A turquoise fluorescence lifetime-based biosensor for quantitative imaging of intracellular calcium

The most successful genetically encoded calcium indicators (GECIs) employ an intensity or ratiometric readout. Despite a large calcium-dependent change in fluorescence intensity, the quantification of calcium concentrations with GECIs is problematic, which is further complicated by the sensitivity of all GECIs to changes in the pH in the biological range. Here, we report on a sensing strategy in which a conformational change directly modifies the fluorescence quantum yield and fluorescence lifetime of a circular permutated turquoise fluorescent protein. The fluorescence lifetime is an absolute parameter that enables straightforward quantification, eliminating intensity-related artifacts. An engineering strategy that optimizes lifetime contrast led to a biosensor that shows a 3-fold change in the calcium-dependent quantum yield and a fluorescence lifetime change of 1.3 ns. We dub the biosensor Turquoise Calcium Fluorescence LIfeTime Sensor (Tq-Ca-FLITS). The response of the calcium sensor is insensitive to pH between 6.2–9. As a result, Tq-Ca-FLITS enables robust measurements of intracellular calcium concentrations by fluorescence lifetime imaging. We demonstrate quantitative imaging of calcium concentrations with the turquoise GECI in single endothelial cells and human-derived organoids.

The H i Column Density Distribution of the Galactic Disk and Halo

We present a census of neutral gas in the Milky Way disk and halo down to limiting column densities of N(H i) ∼ 1014 cm−2 using measurements of H i Lyman series absorption from the Far Ultraviolet Spectroscopic Explorer. Our results are drawn from an analysis of 25 AGN sight lines spread evenly across the sky with Galactic latitude ∣b∣ ≳ 20°. By simultaneously fitting multi-component Voigt profiles to 11 Lyman series absorption transitions covered by FUSE (Lyβ–Lyμ) plus HST measurements of Lyα, we derive the kinematics and column densities of a sample of 152 H i absorption components. While saturation prevents accurate measurements of many components with column densities 17 ≲ log N(H i) ≲ 19, we derive robust measurements at log N(H i) ≲ 17 and log N(H i) ≳ 19. We derive the first ultraviolet H i column density distribution function (CDDF) of the Milky Way, both globally and for low-velocity (ISM), intermediate-velocity clouds (IVCs), and high-velocity clouds (HVCs). We find that IVCs and HVCs show statistically indistinguishable CDDF slopes, with β IVC = − 1.01 − 0.14 + 0.15 and β HVC = − 1.05 − 0.06 + 0.07 . Overall, the CDDF of the Galactic disk and halo appears shallower than that found by comparable extragalactic surveys, suggesting a relative abundance of high column density gas in the Galactic halo. We derive the sky-covering fractions as a function of H i column density, finding an enhancement of IVC gas in the northern hemisphere compared to the south. We also find evidence for an excess of inflowing H i over outflowing H i, with −0.88 ± 0.40 M ⊙ yr−1 of HVC inflow versus ≈0.20 ± 0.10 M ⊙ yr−1 of HVC outflow, confirming an excess of inflowing HVCs seen in UV metal lines.

Evaluation of Chip Formation Mechanisms in the Turning of Sintered ZnO Electro-Ceramics

The sintered zinc oxide (ZnO) electro-ceramics are a brittle class of hard-to-cut materials such that shaping them with the post-finishing operations necessitates careful handling and precision machining. The conventional machining approach using the grinding and lapping processes represents limited productivity, an inability to produce the required geometries and frequent uncontrolled chipping of the edges of the final products. This study thus investigates the turning performance of dense sintered ZnO varistors and chip formations to obtain the parametric range (cutting mechanism) which causes the chipping or the trans-granular/sudden failure in these brittle materials. With the analysis of the cutting tool vibration in relation to the machining parameters (f and VC), the vibration-induced chipping correlations are made and interlinked with the occurrence of grain pull-out during the turning operation. The results show that the reflected vibratory motion of the tools is directly correlated with the chip formation mechanisms in the turning of ZnO ceramics and thus provide robust measurements for quality assurance in final products.

A turquoise fluorescence lifetime-based biosensor for quantitative imaging of intracellular calcium

The most successful genetically encoded calcium indicators (GECIs) employ an intensity or intensiometric readout. Despite a large calcium-dependent change in fluorescence intensity, the quantification of calcium concentrations with GECIs is problematic, which is further complicated by the sensitivity of all GECIs to changes in the pH in the biological range. Here, we report on a novel sensing strategy in which a conformational change directly modifies the fluorescence quantum yield and fluorescence lifetime of a circular permutated turquoise fluorescent protein. The fluorescence lifetime is an absolute parameter that enables straightforward quantification, eliminating intensity-related artifacts. A new engineering strategy that optimizes lifetime contrast led to a biosensor that shows a 3-fold change in the calcium-dependent quantum yield and a fluorescence lifetime change of 1.3 ns. Additionally, the response of the calcium sensor is insensitive to pH between 6.2-9. As a result, the turquoise GECI enables robust measurements of intracellular calcium concentrations by fluorescence lifetime imaging. We demonstrate quantitative imaging of calcium concentration with the turquoise GECI in single endothelial cells and human-derived organoids.

Identification of Candidate Protein Biomarkers for CIN2+ Lesions from Self-Sampled, Dried Cervico–Vaginal Fluid Using LC-MS/MS

Molecular screening programs for cervical cancer detect the presence of human papilloma virus (HPV) in cell material or vaginal fluids. Persistent infection with high-risk HPV is a necessary pre-requisite, but the majority of infections do not lead to pathological states. Additional biomarkers are needed to increase the specificity of the molecular tests. Here, we have investigated the possibility of detecting protein biomarkers using mass spectrometry from dried self-sampled cervico–vaginal fluid deposited on FTA cards. We found significant intra-individual correlations (p < 2.2 × 10−16), although heterogenous protein profiles were obtained between individuals. Out of 3699 proteins found in total, 169 were detected in at least 95% of the samples. Using a discovery/replication design, 18 proteins were found to be significant in the discovery cohort, with higher values in those cases compared to controls. All of these were found to also have higher levels among the cases in the replication cohort, with one protein (DEAD-Box Helicase) remaining statistically significant. Finally, a predictive 7-protein multivariate model was developed with a sensitivity and specificity of 0.90 and 0.55, respectively. Our results demonstrate that robust measurements of protein biomarkers can be obtained from self-sampled dried CVF and that these could be used to predict cervical cancer pre-stages.

Online process monitoring of a batch distillation by medium field NMR spectroscopy

Medium field NMR spectrometers are attractive for online process monitoring. Therefore, in the present work, a single-stage laboratory batch distillation still was coupled online with a medium field NMR spectrometer. This enables quantitative non-invasive measurements without calibration. The technique was used for studying isobaric and isothermal residue curves in two ternary systems: (dimethyl sulfoxide + acetonitrile + ethyl formate) and (ethyl acetate + acetone + diethyl ether) and boiling curves and high-boiling azeotropes in two binary systems: (acetic acid + pyridine) and (methanol + diethylamine). The results of the online NMR spectroscopic analysis were compared to results from offline analysis as well as to results from thermodynamic modeling using NRTL parameters that were parametrized with literature data. The new method for online process monitoring gives reliable results and is well-suited for fast and robust measurements of residue curves.

Natural abundance isotope ratios to differentiate sources of carbon used during tumor growth in vivo

Background Radioactive or stable isotopic labeling of metabolites is a strategy that is routinely used to map the cellular fate of a selected labeled metabolite after it is added to cell culture or to the circulation of an animal. However, a labeled metabolite can be enzymatically changed in cellular metabolism, complicating the use of this experimental strategy to understand how a labeled metabolite moves between organs. These methods are also technically demanding, expensive and potentially toxic. To allow quantification of the bulk movement of metabolites between organs, we have developed a novel application of stable isotope ratio mass spectrometry (IRMS). Results We exploit natural differences in 13C/12C ratios of plant nutrients for a low-cost and non-toxic carbon labeling, allowing a measurement of bulk carbon transfer between organs in vivo. IRMS measurements were found to be sufficiently sensitive to measure organs from individual Drosophila melanogaster larvae, giving robust measurements down to 2.5 μg per sample. We apply the method to determine if carbon incorporated into a growing solid tumor is ultimately derived from food or host tissues. Conclusion Measuring tumor growth in a D. melanogaster larvae tumor model reveals that these tumors derive a majority of carbon from host sources. We believe the low cost and non-toxic nature of this methodology gives it broad applicability to study carbon flows between organs also in other animals and for a range of other biological questions.

UAB-UCSD O'Brien Center for Acute Kidney Injury Research

Acute kidney injury (AKI) remains a significant clinical problem through its diverse etiologies, the challenges of robust measurements of injury and recovery and its progression to chronic kidney disease. Bridging the gap in knowledge of this disorder requires bringing together not only the technical resources for research, but also the investigators endeavoring to expand our knowledge and those who might bring novel ideas and expertise to this important challenge. The UAB-UCSD O'Brien Center for AKI Research brings together technical expertise and programmatic and educational efforts to advance our knowledge in these diverse issues and the required infrastructure to develop areas of novel exploration. Since inception, this Center has grown its impact by providing state-of-the-art resources in clinical and pre-clinical modeling of AKI, a bioanalytical core that facilitates measurement of critical biomarkers, including serum creatinine via LC-MS/MS among others, and a biostatistical resource. Through these resources and with educational efforts, our Center has grown the Investigator Base to include >200 members from 51 institutions. Importantly, this Center has translated its pilot and catalyst funding program with a $37 return/dollar invested. Over 500 publications have resulted from the support provided with a relative citation ratio of 2.18 ± 0.12. Through its efforts, this disease-centric O'Brien Center is providing the infrastructure and focus to help the development of the next generation of researchers in the basic and clinical science of AKI. This Center creates the promise of the application at the bedside of the advances in AKI made by current and future investigators.

Sentinel-1 A-DInSAR Approaches to Map and Monitor Ground Displacements

Persistent scatterer interferometry (PSI) is a group of advanced interferometric synthetic aperture radar (SAR) techniques used to measure and monitor terrain deformation. Sentinel-1 has improved the data acquisition throughout and, compared to previous sensors, increased considerably the differential interferometric SAR (DInSAR) and PSI deformation monitoring potential. The low density of persistent scatterer (PS) in non-urban areas is a critical issue in DInSAR and has inspired the development of alternative approaches and refinement of the PS chains. This paper proposes two different and complementary data-driven procedures to obtain terrain deformation maps. These approaches aim to exploit Sentinel-1 highly coherent interferograms and their short revisit time. The first approach, called direct integration (DI), aims at providing a very fast and straightforward approach to screen-wide areas and easily detects active areas. This approach fully exploits the coherent interferograms from consecutive images provided by Sentinel-1, resulting in a very high sampling density. However, it lacks robustness and its usability lays on the operator experience. The second method, called persistent scatterer interferometry geomatics (PSIG) short temporal baseline, provides a constrained application of the PSIG chain, the CTTC approach to the PSI. It uses short temporal baseline interferograms and does not assume any deformation model for point selection. It is also quite a straightforward approach, which improves the performances of the standard PSIG approach, increasing the PS density and providing robust measurements. The effectiveness of the approaches is illustrated through analyses performed on different test sites.