Development of Company-Specific Emission Factors with Confidence Intervals for Natural Gas Customer Meters in Southern California.

Methane is both a significant and short-lived greenhouse gas compared to CO2, and reducing methane emissions from natural gas distribution systems may offer cost-effective reduction opportunities. We report substantial new direct leak rate measurements from customer meter set assemblies (MSAs) in Southern California. In a novel way, emission factors are defined in terms of aboveground Hazardous and Nonhazardous leak categories, which take direct advantage of readily available industry leak data. We also studied leaks that were not detected as part of normal leak survey procedures. As a result, this yields company-specific emission factors that can be used to track progress in reducing methane emissions. This approach also has the advantage of explicitly accounting for the skewed or fat-tail distribution of leak rates by treating high flow rate MSA leaks separately from low flow rate MSA leaks. The Southern California Gas (SoCalGas) methane emission factors, based on 485 leak rate measurements by direct enclosure, were 4.55 (95% confidence interval: 2.32 to 7.14) kg/day for Hazardous leaks, 0.149 (0.119 to 0.183) kg/day for Nonhazardous leaks, and 0.0039 (0.0003 to 0.0198) kg/day for Non-Detected leaks. The percentage of surveyed meters with nondetected leaks was 29.1% (24.3 to 34.6%). Based on a robust Monte Carlo analysis, total leak emissions from MSAs for the SoCalGas system were reduced by 35% based on data from 2015 to 2022. These reductions were attributed to surveying a larger number of MSAs and accelerated leak repair rates. In traditional population-based emission inventories, an individual emission factor for a given asset category is multiplied by the total population of MSAs within the category. This approach simply cannot capture the reduction in leak numbers and methane emissions resulting from leak mitigation and prevention programs.

Reliability Testing of a Low-Cost, Multi-Purpose Arduino-Based Data Logger Deployed in Several Applications Such as Outdoor Air Quality, Human Activity, Motion, and Exhaust Gas Monitoring.

This contribution shows the possibilities of applying a low-cost, multi-purpose data logger built around an Arduino Mega 2560 single-board computer. Most projects use this kind of hardware to develop single-purpose data loggers. In this work, a data logger with a more general hardware and software architecture was built to perform measurement campaigns in very different domains. The wide applicability of this data logger was demonstrated with short-term monitoring campaigns in relation to outdoor air quality, human activity in an office, motion of a journey on a bike, and exhaust gas monitoring of a diesel generator. In addition, an assessment process and corresponding evaluation framework are proposed to assess the credibility of low-cost scientific devices built in-house. The experiences acquired during the development of the system and the short measurement campaigns were used as inputs in the assessment process. The assessment showed that the system scores positively on most product-related targets. However, unexpected events affect the assessment over the longer term. This makes the development of low-cost scientific devices harder than expected. To assure stability and long-term performance of this type of design, continuous evaluation and regular engineering corrections are needed throughout longer testing periods.

The Growing and Glowing Toolbox of Fluorescent and Photoactive Proteins.

Over the past 20 years, protein engineering has been extensively used to improve and modify the fundamental properties of fluorescent proteins (FPs) with the goal of adapting them for a fantastic range of applications. FPs have been modified by a combination of rational design, structure-based mutagenesis, and countless cycles of directed evolution (gene diversification followed by selection of clones with desired properties) that have collectively pushed the properties to photophysical and biochemical extremes. In this review, we provide both a summary of the progress that has been made during the past two decades, and a broad overview of the current state of FP development and applications in mammalian systems.

Fluorescent proteins for in vivo imaging, where's the biliverdin?

Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deeper imaging. Near-infrared light penetrates biological tissue with blood vessels due to low absorbance, scattering, and reflection of light and has a greater signal-to-noise due to less autofluorescence. Far-red and near-infrared fluorescent proteins absorb light >600 nm to expand the color palette for imaging multiple biosensors and noninvasive in vivo imaging. The ideal fluorescent proteins are bright, photobleach minimally, express well in the desired cells, do not oligomerize, and generate or incorporate exogenous fluorophores efficiently. Coral-derived red fluorescent proteins require oxygen for fluorophore formation and release two hydrogen peroxide molecules. New fluorescent proteins based on phytochrome and phycobiliproteins use biliverdin IXα as fluorophores, do not require oxygen for maturation to image anaerobic organisms and tumor core, and do not generate hydrogen peroxide. The small Ultra-Red Fluorescent Protein (smURFP) was evolved from a cyanobacterial phycobiliprotein to covalently attach biliverdin as an exogenous fluorophore. The small Ultra-Red Fluorescent Protein is biophysically as bright as the enhanced green fluorescent protein, is exceptionally photostable, used for biosensor development, and visible in living mice. Novel applications of smURFP include in vitro protein diagnostics with attomolar (10-18 M) sensitivity, encapsulation in viral particles, and fluorescent protein nanoparticles. However, the availability of biliverdin limits the fluorescence of biliverdin-attaching fluorescent proteins; hence, extra biliverdin is needed to enhance brightness. New methods for improved biliverdin bioavailability are necessary to develop improved bright far-red and near-infrared fluorescent proteins for noninvasive imaging in vivo.

An Automated Pipeline for Image Processing and Data Treatment to Track Activity Rhythms of Paragorgia arborea in Relation to Hydrographic Conditions.

Imaging technologies are being deployed on cabled observatory networks worldwide. They allow for the monitoring of the biological activity of deep-sea organisms on temporal scales that were never attained before. In this paper, we customized Convolutional Neural Network image processing to track behavioral activities in an iconic conservation deep-sea species-the bubblegum coral Paragorgia arborea-in response to ambient oceanographic conditions at the Lofoten-Vesterålen observatory. Images and concomitant oceanographic data were taken hourly from February to June 2018. We considered coral activity in terms of bloated, semi-bloated and non-bloated surfaces, as proxy for polyp filtering, retraction and transient activity, respectively. A test accuracy of 90.47% was obtained. Chronobiology-oriented statistics and advanced Artificial Neural Network (ANN) multivariate regression modeling proved that a daily coral filtering rhythm occurs within one major dusk phase, being independent from tides. Polyp activity, in particular extrusion, increased from March to June, and was able to cope with an increase in chlorophyll concentration, indicating the existence of seasonality. Our study shows that it is possible to establish a model for the development of automated pipelines that are able to extract biological information from times series of images. These are helpful to obtain multidisciplinary information from cabled observatory infrastructures.

A Flexible Autonomous Robotic Observatory Infrastructure for Bentho-Pelagic Monitoring.

This paper presents the technological developments and the policy contexts for the project "Autonomous Robotic Sea-Floor Infrastructure for Bentho-Pelagic Monitoring" (ARIM). The development is based on the national experience with robotic component technologies that are combined and merged into a new product for autonomous and integrated ecological deep-sea monitoring. Traditional monitoring is often vessel-based and thus resource demanding. It is economically unviable to fulfill the current policy for ecosystem monitoring with traditional approaches. Thus, this project developed platforms for bentho-pelagic monitoring using an arrangement of crawler and stationary platforms at the Lofoten-Vesterålen (LoVe) observatory network (Norway). Visual and acoustic imaging along with standard oceanographic sensors have been combined to support advanced and continuous spatial-temporal monitoring near cold water coral mounds. Just as important is the automatic processing techniques under development that have been implemented to allow species (or categories of species) quantification (i.e., tracking and classification). At the same time, real-time outboard processed three-dimensional (3D) laser scanning has been implemented to increase mission autonomy capability, delivering quantifiable information on habitat features (i.e., for seascape approaches). The first version of platform autonomy has already been tested under controlled conditions with a tethered crawler exploring the vicinity of a cabled stationary instrumented garage. Our vision is that elimination of the tether in combination with inductive battery recharge trough fuel cell technology will facilitate self-sustained long-term autonomous operations over large areas, serving not only the needs of science, but also sub-sea industries like subsea oil and gas, and mining.

A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein.

Far-red fluorescent proteins (FPs) are desirable for in vivo imaging because with these molecules less light is scattered, absorbed, or re-emitted by endogenous biomolecules compared with cyan, green, yellow, and orange FPs. We developed a new class of FP from an allophycocyanin α-subunit (APCα). Native APC requires a lyase to incorporate phycocyanobilin. The evolved FP, which we named small ultra-red FP (smURFP), covalently attaches a biliverdin (BV) chromophore without a lyase, and has 642/670-nm excitation-emission peaks, a large extinction coefficient (180,000 M(-1)cm(-1)) and quantum yield (18%), and photostability comparable to that of eGFP. smURFP has significantly greater BV incorporation rate and protein stability than the bacteriophytochrome (BPH) FPs. Moreover, BV supply is limited by membrane permeability, and smURFPs (but not BPH FPs) can incorporate a more membrane-permeant BV analog, making smURFP fluorescence comparable to that of FPs from jellyfish or coral. A far-red and near-infrared fluorescent cell cycle indicator was created with smURFP and a BPH FP.

Efficient non-degenerate two-photon excitation for fluorescence microscopy.

Non-degenerate two-photon excitation (ND-TPE) has been explored in two-photon excitation microscopy. However, a systematic study of the efficiency of ND-TPE to guide the selection of fluorophore excitation wavelengths is missing. We measured the relative non-degenerate two-photon absorption cross-section (ND-TPACS) of several commonly used fluorophores (two fluorescent proteins and three small-molecule dyes) and generated 2-dimensional ND-TPACS spectra. We observed that the shape of a ND-TPACS spectrum follows that of the corresponding degenerate two-photon absorption cross-section (D-TPACS) spectrum, but is higher in magnitude. We found that the observed enhancements are higher than theoretical predictions.

New Dioxaborolane Chemistry Enables [(18)F]-Positron-Emitting, Fluorescent [(18)F]-Multimodality Biomolecule Generation from the Solid Phase.

New protecting group chemistry is used to greatly simplify imaging probe production. Temperature and organic solvent-sensitive biomolecules are covalently attached to a biotin-bearing dioxaborolane, which facilitates antibody immobilization on a streptavidin-agarose solid-phase support. Treatment with aqueous fluoride triggers fluoride-labeled antibody release from the solid phase, separated from unlabeled antibody, and creates [(18)F]-trifluoroborate-antibody for positron emission tomography and near-infrared fluorescent (PET/NIRF) multimodality imaging. This dioxaborolane-fluoride reaction is bioorthogonal, does not inhibit antigen binding, and increases [(18)F]-specific activity relative to solution-based radiosyntheses. Two applications are investigated: an anti-epithelial cell adhesion molecule (EpCAM) monoclonal antibody (mAb) that labels prostate tumors and Cetuximab, an anti-epidermal growth factor receptor (EGFR) mAb (FDA approved) that labels lung adenocarcinoma tumors. Colocalized, tumor-specific NIRF and PET imaging confirm utility of the new technology. The described chemistry should allow labeling of many commercial systems, diabodies, nanoparticles, and small molecules for dual modality imaging of many diseases.

Directed Evolution of Fluorescent Proteins in Bacteria.

Directed evolution has revolutionized the way scientists create new biomolecules not found in nature. Error-prone polymerase chain reaction (PCR) introduces random mutations and was used to evolve jellyfish and coral fluorescent proteins in bacteria. We describe a novel method for the directed evolution of a far-red fluorescent protein in E. coli. The new method used genes to produce fluorophores inside E. coli and allowed changing the native fluorophore, phycocyanobilin, for a second small-molecule fluorophore, biliverdin. The directed evolution blueshifted the fluorescence, which enhanced the quantum yield to produce a brighter fluorescent protein. Finally, the evolution selected fluorescent proteins that expressed in large quantities in E. coli. The evolved fluorescent protein was named the small ultra-red fluorescent protein (smURFP) and was biophysically as bright as the enhanced green fluorescent protein (EGFP). This chapter describes the materials and methods used to evolve a far-red fluorescent protein in bacteria. While the focus is a fluorescent protein, the protocol is adaptable for the evolution of other biomolecules in bacteria when using a proper selection strategy.

Structural and photophysical characterization of the small ultra-red fluorescent protein.

The small Ultra-Red Fluorescent Protein (smURFP) represents a new class of fluorescent protein with exceptional photostability and brightness derived from allophycocyanin in a previous directed evolution. Here, we report the smURFP crystal structure to better understand properties and enable further engineering of improved variants. We compare this structure to the structures of allophycocyanin and smURFP mutants to identify the structural origins of the molecular brightness. We then use a structure-guided approach to develop monomeric smURFP variants that fluoresce with phycocyanobilin but not biliverdin. Furthermore, we measure smURFP photophysical properties necessary for advanced imaging modalities, such as those relevant for two-photon, fluorescence lifetime, and single-molecule imaging. We observe that smURFP has the largest two-photon cross-section measured for a fluorescent protein, and that it produces more photons than organic dyes. Altogether, this study expands our understanding of the smURFP, which will inform future engineering toward optimal FPs compatible with whole organism studies.

Comparative study of convolutional neural network architectures for gastrointestinal lesions classification.

The gastrointestinal (GI) tract can be affected by different diseases or lesions such as esophagitis, ulcers, hemorrhoids, and polyps, among others. Some of them can be precursors of cancer such as polyps. Endoscopy is the standard procedure for the detection of these lesions. The main drawback of this procedure is that the diagnosis depends on the expertise of the doctor. This means that some important findings may be missed. In recent years, this problem has been addressed by deep learning (DL) techniques. Endoscopic studies use digital images. The most widely used DL technique for image processing is the convolutional neural network (CNN) due to its high accuracy for modeling complex phenomena. There are different CNNs that are characterized by their architecture. In this article, four architectures are compared: AlexNet, DenseNet-201, Inception-v3, and ResNet-101. To determine which architecture best classifies GI tract lesions, a set of metrics; accuracy, precision, sensitivity, specificity, F1-score, and area under the curve (AUC) were used. These architectures were trained and tested on the HyperKvasir dataset. From this dataset, a total of 6,792 images corresponding to 10 findings were used. A transfer learning approach and a data augmentation technique were applied. The best performing architecture was DenseNet-201, whose results were: 97.11% of accuracy, 96.3% sensitivity, 99.67% specificity, and 95% AUC.

Therapeutic Ultrasound for Topical Corneal Delivery of Macromolecules.

Purpose: The objective of this study was to utilize therapeutic ultrasound in enhancing delivery of topical macromolecules into the cornea. Methods: Rabbit corneas were dissected and placed in a diffusion cell with a small ultra-red fluorescent protein (smURFP; molecular weight of 32,000 Da) as a macromolecule solution. The corneas were treated with continuous ultrasound application at frequencies of 400 or 600 kHz and intensities of 0.8 to 1.0 W/cm2 for 5 minutes, or sham-treated. Fluorescence imaging of the cornea sections was used to observe the delivery of macromolecules into individual epithelial cells. Spectrophotometric analysis at smURFP maximal absorbance of 640 nm was done to determine the presence of macromolecules in the receiver compartment. Safety of ultrasound application was studied through histology analysis. Results: Ultrasound-treated corneas showed smURFP delivery into epithelial cells by fluorescence in the cytoplasm, whereas sham-treated corneas lacked any appreciable fluorescence in the individual cells. The sham group showed 0% of subcellular penetration, whereas the 400 kHz ultrasound-treated group and 600 kHz ultrasound-treated group showed 31% and 57% of subcellular penetration, respectively. Spectrophotometry measurements indicated negligible presence of smURFP macromolecules in the receiver compartment solution in both the sham and ultrasound treatment groups, and these macromolecules did not cross the entire depth of the cornea. Histological studies showed no significant corneal damage due to ultrasound application. Conclusions: Therapeutic ultrasound application was shown to increase the delivery of smURFP macromolecules into the cornea. Translational Relevance: Our study offers a clinical potential for a minimally invasive macromolecular treatment of corneal diseases.

Correlation between the cervical sagittal alignment and spine - pelvic sagittal alignment in asymptomatic adults.

Background: Although there are studies that adequately document the linear correlation between pelvic incidence (PI), sacral slope, lumbar lordosis, and thoracic kyphosis, few have analyzed the pelvic-spine correlation including the cervical spine. Methods: This is a cross-sectional study, wherein the cervical spine was evaluated using radiography and computed tomography (CT) scans, the lumbosacral spine and the pelvis was evaluated using radiography, in adult patients without spinal pathology. Using the Surgimap tool, cervical and spinopelvic parameters were calculated by several investigators. To evaluate the correlation between cervical and spinopelvic parameters, Spearman's coefficient was calculated. To evaluate the concordance correlation of the measured parameters of cervical sagittal alignment on tomography and conventional radiography, Lin's coefficient was calculated and Bland-Altman plots were performed. Results: A total of 51 healthy adults were included in a follow-up from January 2019 to December 2020. Cervical sagittal alignment and sagittal spinopelvic alignment were assessed using radiography, and a correlation was observed between T1 slope (T1S) and lumbar mismatch (coefficient of 0.28, P = 0.047). Then, cervical sagittal alignment was evaluated using CT and sagittal spinopelvic alignment using radiography, and no correlation was observed between PI and thoracic inlet angle or cervical mismatch with lumbar mismatch. Conclusion: In asymptomatic patients, in whom cervical sagittal alignment and spinal-pelvic alignment were evaluated, only a positive correlation was found between lumbar mismatch and T1S, which lacks clinical significance. No concordance was identified between lumbar mismatch and cervical mismatch. Therefore, it is inferred that there is an independence between the sagittal spine-pelvic alignment with respect to the sagittal cervical alignment.

A self-labeling protein based on the small ultra-red fluorescent protein, smURFP.

Self-labeling proteins have revolutionized super-resolution and sensor imaging. Tags recognize a bioorthogonal substrate for covalent attachment. We show the small Ultra-Red Fluorescent Protein (smURFP) is a self-labeling protein. The substrate is fluorogenic, fluoresces when attached, and quenches fluorescent cargo. The smURFP-tag has novel properties for tool development.

A rationally enhanced red fluorescent protein expands the utility of FRET biosensors.

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.

Small ultra-red fluorescent protein nanoparticles as exogenous probes for noninvasive tumor imaging in vivo.

Nanoparticles are excellent imaging agents for cancer, but variability in chemical structure, racemic mixtures, and addition of heavy metals hinders FDA approval in the United States. We developed a small ultra-red fluorescent protein, named smURFP, to have optical properties similar to the small-molecule Cy5, a heptamethine subclass of cyanine dyes (Ex/Em = 642/670 nm). smURFP has a fluorescence quantum yield of 18% and expresses so well in E. coli, that gram quantities of fluorescent protein are purified from cultures in the laboratory. In this research, the fluorescent protein smURFP was combined with bovine serum albumin into fluorescent protein nanoparticles. These nanoparticles are fluorescent with a quantum yield of 17% and 12-14 nm in diameter. The far-red fluorescent protein nanoparticles noninvasively image tumors in living mice via the enhanced permeation and retention (EPR) mechanism. This manuscript describes the use of a new fluorescent protein nanoparticle for in vivo fluorescent imaging. This protein nanoparticle core should prove useful as a biomacromolecular scaffold, which could bear extended chemical modifications for studies, such as the in vivo imaging of fluorescent protein nanoparticles targeted to primary and metastatic cancer, theranostic treatment, and/or dual-modality imaging with positron emission tomography for entire human imaging.

Single-molecule imaging of a fluorescent unnatural amino acid incorporated into nicotinic receptors.

We report on the first, to our knowledge, successful detection of a fluorescent unnatural amino acid (fUAA), Lys(BODIPYFL), incorporated into a membrane protein (the muscle nicotinic acetylcholine receptor, nAChR) in a living cell. Xenopus oocytes were injected with a frameshift-suppressor tRNA, amino-acylated with Lys(BODIPYFL) and nAChR (alpha/beta19'GGGU/gamma/delta) mRNAs. We measured fluorescence from oocytes expressing nAChR beta19'Lys(BODIPYFL), using time-resolved total internal reflection fluorescence microscopy. Under conditions of relatively low receptor density (<0.1 receptors/microm(2)), we observed puncta with diffraction-limited profiles that were consistent with the point-spread function of our microscope. Furthermore, diffraction-limited puncta displayed step decreases in fluorescence intensity, consistent with single-molecule photobleaching. The puncta densities agreed with macroscopic ACh-induced current densities, showing that the fUAA was incorporated, and that receptors were functional. Dose-response relations for the nAChR beta19'Lys(BODIPYFL) receptors were similar to those for wild-type receptors. We also studied nAChR beta19'Lys(BODIPYFL) receptors labeled with alpha-bungarotoxin monoconjugated with Alexa488 (alphaBtxAlexa488). The nAChR has two alphaBtx binding sites, and puncta containing the Lys(BODIPYFL) labeled with alphaBtxAlexa488 yielded the expected three discrete photobleaching steps. We also performed positive control experiments with a nAChR containing enhanced green fluorescent protein in the gamma-subunit M3-M4 loop, which confirmed our nAChR beta19'Lys(BODIPYFL) measurements. Thus, we report on the cell-based single-molecule detection of nAChR beta19'Lys(BODIPYFL).

A Fluorescent, [18F]-Positron-Emitting Agent for Imaging Prostate-Specific Membrane Antigen Allows Genetic Reporting in Adoptively Transferred, Genetically Modified Cells.

Clinical trials involving genome-edited cells are growing in popularity, where CAR-T immunotherapy and CRISPR/Cas9 editing are more recognized strategies. Genetic reporters are needed to localize the molecular events inside these cells in patients. Specifically, a nonimmunogenic genetic reporter is urgently needed as current reporters are immunogenic due to derivation from nonhuman sources. Prostate-specific membrane antigen (PSMA) is potentially nonimmunogenic due to its natural, low-level expression in select tissues (self-MHC display). PSMA overexpression on human prostate adenocarcinoma is also visible with excellent contrast. We exploit these properties in a transduced, two-component, Human-Derived, Genetic, Positron-emitting, and Fluorescent (HD-GPF) reporter system. Mechanistically analogous to the luciferase and luciferin reporter, PSMA is genetically encoded into non-PSMA expressing 8505C cells and tracked with ACUPA-Cy3-BF3, a single, systemically injected small molecule that delivers positron emitting fluoride (18F) and a fluorophore (Cy3) to report on cells expressing PSMA. PSMA-lentivirus transduced tissues become visible by Cy3 fluorescence, [18F]-positron emission tomography (PET), and γ-scintillated biodistribution. HD-GPF fluorescence is visible at subcellular resolution, while a reduced PET background is achieved in vivo, due to rapid ACUPA-Cy3-BF3 renal excretion. Co-transduction with luciferase and GFP show specific advantages over popular genetic reporters in advanced murine models including, a "mosaic" model of solid-tumor intratumoral heterogeneity and a survival model for observing postsurgical recurrence. We report an advanced genetic reporter that tracks genetically modified cells in entire animals and with subcellular resolution with PET and fluorescence, respectively. This reporter system is potentially nonimmunogenic and will therefore be useful in human studies. PSMA is a biomarker of prostate adenocarcinoma and ACUPA-Cy3-BF3 potential in radical prostatectomy is demonstrated.