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Gene therapy approaches that utilize Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) ribonucleases have tremendous potential to treat human disease. However, CRISPR therapies delivered by integrating viral vectors are limited by potential off-target genome editing caused by constitutive activation of ribonuclease functions. Thus, biomaterial formulations are being used for the delivery of purified CRISPR components to increase the efficiency and safety of genome editing approaches. We previously demonstrated that a novel peptide identified by phage display, TAxI-peptide, mediates delivery of recombinant proteins into neurons. In this report we utilized NeutrAvidin protein to formulate neuron-targeted genome-editing nanoparticles. Cas12a ribonucleases was loaded with biotinylated guide RNA and biotinylated TAxI-peptide onto NeutrAvidin protein to coordinate the formation a targeted ribonuclease protein (RNP) complex. TAxI-RNP complexes are polydisperse with a 14.3 nm radius. The nanoparticles are stable after formulation and show good stability in the presence of normal mouse serum. TAxI-RNP nanoparticles increased neuronal delivery of Cas12a in reporter mice, resulting in induced tdTomato expression after direct injection into the dentate gyrus of the hippocampus. TAxI-RNP nanoparticles also increased genome editing efficacy in hippocampal neurons versus glia. These studies demonstrate the ability to assemble RNP nanoformulations with NeutrAvidin by binding biotinylated peptides and gRNA-loaded Cas12a ribonucleases into protein nanoparticles that target CRISPR delivery to specific cell-types in vivo. The potential to deliver CRISPR nanoparticles to specific cell-types and control off-target delivery to further reduce deleterious genome editing is essential for the creation of viable therapies to treat nervous system disease.

Traumatic brain injury (TBI) is largely non-preventable and often kills or permanently disables its victims. Because current treatments for TBI merely ameliorate secondary effects of the initial injury like swelling and hemorrhaging, strategies for the induction of neuronal regeneration are desperately needed. Recent discoveries regarding the TBI-responsive migratory behavior and differentiation potential of neural progenitor cells (NPCs) found in the subventricular zone (SVZ) have prompted strategies targeting gene therapies to these cells to enhance neurogenesis after TBI. We have previously shown that plasmid polyplexes can non-virally transfect SVZ NPCs when directly injected in the lateral ventricles of uninjured mice. We describe the first reported intracerebroventricular transfections mediated by polymeric gene carriers in a murine TBI model and investigate the anatomical parameters that dictate transfection through this route of administration. Using both luciferase and GFP plasmid transfections, we show that the time delay between injury and polyplex injection directly impacts the magnitude of transfection efficiency, but that overall trends in the location of transfection are not affected by injury. Confocal microscopy of quantum dot-labeled plasmid uptake in vivo reveals association between our polymers and negatively charged NG2 chondroitin sulfate proteoglycans of the SVZ extracellular matrix. We further validate that glycosaminoglycans but not sulfate groups are required for polyplex uptake and transfection in vitro. These studies demonstrate that non-viral gene delivery is impacted by proteoglycan interactions and suggest the need for improved polyplex targeting materials that penetrate brain extracellular matrix to increase transfection efficiency in vivo.

This review summarizes the relevant literature for the initial imaging of patients with symptoms of dysphagia. For patients with oropharyngeal dysphagia who have an underlying attributable cause, a modified barium swallow is usually appropriate for initial imaging but for those who have unexplained dysphagia a fluoroscopic biphasic esophagram is usually appropriate. Fluoroscopic biphasic esophagram is usually appropriate for initial imaging in both immunocompetent and immunocompromised patients who have retrosternal dysphagia. For postoperative patients with dysphagia, fluoroscopic single-contrast esophagram and CT neck and chest with intravenous (IV) contrast are usually appropriate for oropharyngeal or retrosternal dysphagia occurring in the early postoperative period where water-soluble contrast is usually preferred rather than barium sulfate. In the later postoperative period (greater than 1 month), CT neck and chest with IV contrast and fluoroscopic single-contrast esophagram are usually appropriate. The American College of Radiology Appropriateness Criteria are evidence-based guidelines for specific clinical conditions that are reviewed annually by a multidisciplinary expert panel. The guideline development and revision include an extensive analysis of current medical literature from peer reviewed journals and the application of well-established methodologies (RAND/UCLA Appropriateness Method and Grading of Recommendations Assessment, Development, and Evaluation or GRADE) to rate the appropriateness of imaging and treatment procedures for specific clinical scenarios. In those instances where evidence is lacking or equivocal, expert opinion may supplement the available evidence to recommend imaging or treatment.

Quantifying and representing uncertainty for geothermal systems is often ignored, in practice, during the exploration phase of a geothermal development project. We propose that this occurs potentially because the task seems so formidable. The primary goal of this paper is to initiate a dialogue within the geothermal community about: which geothermal uncertainties should receive the most attention and which uncertainty analysis methods could provide the greatest benefit for the advancement of the geothermal energy industry. Specifically, in this paper, we review uncertainty quantification techniques that are applicable to geothermal exploration. In general, uncertainty associated with data acquisition/processing (i.e., objective uncertainty) is small compared to the uncertainty in interpretational space (i.e., subjective uncertainty) that lies between data points where extrapolation is required. Therefore, it is important to classify, assess, and quantify uncertainty to help select strategies to reduce uncertainty and to better gauge the impact that separate uncertainties have on the overall likelihood of project success. The discipline of geostatistics provides multiple quantitative methods for producing stochastic models which adhere to measured data and spatial correlation. The petroleum industry has successfully used both geostatistics and decision analysis methods to combine diverse and multiple types of uncertainties. We argue that instead of one single and final interpretation of the geothermal system, numerous interpretations may be more indicative of the possible subsurface scenarios, and these different scenarios can be evaluated using decision analyses and value of information methodologies. Finally, we recommend that the potential power generation of a geothermal reservoir should be grounded in the geologic data and modeling for a specific field and their estimated uncertainties.

Endocytosed biomacromolecule delivery systems must escape the endosomal trafficking pathway in order for their cargo to exert effects in other cellular compartments. Although endosomal release is well-recognized as one of the greatest barriers to efficacy of biologic drugs with intracellular targets, most drug carriers have relied on cationic materials that passively induce endosomal swelling and membrane rupture with low efficiency. To address the endosome release challenge, our lab has developed a diblock copolymer system for nucleic acid delivery that selectively displays a potent membrane-lytic peptide (melittin) in response to the pH drop during the endosomal maturation. To further optimize this system, we evaluated a panel of peptides with reported lytic activity in comparison to melittin. Nineteen different lytic peptides were synthesized and their membrane-lytic properties at both neutral and acidic pH characterized using a red blood cell hemolysis assay. The top five performing peptides were then conjugated to our pH-sensitive diblock copolymer via disulfide linkers and used to deliver a variety of nucleic acids to cultured mammalian cells as well as in vivo to the mouse brain. We demonstrate that the sharp pH-transition of VIPER compensates for potential advantages from pH-sensitive peptides, such that polymer-peptide conjugates with poorly selective but highly lytic peptides achieve safe and effective transfection both in vitro and in vivo. In addition, peptides that require release from polymer backbones for lysis were less effective in the VIPER system, likely due to limited endosomal reducing power of target cells. Finally, we show that certain peptides are potentiated in lytic ability by polymer conjugation and that these peptide-polymer constructs are most effective in vivo.

The range of pathology in adults that can produce abdominal pain is broad and necessitates an imaging approach to evaluate many different organ systems. Although localizing pain prompts directed imaging/management, clinical presentations may vary and result in nonlocalized symptoms. This review focuses on imaging the adult population with nonlocalized abdominal pain, including patients with fever, recent abdominal surgery, or neutropenia. Imaging of the entire abdomen and pelvis to evaluate for infectious or inflammatory processes of the abdominal viscera and solid organs, abdominal and pelvic neoplasms, and screen for ischemic or vascular etiologies is essential for prompt diagnosis and treatment. Often the first-line modality, CT quickly evaluates the abdomen/pelvis, providing for accurate diagnoses and management of patients with abdominal pain. Ultrasound and tailored MRI protocols may be useful as first-line imaging studies, especially in pregnant patients. In the postoperative abdomen, fluoroscopy may help detect anastomotic leaks/abscesses. While often performed, abdominal radiographs may not alter management. The American College of Radiology Appropriateness Criteria are evidence-based guidelines for specific clinical conditions that are reviewed annually by a multidisciplinary expert panel. The guideline development and revision include an extensive analysis of current medical literature from peer reviewed journals and the application of well-established methodologies (RAND/UCLA Appropriateness Method and Grading of Recommendations Assessment, Development, and Evaluation or GRADE) to rate the appropriateness of imaging and treatment procedures for specific clinical scenarios. In those instances where evidence is lacking or equivocal, expert opinion may supplement the available evidence to recommend imaging or treatment.

Targeting ligands are used in drug delivery to improve drug distribution to desired cells or tissues and to facilitate cellular entry. In vivo biopanning, whereby billions of potential ligand sequences are screened in biologically-relevant and complex conditions, is a powerful method for identification of novel target ligands. This tool has impacted drug delivery technologies and expanded our arsenal of therapeutics and diagnostics. Within this review we will discuss current in vivo panning technologies and ways that these technologies can be improved to advance next-generation drug delivery strategies.

This study assessed the potential for a high-temperature blind geothermal system in southeastern Gabbs Valley, an area with no previous geothermal exploration, by utilizing geothermal play fairway analysis. Gabbs Valley is a structurally complex, tectonically active basin occupying a transtensional displacement transfer zone. Multiple lines of evidence suggest the presence of a blind geothermal system, including collocated intersecting gravity gradients, magnetic-low, low-resistivity, 2-m temperature anomaly, and 130–140 °C geothermometers obtained from nearby agricultural wells. Six 150 m-deep temperature-gradient holes were drilled to target the geophysical and thermal anomalies. Two holes produced bottom-hole temperatures of 114.5 °C and 124.9 °C. Drilling results establish the discovery of a blind geothermal system. Power estimates based on resource conceptual models of the geothermal system suggest a median (P50) capacity of ∼16 MWe.

Financial risk due to geological uncertainty is a major barrier for geothermal development. Production from a geothermal well depends on the unknown location of subsurface geological structures, such as faults that contain hydrothermal fluids. Traditionally, geoscientists collect many different datasets, interpret the datasets manually, and create a single model estimating faults' locations. This method, however, does not provide information about the uncertainty regarding the location of faults and often does not fully respect all observed datasets. Previous researchers investigated the use of stochastic inversion schemes for addressing geological uncertainty, but often at the expense of geologic realism. In this paper, we present algorithms and open-source code to stochastically invert five typical datasets for creating geologically realistic structural models. Using a case study with real data from the Patua Geothermal Field, we show that these inversion algorithms are successful in finding an ensemble of structural models that are geologically realistic and match the observed data sufficiently. Geoscientists can use this ensemble of models to optimize reservoir management decisions given structural uncertainty.

Upper gastrointestinal bleeding (UGIB) remains a significant cause of morbidity and mortality with mortality rates as high as 14%. This document addresses the indications for imaging UGIB that is nonvariceal and unrelated to portal hypertension. The four variants are derived with respect to upper endoscopy. For the first three, it is presumed that upper endoscopy has been performed, with three potential initial outcomes: endoscopy reveals arterial bleeding source, endoscopy confirms UGIB without a clear source, and negative endoscopy. The fourth variant, "postsurgical and traumatic causes of UGIB; endoscopy contraindicated" is considered separately because upper endoscopy is not performed. When endoscopy identifies the presence and location of bleeding but bleeding cannot be controlled endoscopically, catheter-based arteriography with treatment is an appropriate next study. CT angiography (CTA) is comparable with angiography as a diagnostic next step. If endoscopy demonstrates a bleed but the endoscopist cannot identify the bleeding source, angiography or CTA can be typically performed and both are considered appropriate. In the event of an obscure UGIB, angiography and CTA have been shown to be equivalent in identifying the bleeding source; CT enterography may be an alternative to CTA to find an intermittent bleeding source. In the postoperative or traumatic setting when endoscopy is contraindicated, primary angiography, CTA, and CT with intravenous contrast are considered appropriate. The American College of Radiology Appropriateness Criteria are evidence-based guidelines for specific clinical conditions that are reviewed annually by a multidisciplinary expert panel. The guideline development and revision include an extensive analysis of current medical literature from peer reviewed journals and the application of well-established methodologies (RAND/UCLA Appropriateness Method and Grading of Recommendations Assessment, Development, and Evaluation or GRADE) to rate the appropriateness of imaging and treatment procedures for specific clinical scenarios. In those instances where evidence is lacking or equivocal, expert opinion may supplement the available evidence to recommend imaging or treatment.

Cationic polymer gene delivery vehicles that effectively resist premature serum degradation often have difficulty releasing their nucleic acid cargoes. In this work, we report a pH-sensitive polymer (SP), poly(oligo(ethylene glycol) monomethyl ether methacrylate)-co-poly(2-(dimethylamino)ethyl methacrylate)-block- poly(propargyl methacrylate-graft-propyl-(4-methoxy-benzylidene)-amine) (p(PMA-PMBA)–b-(p(OEGMA-DMAEMA)), for successful in vitro and in vivo gene transfer. In the physiological condition, the hydrophobization of p(OEGMA-DMAEMA) polycations by p(PMA-PMBA) significantly enhanced the stability of its polyplexes counterpart. In endosomes, the polymer undergoes an acid-triggered hydrophilic transition through the cleavage of benzoic imines, thus allowing the vector to quickly release nucleic acid cargo due to the loss of hydrophobic functionalization. Compared to a pH-insensitive polymer (IP), SP exhibited more significant luciferase plasmid delivery efficiency with HeLa cells in vitro and with in vivo intraventricular brain injections. Therefore, the polymer designed here is a good solution to address the dilemma of stability and cargo release in gene delivery, and may have broad potential applications in therapeutic agent delivery.

In many hydrothermal systems, fracture permeability along faults provides pathways for groundwater to transport heat from depth. Faulting generates a range of deformation styles that cross-cut heterogeneous geology, resulting in complex patterns of permeability, porosity, and hydraulic conductivity. Vertical connectivity (a throughgoing network of permeable areas that allows advection of heat from depth to the shallow subsurface) is rare and is confined to relatively small volumes that have highly variable spatial distribution. This local compartmentalization of connectivity represents a significant challenge to understanding hydrothermal circulation and for exploring, developing, and managing hydrothermal resources. Here, we present an evaluation of the geologic characteristics that control this compartmentalization in hydrothermal systems through 3-D analysis of the Brady geothermal field in western Nevada. A published 3-D geologic map of the Brady area is used as a basis to develop structural and geological variables that are hypothesized to control or effect permeability or connectivity. The 3-D distribution of these variables is compared to the distribution of productive and non-productive fluid flow intervals along production wells and non-productive wells via principal component analysis (PCA). This comparison elucidates which geologic and structural variables are most closely associated with productive fluid flow intervals. Results indicate that production intervals at Brady are located: (1) within or near to known and stress-loaded macro-scale faults, and (2) in areas of high fault and fracture density.

As geothermal exploration increasingly focuses on blind or hidden systems, precise geologic characterization of the sub-surface at potential development sites becomes essential. Geothermal circulation requires elevated heat, relatively high permeability, and ample fluid flow. Evidence for the collocation of these characteristics occur in areas where geothermal circulation is most likely to occur and where exploration activities should be focused. Employing a 3D geologic framework constructed through integration of many separate datasets, we demonstrate a methodology for analyzing the data types that can be used as proxies for these three key characteristics. This methodology is applied at the Astor Pass geothermal prospect in northwestern Nevada, western USA. Based on geologic structure modeled in 3D, several proxies for heat, fluids and permeability are compared in order to identify areas within the field with the highest favorability for geothermal fluid flow. Geological and conceptual models constructed through these methodologies can be used to develop exploration strategies and subsequently site wells. Such models can be iteratively adapted with newly acquired data, as prospects evolve into mature geothermal developments. If developed prior to expensive drilling programs, these techniques allow for more efficient use of limited drilling budgets, ultimately lowering the risks and costs of geothermal exploration and development.

Geothermal fluids produced from two of the largest production geothermal fields in the Great Basin have helium isotope ratios that are anomalously high relative to basin-wide trends. These data indicate that the geothermal systems, Dixie Valley, Nevada and McGinness Hills, Nevada have an anomalously high fraction of mantle derived fluid. These connections to deeply derived fluid and heat may supplement crustal heat production and be responsible, in part, for the anomalously high production capacity, relative to other Great Basin geothermal fields, that Dixie Valley and McGinness Hills support. Deep-seated crustal structures across the Great Basin and around the world are known to be associated with structural reactivation, can have relatively high permeability, and can act as fluid flow conduits. These deep seated structures across the Great Basin control upflow of deeply derived heat and fluids into the shallow geothermal systems at Dixie Valley and McGinness Hills, contributing to their productivity.

Peroxiredoxin 1 (Prx 1), also known as natural killer enhancing factor A (NKEF A), has been implicated in the immune response of both mammals and fish. Amoebic gill disease (AGD), caused by Neoparamoeba perurans, is a significant problem for the Atlantic salmon (Salmo salar L.) aquaculture industry based in Tasmania, Australia. Here we have cloned and functionally characterized a Prx 1 open reading frame (ORF) from Atlantic salmon liver and shown that Prx 1 gene expression was down-regulated in the gills of Atlantic salmon displaying symptoms of AGD. The Prx 1 ORF encoded all of the residues and motifs characteristic of typical 2-Cys Prx proteins from eukaryotes and the recombinant protein expressed in Escherichia coli catalyzed thioredoxin (Trx)-dependent reduction of H 2O 2, cumene hydroperoxide (CuOOH) and t-butyl hydroperoxide (t-bOOH) with K m values of 122, 77 and 91 μM, respectively, confirming that it was a genuine 2-Cys Prx. The recombinant protein also displayed a double displacement reaction mechanism and a catalytic efficiency (k cat/K m) with H 2O 2 of 1.5 × 10 5 M -1 s -1 which was consistent with previous reports for the 2-Cys Prx family of proteins. This is the first time that a Prx 1 protein has been functionally characterized from any fish species and it paves the way for further investigation of this important 2-Cys Prx family member in fish. Refereed/Peer-reviewed

Components of the blood have been proposed as potential therapeutic targets for improving cellular regeneration after injury and neurodegenerative disease. In this work, thrombin is shown to increase endogenous neural progenitor proliferation in the intact murine spinal cord. A local injection of heparin before a spinal cord injury reduces cell proliferation and astrogliogenesis associated with scarring. We sought to create depot-formulations of PLGA microsphere and Pluronic F-127 for sustained local delivery of two thrombin inhibitors, heparin and hirudin. Each hydrogel depot-formulation showed delayed drug release compared to microspheres or hydrogel alone. Animals with a lateral demyelination lesion showed a reduction in CD68+ macrophages when treated with hirudin-loaded PLGA/F-127 gels compared to control and heparin-treated animals. Moreover, hirudin-loaded materials showed an accelerated recovery in coordinated stepping and increased oligodendrocyte densities. Together, these data demonstrate that controlled delivery of hirudin accelerates functional recovery from a demyelination lesion in the spinal cord.

Background: Tuberculosis (TB) Regional Training and Medical Consultation Centers (RTMCCs) were established in 2005 for TB medical consultation, training and education in the United States. A medical consultation database (MCD) captured all consultations provided by RTMCCs; we report on those provided from June 1, 2010 to May 31, 2014. Methods: All MCD consultations during 2010–2014 were categorized into: provider type, setting, consultation topic, and patient age. We analyzed data frequencies and performed subgroup analyses by RTMCC, by TB incidence for the geographical area, and by year of consultation. End-user satisfaction was assessed by a 2016 telephone evaluation of RTMCC services. Results: A total of 11,074 consultations were delivered, with 10,754 (97.1%) in the U.S. and its current or former territories. Of these, 6018 (56%) were for high, 2443 (22.7%) for medium, and 2293 (21.3%) for low TB incidence settings. Most were for adults (81.3%) and answered within 24 h (96.2%). Nearly 2/3 consultations originated from health departments; providers included mostly physicians (44.3%) or nurses (37.6%). Common consult categories included TB disease (47.7%), case management (29.8%), latent TB infection (19.3%), diagnosis (16.1%), pharmacology (14.7%) and adverse side effects (14.3%). Among adverse side effects, hepatotoxicity was most common (39.6%). Volume and nature of consult requests remained relatively stable over the four-year period. Feedback from a 2016 CDC evaluation indicated overall satisfaction with RTMCC medical consultation services. Conclusion: RTMCCS were an important source of TB medical consultation over the time-frame of this assessment and provided quality expert consultation within 24 h. RMTCCs represent a reservoir of TB subject-matter expertise in the United States. Keywords: Tuberculosis, Database, Consultation

Base excision repair is the system used from bacteria to man to remove the tens of thousands of endogenous DNA damages produced daily in each human cell. Base excision repair is required for normal mammalian development and defects have been associated with neurological disorders and cancer. In this paper we provide an overview of short patch base excision repair in humans and summarize current knowledge of defects in base excision repair in mouse models and functional studies on short patch base excision repair germ line polymorphisms and their relationship to cancer. The biallelic germ line mutations that result in MUTYH-associated colon cancer are also discussed.

Rapidly advancing technology has resulted in the generation of the genomic sequences of several human tumors. We have identified several mutations of the DNA polymerase β (pol β) gene in human colorectal cancer. We have demonstrated that the expression of the pol β G231D variant increased chromosomal aberrations and induced cellular transformation. The transformed phenotype persisted in the cells even once the expression of G231D was extinguished, suggesting that it resulted as a consequence of genomic instability. Biochemical analysis revealed that its catalytic rate was 140-fold slower than WT pol β, and this was a result of the decreased binding affinity of nucleotides by G231D. Residue 231 of pol β lies in close proximity to the template strand of the DNA. Molecular modeling demonstrated that the change from a small and nonpolar glycine to a negatively charged aspartate resulted in a repulsion between the template and residue 231 leading to the distortion of the dNTP binding pocket. In addition, expression of G231D was insufficient to rescue pol β-deficient cells treated with chemotherapeutic agents suggesting that these agents may be effectively used to treat tumors harboring this mutation. More importantly, this suggests that the G231D variant has impaired base excision repair. Together, these data indicate that the G231D variant plays a role in driving cancer. Background: Mutations in the POLB gene are present in 40% of human colorectal tumors. Results: The G231D variant is a slow polymerase that induces genomic instability and cellular transformation. Conclusion: The slow G231D variant induces cellular transformation due to its inability to fill in single nucleotide gaps. Significance: Slow pol β variants may drive tumorigenesis.

Inward-dipping (cone) sheet swarms and an associated central volcano are well-exposed in the deeply-eroded Tertiary crust of Vatnsdalur, Skagi Peninsula region, northern Iceland. Spatially registered orientations of 389 mafic sheets, mapped in three distinct sheet swarms define both the overall shape and magmatic source of each swarm. The Vatnsdalur sheet swarms consist of planar inward-dipping sheets that collectively define a conical shape rather than a bowl- or trumpet-shape as have been found in swarms in other locations. In the best exposed swarm, three-dimensional projection of mafic sheets into the subsurface defines two distinct foci, which are interpreted as the magmatic sources of two temporally distinct sub-swarms. These results help to establish the influence of inclined sheet intrusion on crustal accretion at central volcanoes. The geometry of the swarm constrains the thickness of material that was added to the crust during sheet intrusion. When combined with estimates of surface relief, we calculate that 2.2 to 4.1 km of subsidence were required beneath the central volcano in order to accommodate the intrusion of the sheet swarm. Similar processes of crustal thickening and subsidence likely occur in a wide variety of both continental rift and mid-ocean ridge systems where magmatic activity is focused at central volcanoes.

The Second Pacific-Rim Colloquium (January 2015) had its focus on the business structure for warehouses and warehouse receipts and discussions about creating an Electronic Warehouse Receipts (EWR) system, particularly on an “open” or a “closed” system. The Colloquium identified two legal concepts – “negotiable” and “duly negotiated” – as the ultimate goals for a functioning EWR system. However, foundations for an EWR system must be in place before legal concepts can become functionally meaningful.

This article presents a regulatory model that would regulate agricultural biotechnology on the basis of the product created and, then, on a tiered risk-based analysis. The article presents this model because United States regulatory agencies are currently reviewing their agricultural biotechnology regulations. This article joins that review conversation by proposing a risk-based model as the appropriate regulatory approach.