Transcriptome analysis identifies an ASD-Like phenotype in oligodendrocytes and microglia from C58/J amygdala that is dependent on sex and sociability.

BACKGROUND: Autism Spectrum Disorder (ASD) is a group of neurodevelopmental disorders with higher incidence in males and is characterized by atypical verbal/nonverbal communication, restricted interests that can be accompanied by repetitive behavior, and disturbances in social behavior. This study investigated brain mechanisms that contribute to sociability deficits and sex differences in an ASD animal model. METHODS: Sociability was measured in C58/J and C57BL/6J mice using the 3-chamber social choice test. Bulk RNA-Seq and snRNA-Seq identified transcriptional changes in C58/J and C57BL/6J amygdala within which DMRseq was used to measure differentially methylated regions in amygdala. RESULTS: C58/J mice displayed divergent social strata in the 3-chamber test. Transcriptional and pathway signatures revealed immune-related biological processes differ between C58/J and C57BL/6J amygdala. Hypermethylated and hypomethylated genes were identified in C58/J versus C57BL/6J amygdala. snRNA-Seq data in C58/J amygdala identified differential transcriptional signatures within oligodendrocytes and microglia characterized by increased ASD risk gene expression and predicted impaired myelination that was dependent on sex and sociability. RNA velocity, gene regulatory network, and cell communication analysis showed diminished oligodendrocyte/microglia differentiation. Findings were verified using Bulk RNA-Seq and demonstrated oxytocin's beneficial effects on myelin gene expression. LIMITATIONS: Our findings are significant. However, limitations can be noted. The cellular mechanisms linking reduced oligodendrocyte differentiation and reduced myelination to an ASD phenotype in C58/J mice need further investigation. Additional snRNA-Seq and spatial studies would determine if effects in oligodendrocytes/microglia are unique to amygdala or if this occurs in other brain regions. Oxytocin's effects need further examination to understand its' potential as an ASD therapeutic. CONCLUSIONS: Our work demonstrates the C58/J mouse model's utility in evaluating the influence of sex and sociability on the transcriptome in concomitant brain regions involved in ASD. Our single-nucleus transcriptome analysis elucidates potential pathological roles of oligodendrocytes and microglia in ASD. This investigation provides details regarding regulatory features disrupted in these cell types, including transcriptional gene dysregulation, aberrant cell differentiation, altered gene regulatory networks, and changes to key pathways that promote microglia/oligodendrocyte differentiation. Our studies provide insight into interactions between genetic risk and epigenetic processes associated with divergent affiliative behavior and lack of positive sociability.

A neural tract-inspired conduit for facile, on-demand biopsy of glioblastoma.

Background: A major hurdle to effectively treating glioblastoma (GBM) patients is the lack of longitudinal information about tumor progression, evolution, and treatment response. Methods: In this study, we report the use of a neural tract-inspired conduit containing aligned polymeric nanofibers (i.e., an aligned nanofiber device) to enable on-demand access to GBM tumors in 2 rodent models. Depending on the experiment, a humanized U87MG xenograft and/or F98-GFP+ syngeneic rat tumor model was chosen to test the safety and functionality of the device in providing continuous sampling access to the tumor and its microenvironment. Results: The aligned nanofiber device was safe and provided a high quantity of quality genomic materials suitable for omics analyses and yielded a sufficient number of live cells for in vitro expansion and screening. Transcriptomic and genomic analyses demonstrated continuity between material extracted from the device and that of the primary, intracortical tumor (in the in vivo model). Conclusions: The results establish the potential of this neural tract-inspired, aligned nanofiber device as an on-demand, safe, and minimally invasive access point, thus enabling rapid, high-throughput, longitudinal assessment of tumor and its microenvironment, ultimately leading to more informed clinical treatment strategies.

UBE2N Is Essential for Maintenance of Skin Homeostasis and Suppression of Inflammation.

UBE2N, a Lys63 ubiquitin-conjugating enzyme, plays critical roles in embryogenesis and immune system development and function. However, its roles in adult epithelial tissue homeostasis and pathogenesis are unclear. We generated conditional mouse models that deleted Ube2n in skin cells in a temporally and spatially controlled manner. We found that Ube2n knockout in the adult skin keratinocytes induced a range of inflammatory skin defects characteristic of psoriatic and actinic keratosis. These included inflammation, epidermal and dermal thickening, parakeratosis, and increased immune cell infiltration as well as signs of edema and blistering. Single-cell transcriptomic analyses and RT-qPCR showed that Ube2n-knockout keratinocytes expressed elevated myeloid cell chemoattractants such as Cxcl1 and Cxcl2 and decreased the homeostatic T lymphocyte chemoattractant Ccl27a. Consistently, the infiltrating immune cells were predominantly myeloid-derived cells, including neutrophils and M1-like macrophages, which expressed high levels of inflammatory cytokines such as Il1ß and Il24. Pharmacological blockade of the IL-1 receptor associated kinases (IRAK1/4) alleviated inflammation, epidermal and dermal thickening, and immune infiltration of the Ube2n-mutant skin. Together, these findings highlight a key role of keratinocyte UBE2N in maintenance of epidermal homeostasis and skin immunity and identify IRAK1/4 as potential therapeutic target for inflammatory skin disorders.

Accelerated epigenetic age is associated with whole-brain functional connectivity and impaired cognitive performance in older adults.

While chronological age is a strong predictor for health-related risk factors, it is an incomplete metric that fails to fully characterize the unique aging process of individuals with different genetic makeup, neurodevelopment, and environmental experiences. Recent advances in epigenomic array technologies have made it possible to generate DNA methylation-based biomarkers of biological aging, which may be useful in predicting a myriad of cognitive abilities and functional brain network organization across older individuals. It is currently unclear which cognitive domains are negatively correlated with epigenetic age above and beyond chronological age, and it is unknown if functional brain organization is an important mechanism for explaining these associations. In this study, individuals with accelerated epigenetic age (i.e. AgeAccelGrim) performed worse on tasks that spanned a wide variety of cognitive faculties including both fluid and crystallized intelligence (N = 103, average age = 68.98 years, 73 females, 30 males). Additionally, fMRI connectome-based predictive models suggested a mediating mechanism of functional connectivity on epigenetic age acceleration-cognition associations primarily in medial temporal lobe and limbic structures. This research highlights the important role of epigenetic aging processes on the development and maintenance of healthy cognitive capacities and function of the aging brain.

Spatial transcriptomics reveals segregation of tumor cell states in glioblastoma and marked immunosuppression within the perinecrotic niche.

Glioblastoma (GBM) remains an untreatable malignant tumor with poor patient outcomes, characterized by palisading necrosis and microvascular proliferation. While single-cell technology made it possible to characterize different lineage of glioma cells into neural progenitor-like (NPC-like), oligodendrocyte-progenitor-like (OPC-like), astrocyte-like (AC-like) and mesenchymal like (MES-like) states, it does not capture the spatial localization of these tumor cell states. Spatial transcriptomics empowers the study of the spatial organization of different cell types and tumor cell states and allows for the selection of regions of interest to investigate region-specific and cell-type-specific pathways. Here, we obtained paired 10x Chromium single-nuclei RNA-sequencing (snRNA-seq) and 10x Visium spatial transcriptomics data from three GBM patients to interrogate the GBM microenvironment. Integration of the snRNA-seq and spatial transcriptomics data reveals patterns of segregation of tumor cell states. For instance, OPC-like tumor and NPC-like tumor significantly segregate in two of the three samples. Our differentially expressed gene and pathway analyses uncovered significant pathways in functionally relevant niches. Specifically, perinecrotic regions were more immunosuppressive than the endogenous GBM microenvironment, and perivascular regions were more pro-inflammatory. Our gradient analysis suggests that OPC-like tumor cells tend to reside in areas closer to the tumor vasculature compared to tumor necrosis, which may reflect increased oxygen requirements for OPC-like cells. In summary, we characterized the localization of cell types and tumor cell states, the gene expression patterns, and pathways in different niches within the GBM microenvironment. Our results provide further evidence of the segregation of tumor cell states and highlight the immunosuppressive nature of the necrotic and perinecrotic niches in GBM.

Subventricular zone stem cell niche injury is associated with intestinal perforation in preterm infants and predicts future motor impairment.

Brain injury is highly associated with preterm birth. Complications of prematurity, including spontaneous or necrotizing enterocolitis (NEC)-associated intestinal perforations, are linked to lifelong neurologic impairment, yet the mechanisms are poorly understood. Early diagnosis of preterm brain injuries remains a significant challenge. Here, we identified subventricular zone echogenicity (SVE) on cranial ultrasound in preterm infants following intestinal perforations. The development of SVE was significantly associated with motor impairment at 2 years. SVE was replicated in a neonatal mouse model of intestinal perforation. Examination of the murine echogenic subventricular zone (SVZ) revealed NLRP3-inflammasome assembly in multiciliated FoxJ1+ ependymal cells and a loss of the ependymal border in this postnatal stem cell niche. These data suggest a mechanism of preterm brain injury localized to the SVZ that has not been adequately considered. Ultrasound detection of SVE may serve as an early biomarker for neurodevelopmental impairment after inflammatory disease in preterm infants.

Radioactive waste microbiology: predicting microbial survival and activity in changing extreme environments.

The potential for microbial activity to occur within the engineered barrier system (EBS) of a geological disposal facility (GDF) for radioactive waste is acknowledged by waste management organizations as it could affect many aspects of the safety functions of a GDF. Microorganisms within an EBS will be exposed to changing temperature, pH, radiation, salinity, saturation, and availability of nutrient and energy sources, which can limit microbial survival and activity. Some of the limiting conditions are incorporated into GDF designs for safety reasons, including the high pH of cementitious repositories, the limited pore space of bentonite-based repositories, or the high salinity of GDFs in evaporitic geologies. Other environmental conditions such as elevated radiation, temperature, and desiccation, arise as a result of the presence of high heat generating waste (HHGW). Here, we present a comprehensive review of how environmental conditions in the EBS may limit microbial activity, covering HHGW and lower heat generating waste (LHGW) in a range of geological environments. We present data from the literature on the currently recognized limits to life for each of the environmental conditions described above, and nutrient availability to establish the potential for life in these environments. Using examples where each variable has been modelled for a particular GDF, we outline the times and locations when that variable can be expected to limit microbial activity. Finally, we show how this information for multiple variables can be used to improve our understanding of the potential for microbial activity to occur within the EBS of a GDF and, more broadly, to understand microbial life in changing environments exposed to multiple extreme conditions.

Transcriptome Analysis Identifies An ASD-Like Phenotype In Oligodendrocytes And Microglia From C58/J Amygdala That Is Dependent On Sex and Sociability.

Background: Autism Spectrum Disorder (ASD) is a group of neurodevelopmental disorders with higher incidence in males and is characterized by atypical verbal/nonverbal communication, restricted interests that can be accompanied by repetitive behavior, and disturbances in social behavior. This study investigated brain mechanisms that contribute to sociability deficits and sex differences in an ASD animal model. Methods: Sociability was measured in C58/J and C57BL/6J mice using the 3-chamber social choice test. Bulk RNA-Seq and snRNA-Seq identified transcriptional changes in C58/J and C57BL/6J amygdala within which DMRseq was used to measure differentially methylated regions in amygdala. Results: C58/J mice displayed divergent social strata in the 3-chamber test. Transcriptional and pathway signatures revealed immune-related biological processes differ between C58/J and C57BL/6J amygdala. Hypermethylated and hypomethylated genes were identified in C58/J versus C57BL/6J amygdala. snRNA-Seq data in C58/J amygdala identified differential transcriptional signatures within oligodendrocytes and microglia characterized by increased ASD risk gene expression and predicted impaired myelination that was dependent on sex and sociability. RNA velocity, gene regulatory network, and cell communication analysis showed diminished oligodendrocyte/microglia differentiation. Findings were verified using bulk RNA-Seq and demonstrated oxytocin's beneficial effects on myelin gene expression. Limitations: Our findings are significant. However, limitations can be noted. The cellular mechanisms linking reduced oligodendrocyte differentiation and reduced myelination to an ASD phenotype in C58/J mice need further investigation. Additional snRNA-Seq and spatial studies would determine if effects in oligodendrocytes/microglia are unique to amygdala or if this occurs in other brain regions. Oxytocin's effects need further examination to understand its potential as an ASD therapeutic. Conclusions: Our work demonstrates the C58/J mouse model's utility in evaluating the influence of sex and sociability on the transcriptome in concomitant brain regions involved in ASD. Our single-nucleus transcriptome analysis elucidates potential pathological roles of oligodendrocytes and microglia in ASD. This investigation provides details regarding regulatory features disrupted in these cell types, including transcriptional gene dysregulation, aberrant cell differentiation, altered gene regulatory networks, and changes to key pathways that promote microglia/oligodendrocyte differentiation. Our studies provide insight into interactions between genetic risk and epigenetic processes associated with divergent affiliative behavior and lack of positive sociability.

UBE2N is essential for maintenance of skin homeostasis and suppression of inflammation.

UBE2N, a Lys63-ubiquitin conjugating enzyme, plays critical roles in embryogenesis and immune system development and function. However, its roles in adult epithelial tissue homeostasis and pathogenesis are unclear. We generated conditional mouse models that deleted Ube2n in skin cells in a temporally and spatially controlled manner. We found that Ube2n-knockout (KO) in the adult skin keratinocytes induced a range of inflammatory skin defects characteristic of psoriatic and actinic keratosis. These included eczematous inflammation, epidermal and dermal thickening, parakeratosis, and increased immune cell infiltration, as well as signs of edema and blistering. Single cell transcriptomic analyses and RT-qPCR showed that Ube2n KO keratinocytes expressed elevated myeloid cell chemo-attractants such as Cxcl1 and Cxcl2 and decreased the homeostatic T lymphocyte chemo-attractant, Ccl27a. Consistently, the infiltrating immune cells of Ube2n-KO skin were predominantly myeloid-derived cells including neutrophils and M1-like macrophages that were highly inflammatory, as indicated by expression of Il1ß and Il24. Pharmacological blockade of the IL-1 receptor associated kinases (IRAK1/4) alleviated eczema, epidermal and dermal thickening, and immune infiltration of the Ube2n mutant skin. Together, these findings highlight a key role of keratinocyte-UBE2N in maintenance of epidermal homeostasis and skin immunity and identify IRAK1/4 as potential therapeutic target for inflammatory skin disorders.

Ataxia-telangiectasia mutated ( Atm ) disruption sensitizes spatially-directed H3.3K27M/TP53 diffuse midline gliomas to radiation therapy.

Diffuse midline gliomas (DMGs) are lethal brain tumors characterized by p53-inactivating mutations and oncohistone H3.3K27M mutations that rewire the cellular response to genotoxic stress, which presents therapeutic opportunities. We used RCAS/tv-a retroviruses and Cre recombinase to inactivate p53 and induce K27M in the native H3f3a allele in a lineage- and spatially-directed manner, yielding primary mouse DMGs. Genetic or pharmacologic disruption of the DNA damage response kinase Ataxia-telangiectasia mutated (ATM) enhanced the efficacy of focal brain irradiation, extending mouse survival. This finding suggests that targeting ATM will enhance the efficacy of radiation therapy for p53-mutant DMG but not p53-wildtype DMG. We used spatial in situ transcriptomics and an allelic series of primary murine DMG models with different p53 mutations to identify transactivation-independent p53 activity as a key mediator of such radiosensitivity. These studies deeply profile a genetically faithful and versatile model of a lethal brain tumor to identify resistance mechanisms for a therapeutic strategy currently in clinical trials.

Tackling differential attainment in specialist GP training in England and Scotland.

In this article NHS England and NHS Education for Scotland describe practical ways we are tackling differences in the attainment of people training as general practitioners (GPs).Trainees from minority ethnic groups and international medical graduates are less likely than others to qualify as GPs. It is difficult to change systemic inequalities, but over the past five years we have made practical changes to GP speciality training. Educators recognise there is an issue and are trying to tackle it.For example, people who had not successfully qualified had an opportunity to return to GP training. When we provided individualised targeted support, the proportion who completed training significantly increased (76%).This was a catalyst for reviewing unconscious bias in GP training. We implemented a national programme to tackle differential attainment and system-level bias. Educators now work with all GP trainees to identify their individual needs. Supervisors are trained to recognise bias and provide targeted support. There is mental health support and regular reviews to see whether trainees are ready to sit exams. Trainee representatives are championing the learner voice in national committees. Exams are being altered to reduce unconscious bias. We are monitoring attainment over time.The key message is that differential attainment should not be in the 'too hard basket'. The narrative is changing from 'can't do' to 'must do', supported by appropriate leadership, promotion and resourcing. There is much more to do, but we are making changes, evaluating and applying our learning. We have moved from talking to taking action.

20-αHydroxycholesterol, an oxysterol in human breast milk, reverses mouse neonatal white matter injury through Gli-dependent oligodendrogenesis.

White matter injuries (WMIs) are the leading cause of neurologic impairment in infants born premature. There are no treatment options available. The most common forms of WMIs in infants occur prior to the onset of normal myelination, making its pathophysiology distinctive, thus requiring a tailored approach to treatment. Neonates present a unique opportunity to repair WMIs due to a transient abundance of neural stem/progenitor cells (NSPCs) present in the germinal matrix with oligodendrogenic potential. We identified an endogenous oxysterol, 20-αHydroxycholesterol (20HC), in human maternal breast milk that induces oligodendrogenesis through a sonic hedgehog (shh), Gli-dependent mechanism. Following WMI in neonatal mice, injection of 20HC induced subventricular zone-derived oligodendrogenesis and improved myelination in the periventricular white matter, resulting in improved motor outcomes. Targeting the oligodendrogenic potential of postnatal NSPCs in neonates with WMIs may be further developed into a novel approach to mitigate this devastating complication of preterm birth.

A simple method for testing and controlling inhibition in soil and sediment samples for qPCR.

The presence of polymerase-chain-reaction (PCR) inhibitors in many environmental samples can make reliable and repeatable quantitative-polymerase-chain-reaction (qPCR) analysis difficult without sample dilution. To estimate an optimal sample dilution for qPCR and reduce effects of inhibition, a simple test based on multiple dilution series of samples is presented that avoids the use of internal controls and standards reducing complexity and cost.

The RNA helicase DDX39B activates FOXP3 RNA splicing to control T regulatory cell fate.

Genes associated with increased susceptibility to multiple sclerosis (MS) have been identified, but their functions are incompletely understood. One of these genes codes for the RNA helicase DExD/H-Box Polypeptide 39B (DDX39B), which shows genetic and functional epistasis with interleukin-7 receptor-α gene (IL7R) in MS-risk. Based on evolutionary and functional arguments, we postulated that DDX39B enhances immune tolerance thereby decreasing MS risk. Consistent with such a role we show that DDX39B controls the expression of many MS susceptibility genes and important immune-related genes. Among these we identified Forkhead Box P3 (FOXP3), which codes for the master transcriptional factor in CD4+/CD25+ T regulatory cells. DDX39B knockdown led to loss of immune-regulatory and gain of immune-effector expression signatures. Splicing of FOXP3 introns, which belong to a previously unrecognized type of introns with C-rich polypyrimidine tracts, was exquisitely sensitive to DDX39B levels. Given the importance of FOXP3 in autoimmunity, this work cements DDX39B as an important guardian of immune tolerance.

JAK-STAT activation contributes to cytotoxic T cell-mediated basal cell death in human chronic lung allograft dysfunction.

Chronic lung allograft dysfunction (CLAD) is the leading cause of death in lung transplant recipients. CLAD is characterized clinically by a persistent decline in pulmonary function and histologically by the development of airway-centered fibrosis known as bronchiolitis obliterans. There are no approved therapies to treat CLAD, and the mechanisms underlying its development remain poorly understood. We performed single-cell RNA-Seq and spatial transcriptomic analysis of explanted tissues from human lung recipients with CLAD, and we performed independent validation studies to identify an important role of Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling in airway epithelial cells that contributes to airway-specific alloimmune injury. Specifically, we established that activation of JAK-STAT signaling leads to upregulation of major histocompatibility complex 1 (MHC-I) in airway basal cells, an important airway epithelial progenitor population, which leads to cytotoxic T cell-mediated basal cell death. This study provides mechanistic insight into the cell-to-cell interactions driving airway-centric alloimmune injury in CLAD, suggesting a potentially novel therapeutic strategy for CLAD prevention or treatment.

Ganglioglioma deep transcriptomics reveals primitive neuroectoderm neural precursor-like population.

Gangliogliomas are brain tumors composed of neuron-like and macroglia-like components that occur in children and young adults. Gangliogliomas are often characterized by a rare population of immature astrocyte-appearing cells expressing CD34, a marker expressed in the neuroectoderm (neural precursor cells) during embryogenesis. New insights are needed to refine tumor classification and to identify therapeutic approaches. We evaluated five gangliogliomas with single nucleus RNA-seq, cellular indexing of transcriptomes and epitopes by sequencing, and/or spatially-resolved RNA-seq. We uncovered a population of CD34+ neoplastic cells with mixed neuroectodermal, immature astrocyte, and neuronal markers. Gene regulatory network interrogation in these neuroectoderm-like cells revealed control of transcriptional programming by TCF7L2/MEIS1-PAX6 and SOX2, similar to that found during neuroectodermal/neural development. Developmental trajectory analyses place neuroectoderm-like tumor cells as precursor cells that give rise to neuron-like and macroglia-like neoplastic cells. Spatially-resolved transcriptomics revealed a neuroectoderm-like tumor cell niche with relative lack of vascular and immune cells. We used these high resolution results to deconvolute clinically-annotated transcriptomic data, confirming that CD34+ cell-associated gene programs associate with gangliogliomas compared to other glial brain tumors. Together, these deep transcriptomic approaches characterized a ganglioglioma cellular hierarchy-confirming CD34+ neuroectoderm-like tumor precursor cells, controlling transcription programs, cell signaling, and associated immune cell states. These findings may guide tumor classification, diagnosis, prognostication, and therapeutic investigations.

Aging and obesity prime the methylome and transcriptome of adipose stem cells for disease and dysfunction.

The epigenome of stem cells occupies a critical interface between genes and environment, serving to regulate expression through modification by intrinsic and extrinsic factors. We hypothesized that aging and obesity, which represent major risk factors for a variety of diseases, synergistically modify the epigenome of adult adipose stem cells (ASCs). Using integrated RNA- and targeted bisulfite-sequencing in murine ASCs from lean and obese mice at 5- and 12-months of age, we identified global DNA hypomethylation with either aging or obesity, and a synergistic effect of aging combined with obesity. The transcriptome of ASCs in lean mice was relatively stable to the effects of age, but this was not true in obese mice. Functional pathway analyses identified a subset of genes with critical roles in progenitors and in diseases of obesity and aging. Specifically, Mapt, Nr3c2, App, and Ctnnb1 emerged as potential hypomethylated upstream regulators in both aging and obesity (AL vs. YL and AO vs. YO), and App, Ctnnb1, Hipk2, Id2, and Tp53 exhibited additional effects of aging in obese animals. Furthermore, Foxo3 and Ccnd1 were potential hypermethylated upstream regulators of healthy aging (AL vs. YL), and of the effects of obesity in young animals (YO vs. YL), suggesting that these factors could play a role in accelerated aging with obesity. Finally, we identified candidate driver genes that appeared recurrently in all analyses and comparisons undertaken. Further mechanistic studies are needed to validate the roles of these genes capable of priming ASCs for dysfunction in aging- and obesity-associated pathologies.

Groundwater Microbiology of an Urban Open-Loop Ground Source Heat Pump with High Methane.

Ground source heat pumps (GSHPs) are low-carbon alternatives to gas boilers for decarbonizing heating. Open-loop GSHP systems abstract groundwater, pass it through a heat exchanger, and return it to ground or surface water. Groundwater samples from the top and base of an abstraction and a recharge borehole of an open-loop GSHP system in Cardiff, UK were assessed, and compared to two local boreholes in the same aquifer. Groundwater samples were taken when the GSHP system was active (once) and inactive (twice) and analyzed for changes in geochemistry, viable cell counts, and microbial community (16S rRNA gene sequencing). The GSHP had a distinct geochemistry and microbial community compared to the control boreholes, and the abstraction borehole showed greater variability than the recharge borehole. The microbial community of the GSHP system showed an increase in relative abundance of genera involved in oxidation of methane and methylated compounds, of which Methylotenera was the most abundant (up to 83.9% of 16S rRNA gene sequences). There were also changes in genera associated with nitrification (Nitrospira, Nitrosomonas) and those with potential for sulfur and iron cycling (Rhodoferax). Methane concentration was analyzed after identification of methylotrophs and found that methane concentrations were up to 2855 µg L-1 , thus likely having had a significant impact on the bacterial communities present. Understanding the microbiology and biogeochemistry of GSHP systems provides insight into potential issues with local infrastructure and long-term system performance, and supports modeling to maximize efficient and sustainable use of the subsurface.