Deep learning is widely applicable to phenotyping embryonic development and disease.

Genome editing simplifies the generation of new animal models for congenital disorders. However, the detailed and unbiased phenotypic assessment of altered embryonic development remains a challenge. Here, we explore how deep learning (U-Net) can automate segmentation tasks in various imaging modalities, and we quantify phenotypes of altered renal, neural and craniofacial development in Xenopus embryos in comparison with normal variability. We demonstrate the utility of this approach in embryos with polycystic kidneys (pkd1 and pkd2) and craniofacial dysmorphia (six1). We highlight how in toto light-sheet microscopy facilitates accurate reconstruction of brain and craniofacial structures within X. tropicalis embryos upon dyrk1a and six1 loss of function or treatment with retinoic acid inhibitors. These tools increase the sensitivity and throughput of evaluating developmental malformations caused by chemical or genetic disruption. Furthermore, we provide a library of pre-trained networks and detailed instructions for applying deep learning to the reader's own datasets. We demonstrate the versatility, precision and scalability of deep neural network phenotyping on embryonic disease models. By combining light-sheet microscopy and deep learning, we provide a framework for higher-throughput characterization of embryonic model organisms. This article has an associated 'The people behind the papers' interview.

HNF1B Alters an Evolutionarily Conserved Nephrogenic Program of Target Genes.

SIGNIFICANCE STATEMENT: Mutations in hepatocyte nuclear factor-1 ß ( HNF1B ) are the most common monogenic causes of congenital renal malformations. HNF1B is necessary to directly reprogram fibroblasts to induced renal tubule epithelial cells (iRECs) and, as we demonstrate, can induce ectopic pronephric tissue in Xenopus ectodermal organoids. Using these two systems, we analyzed the effect of HNF1B mutations found in patients with cystic dysplastic kidney disease. We found cross-species conserved targets of HNF1B, identified transcripts that are differentially regulated by the patient-specific mutant protein, and functionally validated novel HNF1B targets in vivo . These results highlight evolutionarily conserved transcriptional mechanisms and provide insights into the genetic circuitry of nephrogenesis. BACKGROUND: Hepatocyte nuclear factor-1 ß (HNF1B) is an essential transcription factor during embryogenesis. Mutations in HNF1B are the most common monogenic causes of congenital cystic dysplastic renal malformations. The direct functional consequences of mutations in HNF1B on its transcriptional activity are unknown. METHODS: Direct reprogramming of mouse fibroblasts to induced renal tubular epithelial cells was conducted both with wild-type HNF1B and with patient mutations. HNF1B was expressed in Xenopus ectodermal explants. Transcriptomic analysis by bulk RNA-Seq identified conserved targets with differentially regulated expression by the wild-type or R295C mutant. CRISPR/Cas9 genome editing in Xenopus embryos evaluated transcriptional targets in vivo . RESULTS: HNF1B is essential for reprogramming mouse fibroblasts to induced renal tubular epithelial cells and induces development of ectopic renal organoids from pluripotent Xenopus cells. The mutation R295C retains reprogramming and inductive capacity but alters the expression of specific sets of downstream target genes instead of diminishing overall transcriptional activity of HNF1B. Surprisingly, targets associated with polycystic kidney disease were less affected than genes affected in congenital renal anomalies. Cross-species-conserved transcriptional targets were dysregulated in hnf1b CRISPR-depleted Xenopus embryos, confirming their dependence on hnf1b . CONCLUSIONS: HNF1B activates an evolutionarily conserved program of target genes that disease-causing mutations selectively disrupt. These findings provide insights into the renal transcriptional network that controls nephrogenesis.

Rates of repeated colonoscopies to clean the colon from low-risk and high-risk adenomas: results from the EPoS trials.

OBJECTIVE: High-quality colonoscopy (adequate bowel preparation, whole-colon visualisation and removal of all neoplastic polyps) is a prerequisite to start polyp surveillance, and is ideally achieved in one colonoscopy. In a large multinational polyp surveillance trial, we aimed to investigate clinical practice variation in number of colonoscopies needed to enrol patients with low-risk and high-risk adenomas in polyp surveillance. DESIGN: We retrieved data of all patients with low-risk adenomas (one or two tubular adenomas <10 mm with low-grade dysplasia) and high-risk adenomas (3-10 adenomas, ≥1 adenoma ≥10 mm, high-grade dysplasia or villous components) in the European Polyp Surveillance trials fulfilling certain logistic and methodologic criteria. We analysed variations in number of colonoscopies needed to achieve high-quality colonoscopy and enter polyp surveillance by endoscopy centre, and by endoscopists who enrolled ≥30 patients. RESULTS: The study comprised 15 581 patients from 38 endoscopy centres in five European countries; 6794 patients had low-risk and 8787 had high-risk adenomas. 961 patients (6.2%, 95% CI 5.8% to 6.6%) underwent two or more colonoscopies before surveillance began; 101 (1.5%, 95% CI 1.2% to 1.8%) in the low-risk group and 860 (9.8%, 95% CI 9.2% to 10.4%) in the high-risk group. Main reasons were poor bowel preparation (21.3%) or incomplete colonoscopy/polypectomy (14.4%) or planned second procedure (27.8%). Need of repeat colonoscopy varied between study centres ranging from 0% to 11.8% in low-risk adenoma patients and from 0% to 63.9% in high-risk adenoma patients. On the second colonoscopy, the two most common reasons for a repeat (third) colonoscopy were piecemeal resection (26.5%) and unspecified reason (23.9%). CONCLUSION: There is considerable practice variation in the number of colonoscopies performed to achieve complete polyp removal, indicating need for targeted quality improvement to reduce patient burden. TRIAL REGISTRATION NUMBER: NCT02319928.

Optimizing the Quality of Colorectal Cancer Screening Worldwide.

Screening, followed by colonoscopic polypectomy (or surgery for malignant lesions), prevents incident colorectal cancer and mortality. However, there are variations in effective application of nearly every aspect of the screening process. Screening is a multistep process, and failure in any single step could result in unnecessary morbidity and mortality. Awareness of variations in operator- and system-dependent performance has led to detailed, comprehensive recommendations in the United States and Europe on how colonoscopy screening should be performed and measured. Likewise, guidance has been provided on quality assurance for nonprimary colonoscopy-based screening programs, including strategies to maximize adherence. Quality improvement is now a validated science, and there is clear evidence that higher quality prevents incident cancer and cancer death. Quality must be addressed at the levels of the system, provider, and individuals, to maximize the benefits of screening for any population. We review the important aspects of measuring and improving the quality of colorectal cancer screening.

Mutations in transcription factor CP2-like 1 may cause a novel syndrome with distal renal tubulopathy in humans.

BACKGROUND: An underlying monogenic cause of early-onset chronic kidney disease (CKD) can be detected in ∼20% of individuals. For many etiologies of CKD manifesting before 25 years of age, >200 monogenic causative genes have been identified to date, leading to the elucidation of mechanisms of renal pathogenesis. METHODS: In 51 families with echogenic kidneys and CKD, we performed whole-exome sequencing to identify novel monogenic causes of CKD. RESULTS: We discovered a homozygous truncating mutation in the transcription factor gene transcription factor CP2-like 1 (TFCP2L1) in an Arabic patient of consanguineous descent. The patient developed CKD by the age of 2 months and had episodes of severe hypochloremic, hyponatremic and hypokalemic alkalosis, seizures, developmental delay and hypotonia together with cataracts. We found that TFCP2L1 was localized throughout kidney development particularly in the distal nephron. Interestingly, TFCP2L1 induced the growth and development of renal tubules from rat mesenchymal cells. Conversely, the deletion of TFCP2L1 in mice was previously shown to lead to reduced expression of renal cell markers including ion transporters and cell identity proteins expressed in different segments of the distal nephron. TFCP2L1 localized to the nucleus in HEK293T cells only upon coexpression with its paralog upstream-binding protein 1 (UBP1). A TFCP2L1 mutant complementary DNA (cDNA) clone that represented the patient's mutation failed to form homo- and heterodimers with UBP1, an essential step for its transcriptional activity. CONCLUSION: Here, we identified a loss-of-function TFCP2L1 mutation as a potential novel cause of CKD in childhood accompanied by a salt-losing tubulopathy.

Mutations in TBX18 Cause Dominant Urinary Tract Malformations via Transcriptional Dysregulation of Ureter Development.

Congenital anomalies of the kidneys and urinary tract (CAKUT) are the most common cause of chronic kidney disease in the first three decades of life. Identification of single-gene mutations that cause CAKUT permits the first insights into related disease mechanisms. However, for most cases the underlying defect remains elusive. We identified a kindred with an autosomal-dominant form of CAKUT with predominant ureteropelvic junction obstruction. By whole exome sequencing, we identified a heterozygous truncating mutation (c.1010delG) of T-Box transcription factor 18 (TBX18) in seven affected members of the large kindred. A screen of additional families with CAKUT identified three families harboring two heterozygous TBX18 mutations (c.1570C>T and c.487A>G). TBX18 is essential for developmental specification of the ureteric mesenchyme and ureteric smooth muscle cells. We found that all three TBX18 altered proteins still dimerized with the wild-type protein but had prolonged protein half life and exhibited reduced transcriptional repression activity compared to wild-type TBX18. The p.Lys163Glu substitution altered an amino acid residue critical for TBX18-DNA interaction, resulting in impaired TBX18-DNA binding. These data indicate that dominant-negative TBX18 mutations cause human CAKUT by interference with TBX18 transcriptional repression, thus implicating ureter smooth muscle cell development in the pathogenesis of human CAKUT.

A Dominant Mutation in Nuclear Receptor Interacting Protein 1 Causes Urinary Tract Malformations via Dysregulation of Retinoic Acid Signaling.

Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of CKD in the first three decades of life. However, for most patients with CAKUT, the causative mutation remains unknown. We identified a kindred with an autosomal dominant form of CAKUT. By whole-exome sequencing, we identified a heterozygous truncating mutation (c.279delG, p.Trp93fs*) of the nuclear receptor interacting protein 1 gene (NRIP1) in all seven affected members. NRIP1 encodes a nuclear receptor transcriptional cofactor that directly interacts with the retinoic acid receptors (RARs) to modulate retinoic acid transcriptional activity. Unlike wild-type NRIP1, the altered NRIP1 protein did not translocate to the nucleus, did not interact with RARα, and failed to inhibit retinoic acid-dependent transcriptional activity upon expression in HEK293 cells. Notably, we also showed that treatment with retinoic acid enhanced NRIP1 binding to RARα RNA in situ hybridization confirmed Nrip1 expression in the developing urogenital system of the mouse. In explant cultures of embryonic kidney rudiments, retinoic acid stimulated Nrip1 expression, whereas a pan-RAR antagonist strongly reduced it. Furthermore, mice heterozygous for a null allele of Nrip1 showed a CAKUT-spectrum phenotype. Finally, expression and knockdown experiments in Xenopus laevis confirmed an evolutionarily conserved role for NRIP1 in renal development. These data indicate that dominant NRIP1 mutations can cause CAKUT by interference with retinoic acid transcriptional signaling, shedding light on the well documented association between abnormal vitamin A levels and renal malformations in humans, and suggest a possible gene-environment pathomechanism in this disease.

Engineering kidney cells: reprogramming and directed differentiation to renal tissues.

Growing knowledge of how cell identity is determined at the molecular level has enabled the generation of diverse tissue types, including renal cells from pluripotent or somatic cells. Recently, several in vitro protocols involving either directed differentiation or transcription-factor-based reprogramming to kidney cells have been established. Embryonic stem cells or induced pluripotent stem cells can be guided towards a kidney fate by exposing them to combinations of growth factors or small molecules. Here, renal development is recapitulated in vitro resulting in kidney cells or organoids that show striking similarities to mammalian embryonic nephrons. In addition, culture conditions are also defined that allow the expansion of renal progenitor cells in vitro. Another route towards the generation of kidney cells is direct reprogramming. Key transcription factors are used to directly impose renal cell identity on somatic cells, thus circumventing the pluripotent stage. This complementary approach to stem-cell-based differentiation has been demonstrated to generate renal tubule cells and nephron progenitors. In-vitro-generated renal cells offer new opportunities for modelling inherited and acquired renal diseases on a patient-specific genetic background. These cells represent a potential source for developing novel models for kidney diseases, drug screening and nephrotoxicity testing and might represent the first steps towards kidney cell replacement therapies. In this review, we summarize current approaches for the generation of renal cells in vitro and discuss the advantages of each approach and their potential applications.

Design, synthesis and evaluation of indole-2-carboxamides with pan anti-mycobacterial activity.

Current treatment regimens for non-tuberculous mycobacteria (NTM) and tuberculosis (TB) generally require long duration of therapy with multiple drugs, some of which are broad spectrum antibiotics. Despite some advances in antimicrobial compounds, there remains a need in therapy for antibiotics with specific mycobacterial targets. It has been shown that MmpL3 is an essential transporter required for the translocation of mycolic acids to the mycobacterial cell envelope. Here, we synthesized a series of indole-2-carboxamides that inhibit MmpL3 and have potent pan-activity against mycobacterial species. The compounds were tested against several fast and slow-growing Mycobacterium species, including M. abscessus, M. massiliense, M. bolletii, M. chelonae, M. tuberculosis, M. avium, M. xenopi and M. smegmatis. The target of these indole-based compounds makes them selective for mycobacteria, while showing no clinically relevant bactericidal activity against S. aureus or P. aeruginosa. These compounds were tested against THP-1, a human-cell line, and showed minimal in vitro cytotoxicity and good selectivity indices. The data shown and discussed suggest that lead indole-2-carboxamides are strong contenders for further preclinical testing as NTM therapeutics.

Pandemic 2009 H1N1 influenza A virus carrying a Q136K mutation in the neuraminidase gene is resistant to zanamivir but exhibits reduced fitness in the guinea pig transmission model.

Resistance of influenza A viruses to neuraminidase inhibitors can arise through mutations in the neuraminidase (NA) gene. We show here that a Q136K mutation in the NA of the 2009 pandemic H1N1 virus confers a high degree of resistance to zanamivir. Resistance is accompanied by reduced numbers of NA molecules in viral particles and reduced intrinsic enzymatic activity of mutant NA. Interestingly, the Q136K mutation strongly impairs viral fitness in the guinea pig transmission model.

A Review of the Resuspension of Radioactively Contaminated Particles by Vehicle and Pedestrian Traffic-Current Theory, Practice, Gaps, and Needs.

ABSTRACT: The resuspension of radioactively contaminated particles in a built environment, such as from urban surfaces like foliage, building exteriors, and roadways, is described empirically by current plume and dosimetry models used for hazard assessment and long-term risk purposes. When applying these models to radiological contamination emergencies affecting urban areas, the accuracy of the results for recent contamination deposition is impacted in two main ways. First, the data supporting the underlying resuspension equations was acquired for open, quiescent conditions with no vehicle traffic or human activities, so it is not necessarily representative of the urban environment. Second, mechanical disturbance by winds in urban canyons and during emergency operations caused by vehicle traffic and human activities are not directly considered by the equations. Accordingly, plume and dosimetry models allow the user to input certain compensating values, but the models do not necessarily supply users instructions on what values to use. This manuscript reviews the available literature to comprehensively and consistently pool data for resuspension due to mechanically induced resuspension applicable to urban contamination. Because there are few studies that directly measured radioactive resuspension due to vehicles and pedestrians, this review novelly draws on a range of other studies involving non-radioactive particles, ranging from outdoor air pollution emissions to indoor allergen transport. The results lead to tabulated, recommended values for specific conditions in the emergency phase to help users of plume and dosimetry models maintain the conservativeness needed to properly capture the potential radiation dose posed by mechanically induced resuspension. These values are of benefit to model users until better data are available. The results also suggest the types of data that may result in improved plume and dose modeling.

Effects of aging of radioactive fallout on timely decontamination of concrete using low and mild pressure washing.

Timely decontamination will reduce the consequences of a radiological contamination event. For this purpose, pressure washing can be rapidly deployed, but its effectiveness will change if the interactions between the surface and radionuclides changes as the contamination "ages" under the influence of time and precipitation. While effects of this aging have been reported for dissolved cesium, they have not been studied for radionuclides present as particulate, e.g., fallout. This work studied the effects of aging on decontamination with low (<280 kPa/40 psi) and mild (14,000 kPa/2000 psi) pressure washing, on concrete contaminated with surrogate fallout consisting of soluble Cs-137, 0.5 µm silica particles, and 2 µm silica particles. The samples were aged up to 59 days (time between contamination and decontamination) with and without simulated precipitation. The percent removal following decontamination of the soluble cesium decreased over the first ten days of aging until the removals were less than 10 % for both low and mild pressure washing. The particle decontamination was independent of aging time but decontaminating via mild pressure washing (>80 % particle removal) significantly outperformed decontaminating by low pressure washing by flowing solution across (parallel to) the contaminated surface (<25 % particle removal). The observed changes in decontamination efficacy are explained via measurements of the penetration depth of contaminants. For soluble cesium, the results compared favorably with prior studies and theoretical treatment of cesium penetration, and they yielded additional insight into the effect of washing pressures on decontamination. There are no comparable studies for particulate contamination, so this study resulted in several novel observations which are operationally important for timely decontamination of surfaces following a radiological incident. It also suggests an evidence-based pressure washing procedure for timely decontamination of soluble and insoluble radionuclides which can be used throughout the emergency phase and into the early recovery phase.

Association of patient socioeconomic status with outcomes after palliative treatment for disseminated cancer.

BACKGROUND: Palliative treatment has been associated with improved quality of life and survival for a wide variety of metastatic cancers. However, it is unclear whether the benefits of palliative treatment are uniformly experienced across the US cancer population. We evaluated patterns and outcomes of palliative treatment based on socioeconomic, sociodemographic and treating facility characteristics. METHODS: Patients diagnosed between 2008 and 2019 with Stage IV primary cancer of nine organ sites were analyzed in the National Cancer Database. The association between identified variables, and outcomes concerning the administration of palliative treatment were analyzed with multivariable logistic regression and Cox proportional hazard models. RESULTS: Overall 238,995 (23.6%) of Stage IV patients received palliative treatment, which increased over time for all cancers (from 20.7% in 2008 to 25.6% in 2019). Palliative treatment utilization differed significantly by region (West less than Northeast, OR: 0.55 [0.54-0.56], p < 0.001) and insurance payer status (uninsured greater than private insurance, OR: 1.35 [1.32-1.39], p < 0.001). Black race and Hispanic ethnicity were also associated with lower rates of palliative treatment compared to White and non-Hispanics respectively (OR for Blacks: 0.91 [0.90-0.93], p < 0.001 and OR for Hispanics: 0.79 [0.77-0.81] p < 0.001). CONCLUSIONS: There are important differences in the utilization of palliative treatment across different populations in the United States. A better understanding of variability in palliative treatment use and outcomes may identify opportunities to improve informed decision making and optimize quality of care at the end-of-life.

Synthetic genetic circuits to uncover and enforce the OCT4 trajectories of successful reprogramming of human fibroblasts.

Reprogramming human fibroblasts to induced pluripotent stem cells (iPSCs) is inefficient, with heterogeneity among transcription factor (TF) trajectories driving divergent cell states. Nevertheless, the impact of TF dynamics on reprogramming efficiency remains uncharted. Here, we identify the successful reprogramming trajectories of the core pluripotency TF, OCT4, and design a genetic controller that enforces such trajectories with high precision. By combining a genetic circuit that generates a wide range of OCT4 trajectories with live-cell imaging, we track OCT4 trajectories with clonal resolution and find that a distinct constant OCT4 trajectory is required for colony formation. We then develop a synthetic genetic circuit that yields a tight OCT4 distribution around the identified trajectory and outperforms in terms of reprogramming efficiency other circuits that less accurately regulate OCT4. Our synthetic biology approach is generalizable for identifying and enforcing TF dynamics for cell fate programming applications.

Synthetic genetic circuits to uncover the OCT4 trajectories of successful reprogramming of human fibroblasts.

Reprogramming human fibroblasts to induced pluripotent stem cells (iPSCs) is inefficient, with heterogeneity among transcription factor (TF) trajectories driving divergent cell states. Nevertheless, the impact of TF dynamics on reprogramming efficiency remains uncharted. We develop a system that accurately reports OCT4 protein levels in live cells and use it to reveal the trajectories of OCT4 in successful reprogramming. Our system comprises a synthetic genetic circuit that leverages noise to generate a wide range of OCT4 trajectories and a microRNA targeting endogenous OCT4 to set total cellular OCT4 protein levels. By fusing OCT4 to a fluorescent protein, we are able to track OCT4 trajectories with clonal resolution via live-cell imaging. We discover that a supraphysiological, stable OCT4 level is required, but not sufficient, for efficient iPSC colony formation. Our synthetic genetic circuit design and high-throughput live-imaging pipeline are generalizable for investigating TF dynamics for other cell fate programming applications.

Plasmacytoid dendritic cells and Toll-like receptor 7-dependent signalling promote efficient protection of mice against highly virulent influenza A virus.

Types I and III interferons (IFNs) elicit protective antiviral immune responses during influenza virus infection. Although many cell types can synthesize IFN in response to virus infection, it remains unclear which IFN sources contribute to antiviral protection in vivo. We found that mice carrying functional alleles of the Mx1 influenza virus resistance gene partially lost resistance to infection with a highly pathogenic H7N7 influenza A virus strain if Toll-like receptor 7 (TLR7) signalling was compromised. This effect was achieved by deleting either the TLR7 gene or the gene encoding the TLR7 adaptor molecule MyD88. A similar decrease of influenza virus resistance was observed when animals were deprived of plasmacytoid dendritic cells (pDCs) at day 1 post-infection. Our results provide in vivo proof that pDCs contribute to the protection of the lung against influenza A virus infections, presumably via signals from TLR7.

The Cenozoic palaeoenvironment of the Arctic Ocean.

The history of the Arctic Ocean during the Cenozoic era (0-65 million years ago) is largely unknown from direct evidence. Here we present a Cenozoic palaeoceanographic record constructed from >400 m of sediment core from a recent drilling expedition to the Lomonosov ridge in the Arctic Ocean. Our record shows a palaeoenvironmental transition from a warm 'greenhouse' world, during the late Palaeocene and early Eocene epochs, to a colder 'icehouse' world influenced by sea ice and icebergs from the middle Eocene epoch to the present. For the most recent approximately 14 Myr, we find sedimentation rates of 1-2 cm per thousand years, in stark contrast to the substantially lower rates proposed in earlier studies; this record of the Neogene reveals cooling of the Arctic that was synchronous with the expansion of Greenland ice (approximately 3.2 Myr ago) and East Antarctic ice (approximately 14 Myr ago). We find evidence for the first occurrence of ice-rafted debris in the middle Eocene epoch (approximately 45 Myr ago), some 35 Myr earlier than previously thought; fresh surface waters were present at approximately 49 Myr ago, before the onset of ice-rafted debris. Also, the temperatures of surface waters during the Palaeocene/Eocene thermal maximum (approximately 55 Myr ago) appear to have been substantially warmer than previously estimated. The revised timing of the earliest Arctic cooling events coincides with those from Antarctica, supporting arguments for bipolar symmetry in climate change.

Logistics simulation of a remediation effort for a hypothetical radiological contamination scenario.

To mitigate the effects following a large-scale nuclear or radiological material release in an urban environment and to expedite recovery, the Integrated Wash-Aid Treatment Emergency Reuse System (IWATERS) was developed. IWATERS consists of three operations: washing contaminated surfaces with an ionic wash solution, collecting, and treating the contaminated wash solution on-site to remove contaminants, and reusing the treated solution throughout operations to preserve the clean water resource. This study develops a framework to simulate the logistics of IWATERS deployment, thereby gaining an understanding of the timeline for decontamination operations. For this purpose, the Analysis of Mobility Platform and GoldSim were leveraged for a hypothetical contamination scenario covering 65,200 m2 of an urban center. The framework reveals that remediation progress is limited by several resources, notably the availability of vermiculite, a reactive clay that is required to treat the contaminated wash solution. This study also presents how the simulation approach can be used to characterize alternatives to reduce the influence of limited resources on operational progress. Overall, this work lays the foundation for evaluating different decontamination methods through detailed logistics simulation, i.e., by refining simulation assumptions and expanding the range of scenarios the simulation can depict.

Optimization of the acetic acid method for microfossil extraction from lithified carbonate rocks: Examples from the Jurassic and Miocene limestones of Saudi Arabia.

An optimization experiment with different acid concentrations was carried out to assess the use of acid to minimum sustainable limits for the extraction of microfossils from indurated limestones. Two different limestone formations of Jurassic and Miocene ages were tested. Different concentrations of acid ranging from 50 to 100% and processing times varying from 2 to 10 h were tested for optimal recoveries. The acid residue recoveries show a similar trend for both formations. The weight percentage of residue with particle size >1 mm decreased as the acid concentration increased, especially in the 50-80% acid concentration range. On the other hand, the weight percentage of the smallest size particles > 0.063 mm increased as acid concentration increased. This means that the higher concentrations of acid dissolve more of the unnecessary large particles while the foraminifera, which comprise the sand fraction size, are left in the residue. Although higher acid concentrations with longer reaction times yielded better recoveries than with less reaction time, we recommended a 60% concentration of acetic acid and a reaction time of 10 h for optimal recovery of micropaleontological samples in Saudi Arabian carbonate rocks. By lowering the recommended concentration, the consumption of acid is reduced without compromising the recovery of microfossils.•Acetic acid leaching method is applied on two different age limestone samples to extract foraminifera.•Different concentrations of acetic acid are tried and tested, and consensus is made on an optimum concentration of 60% for a submersion time of 10 h.•The sample recoveries are optimal while using this concentration for a time of 10 h.

Nonregional Lymph Nodes as the Only Metastatic Site in Stage IV Esophageal Cancer.

Introduction: Metastatic involvement of at least one nonregional lymph node currently renders patients with esophageal cancer as having stage IV disease. However, the management and outcomes of patients whose sole determinant of stage IV status is nonregional lymph nodes (abbreviated as "stage IV-nodal" disease) have not been fully characterized. Methods: In this retrospective cohort study, the National Cancer Database was queried to identify patients 18 years of age or older who were diagnosed with stage IV esophageal cancer between 2016 and 2019. Survival was assessed by Kaplan-Meier analysis and Cox models in the overall sample and a propensity-matched sample. Patients with "stage IV-nodal" disease were compared with patients with systemic metastases involving a single organ or multiple organs. Results: Overall, 11,589 patients with clinical stage IV esophageal cancer were identified, including 1331 (11.5%) patients with stage IV-nodal disease. Patients with stage IV-nodal disease were more likely to receive chemotherapy (77%) than those with single systemic organ metastases (64%) and multiorgan metastases (63%) (p < 0.0001); patients with stage IV-nodal disease were also more likely to receive radiation (49%) than those with single systemic organ metastases (40%) and multiorgan metastases (39%) (p < 0.0001). Squamous cell carcinoma (OR = 1.58, 95% confidence interval [CI]: 1.34-1.86, p < 0.0001) and academic facility type (OR = 1.24, 95% CI: 1.09-1.4, p = 0.0009) were associated with higher likelihood of the stage IV-nodal presentation. Patients with stage IV-nodal disease experienced superior survival (hazard ratio = 0.72, 95% CI: 0.66-0.78, p < 0.0001) than those with stage IV-single systemic metastases (reference group) and stage IV-multiorgan disease (hazard ratio = 1.30, 95% CI: 1.24-1.37). Conclusions: Approximately 12% of patients with stage IV esophageal cancer lack systemic metastases at presentation. These patients with stage IV-nodal disease are more likely to receive treatment and experience superior survival. Further study of the stage IV-nodal population and consideration of a potential stage IV subclassification system is justified.