Control of API release with matrix polymorphism in tristearin microspheres.

Glycerides are widely employed as solid matrices in a range of pharmaceutical intermediates and dosage forms. Diffusion-based mechanisms are responsible for drug release, with both chemical and crystal polymorph differences in the solid lipid matrix cited as controlling factors in drug release rates. This work uses model formulations composed of crystalline caffeine embedded in tristearin to study the impacts to drug release from the two primary polymorphic states of tristearin and dependencies on the conversion routes between them. Using contact angles and NMR diffusometry, this work finds that drug release from the meta-stable α-polymorph is rate limited by a diffusive mechanism relating to its porosity and tortuosity, but initial burst release occurs due to ease of initial wetting. Poor wettability resulting from surface blooming can be rate limiting for the ß-polymorph, resulting in slower initial drug release relative to the α-polymorph. The route to achieve the ß-polymorph strongly impacts the bulk release profile due to differences in crystallite size and packing efficiency. API loading enhances the effective porosity, leading to enhanced drug release at high loadings. These findings offer generalizable principles to guide formulators on the types of impacts to drug release rates that one may expect due to triglyceride polymorphism.

Stress induced aging in mouse eye.

Aging, a universal process that affects all cells in an organism, is a major risk factor for a group of neuropathies called glaucoma, where elevated intraocular pressure is one of the known stresses affecting the tissue. Our understanding of molecular impact of aging on response to stress in retina is very limited; therefore, we developed a new mouse model to approach this question experimentally. Here we show that susceptibility to response to stress increases with age and is primed on chromatin level. We demonstrate that ocular hypertension activates a stress response that is similar to natural aging and involves activation of inflammation and senescence. We show that multiple instances of pressure elevation cause aging of young retina as measured on transcriptional and DNA methylation level and are accompanied by local histone modification changes. Our data show that repeated stress accelerates appearance of aging features in tissues and suggest chromatin modifications as the key molecular components of aging. Lastly, our work further emphasizes the importance of early diagnosis and prevention as well as age-specific management of age-related diseases, including glaucoma.

Methane Emissions from Natural Gas Gathering Pipelines in the Permian Basin.

The rapid reduction of methane emissions, especially from oil and gas (O&G) operations, is a critical part of slowing global warming. However, few studies have attempted to specifically characterize emissions from natural gas gathering pipelines, which tend to be more difficult to monitor on the ground than other forms of O&G infrastructure. In this study, we use methane emission measurements collected from four recent aerial campaigns in the Permian Basin, the most prolific O&G basin in the United States, to estimate a methane emission factor for gathering lines. From each campaign, we calculate an emission factor between 2.7 (+1.9/-1.8, 95% confidence interval) and 10.0 (+6.4/-6.2) Mg of CH4 year-1 km-1, 14-52 times higher than the U.S. Environmental Protection Agency's national estimate for gathering lines and 4-13 times higher than the highest estimate derived from a published ground-based survey of gathering lines. Using Monte Carlo techniques, we demonstrate that aerial data collection allows for a greater sample size than ground-based data collection and therefore more comprehensive identification of emission sources that comprise the heavy tail of methane emissions distributions. Our results suggest that pipeline emissions are underestimated in current inventories and highlight the importance of a large sample size when calculating basinwide pipeline emission factors.

Strong methane point sources contribute a disproportionate fraction of total emissions across multiple basins in the United States.

Understanding, prioritizing, and mitigating methane (CH4) emissions requires quantifying CH4 budgets from facility scales to regional scales with the ability to differentiate between source sectors. We deployed a tiered observing system for multiple basins in the United States (San Joaquin Valley, Uinta, Denver-Julesburg, Permian, Marcellus). We quantify strong point source emissions (>10 kg CH4 h-1) using airborne imaging spectrometers, attribute them to sectors, and assess their intermittency with multiple revisits. We compare these point source emissions to total basin CH4 fluxes derived from inversion of Sentinel-5p satellite CH4 observations. Across basins, point sources make up on average 40% of the regional flux. We sampled some basins several times across multiple months and years and find a distinct bimodal structure to emission timescales: the total point source budget is split nearly in half by short-lasting and long-lasting emission events. With the increasing airborne and satellite observing capabilities planned for the near future, tiered observing systems will more fully quantify and attribute CH4 emissions from facility to regional scales, which is needed to effectively and efficiently reduce methane emissions.

Systematic assessment of pathway databases, based on a diverse collection of user-submitted experiments.

A knowledge-based grouping of genes into pathways or functional units is essential for describing and understanding cellular complexity. However, it is not always clear a priori how and at what level of specificity functionally interconnected genes should be partitioned into pathways, for a given application. Here, we assess and compare nine existing and two conceptually novel functional classification systems, with respect to their discovery power and generality in gene set enrichment testing. We base our assessment on a collection of nearly 2000 functional genomics datasets provided by users of the STRING database. With these real-life and diverse queries, we assess which systems typically provide the most specific and complete enrichment results. We find many structural and performance differences between classification systems. Overall, the well-established, hierarchically organized pathway annotation systems yield the best enrichment performance, despite covering substantial parts of the human genome in general terms only. On the other hand, the more recent unsupervised annotation systems perform strongest in understudied areas and organisms, and in detecting more specific pathways, albeit with less informative labels.

Thyroid Eye Disease.

Thyroid eye disease, although rare, is the most common inflammatory orbital disorder and is associated with autoimmune thyroid dysfunction. It is a progressive disorder with symptoms and signs that may cause significant facial disfigurement, visual disability, but rarely blindness. We will review the diagnostic criteria, immunologic basis, clinical course, and medical and surgical treatments for thyroid eye disease. Recent developments in the use of biologic agents to treat this disorder appear to be changing its progression curve and offer the first specific and preventative therapeutic options.

Visual System Hyperexcitability and Compromised V1 Receptive Field Properties in Early-Stage Retinitis Pigmentosa in Mice.

Inherited retinal degenerative diseases are a prominent cause of blindness. Although mutations causing death of photoreceptors are mostly known, the pathophysiology downstream in the inner retina and along the visual pathway is incompletely characterized in the earliest disease stages. Here, we investigated retinal, midbrain and cortical visual function using electroretinography (ERG), the optomotor response (OMR), visual evoked potentials (VEPs), respectively, and single unit electrophysiology at the primary visual cortex (V1) in light-adapted juvenile (approximately one-month-old) and young adult (three-month-old) RhoP23H/WT mice, representative of early-stage retinitis pigmentosa (RP). Photopic ERG revealed up to ∼30% hypersensitivity to light in RhoP23H/WT mice, as measured by the light intensity required to generate half-maximal b-wave (I50 parameter). RhoP23H/WT mice also showed increased OMRs toward low spatial frequency (SF) drifting gratings, indicative of visual overexcitation at the midbrain level. At the V1 level, VEPs and single-cell recordings revealed prominent hyperexcitability in the juvenile RhoP23H/WT mice. Mean VEP amplitudes for light ON stimuli were nearly doubled in one-month-old RhoP23H/WT mice compared with controls, and more than doubled for light OFF. Single-cell recordings showed a significantly increased spontaneous V1 neuron firing in the RhoP23H/WT mice, and persistent contrast and temporal sensitivities. In contrast, direction selectivity was severely compromised. Our data suggest that during early RP, the visual pathway becomes hyperexcited. This could have both compensatory and deleterious consequences for visual behavior. Further studies on the mechanisms of hyperexcitability are warranted as this could lead to therapeutic interventions for RP.

Brain-wide reconstruction of inhibitory circuits after traumatic brain injury.

Despite the fundamental importance of understanding the brain's wiring diagram, our knowledge of how neuronal connectivity is rewired by traumatic brain injury remains remarkably incomplete. Here we use cellular resolution whole-brain imaging to generate brain-wide maps of the input to inhibitory neurons in a mouse model of traumatic brain injury. We find that somatostatin interneurons are converted into hyperconnected hubs in multiple brain regions, with rich local network connections but diminished long-range inputs, even at areas not directly damaged. The loss of long-range input does not correlate with cell loss in distant brain regions. Interneurons transplanted into the injury site receive orthotopic local and long-range input, suggesting the machinery for establishing distant connections remains intact even after a severe injury. Our results uncover a potential strategy to sustain and optimize inhibition after traumatic brain injury that involves spatial reorganization of the direct inputs to inhibitory neurons across the brain.

Methane emissions from US low production oil and natural gas well sites.

Eighty percent of US oil and natural gas (O&G) production sites are low production well sites, with average site-level production ≤15 barrels of oil equivalent per day and producing only 6% of the nation's O&G output in 2019. Here, we integrate national site-level O&G production data and previously reported site-level CH4 measurement data (n = 240) and find that low production well sites are a disproportionately large source of US O&G well site CH4 emissions, emitting more than 4 (95% confidence interval: 3-6) teragrams, 50% more than the total CH4 emissions from the Permian Basin, one of the world's largest O&G producing regions. We estimate low production well sites represent roughly half (37-75%) of all O&G well site CH4 emissions, and a production-normalized CH4 loss rate of more than 10%-a factor of 6-12 times higher than the mean CH4 loss rate of 1.5% for all O&G well sites in the US. Our work suggests that achieving significant reductions in O&G CH4 emissions will require mitigation of emissions from low production well sites.

In vivo base editing rescues cone photoreceptors in a mouse model of early-onset inherited retinal degeneration.

Leber congenital amaurosis (LCA) is the most common cause of inherited retinal degeneration in children. LCA patients with RPE65 mutations show accelerated cone photoreceptor dysfunction and death, resulting in early visual impairment. It is therefore crucial to develop a robust therapy that not only compensates for lost RPE65 function but also protects photoreceptors from further degeneration. Here, we show that in vivo correction of an Rpe65 mutation by adenine base editor (ABE) prolongs the survival of cones in an LCA mouse model. In vitro screening of ABEs and sgRNAs enables the identification of a variant that enhances in vivo correction efficiency. Subretinal delivery of ABE and sgRNA corrects up to 40% of Rpe65 transcripts, restores cone-mediated visual function, and preserves cones in LCA mice. Single-cell RNA-seq reveals upregulation of genes associated with cone phototransduction and survival. Our findings demonstrate base editing as a potential gene therapy that confers long-lasting retinal protection.

A Solanum lycopersicoides reference genome facilitates insights into tomato specialized metabolism and immunity.

Wild relatives of tomato are a valuable source of natural variation in tomato breeding, as many can be hybridized to the cultivated species (Solanum lycopersicum). Several, including Solanum lycopersicoides, have been crossed to S. lycopersicum for the development of ordered introgression lines (ILs), facilitating breeding for desirable traits. Despite the utility of these wild relatives and their associated ILs, few finished genome sequences have been produced to aid genetic and genomic studies. Here we report a chromosome-scale genome assembly for S. lycopersicoides LA2951, which contains 37 938 predicted protein-coding genes. With the aid of this genome assembly, we have precisely delimited the boundaries of the S. lycopersicoides introgressions in a set of S. lycopersicum cv. VF36 × LA2951 ILs. We demonstrate the usefulness of the LA2951 genome by identifying several quantitative trait loci for phenolics and carotenoids, including underlying candidate genes, and by investigating the genome organization and immunity-associated function of the clustered Pto gene family. In addition, syntenic analysis of R2R3MYB genes sheds light on the identity of the Aubergine locus underlying anthocyanin production. The genome sequence and IL map provide valuable resources for studying fruit nutrient/quality traits, pathogen resistance, and environmental stress tolerance. We present a new genome resource for the wild species S. lycopersicoides, which we use to shed light on the Aubergine locus responsible for anthocyanin production. We also provide IL boundary mappings, which facilitated identifying novel carotenoid quantitative trait loci of which one was likely driven by an uncharacterized lycopene ß-cyclase whose function we demonstrate.

Breedbase: a digital ecosystem for modern plant breeding.

Modern breeding methods integrate next-generation sequencing and phenomics to identify plants with the best characteristics and greatest genetic merit for use as parents in subsequent breeding cycles to ultimately create improved cultivars able to sustain high adoption rates by farmers. This data-driven approach hinges on strong foundations in data management, quality control, and analytics. Of crucial importance is a central database able to (1) track breeding materials, (2) store experimental evaluations, (3) record phenotypic measurements using consistent ontologies, (4) store genotypic information, and (5) implement algorithms for analysis, prediction, and selection decisions. Because of the complexity of the breeding process, breeding databases also tend to be complex, difficult, and expensive to implement and maintain. Here, we present a breeding database system, Breedbase (https://breedbase.org/, last accessed 4/18/2022). Originally initiated as Cassavabase (https://cassavabase.org/, last accessed 4/18/2022) with the NextGen Cassava project (https://www.nextgencassava.org/, last accessed 4/18/2022), and later developed into a crop-agnostic system, it is presently used by dozens of different crops and projects. The system is web based and is available as open source software. It is available on GitHub (https://github.com/solgenomics/, last accessed 4/18/2022) and packaged in a Docker image for deployment (https://hub.docker.com/u/breedbase, last accessed 4/18/2022). The Breedbase system enables breeding programs to better manage and leverage their data for decision making within a fully integrated digital ecosystem.

Inhibition of ceramide accumulation in AdipoR1-/- mice increases photoreceptor survival and improves vision.

Adiponectin receptor 1 (ADIPOR1) is a lipid and glucose metabolism regulator that possesses intrinsic ceramidase activity. Mutations of the ADIPOR1 gene have been associated with nonsyndromic and syndromic retinitis pigmentosa. Here, we show that the absence of AdipoR1 in mice leads to progressive photoreceptor degeneration, significant reduction of electroretinogram amplitudes, decreased retinoid content in the retina, and reduced cone opsin expression. Single-cell RNA-Seq results indicate that ADIPOR1 encoded the most abundantly expressed ceramidase in mice and one of the 2 most highly expressed ceramidases in the human retina, next to acid ceramidase ASAH1. We discovered an accumulation of ceramides in the AdipoR1-/- retina, likely due to insufficient ceramidase activity for healthy retina function, resulting in photoreceptor death. Combined treatment with desipramine/L-cycloserine (DC) lowered ceramide levels and exerted a protective effect on photoreceptors in AdipoR1-/- mice. Moreover, we observed improvement in cone-mediated retinal function in the DC-treated animals. Lastly, we found that prolonged DC treatment corrected the electrical responses of the primary visual cortex to visual stimuli, approaching near-normal levels for some parameters. These results highlight the importance of ADIPOR1 ceramidase in the retina and show that pharmacological inhibition of ceramide generation can provide a therapeutic strategy for ADIPOR1-related retinopathy.

Traumatic brain injury to primary visual cortex produces long-lasting circuit dysfunction.

Primary sensory areas of the mammalian neocortex have a remarkable degree of plasticity, allowing neural circuits to adapt to dynamic environments. However, little is known about the effects of traumatic brain injury on visual circuit function. Here we used anatomy and in vivo electrophysiological recordings in adult mice to quantify neuron responses to visual stimuli two weeks and three months after mild controlled cortical impact injury to primary visual cortex (V1). We found that, although V1 remained largely intact in brain-injured mice, there was ~35% reduction in the number of neurons that affected inhibitory cells more broadly than excitatory neurons. V1 neurons showed dramatically reduced activity, impaired responses to visual stimuli and weaker size selectivity and orientation tuning in vivo. Our results show a single, mild contusion injury produces profound and long-lasting impairments in the way V1 neurons encode visual input. These findings provide initial insight into cortical circuit dysfunction following central visual system neurotrauma.

Closing the methane gap in US oil and natural gas production emissions inventories.

Methane (CH4) emissions from oil and natural gas (O&NG) systems are an important contributor to greenhouse gas emissions. In the United States, recent synthesis studies of field measurements of CH4 emissions at different spatial scales are ~1.5-2× greater compared to official greenhouse gas inventory (GHGI) estimates, with the production-segment as the dominant contributor to this divergence. Based on an updated synthesis of measurements from component-level field studies, we develop a new inventory-based model for CH4 emissions, for the production-segment only, that agrees within error with recent syntheses of site-level field studies and allows for isolation of equipment-level contributions. We find that unintentional emissions from liquid storage tanks and other equipment leaks are the largest contributors to divergence with the GHGI. If our proposed method were adopted in the United States and other jurisdictions, inventory estimates could better guide CH4 mitigation policy priorities.

Satellite-based survey of extreme methane emissions in the Permian basin.

Industrial emissions play a major role in the global methane budget. The Permian basin is thought to be responsible for almost half of the methane emissions from all U.S. oil- and gas-producing regions, but little is known about individual contributors, a prerequisite for mitigation. We use a new class of satellite measurements acquired during several days in 2019 and 2020 to perform the first regional-scale and high-resolution survey of methane sources in the Permian. We find an unexpectedly large number of extreme point sources (37 plumes with emission rates >500 kg hour-1), which account for a range between 31 and 53% of the estimated emissions in the sampled area. Our analysis reveals that new facilities are major emitters in the area, often due to inefficient flaring operations (20% of detections). These results put current practices into question and are relevant to guide emission reduction efforts.

Mobile Measurement System for the Rapid and Cost-Effective Surveillance of Methane and Volatile Organic Compound Emissions from Oil and Gas Production Sites.

In this study, a ground-based mobile measurement system was developed to provide rapid and cost-effective emission surveillance of both methane (CH4) and volatile organic compounds (VOCs) from oil and gas (O&G) production sites. After testing in several controlled release experiments, the system was deployed in a field campaign in the Eagle Ford basin, TX. We found fat-tail distributions for both methane and total VOC (C4-C12) emissions (e.g., the top 20% sites ranked according to methane and total VOC (C4-C12) emissions were responsible for ∼60 and ∼80% of total emissions, respectively) and a good correlation between them (Spearman's R = 0.74). This result suggests that emission controls targeting relatively large emitters may help significantly reduce both methane and VOCs in oil and wet gas basins, such as the Eagle Ford. A strong correlation (Spearman's R = 0.84) was found between total VOC (C4-C12) emissions estimated using SUMMA canisters and data reported from a local ambient air monitoring station. This finding suggests that this system has the potential for rapid emission surveillance targeting relatively large emitters, which can help achieve emission reductions for both greenhouse gas (GHG) and air toxics from O&G production well pads in a cost-effective way.

Restoration of visual function in adult mice with an inherited retinal disease via adenine base editing.

Cytosine base editors and adenine base editors (ABEs) can correct point mutations predictably and independent of Cas9-induced double-stranded DNA breaks (which causes substantial indel formation) and homology-directed repair (which typically leads to low editing efficiency). Here, we show, in adult mice, that a subretinal injection of a lentivirus expressing an ABE and a single-guide RNA targeting a de novo nonsense mutation in the Rpe65 gene corrects the pathogenic mutation with up to 29% efficiency and with minimal formation of indel and off-target mutations, despite the absence of the canonical NGG sequence as a protospacer-adjacent motif. The ABE-treated mice displayed restored RPE65 expression and retinoid isomerase activity, and near-normal levels of retinal and visual functions. Our findings motivate the further testing of ABEs for the treatment of inherited retinal diseases and for the correction of pathological mutations with non-canonical protospacer-adjacent motifs.

Projections between visual cortex and pulvinar in the rat.

The extrageniculate visual pathway, which carries visual information from the retina through the superficial layers of the superior colliculus and the pulvinar, is poorly understood. The pulvinar is thought to modulate information flow between cortical areas, and has been implicated in cognitive tasks like directing visually guided actions. In order to better understand the underlying circuitry, we performed retrograde injections of modified rabies virus in the visual cortex and pulvinar of the Long-Evans rat. We found a relatively small population of cells projecting to primary visual cortex (V1), compared to a much larger population projecting to higher visual cortex. Reciprocal corticothalamic projections showed a similar result, implying that pulvinar does not play as big a role in directly modulating rodent V1 activity as previously thought.