3D genomic mapping reveals multifocality of human pancreatic precancers.

Pancreatic intraepithelial neoplasias (PanINs) are the most common precursors of pancreatic cancer, but their small size and inaccessibility in humans make them challenging to study1. Critically, the number, dimensions and connectivity of human PanINs remain largely unknown, precluding important insights into early cancer development. Here, we provide a microanatomical survey of human PanINs by analysing 46 large samples of grossly normal human pancreas with a machine-learning pipeline for quantitative 3D histological reconstruction at single-cell resolution. To elucidate genetic relationships between and within PanINs, we developed a workflow in which 3D modelling guides multi-region microdissection and targeted and whole-exome sequencing. From these samples, we calculated a mean burden of 13 PanINs per cm3 and extrapolated that the normal intact adult pancreas harbours hundreds of PanINs, almost all with oncogenic KRAS hotspot mutations. We found that most PanINs originate as independent clones with distinct somatic mutation profiles. Some spatially continuous PanINs were found to contain multiple KRAS mutations; computational and in situ analyses demonstrated that different KRAS mutations localize to distinct cell subpopulations within these neoplasms, indicating their polyclonal origins. The extensive multifocality and genetic heterogeneity of PanINs raises important questions about mechanisms that drive precancer initiation and confer differential progression risk in the human pancreas. This detailed 3D genomic mapping of molecular alterations in human PanINs provides an empirical foundation for early detection and rational interception of pancreatic cancer.

Conservative Treatments of Adolescent Idiopathic Scoliosis: Physical Therapists' Perspectives.

This study aimed to examine physical therapists' perspectives in conservative treatments of pediatric patients with adolescent idiopathic scoliosis (AIS). A cross-sectional survey design was used. A validated questionnaire was distributed to physical therapists, and the responses were analyzed. Preferred treatment frequency was 60 minutes (53.8%), twice weekly (41.5%), over 3 to 5 months (44.6%). Top 3 clinical interventions were core and trunk stability enhancement (90.8%), abdominal strengthening (83.1%), and postural correction (80.0%). Top 3 therapeutic goal-setting parameters were activity-based (78.5%), quality-of-life measure-based (56.9%), and participation-based (50.8%). The most common quality-of-life survey used was Oswestry low back pain disability questionnaire (15.6%) followed by Scoliosis Research Society-22 instrument (12.5%). According to our data, physical therapists believe that pediatric patients with AIS can benefit with addressing core and trunk stability, a 60-minute per session, twice weekly, over 3 to 5 months based on activity-based goal-setting and quality-of-life measures using Oswestry questionnaire.

Three-dimensional genomic mapping of human pancreatic tissue reveals striking multifocality and genetic heterogeneity in precancerous lesions.

Pancreatic intraepithelial neoplasia (PanIN) is a precursor to pancreatic cancer and represents a critical opportunity for cancer interception. However, the number, size, shape, and connectivity of PanINs in human pancreatic tissue samples are largely unknown. In this study, we quantitatively assessed human PanINs using CODA, a novel machine-learning pipeline for 3D image analysis that generates quantifiable models of large pieces of human pancreas with single-cell resolution. Using a cohort of 38 large slabs of grossly normal human pancreas from surgical resection specimens, we identified striking multifocality of PanINs, with a mean burden of 13 spatially separate PanINs per cm3 of sampled tissue. Extrapolating this burden to the entire pancreas suggested a median of approximately 1000 PanINs in an entire pancreas. In order to better understand the clonal relationships within and between PanINs, we developed a pipeline for CODA-guided multi-region genomic analysis of PanINs, including targeted and whole exome sequencing. Multi-region assessment of 37 PanINs from eight additional human pancreatic tissue slabs revealed that almost all PanINs contained hotspot mutations in the oncogene KRAS, but no gene other than KRAS was altered in more than 20% of the analyzed PanINs. PanINs contained a mean of 13 somatic mutations per region when analyzed by whole exome sequencing. The majority of analyzed PanINs originated from independent clonal events, with distinct somatic mutation profiles between PanINs in the same tissue slab. A subset of the analyzed PanINs contained multiple KRAS mutations, suggesting a polyclonal origin even in PanINs that are contiguous by rigorous 3D assessment. This study leverages a novel 3D genomic mapping approach to describe, for the first time, the spatial and genetic multifocality of human PanINs, providing important insights into the initiation and progression of pancreatic neoplasia.

The Bering Strait was flooded 10,000 years before the Last Glacial Maximum.

The cyclic growth and decay of continental ice sheets can be reconstructed from the history of global sea level. Sea level is relatively well constrained for the Last Glacial Maximum (LGM, 26,500 to 19,000 y ago, 26.5 to 19 ka) and the ensuing deglaciation. However, sea-level estimates for the period of ice-sheet growth before the LGM vary by > 60 m, an uncertainty comparable to the sea-level equivalent of the contemporary Antarctic Ice Sheet. Here, we constrain sea level prior to the LGM by reconstructing the flooding history of the shallow Bering Strait since 46 ka. Using a geochemical proxy of Pacific nutrient input to the Arctic Ocean, we find that the Bering Strait was flooded from the beginning of our records at 46 ka until [Formula: see text] ka. To match this flooding history, our sea-level model requires an ice history in which over 50% of the LGM's global peak ice volume grew after 46 ka. This finding implies that global ice volume and climate were not linearly coupled during the last ice age, with implications for the controls on each. Moreover, our results shorten the time window between the opening of the Bering Land Bridge and the arrival of humans in the Americas.

Influence of sample volume on nitrate N and O isotope ratio analyses with the denitrifier method.

RATIONALE: Analyses of the isotope ratios of nitrogen (15 N/14 N) and oxygen (18 O/16 O) in nitrate (NO3 - ) with the denitrifier method require relatively high sample volumes at low concentrations (≤1 µM) to afford sufficient analyte for mass spectrometry, resulting in isotopic offsets compared to more concentrated samples of the same isotopic composition. METHODS: To uncover the origins of isotopic offsets, we analyzed the N and O isotope ratios of NO3 - reference materials spanning concentrations of 0.5-20 µM. We substantiated the incidence of volume-dependent isotopic offsets, then investigated whether they resulted from (a) incomplete sample recovery during N2 O sparging, (b) blanks - bacterial, atmospheric, or in reference material solutions - and (c) oxygen atom exchange with water during the bacterial conversion of NO3 - to N2 O. RESULTS: Larger sample volumes resulted in modest offsets in δ15 N, but substantial offsets in δ18 O. N2 O recovery from sparging was less complete at higher volumes, resulting in decreases in δ15 N and δ18 O due to associated isotope fractionation. Blanks increased detectably with volume, whereas oxygen atom exchange with water remained constant within batch analyses, being sensitive to neither sample volume nor salinity. The sizeable offsets in δ18 O with volume are only partially explained by the factors considered in our analysis. CONCLUSIONS: Our observations argue for bracketing of NO3 - samples with reference materials that emulate sample volumes (concentrations) to achieve improved measurement accuracy and foster inter-comparability.

Validation of an Age-Appropriate Screening Tool for Female Athlete Triad and Relative Energy Deficiency in Sport in Young Athletes.

Background The purpose of this study was to determine the concurrent validity of a newly created relative energy deficiency in sport (RED-S) specific screening tool (RST) by comparing scores with the validated pre-participation gynecological examination (PPGE). We hypothesized that the investigators would observe no significant difference between the means of the RST and the PPGE survey. Methods This was a crossover study of 39 female subjects who completed both the RST and the PPGE. The survey order was randomized. Results The RST was validated compared with the PPGE (Pearson's r = 0.697, p < 0.001). Conclusion The administration of an RST to middle- and high-school female athletes was validated compared with the PPGE. Formatting limitations of the screening tool were highlighted, leading to changes that improved the accuracy of the screening tool prior to application in a clinical setting. The RST is an age-appropriate screening tool that can be used by coaches, athletic trainers, physical therapists, and other healthcare practitioners to detect RED-S risk and allow for earlier intervention.

The influence of sample matrix on the accuracy of nitrite N and O isotope ratio analyses with the azide method.

RATIONALE: The isotope ratios of nitrogen (15 N/14 N) and oxygen (18 O/16 O) in nitrite (NO2 - ) can be measured by conversion of the nitrite into nitrous oxide (N2 O) with azide, followed by mass spectrometric analysis of N2 O by gas chromatography isotope ratio mass spectrometry (GC/IRMS). While applying this method to brackish samples, we noticed that the N and O isotope ratio measurements of NO2 - are highly sensitive to sample salinity and to the pH at which samples are preserved. METHODS: We investigated the influence of sample salinity and sample preservation pH on the N and O isotope ratios of the N2 O produced from the reaction of NO2 - with azide. The N2 O isotope ratios were measured by GC/IRMS. RESULTS: Under the experimental reaction conditions, the conversion of NO2 - into N2 O was less complete in lower salinity solutions, resulting in respective N and O isotopic offsets of +2.5‰ and -14.0‰ compared with seawater solutions. Differences in salinity were also associated with differences in the fraction of O atoms exchanged between NO2 - and water during the reaction. Similarly, aqueous NO2 - samples preserved at elevated pH values resulted in the incomplete conversion of NO2 - into N2 O by azide, and consequent pH-dependent isotopic offsets, as well as differences in the fraction of O atoms exchanged with water. The addition of sodium chloride to the reaction matrix of samples and standards largely mitigated salinity-dependent isotopic offsets in the N2 O product, and nearly homogenized the fraction of O atom exchange among samples of different salinity. A test of the hypobromite-azide method to measure N isotope ratios of ammonium by conversion into NO2 - then N2 O revealed no influence of sample salinity on the N isotope ratios of the N2 O product. CONCLUSIONS: We outline recommendations to mitigate potential matrix effects among samples and standards, to improve the accuracy of N and O isotope ratios in NO2 - measured with the azide method.

Constraining the Oxygen Isotopic Composition of Nitrate Produced by Nitrification.

Measurements of the stable isotope ratios of nitrogen (15N/14N) and oxygen (18O/16O) in nitrate (NO3-) enable identification of sources, dispersal, and fate of natural and contaminant NO3- in aquatic environments. The 18O/16O of NO3- produced by nitrification is often assumed to reflect the proportional contribution of oxygen atom sources, water, and molecular oxygen, in a 2:1 ratio. Culture and seawater incubations, however, indicate oxygen isotopic equilibration between nitrite (NO2-) and water, and kinetic isotope effects for oxygen atom incorporation, which modulate the NO3- 18O/16O produced during nitrification. To investigate the influence of kinetic and equilibrium effects on the isotopic composition of NO3- produced from the nitrification of ammonia (NH3), we incubated streamwater supplemented with ammonium (NH4+) and increments of 18O-enriched water. Resulting NO3- 18O/16O ratios showed (1) a disproportionate sensitivity to the 18O/16O ratio of water, mediated by isotopic equilibration between water and NO2-, as well as (2) kinetic isotope discrimination during O atom incorporation from molecular oxygen and water. Empirically, the NO3- 18O/16O ratios thus produced fortuitously converge near the 18O/16O ratio of water. More elevated NO3- 18O/16O values commonly reported in soils and oxic groundwater may thus derive from processes additional to nitrification, including NO3- reduction.

Isotopic overprinting of nitrification on denitrification as a ubiquitous and unifying feature of environmental nitrogen cycling.

Natural abundance nitrogen and oxygen isotopes of nitrate (δ15NNO3 and δ18ONO3) provide an important tool for evaluating sources and transformations of natural and contaminant nitrate (NO3-) in the environment. Nevertheless, conventional interpretations of NO3- isotope distributions appear at odds with patterns emerging from studies of nitrifying and denitrifying bacterial cultures. To resolve this conundrum, we present results from a numerical model of NO3- isotope dynamics, demonstrating that deviations in δ18ONO3 vs. δ15NNO3 from a trajectory of 1 expected for denitrification are explained by isotopic over-printing from coincident NO3- production by nitrification and/or anammox. The analysis highlights two driving parameters: (i) the δ18O of ambient water and (ii) the relative flux of NO3- production under net denitrifying conditions, whether catalyzed aerobically or anaerobically. In agreement with existing analyses, dual isotopic trajectories >1, characteristic of marine denitrifying systems, arise predominantly under elevated rates of NO2- reoxidation relative to NO3- reduction (>50%) and in association with the elevated δ18O of seawater. This result specifically implicates aerobic nitrification as the dominant NO3- producing term in marine denitrifying systems, as stoichiometric constraints indicate anammox-based NO3- production cannot account for trajectories >1. In contrast, trajectories <1 comprise the majority of model solutions, with those representative of aquifer conditions requiring lower NO2- reoxidation fluxes (<15%) and the influence of the lower δ18O of freshwater. Accordingly, we suggest that widely observed δ18ONO3 vs. δ15NNO3 trends in freshwater systems (<1) must result from concurrent NO3- production by anammox in anoxic aquifers, a process that has been largely overlooked.

The contamination of commercial 15N2 gas stocks with 15N-labeled nitrate and ammonium and consequences for nitrogen fixation measurements.

We report on the contamination of commercial 15-nitrogen (15N) N2 gas stocks with 15N-enriched ammonium, nitrate and/or nitrite, and nitrous oxide. 15N2 gas is used to estimate N2 fixation rates from incubations of environmental samples by monitoring the incorporation of isotopically labeled 15N2 into organic matter. However, the microbial assimilation of bioavailable 15N-labeled N2 gas contaminants, nitrate, nitrite, and ammonium, is liable to lead to the inflation or false detection of N2 fixation rates. 15N2 gas procured from three major suppliers was analyzed for the presence of these 15N-contaminants. Substantial concentrations of 15N-contaminants were detected in four Sigma-Aldrich 15N2 lecture bottles from two discrete batch syntheses. Per mole of 15N2 gas, 34 to 1900 µmoles of 15N-ammonium, 1.8 to 420 µmoles of 15N-nitrate/nitrite, and ≥21 µmoles of 15N-nitrous oxide were detected. One 15N2 lecture bottle from Campro Scientific contained ≥11 µmoles of 15N-nitrous oxide per mole of 15N2 gas, and no detected 15N-nitrate/nitrite at the given experimental 15N2 tracer dilutions. Two Cambridge Isotopes lecture bottles from discrete batch syntheses contained ≥0.81 µmoles 15N-nitrous oxide per mole 15N2, and trace concentrations of 15N-ammonium and 15N-nitrate/nitrite. 15N2 gas equilibrated cultures of the green algae Dunaliella tertiolecta confirmed that the 15N-contaminants are assimilable. A finite-differencing model parameterized using oceanic field conditions typical of N2 fixation assays suggests that the degree of detected 15N-ammonium contamination could yield inferred N2 fixation rates ranging from undetectable, <0.01 nmoles N L(-1) d(-1), to 530 nmoles N L(-1) d(-1), contingent on experimental conditions. These rates are comparable to, or greater than, N2 fixation rates commonly detected in field assays. These results indicate that past reports of N2 fixation should be interpreted with caution, and demonstrate that the purity of commercial 15N2 gas must be ensured prior to use in future N2 fixation rate determinations.

Eukaryotic assimilatory nitrate reductase fractionates N and O isotopes with a ratio near unity.

In order to (i) establish the biological systematics necessary to interpret nitrogen (N) and oxygen (O) isotope ratios of nitrate ((15)N/(14)N and (18)O/(16)O) in the environment and (ii) investigate the potential for isotopes to elucidate the mechanism of a key N cycle enzyme, we measured the nitrate N and O isotope effects ((15)ε and (18)ε) for nitrate reduction by two assimilatory eukaryotic nitrate reductase (eukNR) enzymes. The (15)ε for purified extracts of NADPH eukNR from the fungus Aspergillus niger and the (15)ε for NADH eukNR from cell homogenates of the marine diatom Thalassiosira weissflogii were indistinguishable, yielding a mean (15)ε for the enzyme of 26.6 ± 0.2‰. Both forms of eukNR imparted near equivalent fractionation on N and O isotopes. The increase in (18)O/(16)O versus the increase in (15)N/(14)N (relative to their natural abundances) was 0.96 ± 0.01 for NADPH eukNR and 1.09 ± 0.03 for NADH eukNR. These results are the first reliable measurements of the coupled N and O isotope effects for any form of eukNR. They support the prevailing view that intracellular reduction by eukNR is the dominant step in isotope fractionation during nitrate assimilation and that it drives the (18)ε:(15)ε ≈ 1 observed in phytoplankton cultures, suggesting that this O-to-N isotope signature will apply broadly in the environment. Our measured (15)ε and (18)ε may represent the intrinsic isotope effects for eukNR-mediated N-O bond rupture, a potential constraint on the nature of the enzyme's transition state.

Removal of nitrite with sulfamic acid for nitrate N and O isotope analysis with the denitrifier method.

In environmental water samples that contain both nitrate (NO3-) and nitrite (NO2-), isotopic analysis of nitrate alone by all currently available methods requires pretreatment to remove nitrite. Sulfamic acid addition, used previously for this purpose (Wu JP, Calvert SE, Wong CS. Deep-Sea Research Part I - Oceanographic Research Papers 1997; 44: 287), is shown here to be compatible with the denitrifier method for both N and O isotope analysis of nitrate. Sulfamic acid at a pH of approximately 1.7 reduces nitrite to N2. Samples are then neutralized with base prior to isotope analysis, to alleviate the buffering demands of the bacterial media and as a precaution to prevent modification of nitrate during storage with the residual sulfamic acid at low pH. Under appropriate reaction conditions, nitrite is completely removed within minutes. Sulfamic acid treatment does not compromise the completeness of the conversion of nitrate into N2O or the precision and accuracy of N and O isotope measurements by the denitrifier method. Nitrite concentrations upwards of 7 times the ambient nitrate can be removed without affecting the isotope composition of nitrate. The method is applied to analyses of the coupled N and O isotopes of nitrate and nitrite in waters of the Mexican Margin, to illustrate its efficacy and utility when employed either in the field upon sample collection or in the lab after months of frozen sample storage.