Contiguous Silver(I)-Mediated Base Pairs of Imidazophenanthroline and Canonical Nucleobases in DNA Duplexes: Formation of Classical Duplexes versus Homodimer Formation.

Metal-mediated base pairs represent a topical alternative to canonical hydrogen-bonded base pairs. In this context, the ligand 1H-imidazo[4,5-f][1,10]phenanthroline (P) was introduced as an artificial nucleobase in a glycol nucleic acid-based nucleoside analogue into a DNA oligonucleotide in a way that the oligonucleotide contains a central block of six contiguous P residues. The ability to engage in Ag+-mediated base pairing was evaluated with respect to the four canonical nucleosides in positions complementary to P. Highly stabilizing Ag+-mediated base pairs were formed with cytosine and guanine (i.e., P-Ag+-C and P-Ag+-G base pairs), whereas the analogous base pairs with thymine and adenine were much less stable and hence formed incompletely. Surprisingly, the intermediate formation of a homodimeric duplex of the P-containing oligonucleotide was observed in all cases, albeit to a different extent. The homodimer is composed of P-Ag+-P base pairs and 18 overhanging mismatched canonical nucleobases. It demonstrates the obstacles present when designing metal-mediated base pairs as metal complexation may take place irrespective of the surrounding natural base pairs. Homodimer formation was found to be particularly prominent when the designated metal-mediated base pairs are of low stability, suggesting that homodimers and regular duplexes are formed in a competing manner.

Endoplasmic reticulum calnexins participate in the primary root growth response to phosphate deficiency.

Accumulation of incompletely folded proteins in the endoplasmic reticulum (ER) leads to ER stress, activates ER protein degradation pathways, and upregulates genes involved in protein folding. This process is known as the unfolded protein response (UPR). The role of ER protein folding in plant responses to nutrient deficiencies is unclear. We analyzed Arabidopsis (Arabidopsis thaliana) mutants affected in ER protein quality control and established that both CALNEXIN (CNX) genes function in the primary root response to phosphate (Pi) deficiency. CNX1 and CNX2 are homologous ER lectins promoting protein folding of N-glycosylated proteins via the recognition of the GlcMan9GlcNAc2 glycan. Growth of cnx1-1 and cnx2-2 single mutants was similar to that of the wild type under high and low Pi conditions, but the cnx1-1 cnx2-2 double mutant showed decreased primary root growth under low Pi conditions due to reduced meristematic cell division. This phenotype was specific to Pi deficiency; the double mutant responded normally to osmotic and salt stress. Expression of CNX2 mutated in amino acids involved in binding the GlcMan9GlcNAc2 glycan failed to complement the cnx1-1 cnx2-2 mutant. The root growth phenotype was Fe-dependent and was associated with root apoplastic Fe accumulation. Two genes involved in Fe-dependent inhibition of primary root growth under Pi deficiency, the ferroxidase LOW PHOSPHATE 1 (LPR1) and P5-type ATPase PLEIOTROPIC DRUG RESISTANCE 2 (PDR2) were epistatic to CNX1/CNX2. Overexpressing PDR2 failed to complement the cnx1-1 cnx2-2 root phenotype. The cnx1-1 cnx2-2 mutant showed no evidence of UPR activation, indicating a limited effect on ER protein folding. CNX might process a set of N-glycosylated proteins specifically involved in the response to Pi deficiency.

Two-center validation of assays for the detection of binding and neutralizing anti-factor VIII antibodies.

INTRODUCTION: Patients with hemophilia A treated with coagulation Factor VIII (FVIII) products are at risk for developing anti-FVIII antibodies. The ABIRISK Consortium aimed to provide knowledge on the formation and detection of anti-drug antibodies against biopharmaceutical products, including FVIII. Accordingly, standardized and validated assays for the detection of binding (total) and neutralizing antibodies are needed. AIM: Two-center validation of an ELISA for the detection of total FVIII-binding IgG-antibodies and Nijmegen-Bethesda assays for the quantification of FVIII-neutralizing antibodies according to consensus validation guidelines. METHODS: Validation of assays at both sites was done according to published recommendations and included preanalytics, the determination of key assay parameters, including cut-points, assay sensitivity, precision, and FVIII interference. RESULTS: The validated assays reproducibly detected FVIII-binding and -neutralizing antibodies with comparable performance in both laboratories. Floating screening cut-points were established for both assays. Determined mass-based sensitivity of both assays (all values ≤66 ng/mL) complied with the minimum sensitivity for the detection of anti-drug antibodies as recommended by the FDA (<100 ng/mL). Intra- and inter-assay coefficients of variation did not exceed 25%. Assay validation further revealed that pre-analytical heat treatment led to potentially false-positive ELISA results, while up to 0.15 IU/mL, residual FVIII showed no significant impact. Overall, good agreement of results was found for patient samples analyzed at both study sites. CONCLUSION: Comprehensive validation of different anti-FVIII-antibody assays in two laboratories gave novel insights into the impact of pre-analytical sample treatment as well as the comparability of test results generated by the use of methodically different assays.

Ex Vivo Modeling of the PC (Protein C) Pathway Using Endothelial Cells and Plasma: A Personalized Approach.

BACKGROUND: The endothelial cell-dependent PC (protein C) pathway is critically involved in the regulation of coagulation, anti-inflammatory, and cytoprotective signaling. Its reactivity shows high interindividual variability, and it contributes to prothrombotic disorders, such as the FVL (factor V Leiden) mutation. METHODS: Endothelial colony-forming cells (ECFCs) were isolated from heparinized peripheral blood from healthy individuals and FVL carriers. Confluent monolayers of ECFCs were overlaid with plasma, and thrombin formation was initiated by addition of tissue factor (1 pmol/L). Subsequently, thrombin and APC (activated PC) formation rates were measured over time using oligonucleotide-based enzyme capture assays. To induce downregulation of TM (thrombomodulin) expression, ECFCs were stimulated with IL-1ß (interleukin 1ß). In vivo APC response rates were monitored in study participants after infusion of low-dose rFVIIa (recombinant activated factor VII). RESULTS: The median peak APC concentration was 1.12 nmol/L in experiments with IL-1ß stimulated ECFCs and 3.66 nmol/L without IL-1ß. Although thrombin formation rates were comparable, APC formation rates were significantly higher in FVL carriers (n=6) compared to noncarriers (n=5) as evidenced by a higher ratio between the area under the curve of APC generation to the area under the curve of thrombin generation (median 0.090 versus 0.031, P=0.017). These ex vivo results were correlated with an increased APC response to rFVIIa-induced thrombin formation in FVL carriers in vivo. CONCLUSIONS: Patient-specific ex vivo modeling of the PC pathway was achieved using blood-derived ECFCs. The correlation between in and ex vivo APC response rates confirms that the autologous PC model accurately depicts the in vivo situation.

6-Pyrazolylpurine and its deaza derivatives as nucleobases for silver(I)-mediated base pairing with pyrimidines.

The artificial nucleobase 6-pyrazolylpurine (6PP) and its deaza derivatives 1-deaza-6-pyrazolylpurine (1D6PP), 7-deaza-6-pyrazolylpurine (7D6PP), and 1,7-dideaza-6-pyrazolylpurine (1,7D6PP) were investigated with respect to their ability to differentiate between the canonical nucleobases cytosine and thymine by means of silver(I)-mediated base pairing. As shown by temperature-dependent UV spectroscopy and by circular dichroism spectroscopy, 6PP and (to a lesser extent) 7D6PP form stable silver(I)-mediated base pairs with cytosine, but not with thymine. 1D6PP and 1,7D6PP do not engage in the formation of stabilizing silver(I)-mediated base pairs with cytosine or thymine. The different behavior of 1D6PP, 7D6PP, and 1,7D6PP indicates that silver(I) binding occurs via the N1 position of the purine derivative, i.e. via the Watson-Crick face. The data show that 6PP is capable of differentiating between cytosine and thymine, which is potentially relevant in the context of detecting single-nucleotide polymorphisms.

Unexpectedly Large Structural Variations in Benzyl-Substituted Sila[1]ferrocenophanes.

A set of four planar-chiral sila[1]ferrocenophanes equipped with a benzyl group in the α-position, either on one or both Cp rings, and substituted on the bridging silicon atom, either by methyl or phenyl groups, were prepared. While NMR, UV/Vis, and DSC measurements did not show anything uncommon, single crystal X-ray analyses revealed unexpectedly large variations of the dihedral angles between both Cp rings (α tilt angle). While DFT calculations predicted α between 19.6 and 20.8°, measured values were found between 16.6(2) and 21.45(14)°. However, experimentally determined conformers differ significantly from those calculated for the gas phase. For the sila[1]ferrocenophane with the largest mismatch between the experimental and predicted α angle, it was shown that the orientation of benzyl groups have a significant influence on the ring-tilted structure. Packing of molecules in the crystal lattice forces benzyl groups into unusual orientations, resulting in a significantly reduced α angle through steric repulsions.

Sila[1]ruthenocenophanes: Long Sought-After Family Members.

During the last three decades, silicon-bridged [1]ferrocenophanes have been developed into an important class of monomers with the resulting polymers being the subject of hundreds of publications. For unknown reasons, not a single example of a silicon-bridged [1]ruthenocenophane is described in the literature. We set out to address this mystery and could synthesize the first sila[1]ruthenocenophanes. Their molecular structures as well as their heat release in the melt, render them as highly strained sandwich compounds that are polymerizable. Why did it take so long to prepare the first sila[1]ruthenocenophanes? This question is addressed by a detailed analysis of the kinetics of the salt-metathesis approach, which reveals a striking difference between ferrocene and ruthenocene.

Chiral-at-Metal Silver-Mediated Base Pairs: Metal-Centred Chirality versus DNA Helical Chirality.

When covalently incorporating ligands capable of forming chiral metal complexes into a DNA oligonucleotide duplex, an enantiospecific formation of metal-mediated base pairs is possible. We have been investigating the chirality of the silver-mediated base pair P-AgI -P (P, 1H-imidazo[4,5-f][1,10]phenanthroline) depending on the number of consecutive P : P pairs within a series of duplexes. Towards this end, both enantiomers of the nucleoside analogue 3-(1H-imidazo[4,5-f][1,10]phenanthrolin-1-yl)propane-1,2-diol comprising an acyclic backbone were introduced into DNA duplexes, resulting in diastereomeric metal-mediated base pairs. The same chiral-at-metal complex is formed inside the duplex for up to five neighbouring P-AgI -P pairs, irrespective of whether (S)-P or (R)-P is used. With six silver-mediated base pairs, the chirality of the metal complex is inverted for (S)-P but not for (R)-P. This indicates an intricate balance of what determines the configuration of the metal complex, the intrinsically preferred metal-centred chirality or the DNA helical chirality.

Open-Source Implementations of the Reactive Asset Administration Shell: A Survey.

The use of open-source software is crucial for the digitalization of manufacturing, including the implementation of Digital Twins as envisioned in Industry 4.0. This research paper provides a comprehensive comparison of free and open-source implementations of the reactive Asset Administration Shell (AAS) for creating Digital Twins. A structured search on GitHub and Google Scholar was conducted, leading to the selection of four implementations for detailed analysis. Objective evaluation criteria were defined, and a testing framework was created to test support for the most common AAS model elements and API calls. The results show that all implementations support at least a minimal set of required features while none implement the specification in all details, which highlights the challenges of implementing the AAS specification and the incompatibility between different implementations. This paper is therefore the first attempt at a comprehensive comparison of AAS implementations and identifies potential areas for improvement in future implementations. It also provides valuable insights for software developers and researchers in the field of AAS-based Digital Twins.

GGCX variants leading to biallelic deficiency to γ-carboxylate GRP cause skin laxity in VKCFD1 patients.

γ-Glutamyl carboxylase (GGCX) catalyzes the γ-carboxylation of 15 different vitamin K dependent (VKD) proteins. Pathogenic variants in GGCX cause a rare hereditary bleeding disorder called Vitamin K dependent coagulation factor deficiency type 1 (VKCFD1). In addition to bleedings, some VKCFD1 patients develop skin laxity and skeletal dysmorphologies. However, the pathophysiological mechanisms underlying these non-hemorrhagic phenotypes remain elusive. Therefore, we have analyzed 20 pathogenic GGCX variants on their ability to γ-carboxylate six non-hemostatic VKD proteins in an in vitro assay, where GGCX variants were expressed in GGCX-/- cells and levels of γ-carboxylated co-expressed VKD proteins were detected by a functional ELISA. We observed that GGCX variants causing markedly reduced γ-carboxylation of Gla rich protein (GRP) in vitro were reported in patients with skin laxity. Reduced levels of γ-carboxylated Matrix gla protein (MGP) are not exclusive for causing skeletal dysmorphologies in VKCFD1 patients. In silico docking of vitamin K hydroquinone on a GGCX model revealed a binding site, which was validated by in vitro assays. GGCX variants affecting this site result in disability to γ-carboxylate VKD proteins and hence are involved in the most severe phenotypes. This genotype-phenotype analysis will help to understand the development of non-hemorrhagic phenotypes and hence improve treatment in VKCFD1 patients.

Monoanionic C

A family of Pt(II) complexes bearing monoanionic C^N^N ligands as luminophoric units as well as a set of monodentate ligands derived from allenylidene and carbene species were synthesized and characterized in terms of structure and photophysical properties. In addition, we present the extraordinary molecular structure of a phosphorescent complex carrying an allenylidene ligand. Depending on the co-ligand, an effect can be observed in the photoluminescence lifetimes and quantum yields as well as in the radiative and radiation less deactivation rate constants. Their correlation with the substitution pattern was analyzed by comparing the photoluminescence in fluid solution at room temperature and in frozen glassy matrices at 77 K. Moreover, in order to gain a deeper understanding of the electronic states responsible for the optical properties, density functional theory calculations were performed. Finally, the cytotoxicity of the complexes was evaluated in vitro, showing that the cationic complexes exhibit strong effects at low micromolar concentrations. The calculated half-maximum effective concentrations (EC50 values) were 4 times lower in comparison to the established antitumor agent oxaliplatin. In contrast, the neutral species are less toxic, rendering them as potential bioimaging agents.

Modulating aptamer function by copper(II)-mediated base pair formation.

Two aptamers, one for ATP and one for arginine, were modified using an artificial 2'-dexoyribonucleoside based on the nucleobase surrogate imidazole-4-carboxylate. This synthetic nucleoside substitute does not engage in hydrogen bonding but is capable of forming Cu(II)-mediated base pairs instead. Hence, the addition of Cu(II) can be used to influence the ability of the aptamer derivatives to adopt the correct fold necessary for binding their respective target molecule. As a result, aptamer function can be modulated via the addition of Cu(II). The extent of modulation ability depends on the identity of the aptamer and on the exact location of the artificial nucleosides within the oligonucleotide sequence.

Metal-dependent electrochemical discrimination of DNA quadruplex sequences.

Films of four different DNA quadruplex-forming (G4) sequences (c-KIT, c-MYC, HTelo, and BCL2) on gold surfaces were investigated by electrochemical impedance spectroscopy (EIS) to evaluate whether they evoke unique electrochemical responses that can be used for their identification. This could render EIS an alternative means for the determination of G4 sequences of unknown structure. Towards, this end, cation-dependent topology changes in the presence of either K+, K+ in combination with Li+, or Pb2+ in the presence of Li+ were first evaluated by circular dichroism (CD) spectroscopy, and electrochemical studies were performed subsequently. As a result, G4-sequence specific charge transfer resistance (RCT) patterns were in fact observed for each G4 sequence, allowing their discrimination by EIS.

Antibody-mediated procoagulant platelets in SARS-CoV-2-vaccination associated immune thrombotic thrombocytopenia.

The COVID-19 pandemic has resulted in significant morbidity and mortality worldwide. To prevent severe infection, mass COVID-19 vaccination campaigns with several vaccine types are currently underway. We report pathological and immunological findings in 8 patients who developed vaccine-induced immune thrombotic thrombocytopenia (VITT) after administration of SARS-CoV-2 vaccine ChAdOx1 nCoV-19. We analyzed patient material using enzyme immune assays, flow cytometry and heparin-induced platelet aggregation assay and performed autopsies on two fatal cases. Eight patients (5 female, 3 male) with a median age of 41.5 years (range, 24 to 53) were referred to us with suspected thrombotic complications 6 to 20 days after ChAdOx1 nCoV-19 vaccination. All patients had thrombocytopenia at admission. Patients had a median platelet count of 46.5 x109/L (range, 8 to 92). Three had a fatal outcome and 5 were successfully treated. Autopsies showed arterial and venous thromboses in various organs and the occlusion of glomerular capillaries by hyaline thrombi. Sera from VITT patients contain high titer antibodies against platelet factor 4 (PF4) (OD 2.59±0.64). PF4 antibodies in VITT patients induced significant increase in procoagulant markers (P-selectin and phosphatidylserine externalization) compared to healthy volunteers and healthy vaccinated volunteers. The generation of procoagulant platelets was PF4 and heparin dependent. We demonstrate the contribution of antibody-mediated platelet activation in the pathogenesis of VITT.

Increased Activated Protein C Response Rates Reduce the Thrombotic Risk of Factor V Leiden Carriers But Not of Prothrombin 20210G>A Carriers.

RATIONALE: Carriers of the most common prothrombotic mutations FVL (factor V Leiden) and FII (prothrombin) 20210G>A show a highly variable clinical phenotype. Using standardized in vivo coagulation activation followed by activity pattern analysis we have recently shown, that the FVL mutation accelerates thrombin and APC (activated protein C) formation in carriers without a history of venous thromboembolism (VTE). OBJECTIVE: The aim of this prospective cohort study was to investigate, if the FII 20210G>A mutation induces a similar reaction pattern, and if the response rates differ in FVL and FII 20210G>A mutation carriers with prior VTE (VTE+). METHODS AND RESULTS: We comparatively analyzed 30 FVL carriers, 28 FII 20210G>A carriers (thereof 13 VTE+ each) and 15 healthy controls. Changes in plasma levels of thrombin, prothrombin activation fragment 1+2 (F1+2), TAT (thrombin-antithrombin complex), APC, and D-dimer were monitored over 8 hours after infusion of recombinant factor VIIa (15 µg/kg). An increase of F1+2 and TAT levels was observed, that did neither differ between FVL and FII 20210G>A carriers nor between asymptomatic and VTE+ carriers of these mutations. Median plasma levels of APC increased more (P=0.008) in FVL carriers (from 1.39 to 7.79 pmol/L) than in FII 20210G>A carriers (from 1.03 to 5.79 pmol/L), and more in FII 20210G>A carriers (P=2×10-4) than in healthy controls (from 0.86 to 3.00 pmol/L). Most importantly, however, the APC response was greater (P=0.015) in asymptomatic (n=13) than in VTE+ (n=12) heterozygous FVL carriers, with an increase of APC levels from 1.44 to 8.11 pmol/L versus 1.27 to 5.62 pmol/L. CONCLUSIONS: These in vivo data demonstrate that the FII 20210G>A and FVL mutations share an intermediate phenotype that is characterized by increased thrombin formation after coagulation activation. Furthermore, our data support the conclusion that the APC activating capacity of FVL carriers modifies the thrombotic risk of this common prothrombotic mutation.

Triggering Copper(II)-Mediated Base Pair Formation by Light.

Metal-mediated base pairs enable a site-specific incorporation of transition metal ions into nucleic acid structures. The resulting nucleic acid-metal complex conjugates are of interest in the context of functionalized nucleic acids, as they bear metal-based functionality. It is desirable to devise nucleic acids with an externally triggered metal-binding affinity, as this may allow regulating this functionality. Toward this end, a caged deoxyribonucleoside analog HNPP was devised for the site-specific binding of copper(II) ions upon irradiation by light, based on the ligand 3-hydroxy-2-methylpyridin-4(1H)-one (H) and the photocleavable 2-(2-nitrophenyl)propoxy protecting group (NPP). The formation of both H-Cu(II)-H homo base pairs and H-Cu(II)-X hetero base pairs (involving a second artificial deoxyribonucleoside X, based on imidazole-4-carboxylate) was achieved upon irradiation of DNA duplexes bearing the respective HNPP:HNPP or HNPP:X mispairs in the presence of copper(II) ions. The H-Cu(II)-X pair shows an exceptional DNA duplex stabilization of up to 43 °C upon its formation, exceeding that of the H-Cu(II)-H pair. It therefore represents one of the most stabilizing Cu(II)-mediated base pairs reported so far. Our findings expand the scope of light-triggered metal-mediated base pair formation by introducing a copper(II)-binding ligand.

Functional Characterization of Antithrombin Mutations by Monitoring of Thrombin Inhibition Kinetics.

Inactivation of thrombin by the endogenous inhibitor antithrombin (AT) is a central mechanism in the regulation of hemostasis. This makes hereditary AT deficiency, which is caused by SERPINC1 gene mutations, a major thrombophilic risk factor. Aim of this study was to assess to what extent AT mutations impair thrombin inhibition kinetics. The study population included 36 thrombophilic patients with 19 different mutations and mean AT levels of 65% in a thrombin-based functional assay, and 26 healthy controls. To assess thrombin inhibition kinetics, thrombin (3.94 mU/mL final concentration) was added to citrated plasma. Subsequently, endogenous thrombin inhibition was stopped by addition of the reversible thrombin inhibitor argatroban and the amount of argatroban-complexed thrombin quantified using an oligonucleotide-based enzyme capture assay. The plasma half-life of human thrombin was significantly longer in patients with AT mutations than in the controls (119.9 versus 55.9 s). Moreover, it was disproportionately prolonged when compared with preparations of wild type AT in plasma, in whom a comparable thrombin half-life of 120.8 s was reached at a distinctly lower AT level of 20%. These findings may help to better understand the increased thrombotic risk of SERPINC1 mutations with near normal AT plasma levels in functional assays.

Controlling Coagulation in Blood with Red Light.

Precise control of blood clotting and rapid reversal of anticoagulation are essential in many clinical situations. We were successful in modifying a thrombin-binding aptamer with a red-light photocleavable linker derived from Cy7 by Cu-catalyzed Click chemistry. We were able to show that we can successfully deactivate the modified aptamer with red light (660 nm) even in human blood-restoring the blood's natural coagulation capability.

Enzymatic Construction of Artificial Base Pairs: The Effect of Metal Shielding.

Th formation of metal base pairs is a versatile method for the introduction of metal cations into nucleic acids that has been used in numerous applications including the construction of metal nanowires, development of energy, charge-transfer devices and expansion of the genetic alphabet. As an alternative, enzymatic construction of metal base pairs is an alluring strategy that grants access to longer sequences and offers the possibility of using such unnatural base pairs (UBPs) in SELEX experiments for the identification of functional nucleic acids. This method remains rather underexplored, and a better understanding of the key parameters in the design of efficient nucleotides is required. We have investigated the effect of methylation of the imidazole nucleoside (dImnMe TP) on the efficiency of the enzymatic construction of metal base pairs. The presence of methyl substituents on dImTP facilitates the polymerase-driven formation of dIm4Me -AgI -dIm and dIm2Me TP-CrIII -dIm base pairs. Steric factors rather than the basicity of the imidazole nucleobase appear to govern the enzymatic formation of such metal base pairs. We also demonstrate the compatibility of other metal cations rarely considered in the construction of artificial metal bases by enzymatic DNA synthesis under both primer extension reaction and PCR conditions. These findings open up new directions for the design of nucleotide analogues for the development of metal base pairs.

The Local Phosphate Deficiency Response Activates Endoplasmic Reticulum Stress-Dependent Autophagy.

Inorganic phosphate (Pi) is often a limiting plant nutrient. In members of the Brassicaceae family, such as Arabidopsis (Arabidopsis thaliana), Pi deprivation reshapes root system architecture to favor topsoil foraging. It does so by inhibiting primary root extension and stimulating lateral root formation. Root growth inhibition from phosphate (Pi) deficiency is triggered by iron-stimulated, apoplastic reactive oxygen species generation and cell wall modifications, which impair cell-to-cell communication and meristem maintenance. These processes require LOW PHOSPHATE RESPONSE1 (LPR1), a cell wall-targeted ferroxidase, and PHOSPHATE DEFICIENCY RESPONSE2 (PDR2), the single endoplasmic reticulum (ER)-resident P5-type ATPase (AtP5A), which is thought to control LPR1 secretion or activity. Autophagy is a conserved process involving the vacuolar degradation of cellular components. While the function of autophagy is well established under nutrient starvation (C, N, or S), it remains to be explored under Pi deprivation. Because AtP5A/PDR2 likely functions in the ER stress response, we analyzed the effect of Pi limitation on autophagy. Our comparative study of mutants defective in the local Pi deficiency response, ER stress response, and autophagy demonstrated that ER stress-dependent autophagy is rapidly activated as part of the developmental root response to Pi limitation and requires the genetic PDR2-LPR1 module. We conclude that Pi-dependent activation of autophagy in the root apex is a consequence of local Pi sensing and the associated ER stress response, rather than a means for systemic recycling of the macronutrient.