The discovery of drug-like molecules that bind pockets in proteins that are not present in crystallographic structures yet exert allosteric control over activity has generated great interest in ...designing pharmaceuticals that exploit allosteric effects. However, there have only been a small number of successes, so the therapeutic potential of these pockets—called hidden allosteric sites—remains unclear. One challenge for assessing their utility is that rational drug design approaches require foreknowledge of the target site, but most hidden allosteric sites are only discovered when a small molecule is found to stabilize them. We present a means of decoupling the identification of hidden allosteric sites from the discovery of drugs that bind them by drawing on new developments in Markov state modeling that provide unprecedented access to microsecond- to millisecond-timescale fluctuations of a protein’s structure. Visualizing these fluctuations allows us to identify potential hidden allosteric sites, which we then test via thiol labeling experiments. Application of these methods reveals multiple hidden allosteric sites in an important antibiotic target—TEM-1 β-lactamase. This result supports the hypothesis that there are many as yet undiscovered hidden allosteric sites and suggests our methodology can identify such sites, providing a starting point for future drug design efforts. More generally, our results demonstrate the power of using Markov state models to guide experiments.
Significance Rational drug design efforts typically focus on identifying inhibitors that bind to protein active sites. Pockets that are not present in crystallographic structures yet can exert allosteric (i.e., long-range) control over distant active sites present an exciting alternative. However, identifying these hidden allosteric sites is extremely challenging because one usually has to simultaneously find a small molecule that binds to and stabilizes the open conformation of the pocket. Here, we present a means of combining advances in computer modeling—using Markov state models to capture long timescale dynamics—with biophysical experiments to identify hidden allosteric sites without requiring the simultaneous discovery of drug-like compounds that bind them. Using this technology, we discover multiple hidden allosteric sites in a single protein.
Palaeontologists characterize mass extinctions as times when the Earth loses more than three-quarters of its species in a geologically short interval, as has happened only five times in the past 540 ...million years or so. Biologists now suggest that a sixth mass extinction may be under way, given the known species losses over the past few centuries and millennia. Here we review how differences between fossil and modern data and the addition of recently available palaeontological information influence our understanding of the current extinction crisis. Our results confirm that current extinction rates are higher than would be expected from the fossil record, highlighting the need for effective conservation measures.
Solid tumors, including the aggressive primary brain cancer glioblastoma multiforme, develop resistance to cell death, in part as a result of a switch from mitochondrial oxidative phosphorylation to ...cytoplasmic glycolysis. This metabolic remodeling is accompanied by mitochondrial hyperpolarization. We tested whether the small-molecule and orphan drug dichloroacetate (DCA) can reverse this cancer-specific metabolic and mitochondrial remodeling in glioblastoma. Freshly isolated glioblastomas from 49 patients showed mitochondrial hyperpolarization, which was rapidly reversed by DCA. In a separate experiment with five patients who had glioblastoma, we prospectively secured baseline and serial tumor tissue, developed patient-specific cell lines of glioblastoma and putative glioblastoma stem cells (CD133(+), nestin(+) cells), and treated each patient with oral DCA for up to 15 months. DCA depolarized mitochondria, increased mitochondrial reactive oxygen species, and induced apoptosis in GBM cells, as well as in putative GBM stem cells, both in vitro and in vivo. DCA therapy also inhibited the hypoxia-inducible factor-1alpha, promoted p53 activation, and suppressed angiogenesis both in vivo and in vitro. The dose-limiting toxicity was a dose-dependent, reversible peripheral neuropathy, and there was no hematologic, hepatic, renal, or cardiac toxicity. Indications of clinical efficacy were present at a dose that did not cause peripheral neuropathy and at serum concentrations of DCA sufficient to inhibit the target enzyme of DCA, pyruvate dehydrogenase kinase II, which was highly expressed in all glioblastomas. Metabolic modulation may be a viable therapeutic approach in the treatment of glioblastoma.
Simulation for Vascular Trainees Maguire, Seán C.; Kavanagh, Dara O.
European journal of vascular and endovascular surgery,
06/2021, Letnik:
61, Številka:
6
Journal Article
Gene therapy is emerging as a therapeutic modality for treating disorders of the retina. Photoreceptor cells are the primary cell type affected in many inherited diseases of retinal degeneration. ...Successfully treating these diseases with gene therapy requires the identification of efficient and safe targeting vectors that can transduce photoreceptor cells. One serotype of adeno-associated virus, AAV2, has been used successfully in clinical trials to treat a form of congenital blindness that requires transduction of the supporting cells of the retina in the retinal pigment epithelium (RPE). Here, we determined the dose required to achieve targeting of AAV2 and AAV8 vectors to photoreceptors in nonhuman primates. Transgene expression in animals injected subretinally with various doses of AAV2 or AAV8 vectors carrying a green fluorescent protein transgene was correlated with surgical, clinical, and immunological observations. Both AAV2 and AAV8 demonstrated efficient transduction of RPE, but AAV8 was markedly better at targeting photoreceptor cells. These preclinical results provide guidance for optimal vector and dose selection in future human gene therapy trials to treat retinal diseases caused by loss of photoreceptors.
Concerns about the long-term durability of endovascular aortic aneurysm repair and the requirement for explantation of stents in the case of infection demonstrate the continued need for open ...abdominal aortic aneurysm (AAA) repair. However, with the increased complexity and decreasing volume of open cases performed, maintenance of skills and training of younger surgeons are challenging. The aim of this review was to identify and to examine studies pertaining to open AAA simulation, with focus on methods and outcomes.
We performed a systematic review of the literature to identify primary research pertaining to open AAA repair through the use of simulators. The primary outcome was to identify predominant modes of simulator design and validated assessment tools that could demonstrate improvement in trainee skills. Secondary outcomes included identifying participant numbers needed to power studies and whether tools not validated externally contributed to the studies.
There were 309 unique papers identified, from which five papers met the inclusion criteria. The selected papers used a combination of synthetic (commercial and homemade) and cadaveric simulators. A variety of validated and nonvalidated assessment metrics were used, including Objective Structured Assessment of Technical Skills, global rating scales, and realism surveys. Three of the five papers used blinding as part of their assessments. Mean participant numbers were 30.8 ± 25.7 and with the exception of one paper consisted entirely of surgical trainees in dedicated general or vascular surgery training programs.
Several options are currently available for open AAA simulation, all of which demonstrate improved scoring metrics after simulator use. Validated scoring systems, the Objective Structured Assessment of Technical Skills in particular, were most frequently used to deliver objective results. Whereas junior trainees derive the most benefit, senior trainees also showed significant improvements, demonstrating that simulation benefits all levels of surgical trainees. Low numbers of participants were sufficient to achieve statistical benefit within individual studies.
Species distribution models (SDMs) assume species exist in isolation and do not influence one another's distributions, thus potentially limiting their ability to predict biodiversity patterns. ...Community-level models (CLMs) capitalize on species co-occurrences to fit shared environmental responses of species and communities, and therefore may result in more robust and transferable models. Here, we conduct a controlled comparison of five paired SDMs and CLMs across changing climates, using palaeoclimatic simulations and fossil-pollen records of eastern North America for the past 21 000 years. Both SDMs and CLMs performed poorly when projected to time periods that are temporally distant and climatically dissimilar from those in which they were fit; however, CLMs generally outperformed SDMs in these instances, especially when models were fit with sparse calibration datasets. Additionally, CLMs did not over-fit training data, unlike SDMs. The expected emergence of novel climates presents a major forecasting challenge for all models, but CLMs may better rise to this challenge by borrowing information from co-occurring taxa.
Colorectal cancer (CRC) is the third most common cancer in the UK: incidence increases with age, median age at diagnosis being over 70 years. Approximately 25% of cases occur in individuals with a ...family history of CRC, including 5% caused by familial adenomatous polyposis (FAP) or hereditary non-polyposis CRC (HNPCC). Most develop from adenomatous polyps arising from the intestine lining. Individuals with these polyps undergo polypectomy and are invited for endoscopic surveillance. Screening via faecal occult blood testing has been rolled out across the UK.
To evaluate the clinical effectiveness and cost-effectiveness of drug and micronutrient interventions for the prevention of CRC and/or adenomatous polyps. Interventions considered include: non-steroidal anti-inflammatory drugs (NSAIDs), including aspirin and cyclo-oxygenase-2 (COX-2) inhibitors; folic acid; calcium; vitamin D and antioxidants (including vitamin A, vitamin C, vitamin E, selenium and beta-carotene). Chemoprevention was assessed in the general population, in individuals at increased risk of CRC, and in individuals with FAP or HNPCC.
A systematic review identified randomised controlled trials (RCTs) assessing drug and nutritional agents for the prevention of CRC or adenomatous polyps. A separate search identified qualitative studies relating to individuals' views, attitudes and beliefs about chemoprevention. MEDLINE, MEDLINE In-Process & Other Non-Indexed Citations, EMBASE, CINAHL, the Cochrane Database of Systematic Reviews, Cochrane CENTRAL Register of Controlled Trials, DARE, NHS-EED (NHS Economic Evaluation Database), HTA database, Science Citation Index, BIOSIS previews and the Current Controlled Trials research register were searched in June 2008. Data were extracted by one reviewer and checked by a second.
The synthesis methods used were systematic review and meta-analysis for RCTs and qualitative framework synthesis for qualitative studies. A health economic model was developed to assess the cost-effectiveness of chemoprevention for two populations with different levels of risk of developing CRC: the general population and an intermediate-risk population.
The search identified 44 relevant RCTs and six ongoing studies. A small study of aspirin in FAP patients produced no statistically significant reduction in polyp number but a possible reduction in polyp size. There was a statistically significant 21% reduction in risk of adenoma recurrence relative risk (RR) 0.79, 95% confidence interval (CI) 0.68 to 0.92 in an analysis of aspirin versus no aspirin in individuals with a history of adenomas or CRC. In the general population, a significant 26% reduction in CRC incidence was demonstrated in studies with a 23-year follow-up (RR 0.74, 95% CI 0.57 to 0.97). Non-aspirin NSAID use in FAP individuals produced a non-statistically significant reduction in adenoma incidence after 4 years of treatment and follow-up and reductions in polyp number and size. In individuals with a history of adenomas there was a statistically significant 34% reduction in adenoma recurrence risk (RR 0.66, 95% CI 0.60 to 0.72) and a statistically significant 55% reduction in advanced adenoma incidence (RR 0.45, 95% CI 0.35 to 0.58). No studies assessed the effect of non-aspirin NSAIDs in the general population. There were no studies of folic acid in individuals with FAP or HNPCC. There was no significant effect of folic acid versus placebo on adenoma recurrence (RR 1.16, 95% CI 0.97 to 1.39) or advanced adenoma incidence in individuals with a history of adenomas. In the general population there was no significant effect of folic acid on risk of CRC (RR 1.13, 95% CI 0.77 to 1.64), although studies were of relatively short duration. Calcium use by FAP patients produced no significant reduction in polyp number or disease progression. In individuals with a history of adenomas there was a statistically significant 18% reduction in risk of adenoma recurrence (RR 0.82, 95% CI 0.69 to 0.98) and a non-significant reduction in risk of advanced adenomas (RR 0.77, 95% CI 0.50 to 1.17). In the general population there was no significant effect of calcium on risk of CRC (RR 1.08, 95% CI 0.87 to 1.34), although studies were of relatively short duration. There were no studies of antioxidant use in individuals with FAP or HNPCC, and in individuals with a history of adenomas no statistically significant differences in relative risk of adenoma recurrence were found. In the general population there was no difference in incidence of CRC (RR 1.00, 95% CI 0.88 to 1.13) with antioxidant use compared with no antioxidant use. Twenty studies reported qualitative findings concerning chemoprevention. People are more likely to use NSAIDs if there is a strong perceived need. Perceptions of risk and benefit also influence decision-making and use. People have fewer concerns about using antioxidants or other supplements, but their perception of the benefits of these agents is less well-defined. The model analysis suggested that the most cost-effective age-range policy in the general population would be to provide chemoprevention to all individuals within the general population from age 50 to 60 years. The use of aspirin in addition to screening within the general population is likely to result in a discounted cost per life-year gained of around 10,000 pounds and a discounted cost per quality-adjusted life-year (QALY) gained of around 23,000 pounds compared with screening alone. In the intermediate-risk group the most economically viable age-range policy would be to provide chemoprevention to individuals following polypectomy aged 61 to 70 years. Calcium is likely to have a discounted cost per QALY gained of around 8000 pounds compared with screening alone. Although aspirin in addition to screening should be more effective and less costly than screening alone, under the current assumptions of benefits to harms of aspirin and calcium, aspirin is expected to be extendedly dominated by calcium.
Whilst a number of studies were included in the review, the duration of follow-up was generally insufficient to detect an effect on cancer incidence. Given the uncertainties and ambiguities in the evidence base, the results of the health economic analysis should be interpreted with caution.
Aspirin and celecoxib may reduce recurrence of adenomas and incidence of advanced adenomas in individuals with an increased risk of CRC and calcium may reduce recurrence of adenomas in this group. COX-2 inhibitors may decrease polyp number in patients with FAP. There is some evidence for aspirin reducing the incidence of CRC in the general population. Both aspirin and NSAIDs are associated with adverse effects so it will be important to consider the risk-benefit ratio before recommending these agents for chemoprevention. The economic analysis suggests that chemoprevention has the potential to represent a cost-effective intervention, particularly when targeted at intermediate-risk populations following polypectomy.
Adeno-associated virus (AAV) vectors are showing promise in gene therapy trials and have proven to be extremely efficient biological tools in basic neuroscience research. One major limitation to ...their widespread use in the neuroscience laboratory is the cost, labor, skill and time-intense purification process of AAV. We have recently shown that AAV can associate with exosomes (exo-AAV) when the vector is isolated from conditioned media of producer cells, and the exo-AAV is more resistant to neutralizing anti-AAV antibodies compared with standard AAV. Here, we demonstrate that simple pelleting of exo-AAV from media via ultracentrifugation results in high-titer vector preparations capable of efficient transduction of central nervous system (CNS) cells after systemic injection in mice. We observed that exo-AAV is more efficient at gene delivery to the brain at low vector doses relative to conventional AAV, even when derived from a serotype that does not normally efficiently cross the blood-brain barrier. Similar cell types were transduced by exo-AAV and conventionally purified vector. Importantly, no cellular toxicity was noted in exo-AAV-transduced cells. We demonstrated the utility and robustness of exo-AAV-mediated gene delivery by detecting direct GFP fluorescence after systemic injection, allowing three-dimensional reconstruction of transduced Purkinje cells in the cerebellum using ex vivo serial two-photon tomography. The ease of isolation combined with the high efficiency of transgene expression in the CNS, may enable the widespread use of exo-AAV as a neuroscience research tool. Furthermore, the ability of exo-AAV to evade neutralizing antibodies while still transducing CNS after peripheral delivery is clinically relevant.
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