model using sedimentary porewater and solid-phase data collected from a well-characterized modern shallow marine site  and the deep-sea ODP site 1226 14, shifts in dissolved calcium concentrations are likely to have been a key environmental factor controlling P burial efficiency over this interval.Despite significant increases in the complexity of our extended diagenetic model, we find, in agreement with the results of our basic diagenetic model, that marine Ca concentrations markedly alter the efficiency of P burial. Calcium concentrations, by strongly influencing CFA saturation state, have a strong impact on overall P burial relative to other environmental factors Figs. –7. Unlik32−and PO43− in CFA is low, relative to that of Ca2+ (ref. 11). Moreover, porewater DIC and dissolved inorganic phosphorus (DIP) are mainly generated by the decomposition of organic matter and/or the reduction of iron oxides8, rather than diffusion from seawater. The Mg concentration of seawater also does not appear to exercise a strong control on CFA burial, likely because Mg is a minor component of CFA. Bottom-water pH also appears to not strongly influence CFA burial , as well as dissolved sulfate, magnesium, and phosphate concentrations appear to have relatively little impact on CFA burial Fig. . There a14, Fig. A Ca control on marine P burial is also supported by P speciation data compiled from Cenozoic deep-sea sediment cores across the Pacific and Atlantic Ocean basins Fig. . After fpO2, we coupled our extended diagenetic model to a simple global carbon cycle mass balance model. Specifically, we used the outputs of our extended diagenetic model to parameterize how P burial efficiencies vary as a function of bottom-water oxygen, extent of organic matter loading, marine Ca concentrations, and bioturbation in a global carbon cycle mass balance model .Given the large swings in Phanerozoic seawater Ca concentration recorded by geologic archivespO2. A compilation of deep-sea P speciation data from the past 80 million years provides empirical support for the effect of seawater dissolved Ca concentrations on P burial and bolster the case that this Ca-driven feedback strongly impacts the global ocean-atmosphere system. These results provide a new view of the processes linking marine elemental cycles, tectonics and atmospheric pO2, and suggest that the major ion composition of seawater has been an important driver of biospheric change and atmospheric evolution throughout Earth’s history.Our modeling results indicate that seawater Ca concentration is likely a major driver of total P burial, such that, over Earth’s history, high seawater dissolved Ca concentrations should correspond to significant increases in P burial efficiency and relatively low atmospheric 2+], [DIC], [DIP], and [F−]. The mass balance functions for this basic model are31:Cl is the concentration of solute, Cs is the concentration of solid, ϕ is porosity, D is molecular diffusion, ʋ is the solute advection rate, ω is solid advection rate, and Rs and Rl are, respectively, the solid and solute reaction rates. The effect of molecular diffusion is calibrated to tortuosity through m is the intensity of molecular diffusion. The effect of compaction on the solute and solid advection rates was also considered using the method described in ref. 30. The depth dependence of porosity was represented by ϕ(x) = ϕ∞ + (ϕ0 − ϕ∞) exp(−x/λ), where λ is the porosity attenuation length, and ϕ0 and ϕ∞ represent porosity at the sediment–seawater interface and at depth, respectively. The boundary conditions for these species and the above model parameters were also used for the extended, multicomponent version of the diagenetic model 4.8(CO3)1.2F2.48—although the stoichiometry of CFA can be variable. Previous studies have simplified the precipitation rate of CFA by presuming a linear relationship with excess dissolved phosphate . However, experimental studies have shown that the rates of the crystal growth of calcium hydroxylapatite and calcium fluorapatite are functions of saturation state16. Thus, in this study, we more completely parameterize the kinetics of CFA formation by assuming that the formation rate of CFA is characterized by a first-order relationship with its saturation state. When the saturation state of CFA (ΩCFA) is higher than 1, the precipitation rate of CFA can be expressed as:kCFA is a kinetic constant for CFA, obtained by fitting to FOAM porewater and sedimentary CFA profiles. ΩCFA is expressed as:rCa, rNa, rMg, rPO4, rCO3, and rF represent the activity coefficients of each ion. KspCFA is the equilibrium constant of CFA, which was found to be a function of carbonate activity11:KspCFA (10−99.7) without a relationship with carbonate activity in a series of runs . Following the approach of previous studies10, the dissolution of CFA under ΩCFA < 1 is not included, as CFA is highly insoluble in marine sediments.CFA is the largest P sink in the global marine P cycle36, we also built an extended 1D multicomponent model incorporating the biogeochemical cycles of C, N, P, S, Fe, and Mn to more fully simulate the diagenetic P cycle in marine sediments (SEDCHEM). The model includes 15 solutes and 21 solids . Model components and boundary conditions are shown in Supplementary Table Building from previous efforts36). Three solid P species—organic phosphorus (orgP), iron-bound phosphorus (PFe), and CFA—as well as dissolved inorganic phosphate (ƩPO43−) are included in the extended version of our diagenetic model. In contrast to previous studies, however, our model also includes a more intrinsic reaction rate law for CFA formation, which involves all the major components of CFA and parameterizes the effect of pH on P speciation and burial. To simulate pH, our model also includes proper descriptions of adsorption, the diagenetic Fe cycle, and authigenic carbonate precipitation and dissolution.Parameterizations of the diagenetic P cycle explored in this study share many features with previously published model exercises  of these pathways is dictated by their respective energy yields40, a Monod scheme was used to describe the operation of these reactions36.Decomposition of organic matter drives diagenetic reactions. The chemical formula of organic matter can be simplified as CN41 developed from a reactive continuum-type model32. The advantage of this model is that it not only can represent the reactive continuum of organic matter, but also is suitable to apply to the bioturbated zone of the sediment pile41. We divided organic matter into 12 G in this study. Although each G has its own first-order rate constant, their fractions in total organic matter are determined by a gamma distribution41. The rate constant for each Gi isi isa is the average lifetime of more reactive orgC and v is the shape of orgC distribution32.For the mineralization of orgC in the extended model, we used a multi-G model42, we assume that this variation is generated by different C/P ratios in different organic components, with less reactive (more refractory) organic matter having higher C/P ratios. Thus, we further divided the 12 G fractions into two pools (α and β) with different C/P ratios. With this procedure, it is possible to reproduce empirical sedimentary profiles of organic phosphorus and dissolved phosphate , whereas the β pool includes the remaining 10 G fractions (G3–G12). The C/P ratios of the two pools are shown in Supplementary Table The C/P ratio of particulate organic matter typically increases with depth in the sediment pile. Following previous methods3α); (2) less reactive iron hydroxides (Fe(OH)3β); (3) unreactive iron hydroxides (Fe(OH)3γ); (4) magnetite (Fe3O4); and (5) biotite (Biot). Demarcation of iron hydroxides by reactivity is similar to the treatment employed by previous models36. The rate law for magnetite dissolution is reasonably well established43. Iron may also occur as other silicate-bound phases, but we use biotite as a representation of silicate iron (see ref. 44). At FOAM, biotite dissolution appears to be closely linked to porewater pH. As for previous models8, iron-bound phosphorus is assumed to be associated with iron hydroxides, and the P/Fe molar ratio is described using a constant γ and a variable θ (Supplementary Tables Five iron phases are included in the modeled flux to the sediment–seawater interface: 1) highly reactive iron hydroxides (Fe(OH) highly rO4); and  biotite 3α);  less rea10 is that we have parameterized the formation rate of CFA using its saturation state (see above), which is consistent with experimental results16. A full description of this method can be found above, as well as in the A major difference of this study from previous studies45. Following ref. 45, we have used the Michaelis–Menten kinetics for dissolved phosphate and iron in marine porewater to describe the formation of vivianite. Details of the formulation and parameters for vivianite can be found in Supplementary Tables We have also included the iron phosphate mineral vivianite in the extended model. Although it has not been extensively documented in marine sediments, it is commonly found in restricted settings46 to simulate pH variation during diagenetic processes. Instead of total alkalinity, we used total proton balance (TP) as an equilibrium-invariant and implicit differential variable  are listed in Supplementary Tables Our coupled carbon–phosphorus–oxygen cycle model integrates our newly developed diagenetic model with a global biogeochemical cycling model modified from the carbon–phosphorus cycle model of Van Cappellen and Ingall47, in our coupled model the marine sedimentary burial of calcium-bound phosphate (F58) is calculated from our diagenetic model (interpolated between time slices of 1 million years). Rates of organic carbon burial  and organic carbon weathering are, in our model, influenced by atmospheric O2 level, following the parameterization of the COPSE model48. In particular, we have also, for these runs, parameterized the organic C/P ratio as a function of seawater oxygen level in our diagenetic model. This is achieved by parameterizing two factors rP1 and rP2 using the following functions:In contrast to the approach of Van Cappellen and IngallSupplementary InformationPeer Review FileSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4


Among the different platforms for quantum information processing, individual electron spins in semiconductor quantum dots stand out for their long coherence times and potential for scalable fabrication. The past years have witnessed substantial progress in the capabilities of spin qubits. However, coupling between distant electron spins, which is required for quantum error correction, presents a challenge, and this goal remains the focus of intense research. Quantum teleportation is a canonical method to transmit qubit states, but it has not been implemented in quantum-dot spin qubits. Here, we present evidence for quantum teleportation of electron spin qubits in semiconductor quantum dots. Although we have not performed quantum state tomography to definitively assess the teleportation fidelity, our data are consistent with conditional teleportation of spin eigenstates, entanglement swapping, and gate teleportation. Such evidence for all-matter spin-state teleportation underscores the capabilities of exchange-coupled spin qubits for quantum-information transfer. Despite recent demonstrations of coherent spin-state transfer in arrays of spin qubits via exchange interaction, all-matter spin-state teleportation is still out of reach. Here the authors provide evidence for conditional teleportation of quantum-dot spin states, entanglement swapping, and gate teleportation. Teleportation has been demonstrated in many experimental quantum information processing platforms7, and it is an essential tool for quantum error correction8, measurement-based quantum computing9, and quantum gate teleportation10. However, quantum teleportation has not previously been demonstrated in quantum-dot spin qubits. Separating entangled pairs of spins to remote locations, as required for quantum teleportation, has previously presented the main challenge to teleportation in quantum dots.Quantum teleportation11. This technique does not involve the motion of electrons, greatly simplifying the teleportation procedure. Our teleportation method also leverages Pauli spin blockade, a unique feature of electrons in quantum dots, to generate and measure entangled pairs of spins. We combine these concepts to perform conditional teleportation in a system of four GaAs quantum-dot spin qubits. Our data are consistent with conditional teleportation of quantum-dot spin states, entanglement swapping, and gate teleportation. Entanglement swapping12 goes beyond teleportation of single-qubit states to create entanglement between uncorrelated particles via measurements, and demonstrations of entanglement swapping in matter qubits are rare14. Our technique is fully compatible with all gate-defined quantum-dot types, including Si quantum dots. Although we use coherent spin-state transfer via Heinseberg exchange11 to distribute entangled pairs of spins, other methods to create long-range-entangled states of spins including tunneling17 and coupling via superconducting resonators18 could be used as well.Here, we overcome this challenge using a recently demonstrated technique to distribute entangled spin states via Heisenberg exchange20. Furthermore, spin-to-charge conversion via Pauli spin blockade21 enables rapid single-shot measurement of pairs of electron spins in the We implement our teleportation method in a four-qubit quantum processor, which consists of a quadruple quantum dot fabricated in a GaAs/AlGaAs heterostructure Fig. a. Becaus11. To transfer a spin state from one electron to another, we induce exchange coupling between electrons by applying a voltage pulse to the barrier gate between them [Fig. 23. Because exchange coupling generates a SWAP operation, this procedure interchanges the two states. This procedure can be repeated for different pairs of spins to enable long-distance spin-state transfer. Importantly, exchange-based spin swaps preserve entangled states11.We achieve separation and distribution of entangled pairs of spins through coherent spin-state transfer based on Heisenberg exchange11 [Fig.  Figure mentclass2pt{minim2 or through process tomography5. Because our four-qubit device does not incorporate a micromagnet or antenna for magnetic resonance, we are not able to prepare superposition states of single spins. Therefore, to illustrate the operation of the teleportation procedure, we first teleport a classical spin state from qubit 1 to qubit 4. Later, we demonstrate entanglement swapping in our four-qubit processor, which conclusively demonstrates non-local manipulation of quantum states via measurements. In the future, quantum state tomography will be required to establish that the teleportation fidelity exceeds the classical bound, as discussed below.The utility of quantum teleportation lies in its ability to transmit unknown quantum states. Usually, teleportation of unknown states is experimentally demonstrated by verifying teleportation of a complete set of single-qubit basis states1. Because qubit 3 has the To demonstrate the basic operation of our teleportation method using entclass1pt{minimaentclass1pt{minimaentclass1pt{minimaentclass1pt{minimaentclass1pt{minimaTo verify conditional teleportation of the classical state, we perform an exchange gate on qubits 3 and 4 following the teleport [Fig. entclass1pt{minimap(SL∣SR) and p(SR∣SL)  Fig. , which l12 uses teleportation to generate entanglement between uncorrelated particles via measurements. In this case, we prepare the EPR state between qubits 1 and 2 via a B34 for a variable time t. This evolution generates an effective z-rotation on qubit 4 relative to qubit 3 by an angle θ = gμBΔB34t/ℏ, where g is the electron g factor in GaAs, and μB is the Bohr magneton. The z-rotation on qubit 4 coherently rotates the joint state of qubits 3 and 4 to 20. During this evolution time, qubits 3 and 4 remain maximally entangled.Having illustrated the basic operation of the teleport procedure, we now present evidence for conditional entanglement swapping, which confirms that the four-qubit processor indeed performs non-local coherent manipulation of quantum information using measurements Fig. a. Entang20 (see “Methods”) following another After a SWAP gate between qubits 2 and 3, qubits 1 and 3 are entangled, and qubits 2 and 4 are entangled. After projection to a singlet on the right side, entanglements have been swapped, because the entangled state of qubit 4 is teleported to qubit 1. Qubits 1 and 2, which were not entangled immediately before the measurement, become entangled, provided qubits 3 and 4 project onto the singlet state. Moreover, the coherent singlet–triplet evolution that occurred on qubits 3 and 4 should appear on qubits 1 and 2, given a singlet outcome on the right pair . However, prominent oscillations are visible in the probability of a singlet on the left given a singlet on the right p(SL∣SR) and also in p(SL∣TR), in good agreement with our simulations [Fig. To observe the anticipated oscillations, we sweep ons Fig. . These oB12) and dots 3 and 4 (ΔB34) , which is applied to one member of the EPR pair before teleportation, appears on qubit 4 after teleportation. The initial entangled state of qubits 1 and 2 is U has been applied to qubit 4. The added z rotation on qubit 4 occurs because of the additional A similar circuit Fig. d also im11. Post-selecting based on singlet outcomes on the left side yields prominent oscillations in time, consistent with our simulations. The extracted oscillation frequency versus repetition number agrees well with the data from Fig. We measure the right pair of qubits via diabatic charge transfer to verify teleportation Fig. e. The un11. The simulations shown in Figs. The fidelity of the teleport operation is limited by readout fidelity, relaxation during readout, state preparation, charge noise, and the hyperfine coupling between the electron spins and Ga and As nuclear spins in the substrate. Readout fidelity and relaxation both limit the probability that we will correctly measure the Bell state of one of the EPR pair and the qubit to be teleported. Readout fidelities are 0.93 for the left pair and 0.87 for the right pair  charge configuration20. The orientation of the two spins in this product state depends on the orientation of the local hyperfine field.We can initialize either pair of electrons as 23. Exchange coupling generated in this way is first-order insensitive to charge noise associated with the plunger gates. Barrier-gate pulses are accompanied by compensation pulses on the plunger gates to keep the dot chemical potentials fixed. For the exchange gates used in this work, we used a combination of barrier-23, and tilt-controlled19 exchange. Empirically, we found that using this combination helps us to boost the exchange strength and improves the fidelity of the SWAP operation. All exchange pulses are optimized at the same tuning used to acquire all data in this work with one electron in each dot. We do not observe that pulsing exchange between two spins generates spurious enhanced exchange coupling elsewhere in the array.We induce exchange coupling between pairs of qubits by applying a voltage pulse to the barrier between the respective pair of dots20. Adiabatic charge transfer into the outer dots maps either 20. Here, “diabatic” or “adiabatic” refer to the speed with which the electrons are recombined relative to the size of the hyperfine gradient. We represent readout by diabatic charge transfer with an “ST” in figures, and we represent readout by adiabatic charge transfer with a “↓↑” in figures. When used to verify teleportation, diabatic charge transfer can only verify teleportation when B34 were such that Diabatic charge transfer into the outer dots projects the spin state of the separated pair onto the 39 to enhance the readout visibility. Using the two sensor quantum dots configured for rf-reflectometry /3. Also, fs = 0.89 is the singlet load fidelity. All coefficients are given random phases for each realization of the simulation. To simulate loading error during adiabatic separation of electrons, we set38. We directly extract the population coefficients of the other three two-electron spin states. We create a state which is a sum of all two-electron spin states:ti∣2 is determined as discussed above. We assign random phases to each of the coefficients during each realization of the simulation.Our simulations include errors associated with state preparation, readout fidelity, relaxation during readout, charge noise, and the fluctuating magnetic gradient. We approximate singlet loading error by creating a two-electron stateTo simulate the spin-eigenstate teleport operation, we set the initial state of the four-qubit system asJ23 of 250 MHz between spins 2 and 3, and we adjust the time TS for the SWAP operation to give a π pulse. These parameters correspond closely to the actual experiments. To account for charge noise, we allow the value of J23 to fluctuate by 1% between simulation runs. We arrive at this level of charge noise via the expression 41, where Bk of spin k to be  MHz We incorporate charge noise and the hyperfine magnetic field and their effects on the SWAP operation by directly solving the Schrödinger equation for a four spin system. We generated a simulated SWAP operation from the following Hamiltonian:entclass1pt{minimaTB = 2 ns. To simulate the spin-eigenstate teleport experiment, the simulated final state after the teleport operation is thus The voltage pulses in our setup have finite rise times, which cause the four-qubit system to evolve under the magnetic gradient in the absence of exchange. To simulate this effect, we definey axis in the effective S − T0 subspace for each pair of qubits. The y axis is defined by the usual S − T0 Hamiltonian: Jσz + ΔBσx. The rotation angle of 7° was chosen to match an additional control data set in which we adiabatically measured a singlet prepared via diabatic separation. Ideally, this measurement yields a singlet probability of 0.5. In practice, the measured singlet probability is slightly larger than this due to pulse errors, and 7 degrees was chosen to match the observed return probability.For the simulations presented in Supplementary Figs. Cα,β = 〈ψ∣α ⊗ β〉〈α ⊗ β∣ψ〉, where α (qubits 1 and 2) and β (qubits 3 and 4) can be any of To compute the expected probabilities in Fig. rL(R) = 1 − fL(R) as the probability that singlet or triplet on the left (right) side will be misidentified due to noise. Here fL(R) is the measurement fidelity due to random noise on the left (right) pair. The experimentally measured probabilities areTo simulate readout errors, we define the entclass1pt{minimaU12 and U34 using Hamiltonians analogous to Eq. (Cα,β, where α is any of β is any of To simulate the data shown in Fig. s to Eq.  for excht in their magnetic gradient ΔB34. We compute all possible correlators Cα,β, where α is any of β is any of To simulate the data in Fig. To simulate the ideal results in the absence of noise in Fig. To extract the oscillation frequencies of the data in Fig. 23, and readout and initialization via Pauli spin-blockade42, have already been demonstrated in Si quantum dots. In general, we expect teleportation to work even better in Si, where magnetic gradients and noise can be reduced. One potential challenge is the requirement for spin blockade; small valley splittings in Si can easily lift spin-blockade43. However, this challenge is easily overcome by operating the quantum dots at larger occupation numbers where the singlet–triplet energy splitting is dominated by the orbital energy spacing45. Another potential complication for Si qubits is the frequent use of micromagnets, which generate intense magnetic field gradients, for single-spin control. In particular, strong magnetic gradients make pure exchange rotations challenging. However, resonant exchange gates26 or dynamically corrected gates27 can still generate high-fidelity SWAP operations in large magnetic gradients.All of the necessary steps for conditional teleportation, including barrier-controlled exchange32. High-fidelity single-qubit31 and CNOT gates30 have already been demonstrated in Si. Single-spin readout can be achieved via Pauli spin-blockade measurements with a known ancilla spin46, as discussed in the Readout section above, and SWAP operations. Alternatively, spin-selective tunneling47 can be used. Fast spin measurements48 together with real-time adaptive control33 could be used to complete the deterministic state transfer process.Deterministic teleportation of arbitrary states requires the ability to distinguish all four Bell states and the ability to generate arbitrary input states to the teleport. Achieving complete readout in the Bell-state basis is most easily achieved with single-qubit and CNOT gates together with single-qubit readoutSupplementary Information


Recent studies have demonstrated that embryonic stem cells (ESCs) are deficient in expressing type I interferons (IFN), the cytokines that play key roles in antiviral responses. However, the underlying molecular mechanisms and biological implications of this finding are poorly understood. In this study, we developed a synthetic RNA-based assay that can simultaneously assess multiple forms of antiviral responses. Dicer is an enzyme essential for RNA interference (RNAi), which is used as a major antiviral mechanism in invertebrates. RNAi activity is detected in wild-type ESCs but is abolished in Dicer knockout ESCs (D−/−ESCs) as expected. Surprisingly, D−/−ESCs have gained the ability to express IFN, which is otherwise deficient in wild-type ESCs. Furthermore, D−/−ESCs have constitutively active double-stranded RNA (dsRNA)-activated protein kinase (PKR), an enzyme that is also involved in antiviral response. D−/−ESCs show increased sensitivity to the cytotoxicity resulting from RNA transfection. The effects of dsRNA can be partly replicated with a synthetic B2RNA corresponding to the retrotransposon B2 short interspersed nuclear element. B2RNA has secondary structure features of dsRNA and accumulates in D−/−ESCs, suggesting that B2RNA could be a cellular RNA that activates PKR and contributes to the decreased cell proliferation and viability of D−/−ESCs. Treatment of D−/−ESCs with a PKR inhibitor and IFNβ-neutralizing antibodies increased cell proliferation rate and cell viability. Based on these findings, we propose that, in ESCs, Dicer acts as a repressor of antiviral responses and plays a key role in the maintenance of proliferation, viability, and pluripotency of ESCs. The innate immune system is the first line of an organism’s defense against a broad range of pathogen invasions. Although innate immunity consists of different mechanisms, the antiviral response is one of the most critical components and is presumably developed in most, if not all, mammalian cells , 2. HoweThe biological implications of the attenuated innate immune responses in ESCs have been speculated from different perspectives. Immune response is a double-edged sword: it serves as a critical part of the defense mechanism, but it can also cause immunologic toxicity to tissues since IFN and inflammatory cytokines negatively impact cell proliferation and viability , 6, 7. Win vitro assay to determine RNAi activity in this study. RNAi activity was indeed detected in wild-type ESCs, but not in D−/−ESCs, as expected. However, D−/−ESCs showed increased antiviral responses to RNA transfection.Although RNAi is widely recognized as a major antiviral mechanism in invertebrates, such a function has not been convincingly demonstrated in mammals. Interestingly, RNAi activity was detected in viral infected mouse ESCs, pointing to the possibility that RNAi could be an alternative antiviral mechanism in ESCs, in which the IFN system is deficient . HoweverViral RNA induces IFN response by interacting with toll-like receptors (TLRs) and retinoic acid–inducible gene I (RIG-I)-like receptors (RLRs), leading to IFN transcription through activation of NFκB and IRFs , 20, 21.While it is apparent that the deficiency of ESCs in expressing IFN is closely related to pluripotency, the underlying molecular basis of this deficiency is poorly understood. In this study, we demonstrate that D−/−ESCs have gained the ability to express not only the type I IFN system, but also constitutively active PKR, which together contribute to the reduced cell proliferation and cell viability of D−/−ESCs. Our data revealed a critical role of Dicer as a repressor of antiviral responses in ESCs, which represents a novel mechanism essential for ESCs to maintain rapid proliferation and to prevent potential cell damage resulting from dysregulated endogenous RNA transcripts.A). Quantitative analysis by flow cytometry indicated that GFP fluorescence intensity in ESCs was reduced more by dsGFP than by dsLuc . However, in D−/−ESCs, both dsGFP and dsLuc reduced the expression of GFP to a similar level . A logical explanation for these results would be that the different effect between dsGFP and dsLuc in ESCs is due to sequence-specific reduction of GFP-mRNA by dsGFP via Dicer-dependent RNAi activity, which is abolished in D−/−ESCs. The non-sequence-specific effects in the reduction of GFP fluorescence caused by dsGFP and dsLuc in both ESCs and D−/−ESCs are likely due to the activation of other pathways.This assay was initially intended to determine the RNAi activity in ESCs. Functionalized GFP-mRNA was first transfected into cells where it was translated to GFP. The cells were then transfected with a synthetic dsRNA corresponding to the sequence of GFP (designated as dsGFP). Based on the principle of RNAi, dsGFP would be processed to yield siRNA that will specifically target GFP-mRNA, thereby reducing GFP expression. GFP was detected as early as 3 h after GFP-mRNA transfection. Subsequent transfection with either dsGFP or dsLuc (a control luciferase dsRNA of similar length with a sequence unrelated to GFP-mRNA) reduced GFP expression as indicated by diminished green fluorescence A. QuantiA, D−/−ESC1), whereas the cytotoxicity caused by dsRNA transfection in ESCs is low and limited to reduced colony size . These results suggested that D−/−ESCs have increased susceptibility to the cytotoxicity of transfected RNA. The GFP-mRNA contains some uncapped GFP-mRNA, which has a 5’ppp-group that can activate RIG-I , a synthetic dsRNA used as a viral RNA analog in our previous studies  showed patterns similar to dsGFP and dsLuc in inducing IFNβ and ISG15 expression in ESCs and D−/−ESCs. It is noted that the endogenous B2RNA level in D−/−ESCs was significantly higher than in ESCs, indicating its accumulation in D−/−ESCs (C). If Dicer is responsible for processing endogenous B2RNA, it could target the transfected B2 RNA as well. To test this possibility, we transfected ESCs and D−/−ESCs with Cy3-labeled fluorescent B2RNA (Cy3-B2) and analyzed the levels of Cy3-B2 by Cy3 fluorescence intensity. Our results indicated that Cy3-B2 was detected at higher levels in D−/−ESCs than in ESCs (C). We also compared the basal mRNA levels of the RNA receptors that mediate the effects of viral RNA. With the exception of MDA5, which is expressed at higher levels in D−/−ESCs than in ESCs, the other tested dsRNA receptors are expressed at comparable levels (D).It is known that certain cellular RNA with dsRNA structures, including those from apoptotic cells, misprocessed RNA, and transcripts of transposable elements (TEs), can induce antiviral responses and cause cellular damage in the absence of infection , 28, 29.Figure 1D−/−ESCs C. If Dic in ESCs , consist in ESCs C. We alse levels D.A). It is also noted that the basal mRNA levels of all three ISGs are about threefold higher in D−/−ESCs than in ESCs. At the protein level, STAT1 was detected in unstimulated cells, but it was induced by IFNα in both ESCs and D−/−ESCs. IFNα-induced PKR was also apparent in ESCs and D−/−ESCs .ESCs are able to respond to IFNα and IFNβ, but they are unable to express the two cytokines , 26. To Fig. 2B). In this experiment, we used ESC-differentiated fibroblasts (ESC-FBs) since they are much more responsive to IFN than ESCs , induced the expression of STAT1 in ESC-FBs. This result indicated that dsGFP[CM] only from D−/−ESCs contains secreted IFN, as confirmed by ELISA analysis of IFNβ in the CMs . Together, these data demonstrate that D−/−ESCs have a fully functional type I IFN system.For a functional analysis of the IFN system, we prepared conditioned medium (CM) from dsGFP-transfected ESCs and D−/−ESCs (dsGFP[CM]). The rationale is that if the CM contains IFN, it will induce ISGs, such as STAT1, resembling the effects of IFNα B. In thihan ESCs . As showD, RT-qPCR analysis indicated that most of these molecules were expressed at comparable mRNA levels in ESCs and D−/−ESCs although some variations were noted for JAK1, IFNAR2, and STAT1 between the two different D−/−ESC lines.We further analyzed the expression levels of the major signaling molecules that mediate the effects of IFN. As shown in Figure 2The PKR pathway is functional in ESCs as we demonstrated with polyIC transfection and viral infection . To deteA). RT-qPCR analysis indicated that the mRNA level of cyclin E and cyclin B appeared to be lower in D−/−ESCs than in ESCs while cyclin A was not significantly altered. However, three major cell cycle inhibitors, p21, p19, and p16, which are expressed at very low levels in ESCs, are expressed at substantially higher levels in D−/−ESCs . These results explain, at least partially, the reduced cell proliferation in D−/−ESCs.ESCs are characterized by their rapid cell proliferation rate due to constitutively activated cyclin-dependent kinases (CDKs) driven by high expression levels of cyclins A, B, and E . D−/−ESCFig. 4A, C16 treatment significantly increased the cell number of D−/−ESCs in a dose-dependent manner with the maximal effect at the concentration of 0.75 μM, but this dose dependence was not seen in ESCs. We further analyzed the cells by flow cytometry. ESCs have large populations at the S phase and similar cell populations at the G1 and G2 phases when they are grown at low density. However, in comparison, D−/−ESCs have increased G1 cell populations and reduced S phase cell populations, which are indicators of slowed cell cycle progression. Treatment of D−/−ESCs with C16 reduced the G1 cell population. At 0.75 μM and 1 μM, C16-treated D−/−ESCs have a cell cycle profile with a ratio of G1 and G2 cells similar to ESCs. Although the S phase cell population in ESCs was slightly reduced by C16, the overall cell cycle profiles were not altered (B), consistent with the unchanged cell proliferation rate (A). The effect of C16 on the inhibition of PKR was confirmed by knocking down PKR expression with its specific siRNA, leading to increased cell proliferation similar to the effect of C16 (C).PKR activation is known to inhibit cell proliferation, including in ESCs as we previously demonstrated with polyIC-activated PKR . To testFigure 5 altered B, consision rate A. The eft of C16 C.Figure B, western blot analysis indicated that p21 and p19 proteins were expressed at substantially higher levels in D−/−ESCs than in ESCs, but their relative levels were not affected by C16 (A). The relative levels of CDK2 (B) and cyclin E and A were not significantly altered in either D−/−ESCs or ESCs by C16 treatment (data not shown). However, CDC25A, a protein phosphatase that activates CDKs and leads to G1/S cell cycle progression (B). This result could contribute to the reduction of G1 phase cells by C16 in D−/−ESCs.In line with the results from RT-qPCR analysis shown in Figure 4d by C16 A. The re of CDK2 B and cycgression , is upregression B. This rA. By quantitatively analyzing the number of viable cells after transfection with different concentrations of dsRNA, our results demonstrated that D−/−ESCs are more sensitive to the cytotoxicity of dsGFP and dsLuc at all concentrations tested (A). To determine the contribution of PKR to the cytotoxicity caused by RNA transfection, we pretreated cells with C16 to block PKR activation prior to RNA transfection. As shown in Figure 6B, the cytotoxicity caused by both dsGFP and B2RNA can be partially reversed by C16 treatment.In routine cell culture, it is notable that there are more cells undergoing spontaneous cell death in D−/−ESCs than in ESCs. In particular, D−/−ESCs are more susceptible than ESCs to the cytotoxicity of RNA transfection as shown in Fig. S1s tested A. To detC, neither IFNβ-neutralizing antibodies nor control antibodies affect the effect of dsRNA on ESC viability. However, IFNβ-neutralizing antibodies, but not control antibodies, significantly reduced the cytotoxicity of dsGFP and dsLuc on D−/−ESCs (C). Similar results were observed when the cells were transfected with polyIC or B2RNA (data not shown). These results suggested that IFNβ contributes to the cytotoxicity only in D−/−ESCs in which it can be produced, but not in ESCs that are deficient in expressing this cytokine. Together, these data suggest that both PKR activation and IFNβ production contribute to the cytotoxicity associated with antiviral responses in D−/−ESCs.We have previously reported that IFNα or IFNβ alone does not have detectable effects on proliferation and viability of ESCs , 26 or DFigure 7D−/−ESCs C. SimilaUsing GFP expressed from its synthetic mRNA as an siRNA target, we detected Dicer-dependent RNAi activity in ESCs, a conclusion similar to the results reported in a study using GFP expressed from a plasmid . HoweverSince the deficiency in expressing type I IFN is a common feature of all types of pluripotent cells, it appears that this is an intrinsic feature inherently related to the pluripotent state . HoweverThe constitutively activated PKR in D−/−ESCs may have several biological implications. The PKR pathway is functional in ESCs and is activated during mitosis by cellular dsRNA in a highly regulated manner . We prevWhile the molecular mechanisms underlying how D−/−ESCs have gained the ability to produce type I IFN remain to be determined, we can logically assume that deficiency in miRNA biogenesis would be a primary reason. In particular, ESCs express a distinct set of miRNA, known as ESC-specific miRNA (ESC-miRNA), that are critical for the maintenance of the stem cell state . Althougvia phosphorylation of eIF2α  in our previous studies . Two paiin vitro transcription according to the methods that have been previously described  to generate 5′-capped GFP-mRNA transcripts. The purified RNA transcripts were polyadenylated by E. coli Poly(A) polymerase (New England Biolabs), resulting in functional mRNA, m7GpppA-GFP-polyA (designated as GFP-mRNA).Synthetic RNA were prepared by escribed . Brieflycription , 50, thecription , and thein vivo cloning method (To prepare double-stranded RNA (dsRNA), dsRNA containing 650 nucleotides that nearly encompass the entire length of GFP-mRNA and dsRNA containing 606 nucleotides of Gausia luciferase mRNA  were prepared by annealing the sense and antisense RNA transcribed from separate templates of the same sequences but with a T7 φ2.5 promoter in opposite directions, therefore having a perfect dsRNA structure. B2RNA is a 178 nucleotide RNA sequence corresponding to the B2 SINE that is abundantly distributed in the mouse genome . It can g method . Restricg method ESCs and D−/−ESCs were plated at 40 to 50% confluence and were usually cultured for 24 h before the experiments. Synthetic RNA were transfected into the cells with Endofectin Max (Genecopoeia) at 300 ng/ml for GFP-mRNA, B2RNA, and polyIC (Sigma-Aldrich) and 200 ng/ml for dsGFP and dsLuc, or at the concentrations specified in individual experiments. The imidazolo-oxindole PKR inhibitor  was used to inhibit PKR activity as previously described . To deteg for 15 min, and the supernatants were collected and used for the treatment of ESC-FBs. To analyze IFNβ secreted to the culture medium, ESCs and D−/−ESCs were transfected with dsGFP (1 μg/ml) in serum free medium with 0.2% BSA. After 24 h, the culture medium was collected, concentrated with a centrifugal concentrator (10 kd cutoff), and analyzed for IFNβ protein using a quantitative mouse ELISA kit (BioLegend) according to the manufacturer’s instruction.CM preparation was carried out with protocols previously described . Brieflyversus side scatter (FSC versus SSC) dot plots to exclude debris. Histograms that represent GFP expression levels were generated from median fluorescence intensities of analyzed samples with CFlow software (BD Biosciences) as previously described . The images were acquired with a digital camera mounted on the microscope. The cell population expressing GFP was quantitatively determined by flow cytometry with an Accuri C6 flow cytometer (BD Biosciences) . Cell gaescribed .The viability of ESCs and D−/−ESCs was determined by cell number and by cell morphology after toluidine blue (TB) staining. The absorbance at 630 nm of TB-stained cells was measured with a microtiter plate reader. The values, which correlate with the number of viable cells, were used as an indirect measurement of cell proliferation or viability. Cell cycle analysis was performed with an LSRFortessa flow cytometer (BD Biosciences) after the cells were stained with 50 μg/ml propidium iodide. The cell cycle profiles were generated using FlowJo software.siRNA targeting PKR and negative control siRNA (Santa Cruz Biotechnology) were transfected to the cells with Endofectin Max at a final concentration of 100 nM. The cells were then analyzed for siRNA knockdown efficiency and the effect on cell proliferation under the specified experimental conditions.Protein analysis by Western blot was performed according to our published method . The antTotal RNA was extracted using Tri-reagent (Sigma). cDNA was prepared by MMLV reverse transcriptase (Promega). RT-qPCR was performed using SYBR green ready mix on an MX3000PTM RT-PCR system (Stratagene), as previously reported . The mRNt-test. Data are presented as the mean ± SD under specified experimental conditions. Statistical differences are indicated by p-values. p < 0.05∗ was considered statistically significant. p values up to four significant digits are shown as specified in figure legends.Statistical analysis was performed with Microsoft Excel using a two-tailed and unpaired Student’s All data described in this study are either presented in the main article or in the supporting information.The authors declare that they have no conflicts of interest with the contents of this article.


Sponges are the oldest known extant animal-microbe symbiosis. These ubiquitous benthic animals play an important role in marine ecosystems in the cycling of dissolved organic matter (DOM), the largest source of organic matter on Earth. The conventional view on DOM cycling through microbial processing has been challenged by the interaction between this efficient filter-feeding host and its diverse and abundant microbiome. Here we quantify, for the first time, the role of host cells and microbial symbionts in sponge heterotrophy. We combined stable isotope probing and nanoscale secondary ion mass spectrometry to compare the processing of different sources of DOM  and particulate organic matter (POM) by a high-microbial abundance (HMA) and low-microbial abundance (LMA) sponge with single-cell resolution. Contrary to common notion, we found that both microbial symbionts and host choanocyte (i.e. filter) cells and were active in DOM uptake. Although all DOM sources were assimilated by both sponges, higher microbial biomass in the HMA sponge corresponded to an increased capacity to process a greater variety of dissolved compounds. Nevertheless, in situ feeding data demonstrated that DOM was the primary carbon source for both the LMA and HMA sponge, accounting for ~90% of their heterotrophic diets. Microbes accounted for the majority (65–87%) of DOM assimilated by the HMA sponge  but <5% in the LMA sponge. We propose that the evolutionary success of sponges is due to their different strategies to exploit the vast reservoir of DOM in the ocean. As the oldest extant animal phyla, sponges (phylum Porifera) have thrived on Earth for more than 600 million years , 2. ThesThe largest potential source of heterotrophic food in the oceans is dissolved organic matter (DOM) , but thiThe microbial communities associated with sponges are exceptionally diverse , formingAplysina aerophoba  bulk sponge tissue, (ii) separated sponge and microbial cell fractions, and (iii) single cells with subcellular resolution NanoSIMS imaging. SIP was complemented with in situ measurements of the natural sponge diet to determine the total heterotrophic contribution of sponge symbionts to the sponge diet.To quantify host and symbiont contributions to sponge DOM uptake, we performed SIP experiments to compare the processing of three dissolved food sources  and one particulate food source (heterotrophic bacteria) in the HMA sponge oba Fig.  and LMA ara Fig. . These tant Fig.  and moreant Fig.  microbiaAplysina aerophoba and Dysidea avara (n = 20) were collected by SCUBA from the coast of Girona, Spain  at depths of 3–15 m during April and May 2017. Sponges were transferred to the aquaria facilities at the Institute of Marine Sciences (ICM-CSIC) in Barcelona and maintained in individual 6 L aquaria supplied with fresh flowing seawater at a rate of ~30 L h−1. Each sponge individual was divided into five fragments of a similar size, each with a single fully functional osculum, and attached to PVC plates. Sponges were acclimated for 5 days and only healthy, actively pumping individuals were used in experiments.Specimens of 13C-glucose, 13C- and 15N-amino acids, 13C- and 15N-algal DOM) and one particulate food source (13C- and 15N-labeled bacteria). Details of the preparation of the four food sources are described in the −1 C, approximately equivalent to the background concentrations of DOC in the surrounding seawater (60–120 µmol L−1 C). Total amounts C and N added and enrichment of the four food sources is listed in Table n = 20 replicates per treatment). Control samples were collected before (n = 4) and after (n = 4) the experiments. The pulse-chase design was conducted to test for potential translocation of C and N from symbiont to host cells. However, since no significant differences in host cell enrichment were detected in the chase samples  were rinsed in filtered seawater followed by a brief rinse with MQ to remove excess salt and frozen at −80 °C. The remainder of the sponge tissue was cut into small pieces and placed in ice-cold calcium- and magnesium-free artificial seawater with 10% EDTA at 4 °C for separation into sponge cell and microbial cell fractions (details in Sponge tissue samples were collected for (1) stable isotope analysis of bulk sponge tissue, (2) stable isotope analysis of separated sponge and microbial cell fractions, and (3) SEM and NanoSIMS. Samples for SEM and NanoSIMS were sampled with a 2-mm tissue biopsy punch and immediately fixed in 4% paraformaldehyde in PBS buffer for 12 h at 4 °C and then transferred to PBS and stored at 4 °C until further processing. Samples for isotope analysis of the bulk sponge tissue  and atmospheric nitrogen for N (Rref = 0.00368 N). Bulk tissue uptake rates presented in Fig. Bulk tissue samples were lyophilized, homogenized, and sub-samples weighed into silver (C) and tin (N) cups for stable isotope analysis of δN = 56). Purity of cell fractions was >99% for the microbial cell fractions and >85% for the sponge cell fractions . Cell counts indicated that >99.5% of the microbial cells were effectively captured on the filters. The C and N content of the filters was divided by the number of cells filtered to calculate the mean C and N content per sponge and microbial cell for each of the two species. The mean C and N contents were multiplied by the total number of sponge and microbial cells in the initial homogenate (pre-separation) to calculate the percent biomass of sponge and microbial C and N in the two sponge species.To determine C and N contents of the sponge and microbial cells for biomass calculations, known volumes of the separated cell fractions were filtered onto a pre-combusted GF/F filter for elemental CN analysis  were used to calculate the percent contributions of host cells and microbial symbionts to the total uptake rates of the three dissolved food sources shown in Fig. The C/N biomass of sponge and microbial cells and the Fixed tissue samples from the pulse-chase experiment were dehydrated in a series of ethanol and embedded in LR-white for correlative SEM and NanoSIMS. Ultrathin tissue sections (~120 nm) were mounted onto silicon wafers and stained with uranyl acetate and lead citrate before imaging on a Zeiss Gemini 500 field emission variable pressure SEM equipped with energy selective backscatter detector and secondary ion detector at 5 kv. Images with ~50 × 50 µm field-of-view containing cellular structures of interest were mapped for subsequent NanoSIMS analysis.13C and 15N enrichment in the two sponges at single-cell resolution, the selected areas mapped by SEM were analyzed for 13C and 15N enrichment using a NanoSIMS 50 L ion probe (CAMECA). SEM sections on silicon wafers were gold-coated and bombarded with a 16 keV primary Cs+ ion beam focused to a spot size of ~120 nm. Raster scans  of the areas of interest were performed with a beam dwell time of 5 ms per pixel and repeated ten times. The secondary ions 12C2− (mass 24), 13C12C− (mass 25), 12C14N− (mass 26), and 12C15N− (mass 27) were simultaneously collected using electron multipliers at a mass resolution (M/ΔM) of 9000. Unlabeled sponges were measured twice daily as controls.To examine https://github.com/BWHCNI/OpenMIMS/wiki). Maps of 13C/12C and 15N/14N enrichment were obtained by taking the ratio of the drift corrected and stacked 13C12C− and 12C2− or 12C15N− and 12C14N− images, respectively. Quantification of isotope ratios in the different cell types was achieved by manually drawing regions of interest (ROIs) on the 12C14N-image using the corresponding SEM maps for reference. These SEM images were also used to identify the cell types present in the NanoSIMS images. The analysis focused on three main cell types (1) symbiont bacteria (0.2–2 µm), (2) host choanocyte cells (3–10 µm), and (3) all other host cells in the sponge mesophyll and pinacoderm, including ameobocytes, archeocytes, spherulous cells, and pinacocytes (7–30 µm). Hotspots of enrichment in the host choanocyte cells were extracted from the isotope ratio images separately. A total of 16 samples from the 3-h pulse time point were analyzed: two samples per species from each treatment (apart from the glucose treatment where enrichment levels were low and only one sample was measured) and two controls (one for each species). Approximately 4–8 images were obtained per sample in order to capture a minimum of n = 20 for each cell or ROI type, and in total 4354 ROIs were defined  were calculated as above replacing the reference values with the measured 13C/12C and 15N/14N ratios of the control samples .NanoSIMS data were processed using the ImageJ plugin OpenMIMS in Fiji  between 5 and 15 m water depth. Data for D. avara was taken from Morganti et al. ) −1 compared with 5 µmol C L−1 by the LMA sponge, the higher pumping rate in the LMA sponge actually resulted in an overall higher DOC uptake rate  higher symbiont densities and (2) slower pumping rates which increase the residence time of water in the sponge aquiferous system and therefore the contact time for the cells to access DOM . Howeverate Fig. . These aGeodia barretti are estimated to be sufficient to allow for a significant proportion of microbial-assimilated DOM to be transferred to the host [Microbes not only accounted for the majority of DOM uptake in the HMA sponge, but also accounted for more than half of the total heterotrophic C assimilated by the HMA sponge. For some sponge species, chemo- and photosynthetic microbes are known to provide the sponge host with autotrophically fixed C , 78–82, the host , which ithe host , 68, 84,the host , 86. AltWe quantitatively demonstrate that both microbial symbionts and host cells are actively involved in DOM processing, enabling both HMA and LMA sponges to effectively utilize DOM. The relative contribution of symbionts differs along the HMA–LMA dichotomy due to variations in microbial biomass; while microbes accounted for the majority of DOM uptake in the HMA sponge, they made a minimal contribution in the LMA sponge, which instead relied on uptake by host choanocyte cells. A complex interplay between DOM quality and quantity, host physiology, and symbiont abundance is likely to influence DOM uptake in marine sponges. We show that, similar to autotrophic symbionts, heterotrophic symbionts can play an important role in nutrient acquisition in marine sponges—accounting for more than half of the total C assimilated by the HMA sponge. Further studies are needed to establish the exact mechanisms by which DOM is taken up, whether the host can regulate symbiont access to DOM, and the degree to which assimilated C and N is exchanged between host and symbionts. The fact that both types of marine sponge have evolved different strategies to capitalize on DOM, the largest reservoir of organic carbon in the ocean, underscores its importance to the sponge diet.Supplemental Material


This enzyme, which belongs to the CaMKII family, is the predominant family member in the heart and functions as a messenger toward adaptive or detrimental signaling in cardiomyocytes. Classically, the nuclear CaMKIIδB and cytoplasmic CaMKIIδC splice variants are described as mediators of arrhythmias, contractile function, Ca2+ handling, and gene transcription. Recent findings also put CaMKIIδA and CaMKIIδ9 as cardinal players in the global CaMKII response in the heart. In this review, we discuss and summarize the new insights into CaMKIIδ splice variants and their (proposed) functions, as well as CaMKII-engineered mouse phenotypes and cardiac dysfunction related to CaMKIIδ missplicing. We also discuss RNA splicing factors affecting CaMKII splicing. Finally, we discuss the translational perspective derived from these insights and future directions on CaMKIIδ splicing research in the healthy and diseased heart.RNA splicing has been recognized in recent years as a pivotal player in heart development and disease. The Ca Heart disease is a major cause of death around the world, but the mechanisms underlying its development are not completely understood . In receIn the heart, alternative splicing participates in pre- and postnatal development, as well as in the development and progression of heart disease . The funTTN gene, is a sarcomeric protein that acts as a scaffolding filament, a signaling platform, and is a principal regulator of contraction in striated muscle. Mutations located in the RBM20 gene are linked to Titin missplicing toward a larger isoform termed N2BA-Giant, both in animal models and human dilated cardiomyopathy (2+ channel ryanodine receptor type 2 (RYR2), the L-type Ca2+ channel (LTCC) alpha 1C subunit (CACNA1C/Cav1.2), and the Ca2+/calmodulin-dependent protein kinase II delta (CAMK2δ). Missplicing of these genes may contribute to the severity of the cardiomyopathy  in the heart, from the CaMKIIδB and CaMKIIδC isoform to the CaMKIIδA and CaMKIIδ9 isoform  belongs to the multifunctional CaMK family. In mammals, CaMKs are divided into 3 classes: CaMKI, CaMKII, and CaMKIV; which in total comprise more than 27 proteins  maladaptive and detrimental effects on cardiac integrity , a regulatory segment (exons 11–12), a variable linker (exons 13–19), and a hub domain  gene reporter as well as Anf protein expression  and RyR2 (Ser-2814) phosphorylation  increases both SR Cad by TAC . Notablyd by TAC . These efunction . Howeverfunction . This su2+ handling defects seen in transgenic mice overexpressing CaMKIIδC, which is in line with the hypothesis that the decreased SR Ca2+ uptake through SERCA underlies the Ca2+ handling defects. However, while the SR Ca2+content and Ca2+ transient amplitude were normalized, left ventricular dilation, ventricular function, apoptosis, and mortality were exacerbated  attenuates the Cacerbated . This capoptosis . On the poptosis .2+ handling are distinct from the effects of CaMKIIδB overexpression, but overexpression of both splice variants similarly leads to HDAC4 phosphorylation (Ser-632), which inhibits nuclear import, and subsequently induces MEF2 transcriptional activity , which is the transmembrane protein that allows the passage of Ca2+ from cytoplasm into the inner mitochondria  opening, triggering programmed cell death . ASOs in their original form are short, single-stranded, synthetically produced oligodeoxynucleotides that bind to mRNA targets in a complementary manner and thus modulate mRNA and protein expression . The firin vivo, since ASOs circulate in the bloodstream after intravenous or subcutaneous injection and can accumulate in the liver, kidney or spleen  its levels are upregulated in both experimental models of heart failure and human samples which suggests a causal role for increased CaMKIIδ activation in disease, (2) the transgenic overexpression of CaMKIIδA, -δC and -δ9 leads to adverse cardiac remodeling accompanied by contractile dysfunction, and most importantly (3) CaMKIIδ inhibition is generally associated with protection against cardiac dysfunction and a reduction in cardiac damage in animal models subjected to detrimental stimuli. Nevertheless, the importance of CaMKIIδ in physiological processes, as well as the proposed protective effects of, e.g., CaMKIIδB suggest that CaMKIIδ-directed therapeutics require high specificity [For further details see ].2+ in the heart came from work of Ringer in the nineteenth century who accidentally used tap water instead of distilled water in his NaCl solution, and then found out that Ca2+ was necessary for contraction  studies for decades  could clarify why these isoforms exert different and/or redundant functions. Moreover, the 4 isoforms described in this review are not the only CaMKIIδ splice isoforms expressed in the heart. Even though less highly expressed, other splice isoforms such as CaMKIIδ4 are also found in the heart, and the function and relevance of these isoforms has not been explored. The fact that CaMKIIδ9, after being overlooked for a long time, turns out to be one of the highest expressed isoforms with a relevant function in the heart, illustrates that lesser known isoforms should not be neglected (The first report of the role of Catraction . Since t decades . These s decades . Based o decades  are only decades . It wouleglected . AnotherAll authors contributed to writing and revising the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Southern India lies in an area of Gondwana where multiple blocks are juxtaposed along Moho-penetrating structures, the significance of which are not well understood. Adequate geochronological data that can be used to differentiate the various blocks are also lacking. We present a newly acquired SIMS U–Pb, Lu–Hf, O isotopic and trace element geochemical dataset from zircon and garnet from the protoliths of the Nagercoil Block at the very tip of southern India. The data indicate that the magmatic protoliths of the rocks in this block formed at c. 2040 Ma with Lu–Hf, O-isotope and trace element data consistent with formation in a magmatic arc environment. The zircon data from Nagercoil Block are isotopically and temporally distinct from those in all the other blocks in southern India, but remarkably correspond to rocks in East Africa that are exposed on the southern margin of the Tanzania–Bangweulu Block. The new data suggest that the tip of southern India has an African affinity and a major suture zone must lie along its northern margin. All of these blocks were finally brought together during the Ediacaran-Cambrian amalgamation of Gondwana where they underwent high to ultrahigh temperature metamorphism. However, the southernmost tip of India (the Nagercoil Block) remains an enigma, as it has been included as part of Proterozoic India3, with correlation and connection to Africa5, or Sri Lanka7.Southern peninsular India is the meeting place of two major strands of the East African Orogen (EAO) and has been referred to as the “Gondwana Junction” e.g. Santosh, et al.8 and Plavsa, et al.9 with Tucker, et al.2 and Boger et al.3). Secondly, this area forms one of the largest exposed regions of extreme crustal metamorphism found on the planet8, the drivers for which have been challenging to model11. Initial parameters in developing models to explain this process requires knowledge of the lithospheric framework, something that in the EAO is still unresolved. Here we present new SIMS U–Pb zircon, Lu–Hf and O isotopic data that strongly point to the pre-Gondwana provenance of the Nagercoil Block and constrain the lithospheric framework of this significant part of the Gondwana amalgam.Understanding the crustal framework and sequence of events that occurred during the final stages of Gondwana assembly in this region is important for a number of reasons. Firstly, considerable controversy surrounds the tectonic evolution of the India-Africa collision, the largest of the Gondwana-forming orogens  history of the Nagercoil Block rocks to be comparable to those recorded within the adjoining Madurai and Trivandrum Blocks27.The southern tip of India is dominated by massive charnockites  that previous studies have identified to be distinct from the adjoining Trivandrum and Madurai Blocks and is referred to as the Nagercoil Block16 Fig. . GeochroSamples were collected from three locations within the Nagercoil Block to constrain the age of magmatism and metamorphism. The field relationships, petrography, results for SHRIMP U–Pb, Lu–Hf and oxygen isotope zircon analyses and LA-ICPMS rare earth element (REE) compositions of zircon, garnet and orthopyroxene are reported below. Complete data sets are reported in Supplementary Tables The quarry at Arakakulam contains exposures dominated by garnet-absent charnockite and with subordinate garnet-bearing charnockite Fig. . The chaCharnockite bands are associated with ~20 m long metapelitic bands, charnockite proximal to the metapeltic bands and extending up to several metres from the contact contain higher modal proportions of garnet. The boundary between the metapelite raft and the charnockite is quite diffuse suggesting partial assimilation of metapelite during either the incorporation of the metapelite into the original granitic magma as a raft or during the high-temperature metamorphism and associated partial melting during the Neoproterozoic-Ediacaran. The garnet-enriched charnockite zone has a reddish color and extends up to ~10 m from the metapelite. At a distances greater than 10 m from the contact the rock is a homogeneous green-grey equigranular garnet-absent charnockite that shows no evidence of a pervasive foliation. At the eastern end of the quarry a sub-horizontal undeformed mafic dyke intrudes the charnockite. A summary of the field and petrographic relationships are shown in Fig. Two charnockite styles, a garnet-absent and garnet-bearing, occur at Kozokodupothai, with the garnet-absent variety being the dominant type. The charnockites are cross-cut by an extensive network of garnet-bearing leucogranites. One of these granites contains evidence for the development of incipient charnockite. A summary of the field and petrographic relationships are shown in Fig. 207Pb/206Pb average age of the least discordant analyses (<10% discordant) of 2039 ± 20 Ma . A single analysis at the rim of a zircon from this sample gave a 206Pb/238U age of 515 ± 12 Ma that is within uncertainty of the lower intercept age of 550 ± 60 Ma  were analysed at this location. Zircon grains from the garnet bearing charnockite (IND12–004B) yielded an upper intercept age of 2038 ± 45 Ma  was sampled at this locality. Analyses of the oscillatory-zoned cores of zircon from the charnockite yield a discordia with an upper intercept of 2027 ± 41 Ma and a lower intercept of 585 ± 17 Ma Fig.  (MSWD = Three samples (two garnet-leucogranites one with patchy charnockitisation and a garnet-bearing charnockite) were analysed at this location. The garnet leucogranites (IND12-006B and IND12-006C) both gave discordant arrays of analyses with poorly defined upper intercept ages of 2012 ± 53 Ma and 2029 ± 65 Ma and lower intercept ages of 571 ± 31 Ma and 536 ± 48 Ma Fig. . There wDM and TDMC, the latter assumes derivation of magma from average continental crust28. The evolution of Lu-Hf in a closed system zircon will be different to that in a piece of crust due to the differing proportions of these elements. On Fig. Hafnium isotopic analyses were carried out on <10% discordant zircon grains and the results are presented in Supplementary Table Hafnium isotopic data from charnockites and leucogranites throughout the Nagercoil Block yield two distinct populations in εHf versus U-Pb age space Fig.  that refSIMS oxygen isotope analyses of zircon from all samples falls in the range 7 to 8 per mill (V-SMOW)  from these rocks are significantly more evolved than the Nagercoil Block data , then with Neoproterozoic India.Considerable controversy surrounds the tectonic framework of this key orogen in the Gondwana amalgam. Fitzsimons and Hulscher8 and within the southern Madurai Block/Achancovil Zone1 with the remnants of the Neoproterozoic ocean-basin sediments and associated magmatism preserved33. In addition, the rocks of the Palghat-Cauvery shear zone contain evidence of Neoproterozoic high-pressure metamorphism35, interpreted ophiolitic rocks38 and has the geohysical characteristics of a mantle penetrating structure39. All of these observations are consistent with the PCSS representing a suture zone along which the remnants of the Mozambique ocean were consumed. In addition, these findings reinforce the notion that presented by various workers and summarized by Collins et al.8 on the detrital provenance of the Palaeoproterozoic sedimentary units that make up the bulk of the Trivandrum and Madurai Blocks are sourced from African protoliths. The Nagercoil Block could be considered the remnant African basement upon which these sediments were deposited.The data presented here demonstrate that southernmost India has a considerably greater pre-Gondwana affinity with East Africa, than any other block with a magmatic protolith in the central East African Orogen. The major implication of this link is that southernmost India (and Madagascar) is derived from pre-Gondwana Africa and a major strand of the Mozambique Ocean lay to the north-east of the Nagercoil Block. Potential sites of this suture lie in the Palghat-Cauvery shear zone, along the northern margin of the Madurai Block8 rather than in Madagascar e.g.3 or to the west of Madagascar31.The Nagercoil Block was part of the Congo-Tanzania-Bangweulu continent (Africa) that was subsequently welded to India during Gondwana amalgamation where it was metamorphosed to granulite-facies resulting in the formation of orthopyroxene (+/− garnet)-bearing gneisses. The African affinity of southernmost India requires a Neoproterozoic oceanic suture to lie within southern India e.g.Zircon was separated from crushed rock samples using traditional magnetic and methylene iodide heavy liquid separation techniques. Grains were hand picked and mounted in 25 mm diameter epoxy resin discs. Mounts were carbon coated for imaging on a Tescan MIRA3 scanning electron microscope (SEM) with zircon CL images taken at a working distance of 15 mm and using an accelerating voltage of 10 kV. For SHRIMP analyses the samples were coated with a thin membrane of gold that produced a resistivity of 10–15 Ω across the mount surface.40 and41 and are similar to those described by42 and43. For zircon analysis a 25–30 μm diameter spot was used, with a mass-filtered O2− primary beam of ~2 nA. Data for each spot were collected in sets of 6 scans through the mass range of 196Zr2O+, 204Pb+, Background, 206Pb+, 207Pb+, 208Pb+, 238U+, 248ThO+, 254UO+. The 206Pb/238U age standards used were BR266, a Sri Lankan gem zircon44, and Temora-2 a zircon grain separate45. The 207Pb/206Pb standard used to enable correction for instrument induced mass fractionation was OG1 zircon46. The common Pb correction was based on the measured 204Pb42. The correction formula for Pb/U fractionation is 206Pb+/238U+ = a(238U16O+/238U+)b 47 using the parameter values of45.U-Pb isotopes were analysed on the SHRIMP II at the John de Laeter Centre SHRIMP Facility, Curtin University, Perth, Western Australia. The analytical procedures for the Curtin consortium SHRIMP II have been described byExternal spot-to-spot errors on zircon U-Pb calibration sessions were <1% for both sessions, a minimum error of 1% was applied which reflects the long-term performance of the SHRIMP II facility. Uncertainty cited for individual spot analysis in the text and data tables include errors from counting statistics, the common-Pb correction, and the U–Pb calibration error based on reproducibility of U–Pb measurements of the standard, and are at the 2σ level. Uncertainties of weighted mean values for pooled analyses and upper and lower intercepts in the figures are at the 95% confidence level, with MSWD calculated for concordance and equivalence  and garnet (18%). Precision based on repeated analysis of standards is approximately 5–10%, with detection limits for REE in this study ranging from 0.1 to 0.5 ppm. Due to the depth of the laser ablation pit relative to those associated with SHRIMP analysis, several analyses had to be rejected as they intersected heterogeneous material and/or inclusions of other phases.Rare earth element (REE) analyses of zircon and garnet were performed at the Curtin University LA–ICP–MS facility using a Resonetics M-50 193 nm excimer laser with an Agilent 7700 mass spectrometer. Zircon was analysed in the grain separate mount used for SHRIMP analysis, while garnet was analysed in thin section. Beam diameter was 23 μm using a repetition rate of 5 Hz which produced a laser power density of ~3 J/cm176Lu on 176Hf was corrected by measurement of interference-free 175Lu, and using the invariant 176Lu/175Lu correction factor 1/40.02669 . Interference of 176Yb on 176Hf was corrected by measuring the interference-free 172Yb isotope, and using the 176Yb/172Yb ratio to obtain the interference-free 176Yb/177Hf ratio. The appropriate value of 176Yb/172Yb was determined through spiking of the JMC475 hafnium standard solution with ytterbium, and finding the value of 176Yb/172Yb (0.58669) required to yield the 176Hf/177Hf value for the un-spiked solution. The typical 2σ precision of the 176Hf/177Hf ratios is +0.00002, equivalent to +0.7 ɛHf unit.Hafnium isotope analyses were conducted on previously dated zircons mounted in epoxy resin using a New Wave/Merchantek LUV213 laser-ablation microprobe, attached to a Nu Plasma multi-collector inductively coupled plasma mass spectrometer (LA-MC-ICPMS). The analyses employed a beam diameter of ∼55 μm and a 5 Hz repetition rate which resulted in ablation pits typically 40–60 μm deep. The ablated sample material was transported from the laser cell to the ICP-MS torch by a helium carrier gas. Interference of 176Hf/177Hf value  are within 2 standard deviations of the recommended value (0.282522 ± 0.000042 (2σ); Griffin et al., 2007). Six analyses of the 91500 zircon standard analysed during this study indicated 176Hf/177Hf = 0.282320 ± 0.000021 (2σ), which is well within the range of values reported by Griffin et al. (2006).Thirty zircons from the Mud Tank carbonatite locality were analysed, together with the samples, as a measure of the accuracy of the results. Most of the data and the mean 176Hf/177Hf is based on the 176Lu decay constant of Scherer et al.  and ɛHf values employed the present day chondritic measurement of Blichert-Toft and Albarède, . Calculation of model ages (TDM) is based on a depleted-mantle source with (176Hf/177Hf)i = 0.279718 at 4.56 Ga and 176Lu/177Hf = 0.0384 . TDM  ages were calculated assuming that the Hf within each zircon resided within a reservoir with 176Lu/177Hf ratio of 0.015, corresponding to an average Continental Crust28 .Calculation of initial 18O/16O) were determined using a Cameca IMS 1280 multi-collector ion microprobe located at the Centre for Microscopy, Characterisation and Analysis (CMCA), University of Western Australia (UWA). Oxygen isotope analyses were performed with a ca. 3 nA Cs+ beam with an impact energy of 20 keV focused to a 10–15 µm spot on the sample surface. Instrument parameters included a magnification of 130 × between the sample and field aperture (FA), 400 μm contrast aperture (CA), 4000 μm FA, 110 μm entrance slit, 500 μm exit slits, and a 40 eV band pass for the energy slit with a 5 eV gap toward the high energy side. Secondary O− ions were accelerated to 10 keV and analyzed with a mass resolving power of approximately 2400 using dual Faraday Cup detectors. A normal-incidence electron gun was used to provide charge compensation and NMR regulation was used for magnetic field control.Oxygen isotope ratios  was corrected using Temora II following the procedure described in Kita, et al.53). The spot-to-spot reproducibility  was better than 0.3‰ (2 SD) on Temora II during the analytical session. Propagated uncertainty on each δ18O spot has been calculated by propagating the errors on instrumental mass fractionation determination, including the error on the reference value of the standard, and internal error on each sample data point. The resulting uncertainty was typically between 0.2 and 0.3‰ (2 SD).Ten seconds of pre-sputtering was followed by automatic centering of the secondary beam in the FA and CA. Each analysis consisted of 20 four-second cycles, which gave an average internal precision of 0.2‰ (2 SE). Analytical sessions were monitored in terms of drift and precision using at least four bracketing standards (Temora II; 8.2‰Supplementary Tables