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Genetically encoded Förster Resonance Energy Transfer (FRET)-based biosensors are powerful tools to illuminate spatiotemporal regulation of cell signaling in living cells, but the utility of the red spectrum for biosensing was limited due to a lack of bright and stable red fluorescent proteins. Here, we rationally improve the photophysical characteristics of the coral-derived fluorescent protein TagRFP-T. We show that a new single-residue mutant, super-TagRFP (stagRFP) has nearly twice the molecular brightness of TagRFP-T and negligible photoactivation. stagRFP facilitates significant improvements on multiple green-red biosensors as a FRET acceptor and is an efficient FRET donor that supports red/far-red FRET biosensing. Capitalizing on the ability of stagRFP to couple with multiple FRET partners, we develop a novel multiplex method to examine the confluence of signaling activities from three kinases simultaneously in single living cells, providing evidence for a role of Src family kinases in regulating growth factor induced Akt and ERK activities. TagRFP is a bright red fluorescent protein, but undergoes photoconversion to a dark state, making it undesirable for conventional fluorescence microscopy. Here, the authors introduce stabilising mutations to create super-TagRFP (stagRFP) and demonstrate its application as both a FRET acceptor and FRET donor.

Gasdermin D forms large, ~21 nm diameter pores in the plasma membrane to drive the cell death program pyroptosis. These pores are thought to be permanently open, and the resultant osmotic imbalance is thought to be highly damaging. Yet some cells mitigate and survive pore formation, suggesting an undiscovered layer of regulation over the function of these pores. However, no methods exist to directly reveal these mechanistic details. Here, we combine optogenetic tools, live cell fluorescence biosensing, and electrophysiology to demonstrate that gasdermin pores display phosphoinositide-dependent dynamics. We quantify repeated and fast opening-closing of these pores on the tens of seconds timescale, visualize the dynamic pore geometry, and identify the signaling that controls dynamic pore activity. The identification of this circuit allows pharmacological tuning of pyroptosis and control of inflammatory cytokine release by living cells. During pyroptosis, gasdermin D (GSDMD) forms plasma membrane pores that initiate cell lysis. Here, the authors develop optogenetically activatable human GSDMD to assess GSDMD pore behavior and show that they are dynamic and can close, which can be a pyroptosis regulatory mechanism.

Superresolution fluorescence microscopy overcomes the diffraction resolution barrier and allows the molecular intricacies of life to be revealed with greatly enhanced detail. However, many current superresolution techniques still face limitations and their implementation is typically associated with a steep learning curve. Patterned illumination-based superresolution techniques [e.g., stimulated emission depletion (STED), reversible optically-linear fluorescence transitions (RESOLFT), and saturated structured illumination microscopy (SSIM)] require specialized equipment whereas single-molecule-based approaches [e.g., stochastic optical reconstruction microscopy (STORM), photo-activation localization microscopy (PALM), and fluorescence-PALM (F-PALM)] involve repetitive single-molecule localization, which requires its own set of expertise and is also temporally demanding. Here we present a superresolution fluorescence imaging method, photochromic stochastic optical fluctuation imaging (pcSOFI). In this method, irradiating a reversibly photoswitching fluorescent protein at an appropriate wavelength produces robust single-molecule intensity fluctuations, from which a superresolution picture can be extracted by a statistical analysis of the fluctuations in each pixel as a function of time, as previously demonstrated in SOFI. This method, which uses off-the-shelf equipment genetically encodable labels, and simple and rapid data acquisition, is capable of providing two-to threefold-enhanced spatial resolution, significant background rejection, markedly improved contrast, and favorable temporal resolution in living cells. Furthermore, both 3D and multicolor imaging are readily achievable. Because of its ease of use and high performance, we anticipate that pcSOFI will prove an attractive approach for superresolution imaging.

New fluorescent biosensors enable the first super-resolution imaging of enzyme activity in live cells via fluorescence fluctuation increase by contact (FLINC). Compartmentalized biochemical activities are essential to all cellular processes, but there is no generalizable method to visualize dynamic protein activities in living cells at a resolution commensurate with cellular compartmentalization. Here, we introduce a new class of fluorescent biosensors that detect biochemical activities in living cells at a resolution up to threefold better than the diffraction limit. These 'FLINC' biosensors use binding-induced changes in protein fluorescence dynamics to translate kinase activities or protein–protein interactions into changes in fluorescence fluctuations, which are quantifiable through stochastic optical fluctuation imaging. A protein kinase A (PKA) biosensor allowed us to resolve minute PKA activity microdomains on the plasma membranes of living cells and to uncover the role of clustered anchoring proteins in organizing these activity microdomains. Together, these findings suggest that biochemical activities of the cell are spatially organized into an activity architecture whose structural and functional characteristics can be revealed by these new biosensors.

This paper reports a new strategy for the rapid and efficient generation of ratiometric fluorescent sensors for a variety of cellular processes. We have developed a versatile new class of genetically encoded fluorescent biosensor based on reversible exchange of the heterodimeric partners of green and red dimerization-dependent fluorescent proteins. We demonstrate the use of this strategy to construct both intermolecular and intramolecular ratiometric biosensors for qualitative imaging of caspase activity, Ca 2+ concentration dynamics and other second-messenger signaling activities.

Drought indices derived from the North American Land Data Assimilation System (NLDAS) Variable Infiltration Capacity (VIC) and Noah models from 1950 to 2000 are intercompared and evaluated for their ability to classify drought across the United States. For meteorological drought, the standardized precipitation index (SPI) is used to measure precipitation deficits. The standardized runoff index (SRI), which is similar to the SPI, is used to classify hydrological drought. Agricultural drought is measured by monthly-mean soil moisture (SM) anomaly percentiles based on probability distributions (PDs). The PDs for total SM are regionally dependent and influenced by the seasonal cycle, but the PDs for SM monthly-mean anomalies are unimodal and Gaussian. Across the eastern United States (east of 95°W), the indices derived from VIC and Noah are similar, and they are able to detect the same drought events. Indices are also well correlated. For river forecast centers (RFCs) across the eastern United States, different drought indices are likely to detect the same drought events. The monthly-mean soil moisture (SM) percentiles and runoff indices between VIC and Noah have large differences across the western interior of the United States. For small areas with a horizontal resolution of 0.5° on the time scales of one to three months, the differences of SM percentiles and SRI between VIC and Noah are larger than the thresholds used to classify drought. For the western RFCs, drought events selected according to SM percentiles or SRI derived from different NLDAS systems do not always overlap.

Data from observations and the Intergovernmental Panel on Climate Change (IPCC) twentieth-century climate change model [phase 3 of the Coupled Model Intercomparison Project (CMIP3)] simulations were analyzed to examine the decadal changes of the impact of ENSO on air temperatureT airand precipitationPover the United States. The comparison of composites for the early period (1915–60) and the recent period (1962–2006) indicates that cooling (warming) over the south and warming (cooling) over the north during ENSO warm (cold) winters have been weakening. The ENSO influence on winterPover the Southwest is strengthening, while the impact onPover the Ohio Valley is weakening for the recent decades. These differences are not due to the long-term trends inT airorP; they are attributed to the occurrence of the central Pacific (CPAC) ENSO events in the recent years. The CPAC ENSO differs from the canonical eastern Pacific (EPAC) ENSO. The EPAC ENSO has a sea surface temperature anomaly (SSTA) maximum in the eastern Pacific. Enhanced convection extends from the date line to the eastern Pacific, with negative anomalies in the western Pacific. The atmospheric responses resemble a tropical Northern Hemisphere pattern. The wave train is consistent with the north–southT aircontrast over North America during the EPAC ENSO winters. The CPAC ENSO has enhanced convection in the central Pacific. The atmospheric responses show a Pacific–North American pattern. It is consistent with west–east contrast inT airand more rainfall over the Southwest during the CPAC ENSO winters.

PurposeCancer survivors experience significant health concerns compared to the general population. Sydney Survivorship Clinic (SSC) is a multi-disciplinary clinic aiming to help survivors treated with curative intent manage side effects, and establish a healthy lifestyle. Here, we determine the health concerns of survivors post-primary treatment.MethodsSurvivors completed questionnaires assessing symptoms, quality of life (QOL), distress, diet, and exercise before attending SSC, and a satisfaction survey after. Body mass index (BMI), clinical findings and recommendations were reviewed. Descriptive statistical methods were used.ResultsOverall, 410 new patients attended SSC between September 2013 and April 2018, with 385 survivors included in analysis: median age 57 years (range 18–86); 69% female; 43% breast, 31% colorectal and 19% haematological cancers. Median time from diagnosis, 12 months. Common symptoms of at least moderate severity: fatigue (45%), insomnia (37%), pain (34%), anxiety (31%) and with 56% having > 5 moderate-severe symptoms. Overall, 45% scored distress ≥ 4/10 and 62% were rated by clinical psychologist as having ‘fear of cancer recurrence’. Compared to population mean of 50, mean global QOL T-score was 47.2, with physical and emotional well-being domains most affected. Average BMI was 28.2 kg/m2 (range 17.0–59.1); 61% overweight/obese. Only 31% met aerobic exercise guidelines. Overall, 98% ‘agreed’/‘completely agreed’ attending the SSC was worthwhile, and 99% would recommend it to others.ConclusionDistress, fear of cancer recurrence, fatigue, obesity and sedentary lifestyle are common in cancer survivors attending SSC and may best be addressed in a multi-disciplinary Survivorship Clinic to minimise longer-term effects. This model is well-rated by survivors.

Next-generation neutrino telescopes with substantially improved sensitivity are required to pinpoint the sources of the diffuse astrophysical neutrino flux detected by IceCube and uncover the century-old puzzle of cosmic-ray origins. A detector near the Equator will provide a unique viewpoint of the neutrino sky, complementing IceCube and other neutrino telescopes in the Northern Hemisphere. Here we present results from an expedition to the northeastern region of the South China Sea, in the western Pacific Ocean. A favourable neutrino telescope site was found on an abyssal plain at a depth of ~3.5 km. At depths below 3 km, the sea current speed, water absorption and scattering lengths for Cherenkov light were measured to be vc < 10 cm s−1, λabs ≈ 27 m and λsca ≈ 63 m, respectively. Accounting for these measurements, we present the design and expected performance of a next-generation neutrino telescope, Tropical Deep-sea Neutrino Telescope (TRIDENT). With its advanced photon-detection technology and large dimensions, TRIDENT expects to observe the IceCube steady source candidate NGC 1068 with 5σ significance within 1 year of operation. This level of sensitivity will open a new arena for diagnosing the origin of cosmic rays and probing fundamental physics over astronomical baselines.A South China Sea expedition in 2021 identified a 3.5-km-deep site close to the Equator for a next-generation neutrino telescope: TRIDENT. A large array of advanced detectors will be arrayed on the seabed to probe fundamental physics and explore the extreme Universe.