CT perfusion for Assessment of poor Neurological outcome in Comatose Cardiac Arrest Patients (CANCCAP): protocol for a prospective study.

INTRODUCTION: Cardiac arrest remains one of the most common causes of death with the majority occurring outside of hospitals (out of hospital cardiac arrest). Despite advancements in resuscitation management, approximately 50% of comatose cardiac arrest patients (CCAP) will suffer a severe unsurvivable brain injury. To assess brain injury, a neurological examination is conducted, however, its reliability in predicting outcomes in the first days following cardiac arrest is limited. Non-contrast CT is the most employed scan to assess hypoxic changes, even though it is not sensitive to early hypoxic-ischaemic changes in the brain. CT perfusion (CTP) has shown high sensitivity and specificity in brain death patients, although its use in predicting poor neurological outcome in CCAP has not yet been explored. The purpose of this study is to validate CTP for predicting poor neurological outcome (modified Rankin scale, mRS≥4) at hospital discharge in CCAP. METHODS AND ANALYSIS: The CT Perfusion for Assessment of poor Neurological outcome in Comatose Cardiac Arrest Patients study is a prospective cohort study funded by the Manitoba Medical Research Foundation. Newly admitted CCAP receiving standard Targeted Temperature Management are eligible. Patients undergo a CTP at the same time as the admission standard of care head CT. Admission CTP findings will be compared with the reference standard of an accepted bedside clinical assessment at the time of admission. Deferred consent will be used. The primary outcome is a binary outcome of good neurological status, defined as mRs<4 or poor neurological status (mRs≥4) at hospital discharge. A total of 90 patients will be enrolled. ETHICS AND DISSEMINATION: This study has been approved by the University of Manitoba Health Research Ethics Board. The findings from our study will be disseminated through peer-reviewed journals and presentations at local rounds, national and international conferences. The public will be informed at the end of the study. TRIAL REGISTRATION NUMBER: NCT04323020.

Structural snapshots of base excision by the cancer-associated variant MutY N146S reveal a retaining mechanism.

DNA glycosylase MutY plays a critical role in suppression of mutations resulted from oxidative damage, as highlighted by cancer-association of the human enzyme. MutY requires a highly conserved catalytic Asp residue for excision of adenines misinserted opposite 8-oxo-7,8-dihydroguanine (OG). A nearby Asn residue hydrogen bonds to the catalytic Asp in structures of MutY and its mutation to Ser is an inherited variant in human MUTYH associated with colorectal cancer. We captured structural snapshots of N146S Geobacillus stearothermophilus MutY bound to DNA containing a substrate, a transition state analog and enzyme-catalyzed abasic site products to provide insight into the base excision mechanism of MutY and the role of Asn. Surprisingly, despite the ability of N146S to excise adenine and purine (P) in vitro, albeit at slow rates, N146S-OG:P complex showed a calcium coordinated to the purine base altering its conformation to inhibit hydrolysis. We obtained crystal structures of N146S Gs MutY bound to its abasic site product by removing the calcium from crystals of N146S-OG:P complex to initiate catalysis in crystallo or by crystallization in the absence of calcium. The product structures of N146S feature enzyme-generated ß-anomer abasic sites that support a retaining mechanism for MutY-catalyzed base excision.

G-quadruplexes sense natural porphyrin metabolites for regulation of gene transcription and chromatin landscapes.

BACKGROUND: G-quadruplexes (G4s) are unique noncanonical nucleic acid secondary structures, which have been proposed to physically interact with transcription factors and chromatin remodelers to regulate cell type-specific transcriptome and shape chromatin landscapes. RESULTS: Based on the direct interaction between G4 and natural porphyrins, we establish genome-wide approaches to profile where the iron-liganded porphyrin hemin can bind in the chromatin. Hemin promotes genome-wide G4 formation, impairs transcription initiation, and alters chromatin landscapes, including decreased H3K27ac and H3K4me3 modifications at promoters. Interestingly, G4 status is not involved in the canonical hemin-BACH1-NRF2-mediated enhancer activation process, highlighting an unprecedented G4-dependent mechanism for metabolic regulation of transcription. Furthermore, hemin treatment induces specific gene expression profiles in hepatocytes, underscoring the in vivo potential for metabolic control of gene transcription by porphyrins. CONCLUSIONS: These studies demonstrate that G4 functions as a sensor for natural porphyrin metabolites in cells, revealing a G4-dependent mechanism for metabolic regulation of gene transcription and chromatin landscapes, which will deepen our knowledge of G4 biology and the contribution of cellular metabolites to gene regulation.

Ligand-induced native G-quadruplex stabilization impairs transcription initiation.

G-quadruplexes (G4s) are noncanonical DNA secondary structures formed through the self-association of guanines, and G4s are distributed widely across the genome. G4 participates in multiple biological processes including gene transcription, and G4-targeted ligands serve as potential therapeutic agents for DNA-targeted therapies. However, genome-wide studies of the exact roles of G4s in transcriptional regulation are still lacking. Here, we establish a sensitive G4-CUT&Tag method for genome-wide profiling of native G4s with high resolution and specificity. We find that native G4 signals are cell type-specific and are associated with transcriptional regulatory elements carrying active epigenetic modifications. Drug-induced promoter-proximal RNA polymerase II pausing promotes nearby G4 formation. In contrast, G4 stabilization by G4-targeted ligands globally reduces RNA polymerase II occupancy at gene promoters as well as nascent RNA synthesis. Moreover, ligand-induced G4 stabilization modulates chromatin states and impedes transcription initiation via inhibition of general transcription factors loading to promoters. Together, our study reveals a reciprocal genome-wide regulation between native G4 dynamics and gene transcription, which will deepen our understanding of G4 biology toward therapeutically targeting G4s in human diseases.

Hydrogels as Durable Anti-Icing Coatings Inhibit and Delay Ice Nucleation.

The accumulation of ice on surfaces brings dangerous and costly problems to our daily life. Thus, it would be desirable to design anti-icing coatings for various surfaces. We report a durable anti-icing coating based on mussel-inspired chemistry, which is enabled via fabricating a liquid water layer, achieved by modifying solid substrates with the highly water absorbing property of sodium alginate. Dopamine, the main component of the mussel adhesive protein, is introduced to anchor the sodium alginate in order to render the coating applicable to all types of solid surfaces. Simultaneously, it serves as the cross-linking agent for sodium alginate; thus, the cross-linking degree of the coatings could be easily varied. The non-freezable and freezable water in the coatings with different cross-link degrees all remain liquid-like at subzero conditions and synergistically fulfill the aim of decreasing the temperature of ice nucleation. These anti-icing coatings display excellent stability even under harsh conditions. Furthermore, these coatings can be applied to almost all types of solid surfaces and have great promise in practical applications.

Metal-catechol complexes mediate ice nucleation.

The capability of mediating ice nucleation is pertinent to a broad range of fields. Herein, inspired by metal-catechol coordination found in adhesive proteins in which catechol moieties can construct strong complexes with a diverse array of metal ions, we develop a platform for mediating ice nucleation based on metal-catechol complexes and demonstrate that ice nucleation can be successively mediated by varying the characteristics and valence of the metal in metal-catechol complexes.

Molecular evolution of paclitaxel biosynthetic genes TS and DBAT of Taxus species.

Evolutionary patterns of sequence divergence were analyzed in genes from the conifer genus Taxus (yew), encoding paclitaxel biosynthetic enzymes taxadiene synthase (TS) and 10-deacetylbaccatin III-10 beta-O-acetyltransferase (DBAT). N-terminal fragments of TS, full-length DBAT and internal transcribed spacer (ITS) were amplified from 15 closely related Taxus species and sequenced. Premature stop codons were not found in TS and DBAT sequences. Codon usage bias was not found, suggesting that synonymous mutations are selectively neutral. TS and DBAT gene trees are not consistent with the ITS tree, where species formed monophyletic clades. In fact, for both genes, alleles were sometimes shared across species and parallel amino acid substitutions were identified. While both TS and DBAT are, overall, under purifying selection, we identified a number of amino acids of TS under positive selection based on inference using maximum likelihood models. Positively selected amino acids in the N-terminal region of TS suggest that this region might be more important for enzyme function than previously thought. Moreover, we identify lineages with significantly elevated rates of amino acid substitution using a genetic algorithm. These findings demonstrate that the pattern of adaptive paclitaxel biosynthetic enzyme evolution can be documented between closely related Taxus species, where species-specific taxane metabolism has evolved recently.

Phylogenetic relationships of the genus Taxus inferred from chloroplast intergenic spacer and nuclear coding DNA.

A cladistic analysis of the medicinal plant Taxus, using the sequences of one chloroplast (trnS-trnQ spacer) and three nuclear taxadiene synthase (TS), 10-deacetylbaccatin III-10beta-O-acetyltransferase (DBAT), and 18S rDNA) molecular markers, was carried out by distance, parsimony, likelihood, and Bayesian methods. Three of the four New World species (T. brevifolia, T. floridana and T. globosa) form a well-supported clade, whereas T. canadensis initially branches-appearing distantly related to both Old World taxa and New World species. In Asia, Taxus chinensis, T. mairei, T. sumatrana and T. wallichiana cluster together and are sister to a clade containing T. baccata and T. contorta. Taxus yunnanensis is more closely related to T. wallichiana than to four other Taxus species in our study from China; T. contorta is closer to the Euro-Mediterranean T. baccata than to the Asian species. This study provides a genetic method for authentication of economically important Taxus species and proposes a robust phylogenetic hypothesis for the genus. Using trnS-trnQ spacer sequences, we were able to distinguish T. mairei from all other species of Taxus.