Development of the next-generation functional neuro-cognitive imaging protocol - Part 1: A 3D sliding-window convolutional neural net for automated brain parcellation.

Functional MRI has emerged as a powerful tool to assess the severity of Post-concussion syndrome (PCS) and to provide guidance for neuro-cognitive therapists during treatment. The next-generation functional neuro-cognitive imaging protocol (fNCI2) has been developed to provide this assessment. This paper covers the first step in the analysis process, the development of a rapidly re-trainable, machine-learning, brain parcellation tool. The use of a sufficiently deep U-Net architecture encompassing a small (39 × 39 × 39 voxel input, 27 × 27 × 27 voxel output) sliding window to sample the entirety of the 3D image allows for the prediction of the entire image using only a single trained network. A large number of training, validating, and testing windows are thus generated from the 101 manually-labeled Mindboggle images, and full-image prediction is provided via a voxel-vote method using overlapping windows. Our method produces parcellated images that are highly consistent with standard atlas-based methods in under 3 min on a modern GPU, and the single network architecture allows for rapid retraining (<36 hr) as needed.

Characterization and engineering of a two-enzyme system for plastics depolymerization.

Plastics pollution represents a global environmental crisis. In response, microbes are evolving the capacity to utilize synthetic polymers as carbon and energy sources. Recently, Ideonella sakaiensis was reported to secrete a two-enzyme system to deconstruct polyethylene terephthalate (PET) to its constituent monomers. Specifically, the I. sakaiensis PETase depolymerizes PET, liberating soluble products, including mono(2-hydroxyethyl) terephthalate (MHET), which is cleaved to terephthalic acid and ethylene glycol by MHETase. Here, we report a 1.6 Å resolution MHETase structure, illustrating that the MHETase core domain is similar to PETase, capped by a lid domain. Simulations of the catalytic itinerary predict that MHETase follows the canonical two-step serine hydrolase mechanism. Bioinformatics analysis suggests that MHETase evolved from ferulic acid esterases, and two homologous enzymes are shown to exhibit MHET turnover. Analysis of the two homologous enzymes and the MHETase S131G mutant demonstrates the importance of this residue for accommodation of MHET in the active site. We also demonstrate that the MHETase lid is crucial for hydrolysis of MHET and, furthermore, that MHETase does not turnover mono(2-hydroxyethyl)-furanoate or mono(2-hydroxyethyl)-isophthalate. A highly synergistic relationship between PETase and MHETase was observed for the conversion of amorphous PET film to monomers across all nonzero MHETase concentrations tested. Finally, we compare the performance of MHETase:PETase chimeric proteins of varying linker lengths, which all exhibit improved PET and MHET turnover relative to the free enzymes. Together, these results offer insights into the two-enzyme PET depolymerization system and will inform future efforts in the biological deconstruction and upcycling of mixed plastics.

Characterization and engineering of a plastic-degrading aromatic polyesterase.

Poly(ethylene terephthalate) (PET) is one of the most abundantly produced synthetic polymers and is accumulating in the environment at a staggering rate as discarded packaging and textiles. The properties that make PET so useful also endow it with an alarming resistance to biodegradation, likely lasting centuries in the environment. Our collective reliance on PET and other plastics means that this buildup will continue unless solutions are found. Recently, a newly discovered bacterium, Ideonella sakaiensis 201-F6, was shown to exhibit the rare ability to grow on PET as a major carbon and energy source. Central to its PET biodegradation capability is a secreted PETase (PET-digesting enzyme). Here, we present a 0.92 Å resolution X-ray crystal structure of PETase, which reveals features common to both cutinases and lipases. PETase retains the ancestral α/ß-hydrolase fold but exhibits a more open active-site cleft than homologous cutinases. By narrowing the binding cleft via mutation of two active-site residues to conserved amino acids in cutinases, we surprisingly observe improved PET degradation, suggesting that PETase is not fully optimized for crystalline PET degradation, despite presumably evolving in a PET-rich environment. Additionally, we show that PETase degrades another semiaromatic polyester, polyethylene-2,5-furandicarboxylate (PEF), which is an emerging, bioderived PET replacement with improved barrier properties. In contrast, PETase does not degrade aliphatic polyesters, suggesting that it is generally an aromatic polyesterase. These findings suggest that additional protein engineering to increase PETase performance is realistic and highlight the need for further developments of structure/activity relationships for biodegradation of synthetic polyesters.

Crystal Structures and Nuclear Magnetic Resonance Studies of the Apo Form of the c-MYC:MAX bHLHZip Complex Reveal a Helical Basic Region in the Absence of DNA.

The c-MYC transcription factor is a master regulator of cell growth and proliferation and is an established target for cancer therapy. This basic helix-loop-helix Zip protein forms a heterodimer with its obligatory partner MAX, which binds to DNA via the basic region. Considerable research efforts are focused on targeting the heterodimerization interface and the interaction of the complex with DNA. The only available crystal structure is that of a c-MYC:MAX complex artificially tethered by an engineered disulfide linker and prebound to DNA. We have carried out a detailed structural analysis of the apo form of the c-MYC:MAX complex, with no artificial linker, both in solution using nuclear magnetic resonance (NMR) spectroscopy and by X-ray crystallography. We have obtained crystal structures in three different crystal forms, with resolutions between 1.35 and 2.2 Å, that show extensive helical structure in the basic region. Determination of the α-helical propensity using NMR chemical shift analysis shows that the basic region of c-MYC and, to a lesser extent, that of MAX populate helical conformations. We have also assigned the NMR spectra of the c-MYC basic helix-loop-helix Zip motif in the absence of MAX and showed that the basic region has an intrinsic helical propensity even in the absence of its dimerization partner. The presence of helical structure in the basic regions in the absence of DNA suggests that the molecular recognition occurs via a conformational selection rather than an induced fit. Our work provides both insight into the mechanism of DNA binding and structural information to aid in the development of MYC inhibitors.

Structural basis for Pan3 binding to Pan2 and its function in mRNA recruitment and deadenylation.

The conserved eukaryotic Pan2-Pan3 deadenylation complex shortens cytoplasmic mRNA 3' polyA tails to regulate mRNA stability. Although the exonuclease activity resides in Pan2, efficient deadenylation requires Pan3. The mechanistic role of Pan3 is unclear. Here, we show that Pan3 binds RNA directly both through its pseudokinase/C-terminal domain and via an N-terminal zinc finger that binds polyA RNA specifically. In contrast, isolated Pan2 is unable to bind RNA. Pan3 binds to the region of Pan2 that links its N-terminal WD40 domain to the C-terminal part that contains the exonuclease, with a 2:1 stoichiometry. The crystal structure of the Pan2 linker region bound to a Pan3 homodimer shows how the unusual structural asymmetry of the Pan3 dimer is used to form an extensive high-affinity interaction. This binding allows Pan3 to supply Pan2 with substrate polyA RNA, facilitating efficient mRNA deadenylation by the intact Pan2-Pan3 complex.

Molecular mechanism of sequence-directed DNA loading and translocation by FtsK.

Dimeric circular chromosomes, formed by recombination between monomer sisters, cannot be segregated to daughter cells at cell division. XerCD site-specific recombination at the Escherichia coli dif site converts these dimers to monomers in a reaction that requires the DNA translocase FtsK. Short DNA sequences, KOPS (GGGNAGGG), which are polarized toward dif in the chromosome, direct FtsK translocation. FtsK interacts with KOPS through a C-terminal winged helix domain gamma. The crystal structure of three FtsKgamma domains bound to 8 bp KOPS DNA demonstrates how three gamma domains recognize KOPS. Using covalently linked dimers of FtsK, we infer that three gamma domains per hexamer are sufficient to recognize KOPS and load FtsK and subsequently activate recombination at dif. During translocation, FtsK fails to recognize an inverted KOPS sequence. Therefore, we propose that KOPS act solely as a loading site for FtsK, resulting in a unidirectionally oriented hexameric motor upon DNA.

Acetylation of lysine 120 of p53 endows DNA-binding specificity at effective physiological salt concentration.

Lys120 in the DNA-binding domain (DBD) of p53 becomes acetylated in response to DNA damage. But, the role and effects of acetylation are obscure. We prepared p53 specifically acetylated at Lys120, AcK120p53, by in vivo incorporation of acetylated lysine to study biophysical and structural consequences of acetylation that may shed light on its biological role. Acetylation had no affect on the overall crystal structure of the DBD at 1.9-Å resolution, but significantly altered the effects of salt concentration on specificity of DNA binding. p53 binds DNA randomly in vitro at effective physiological salt concentration and does not bind specifically to DNA or distinguish among its different response elements until higher salt concentrations. But, on acetylation, AcK120p53 exhibited specific DNA binding and discriminated among response elements at effective physiological salt concentration. AcK120p53 and p53 had the highest affinity to the same DNA sequence, although acetylation reduced the importance of the consensus C and G at positions 4 and 7, respectively. Mass spectrometry of p53 and AcK120p53 DBDs bound to DNA showed they preferentially segregated into complexes that were either DNA(p53DBD)(4) or DNA(AcK120DBD)(4), indicating that the different DBDs prefer different quaternary structures. These results are consistent with electron microscopy observations that p53 binds to nonspecific DNA in different, relaxed, quaternary states from those bound to specific sequences. Evidence is accumulating that p53 can be sequestered by random DNA, and target search requires acetylation of Lys120 and/or interaction with other factors to impose specificity of binding via modulating changes in quaternary structure.

Concentration-Dependent Inhibition of Mesophilic PETases on Poly(ethylene terephthalate) Can Be Eliminated by Enzyme Engineering.

Enzyme-based depolymerization is a viable approach for recycling of poly(ethylene terephthalate) (PET). PETase from Ideonella sakaiensis (IsPETase) is capable of PET hydrolysis under mild conditions but suffers from concentration-dependent inhibition. In this study, this inhibition is found to be dependent on incubation time, the solution conditions, and PET surface area. Furthermore, this inhibition is evident in other mesophilic PET-degrading enzymes to varying degrees, independent of the level of PET depolymerization activity. The inhibition has no clear structural basis, but moderately thermostable IsPETase variants exhibit reduced inhibition, and the property is completely absent in the highly thermostable HotPETase, previously engineered by directed evolution, which simulations suggest results from reduced flexibility around the active site. This work highlights a limitation in applying natural mesophilic hydrolases for PET hydrolysis and reveals an unexpected positive outcome of engineering these enzymes for enhanced thermostability.

Assessment of brain activity during memory encoding in a narcolepsy patient on and off modafinil using normative fMRI data.

We present behavioral and functional magnetic resonance imaging (fMRI) findings of a 20-year-old female with narcolepsy who completed a standardized fMRI-adapted face memory task both 'off' and 'on' modafinil compared to a normative sample (N = 38). The patient showed poor recognition performance off modafinil (z = -2.03) but intact performance on modafinil (z = 0.78). fMRI results showed atypical activation during memory encoding off modafinil, with frontal lobe hypoactivity, but hippocampal hyperactivity, whereas all brain regions showed more normalized activation on modafinil. Results from this limited study suggest hippocampal and frontal alterations in individuals with narcolepsy. Further, the results suggest the hypothesis that modafinil may affect brain activation in some people with narcolepsy.

The functional magnetic resonance imaging-based verbal fluency test in obsessive-compulsive disorder.

Clinical use of functional magnetic resonance imaging (fMRI) in obsessive-compulsive disorder (OCD) is limited by a relative absence of fMRI task development, standardization, and normative performance databases. We investigated the fMRI-based verbal fluency test (f-VFT) by quantitatively evaluating brain activation patterns in OCD participants (8 females and 4 males) compared with a normative database (16 females and 16 males). At the group level, OCD participants and references had highly similar activation in left-hemisphere language regions, including the precentral/premotor cortex, thalamus, basal ganglia, and inferior frontal gyrus/frontal operculum. At the interindividual level, however, the OCD group had highly variable activation patterns in the dorsal and ventral regions of the pre-supplementary motor area (pre-SMA) that may correspond with differences in demographic and clinical variables. Further, there were significant correlations in the OCD participants between pre-SMA dorsal and ventral activation and between dorsal pre-SMA activation and perfectionism. Our findings suggest considerable functional anatomical overlap in left-hemisphere language regions between OCD participants and references but significantly higher pre-SMA interindividual variability in OCD compared to the reference group that may be relevant in clinical fMRI application and the theoretical understanding of OCD.

An fMRI investigation of emotional processing of body shape in bulimia nervosa.

OBJECTIVE: Cognitive-behavioral theories of eating disorder etiology emphasize the role of body-oriented self-schemas. Examination of brain regions associated with self-referencing, such as medial prefrontal cortex (mPFC), during processing of body-related stimuli can thus be utilized to evaluate such theories. METHOD: Twelve women with bulima nervosa (BN) and 12 comparison women underwent functional brain imaging while viewing images of women with either thin or overweight bodies in a self-referencing context. RESULTS: For thin bodies, there was no significant mPFC activation for either group. For overweight bodies, mPFC activation was significantly greater for BN patients, with a focus in subregions associated with emotional processing. DISCUSSION: These findings are consistent with cognitive models of eating disorders which posit that negative body-related stimuli are more central to self-schemas and more emotionally provocative in persons with eating disorders, lending support to treatment and prevention interventions that emphasize body overvaluation as a primary target of change.

The FtsK gamma domain directs oriented DNA translocation by interacting with KOPS.

The bacterial septum-located DNA translocase FtsK coordinates circular chromosome segregation with cell division. Rapid translocation of DNA by FtsK is directed by 8-base-pair DNA motifs (KOPS), so that newly replicated termini are brought together at the developing septum, thereby facilitating completion of chromosome segregation. Translocase functions reside in three domains, alpha, beta and gamma. FtsKalphabeta are necessary and sufficient for ATP hydrolysis-dependent DNA translocation, which is modulated by FtsKgamma through its interaction with KOPS. By solving the FtsKgamma structure by NMR, we show that gamma is a winged-helix domain. NMR chemical shift mapping localizes the DNA-binding site on the gamma domain. Mutated proteins with substitutions in the FtsKgamma DNA-recognition helix are impaired in DNA binding and KOPS recognition, yet remain competent in DNA translocation and XerCD-dif site-specific recombination, which facilitates the late stages of chromosome segregation.

SWI/SNF subunit BAF155 N-terminus structure informs the impact of cancer-associated mutations and reveals a potential drug binding site.

SWI/SNF (BAF) chromatin remodelling complexes are key regulators of gene expression programs, and attractive drug targets for cancer therapies. Here we show that the N-terminus of the BAF155/SMARCC1 subunit contains a putative DNA-binding MarR-like domain, a chromodomain and a BRCT domain that are interconnected to each other to form a distinct module. In this structure the chromodomain makes interdomain interactions and has lost its canonical function to bind to methylated lysines. The structure provides new insights into the missense mutations that target this module in cancer. This study also reveals two adjacent, highly-conserved pockets in a cleft between the domains that form a potential binding site, which can be targeted with small molecules, offering a new strategy to target SWI/SNF complexes.

Neuroimaging in Pediatric Patients with Mild Traumatic Brain Injury: Relating the Current 2018 Centers for Disease Control Guideline and the Potential of Advanced Neuroimaging Modalities for Research and Clinical Biomarker Development.

The Center for Disease Control and Prevention (CDC)'s 2018 Guideline for current practices in pediatric mild traumatic brain injury (mTBI; also referred to as concussion herein) systematically identified the best up-to-date practices based on current evidence and, specifically, identified recommended practices regarding computed tomography (CT), magnetic resonance imaging (MRI), and skull radiograph imaging. In this article, we discuss types of neuroimaging not discussed in the guideline in terms of their safety for pediatric populations, their potential application, and the research investigating the future use of certain modalities to aid in the diagnosis and treatment of mTBI in children. The role of neuroimaging in pediatric mTBI cases should be considered for the potential contribution to children's neural and social development, in addition to the immediate clinical value (as in the case of acute structural findings). Selective use of specific neuroimaging modalities in research has already been shown to detect aspects of diffuse brain injury, disrupted cerebral blood flow, and correlate physiological factors with persistent symptoms, such as fatigue, cognitive decline, headache, and mood changes, following mTBI. However, these advanced neuroimaging modalities are currently limited to the research arena, and any future clinical application of advanced imaging modalities in pediatric mTBI will require robust evidence for each modality's ability to provide measurement of the subtle conditions of brain development, disease, damage, or degeneration, while accounting for variables at both non-injury and time-post-injury epochs. Continued collaboration and communication between researchers and healthcare providers is essential to investigate, develop, and validate the potential of advanced imaging modalities in pediatric mTBI diagnostics and management.

Engineering a Cytochrome P450 for Demethylation of Lignin-Derived Aromatic Aldehydes.

Biological funneling of lignin-derived aromatic compounds is a promising approach for valorizing its catalytic depolymerization products. Industrial processes for aromatic bioconversion will require efficient enzymes for key reactions, including demethylation of O-methoxy-aryl groups, an essential and often rate-limiting step. The recently characterized GcoAB cytochrome P450 system comprises a coupled monoxygenase (GcoA) and reductase (GcoB) that catalyzes oxidative demethylation of the O-methoxy-aryl group in guaiacol. Here, we evaluate a series of engineered GcoA variants for their ability to demethylate o-and p-vanillin, which are abundant lignin depolymerization products. Two rationally designed, single amino acid substitutions, F169S and T296S, are required to convert GcoA into an efficient catalyst toward the o- and p-isomers of vanillin, respectively. Gain-of-function in each case is explained in light of an extensive series of enzyme-ligand structures, kinetic data, and molecular dynamics simulations. Using strains of Pseudomonas putida KT2440 already optimized for p-vanillin production from ferulate, we demonstrate demethylation by the T296S variant in vivo. This work expands the known aromatic O-demethylation capacity of cytochrome P450 enzymes toward important lignin-derived aromatic monomers.

Different patterns of cerebral activation in genuine and malingered cognitive effort during performance on the Word Memory Test.

PRIMARY OBJECTIVE: The Word Memory Test (WMT) is a popular symptom validity test in which individuals are required to remember and recall semantically-related word pairs. Research shows successful WMT completion employs a wide neural network which is involved in tasks requiring high cognitive effort. The primary purpose of this study was to replicate earlier fMRI findings using a larger sample and extend previous findings by including male and female subjects. The second purpose was to investigate the neural networks involved during intentional malingering on the WMT. RESEARCH DESIGN: For all trials, a time-series ANCOVA design was implemented using SPM5 software. METHODS AND PROCEDURES: Ten subjects (five male and five female) underwent fMRI imaging while completing the WMT in full-effort and simulated poor effort conditions. MAIN OUTCOME AND RESULTS: Full-effort trials found activation peaks in dorsolateral prefrontal cortex, superior parietal lobe, anterior cingulate, bilateral lingual cortices and anterior insula/frontal operculum, supporting earlier findings. Simulated poor effort trials had similar foci of activation, with additional peak strength in surrounding cortical regions identified previously as relevant to simulated malingering. No sex differences were observed in either condition. CONCLUSIONS: These findings demonstrate the neural underpinnings of WMT performance in normal and simulated performance.

Structure of the BRK domain of the SWI/SNF chromatin remodeling complex subunit BRG1 reveals a potential role in protein-protein interactions.

BRG1/SMARCA4 and its paralog BRM/SMARCA2 are the ATPase subunits of human SWI/SNF chromatin remodeling complexes. These multisubunit assemblies can act as either tumor suppressors or drivers of cancer, and inhibiting both BRG1 and BRM, is emerging as an effective therapeutic strategy in diverse cancers. BRG1 and BRM contain a BRK domain. The function of this domain is unknown, but it is often found in proteins involved in transcription and developmental signaling in higher eukaryotes, in particular in proteins that remodel chromatin. We report the NMR structure of the BRG1 BRK domain. It shows similarity to the glycine-tyrosine-phenylalanine (GYF) domain, an established protein-protein interaction module. Computational peptide-binding-site analysis of the BRK domain identifies a binding site that coincides with a highly conserved groove on the surface of the protein. This sets the scene for experiments to elucidate the role of this domain, and evaluate the potential of targeting it for cancer therapy.

An fMRI study of sex differences in brain activation during object naming.

We present a neuroimaging experiment that examines whether males and females use distinct brain systems while performing a confrontational naming task, with specific attention to the possibility of laterality differences, as suggested by some theories of sex differences in language processing. We further address whether sex-based differences in functional brain organization might interact with object category distinctions, given that previous behavioral studies have shown some consistent processing differences between the sexes with respect to tools versus plants. Functional magnetic resonance imaging (fMRI) data were collected from 26 participants (13 males and 13 females). Main effect and interaction analyses reveal no discernable laterality differences between the sexes. All other results, however, were consistent with previous object-naming studies. Global effects revealed dominant foci in fusiform gyrus, left posterior middle temporal gyrus, left basal ganglia/thalamus, left middle/inferior frontal gyri, left frontal operculum, left supplementary motor area/dorsal anterior cingulate, and left pre-central gyrus. Main contrasts for tools versus plants were likewise consistent with previous fMRI studies. Although men and women showed no discernable activation differences, hemispheric or otherwise, when collapsed across object categories, sex-by-category analyses showed selective activation for females in dorsal anterior cingulate gyrus and left posterior middle temporal gyrus for tools, and selective activation for males in left posterior middle temporal gyrus for plants. We discuss the relevance of these sex-by-category effects to previous behavioral findings and theories that relate to vocabulary differences between the sexes.

Solution structure of the BRK domains from CHD7.

CHD7 is a member of the chromodomain helicase DNA binding domain (CHD) family of ATP-dependent chromatin remodelling enzymes. It is mutated in CHARGE syndrome, a multiple congenital anomaly condition. CHD7 is one of a subset of CHD proteins, unique to metazoans that contain the BRK domain, a protein module also found in the Brahma/BRG1 family of helicases. We describe here the NMR solution structure of the two BRK domains of CHD7. Each domain has a compact betabetaalphabeta fold. The second domain has a C-terminal extension consisting of two additional helices. The structure differs from those of other domains present in chromatin-associated proteins.

The structure of INI1/hSNF5 RPT1 and its interactions with the c-MYC:MAX heterodimer provide insights into the interplay between MYC and the SWI/SNF chromatin remodeling complex.

c-MYC and the SWI/SNF chromatin remodeling complex act as master regulators of transcription, and play a key role in human cancer. Although they are known to interact, the molecular details of their interaction are lacking. We have determined the structure of the RPT1 region of the INI1/hSNF5/BAF47/SMARCB1 subunit of the SWI/SNF complex that acts as a c-MYC-binding domain, and have localized the interaction regions on both INI1 and on the c-MYC:MAX heterodimer. c-MYC interacts with a highly conserved groove on INI1, while INI1 binds to the c-MYC helix-loop-helix region. The binding site overlaps with the c-MYC DNA-binding region, and we show that binding of INI1 and E-box DNA to c-MYC:MAX are mutually exclusive.