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Early-stage aggregates of amyloid-forming proteins, specifically soluble oligomers, are implicated in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. Protein aggregation is typically monitored by fluorescence using the amyloid-binding fluorophore thioflavin T (ThT). Thioflavin T interacts, however, preferentially with fibrillar amyloid structures rather than with soluble, early-stage aggregates. In contrast, the two fluorophores, aminonaphthalene 2-cyanoacrylate-spiropyran (AN-SP) and triazole-containing boron-dipyrromethene (taBODIPY), were reported to bind preferentially to early-stage aggregates of amyloidogenic proteins. The present study compares ThT with AN-SP and taBODIPY with regard to their ability to monitor early stages of aggregation of four different amyloid-forming proteins, including amyloid-ß (Aß), tau protein, amylin, and α-synuclein. The results show that the three fluorophores vary in their suitability to monitor the early aggregation of different amyloid-forming proteins. For instance, in the presence of Aß and amylin, the fluorescence intensity of AN-SP increased at an earlier stage of aggregation than the fluorescence of ThT, albeit with only a small fluorescence increase in the case of AN-SP. In contrast, in the presence of tau and amylin, the fluorescence intensity of taBODIPY increased at an earlier stage of aggregation than the fluorescence of ThT. Finally, α-synuclein aggregation could only be monitored by ThT fluorescence; neither AN-SP nor taBODIPY showed a significant increase in fluorescence over the course of aggregation of α-synuclein. These results demonstrate the ability of AN-SP and taBODIPY to monitor the formation of early-stage aggregates from specific amyloid-forming proteins at an early stage of aggregation, although moderate increases in fluorescence intensity, relatively large uncertainties in fluorescence values, and limited solubility of both fluorophores limit their usefulness for some amyloid proteins. The capability to monitor early aggregation of some amyloid proteins, such as amylin, might accelerate the discovery of aggregation inhibitors to minimize the formation of toxic oligomeric species for potential therapeutic use.
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Membranes are widely used for separation processes in applications such as water desalination, batteries and dialysis, and are crucial in key sectors of our economy and society1. The majority of technologically exploited membranes are based on solid polymers and function as passive barriers, whose transport characteristics are governed by their chemical composition and nanostructure. Although such membranes are ubiquitous, it has proved challenging to maximize selectivity and permeability independently, leading to trade-offs between these pertinent characteristics2. Self-assembled biological membranes, in which barrier and transport functions are decoupled3,4, provide the inspiration to address this problem5,6. Here we introduce a self-assembly strategy that uses the interface of an aqueous two-phase system to template and stabilize molecularly thin (approximately 35 nm) biomimetic block copolymer bilayers of scalable area that can exceed 10 cm2 without defects. These membranes are self-healing, and their barrier function against the passage of ions (specific resistance of approximately 1 MΩ cm2) approaches that of phospholipid membranes. The fluidity of these membranes enables straightforward functionalization with molecular carriers that shuttle potassium ions down a concentration gradient with exquisite selectivity over sodium ions. This ion selectivity enables the generation of electric power from equimolar solutions of NaCl and KCl in devices that mimic the electric organ of electric rays.
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To produce more powerful compact ultrafast lasers, research aims at improving the quality of bonds between components inside the laser cavity. Increasing bond robustness under optical irradiation helps the bonds to survive the high energy pulses that these lasers are designed to produce. A measure for such robustness is reported here to support work toward improved bonding processes for such lasers. We produced bonds between pairs of optical grade fused silica glass cylinders using a wet direct bonding procedure. We evaluated these bonds using conventional microscopy, including scanning electron microscopy (SEM) and optical microscopy, without quantifiable results. The bond interface was not discernible through conventional SEM imaging, even after cross sectioning and polishing. The majority of the interface was also undetectable in optical micrographs, except for some limited areas of interfacial disturbance. To obtain quantifiable results for optical robustness, we used an 800 nm femtosecond laser to produce filament-shaped damage from a focal spot moving across the interface. Microscopy of the damage showed its interaction with the interface, the presence of which caused a ≈0.130 to ≈0.230 mm long interruption in the damage line. The exact value depended not only on laser power but also interface quality, and thereby quantified the optical robustness. The reported method proved more sensitive in detecting bonds of fused silica samples compared to other visualization techniques used. Our results suggest a nuanced understanding of bonded glass joints-mechanically sound, yet with limited optical robustness under specific laser conditions.
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Polymers that degrade on demand have the potential to facilitate chemical recycling, reduce environmental pollution and are useful in implant immolation, drug delivery or as adhesives that debond on demand. However, polymers made by radical polymerization, which feature all carbon-bond backbones and constitute the most important class of polymers, have proven difficult to render degradable. Here we report cyclobutene-based monomers that can be co-polymerized with conventional monomers and impart the resulting polymers with mechanically triggered degradability. The cyclobutene residues act as mechanophores and can undergo a mechanically triggered ring-opening reaction, which causes a rearrangement that renders the polymer chains cleavable by hydrolysis under basic conditions. These cyclobutene-based monomers are broadly applicable in free radical and controlled radical polymerizations, introduce functional groups into the backbone of polymers and allow the mechanically gated degradation of high-molecular-weight materials or cross-linked polymer networks into low-molecular-weight species.
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BACKGROUND: Ischemia with no obstructive coronary arteries is frequently caused by coronary microvascular dysfunction (CMD). Consensus diagnostic criteria for CMD include baseline angiographic slow flow by corrected TIMI (Thrombolysis In Myocardial Infarction) frame count (cTFC), but correlations between slow flow and CMD measured by invasive coronary function testing (CFT) are uncertain. OBJECTIVES: The aim of this study was to investigate relationships between cTFC and invasive CFT for CMD. METHODS: Adults with ischemia with no obstructive coronary arteries underwent invasive CFT with thermodilution-derived baseline coronary blood flow, coronary flow reserve (CFR), and index of microcirculatory resistance (IMR). CMD was defined as abnormal CFR (<2.5) and/or abnormal IMR (≥25). cTFC was measured from baseline angiography; slow flow was defined as cTFC >25. Correlations between cTFC and baseline coronary flow and between CFR and IMR and associations between slow flow and invasive measures of CMD were evaluated, adjusted for covariates. All patients provided consent. RESULTS: Among 508 adults, 49% had coronary slow flow. Patients with slow flow were more likely to have abnormal IMR (36% vs 26%; P = 0.019) but less likely to have abnormal CFR (28% vs 42%; P = 0.001), with no difference in CMD (46% vs 51%). cTFC was weakly correlated with baseline coronary blood flow (r = -0.35; 95% CI: -0.42 to -0.27), CFR (r = 0.20; 95% CI: 0.12 to 0.28), and IMR (r = 0.16; 95% CI: 0.07-0.24). In multivariable models, slow flow was associated with lower odds of abnormal CFR (adjusted OR: 0.53; 95% CI: 0.35 to 0.80). CONCLUSIONS: Coronary slow flow was weakly associated with results of invasive CFT and should not be used as a surrogate for the invasive diagnosis of CMD.
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Enfermedad de la Arteria Coronaria , Cisteína/análogos & derivados , Infarto del Miocardio , Isquemia Miocárdica , Adulto , Humanos , Microcirculación/fisiología , Resistencia Vascular/fisiología , Resultado del Tratamiento , Vasos Coronarios/diagnóstico por imagen , Circulación Coronaria/fisiología , Angiografía Coronaria , Enfermedad de la Arteria Coronaria/diagnóstico por imagen , Enfermedad de la Arteria Coronaria/terapiaRESUMEN
The interannual variability of the Equatorial Eastern Indian Ocean (EEIO) is highly relevant for the climate anomalies on adjacent continents and affects global teleconnection patterns. Yet, this is an area where seasonal forecasting systems exhibit large errors. Here we investigate the reasons for these errors in the ECMWF seasonal forecasting system SEAS5 using tailored diagnostics and a series of numerical experiments. Results indicate that there are two fundamental and independent sources of forecast errors in the EEIO. The first one is of atmospheric nature and is largely related with too strong and stable easterly atmospheric circulation present in the equatorial Indian Ocean. This induces an easterly bias which leaves the coupled model predominantly in a state with a shallow thermocline and cold SSTs in the EEIO. The second error is of oceanic origin, associated with a too shallow thermocline, which enhances the SST errors arising from errors in the wind. Ocean initial conditions, which depend on both the quality of the assimilation and the ocean model, play an important role in this context. Nevertheless, it is found that the version of the ocean model used for the forecast can also play a non-negligible role at the seasonal time scales, by amplifying or damping the subsurface errors in the initial conditions. Errors in the EEIO are regime-dependent, having different causes in the warm (deep thermocline) regime with strong atmospheric convection and in the cold (shallow thermocline) regime. Errors also exhibit decadal variations, which challenges the calibration methods used in seasonal forecasts. Supplementary Information: The online version contains supplementary material available at 10.1007/s00382-023-06985-3.
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The number of people experiencing homelessness in tent encampments in the US has increased significantly. Citing concerns over health and safety, many cities have pursued highly visible encampment removals. In January 2022, a major tent encampment in Boston, Massachusetts, was cleared using a unique approach: Most encampment residents were placed in transitional harm reduction housing. We conducted interviews between July 2022 and February 2023 with thirty former encampment residents to explore how the encampment clearing affected their health and sense of safety. We also explored participants' perspectives on harm reduction housing. Of those interviewed, fourteen people had been placed in such housing. Among those not placed, the encampment clearing tended to exacerbate health and safety concerns, especially those related to mental health conditions and risk for violence. Among people successfully placed, harm reduction housing improved health and safety and allowed participants to make meaningful progress toward long-term goals such as addiction recovery, management of chronic health conditions, and permanent housing. Our findings suggest that encampments can have safety-promoting characteristics, but if encampment removal is pursued, offering harm reduction housing after removal can be beneficial.
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Conducta Adictiva , Personas con Mala Vivienda , Trastornos Mentales , Humanos , Vivienda , CiudadesRESUMEN
Proteins unfold in chaotropic salt solutions, a process that is difficult to observe at the single protein level. The work presented here demonstrates that a liquid-based atomic force microscope and graphene liquid-cell-based scanning transmission electron microscope make it possible to observe chemically induced protein unfolding. To illustrate this capability, ferritin proteins were deposited on a graphene surface, and the concentration-dependent urea- or guanidinium-induced changes of morphology were monitored for holo-ferritin with its ferrihydrite core as well as apo-ferritin without this core. Depending on the chaotropic agent the liquid-based imaging setup captured an unexpected transformation of natively folded holo-ferritin proteins into rings after urea treatment but not after guanidinium treatment. Urea treatment of apo-ferritin did not result in nanorings, confirming that nanorings are a specific signature of denaturation of holo-ferritins after exposture to sufficiently high urea concentrations. Mapping the in situ images with molecular dynamics simulations of ferritin subunits in urea solutions suggests that electrostatic destabilization triggers denaturation of ferritin as urea makes direct contact with the protein and also disrupts the water H-bonding network in the ferritin solvation shell. Our findings deepen the understanding of protein denaturation studied using label-free techniques operating at the solid-liquid interface.
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Grafito , Guanidina/química , Desnaturalización Proteica , Ferritinas , Urea/químicaRESUMEN
PURPOSE OF REVIEW: In this article, we discuss the relationship between emotional distress and common cardiovascular disease condition, including coronary artery disease, atrial fibrillation, congestive heart failure, mechanical circulatory support, and heart transplant. We review screening measures that have been studied and used in clinical practice for each condition, as well as priorities for future research. RECENT FINDINGS: Studies consistently demonstrate failing to identify and treat emotional distress in patients with cardiovascular disease is associated with adverse outcomes. However, routine emotional distress screening is not formally recommended for all cardiovascular disease conditions and is limited to depression screening in select patient populations. Future research should focus on evaluating the validity and reliability of standardized screening measures across the scope of emotional distress in patients with or at risk for cardiovascular disease. Other areas of future research include implementation of evidence-based pharmaceutical treatments and integrated behavioral health approaches and interventions.
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Fibrilación Atrial , Enfermedades Cardiovasculares , Distrés Psicológico , Humanos , Enfermedades Cardiovasculares/diagnóstico , Reproducibilidad de los Resultados , Emociones , Fibrilación Atrial/diagnósticoRESUMEN
Several studies have investigated a potential positive association between agentic narcissism and general dishonesty, revealing both supportive and contradicting evidence. Few have focused on dishonesty within romantic relationships, a phenomenon that occurs in almost all partnerships. With the present research, we first aimed to extend existing literature on narcissism by including its two complementary facets (i.e., agentic and communal narcissism). Second, we aimed to improve the understanding of narcissists' lying behavior in the context of partnerships by distinguishing between two different types of lies (i.e., self-centered and other-oriented lies). We hypothesized that both, people higher in agentic and communal narcissism, will report increased dishonesty toward their romantic partners (Hypothesis 1). Given the self-benefit function of self-centered lies and given that agentic narcissists aim to fulfill their relationship-based motives by agentic means, we predict agentic narcissism (compared with communal narcissism) will be a stronger predictor for self-centered lies (Hypothesis 1a). Given the other-benefiting function of other-oriented lies and given that communal narcissists aim to fulfill their motive of self-enhancement by communal means, we predict that communal narcissism (compared with agentic narcissism) will be a stronger predictor for other-oriented lies (Hypothesis 1b). In two preregistered online studies (N = 298: N = 256) we showed that people higher in agentic narcissism believed to be good liars, but this does not lead to higher self-reported frequencies of other-oriented and self-centered lies within romantic relationships historically; communal narcissism was also not related to self-reported deception. Limitations and directions for future research are discussed.
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Soluble oligomers of amyloid-forming proteins are implicated as toxic species in the context of several neurodegenerative diseases. Since the size and shape of these oligomers influence their toxicity, their biophysical characterization is essential for a better understanding of the structure-toxicity relationship. Amyloid oligomers are difficult to characterize by conventional approaches due to their heterogeneity in size and shape, their dynamic aggregation process, and their low abundance. This work demonstrates that resistive pulse measurements using polymer-coated solid-state nanopores enable single-particle-level characterization of the size and shape of individual αSyn oligomers in solution within minutes. A comparison of the resulting size distribution with single-particle analysis by transmission electron microscopy and mass photometry reveals good agreement with superior resolution by nanopore-based characterization. Moreover, nanopore-based analysis has the capability to combine rapid size analysis with an approximation of the oligomer shape. Applying this shape approximation to putatively toxic oligomeric species that range in size from 18 ± 7 aggregated monomers (10S) to 29 ± 10 aggregated monomers (15S) and in concentration from picomolar to nanomolar revealed oligomer shapes that agree well with previous estimates by cryo-EM with the added advantage that nanopore-based analysis occurs rapidly, in solution, and has the potential to become a widely accessible technique.
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Enfermedad de Parkinson , alfa-Sinucleína , Humanos , alfa-Sinucleína/metabolismo , Enfermedad de Parkinson/metabolismo , Microscopía Electrónica de Transmisión , Amiloide , Proteínas AmiloidogénicasRESUMEN
Ferritin is a protein that stores and releases iron to prevent diseases associated with iron dysregulation in plants, animals, and bacteria. The conversion between iron-loaded holo-ferritin and empty apo-ferritin is an important process for iron regulation. To date, studies of ferritin have used either ensemble measurements to quantify the characteristics of a large number of proteins or single-molecule approaches to interrogate labeled or modified proteins. Here we demonstrate the first real-time study of the dynamics of iron ion loading and biomineralization within a single, unlabeled ferritin protein. Using optical nanotweezers, we trapped single apo- and holo-ferritins indefinitely, distinguished one from the other, and monitored their structural dynamics in real time. The study presented here deepens the understanding of the iron uptake mechanism of ferritin proteins, which may lead to new therapeutics for iron-related diseases.
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Ferritinas , Hierro , Animales , Hierro/química , Ferritinas/química , Transporte BiológicoRESUMEN
This paper presents a theoretical investigation of the design of a new actuator type made of anisotropic colloidal particles grafted with stimuli-responsive polymer chains. These artificial muscles combine the osmotic actuation principle of stimuli-responsive hydrogels with the structural alignment of colloidal liquid crystals to achieve directional motion. The solubility of the stimuli-responsive polymer in the neutral state, its degree of polymerization, the salt concentration, and the grafting density of the polymer chains on the surface of the colloidal particles are investigated and identified as important for actuator performance and tunability. The computational results suggest that the proposed mechanically active material matches or exceeds the performances of natural muscles and provide the guidelines for the realization of artificial muscles with predetermined actuation properties.
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Nanocompuestos , Polímeros de Estímulo Receptivo , Polímeros/química , Hidrogeles/químicaRESUMEN
PURPOSE: Posterior instrumentation is the state-of-the-art surgical treatment for fractures of the thoracic and lumbar spine. Options for pedicle screw placement comprise open or minimally invasive techniques. Open instrumentation causes large approach related muscle detachment, which minimally invasive techniques aim to reduce. However, concerns of accurate pedicle screw placement are still a matter of debate. Beside neurological complications due to pedicle screw malplacement, also affection of the facet joints and thus motion dependent pain is known as a complication. The aim of this study was to assess accuracy of pedicle screw placement concerning facet joint violation (FJV) after open- and minimally invasive posterior instrumentation. METHODS: A retrospective data analysis of postoperative computer tomographic scans of 219 patients (1124 pedicle screws) was conducted. A total of 116 patients underwent open screw insertion (634 screws) and 103 patients underwent minimally invasive, percutaneous screw insertion (490 screws). RESULTS: In the lumbar spine (segments L3, L4, L5), there were significantly more and higher grade (open = 0.55 vs. percutaneous = 1.2; p = 0.001) FJV's after percutaneously compared to openly inserted screws. In the thoracic spine, no significant difference concerning rate and grade of FJV was found (p > 0.56). CONCLUSION: FJV is more likely to occur in percutaneously placed pedicle screws. Additionally, higher grade FJV's occur after percutaneous instrumentation. However, in the thoracic spine we didn't find a significant difference between open and percutaneous technique. Our results suggest a precise consideration concerning surgical technique according to the fractured vertebrae in the light of the individual anatomic structures in the preop CT.
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Tornillos Pediculares , Fracturas de la Columna Vertebral , Fusión Vertebral , Articulación Cigapofisaria , Humanos , Articulación Cigapofisaria/diagnóstico por imagen , Articulación Cigapofisaria/cirugía , Estudios Retrospectivos , Vértebras Lumbares/diagnóstico por imagen , Vértebras Lumbares/cirugía , Fracturas de la Columna Vertebral/diagnóstico por imagen , Fracturas de la Columna Vertebral/cirugía , Tornillos Pediculares/efectos adversos , Fusión Vertebral/efectos adversosRESUMEN
PURPOSE: Measurement of neck rotation is currently reliant on radiologic imaging. Given the radiation exposure for CT imaging and the additional inconvenience for the patients, an alternative assessment is needed. Goniometers are comfortably to use and easy to access, also for private consulting. The aim of this study was the assessment of whether a handheld goniometer can be used for accurately measuring the rotation of C1-C2. METHODS: Clinical measurement of rotation was taken in flexed position of the neck. As comparison functional MRI was used. The measured rotation of C1-C2 was compared to identify the accuracy of the goniometer, in comparison to functional MRI scan. RESULTS: Analysis of accuracy using a goniometer and dynamic MRI to assess C1-2 axial rotation showed significant differences for absolute values, but not regarding the percentage of rotation compared to total neck rotation. CONCLUSION: The goniometer is exact to impartially determine the percentage contribution of C1-2 rotation to total neck rotation.
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Articulación Atlantoaxoidea , Vértebras Cervicales , Humanos , Vértebras Cervicales/diagnóstico por imagen , Rotación , Articulación Atlantoaxoidea/diagnóstico por imagen , Imagen por Resonancia Magnética , Rango del Movimiento Articular , Fenómenos BiomecánicosRESUMEN
Formation of Tau protein aggregates in neurons is a pathological hallmark of several neurodegenerative diseases, including Alzheimer's disease. Fluorescently labeled Tau protein is therefore useful to study the aggregation of these pathological proteins and to identify potential therapeutic targets. Conventionally, cysteine residues are used for labeling Tau proteins; however, the full-length Tau isoform contains two cysteine residues in the microtubule-binding region, which are implicated in Tau aggregation by forming intermolecular disulfide bonds. To prevent the fluorescent label from disturbing the microtubule binding region, we developed a strategy to fluorescently label Tau at its C-terminus while leaving cysteine residues unperturbed. We took advantage of a Sortase A-mediated transpeptidation approach to bind a short peptide (GGGH6-Alexa647) with a His-tag and a covalently attached Alexa 647 fluorophore to the C-terminus of Tau. This reaction relies on the presence of a Sortase recognition motif (LPXTG), which we attached to the C-terminus of recombinantly expressed Tau. We demonstrate that C-terminal modification of Tau protein results in no significant differences between the native and C-terminally labeled Tau monomer with regard to aggregation kinetics, secondary structure, and fibril morphology.
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Direct glass-to-glass bonding is important for high-technology components in optics, microfluidics, and micro-electromechanical systems applications. We studied direct bonding of 1 mm thick soda-lime float glass substrates. The process is based on the classic RCA-1 cleaning procedure from the semiconductor industry modified with an ammonium hydroxide rinse, followed by a thermal treatment under unidirectional pressure without the need for a dedicated drying step. RCA-1 uses a solution of ammonium hydroxide and hydrogen peroxide to clean contaminants off the surface of silicon and enable subsequent bonding. Bond quality was evaluated using destructive shear testing. Strong bonds (≈7.81 MPa on average) were achieved using unidirectional pressure of approximately 0.88 MPa and bonding temperatures between 160 °C and 300 °C applied for 30 min. Surface roughness and chemistry was characterized before and after cleaning. The optical robustness of the bonds was tested and shown to be capable of surviving high powered continuous wave (CW) fiber laser irradiation of at least 375 W focused for 2 s without delamination. Melting of the substrate was observed at higher powers and longer exposure times.
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Breast cancer is the second type of cancer with a high probability of brain metastasis and has always been one of the main problems of breast cancer research due to the lack of effective treatment methods. Demand for developing an effective drug against breast cancer brain metastasis and finding molecular mechanisms that play a role in effective treatment are gradually increasing. However, there is no effective anticancer therapeutic drug or treatment method specific to breast cancer, in particular, for patients with a high risk of brain metastases. It is known that mTOR and HDAC enzymes play essential roles in the development of breast cancer brain metastasis. Therefore, it is vital to develop some new drugs and conduct studies toward the inhibition of these enzymes that might be a possible solution to treat breast cancer brain metastasis. In this study, a series of 1,10-phenanthroline and Prodigiosin derivatives consisting of their copper(I) complexes have been synthesized and characterized. Their biological activities were tested in vitro on six different cell lines (including the normal cell line). To obtain additional parallel validations of the experimental data, some in silico modeling studies were carried out with mTOR and HDAC1 enzymes, which are very crucial drug targets, to discover novel and potent drugs for breast cancer and related brain metastases disease.
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Station RN33 on Mount Schauinsland near Freiburg, Germany, is part of the International Monitoring System monitoring radioxenon in air (131mXe, 133Xe, 133mXe, and 135Xe) for verification of the Comprehensive Nuclear Test Ban Treaty. Here, we present data from phase II testing of a new system, Xenon International at RN33, July 14th, 2021 to Jan 22nd, 2022, together with SPALAX data from the same time period. Radioxenon could be detected in 473 of 719 samples, among them many multiple isotope detections. Activity concentrations of spiked and selected environmental samples were verified by laboratory reanalysis. The sensitivity of Xenon International for radioxenons is up to one order of magnitude better for the metastable isotopes than that of the SPALAX, with a shorter sampling duration of 6 h.
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Contaminantes Radiactivos del Aire , Monitoreo de Radiación , Spalax , Animales , Contaminantes Radiactivos del Aire/análisis , Alemania , Isótopos/análisis , Xenón/análisis , Radioisótopos de Xenón/análisisRESUMEN
There exists an urgent demand for the advancement of technologies that reduce and capture carbon dioxide (CO2) emissions to mitigate anthropogenic contributions to climate change. This paper compares the maximum power densities achieved from the combination of reverse electrodialysis (RED) with carbon capture (CC) using various CC solvents. Carbon capture reverse electrodialysis (CCRED) harvests energy from the salinity gradients generated from the reaction of CO2 with specific solvents, generally amines. To eliminate the requirement of freshwater as an external resource, we took advantage of a semiclosed system that would allow the inputs to be industrial emissions and heat and the outputs to be electrical power, clean emissions, and captured CO2. We assessed the power density that can be attained using CCRED with five commonly studied CC solvents: monoethanolamine (MEA), diethanolamine (DEA), N-methyldiethanolamine (MDEA), 2-amino-2-methyl-2-propanol (AMP), and ammonia. We achieved the highest power density, 0.94 W m-2 cell-1, using ammonia. This work provides a foundation for future iterations of CCRED that may help to incentivize adoption of CC technology.