First-principles calculation of HubbardUfor Terbium metal under high pressure.

Using density functional theory (DFT) and linear response approaches, we compute the on-site Hubbard interactionUof elemental Terbium (Tb) metal in the pressure range ∼ 0-65 GPa. The resulting first-principlesUvalues with experimental crystal structures enable us to examine the magnetic properties of Tb using a DFT+U method. The lowest-energy magnetic states in our calculations for different high-pressure Tb phases-including hcp,α-Sm, and dhcp-are found to be compatible with the corresponding magnetic ordering vectors reported in experiments. The result shows that the inclusion of HubbardUsubstantially improves the accuracy and efficiency in modeling correlated rare-earth materials. Our study also provides the necessaryUinformation for other quantum many-body techniques to study Tb under extreme pressure conditions.

Novel modelling approaches to predict the role of antivirals in reducing influenza transmission.

To aid understanding of the effect of antiviral treatment on population-level influenza transmission, we used a novel pharmacokinetic-viral kinetic transmission model to test the correlation between nasal viral load and infectiousness, and to evaluate the impact that timing of treatment with the antivirals oseltamivir or baloxavir has on influenza transmission. The model was run under three candidate profiles whereby infectiousness was assumed to be proportional to viral titer on a natural-scale, log-scale, or dose-response model. Viral kinetic profiles in the presence and absence of antiviral treatment were compared for each individual (N = 1000 simulated individuals); subsequently, viral transmission mitigation was calculated. The predicted transmission mitigation was greater with earlier administration of antiviral treatment, and with baloxavir versus oseltamivir. When treatment was initiated 12-24 hours post symptom onset, the predicted transmission mitigation was 39.9-56.4% for baloxavir and 26.6-38.3% for oseltamivir depending on the infectiousness profile. When treatment was initiated 36-48 hours post symptom onset, the predicted transmission mitigation decreased to 0.8-28.3% for baloxavir and 0.8-19.9% for oseltamivir. Model estimates were compared with clinical data from the BLOCKSTONE post-exposure prophylaxis study, which indicated the log-scale model for infectiousness best fit the observed data and that baloxavir affords greater reductions in secondary case rates compared with neuraminidase inhibitors. These findings suggest a role for baloxavir and oseltamivir in reducing influenza transmission when treatment is initiated within 48 hours of symptom onset in the index patient.

Turbulence is an Ineffective Mixer when Schmidt Numbers Are Large.

We solve the advection-diffusion equation for a stochastically stationary passive scalar θ, in conjunction with forced 3D Navier-Stokes equations, using direct numerical simulations in periodic domains of various sizes, the largest being 8192^{3}. The Taylor-scale Reynolds number varies in the range 140-650 and the Schmidt number Sc≡ν/D in the range 1-512, where ν is the kinematic viscosity of the fluid and D is the molecular diffusivity of θ. Our results show that turbulence becomes an ineffective mixer when Sc is large. First, the mean scalar dissipation rate ⟨χ⟩=2D⟨|∇θ|^{2}⟩, when suitably nondimensionalized, decreases as 1/logSc. Second, 1D cuts through the scalar field indicate increasing density of sharp fronts on larger scales, oscillating with large excursions leading to reduced mixing, and additionally suggesting weakening of scalar variance flux across the scales. The scaling exponents of the scalar structure functions in the inertial-convective range appear to saturate with respect to the moment order and the saturation exponent approaches unity as Sc increases, qualitatively consistent with 1D cuts of the scalar.

Small-Scale Isotropy and Ramp-Cliff Structures in Scalar Turbulence.

Passive scalars advected by three-dimensional Navier-Stokes turbulence exhibit a fundamental anomaly in odd-order moments because of the characteristic ramp-cliff structures, violating small-scale isotropy. We use data from direct numerical simulations with grid resolution of up to 8192^{3} at high Péclet numbers to understand this anomaly as the scalar diffusivity, D, diminishes, or as the Schmidt number, Sc=ν/D, increases; here ν is the kinematic viscosity of the fluid. The microscale Reynolds number varies from 140 to 650 and Sc varies from 1 to 512. A simple model for the ramp-cliff structures is developed and shown to characterize the scalar derivative statistics very well. It accurately captures how the small-scale isotropy is restored in the large-Sc limit, and additionally suggests a possible correction to the Batchelor length scale as the relevant smallest scale in the scalar field.

IL-6 induced upregulation of T-type Ca2+ currents and sensitization of DRG nociceptors is attenuated by MNK inhibition.

Phosphorylation of the 5' cap-binding protein eIF4E by MAPK-interacting kinases (MNK1/2) is important for nociceptor sensitization and the development of chronic pain. IL-6-induced dorsal root ganglion (DRG) nociceptor excitability is attenuated in mice lacking eIF4E phosphorylation, in MNK1/2-/- mice, and by the nonselective MNK1/2 inhibitor cercosporamide. Here, we sought to better understand the neurophysiological mechanisms underlying how IL-6 causes nociceptor excitability via MNK-eIF4E signaling using the highly selective MNK inhibitor eFT508. DRG neurons were cultured from male and female ICR mice, 4-7 wk old. DRG cultures were treated with vehicle, IL-6, eFT508 (pretreat) followed by IL-6, or eFT508 alone. Whole cell patch-clamp recordings were done on small-diameter neurons (20-30 pF) to measure membrane excitability in response to ramp depolarization. IL-6 treatment (1 h) resulted in increased action potential firing compared with vehicle at all ramp intensities, an effect that was blocked by pretreatment with eFT508. Basic membrane properties, including resting membrane potential, input resistance, and rheobase, were similar across groups. Latency to the first action potential in the ramp protocol was lower in the IL-6 group and rescued by eFT508 pretreatment. We also found that the amplitudes of T-type voltage-gated calcium channels (VGCCs) were increased in the DRG following IL-6 treatment, but not in the eFT508 cotreatment group. Our findings are consistent with a model wherein MNK-eIF4E signaling controls the translation of signaling factors that regulate T-type VGCCs in response to IL-6 treatment. Inhibition of MNK with eFT508 disrupts these events, thereby preventing nociceptor hyperexcitability.NEW & NOTEWORTHY In this study, we show that the MNK inhibitor and anti-tumor agent eFT508 (tomivosertib) is effective in attenuating IL-6 induced sensitization of dorsal root ganglion (DRG) nociceptors. Pretreatment with eFT508 in DRG cultures from mice helps mitigate the development of hyperexcitability in response to IL-6. Furthermore, our data reveal that the upregulation of T-type voltage-gated calcium channels following IL-6 application can be blocked by eFT508, implicating the MNK-eIF4E signaling pathway in membrane trafficking of ion channels.

SINE Retrotransposon variation drives Ecotypic disparity in natural populations of Coilia nasus.

BACKGROUND: SINEs are a type of nonautonomous retrotransposon that can transpose from one site to be integrated elsewhere in an organism genome. SINE insertion can give rise to genetic variants and regulate gene expression, allowing organisms to acquire new adaptive capacity. Studies on this subject have focused on the impacts of SINEs on genes. However, ecological disparities in fish have not yet been explained by SINEs. RESULTS: New SINEs were isolated from Coilia nasus, which has two ecotypes-migratory and resident-that differ in their spawning and migration behaviors. The SINEs possess two structures that resemble a tRNA gene and a LINE retrotransposon tail. Comparison of olfactory tissue transcriptomes, intact SINE transcript copies were detected in only the migratory fish at the initial retrotransposition stage. The SINE DNA copy numbers were higher in the resident type than in the migratory type, while the frequency of SINE insertion was higher in the migratory type than in the resident type. Furthermore, SINE insertions can lead to new repeats of short DNA fragments in the genome, along with target site duplications. SINEs in the resident type have undergone excision via a mechanism in which predicted cleavage sites are formed by mutations, resulting in gaps that are then filled by microsatellites via microhomology-induced replication. CONCLUSIONS: Notably, SINEs in the resident type have undergone strong natural selection, causing genomic heteroplasmy and driving ecological diversity of C. nasus. Our results reveal possible evolutionary mechanisms underlying the ecological diversity at the interface between SINE mobilization and organism defense.

Modeling Cutaneous Radiation Injury from Fallout.

OBJECTIVE: Beta radiation from nuclear weapons fallout could pose a risk of cutaneous radiation injury (CRI) to evacuating populations but has been investigated only cursorily. This work examines 2 components of CRI necessary for estimating the potential public health consequences of exposure to fallout: dose protraction and depth of dose. METHODS: Dose protraction for dry and moist desquamation was examined by adapting the biological effective dose (BED) calculation to a hazard function calculation similar to those recommended by the National Council on Radiation Protection and Measurements for other acute radiation injuries. Depth of burn was examined using Monte Carlo neutral Particle version 5 to model the penetration of beta radiation from fallout to different skin tissues. RESULTS: Nonlinear least squares analysis of the BED calculation estimated the hazard function parameter θ1 (dose rate effectiveness factors) as 25.5 and 74.5 (Gy-eq)2 h-1 for dry and moist desquamation, respectively. Depth of dose models revealed that beta radiation is primarily absorbed in the dead skin layers and basal layer and that dose to underlying tissues is small (<5% of dose to basal layer). CONCLUSIONS: The low relative dose to tissues below the basal layer suggests that radiation-induced necrosis or deep skin burns are unlikely from direct skin contamination with fallout. These results enable future modeling studies to better examine CRI risk and facilitate effectively managing and treating populations with specialized injuries from a nuclear detonation. (Disaster Med Public Health Preparedness. 2019;13:463-469).

A Sensitized Screen for Genes Promoting Invadopodia Function In Vivo: CDC-42 and Rab GDI-1 Direct Distinct Aspects of Invadopodia Formation.

Invadopodia are specialized membrane protrusions composed of F-actin, actin regulators, signaling proteins, and a dynamically trafficked invadopodial membrane that drive cell invasion through basement membrane (BM) barriers in development and cancer. Due to the challenges of studying invasion in vivo, mechanisms controlling invadopodia formation in their native environments remain poorly understood. We performed a sensitized genome-wide RNAi screen and identified 13 potential regulators of invadopodia during anchor cell (AC) invasion into the vulval epithelium in C. elegans. Confirming the specificity of this screen, we identified the Rho GTPase cdc-42, which mediates invadopodia formation in many cancer cell lines. Using live-cell imaging, we show that CDC-42 localizes to the AC-BM interface and is activated by an unidentified vulval signal(s) that induces invasion. CDC-42 is required for the invasive membrane localization of WSP-1 (N-WASP), a CDC-42 effector that promotes polymerization of F-actin. Loss of CDC-42 or WSP-1 resulted in fewer invadopodia and delayed BM breaching. We also characterized a novel invadopodia regulator, gdi-1 (Rab GDP dissociation inhibitor), which mediates membrane trafficking. We show that GDI-1 functions in the AC to promote invadopodia formation. In the absence of GDI-1, the specialized invadopodial membrane was no longer trafficked normally to the invasive membrane, and instead was distributed to plasma membrane throughout the cell. Surprisingly, the pro-invasive signal(s) from the vulval cells also controls GDI-1 activity and invadopodial membrane trafficking. These studies represent the first in vivo screen for genes regulating invadopodia and demonstrate that invadopodia formation requires the integration of distinct cellular processes that are coordinated by an extracellular cue.

Basement Membranes in the Worm: A Dynamic Scaffolding that Instructs Cellular Behaviors and Shapes Tissues.

The nematode worm Caenorhabditis elegans has all the major basement membrane proteins found in vertebrates, usually with a smaller gene family encoding each component. With its powerful forward genetics, optical clarity, simple tissue organization, and the capability to functionally tag most basement membrane components with fluorescent proteins, C. elegans has facilitated novel insights into the assembly and function of basement membranes. Although basement membranes are generally thought of as static structures, studies in C. elegans have revealed their active properties and essential functions in tissue formation and maintenance. Here, we review discoveries from C. elegans development that highlight dynamic aspects of basement membrane assembly, function, and regulation during organ growth, tissue polarity, cell migration, cell invasion, and tissue attachment. These studies have helped transform our view of basement membranes from static support structures to dynamic scaffoldings that play broad roles in regulating tissue organization and cellular behavior that are essential for development and have important implications in human diseases.

Cadherin 6 promotes neural crest cell detachment via F-actin regulation and influences active Rho distribution during epithelial-to-mesenchymal transition.

The epithelial-to-mesenchymal transition (EMT) is a complex change in cell phenotype that is important for cell migration, morphogenesis and carcinoma metastasis. Loss of epithelial cell adhesion and tight regulation of cadherin adhesion proteins are crucial for EMT. Cells undergoing EMT often display cadherin switching, where they downregulate one cadherin and induce expression of another. However, the functions of the upregulated cadherins and their effects on cell motility are poorly understood. Neural crest cells (NCCs), which undergo EMT during development, lose N-cadherin and upregulate Cadherin 6 (Cdh6) prior to EMT. Cdh6 has been suggested to suppress EMT via cell adhesion, but also to promote EMT by mediating pro-EMT signals. Here, we determine novel roles for Cdh6 in generating cell motility during EMT. We use live imaging of NCC behavior in vivo to show that Cdh6 promotes detachment of apical NCC tails, an important early step of EMT. Furthermore, we show that Cdh6 affects spatiotemporal dynamics of F-actin and active Rho GTPase, and that Cdh6 is required for accumulation of F-actin in apical NCC tails during detachment. Moreover, Cdh6 knockdown alters the subcellular distribution of active Rho, which is known to promote localized actomyosin contraction that is crucial for apical NCC detachment. Together, these data suggest that Cdh6 is an important determinant of where subcellular actomyosin forces are generated during EMT. Our results also identify mechanisms by which an upregulated cadherin can generate cell motility during EMT.

Rho activation is apically restricted by Arhgap1 in neural crest cells and drives epithelial-to-mesenchymal transition.

Epithelial-to-mesenchymal transitions (EMTs) are crucial for morphogenesis and carcinoma metastasis, yet mechanisms controlling the underlying cell behaviors are poorly understood. RhoGTPase signaling has been implicated in EMT; however, previous studies have yielded conflicting results regarding Rho function, and its role in EMT remains poorly understood. Elucidation of precise Rho functions has been challenging because Rho signaling is highly context dependent and its activity is tightly regulated spatiotemporally within the cell. To date, few studies have examined how Rho affects cell motility in intact organisms, and the pattern of Rho activity during motile cell behaviors of EMT has not been determined in any system. Here, we image endogenous active Rho during EMT in vivo, and analyze effects of Rho and Rho-kinase (ROCK) manipulation on cell motility in vivo. We show that Rho is activated in a discrete apical region of premigratory neural crest cells during EMT, and Rho-ROCK signaling is essential for apical detachment and generation of motility within the neuroepithelium, a process that has been poorly understood. Furthermore, we find that Arhgap1 restricts Rho activation to apical areas, and this restriction is necessary for detachment. Our results provide new insight into mechanisms controlling local Rho activation and how it affects dynamic cell behaviors and actomyosin contraction during key steps of EMT in an intact living organism.

The structure of LL-diaminopimelate aminotransferase from Chlamydia trachomatis: implications for its broad substrate specificity.

We have previously reported the structures of the native holo and substrate-bound forms of LL-diaminopimelate aminotransferase from Arabidopsis thaliana (AtDAP-AT). Here, we report the crystal and molecular structures of the LL-diaminopimelate aminotransferase from Chlamydia trachomatis (CtDAP-AT) in the apo-form and the pyridoxal-5'-phosphate-bound form. The molecular structure of CtDAP-AT shows that its overall fold is essentially identical with that of AtDAP-AT except that CtDAP-AT adopts an "open" conformation as opposed to the "closed" conformation of AtDAP-AT. Although AtDAP-AT and CtDAP-AT are approximately 40% identical in their primary sequence, they have major differences in their substrate specificities; AtDAP-AT is highly specific for LL-DAP, whereas CtDAP-AT accepts a wider range of substrates. Since all of the residues involved in substrate recognition are highly conserved between AtDAP-AT and CtDAP-AT, we propose that differences in flexibility of the loops lining the active-site region between the two enzymes likely account for the differences in substrate specificity.

Head and neck cancer stem cells: the side population.

OBJECTIVES/HYPOTHESIS: The cancer stem cell (CSC) theory concludes that a subpopulation of cancer cells, the cancer stem cells, can self-renew and are responsible for tumor growth. Previous studies have identified cells able to efflux Hoechst 33342 dye as the side population (SP). SP cells and CSCs share many characteristics, suggesting the SP isolated from malignant tumors contains CSCs. STUDY DESIGN: Experimental Study. METHODS: The SP was isolated from a head and neck cancer cell line and analyzed for CSC-like characteristics. RESULTS: The SP demonstrated the ability to reproduce both SP and non-side population (NSP) cells from as few as one cell. The SP had lower expression of active ß-catenin and more resistance to 5-fluorouracil; the SP also demonstrated greater expression of Bmi-1 (4.3-fold) and ABCG2 (1.4-fold). SP cells were able to produce tumors in an animal model, whereas NSP were not. SPs were identified in two primary human tumors. CONCLUSIONS: This work adds to the evidence that the SP in head and neck cancer represents cells with CSC properties and provides a method by which CSCs can be isolated and studied.

Regulation of cell adhesions and motility during initiation of neural crest migration.

Accurate neural crest cell (NCC) migration requires tight control of cell adhesions, cytoskeletal dynamics and cell motility. Cadherins and RhoGTPases are critical molecular players that regulate adhesions and motility during initial delamination of NCCs from the neuroepithelium. Recent studies have revealed multiple functions for these molecules and suggest that a precise balance of their activity is crucial. RhoGTPase appears to regulate both cell adhesions and protrusive forces during NCC delamination. Increasing evidence shows that cadherins are multi-functional proteins with novel, adhesion-independent signaling functions that control NCC motility during both delamination and migration. These functions are often regulated by specific proteolytic cleavage of cadherins. After NCC delamination, planar cell polarity signaling acts via RhoGTPases to control NCC protrusions and migration direction.

Control of neural crest cell behavior and migration: Insights from live imaging.

Neural crest cells (NCCs) are a remarkable, dynamic group of cells that travel long distances in the embryo to reach their target sites. They are responsible for the formation of craniofacial bones and cartilage, neurons and glia in the peripheral nervous system, and pigment cells. Live imaging of NCCs as they traverse the embryo has been critical to increasing our knowledge of their biology. NCCs exhibit multiple behaviors and communicate with each other and their environment along each step of their journey. Imaging combined with molecular manipulations has led to insights into the mechanisms controlling these behaviors. In this review, we highlight studies that have used live imaging to provide novel insight into NCC migration and discuss how continued use of such techniques can advance our understanding of NCC biology.

Live imaging of cell motility and actin cytoskeleton of individual neurons and neural crest cells in zebrafish embryos.

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons. Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages. Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells.

Exploration of inhibitors for diaminopimelate aminotransferase.

Bacteria and higher plants make l-lysine from diaminopimelic acid (DAP). In mammals l-lysine is an essential amino acid that must be acquired from the diet as the biosynthetic pathway is absent for this key constituent of proteins. Recently, ll-diaminopimelate aminotransferase (ll-DAP-AT), a pyridoxal-5'-phosphate (PLP)-dependent enzyme, was reported to catalyze a key step in the route to l-lysine in plants and Chlamydia. Specific inhibitors of this enzyme could thus potentially serve as herbicides or antibiotics that are non-toxic to mammals. In this work, 29,201 inhibitors were screened against ll-DAP-AT and the IC(50) values were determined for the top 46 compounds. An aryl hydrazide and rhodanine derivatives were further modified to generate 20 analogues that were also tested against ll-DAP-AT. These analogues provide additional structure-activity relationships (SAR) that are useful in guiding further design of inhibitors.

Single-marker identification of head and neck squamous cell carcinoma cancer stem cells with aldehyde dehydrogenase.

BACKGROUND: In accord with the cancer stem cell (CSC) theory, only a small subset of cancer cells are capable of forming tumors. We previously reported that CD44 isolates tumorigenic cells from head and neck squamous cell cancer (HNSCC). Recent studies indicate that aldehyde dehydrogenase (ALDH) activity may represent a more specific marker of CSCs. METHODS: Six primary HNSCCs were collected. Cells with high and low ALDH activity (ALDH(high)/ALDH(low)) were isolated. ALDH(high) and ALDH(low) populations were implanted into NOD/SCID mice and monitored for tumor development. RESULTS: ALDH(high) cells represented a small percentage of the tumor cells (1% to 7.8%). ALDH(high) cells formed tumors from as few as 500 cells in 24/45 implantations, whereas only 3/37 implantations of ALDH(low) cells formed tumors. CONCLUSIONS: ALDH(high) cells comprise a subpopulation cells in HNSCCs that are tumorigenic and capable of producing tumors at very low numbers. This finding indicates that ALDH activity on its own is a highly selective marker for CSCs in HNSCC.

Rho-kinase and myosin II affect dynamic neural crest cell behaviors during epithelial to mesenchymal transition in vivo.

The induction and migration of neural crest cells (NCCs) are essential to the development of craniofacial structures and the peripheral nervous system. A critical step in the development of NCCs is the epithelial to mesenchymal transition (EMT) that they undergo in order to initiate migration. Several transcription factors are important for the NCC EMT. However, less is known about the effectors regulating changes in cell adhesion, the cytoskeleton, and cell motility associated with the EMT or about specific changes in the behavior of cells undergoing EMT in vivo. We used time-lapse imaging of NCCs in the zebrafish hindbrain to show that NCCs undergo a stereotypical series of behaviors during EMT. We find that loss of cell adhesion and membrane blebbing precede filopodial extension and the onset of migration. Live imaging of actin dynamics shows that actin localizes differently in blebs and filopodia. Moreover, we find that disruption of myosin II or Rho-kinase (ROCK) activity inhibits NCC blebbing and causes reduced NCC EMT. These data reveal roles for myosin II and ROCK in NCC EMT in vivo, and provide a detailed characterization of NCC behavior during EMT that will form a basis for further mechanistic studies.

Mechanism of substrate recognition and PLP-induced conformational changes in LL-diaminopimelate aminotransferase from Arabidopsis thaliana.

LL-Diaminopimelate aminotransferase (LL-DAP-AT), a pyridoxal phosphate (PLP)-dependent enzyme in the lysine biosynthetic pathways of plants and Chlamydia, is a potential target for the development of herbicides or antibiotics. This homodimeric enzyme converts L-tetrahydrodipicolinic acid (THDP) directly to LL-DAP using L-glutamate as the source of the amino group. Earlier, we described the 3D structures of native and malate-bound LL-DAP-AT from Arabidopsis thaliana (AtDAP-AT). Seven additional crystal structures of AtDAP-AT and its variants are reported here as part of an investigation into the mechanism of substrate recognition and catalysis. Two structures are of AtDAP-AT with reduced external aldimine analogues: N-(5'-phosphopyridoxyl)-L-glutamate (PLP-Glu) and N-(5'-phosphopyridoxyl)- LL-Diaminopimelate (PLP-DAP) bound in the active site. Surprisingly, they reveal that both L-glutamate and LL-DAP are recognized in a very similar fashion by the same sets of amino acid residues; both molecules adopt twisted V-shaped conformations. With both substrates, the alpha-carboxylates are bound in a salt bridge with Arg404, whereas the distal carboxylates are recognized via hydrogen bonds to the well-conserved side chains of Tyr37, Tyr125 and Lys129. The distal C(epsilon) amino group of LL-DAP is specifically recognized by several non-covalent interactions with residues from the other subunit (Asn309*, Tyr94*, Gly95*, and Glu97* (Amino acid designators followed by an asterisk (*) indicate that the residues originate in the other subunit of the dimer)) and by three bound water molecules. Two catalytically inactive variants of AtDAP-AT were created via site-directed mutagenesis of the active site lysine (K270N and K270Q). The structures of these variants permitted the observation of the unreduced external aldimines of PLP with L-glutamate and with LL-DAP in the active site, and revealed differences in the torsion angle about the PLP-substrate bond. Lastly, an apo-AtDAP-AT structure missing PLP revealed details of conformational changes induced by PLP binding and substrate entry into the active site.