Thalamic nucleus reuniens coordinates prefrontal-hippocampal synchrony to suppress extinguished fear.

Traumatic events result in vivid and enduring fear memories. Suppressing the retrieval of these memories is central to behavioral therapies for pathological fear. The medial prefrontal cortex (mPFC) and hippocampus (HPC) have been implicated in retrieval suppression, but how mPFC-HPC activity is coordinated during extinction retrieval is unclear. Here we show that after extinction training, coherent theta oscillations (6-9 Hz) in the HPC and mPFC are correlated with the suppression of conditioned freezing in male and female rats. Inactivation of the nucleus reuniens (RE), a thalamic hub interconnecting the mPFC and HPC, reduces extinction-related Fos expression in both the mPFC and HPC, dampens mPFC-HPC theta coherence, and impairs extinction retrieval. Conversely, theta-paced optogenetic stimulation of RE augments fear suppression and reduces relapse of extinguished fear. Collectively, these results demonstrate a role for RE in coordinating mPFC-HPC interactions to suppress fear memories after extinction.

Stable binding to phosphatidylserine-containing membranes requires conserved arginine residues in tandem C domains of blood coagulation factor VIII.

At sites of vascular damage, factor VIII (fVIII) is proteolytically activated by thrombin and binds to activated platelet surfaces with activated factor IX (fIXa) to form the intrinsic "tenase" complex. Previous structural and mutational studies of fVIII have identified the C1 and C2 domains in binding to negatively charged membrane surfaces through ß-hairpin loops with solvent-exposed hydrophobic residues and a ring of positively charged basic residues. Several hemophilia A-associated mutations within the C domains are suggested to disrupt lipid binding, preventing formation of the intrinsic tenase complex. In this study, we devised a novel platform for generating recombinant C1, C2, and C1C2 domain constructs and performed mutagenesis of several charged residues proximal to the putative membrane binding region of each C domain. Binding measurements between phosphatidylserine (PS)-containing lipid membrane surfaces and fVIII C domains demonstrated an ionic strength dependence on membrane binding affinity. Mutations to basic residues adjacent to the surface-exposed hydrophobic regions of C1 and C2 differentially disrupted membrane binding, with abrogation of binding occurring for mutations to conserved arginine residues in the C1 (R2163) and C2 (R2320) domains. Lastly, we determined the X-ray crystal structure of the porcine fVIII C2 domain bound to o-phospho-L-serine, the polar headgroup of PS, which binds to a basic cleft and makes charge-charge contact with R2320. We conclude that basic clefts in the fVIII C domains bind to PS-containing membranes through conserved arginine residues via a C domain modularity, where each C domain possesses modest electrostatic-dependent affinity and tandem C domains are required for high affinity binding.

Structural insights into blood coagulation factor VIII: Procoagulant complexes, membrane binding, and antibody inhibition.

Advances in structural studies of blood coagulation factor VIII (FVIII) have provided unique insight into FVIII biochemistry. Atomic detail models of the B domain-deleted FVIII structure alone and in complex with its circulatory partner, von Willebrand factor (VWF), provide a structure-based rationale for hemophilia A-associated mutations which impair FVIII stability and increase FVIII clearance rates. In this review, we discuss the findings from these studies and their implications toward the design of a recombinant FVIII with improved circulatory half-life. Additionally, we highlight recent structural studies of FVIII bound to inhibitory antibodies that have refined our understanding of FVIII binding to activated platelet membranes and formation of the intrinsic tenase complex. The combination of bioengineering and structural efforts to understand FVIII biochemistry will improve therapeutics for treating hemophilia A, either through FVIII replacement therapeutics, immune tolerance induction, or gene therapy approaches.

SAXS analysis of the intrinsic tenase complex bound to a lipid nanodisc highlights intermolecular contacts between factors VIIIa/IXa.

The intrinsic tenase (Xase) complex, formed by factors (f) VIIIa and fIXa, forms on activated platelet surfaces and catalyzes the activation of factor X to Xa, stimulating thrombin production in the blood coagulation cascade. The structural organization of the membrane-bound Xase complex remains largely unknown, hindering our understanding of the structural underpinnings that guide Xase complex assembly. Here, we aimed to characterize the Xase complex bound to a lipid nanodisc with biolayer interferometry (BLI), Michaelis-Menten kinetics, and small-angle X-ray scattering (SAXS). Using immobilized lipid nanodiscs, we measured binding rates and nanomolar affinities for fVIIIa, fIXa, and the Xase complex. Enzyme kinetic measurements demonstrated the assembly of an active enzyme complex in the presence of lipid nanodiscs. An ab initio molecular envelope of the nanodisc-bound Xase complex allowed us to computationally model fVIIIa and fIXa docked onto a flexible lipid membrane and identify protein-protein interactions. Our results highlight multiple points of contact between fVIIIa and fIXa, including a novel interaction with fIXa at the fVIIIa A1-A3 domain interface. Lastly, we identified hemophilia A/B-related mutations with varying severities at the fVIIIa/fIXa interface that may regulate Xase complex assembly. Together, our results support the use of SAXS as an emergent tool to investigate the membrane-bound Xase complex and illustrate how mutations at the fVIIIa/fIXa dimer interface may disrupt or stabilize the activated enzyme complex.

Structure of Blood Coagulation Factor VIII in Complex With an Anti-C2 Domain Non-Classical, Pathogenic Antibody Inhibitor.

Factor VIII (fVIII) is a procoagulant protein that binds to activated factor IX (fIXa) on platelet surfaces to form the intrinsic tenase complex. Due to the high immunogenicity of fVIII, generation of antibody inhibitors is a common occurrence in patients during hemophilia A treatment and spontaneously occurs in acquired hemophilia A patients. Non-classical antibody inhibitors, which block fVIII activation by thrombin and formation of the tenase complex, are the most common anti-C2 domain pathogenic inhibitors in hemophilia A murine models and have been identified in patient plasmas. In this study, we report on the X-ray crystal structure of a B domain-deleted bioengineered fVIII bound to the non-classical antibody inhibitor, G99. While binding to G99 does not disrupt the overall domain architecture of fVIII, the C2 domain undergoes an ~8 Å translocation that is concomitant with breaking multiple domain-domain interactions. Analysis of normalized B-factor values revealed several solvent-exposed loops in the C1 and C2 domains which experience a decrease in thermal motion in the presence of inhibitory antibodies. These results enhance our understanding on the structural nature of binding non-classical inhibitors and provide a structural dynamics-based rationale for cooperativity between anti-C1 and anti-C2 domain inhibitors.

Structure of blood coagulation factor VIII in complex with an anti-C1 domain pathogenic antibody inhibitor.

Antibody inhibitor development in hemophilia A represents the most significant complication resulting from factor VIII (fVIII) replacement therapy. Recent studies have demonstrated that epitopes present in the C1 domain contribute to a pathogenic inhibitor response. In this study, we report the structure of a group A anti-C1 domain inhibitor, termed 2A9, in complex with a B domain-deleted, bioengineered fVIII construct (ET3i). The 2A9 epitope forms direct contacts to the C1 domain at 3 different surface loops consisting of Lys2065-Trp2070, Arg2150-Tyr2156, and Lys2110-Trp2112. Additional contacts are observed between 2A9 and the A3 domain, including the Phe1743-Tyr1748 loop and the N-linked glycosylation at Asn1810. Most of the C1 domain loops in the 2A9 epitope also represent a putative interface between fVIII and von Willebrand factor. Lastly, the C2 domain in the ET3i:2A9 complex adopts a large, novel conformational change, translocating outward from the structure of fVIII by 20 Å. This study reports the first structure of an anti-C1 domain antibody inhibitor and the first fVIII:inhibitor complex with a therapeutically active fVIII construct. Further structural understanding of fVIII immunogenicity may result in the development of more effective and safe fVIII replacement therapies.

Evidence for water deficit-induced mass increases of raffinose family oligosaccharides (RFOs) in the leaves of three Craterostigma resurrection plant species.

The leaves of the resurrection plant Craterostigma plantagineum accumulate sucrose during dehydration, via a conversion from the unusual C8 ketose-sugar 2-octulose. However, raffinose family oligosaccharides (RFOs) have been shown to be major photosynthetic products in this plant. The tetrasaccharide stachyose is the major phloem-mobile carbohydrate and is used as a carbon store in roots. It has been suggested that this carbon store is remobilized during rehydration, presumably for cellular repair processes. We examined the effects of water deficit on the leaf water-soluble carbohydrate profiles of three Craterostigma species. Apart from the classical 2-octulose-to-sucrose interconversion, there was a strong water deficit-associated mass increase of RFOs up to the pentasaccharide verbascose. However, the activities of three dedicated RFO biosynthetic enzymes (raffinose, stachyose, and verbascose synthase) was not correlated with RFO accumulation, suggesting that biosynthetic enzyme activities measured in the early stages of water-deficit were sufficient to synthesize enough galactinol and lead to RFO accumulation in the leaves. Our findings are suggestive of RFOs providing additional carbohydrate-based stress protection to the leaves of these plants during the desiccated state.

Water deficit induces chlorophyll degradation via the 'PAO/phyllobilin' pathway in leaves of homoio- (Craterostigma pumilum) and poikilochlorophyllous (Xerophyta viscosa) resurrection plants.

Angiosperm resurrection plants exhibit poikilo- or homoiochlorophylly as a response to water deficit. Both strategies are generally considered as effective mechanisms to reduce oxidative stress associated with photosynthetic activity under water deficiency. The mechanism of water deficit-induced chlorophyll (Chl) degradation in resurrection plants is unknown but has previously been suggested to occur as a result of non-enzymatic photooxidation. We investigated Chl degradation during dehydration in both poikilochlorophyllous (Xerophyta viscosa) and homoiochlorophyllous (Craterostigma pumilum) species. We demonstrate an increase in the abundance of PHEOPHORBIDE a OXYGENASE (PAO), a key enzyme of Chl breakdown, together with an accumulation of phyllobilins, that is, products of PAO-dependent Chl breakdown, in both species. Phyllobilins and PAO levels diminished again in leaves from rehydrated plants. We conclude that water deficit-induced poikilochlorophylly occurs via the well-characterized PAO/phyllobilin pathway of Chl breakdown and that this mechanism also appears conserved in a resurrection species displaying homoiochlorophylly. The roles of the PAO/phyllobilin pathway during different plant developmental processes that involve Chl breakdown, such as leaf senescence and desiccation, fruit ripening and seed maturation, are discussed.

Abiotic stress-induced accumulation of raffinose in Arabidopsis leaves is mediated by a single raffinose synthase (RS5, At5g40390).

BACKGROUND: The sucrosylgalactoside oligosaccharide raffinose (Raf, Suc-Gal1) accumulates in Arabidopsis leaves in response to a myriad of abiotic stresses. Whilst galactinol synthases (GolS), the first committed enzyme in Raf biosynthesis are well characterised in Arabidopsis, little is known of the second biosynthetic gene/enzyme raffinose synthase (RS). Conflicting reports suggest the existence of either one or six abiotic stress-inducible RSs (RS-1 to -6) occurring in Arabidopsis. Indirect evidence points to At5g40390 being responsible for low temperature-induced Raf accumulation in Arabidopsis leaves. RESULTS: By heterologously expressing At5g40390 in E.coli, we demonstrate that crude extracts synthesise Raf in vitro, contrary to empty vector controls. Using two independent loss-of-function mutants for At5g40390 (rs 5-1 and 5-2), we confirm that this RS is indeed responsible for Raf accumulation during low temperature-acclimation (4°C), as previously reported. Surprisingly, leaves of mutant plants also fail to accumulate any Raf under diverse abiotic stresses including water-deficit, high salinity, heat shock, and methyl viologen-induced oxidative stress. Correlated to the lack of Raf under these abiotic stress conditions, both mutant plants lack the typical stress-induced RafS activity increase observed in the leaves of wild-type plants. CONCLUSIONS: Collectively our findings point to a single abiotic stress-induced RS isoform (RS5, At5g40390) being responsible for Raf biosynthesis in Arabidopsis leaves. However, they do not support a single RS hypothesis since the seeds of both mutant plants still contained Raf, albeit at 0.5-fold lower concentration than seeds from wild-type plants, suggesting the existence of at least one other seed-specific RS. These results also unambiguously discount the existence of six stress-inducible RS isoforms suggested by recent reports.

An Arabidopsis T-DNA insertion mutant for galactokinase (AtGALK, At3g06580) hyperaccumulates free galactose and is insensitive to exogenous galactose.

Galactokinase (GALK, EC 2.7.1.6) is a cytosolic enzyme with a wide occurrence across the taxonomic kingdoms. It catalyzes the phosphorylation of α-d-galactose (Gal) to α-d-Gal-1-P. The cytotoxicity of free (unphosphorylated) Gal is well documented in plants and causes marked defects. An Arabidopsis GALK (AtGALK, At3g06580) was previously identified, cloned and functionally characterized in Escherichia coli and was suggested to occur as a single copy gene in Arabidopsis. We identified an AtGALK T-DNA insertion mutant (atgalk) that (i) is AtGALK transcript deficient; (ii) displays no GALK activity in vegetative tissues; and (iii) accumulates Gal up to 6.8 mg g(-1) FW in vegetative tissues, in contrast to wild-type plants. By constitutively overexpressing the AtGALK cDNA, atgalk was functionally rescued. Three independent transformed lines showed restored AtGALK transcripts and GALK activity and had low leaf Gal concentrations comparable with those observed in wild-type plants. Surprisingly, in vitro grown atgalk plants were largely insensitive to the exogenous application of up to 100 mM free Gal, while wild-type plants exhibited sensitivity to low Gal concentrations (10 mM). Furthermore, atgalk seedlings retained the capacity for uptake of exogenously supplied Gal (100 mM), accumulating up to 57 mg g(-1) FW in leaves. Leaves from soil-grown atgalk plants that exhibited no growth or morphological defects were used to demonstrate that the accumulating Gal occurred exclusively in the vacuoles of mesophyll protoplasts. Collectively, these findings suggest a novel Gal detoxification pathway that targets free Gal to the vacuole and is active in the atgalk mutant background.

The effect of temperature on soft contact lens modulus and diameter.

PURPOSE: To examine the relative changes in diameter and modulus of soft contact lenses when the temperature is raised from room temperature (RT) to eye temperature (ET). METHODS: Thirteen lens types including 9 silicone hydrogel lenses were measured for diameter and elastic modulus at RT (20 ± 1°C) and ET (34 ± 1°C). Lens diameter measurements were undertaken after equilibration in ISO saline in a temperature-controlled lens analyzer (Optimec, Ltd, Malvern, United Kingdom). Measurements of flexural modulus of elasticity were made using an Instron 3343 tensiometer (Instron, Norwood, MA) with the samples suspended in a temperature-controlled saline bath. RESULTS: All lens types reduced in diameter when raised to ET. The largest mean changes with silicone hydrogel and conventional hydrogel lenses were with Biofinity (Δ0.35 mm) and Acuvue 2 (Δ0.28 mm), respectively. All the silicone hydrogels showed a statistically significant reduction in modulus when raised to ET ranging from Δ0.06 MPa with comfilcon A to Δ0.78 MPa with balafilcon A. All the conventional hydrogels showed relatively small changes (<0.05 MPa) in modulus. Two of the four conventional hydrogels showed a statistically significant change in modulus (etafilcon A and ocufilcon A), but these were small and believed to be clinically insignificant. CONCLUSIONS: This study has highlighted some clinically relevant changes in soft contact lens modulus and diameter when raised from RT to ET. It has also shown the importance of standardizing modulus measurement technique.

Functional identification of Arabidopsis ATSIP2 (At3g57520) as an alkaline α-galactosidase with a substrate specificity for raffinose and an apparent sink-specific expression pattern.

Arabidopsis ATSIP2 has recently been suggested to be a raffinose synthase gene. However, it has high amino acid identity to functionally characterized alkaline α-galactosidases from Cucumis melo and Zea mays. Using the Sf9 insect cell expression system, we demonstrate that recombinant ATSIP2 is a genuine alkaline α-galactosidase with a distinct substrate specificity for raffinose, and not a raffinose synthase. A ß-glucuronidase reporter construct using the ATSIP2 promoter shows that ATSIP2 is strongly expressed in sink tissues of Arabidopsis, i.e. sink leaves and non-xylem parts of the root stele, suggesting a physiological function in raffinose phloem unloading.

Frost tolerance in excised leaves of the common bugle (Ajuga reptans L.) correlates positively with the concentrations of raffinose family oligosaccharides (RFOs).

Mass increases in raffinose family oligosaccharides (RFOs, alpha1,6-galactosyl extensions of sucrose) are well documented in the generative tissues of many plants upon cold acclimation, and they (i.e. mainly the two shortest RFO members, raffinose and stachyose) have been suggested as frost stress protectants. Our focus here was on the longer RFO members as they commonly occur in the frost-hardy evergreen labiate Ajuga reptans in its natural habitat, and accumulate to their highest concentrations in winter when the plant is faced with sub-zero temperatures. We examined the effects of RFO concentration and chain length on frost tolerance using excised leaves which accumulate long-chain RFOs under both cold and warm conditions, thereby uncoupling the acclimation temperature from RFO production. We demonstrated that frost tolerance in excised A. reptans leaves correlates positively with long-chain RFO accumulation under both acclimation temperatures. After 24 d post-excision in the warm, the leaves had increased their RFO concentrations (mainly long-chain RFOs) 22-fold to 78 mg g(-1) fresh weight, and decreased their EL(50) values (temperature at which 50% leakage occurred) from -10.5 to -24.5 degrees C, suggesting a protective role for these oligosaccharides in the natural frost tolerance of A. reptans.

Protection mechanisms in the resurrection plant Xerophyta viscosa: cloning, expression, characterisation and role of XvINO1, a gene coding for a myo-inositol 1-phosphate synthase.

We have used reverse transcription-PCR coupled with 5'- and 3'-RACE to isolate a full length INO1 cDNA (1692 bp with an ORF of 1530) from the resurrection plant Xerophyta viscosa Baker. XvINO1 encodes 510 amino acids, with a predicted MW of 56.7kD and contains four sequence motifs that are highly conserved in plant myo-inositol-1-phosphate synthases (MIPS, EC5.5.1.4), the enzyme that catalyses the first step in the formation of myo-inositol (Ino). Northern and western analyses show that the transcript and protein are constitutively present in leaves but their expression increases, temporarily, in response to both accumulative salt stress (~300 mM NaCl) and desiccation (to 5% relative water content). Leaf Ino concentration increases 40-fold during the first 6 h of salt stress, and levels of this and other carbohydrates (galactinol, sucrose, raffinose, stachyose and hexoses) remain elevated relative to control leaves for the duration of salt stress treatment. The timing and pattern of accumulation of these carbohydrates differ under desiccation stress and we propose that they perform different functions in the respective stresses. These are elaborated in discussion of our data.

Corrigendum to: Protection mechanisms in the resurrection plant Xerophyta viscosa: cloning, expression, characterisation and role of XvINO1, a gene coding for a myo-inositol 1-phosphate synthase.

We have used reverse transcription-PCR coupled with 52- and 32-RACE to isolate a full length INO1 cDNA (1692bp with an ORF of 1530) from the resurrection plant Xerophyta viscosa Baker. XvINO1 encodes 510 amino acids, with a predicted MW of 56.7kD and contains four sequence motifs that are highly conserved in plant myo-inositol-1-phosphate synthases (MIPS, EC5.5.1.4), the enzyme that catalyses the first step in the formation of myo-inositol (Ino). Northern and western analyses show that the transcript and protein are constitutively present in leaves but their expression increases, temporarily, in response to both accumulative salt stress (~300mM NaCl) and desiccation (to 5% relative water content). Leaf Ino concentration increases 40-fold during the first 6h of salt stress, and levels of this and other carbohydrates (galactinol, sucrose, raffinose, stachyose and hexoses) remain elevated relative to control leaves for the duration of salt stress treatment. The timing and pattern of accumulation of these carbohydrates differ under desiccation stress and we propose that they perform different functions in the respective stresses. These are elaborated in discussion of our data.

Protection mechanisms in the resurrection plant Xerophyta viscosa (Baker): both sucrose and raffinose family oligosaccharides (RFOs) accumulate in leaves in response to water deficit.

Changes in water-soluble carbohydrates were examined in the leaves of the resurrection plant Xerophyta viscosa under conditions of water deficit. Sucrose and raffinose family oligosaccharides (RFOs), particularly raffinose, increased under these conditions, with the highest concentrations evident at 5% relative water content [RWC; 23.5 mg g(-1) dry weight (DW) and 17.7 mg g(-1) DW, respectively]. Importantly, these effects were reversible, with concentrations returning to levels comparable with that of the full turgor state 7 d after water deficit conditions were alleviated, providing evidence that both sucrose and RFOs may play a protective role in desiccated leaf tissue of X. viscosa. Further, because the sucrose-to-raffinose mass ratio of 1.3:1 observed in the dehydrated state was very low, compared with published data for other resurrection plants (always >5), it is suggested that, in X. viscosa leaves, RFOs serve the dual purpose of stress protection and carbon storage. XvGolS, a gene encoding a galactinol synthase enzyme responsible for the first catalytic step in RFO biosynthesis, was cloned and functionally expressed. In leaf tissue exposed to water deficit, XvGolS transcript levels were shown to increase at 19% RWC. GolS activity in planta could not be correlated with RFO accumulation, but a negative correlation was observed between RFO accumulation and myo-inositol depletion, during water deficit stress. This correlation was reversed after rehydration, suggesting that during water deficit myo-inositol is channelled into RFO synthesis, but during the rehydration process it is channelled to metabolic pathways related to the repair of desiccation-induced damage.

Identification of a vacuolar sucrose transporter in barley and Arabidopsis mesophyll cells by a tonoplast proteomic approach.

The vacuole is the main cellular storage pool, where sucrose (Suc) accumulates to high concentrations. While a limited number of vacuolar membrane proteins, such as V-type H(+)-ATPases and H(+)-pyrophosphatases, are well characterized, the majority of vacuolar transporters are still unidentified, among them the transporter(s) responsible for vacuolar Suc uptake and release. In search of novel tonoplast transporters, we used a proteomic approach, analyzing the tonoplast fraction of highly purified mesophyll vacuoles of the crop plant barley (Hordeum vulgare). We identified 101 proteins, including 88 vacuolar and putative vacuolar proteins. The Suc transporter (SUT) HvSUT2 was discovered among the 40 vacuolar proteins, which were previously not reported in Arabidopsis (Arabidopsis thaliana) vacuolar proteomic studies. To confirm the tonoplast localization of this Suc transporter, we constructed and expressed green fluorescent protein (GFP) fusion proteins with HvSUT2 and its closest Arabidopsis homolog, AtSUT4. Transient expression of HvSUT2-GFP and AtSUT4-GFP in Arabidopsis leaves and onion (Allium cepa) epidermal cells resulted in green fluorescence at the tonoplast, indicating that these Suc transporters are indeed located at the vacuolar membrane. Using a microcapillary, we selected mesophyll protoplasts from a leaf protoplast preparation and demonstrated unequivocally that, in contrast to the companion cell-specific AtSUC2, HvSUT2 and AtSUT4 are expressed in mesophyll protoplasts, suggesting that HvSUT2 and AtSUT4 are involved in transport and vacuolar storage of photosynthetically derived Suc.