Structural and functional characterization of triketone dioxygenase from Oryza Sativa.

The transgenic expression of rice triketone dioxygenase (TDO; also known as HIS1) can provide protection from triketone herbicides to susceptible dicot crops such as soybean. Triketones are phytotoxic inhibitors of plant hydroxyphenylpyruvate dioxygenases (HPPD). The TDO gene codes for an iron/2-oxoglutarate-dependent oxidoreductase. We obtained an X-ray crystal structure of TDO using SeMet-SAD phasing to 3.16 Å resolution. The structure reveals that TDO possesses a fold like that of Arabidopsis thaliana 2-oxoglutarate­iron-dependent oxygenase anthocyanidin synthase (ANS). Unlike ANS, this TDO structure lacks bound metals or cofactors, and we propose this is because the disordered flexible loop over the active site is sterically constrained from folding properly in the crystal lattice. A combination of mass spectrometry, nuclear magnetic resonance, and enzyme activity studies indicate that rice TDO oxidizes mesotrione in a series of steps; first producing 5-hydroxy-mesotrione and then oxy-mesotrione. Evidence suggests that 5-hydroxy-mesotrione is a much weaker inhibitor of HPPD than mesotrione, and oxy-mesotrione has virtually no inhibitory activity. Of the close homologues which have been tested, only corn and rice TDO have enzymatic activity and the ability to protect plants from mesotrione. Correlating sequence and structure has identified four amino acids necessary for TDO activity. Introducing these four amino acids imparts activity to a mesotrione-inactive TDO-like protein from sorghum, which may expand triketone herbicide resistance in new crop species.

Cryo-EM structure of adeno-associated virus 4 at 2.2†Šresolution.

Adeno-associated virus (AAV) is the vector of choice for several approved gene-therapy treatments and is the basis for many ongoing clinical trials. Various strains of AAV exist (referred to as serotypes), each with their own transfection characteristics. Here, a high-resolution cryo-electron microscopy structure (2.2 Å) of AAV serotype 4 (AAV4) is presented. The receptor responsible for transduction of the AAV4 clade of AAV viruses (including AAV11, AAV12 and AAVrh32.33) is unknown. Other AAVs interact with the same cell receptor, adeno-associated virus receptor (AAVR), in one of two different ways. AAV5-like viruses interact exclusively with the polycystic kidney disease-like 1 (PKD1) domain of AAVR, while most other AAVs interact primarily with the PKD2 domain. A comparison of the present AAV4 structure with prior corresponding structures of AAV5, AAV2 and AAV1 in complex with AAVR provides a foundation for understanding why the AAV4-like clade is unable to interact with either PKD1 or PKD2 of AAVR. The conformation of the AAV4 capsid in variable regions I, III, IV and V on the viral surface appears to be sufficiently different from AAV2 to ablate binding with PKD2. Differences between AAV4 and AAV5 in variable region VII appear to be sufficient to exclude binding with PKD1.

Cross-Species Permissivity: Structure of a Goat Adeno-Associated Virus and Its Complex with the Human Receptor AAVR.

Adeno-associated virus (AAV) is a small ssDNA satellite virus of high interest (in recombinant form) as a safe and effective gene therapy vector. AAV's human cell entry receptor (AAVR) contains polycystic kidney disease (PKD) domains bound by AAV. Seeking understanding of the spectrum of interactions, goat AAVGo.1 is investigated, because its host is the species most distant from human with reciprocal cross-species cell susceptibility. The structure of AAVGo.1, solved by cryo-EM to 2.9 Å resolution, is most similar to AAV5. Through ELISA (enzyme-linked immunosorbent assay) studies, it is shown that AAVGo.1 binds to human AAVR more strongly than do AAV2 or AAV5, and that it joins AAV5 in a class that binds exclusively to PKD domain 1 (PKD1), in contrast to other AAVs that interact primarily with PKD2. The AAVGo.1 cryo-EM structure of a complex with a PKD12 fragment of AAVR at 2.4 Å resolution shows PKD1 bound with minimal change in virus structure. There are only minor conformational adaptations in AAVR, but there is a near-rigid rotation of PKD1 with maximal displacement of the receptor domain by ~1 Å compared to PKD1 bound to AAV5. AAVGo.1 joins AAV5 as the second member of an emerging class of AAVs whose mode of receptor-binding is completely different from other AAVs, typified by AAV2. IMPORTANCE Adeno-associated virus (AAV) is a small ssDNA satellite parvovirus. As a recombinant vector with a protein shell encapsidating a transgene, recombinant AAV (rAAV) is a leading delivery vehicle for gene therapy, with two FDA-approved treatments and 150 clinical trials for 30 diseases. The human entry receptor AAVR has five PKD domains. To date, all serotypes, except AAV5, have interacted primarily with the second PKD domain, PKD2. Goat is the AAV host most distant from human with cross-species cell infectivity. AAVGo.1 is similar in structure to AAV5, the two forming a class with a distinct mode of receptor-binding. Within the two classes, binding interactions are mostly conserved, giving an indication of the latitude available in modulating delivery vectors.

The Candida albicans virulence factor candidalysin polymerizes in solution to form membrane pores and damage epithelial cells.

Candida albicans causes severe invasive candidiasis. C. albicans infection requires the virulence factor candidalysin (CL) which damages target cell membranes. However, the mechanism that CL uses to permeabilize membranes is unclear. We reveal that CL forms membrane pores using a unique mechanism. Unexpectedly, CL readily assembled into polymers in solution. We propose that the basic structural unit in polymer formation is a CL oligomer, which is sequentially added into a string configuration that can close into a loop. CL loops appear to spontaneously insert into the membrane to become pores. A CL mutation (G4W) inhibited the formation of polymers in solution and prevented pore formation in synthetic lipid systems. Epithelial cell studies showed that G4W CL failed to activate the danger response pathway, a hallmark of the pathogenic effect of CL. These results indicate that CL polymerization in solution is a necessary step for the damage of cellular membranes. Analysis of CL pores by atomic force microscopy revealed co-existence of simple depressions and more complex pores, which are likely formed by CL assembled in an alternate oligomer orientation. We propose that this structural rearrangement represents a maturation mechanism that stabilizes pore formation to achieve more robust cellular damage. To summarize, CL uses a previously unknown mechanism to damage membranes, whereby pre-assembly of CL loops in solution leads to formation of membrane pores. Our investigation not only unravels a new paradigm for the formation of membrane pores, but additionally identifies CL polymerization as a novel therapeutic target to treat candidiasis.

The fungus Candida albicans is the most common cause of yeast infections in humans. Like many other disease-causing microbes, it releases several virulent proteins that invade and damage human cells. This includes the peptide candidalysin which has been shown to be crucial for infection. Human cells are surrounded by a protective membrane that separates their interior from their external environment. Previous work showed that candidalysin damages the cell membrane to promote infection. However, how candidalysin does this remained unclear. Similar peptides and proteins cause harm by inserting themselves into the membrane and then grouping together to form a ring. This creates a hole, or 'pore', that weakens the membrane and allows other molecules into the cell's interior. Here, Russell, Schaefer et al. show that candidalysin uses a unique pore forming mechanism to impair the membrane of human cells. A combination of biophysical and cell biology techniques revealed that the peptide groups together to form a chain. This chain of candidalysin proteins then closes in on itself to create a loop structure that can insert into the membrane to form a pore. Once embedded within the membrane, the proteins within the loops rearrange again to make the pores more stable so they can cause greater damage. This type of pore formation has not been observed before, and may open up new avenues of research. For instance, researchers could use this information to develop inhibitors that stop candidalysin from forming chains and harming the membranes of cells. This could help treat the infections caused by C. albicans.

Plasticity within the barrel domain of BamA mediates a hybrid-barrel mechanism by BAM.

In Gram-negative bacteria, the biogenesis of ß-barrel outer membrane proteins is mediated by the ß-barrel assembly machinery (BAM). The mechanism employed by BAM is complex and so far- incompletely understood. Here, we report the structures of BAM in nanodiscs, prepared using polar lipids and native membranes, where we observe an outward-open state. Mutations in the barrel domain of BamA reveal that plasticity in BAM is essential, particularly along the lateral seam of the barrel domain, which is further supported by molecular dynamics simulations that show conformational dynamics in BAM are modulated by the accessory proteins. We also report the structure of BAM in complex with EspP, which reveals an early folding intermediate where EspP threads from the underside of BAM and incorporates into the barrel domain of BamA, supporting a hybrid-barrel budding mechanism in which the substrate is folded into the membrane sequentially rather than as a single unit.

Cryo-ePDF: Overcoming Electron Beam Damage to Study the Local Atomic Structure of Amorphous ALD Aluminum Oxide Thin Films within a TEM.

Atomic layer deposition (ALD) provides uniform and conformal thin films that are of interest for a range of applications. To better understand the properties of amorphous ALD films, we need an improved understanding of their local atomic structure. Previous work demonstrated measurement of how the local atomic structure of ALD-grown aluminum oxide (AlO x ) evolves in operando during growth by employing synchrotron high-energy X-ray diffraction (HE-XRD). In this work, we report on efforts to employ electron diffraction pair distribution function (ePDF) measurements using more broadly available transmission electron microscope (TEM) instrumentation to study the atomic structure of amorphous ALD-AlO x . We observe electron beam damage in the ALD-coated samples during ePDF at ambient temperature and successfully mitigate this beam damage using ePDF at cryogenic temperatures (cryo-ePDF). We employ cryo-ePDF and reverse Monte Carlo (RMC) modeling to obtain structural models of ALD-AlO x coatings formed at a range of deposition temperatures from 150 to 332 °C. From these model structures, we derive structural metrics including stoichiometry, pair distances, and coordination environments in the ALD-AlO x films as a function of deposition temperature. The structural variations we observe with growth temperature are consistent with temperature-dependent changes in the surface hydroxyl density on the growth surface. The sample preparation and cryo-ePDF procedures we report here can be used for the routine measurement of ALD-grown amorphous thin films to improve our understanding of the atomic structure of these materials, establish structure-property relationships, and help accelerate the timescale for the application of ALD to address technological needs.

DRPnet: automated particle picking in cryo-electron micrographs using deep regression.

BACKGROUND: Identification and selection of protein particles in cryo-electron micrographs is an important step in single particle analysis. In this study, we developed a deep learning-based particle picking network to automatically detect particle centers from cryoEM micrographs. This is a challenging task due to the nature of cryoEM data, having low signal-to-noise ratios with variable particle sizes, shapes, distributions, grayscale variations as well as other undesirable artifacts. RESULTS: We propose a double convolutional neural network (CNN) cascade for automated detection of particles in cryo-electron micrographs. This approach, entitled Deep Regression Picker Network or "DRPnet", is simple but very effective in recognizing different particle sizes, shapes, distributions and grayscale patterns corresponding to 2D views of 3D particles. Particles are detected by the first network, a fully convolutional regression network (FCRN), which maps the particle image to a continuous distance map that acts like a probability density function of particle centers. Particles identified by FCRN are further refined to reduce false particle detections by the second classification CNN. DRPnet's first CNN pretrained with only a single cryoEM dataset can be used to detect particles from different datasets without retraining. Compared to RELION template-based autopicking, DRPnet results in better particle picking performance with drastically reduced user interactions and processing time. DRPnet also outperforms the state-of-the-art particle picking networks in terms of the supervised detection evaluation metrics recall, precision, and F-measure. To further highlight quality of the picked particle sets, we compute and present additional performance metrics assessing the resulting 3D reconstructions such as number of 2D class averages, efficiency/angular coverage, Rosenthal-Henderson plots and local/global 3D reconstruction resolution. CONCLUSION: DRPnet shows greatly improved time-savings to generate an initial particle dataset compared to manual picking, followed by template-based autopicking. Compared to other networks, DRPnet has equivalent or better performance. DRPnet excels on cryoEM datasets that have low contrast or clumped particles. Evaluating other performance metrics, DRPnet is useful for higher resolution 3D reconstructions with decreased particle numbers or unknown symmetry, detecting particles with better angular orientation coverage.

The Structure of an AAV5-AAVR Complex at 2.5 Å Resolution: Implications for Cellular Entry and Immune Neutralization of AAV Gene Therapy Vectors.

Adeno-Associated Virus is the leading vector for gene therapy. Although it is the vector for all in vivo gene therapies approved for clinical use by the US Food and Drug Administration, its biology is still not yet fully understood. It has been shown that different serotypes of AAV bind to their cellular receptor, AAVR, in different ways. Previously we have reported a 2.4Å structure of AAV2 bound to AAVR that shows ordered structure for only one of the two AAVR domains with which AAV2 interacts. In this study we present a 2.5Å resolution structure of AAV5 bound to AAVR. AAV5 binds to the first polycystic kidney disease (PKD) domain of AAVR that was not ordered in the AAV2 structure. Interactions of AAV5 with AAVR are analyzed in detail, and the implications for AAV2 binding are explored through molecular modeling. Moreover, we find that binding sites for the antibodies ADK5a, ADK5b, and 3C5 on AAV5 overlap with the binding site of AAVR. These insights provide a structural foundation for development of gene therapy agents to better evade immune neutralization without disrupting cellular entry.

Hunter-Gatherers Harvested and Heated Microbial Biogenic Iron Oxides to Produce Rock Art Pigment.

Red mineral pigment use is recognized as a fundamental component of a series of traits associated with human evolutionary development, social interaction, and behavioral complexity. Iron-enriched mineral deposits have been collected and prepared as pigment for use in rock art, personal adornment, and mortuary practices for millennia, yet little is known about early developments in mineral processing techniques in North America. Microanalysis of rock art pigments from the North American Pacific Northwest reveals a sophisticated use of iron oxide produced by the biomineralizing bacterium Leptothrix ochracea; a keystone species of chemolithotroph recognized in recent advances in the development of thermostable, colorfast biomaterial pigments. Here we show evidence for human engagement with this bacterium, including nanostructural and magnetic properties evident of thermal enhancement, indicating that controlled use of pyrotechnology was a key feature of how biogenic iron oxides were prepared into paint. Our results demonstrate that hunter-gatherers in this area of study prepared pigments by harvesting aquatic microbial iron mats dominated by iron-oxidizing bacteria, which were subsequently heated in large open hearths at a controlled range of 750 °C to 850 °C. This technical gesture was performed to enhance color properties, and increase colorfastness and resistance to degradation. This skilled production of highly thermostable and long-lasting rock art paint represents a specialized technological innovation. Our results contribute to a growing body of knowledge on historical-ecological resource use practices in the Pacific Northwest during the Late Holocene.Figshare link to figures: https://figshare.com/s/9392a0081632c20e9484.

Spatial Restrictions in Chemotaxis Signaling Arrays: A Role for Chemoreceptor Flexible Hinges across Bacterial Diversity.

The chemotactic sensory system enables motile bacteria to move toward favorable environments. Throughout bacterial diversity, the chemoreceptors that mediate chemotaxis are clustered into densely packed arrays of signaling complexes. In these arrays, rod-shaped receptors are in close proximity, resulting in limited options for orientations. A recent geometric analysis of these limitations in Escherichia coli, using published dimensions and angles, revealed that in this species, straight chemoreceptors would not fit into the available space, but receptors bent at one or both of the recently-documented flexible hinges would fit, albeit over a narrow window of shallow bend angles. We have now expanded our geometric analysis to consider variations in receptor length, orientation and placement, and thus to species in which those parameters are known to be, or might be, different, as well as to the possibility of dynamic variation in those parameters. The results identified significant limitations on the allowed combinations of chemoreceptor dimensions, orientations and placement. For most combinations, these limitations excluded straight chemoreceptors, but allowed receptors bent at a flexible hinge. Thus, our analysis identifies across bacterial diversity a crucial role for chemoreceptor flexible hinges, in accommodating the limitations of molecular crowding in chemotaxis core signaling complexes and their arrays.

Angiotensin II suppresses autophagy and disrupts ultrastructural morphology and function of mitochondria in mouse skeletal muscle.

Angiotensin II (ANG II)-induced skeletal muscle wasting is characterized by activation of the ubiquitin-proteasome system. However, the potential involvement of proteolytic system macroautophagy/autophagy in this wasting process remains elusive. Autophagy is precisely regulated to maintain cell survival and homeostasis; thus its dysregulation (i.e., overactivation or persistent suppression) could lead to detrimental outcomes in skeletal muscle. Here we show that infusion of ANG II for 7 days in male FVB mice suppressed autophagy in skeletal muscle. ANG II blunted microtubule-associated protein 1 light chain 3B (LC3B)-I-to-LC3B-II conversion (an autophagosome marker), increased p62/SQSTM1 (an autophagy cargo receptor) protein expression, and decreased the number of autophagic vacuoles. ANG II inhibited UNC-51-like kinase 1 via inhibition of 5'-AMP-activated kinase and activation of mechanistic target of rapamycin complex 1, leading to reduced phosphorylation of beclin-1Ser14 and Autophagy-related protein 14Ser29, suggesting that ANG II impairs autophagosome formation in skeletal muscle. In line with ANG II-mediated suppression of autophagy, ANG II promoted accumulation of abnormal/damaged mitochondria, characterized by swelling and disorganized cristae and matrix dissolution, with associated increase in PTEN-induced kinase 1 protein expression. ANG II also reduced mitochondrial respiration, indicative of mitochondrial dysfunction. Together, these results demonstrate that ANG II reduces autophagic activity and disrupts mitochondrial ultrastructure and function, likely contributing to skeletal muscle wasting. Therefore, strategies that activate autophagy in skeletal muscle have the potential to prevent or blunt ANG II-induced skeletal muscle wasting in chronic diseases. NEW & NOTEWORTHY Our study identified a novel mechanism whereby angiotensin II (ANG II) impairs mitochondrial energy metabolism in skeletal muscle. ANG II suppressed autophagosome formation by inhibiting the UNC-51-like kinase 1(ULK1)-beclin-1 axis, resulting in accumulation of abnormal/damaged and dysfunctional mitochondria and reduced mitochondrial respiratory capacity. Therapeutic strategies that activate the ULK1-beclin-1 axis have the potential to delay or reverse skeletal muscle wasting in chronic diseases characterized by increased systemic ANG II levels.

Comparison of two cleaning and sterilization protocols of diamond burr tips used in debridement for canine superficial chronic corneal epithelial defects.

OBJECTIVES: To serially evaluate morphologic and elemental composition changes to diamond burr tips (DBTs) comparing two sterilization protocols. ANIMALS STUDIED: A total of 300 fresh cadaver porcine globes. PROCEDURES: Six DBTs were randomly, equally assigned into Group 1 or 2, and then analyzed using Scanning Electron Microscopy (SEM) and Energy Dispersive Spectroscopy (EDS) at 0, 25, 50, and 100 cycles. Diamond burr debridement (DBD) was performed for 120 seconds on corneal stroma using the Algerbrush®. DBTs were cleaned, and then: Group 1 was sterilized by Germinator 500™; and Group 2 underwent ultrasonic cleaning and pre-vacuum autoclave. A cycle is defined as one DBD, cleaning and sterilization protocol. Data were quantified using custom MatLab program. RESULTS: Energy Dispersive Spectroscopy revealed minor buildup of sulfur on both groups. Group 1 displayed major buildup of carbon and calcium. All DBTs were stippled with inorganic particulate at baseline. Particulates were no longer present on Group 2 by 25 cycles, but remained on Group 1 at all time points. There was significantly more buildup on Group 1 at all time points (P = 0.0000, 0.0009, and 0.0003 for 25, 50, and 100 cycles, respectively). More damage to Group 2 at all time points (P = 0.003, 0.002, and 0.003 for 25, 50, and 100 cycles, respectively) was observed. CONCLUSIONS: No significant damage to Group 1 DBTs was noted after 100 cycles, however, particulate matter is not adequately removed using this sterilization technique. Ultrasonic cleaning is warranted between DBDs to achieve adequate particulate removal prior to sterilization; greater damage occurs with this technique which supports replacing DBTs regularly.

Multi-Focal Neuronal Ultrastructural Abnormalities and Synaptic Alterations in Mice after Low-Intensity Blast Exposure.

Service members during military actions or combat training are exposed frequently to primary blast generated by explosive weaponry. The majority of military-related neurotrauma are classified as mild and designated as "invisible injuries" that are prevalent during current conflicts. While the previous experimental blast injury studies using moderate- to high-intensity exposures focused mainly on gross and microscopic neuropathology, our previous studies have shown that low-intensity blast (LIB) exposures resulted in nanoscale subcellular myelin and mitochondrial damages and subsequent behavioral disorders in the absence of gross or detectable cellular damage. In this study, we used transmission electron microscopy to delineate the LIB effects at the ultrastructural level specifically focusing on the neuron perikaryon, axons, and synapses in the cortex and hippocampus of mice at seven and 30 days post-injury (DPI). We found dysmorphic dark neuronal perikaryon and "cytoplasmic aeration" of dendritic processes, as well as increased microtubular fragmentation of the myelinated axons along with biochemically measured elevated tau/phosphorylated tau/Aß levels. The number of cortical excitatory synapses decreased along with a compensatory increase of the post-synaptic density (PSD) thickness both at seven and 30 DPI, while the amount of hippocampal CA1 synapses increased with the reduced PSD thickness. In addition, we observed a significant increase in protein levels of PSD95 and synaptophysin mainly at seven DPI indicating potential synaptic reorganization. These results demonstrated that a single LIB exposure can lead to ultrastructural brain injury with accompanying multi-focal neuronal organelle alterations. This pre-clinical study provides key insights into disease pathogenesis related to primary blast exposure.

Inhibition of regrowth of planktonic and biofilm bacteria after peracetic acid disinfection.

Peracetic acid (PAA) is a promising alternative to chlorine for disinfection; however, bacterial regrowth after PAA disinfection is poorly understood. This study compared the regrowth of bacteria (Gram-negative Pseudomonas aeruginosa PAO1 and Gram-positive Bacillus sp.) after disinfection with PAA or free chlorine. In the absence of organic matter, PAA and free chlorine prevented the regrowth of planktonic cells of P. aeruginosa PAO1 at C·t (= disinfectant concentration × contact time) doses of (28.5 ±â€¯9.8) mg PAA·min·L-1 and (22.5 ±â€¯10.6) mg Cl2·min·L-1, respectively, suggesting that they had comparable efficiencies in preventing the regrowth of planktonic bacteria. For comparison, the minimum C·t doses of PAA and free chlorine to prevent the regrowth of P. aeruginosa PAO1 biofilm cells in the absence of organic matter were (14,000 ±â€¯1,732) mg PAA·min·L-1 and (6,500 ±â€¯2,291) mg Cl2·min·L-1, respectively. PAA was less effective than free chlorine in killing bacteria within biofilms in the absence of organic matter most likely because PAA reacts with biofilm matrix constituents slower than free chlorine. In the presence of organic matter, although the bactericidal efficiencies of both disinfectants significantly decreased, PAA was less affected due to its slower reaction with organic matter and/or slower self-decomposition. For instance, in a dilute Lysogeny broth-Miller, the minimum concentrations of PAA and free chlorine to prevent the regrowth of planktonic P. aeruginosa PAO1 were 20 mg PAA·L-1 and 300 mg Cl2·L-1, respectively. While both disinfectants are strong oxidants disrupting cell membrane, environmental scanning electron microscopy (ESEM) revealed that PAA made holes in the center of the cells, whereas free chlorine desiccated the cells. Overall, this study shows that PAA is a powerful disinfectant to prevent bacterial regrowth even in the presence of organic matter.

Ultrastructural Analysis of Chikungunya Virus Dissemination from the Midgut of the Yellow Fever Mosquito, Aedes aegypti.

The transmission cycle of chikungunya virus (CHIKV) requires that mosquito vectors get persistently infected with the virus, following its oral acqsuisition from a vertebrate host. The mosquito midgut is the initial organ that gets infected with orally acquired CHIKV. Following its replication in the midgut epithelium, the virus exits the midgut and infects secondary tissues including the salivary glands before being transmitted to another host. Here, we investigate the pattern of CHIKV dissemination from the midgut of Aedes aegypti at the ultrastructural level. Bloodmeal ingestion caused overstretching of the midgut basal lamina (BL), which was disrupted in areas adjacent to muscles surrounding the midgut as shown by scanning electron microscopy (SEM). Using both transmission electron microscopy (TEM) and focused ion beam scanning electron microscopy (FIB-SEM) to analyze midgut preparations, mature chikungunya (CHIK) virions were found accumulating at the BL and within strands of the BL at 24⁻32 h post-infectious bloodmeal (pibm). From 48 h pibm onwards, virions no longer congregated at the BL and became dispersed throughout the basal labyrinth of the epithelial cells. Ingestion of a subsequent, non-infectious bloodmeal caused mature virions to congregate again at the midgut BL. Our study suggests that CHIKV needs a single replication cycle in the midgut epithelium before mature virions directly traverse the midgut BL during a relatively narrow time window, within 48 h pibm.

NAD+ promotes assembly of the active tetramer of aldehyde dehydrogenase 7A1.

Nicotinamide adenine dinucleotide (NAD) is the redox cofactor of many enzymes, including the vast aldehyde dehydrogenase (ALDH) superfamily. Although the function of NAD(H) in hydride transfer is established, its influence on protein structure is less understood. Herein, we show that NAD+ -binding promotes assembly of the ALDH7A1 tetramer. Multiangle light scattering, small-angle X-ray scattering, and sedimentation velocity all show a pronounced shift of the dimer-tetramer equilibrium toward the tetramer when NAD+ is present. Furthermore, electron microscopy shows that cofactor binding enhances tetramer formation even at the low enzyme concentration used in activity assays, suggesting the tetramer is the active species. Altogether, our results suggest that the catalytically active oligomer of ALDH7A1 is assembled on demand in response to cofactor availability.

The Chaperonin GroEL: A Versatile Tool for Applied Biotechnology Platforms.

The nucleotide-free chaperonin GroEL is capable of capturing transient unfolded or partially unfolded states that flicker in and out of existence due to large-scale protein dynamic vibrational modes. In this work, three short vignettes are presented to highlight our continuing advances in the application of GroEL biosensor biolayer interferometry (BLI) technologies and includes expanded uses of GroEL as a molecular scaffold for electron microscopy determination. The first example presents an extension of the ability to detect dynamic pre-aggregate transients in therapeutic protein solutions where the assessment of the kinetic stability of any folded protein or, as shown herein, quantitative detection of mutant-type protein when mixed with wild-type native counterparts. Secondly, using a BLI denaturation pulse assay with GroEL, the comparison of kinetically controlled denaturation isotherms of various von Willebrand factor (vWF) triple A domain mutant-types is shown. These mutant-types are single point mutations that locally disorder the A1 platelet binding domain resulting in one gain of function and one loss of function phenotype. Clear, separate, and reproducible kinetic deviations in the mutant-type isotherms exist when compared with the wild-type curve. Finally, expanding on previous electron microscopy (EM) advances using GroEL as both a protein scaffold surface and a release platform, examples are presented where GroEL-protein complexes can be imaged using electron microscopy tilt series and the low-resolution structures of aggregation-prone proteins that have interacted with GroEL. The ability of GroEL to bind hydrophobic regions and transient partially folded states allows one to employ this unique molecular chaperone both as a versatile structural scaffold and as a sensor of a protein's folded states.

Redox Modulation of Oligomeric State in Proline Utilization A.

Homooligomerization of proline utilization A (PutA) bifunctional flavoenzymes is intimately tied to catalytic function and substrate channeling. PutA from Bradyrhizobium japonicum (BjPutA) is unique among PutAs in that it forms a tetramer in solution. Curiously, a dimeric BjPutA hot spot mutant was previously shown to display wild-type catalytic activity despite lacking the tetrameric structure. These observations raised the question of what is the active oligomeric state of BjPutA. Herein, we investigate the factors that contribute to tetramerization of BjPutA in vitro. Negative-stain electron microscopy indicates that BjPutA is primarily dimeric at nanomolar concentrations, suggesting concentration-dependent tetramerization. Further, sedimentation-velocity analysis of BjPutA at high (micromolar) concentration reveals that although the binding of active-site ligands does not alter oligomeric state, reduction of the flavin adenine dinucleotide cofactor results in dimeric protein. Size-exclusion chromatography coupled with multiangle light scattering and small-angle x-ray scattering analysis also reveals that reduced BjPutA is dimeric. Taken together, these results suggest that the BjPutA oligomeric state is dependent upon both enzyme concentration and the redox state of the flavin cofactor. This is the first report, to our knowledge, of redox-linked oligomerization in the PutA family.

Immunomodulatory vasoactive intestinal peptide amphiphile micelles.

Two different vasoactive intestinal peptide (VIP) amphiphiles have been formulated which readily form micelles of varying shapes. Interestingly, VIP micelle structure has been found to directly correlate to anti-inflammatory behavior providing evidence that these biomaterials can serve as a promising new therapeutic modality.

Ultrastructural brain abnormalities and associated behavioral changes in mice after low-intensity blast exposure.

Explosive blast-induced mild traumatic brain injury (mTBI), a "signature wound" of recent military conflicts, commonly affects service members. While past blast injury studies have provided insights into TBI with moderate- to high-intensity explosions, the impact of primary low-intensity blast (LIB)-mediated pathobiology on neurological deficits requires further investigation. Our prior considerations of blast physics predicted ultrastructural injuries at nanoscale levels. Here, we provide quantitative data using a primary LIB injury murine model exposed to open field detonation of 350 g of high-energy explosive C4. We quantified ultrastructural and behavioral changes up to 30 days post blast injury (DPI). The use of an open-field experimental blast generated a primary blast wave with a peak overpressure of 6.76 PSI (46.6 kPa) at a 3-m distance from the center of the explosion, a positive phase duration of approximate 3.0 milliseconds (ms), a maximal impulse of 8.7 PSI × ms and a sharp rising time of 9 × 10-3 ms, with no apparent impact/acceleration in exposed animals. Neuropathologically, myelinated axonal damage was observed in blast-exposed groups at 7 DPI. Using transmission electron microscopy, we observed and quantified myelin sheath defects and mitochondrial abnormalities at 7 and 30 DPI. Inverse correlations between blast intensities and neurobehavioral outcomes including motor activities, anxiety levels, nesting behavior, spatial learning and memory occurred. These observations uncover unique ultrastructural brain abnormalities and associated behavioral changes due to primary blast injury and provide key insights into its pathogenesis and potential treatment.