Transcription of HIV-1 at sites of intact latent provirus integration.

HIV-1 antiretroviral therapy is highly effective but fails to eliminate a reservoir of latent proviruses, leading to a requirement for life-long treatment. How the site of integration of authentic intact latent proviruses might impact their own or neighboring gene expression or reservoir dynamics is poorly understood. Here, we report on proviral and neighboring gene transcription at sites of intact latent HIV-1 integration in cultured T cells obtained directly from people living with HIV, as well as engineered primary T cells and cell lines. Proviral gene expression was correlated to the level of endogenous gene expression under resting but not activated conditions. Notably, latent proviral promoters were 100-10,000× less active than in productively infected cells and had little or no measurable impact on neighboring gene expression under resting or activated conditions. Thus, the site of integration has a dominant effect on the transcriptional activity of intact HIV-1 proviruses in the latent reservoir, thereby influencing cytopathic effects and proviral immune evasion.

Fluid shear stress-induced changes in megalin trafficking enhance endocytic capacity in proximal tubule cells.

Proximal tubule (PT) cells maintain a high-capacity apical endocytic pathway to recover essentially all proteins that escape the glomerular filtration barrier. The multi ligand receptors megalin and cubilin play pivotal roles in the endocytic uptake of normally filtered proteins in PT cells but also contribute to the uptake of nephrotoxic drugs, including aminoglycosides. We previously demonstrated that opossum kidney (OK) cells cultured under continuous fluid shear stress (FSS) are superior to cells cultured under static conditions in recapitulating essential functional properties of PT cells in vivo. To identify drivers of the high-capacity, efficient endocytic pathway in the PT, we compared FSS-cultured OK cells with less endocytically active static-cultured OK cells. Megalin and cubilin expression are increased, and endocytic uptake of albumin in FSS-cultured cells is > 5-fold higher compared with cells cultured under static conditions. To understand how differences in receptor expression, distribution, and trafficking rates contribute to increased uptake, we used biochemical, morphological, and mathematical modeling approaches to compare megalin traffic in FSS- versus static-cultured OK cells. Our model predicts that culturing cells under FSS increases the rates of all steps in megalin trafficking. Importantly, the model explains why, despite seemingly counterintuitive observations (a reduced fraction of megalin at the cell surface, higher colocalization with lysosomes, and a shorter half-life of surface-tagged megalin in FSS-cultured cells), uptake of albumin is dramatically increased compared with static-grown cells. We also show that FSS-cultured OK cells more accurately exhibit the mechanisms that mediate uptake of nephrotoxic drugs in vivo compared with static-grown cells. This culture model thus provides a useful platform to understand drug uptake mechanisms, with implications for developing interventions in nephrotoxic injury prevention.

A CRISPR activation screen identifies FBXO22 supporting targeted protein degradation.

Targeted protein degradation (TPD) represents a potent chemical biology paradigm that leverages the cellular degradation machinery to pharmacologically eliminate specific proteins of interest. Although multiple E3 ligases have been discovered to facilitate TPD, there exists a compelling requirement to diversify the pool of E3 ligases available for such applications. Here we describe a clustered regularly interspaced short palindromic repeats (CRISPR)-based transcriptional activation screen focused on human E3 ligases, with the goal of identifying E3 ligases that can facilitate heterobifunctional compound-mediated target degradation. Through this approach, we identified a candidate proteolysis-targeting chimera (PROTAC), 22-SLF, that induces the degradation of FK506-binding protein 12 when the transcription of FBXO22 gene is activated. Subsequent mechanistic investigations revealed that 22-SLF interacts with C227 and/or C228 in F-box protein 22 (FBXO22) to achieve target degradation. Lastly, we demonstrated the versatility of FBXO22-based PROTACs by effectively degrading additional endogenous proteins, including bromodomain-containing protein 4 and the echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase fusion protein.

Baseline tumor-infiltrating lymphocyte patterns and response to immune checkpoint inhibition in metastatic cutaneous melanoma.

INTRODUCTION: The presence of tumor-infiltrating lymphocytes (TILs) in melanoma has been linked to survival. Their predictive capability for immune checkpoint inhibition (ICI) response remains uncertain. Therefore, we investigated the association between treatment response and TILs in the largest cohort to date and analyzed if this association was independent of known clinical predictors. METHODS: In this multicenter cohort study, patients who received first-line anti-PD1 ± anti-CTLA4 for advanced melanoma were identified. TILs were scored on hematoxylin and eosin (H&E) slides of primary melanoma and pre-treatment metastases using the validated TILs-WG, Clark and MIA score. The primary outcome was objective response rate (ORR), with progression free survival and overall survival being secondary outcomes. Univariable and multivariable logistic regression and Cox proportional hazard were performed, adjusting for known clinical predictors. RESULTS: Metastatic melanoma specimens were available for 650 patients and primary specimens for 565 patients. No association was found in primary melanoma specimens. In metastatic specimens, a 10-point increase in the TILs-WG score was associated with a higher probability of response (aOR 1.17, 95 % CI 1.07-1.28), increased PFS (HR 0.93, 95 % CI 0.87-0.996), and OS (HR 0.94, 95 % CI 0.89-0.99). When categorized, patients in the highest tertile TILs-WG score (15-100 %) compared to the lowest tertile (0 %) had a longer median PFS (13.1 vs. 7.3 months, p = 0.04) and OS (49.4 vs. 19.5 months, p = 0.003). Similar results were noted using the MIA and Clark scores. CONCLUSION: In advanced melanoma patients, TIL patterns on H&E slides of pre-treatment metastases, regardless of measurement method, are independently associated with ICI response. TILs are easily scored on readily available H&Es, facilitating the use of this biomarker in clinical practice.

Hypoxia drives HIF2-dependent reversible macrophage cell cycle entry.

Low-oxygen conditions (hypoxia) have been associated primarily with cell-cycle arrest in dividing cells. Macrophages are typically quiescent in G0 but can proliferate in response to tissue signals. Here we show that hypoxia (1% oxygen tension) results in reversible entry into the cell cycle in macrophages. Cell cycle progression is largely limited to G0-G1/S phase transition with little progression to G2/M. This cell cycle transitioning is triggered by an HIF2α-directed transcriptional program. The response is accompanied by increased expression of cell-cycle-associated proteins, including CDK1, which is known to phosphorylate SAMHD1 at T592 and thereby regulate antiviral activity. Prolyl hydroxylase (PHD) inhibitors are able to recapitulate HIF2α-dependent cell cycle entry in macrophages. Finally, tumor-associated macrophages (TAMs) in lung cancers exhibit transcriptomic profiles representing responses to low oxygen and cell cycle progression at the single-cell level. These findings have implications for inflammation and tumor progression/metastasis where low-oxygen environments are common.

Wildfire smoke impacts lake ecosystems.

Wildfire activity is increasing globally. The resulting smoke plumes can travel hundreds to thousands of kilometers, reflecting or scattering sunlight and depositing particles within ecosystems. Several key physical, chemical, and biological processes in lakes are controlled by factors affected by smoke. The spatial and temporal scales of lake exposure to smoke are extensive and under-recognized. We introduce the concept of the lake smoke-day, or the number of days any given lake is exposed to smoke in any given fire season, and quantify the total lake smoke-day exposure in North America from 2019 to 2021. Because smoke can be transported at continental to intercontinental scales, even regions that may not typically experience direct burning of landscapes by wildfire are at risk of smoke exposure. We found that 99.3% of North America was covered by smoke, affecting a total of 1,333,687 lakes ≥10 ha. An incredible 98.9% of lakes experienced at least 10 smoke-days a year, with 89.6% of lakes receiving over 30 lake smoke-days, and lakes in some regions experiencing up to 4 months of cumulative smoke-days. Herein we review the mechanisms through which smoke and ash can affect lakes by altering the amount and spectral composition of incoming solar radiation and depositing carbon, nutrients, or toxic compounds that could alter chemical conditions and impact biota. We develop a conceptual framework that synthesizes known and theoretical impacts of smoke on lakes to guide future research. Finally, we identify emerging research priorities that can help us better understand how lakes will be affected by smoke as wildfire activity increases due to climate change and other anthropogenic activities.

Optical parameters of healthy and tumor breast tissues in mice.

Knowledge of the optical parameters of tumors is important for choosing the correct laser treatment parameters. In this paper, optical properties and refraction indices of breast tissue in healthy mice and a 4T1 model mimicking human breast cancer have been measured. A significant decrease in both the scattering and refractive index of tumor tissue has been observed. The change in tissue morphology has induced the change in the slope of the scattering spectrum. Thus, the light penetration depth into tumor has increased by almost 1.5-2 times in the near infrared "optical windows." Raman spectra have shown lower lipid content and higher protein content in tumor. The difference in the optical parameters of the tissues under study makes it possible to reliably differentiate them. The results may be useful for modeling the distribution of laser radiation in healthy tissues and cancers for deriving optimal irradiation conditions in photodynamic therapy.

High-content analysis of proteostasis capacity in cellular models of amyotrophic lateral sclerosis (ALS).

Disrupted proteome homeostasis (proteostasis) in amyotrophic lateral sclerosis (ALS) has been a major focus of research in the past two decades. However, the proteostasis processes that become disturbed in ALS are not fully understood. Obtaining more detailed knowledge of proteostasis disruption in association with different ALS-causing mutations will improve our understanding of ALS pathophysiology and may identify novel therapeutic targets and strategies for ALS patients. Here we describe the development and use of a novel high-content analysis (HCA) assay to investigate proteostasis disturbances caused by the expression of several ALS-causing gene variants. This assay involves the use of conformationally-destabilised mutants of firefly luciferase (Fluc) to examine protein folding/re-folding capacity in NSC-34 cells expressing ALS-associated mutations in the genes encoding superoxide dismutase-1 (SOD1A4V) and cyclin F (CCNFS621G). We demonstrate that these Fluc isoforms can be used in high-throughput format to report on reductions in the activity of the chaperone network that result from the expression of SOD1A4V, providing multiplexed information at single-cell resolution. In addition to SOD1A4V and CCNFS621G, NSC-34 models of ALS-associated TDP-43, FUS, UBQLN2, OPTN, VCP and VAPB mutants were generated that could be screened using this assay in future work. For ALS-associated mutant proteins that do cause reductions in protein quality control capacity, such as SOD1A4V, this assay has potential to be applied in drug screening studies to identify candidate compounds that can ameliorate this deficiency.

Transcription of HIV-1 at sites of intact latent provirus integration.

HIV-1 anti-retroviral therapy is highly effective but fails to eliminate a reservoir of latent proviruses leading to a requirement for life-long treatment. How the site of integration of authentic intact latent proviruses might impact their own or neighboring gene expression or reservoir dynamics is poorly understood. Here we report on proviral and neighboring gene transcription at sites of intact latent HIV-1 integration in cultured T cells obtained directly from people living with HIV, as well as engineered primary T cells and cell lines. Proviral gene expression was correlated to the level of endogenous gene expression under resting but not activated conditions. Notably, latent proviral promoters were 10010,000X less active than in productively infected cells and had little or no measurable impact on neighboring gene expression under resting or activated conditions. Thus, the site of integration has a dominant effect on the transcriptional activity of intact HIV-1 proviruses in the latent reservoir thereby influencing cytopathic effects and proviral immune evasion.

Mathematical modelling of clonal reduction therapeutic strategies in acute myeloid leukemia.

Over the years, the overall survival of older patients diagnosed with acute myeloid leukemia (AML) has not significantly increased. Although standard cytotoxic therapies that rapidly eliminate dividing myeloblasts are used to induce remission, relapse can occur due to surviving therapy-resistant leukemic stem cells (LSCs). Hence, anti-LSC strategies have become a key target to cure AML. We have recently shown that previously approved cardiac glycosides and glucocorticoids target LSC-enriched CD34+ cells in the primary human AML 8227 model with more efficacy than normal hematopoietic stem cells (HSCs). To translate these in vitro findings into humans, we developed a mathematical model of stem cell dynamics that describes the stochastic evolution of LSCs in AML post-standard-of-care. To this, we integrated population pharmacokinetic-pharmacodynamic (PKPD) models to investigate the clonal reduction potential of several promising candidate drugs in comparison to cytarabine, which is commonly used in high doses for consolidation therapy in AML patients. Our results suggest that cardiac glycosides (proscillaridin A, digoxin and ouabain) and glucocorticoids (budesonide and mometasone) reduce the expansion of LSCs through a decrease in their viability. While our model predicts that effective doses of cardiac glycosides are potentially too toxic to use in patients, simulations show the possibility of mometasone to prevent relapse through the glucocorticoid's ability to drastically reduce LSC population size. This work therefore highlights the prospect of these treatments for anti-LSC strategies and underlines the use of quantitative approaches to preclinical drug translation in AML.

Biodegradable calcium carbonate carriers for the topical delivery of clobetasol propionate.

Inflammatory dermatoses represent a global problem with increasing prevalence and recurrence among the world population. Topical glucocorticoids (GCs) are the most commonly used anti-inflammatory drugs in dermatology due to a wide range of their therapeutic actions, which, however, have numerous local and systemic side effects. Hence, there is a growing need to create new delivery systems for GCs, ensuring the drug localization in the pathological site, thus increasing the effectiveness of therapy and lowering the risk of side effects. Here, we propose a novel topical particulate formulation for the GC clobetasol propionate (CP), based on the use of porous calcium carbonate (CaCO3) carriers in the vaterite crystalline form. The designed carriers contain a substantially higher CP amount than conventional dosage forms used in clinics (4.5% w/w vs. 0.05% w/w) and displayed a good biocompatibility and effective cellular uptake when studied in fibroblasts in vitro. Hair follicles represent an important reservoir for the GC accumulation in skin and house the targets for its action. In this study, we demonstrated successful delivery of the CP-loaded carriers (CP-CaCO3) into the hair follicles of rats in vivo using optical coherent tomography (OCT). Importantly, the OCT monitoring revealed the gradual intrafollicular degradation of the carriers within 168 h with the most abundant follicle filling occurring within the first 48 h. Biodegradability makes the proposed system especially promising when searching for new CP formulations with improved safety and release profile. Our findings evidenced the great potential of the CaCO3 carriers in improving the dermal bioavailability of this poorly water-soluble GC.

Fluid Shear Stress-Induced Changes in Megalin Trafficking Enhance Endocytic Capacity in Proximal Tubule Cells.

Proximal tubule (PT) cells maintain a high-capacity apical endocytic pathway to recover essentially all proteins that escape the glomerular filtration barrier. The multiligand receptors megalin and cubilin play pivotal roles in the endocytic uptake of normally filtered proteins in PT cells but also contribute to the uptake of nephrotoxic drugs, including aminoglycosides. We previously demonstrated that opossum kidney (OK) cells cultured under continuous fluid shear stress (FSS) are superior to cells cultured under static conditions in recapitulating essential functional properties of PT cells in vivo. To identify drivers of the high-capacity, efficient endocytic pathway in the PT, we compared FSS-cultured OK cells with less endocytically active static-cultured OK cells. Megalin and cubilin expression are increased, and endocytic uptake of albumin in FSS-cultured cells is >5-fold higher compared with cells cultured under static conditions. To understand how differences in receptor expression, distribution, and trafficking rates contribute to increased uptake, we used biochemical, morphological, and mathematical modeling approaches to compare megalin traffic in FSS- versus static-cultured OK cells. Our model predicts that culturing cells under FSS increases the rates of all steps in megalin trafficking. Importantly, the model explains why, despite seemingly counterintuitive observations (a reduced fraction of megalin at the cell surface, higher colocalization with lysosomes, and a shorter half-life of surface-tagged megalin in FSS-cultured cells), uptake of albumin is dramatically increased compared with static-grown cells. We also show that FSS-cultured OK cells more accurately exhibit the mechanisms that mediate uptake of nephrotoxic drugs in vivo compared with static-grown cells. This culture model thus provides a useful platform to understand drug uptake mechanisms, with implications for developing interventions in nephrotoxic injury prevention.

The Landscape of Direct-To-Consumer Genetic Testing in Reproductive Health Contexts: An Analytical Framework of Stakeholders and Their Competing Motivations.

We propose a theoretical framework that identifies (a) the different categories of stakeholders and (b) the normative values that drive their attitudes toward direct-to-consumer genetic testing, with an emphasis on the reproductive health contexts. We conducted a literature search using varied combinations of search terms, including direct-to-consumer genetic testing, decision-making, reproductive health, and policy. Using a grounded theory approach to existing literature and in combination with a narrative review, we present a systematic framework of five categories of stakeholders (i.e., genome-driven stakeholders, industry-driven stakeholders, history-driven stakeholders, value-driven stakeholders, and social justice-driven stakeholders) that shape the public's discourse. Moving beyond the dialectical ethics that have governed the public discourse, we also identify the normative values and interests that motivate different stakeholders' attitudes and decision-making through theoretical sampling under the grounded theory. We investigate the competing and conflicting values within the same category of stakeholders. For example, despite being industry-driven stakeholders, medical professionals' attitudes are driven by concerns about standards of care; in contrast, health insurance companies' concerns are centered on profit. We further explore the tensions between these stakeholders that impact their strategic alliances and pose challenges to the practices of direct-to-consumer genetic testing. Finally, we examine how these stakeholders and their corresponding values may shape future development and policies of direct-to-consumer genetic testing in the context of reproductive health.

Functional Connectivity Mechanisms Underlying Symptom Reduction Following Lisdexamfetamine Treatment in Binge-Eating Disorder: A Clinical Trial.

Background: Speculation exists as to whether lisdexamfetamine dimesylate (LDX) acts on the functional connectivity (FC) of brain networks that modulate appetite, reward, or inhibitory control in binge-eating disorder (BED). Better insights into its action may help guide the development of more targeted therapeutics and identify who will benefit most from this medication. Here, we use a comprehensive data-driven approach to investigate the brain FC changes that underlie the therapeutic action of LDX in patients with BED. Methods: Forty-six participants with moderate to severe BED received LDX titrated to 50 or 70 mg for an 8-week period. Twenty age-matched healthy control participants were also recruited. Resting-state functional magnetic resonance imaging was used to probe changes in brain FC pre- and post treatment and correlated with change in clinical measures. Results: Ninety-seven percent of trial completers (n = 31) experienced remission or a reduction to mild BED during the 8-week LDX trial. Widespread neural FC changes occurred, with changes in default mode to limbic, executive control to subcortical, and default mode to executive control networks associated with improvements in clinical outcomes. These connections were not distinct from control participants at pretreatment but were different from control participants following LDX treatment. Pretreatment connectivity did not predict treatment response. Conclusions: FC between networks associated with self-referential processing, executive function, and reward seem to underlie the therapeutic effect of LDX in BED. This suggests that LDX activates change via multiple systems, with most changes in compensatory networks rather than in those characterizing the BED diagnosis.

Draft genome sequences of Arthrobacter sp. AZCC_0090 and Mycobacterium sp. AZCC_0083 isolated from oligotrophic subsurface forest soil in the Santa Catalina mountains of Southern Arizona.

Here, we present the genomes of two soil actinobacteria: Arthrobacter sp. strain AZCC_0090 and Mycobacterium sp. strain AZCC_0083, isolated from oligotrophic subsurface soils in Southern Arizona, USA.

Genome sequence of Arthrobacter globiformis phage MaGuCo.

MaGuCo is a temperate phage isolated from soil collected in Alton, NH, USA, using Arthrobacter globiformis. Its genome is 43,924 base pairs long and contains 63 protein-encoding genes, 44 of which were assigned putative functions. MaCuGo is assigned to cluster AZ2 based on gene content similarity to actinobacteriophages.

Transcriptional consequences of trisomy 21 on neural induction.

Introduction: Down syndrome, caused by trisomy 21, is a complex developmental disorder associated with intellectual disability and reduced growth of multiple organs. Structural pathologies are present at birth, reflecting embryonic origins. A fundamental unanswered question is how an extra copy of human chromosome 21 contributes to organ-specific pathologies that characterize individuals with Down syndrome, and, relevant to the hallmark intellectual disability in Down syndrome, how trisomy 21 affects neural development. We tested the hypothesis that trisomy 21 exerts effects on human neural development as early as neural induction. Methods: Bulk RNA sequencing was performed on isogenic trisomy 21 and euploid human induced pluripotent stem cells (iPSCs) at successive stages of neural induction: embryoid bodies at Day 6, early neuroectoderm at Day 10, and differentiated neuroectoderm at Day 17. Results: Gene expression analysis revealed over 1,300 differentially expressed genes in trisomy 21 cells along the differentiation pathway compared to euploid controls. Less than 5% of the gene expression changes included upregulated chromosome 21 encoded genes at every timepoint. Genes involved in specific growth factor signaling pathways (WNT and Notch), metabolism (including oxidative stress), and extracellular matrix were altered in trisomy 21 cells. Further analysis uncovered heterochronic expression of genes. Conclusion: Trisomy 21 impacts discrete developmental pathways at the earliest stages of neural development. The results suggest that metabolic dysfunction arises early in embryogenesis in trisomy 21 and may affect development and function more broadly.

Effective γδ T-cell clinical therapies: current limitations and future perspectives for cancer immunotherapy.

γδ T cells are a unique subset of T lymphocytes, exhibiting features of both innate and adaptive immune cells and are involved with cancer immunosurveillance. They present an attractive alternative to conventional T cell-based immunotherapy due, in large part, to their lack of major histocompatibility (MHC) restriction and ability to secrete high levels of cytokines with well-known anti-tumour functions. To date, clinical trials using γδ T cell-based immunotherapy for a range of haematological and solid cancers have yielded limited success compared with in vitro studies. This inability to translate the efficacy of γδ T-cell therapies from preclinical to clinical trials is attributed to a combination of several factors, e.g. γδ T-cell agonists that are commonly used to stimulate populations of these cells have limited cellular uptake yet rely on intracellular mechanisms; administered γδ T cells display low levels of tumour-infiltration; and there is a gap in the understanding of γδ T-cell inhibitory receptors. This review explores the discrepancy between γδ T-cell clinical and preclinical performance and offers viable avenues to overcome these obstacles. Using more direct γδ T-cell agonists, encapsulating these agonists into lipid nanocarriers to improve their pharmacokinetic and pharmacodynamic profiles and the use of combination therapies to overcome checkpoint inhibition and T-cell exhaustion are ways to bridge the gap between preclinical and clinical success. Given the ability to overcome these limitations, the development of a more targeted γδ T-cell agonist-checkpoint blockade combination therapy has the potential for success in clinical trials which has to date remained elusive.

National-scale remotely sensed lake trophic state from 1984 through 2020.

Lake trophic state is a key ecosystem property that integrates a lake's physical, chemical, and biological processes. Despite the importance of trophic state as a gauge of lake water quality, standardized and machine-readable observations are uncommon. Remote sensing presents an opportunity to detect and analyze lake trophic state with reproducible, robust methods across time and space. We used Landsat surface reflectance data to create the first compendium of annual lake trophic state for 55,662 lakes of at least 10 ha in area throughout the contiguous United States from 1984 through 2020. The dataset was constructed with FAIR data principles (Findable, Accessible, Interoperable, and Reproducible) in mind, where data are publicly available, relational keys from parent datasets are retained, and all data wrangling and modeling routines are scripted for future reuse. Together, this resource offers critical data to address basic and applied research questions about lake water quality at a suite of spatial and temporal scales.

Multimodal Method for Differentiating Various Clinical Forms of Basal Cell Carcinoma and Benign Neoplasms In Vivo.

Correct classification of skin lesions is a key step in skin cancer screening, which requires high accuracy and interpretability. This paper proposes a multimodal method for differentiating various clinical forms of basal cell carcinoma and benign neoplasms that includes machine learning. This study was conducted on 37 neoplasms, including benign neoplasms and five different clinical forms of basal cell carcinoma. The proposed multimodal screening method combines diffuse reflectance spectroscopy, optical coherence tomography and high-frequency ultrasound. Using diffuse reflectance spectroscopy, the coefficients of melanin pigmentation, erythema, hemoglobin content, and the slope coefficient of diffuse reflectance spectroscopy in the wavelength range 650-800 nm were determined. Statistical texture analysis of optical coherence tomography images was used to calculate first- and second-order statistical parameters. The analysis of ultrasound images assessed the shape of the tumor according to parameters such as area, perimeter, roundness and other characteristics. Based on the calculated parameters, a machine learning algorithm was developed to differentiate the various clinical forms of basal cell carcinoma. The proposed algorithm for classifying various forms of basal cell carcinoma and benign neoplasms provided a sensitivity of 70.6 ± 17.3%, specificity of 95.9 ± 2.5%, precision of 72.6 ± 14.2%, F1 score of 71.5 ± 15.6% and mean intersection over union of 57.6 ± 20.1%. Moreover, for differentiating basal cell carcinoma and benign neoplasms without taking into account the clinical form, the method achieved a sensitivity of 89.1 ± 8.0%, specificity of 95.1 ± 0.7%, F1 score of 89.3 ± 3.4% and mean intersection over union of 82.6 ± 10.8%.