Methods for analyzing chromosome conformation in mammalian cells are either low resolution or low throughput and are technically challenging. In next-generation (NG) Capture-C, we have redesigned the ...Capture-C method to achieve unprecedented levels of sensitivity and reproducibility. NG Capture-C can be used to analyze many genetic loci and samples simultaneously. High-resolution data can be produced with as few as 100,000 cells, and single-nucleotide polymorphisms can be used to generate allele-specific tracks. The method is straightforward to perform and should greatly facilitate the investigation of many questions related to gene regulation as well as the functional dissection of traits examined in genome-wide association studies.
This study aimed to provide an update and compare perioperative outcomes and complications of intracorporeal and extracorporeal urinary diversion following robot-assisted radical cystectomy using ...data from the multi-institutional, prospectively maintained International Robotic Cystectomy Consortium database.
We retrospectively reviewed the records of 2,125 patients from a total of 26 institutions. Intracorporeal urinary diversion was compared with extracorporeal urinary diversion. Multivariate logistic regression models using stepwise variable selection were fit to evaluate preoperative, operative and postoperative predictors of intracorporeal urinary diversion, operative time, high grade complications and 90-day hospital readmissions after robot-assisted radical cystectomy.
In our cohort 1,094 patients (51%) underwent intracorporeal urinary diversion. These patients demonstrated shorter operative time (357 vs 400 minutes), less blood loss (300 vs 350 ml) and fewer blood transfusions (4% vs 19%, all p <0.001). They experienced more high grade complications (13% vs 10%, p = 0.02). Intracorporeal urinary diversion use increased from 9% of all urinary diversions in 2005 to 97% in 2015. Complications after this procedure decreased significantly with time (p <0.001). On multivariable analysis higher annual cystectomy volume (OR 1.02, 95% CI 1.01–1.03, p <0.002), year of robot-assisted radical cystectomy (2013–2016 OR 68, 95% CI 44–105, p <0.001) and American Society of Anesthesiologists® score less than 3 (OR 1.75, 95% CI 1.38–2.22, p <0.001) were associated with undergoing intracorporeal urinary diversion. The procedure was associated with a shorter operative time of 27 minutes (p = 0.001).
The use of intracorporeal urinary diversion has increased in the last decade. A higher annual institutional volume of robot-assisted radical cystectomy was associated with intracorporeal urinary diversion as well as with shorter operative time. Although intracorporeal urinary diversion was associated with higher grade complications than extracorporeal urinary diversion, they decreased with time.
In higher eukaryotes, many genes are regulated by enhancers that are 10
-10
base pairs (bp) away from the promoter. Enhancers contain transcription-factor-binding sites (which are typically around ...7-22 bp), and physical contact between the promoters and enhancers is thought to be required to modulate gene expression. Although chromatin architecture has been mapped extensively at resolutions of 1 kilobase and above; it has not been possible to define physical contacts at the scale of the proteins that determine gene expression. Here we define these interactions in detail using a chromosome conformation capture method (Micro-Capture-C) that enables the physical contacts between different classes of regulatory elements to be determined at base-pair resolution. We find that highly punctate contacts occur between enhancers, promoters and CCCTC-binding factor (CTCF) sites and we show that transcription factors have an important role in the maintenance of the contacts between enhancers and promoters. Our data show that interactions between CTCF sites are increased when active promoters and enhancers are located within the intervening chromatin. This supports a model in which chromatin loop extrusion
is dependent on cohesin loading at active promoters and enhancers, which explains the formation of tissue-specific chromatin domains without changes in CTCF binding.
The function of adult neurogenesis in the rodent brain remains unclear. Ablation of adult born neurons has yielded conflicting results about emotional and cognitive impairments. One hypothesis is ...that adult neurogenesis in the hippocampus enables spatial pattern separation, allowing animals to distinguish between similar stimuli. We investigated whether spatial pattern separation and other putative hippocampal functions of adult neurogenesis were altered in a novel genetic model of neurogenesis ablation in the rat. In rats engineered to express thymidine kinase (TK) from a promoter of the rat glial fibrillary acidic protein (GFAP), ganciclovir treatment reduced new neurons by 98%. GFAP-TK rats showed no significant difference from controls in spatial pattern separation on the radial maze, spatial learning in the water maze, contextual or cued fear conditioning. Meta-analysis of all published studies found no significant effects for ablation of adult neurogenesis on spatial memory, cue conditioning or ethological measures of anxiety. An effect on contextual freezing was significant at a threshold of 5% (P = 0.04), but not at a threshold corrected for multiple testing. The meta-analysis revealed remarkably high levels of heterogeneity among studies of hippocampal function. The source of this heterogeneity remains unclear and poses a challenge for studies of the function of adult neurogenesis.
Phosphorylation sites are hyperabundant in the eukaryotic disordered proteome, suggesting that conformational fluctuations play a major role in determining to what extent a kinase interacts with a ...particular substrate. In biophysical terms, substrate selectivity may be determined not just by the structural–chemical complementarity between the kinase and its protein substrates but also by the free energy difference between the conformational ensembles that are, or are not, recognized by the kinase. To test this hypothesis, we developed a statistical-thermodynamics-based informatics framework, which allows us to probe for the contribution of equilibrium fluctuations to phosphorylation, as evaluated by the ability to predict Ser/Thr/Tyr phosphorylation sites in the disordered proteome. Essential to this framework is a decomposition of substrate sequence information into two types: vertical information encoding conserved kinase specificity motifs and horizontal information encoding substrate conformational equilibrium that is embedded, but often not apparent, within position-specific conservation patterns. We find not only that conformational fluctuations play a major role but also that they are the dominant contribution to substrate selectivity. In fact, the main substrate classifier distinguishing selectivity is the magnitude of change in local compaction of the disordered chain upon phosphorylation of these mostly singly phosphorylated sites. In addition to providing fundamental insights into the consequences of phosphorylation across the proteome, our approach provides a statistical-thermodynamic strategy for partitioning any sequencebased search into contributions from structural–chemical complementarity and those from changes in conformational equilibrium.
Abstract
Background
Cannabidiol (CBD) exhibits anti-inflammatory properties that could improve disease activity in inflammatory bowel disease. This proof-of-concept study assessed efficacy, safety ...and tolerability of CBD-rich botanical extract in ulcerative colitis (UC) patients.
Methods
Patients aged 18 years or older, with left-sided or extensive UC, Mayo scores of 4-10 (endoscopy scores ≥1), and on stable 5-aminosalicylic acid dosing, were randomized to 10-weeks' CBD-rich botanical extract or placebo capsules. The primary endpoint was the percentage of patients in remission after treatment. Statistical testing was 2-sided, using a 10% significance level.
Results
Patients were less tolerant of CBD-rich botanical extract compared with placebo, taking on average one-third fewer capsules, and having more compliance-related protocol deviations (principally insufficient exposure), prompting identification of a per protocol (PP) analysis set. The primary endpoint was negative; end of treatment remission rates were similar for CBD-rich botanical extract (28%) and placebo (26%). However, PP analysis of total and partial Mayo scores favoured CBD-rich botanical extract (P = 0.068 and P = 0.038, respectively). Additionally, PP analyses of the more subjective physician's global assessment of illness severity, subject global impression of change, and patient-reported quality-of-life outcomes were improved for patients taking CBD-rich botanical extract (P = 0.069, P = 0.003, and P = 0.065, respectively). Adverse events (AEs) were predominantly mild/moderate with many in the CBD-rich botanical extract group potentially attributable to the ∆9-tetrahydrocannabinol content. A greater proportion of gastrointestinal-related AEs, indicative of UC worsening, was seen on placebo.
Conclusion
Although the primary endpoint was not reached, several signals suggest CBD-rich botanical extract may be beneficial for symptomatic treatment of UC.
Herein, we present the cathodic paths of the Group-7 metal complex Re(3,3′-DHBPY)(CO)3Cl (3,3′-DHBPY = 3,3′-dihydroxy-2,2′-bipyridine) producing a moderately active catalyst of electrochemical ...reduction of CO2 to CO. The combined techniques of cyclic voltammetry and IR/UV–vis spectroelectrochemistry have revealed significant differences in the chemistry of the electrochemically reduced parent complex compared to the previously published Re/4,4′-DHBPY congener. The initial irreversible cathodic step in weakly coordinating THF is shifted toward much less negative electrode potentials, reflecting facile reductive deprotonation of one hydroxyl group and strong intramolecular hydrogen bonding, O–H···O–. The latter process occurs spontaneously in basic dimethylformamide where Re/4,4′-DHBPY remains stable. The subsequent reduction of singly deprotonated Re(3,3′-DHBPY-H+)(CO)3Cl− under ambient conditions occurs at a cathodic potential close to that of the Re/4,4′-DHBPY-H+ derivative. However, for the stabilized 3,3′-DHBPY-H+ ligand, the latter process at the second cathodic wave is more complex and involves an overall transfer of three electrons. Rapid potential step electrolysis induces 1e–-reductive cleavage of the second O–H bond, triggering dissociation of the Cl– ligand from Re(3,3′-DHBPY-2H+)(CO)3Cl2–. The ultimate product of the second cathodic step in THF was identified as 5-coordinate Re(3,3′-DHBPY-2H+)(CO)33–, the equivalent of classical 2e–-reduced Re(BPY)(CO)3−. Each reductive deprotonation of the DHBPY ligand results in a redshift of the IR ν(CO) absorption of the tricarbonyl complexes by ca. 10 cm–1, facilitating the product assignment based on comparison with the literature data for corresponding Re/BPY complexes. The Cl– dissociation from Re(3,3′-DHBPY-2H+)(CO)3Cl2– was proven in strongly coordinating butyronitrile. The latter dianion is stable at 223 K, converting at 258 K to 6-coordinate Re(3,3′-DHBPY-2H+)(CO)3(PrCN)3–. Useful reference data were obtained with substituted parent Re(3,3′-DHBPY)(CO)3(PrCN)+ that also smoothly deprotonates by the initial reduction to Re(3,3′-DHBPY-H+)(CO)3(PrCN). The latter complex ultimately converts at the second cathodic wave to Re(3,3′-DHBPY-2H+)(CO)3(PrCN)3– via a counterintuitive ETC step generating the 1e– radical of the parent complex, viz., Re(3,3′-DHBPY)(CO)3(PrCN). The same alternative reduction path is also followed by Re(3,3′-DHBPY-H+)(CO)3Cl− at the onset of the second cathodic wave, where the ETC step results in the intermediate Re(3,3′-DHBPY)(CO)3Cl•– further reducible to Re(3,3′-DHBPY-2H+)(CO)33– as the CO2 catalyst.
Electrochemical and photochemical reduction of CO2 are both well‐established, independent catalytic routes toward producing added‐value chemicals. The potential for any cross‐reactivity has, however, ...hardly been explored so far. In this report, we assess a system primarily using spectroelectrochemical monitoring, where photochemistry assists the cathodic activation of precursor complexes fac‐Mn(CO)3(2,2′‐bipyridine)Br and Mo(CO)4(6,6′‐dimethyl‐2,2′‐bipyridine) to lower the catalytic overpotential needed to trigger the electrocatalytic reduction of CO2 to CO. Following the complete initial 1e− reduction of the parent complexes, the key photochemical cleavage of the Mn−Mn and Mo−CO bonds in the reduction products, Mn(CO)3(2,2′‐bipyridine)2 and Mo(CO)4(6,6′‐dimethyl‐2,2′‐bipyridine).−, respectively, generates the 2e−‐reduced, 5‐coordinate catalysts, Mn(CO)3(2,2′‐bipyridine)− and Mo(CO)3(6,6′‐dimethyl‐2,2′‐bipyridine)2− appreciably closer to the initial cathodic wave R1. Experiments under CO2 confirm the activity of both electrocatalysts under the photoirradiation with 405 nm and 365 nm light, respectively. This remarkable achievement corresponds to a ca. 500 mV positive shift of the catalytic onset compared to the exclusive standard electrocatalytic activation.
IR spectroelectrochemistry has unravelled the potential for photo‐assisted electrochemical reduction of Mn(CO)3(bipy)Br and Mo(CO)4(6,6′‐dmbipy) for triggering electrocatalytic reduction of CO2 at lower overpotentials. Utilizing the Mn−Mn and Mo−CO dissociative photochemistry of the 1e−‐reduced species, respectively, the active 5‐coordinate catalysts already operate near the parent cathodic waves, in a marked difference from the purely electrochemical routes.
Adult rat ultrasonic vocalizations (USVs) are a valuable tool for noninvasively assessing an animal's emotional state. USVs are produced in 1 of 2 frequency ranges labeled as 22 kHz or 50 kHz ...vocalizations. One USV subtype within the 50 kHz call category, constant frequency 50 kHz (CF 50 kHz) calls, is not viewed as signaling an emotional state. The current study tested the hypothesis that CF 50 kHz calls are related to a mild negative affective state. In Experiment 1, diazepam (1, 2.5, or 5 mg/kg), or control injections were administered prior to receiving a sequence of mild footshocks (0.5 mA, 0.5 s). Subjects transitioned from producing CF 50 to 22 kHz USVs as footshocks were repeated; a pattern paralleled by a shift from rearing to increased time freezing. USV production was largely absent in the higher diazepam dose groups, whereas the 1 mg/kg dose attenuated CF 50 kHz USV production prior to and immediately following initial footshocks. The higher doses of diazepam similarly reduced rearing activity and overall freezing behavior. In Experiment 2, pre-exposure to the testing environment with or without access to palatable food elicited CF 50 kHz calls and rearing. During re-exposure to the test chamber the following day, CF 50 kHz USV production was reduced prior to footshock onset compared to the prior test day. The pattern of results support an association between CF 50 kHz USVs and a mild negative affective state; dissociating this call type may increase the sensitivity of behavioral measurements of emotion.
Ruthenium(II) polypyridyl complexes Ru(CN-Me-bpy) x (bpy)3–x 2+ (CN-Me-bpy = 4,4′-dicyano-5,5′-dimethyl-2,2′-bipyridine, bpy = 2,2′-bipyridine, and x = 1–3, abbreviated as 1 2+ , 2 2+ , and 3 2+ ) ...undergo four (1 2+ ) or five (2 2+ and 3 2+ ) successive one-electron reduction steps between −1.3 and −2.75 V versus ferrocenium/ferrocene (Fc+/Fc) in tetrahydrofuran. The CN-Me-bpy ligands are reduced first, with successive one-electron reductions in 2 2+ and 3 2+ being separated by 150–210 mV; reduction of the unsubstituted bpy ligand in 1 2+ and 2 2+ occurs only when all CN-Me-bpy ligands have been converted to their radical anions. Absorption spectra of the first three reduction products of each complex were measured across the UV, visible, near-IR (NIR), and mid-IR regions and interpreted with the help of density functional theory calculations. Reduction of the CN-Me-bpy ligand shifts the ν(CN) IR band by ca. −45 cm–1, enhances its intensity ∼35 times, and splits the symmetrical and antisymmetrical modes. Semireduced complexes containing two and three CN-derivatized ligands 2 + , 3 + , and 30 show distinct ν(CN) features due to the presence of both CN-Me-bpy and CN-Me-bpy•–, confirming that each reduction is localized on a single ligand. NIR spectra of 10 , 1 – , and 2 – exhibit a prominent band attributable to the CN-Me-bpy•– moiety between 6000 and 7500 cm–1, whereas bpy•–-based absorption occurs between 4500 and 6000 cm–1; complexes 2 + , 3 + , and 30 also exhibit a band at ca. 3300 cm–1 due to a CN-Me-bpy•– → CN-Me-bpy interligand charge-transfer transition. In the UV–vis region, the decrease of π → π* intraligand bands of the neutral ligands and the emergence of the corresponding bands of the radical anions are most diagnostic. The first reduction product of 1 2+ is spectroscopically similar to the lowest triplet metal-to-ligand charge-transfer excited state, which shows pronounced NIR absorption, and its ν(CN) IR band is shifted by −38 cm–1 and 5–7-fold-enhanced relative to the ground state.
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