Allele-specific chemical genetics enables selective inhibition within families of highly-conserved proteins. The four BET (bromodomain & extra-terminal domain) proteins - BRD2, BRD3, BRD4 and BRDT ...bind acetylated chromatin
their bromodomains and regulate processes such as cell proliferation and inflammation. BET bromodomains are of particular interest, as they are attractive therapeutic targets but existing inhibitors are pan-selective. We previously established a bump-&-hole system for the BET bromodomains, pairing a leucine/alanine mutation with an ethyl-derived analogue of an established benzodiazepine scaffold. Here we optimize upon this system with the introduction of a more conservative and less disruptive leucine/valine mutation. Extensive structure-activity-relationships of diverse benzodiazepine analogues guided the development of potent, mutant-selective inhibitors with desirable physiochemical properties. The active enantiomer of our best compound - 9-ME-1 - shows ∼200 nM potency, >100-fold selectivity for the L/V mutant over wild-type and excellent DMPK properties. Through a variety of
and cellular assays we validate the capabilities of our optimized system, and then utilize it to compare the relative importance of the first and second bromodomains to chromatin binding. These experiments confirm the primacy of the first bromodomain in all BET proteins, but also significant variation in the importance of the second bromodomain. We also show that, despite having a minor role in chromatin recognition, BRD4 BD2 is still essential for gene expression, likely through the recruitment of non-histone proteins. The disclosed inhibitor:mutant pair provides a powerful tool for future cellular and
target validation studies.
The biological activity of scaffolds used in tissue engineering applications hypothetically depends on the density of available ligands, scaffold sites at which specific cell binding occurs. Ligand ...density is characterized by the composition of the scaffold, which defines the surface density of ligands, and by the specific surface area of the scaffold, which defines the total surface of the structure exposed to the cells. It has been previously shown that collagen–glycosaminoglycan (CG) scaffolds used for studies of skin regeneration were inactive when the mean pore size was either lower than 20
μm or higher than 120
μm (Proc. Natl. Acad. Sci., USA 86(3) (1989) 933). To study the relationship between cell attachment and viability in scaffolds and the scaffold structure, CG scaffolds with a constant composition and solid volume fraction (0.005), but with four different pore sizes corresponding to four levels of specific surface area were manufactured using a lyophilization technique. MC3T3-E1 mouse clonal osteogenic cells were seeded onto the four scaffold types and maintained in culture. At the experimental end point (24 or 48
h), the remaining viable cells were counted to determine the percent cell attachment. A significant difference in viable cell attachment was observed in scaffolds with different mean pore sizes after 24 and 48
h; however, there was no significant change in cell attachment between 24 and 48
h for any group. The fraction of viable cells attached to the CG scaffold decreased with increasing mean pore size, increasing linearly (
R
2=0.95, 0.91 at 24 and 48
h, respectively) with the specific surface area of the scaffold. The strong correlation between the scaffold specific surface area and cell attachment indicates that cell attachment and viability are primarily influenced by scaffold specific surface area over this range (95.9–150.5
μm) of pore sizes for MC3T3 cells.
This paper reviews recent advances in integrated waveguide circuits, lithographically fabricated for quantum optics. With the increase in complexity of realizable quantum architectures, the need for ...stability and high quality nonclassical interference within large optical circuits has become a matter of concern in modern quantum optics. Using integrated waveguide structures, we demonstrate a high performance platform from which to further develop quantum technologies and experimental quantum physics using single photons. We review the performance of directional couplers in Hong-Ou-Mandel experiments, together with inherently stable interferometers with controlled phase shifts for quantum state preparation, manipulation, and measurement as well as demonstrating the first on-chip quantum metrology experiments. These fundamental components of optical quantum circuits are used together to construct integrated linear optical realizations of two-photon quantum controlled logic gates. The high quality quantum mechanical performance observed at the single photon level signifies their central role in future optical quantum technologies.
A second mild traumatic brain injury (mTBI) sustained prior to neuropathological recovery can lead to exacerbated effects. Without objective indicators of this neuropathology, individuals may return ...to activities at risk of mTBI when their brain is still vulnerable. With axonal injury recognized as a neuropathological hallmark of mTBI, we hypothesized that serum levels of neurofilament light (NfL), a highly sensitive biomarker of axonal injury, may be predictive of vulnerability to worse outcomes in the event of a second mTBI. Given this hypothesis is difficult to test clinically, we used a two-hit model of mTBI in rats and staggered inter-injury intervals by 1-, 3-, 7-, or 14-days. Repeat-mTBI rats were dichotomized into NfLhigh (NfL>median at the time of re-injury) and NfLlow (NfL<median) groups, with behavior and NfL levels analyzed throughout the 28-days, followed by ex vivo diffusion tensor imaging. NfL levels at the time of the second mTBI were found to be predictive of vulnerability to re-injury, with NfLhigh rats displaying more neurological signs and a greater potentiation of NfL levels after the second mTBI. Importantly, this potentiation phenomenon remained even when limiting analyses to rats with longer inter-injury intervals, providing evidence that vulnerability to re-injury may not be exclusively dependent on inter-injury interval. Finally, NfL levels correlated with, and were predictive of, the severity of neurological signs following the second mTBI. These findings provide evidence that measurement of NfL during mTBI recovery may be reflective of the vulnerability to a second mTBI, and as such may have utility to assist return to sport, duty and work decisions.
Periconceptional nutrition (PCN) can influence foetal hypothalamo-pituitary adrenal (HPA) axis function and alter cortisol secretion with possible consequences for maturation and growth of major ...organs, gestation length and behaviour. We examined effects of PCN on phenotype and survival of the neonatal lamb in 466 Merino ewes allocated to treatments providing 70%, 100% and 150% respectively, of maintenance requirements for 17 days prior and 6 days after insemination. Gestation length and birth weight for lambs in PCN treatment groups was similar (P>0.05) but low PCN decreased the size of the neonate (crown-rump-length and metacarpal length P<0.05). A subset of lambs euthanased at 5 days of age further showed that low PCN decreased the amount of peri-renal fat (P<0.05) and increased liver mass (P<0.05) while high PCN increased neck thymus and ovary mass (P<0.05). Neonatal lambs from low PCN ewes returned faster to their mothers after release (P<0.05) and contacted the udder in the shortest time (P<0.05). Significant interactions between PCN treatment and sex (P<0.05) and between PCN treatment and ewe age (P<0.05) were also observed for time lambs took to follow the ewe. Survival of lambs was similar but potential differences may have been masked by favourable weather conditions. In conclusion, this study provides evidence of significant changes in lamb growth and development dependent on PCN and, for the first time, links these changes with significant changes in behaviour of the neonate. The impact of these effects on lamb survival and potential reproductive capacity of female offspring remains to be determined.
Isolated systolic hypertension occurs in about 15% of people aged 60 years or older. In 1989, the European Working Party on High Blood Pressure in the Elderly investigated whether active treatment ...could reduce cardiovascular complications of isolated systolic hypertension. Fatal and non-fatal stroke combined was the primary endpoint.
All patients (≥60 years) were initially started on masked placebo. At three run-in visits 1 month apart, their average sitting systolic blood pressure was 160–219 mm Hg with a diastolic blood pressure lower than 95 mm Hg. After stratification for centre, sex, and previous cardiovascular complications, 4695 patients were randomly assigned to nitrendipine 10–40 mg daily, with the possible addition of enalapril 5–20 mg daily and hydrochlorothiazide 12·5–25·0 mg daily, or matching placebos. Patients withdrawing from double-blind treatment were still followed up. We compared occurrence of major endpoints by intention to treat.
At a median of 2 years' follow-up, sitting systolic and diastolic blood pressures had fallen by 13 mm Hg and 2 mm Hg in the placebo group (n=2297) and by 23 mm Hg and 7 mm Hg in the active treatment group (n=2398). The between-group differences were systolic 10·1 mm Hg (95% CI 8·8–11·4) and diastolic, 4·5 mm Hg (3·9–5·1). Active treatment reduced the total rate of stroke from 13·7 to 7·9 endpoints per 1000 patient-years (42% reduction; p=0·003). Non-fatal stroke decreased by 44% (p=0·007). In the active treatment group, all fatal and non-fatal cardiac endpoints, including sudden death, declined by 26% (p=0·03). Non-fatal cardiac endpoints decreased by 33% (p=0·03) and all fatal and non-fatal cardiovascular endpoints by 31% (p<0·001). Cardiovascular mortality was slightly lower on active treatment (27%, p=0·07), but all-cause mortality was not influenced (−14%; p=0·22).
Among elderly patients with isolated systolic hypertension, antihypertensive drug treatment starting with nitrendipine reduces the rate of cardiovascular complications. Treatment of 1000 patients for 5 years with this type of regimen may prevent 29 strokes or 53 major cardiovascular endpoints.
Objectives
To assess the relationship of white blood cell count (WBC) and other routinely collected inflammatory and clinical markers including stone size, stone position, and medical expulsive ...therapy use (MET), with spontaneous stone passage (SSP) in a large contemporary cohort of patients with acute ureteric colic, as there are conflicting data on the role of WBC and other inflammatory markers in SSP in patients with acute ureteric colic.
Patients and methods
Multicentre retrospective cohort study coordinated by the British Urology Researchers in Surgical Training (BURST) Research Collaborative at 71 secondary care hospitals across four countries (UK, Republic of Ireland, Australia, and New Zealand). In all, 4170 patients presented with acute ureteric colic and a computed tomography confirmed single ureteric stone. Our primary outcome measure was SSP, as defined by the absence of need for intervention to assist stone passage (SP). Multivariable mixed effects logistic regression was used to explore the relationship between key patient factors and SSP.
Results
In all, 2518 patients were discharged with conservative management and had further follow‐up with a SSP rate of 74% (n = 1874/2518). Sepsis after discharge with conservative management was reported in 0.6% (n = 16/2518). On multivariable analysis neither WBC, neutrophils count, nor C‐reactive protein (CRP) predicted SSP, with an adjusted odds ratio (OR) of 0.97 (95% confidence interval CI 0.91–1.04, P = 0.38), 1.06 (95% CI 0.99–1.13, P = 0.1) and 1.00 (95% CI 0.99–1.00, P = 0.17), respectively. MET also did not predict SSP (adjusted OR 1.11, 95% CI 0.76–1.61). However, stone size and stone position were significant predictors. SSP for stones <5 mm was 89% (95% CI 87–90) compared to 49% (95% CI 44–53) for stones ≥5–7 mm, and 29% (95% CI 23–36) for stones >7 mm. For stones in the upper ureter the SSP rate was 52% (95% CI 48–56), middle ureter was 70% (95% CI 64–76), and lower ureter was 83% (95% CI 81–85).
Conclusion
In contrast to the previously published literature, we found that in patients with acute ureteric colic who are discharged with initial conservative management neither WBC, neutrophil count, nor CRP, helps determine the likelihood of SSP. We also found no overall benefit from the use of MET. Stone size and position are important predictors and our present findings represent the most comprehensive SP rates for each millimetre increase in stone size from a large contemporary cohort adjusting for key potential confounders. We anticipate that these data will aid clinicians managing patients with acute ureteric colic and help guide management decisions and the need for intervention.
Neuromuscular blocking agents are the leading drugs responsible for immediate hypersensitivity reactions during anaesthesia. Most hypersensitivity reactions represent IgE-mediated allergic reactions. ...Their incidence is estimated to be between 1 in 3,000 to 1 in 110,000 general anaesthetics. However striking variations have been reported among countries. The mechanism of sensitisation seems to implicate the presence of a substituted ammonium ion in the molecule. Due to lack of exposure prior to the reaction in a large number of reactors, it has been hypothesised that sensitisation may involve other, as yet undefined, substituted (quaternary and tertiary) ammonium ion containing compounds such as pholcodine, present in the environment of the patient. This hypothesis is still under investigation. The mechanism of non-IgE mediated hypersensitivity reactions is less well known. Identified mechanisms correspond to direct histamine release or interactions with muscarinic and nicotinic receptors. Allergic reactions cannot be clinically distinguished from non-IgE-mediated reactions. Therefore, any suspected hypersensitivity reaction must be investigated using combined pre and postoperative testing. Because of the frequent but not systematic cross-reactivity observed with muscle relaxants, every available neuromuscular blocking agent should be tested, using intradermal tests to confirm the responsibility of the suspected drug which should be definitely excluded. Cross-sensitivity investigation will also try to identify the safety of drugs that can be potentially used in future anaesthesia. The determination of basophil activation investigations using direct leukocyte histamine release test or flow cytometry would be of particular interest to investigate cross sensitisation in complement to skin tests. There is no demonstrated evidence supporting systematic pre-operative screening in the general population at this time. However, since no specific treatment has been shown to reliably prevent anaphylaxis, allergy assessment must be performed in all high-risk patients. In view of the relative complexity of allergy investigation, and of the differences between countries, an active policy to identify patients at risk and to provide any necessary support from expert advice to anaesthetists and allergologists through the constitution of allergo-anaesthesia centres in every country should be promoted.
To assess the impact of immediate versus delayed antihypertensive treatment on the outcome of older patients with isolated systolic hypertension, we extended the double-blind placebo-controlled ...Systolic Hypertension in Europe (Syst-Eur) trial by an open-label follow-up study lasting 4 years.
The Syst-Eur trial included 4695 randomized patients with minimum age of 60 years and an untreated blood pressure of 160-219 mmHg systolic and below 95 mmHg diastolic. The double-blind trial ended after a median follow-up of 2.0 years (range 1-97 months). Of 4409 patients still alive, 3517 received open-label treatment consisting of nitrendipine (10-40 mg daily) with the possible addition of enalapril (5-20 mg daily), hydrochlorothiazide (12.5-25 mg daily), or both add-on drugs. Non-participants (n = 892) were also followed up.
Median follow-up increased to 6.1 years. Systolic pressure decreased to below 150 mmHg (target level) in 2628 participants (75.0%). During the 4-year open-label follow-up, stroke and cardiovascular complications occurred at similar frequencies in patients formerly randomized to placebo and those continuing active treatment. These rates were similar to those previously observed in the active-treatment group during the double-blind trial. Considering the total follow-up of 4695 randomized patients, immediate compared with delayed antihypertensive treatment reduced the occurrence of stroke and cardiovascular complications by 28% (P = 0.01) and 15% (P = 0.03), respectively, with a similar tendency for total mortality (13%, P = 0.09). In 492 diabetic patients, the corresponding estimates of long-term benefit (P < 0.02) were 60, 51 and 38%, respectively.
Antihypertensive treatment can achieve blood pressure control in most older patients with isolated systolic hypertension. Immediate compared with delayed treatment prevented 17 strokes or 25 major cardiovascular events per 1000 patients followed up for 6 years. These findings underscore the necessity of early treatment of isolated systolic hypertension.
PDF
