We introduce a method for optically imaging intracellular proteins at nanometer spatial resolution. Numerous sparse subsets of photoactivatable fluorescent protein molecules were activated, localized (to approximately 2 to 25 nanometers), and then bleached. The aggregate position information from all subsets was then assembled into a superresolution image. We used this method--termed photoactivated localization microscopy--to image specific target proteins in thin sections of lysosomes and mitochondria; in fixed whole cells, we imaged vinculin at focal adhesions, actin within a lamellipodium, and the distribution of the retroviral protein Gag at the plasma membrane.

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Imaging intracellular fluorescent proteins at nanometer resolution.

We have developed a high-resolution fluorescence microscopy method based on high-accuracy localization of photoswitchable fluorophores. In each imaging cycle, only a fraction of the fluorophores were turned on, allowing their positions to be determined with nanometer accuracy. The fluorophore positions obtained from a series of imaging cycles were used to reconstruct the overall image. We demonstrated an imaging resolution of 20 nm. This technique can, in principle, reach molecular-scale resolution.

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Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM).

Summary Background Since December, 2019, Wuhan, China, has experienced an outbreak of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Epidemiological and clinical characteristics of patients with COVID-19 have been reported but risk factors for mortality and a detailed clinical course of illness, including viral shedding, have not been well described. Methods In this retrospective, multicentre cohort study, we included all adult inpatients (≥18 years old) with laboratory-confirmed COVID-19 from Jinyintan Hospital and Wuhan Pulmonary Hospital (Wuhan, China) who had been discharged or had died by Jan 31, 2020. Demographic, clinical, treatment, and laboratory data, including serial samples for viral RNA detection, were extracted from electronic medical records and compared between survivors and non-survivors. We used univariable and multivariable logistic regression methods to explore the risk factors associated with in-hospital death. Findings 191 patients (135 from Jinyintan Hospital and 56 from Wuhan Pulmonary Hospital) were included in this study, of whom 137 were discharged and 54 died in hospital. 91 (48%) patients had a comorbidity, with hypertension being the most common (58 [30%] patients), followed by diabetes (36 [19%] patients) and coronary heart disease (15 [8%] patients). Multivariable regression showed increasing odds of in-hospital death associated with older age (odds ratio 1·10, 95% CI 1·03–1·17, per year increase; p=0·0043), higher Sequential Organ Failure Assessment (SOFA) score (5·65, 2·61–12·23; p Interpretation The potential risk factors of older age, high SOFA score, and d-dimer greater than 1 μg/mL could help clinicians to identify patients with poor prognosis at an early stage. Prolonged viral shedding provides the rationale for a strategy of isolation of infected patients and optimal antiviral interventions in the future. Funding Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences; National Science Grant for Distinguished Young Scholars; National Key Research and Development Program of China; The Beijing Science and Technology Project; and Major Projects of National Science and Technology on New Drug Creation and Development.

Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study

In 1873, Ernst Abbe discovered what was to become a well-known paradigm: the inability of a lens-based optical microscope to discern details that are closer together than half of the wavelength of light. However, for its most popular imaging mode, fluorescence microscopy, the diffraction barrier is crumbling. Here, I discuss the physical concepts that have pushed fluorescence microscopy to the nanoscale, once the prerogative of electron and scanning probe microscopes. Initial applications indicate that emergent far-field optical nanoscopy will have a strong impact in the life sciences and in other areas benefiting from nanoscale visualization.

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Far-Field Optical Nanoscopy

The mechanistic target of rapamycin (mTOR) coordinates eukaryotic cell growth and metabolism with environmental inputs, including nutrients and growth factors. Extensive research over the past two decades has established a central role for mTOR in regulating many fundamental cell processes, from protein synthesis to autophagy, and deregulated mTOR signaling is implicated in the progression of cancer and diabetes, as well as the aging process. Here, we review recent advances in our understanding of mTOR function, regulation, and importance in mammalian physiology. We also highlight how the mTOR signaling network contributes to human disease and discuss the current and future prospects for therapeutically targeting mTOR in the clinic.

mTOR Signaling in Growth, Metabolism, and Disease

Fitting the image of a single molecule to the point spread function of an optical system greatly improves the precision with which single molecules can be located. Centroid localization with nanometer precision has been achieved when a sufficient number of photons are collected. However, if multiple single molecules reside within a diffraction-limited spot, this localization approach does not work. This paper demonstrates nanometer-localized multiple single-molecule (NALMS) fluorescence microscopy by using both centroid localization and photobleaching of the single fluorophores. Short duplex DNA strands are used as nanoscale "rulers" to validate the NALMS microscopy approach. Nanometer accuracy is demonstrated for two to five single molecules within a diffraction-limited area. NALMS microscopy will greatly facilitate single-molecule study of biological systems because it covers the gap between fluorescence resonance energy transfer-based ( 100 nm) measurements of the distance between two fluorophores. Application of NALMS microscopy to DNA mapping with <10-nm (i.e., 30-base) resolution is demonstrated.

https://www.pnas.org/content/pnas/101/31/11298.full.pdf

Nanometer-localized multiple single-molecule fluorescence microscopy.

Heart valve anomalies are some of the most common congenital heart defects, yet neither the genetic nor the epigenetic forces guiding heart valve development are well understood. When functioning normally, mature heart valves prevent intracardiac retrograde blood flow; before valves develop, there is considerable regurgitation, resulting in reversing (or oscillatory) flows between the atrium and ventricle. As reversing flows are particularly strong stimuli to endothelial cells in culture, an attractive hypothesis is that heart valves form as a developmental response to retrograde blood flows through the maturing heart. Here, we exploit the relationship between oscillatory flow and heart rate to manipulate the amount of retrograde flow in the atrioventricular (AV) canal before and during valvulogenesis, and find that this leads to arrested valve growth. Using this manipulation, we determined that klf2a is normally expressed in the valve precursors in response to reversing flows, and is dramatically reduced by treatments that decrease such flows. Experimentally knocking down the expression of this shear-responsive gene with morpholine antisense oligonucleotides (MOs) results in dysfunctional valves. Thus, klf2a expression appears to be necessary for normal valve formation. This, together with its dependence on intracardiac hemodynamic forces, makes klf2a expression an early and reliable indicator of proper valve development. Together, these results demonstrate a critical role for reversing flows during valvulogenesis and show how relatively subtle perturbations of normal hemodynamic patterns can lead to both major alterations in gene expression and severe valve dysgenesis.

/pdf/reversing-blood-flows-act-through-klf2a-to-ensure-normal-53a6syd97j.pdf

Reversing Blood Flows Act through klf2a to Ensure Normal Valvulogenesis in the Developing Heart

Fluorescence microscopy has profoundly changed cell and molecular biology studies by permitting tagged gene products to be followed as they function and interact. The ability of a fluorescent dye to absorb and emit light of different wavelengths allows it to generate startling contrast that, in the best cases, can permit single molecule detection and tracking. However, in many experimental settings, fluorescent probes fall short of their potential due to dye bleaching, dye signal saturation, and tissue autofluorescence. Here, we demonstrate that second harmonic generating (SHG) nanoprobes can be used for in vivo imaging, circumventing many of the limitations of classical fluorescence probes. Under intense illumination, such as at the focus of a laser-scanning microscope, these SHG nanocrystals convert two photons into one photon of half the wavelength; thus, when imaged by conventional two-photon microscopy, SHG nanoprobes appear to generate a signal with an inverse Stokes shift like a fluorescent dye, but with a narrower emission. Unlike commonly used fluorescent probes, SHG nanoprobes neither bleach nor blink, and the signal they generate does not saturate with increasing illumination intensity. The resulting contrast and detectability of SHG nanoprobes provide unique advantages for molecular imaging of living cells and tissues.

https://www.pnas.org/content/pnas/107/33/14535.full.pdf

Second harmonic generating (SHG) nanoprobes for in vivo imaging.

Processivity, the ability of single molecules to move continuously along a track, is a fundamental requirement of cargo-transporting molecular motors. Here, we investigate how cytoplasmic dynein, a homodimeric, microtubule-based motor, achieves processive motion. To do this, we developed a versatile method for assembling Saccharomyces cerevisiae dynein heterodimers, using complementary DNA oligonucleotides covalently linked to dynein monomers labeled with different organic fluorophores. Using two-color, single-molecule microscopy and high-precision, two-dimensional tracking, we find that dynein has a highly variable stepping pattern that is distinct from all other processive cytoskeletal motors, which use 'hand-over-hand' mechanisms. Uniquely, dynein stepping is stochastic when its two motor domains are close together. However, coordination emerges as the distance between motor domains increases, implying that a tension-based mechanism governs these steps. This plasticity may allow tuning of dynein for its diverse cellular functions.

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Dynein achieves processive motion using both stochastic and coordinated stepping.

Background: Anti-inflammatory therapies such as IL-6 inhibition have been proposed for COVID-19 in a vacuum of evidence-based treatment. However, abrogating the inflammatory response in infectious diseases may impair a desired host response and pre-dispose to secondary infections. Methods: We retrospectively reviewed the medical record of critically ill COVID-19 patients during an 8-week span and compared the prevalence of secondary infection and outcomes in patients who did and did not receive tocilizumab. Additionally, we included representative histopathologic post-mortem findings from several COVID-19 cases that underwent autopsy at our institution. Results: One hundred eleven patients were identified, of which 54 had received tocilizumab while 57 had not. Receiving tocilizumab was associated with a higher risk of secondary bacterial (48.1 vs. 28.1%; p = 0.029 and fungal (5.6 vs. 0%; p = 0.112) infections. Consistent with higher number of infections, patients who received tocilizumab had higher mortality (35.2 vs. 19.3%; p = 0.020). Seven cases underwent autopsy. In three cases who received tocilizumab, there was evidence of pneumonia on pathology. Of the four cases that had not been given tocilizumab, two showed evidence of aspiration pneumonia and two exhibited diffuse alveolar damage. Conclusions: Experimental therapies are currently being applied to COVID-19 outside of clinical trials. Anti-inflammatory therapies such as anti-IL-6 therapy have the potential to impair viral clearance, pre-dispose to secondary infection, and cause harm. We seek to raise physician awareness of these issues and highlight the need to better understand the immune response in COVID-19.

/pdf/il-6-inhibition-in-critically-ill-covid-19-patients-is-uz0hrppwbt.pdf

David Wu

Papers

Nanometer-localized multiple single-molecule fluorescence microscopy.

Reversing Blood Flows Act through klf2a to Ensure Normal Valvulogenesis in the Developing Heart

Second harmonic generating (SHG) nanoprobes for in vivo imaging.

Dynein achieves processive motion using both stochastic and coordinated stepping.

IL-6 Inhibition in Critically Ill COVID-19 Patients Is Associated With Increased Secondary Infections.