AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Quantitative mapping of dense microtubule arrays in mammalian neurons

The neuronal microtubule cytoskeleton underlies the polarization and proper functioning of neurons, amongst others by providing tracks for motor proteins that drive intracellular transport. Different subsets of neuronal microtubules, varying in composition, stability, and motor preference, are known to exist, but the high density of microtubules has so far precluded mapping their relative abundance and three-dimensional organization. Here, we use different super-resolution techniques (STED, Expansion Microscopy) to explore the nanoscale organization of the neuronal microtubule network in rat hippocampal neurons. This revealed that in dendrites acetylated microtubules are enriched in the core of the dendritic shaft, while tyrosinated microtubules are enriched near the plasma membrane, thus forming a shell around the acetylated microtubules. Moreover, using a novel analysis pipeline we quantified the absolute number of acetylated and tyrosinated microtubules within dendrites and found that they account for 65-75% and ~20-30% of all microtubules, respectively, leaving only few microtubules that do not fall in either category. Because these different microtubule subtypes facilitate different motor proteins, these novel insights help to understand the spatial regulation of intracellular transport

Quantitative mapping of dense microtubule arrays in mammalian neurons

The neuronal microtubule cytoskeleton underlies the polarization and proper functioning of neurons, amongst others by providing tracks for motor proteins that drive intracellular transport. Different subsets of neuronal microtubules, varying in composition, stability, and motor preference, are known to exist, but the high density of microtubules has so far precluded mapping their relative abundance and three-dimensional organization. Here, we use different super-resolution techniques (STED, Expansion Microscopy) to explore the nanoscale organization of the neuronal microtubule network in rat hippocampal neurons. This revealed that in dendrites acetylated microtubules are enriched in the core of the dendritic shaft, while tyrosinated microtubules are enriched near the plasma membrane, thus forming a shell around the acetylated microtubules. Moreover, using a novel analysis pipeline we quantified the absolute number of acetylated and tyrosinated microtubules within dendrites and found that they account for 65-75% and ~20-30% of all microtubules, respectively, leaving only few microtubules that do not fall in either category. Because these different microtubule subtypes facilitate different motor proteins, these novel insights help to understand the spatial regulation of intracellular transport

Comparing strategies for deep astigmatism-based single-molecule localization microscopy

Single-molecule localization microscopy (SMLM) enables fluorescent microscopy with nanometric resolution. While localizing molecules close to the coverslip is relatively straightforward using high numerical aperture (NA) oil immersion (OI) objectives, optical aberrations impede SMLM deeper in watery samples. Adaptive optics (AO) with a deformable mirror (DM) can be used to correct such aberrations and to induce precise levels of astigmatism to encode the z-position of molecules. Alternatively, the use of water immersion (WI) objectives might be sufficient to limit the most dominant aberrations. Here we compare SMLM at various depths using either WI or OI with or without AO. In addition, we compare the performance of a cylindrical lens and a DM for astigmatism-based z-encoding. We find that OI combined with adaptive optics improves localization precision beyond the performance of WI-based imaging and enables deep (>10 µm) 3D localization

Quantitative mapping of dense microtubule arrays in mammalian neurons

The neuronal microtubule cytoskeleton underlies the polarization and proper functioning of neurons, amongst others by providing tracks for motor proteins that drive intracellular transport. Different subsets of neuronal microtubules, varying in composition, stability, and motor preference, are known to exist, but the high density of microtubules has so far precluded mapping their relative abundance and three-dimensional organization. Here, we use different super-resolution techniques (STED, Expansion Microscopy) to explore the nanoscale organization of the neuronal microtubule network in rat hippocampal neurons. This revealed that in dendrites acetylated microtubules are enriched in the core of the dendritic shaft, while tyrosinated microtubules are enriched near the plasma membrane, thus forming a shell around the acetylated microtubules. Moreover, using a novel analysis pipeline we quantified the absolute number of acetylated and tyrosinated microtubules within dendrites and found that they account for 65-75% and ~20-30% of all microtubules, respectively, leaving only few microtubules that do not fall in either category. Because these different microtubule subtypes facilitate different motor proteins, these novel insights help to understand the spatial regulation of intracellular transport

Comparing strategies for deep astigmatism-based single-molecule localization microscopy

Single-molecule localization microscopy (SMLM) enables fluorescent microscopy with nanometric resolution. While localizing molecules close to the coverslip is relatively straightforward using high numerical aperture (NA) oil immersion (OI) objectives, optical aberrations impede SMLM deeper in watery samples. Adaptive optics (AO) with a deformable mirror (DM) can be used to correct such aberrations and to induce precise levels of astigmatism to encode the z-position of molecules. Alternatively, the use of water immersion (WI) objectives might be sufficient to limit the most dominant aberrations. Here we compare SMLM at various depths using either WI or OI with or without AO. In addition, we compare the performance of a cylindrical lens and a DM for astigmatism-based z-encoding. We find that OI combined with adaptive optics improves localization precision beyond the performance of WI-based imaging and enables deep (>10 µm) 3D localization

Switching of mCherry fluorescence using a novel caging mechanism

status: publishe

Efficient switching of mCherry fluorescence using chemical caging

Fluorophores with dynamic or controllable fluorescence emission have become essential tools for advanced imaging, such as superresolution imaging. These applications have driven the continuing development of photoactivatable or photoconvertible labels, including genetically encoded fluorescent proteins. These new probes work well but require the introduction of new labels that may interfere with the proper functioning of existing constructs and therefore require extensive functional characterization. In this work we show that the widely used red fluorescent protein mCherry can be brought to a purely chemically induced blue-fluorescent state by incubation with β-mercaptoethanol (βME). The molecules can be recovered to the red fluorescent state by washing out the βME or through irradiation with violet light, with up to 80% total recovery. We show that this can be used to perform single-molecule localization microscopy (SMLM) on cells expressing mCherry, which renders this approach applicable to a very wide range of existing constructs. We performed a detailed investigation of the mechanism underlying these dynamics, using X-ray crystallography, NMR spectroscopy, and ab initio quantum-mechanical calculations. We find that the βME-induced fluorescence quenching of mCherry occurs both via the direct addition of βME to the chromophore and through βME-mediated reduction of the chromophore. These results not only offer a strategy to expand SMLM imaging to a broad range of available biological models, but also present unique insights into the chemistry and functioning of a highly important class of fluorophores

Initial invasive or conservative strategy for stable coronary disease

BACKGROUND Among patients with stable coronary disease and moderate or severe ischemia, whether clinical outcomes are better in those who receive an invasive intervention plus medical therapy than in those who receive medical therapy alone is uncertain. METHODS We randomly assigned 5179 patients with moderate or severe ischemia to an initial invasive strategy (angiography and revascularization when feasible) and medical therapy or to an initial conservative strategy of medical therapy alone and angiography if medical therapy failed. The primary outcome was a composite of death from cardiovascular causes, myocardial infarction, or hospitalization for unstable angina, heart failure, or resuscitated cardiac arrest. A key secondary outcome was death from cardiovascular causes or myocardial infarction. RESULTS Over a median of 3.2 years, 318 primary outcome events occurred in the invasive-strategy group and 352 occurred in the conservative-strategy group. At 6 months, the cumulative event rate was 5.3% in the invasive-strategy group and 3.4% in the conservative-strategy group (difference, 1.9 percentage points; 95% confidence interval [CI], 0.8 to 3.0); at 5 years, the cumulative event rate was 16.4% and 18.2%, respectively (difference, 121.8 percentage points; 95% CI, 124.7 to 1.0). Results were similar with respect to the key secondary outcome. The incidence of the primary outcome was sensitive to the definition of myocardial infarction; a secondary analysis yielded more procedural myocardial infarctions of uncertain clinical importance. There were 145 deaths in the invasive-strategy group and 144 deaths in the conservative-strategy group (hazard ratio, 1.05; 95% CI, 0.83 to 1.32). CONCLUSIONS Among patients with stable coronary disease and moderate or severe ischemia, we did not find evidence that an initial invasive strategy, as compared with an initial conservative strategy, reduced the risk of ischemic cardiovascular events or death from any cause over a median of 3.2 years. The trial findings were sensitive to the definition of myocardial infarction that was used