Short-term variation in leaf-level water use efficiency in a tropical forest.

The representation of stomatal regulation of transpiration and CO2 assimilation is key to forecasting terrestrial ecosystem responses to global change. Given its importance in determining the relationship between forest productivity and climate, accurate and mechanistic model representation of the relationship between stomatal conductance (gs ) and assimilation is crucial. We assess possible physiological and mechanistic controls on the estimation of the g1 (stomatal slope, inversely proportional to water use efficiency) and g0 (stomatal intercept) parameters, using diurnal gas exchange surveys and leaf-level response curves of six tropical broadleaf evergreen tree species. g1 estimated from ex situ response curves averaged 50% less than g1 estimated from survey data. While g0 and g1 varied between leaves of different phenological stages, the trend was not consistent among species. We identified a diurnal trend associated with g1 and g0 that significantly improved model projections of diurnal trends in transpiration. The accuracy of modeled gs can be improved by accounting for variation in stomatal behavior across diurnal periods, and between measurement approaches, rather than focusing on phenological variation in stomatal behavior. Additional investigation into the primary mechanisms responsible for diurnal variation in g1 will be required to account for this phenomenon in land-surface models.

The effect of the vertical gradients of photosynthetic parameters on the CO2 assimilation and transpiration of a Panamanian tropical forest.

Terrestrial biosphere models (TBMs) include the representation of vertical gradients in leaf traits associated with modeling photosynthesis, respiration, and stomatal conductance. However, model assumptions associated with these gradients have not been tested in complex tropical forest canopies. We compared TBM representation of the vertical gradients of key leaf traits with measurements made in a tropical forest in Panama and then quantified the impact of the observed gradients on simulated canopy-scale CO2 and water fluxes. Comparison between observed and TBM trait gradients showed divergence that impacted canopy-scale simulations of water vapor and CO2 exchange. Notably, the ratio between the dark respiration rate and the maximum carboxylation rate was lower near the ground than at the top-of-canopy, leaf-level water-use efficiency was markedly higher at the top-of-canopy, and the decrease in maximum carboxylation rate from the top-of-canopy to the ground was less than TBM assumptions. The representation of the gradients of leaf traits in TBMs is typically derived from measurements made within-individual plants, or, for some traits, assumed constant due to a lack of experimental data. Our work shows that these assumptions are not representative of the trait gradients observed in species-rich, complex tropical forests.

Leaves as bottlenecks: The contribution of tree leaves to hydraulic resistance within the soil-plant-atmosphere continuum.

Within vascular plants, the partitioning of hydraulic resistance along the soil-to-leaf continuum affects transpiration and its response to environmental conditions. In trees, the fractional contribution of leaf hydraulic resistance (Rleaf ) to total soil-to-leaf hydraulic resistance (Rtotal ), or fRleaf (=Rleaf /Rtotal ), is thought to be large, but this has not been tested comprehensively. We compiled a multibiome data set of fRleaf using new and previously published measurements of pressure differences within trees in situ. Across 80 samples, fRleaf averaged 0.51 (95% confidence interval [CI] = 0.46-0.57) and it declined with tree height. We also used the allometric relationship between field-based measurements of soil-to-leaf hydraulic conductance and laboratory-based measurements of leaf hydraulic conductance to compute the average fRleaf for 19 tree samples, which was 0.40 (95% CI = 0.29-0.56). The in situ technique produces a more accurate descriptor of fRleaf because it accounts for dynamic leaf hydraulic conductance. Both approaches demonstrate the outsized role of leaves in controlling tree hydrodynamics. A larger fRleaf may help stems from loss of hydraulic conductance. Thus, the decline in fRleaf with tree height would contribute to greater drought vulnerability in taller trees and potentially to their observed disproportionate drought mortality.

An improved representation of the relationship between photosynthesis and stomatal conductance leads to more stable estimation of conductance parameters and improves the goodness-of-fit across diverse data sets.

Stomata play a central role in surface-atmosphere exchange by controlling the flux of water and CO2 between the leaf and the atmosphere. Representation of stomatal conductance (gsw ) is therefore an essential component of models that seek to simulate water and CO2 exchange in plants and ecosystems. For given environmental conditions at the leaf surface (CO2 concentration and vapor pressure deficit or relative humidity), models typically assume a linear relationship between gsw and photosynthetic CO2 assimilation (A). However, measurement of leaf-level gsw response curves to changes in A are rare, particularly in the tropics, resulting in only limited data to evaluate this key assumption. Here, we measured the response of gsw and A to irradiance in six tropical species at different leaf phenological stages. We showed that the relationship between gsw and A was not linear, challenging the key assumption upon which optimality theory is based-that the marginal cost of water gain is constant. Our data showed that increasing A resulted in a small increase in gsw at low irradiance, but a much larger increase at high irradiance. We reformulated the popular Unified Stomatal Optimization (USO) model to account for this phenomenon and to enable consistent estimation of the key conductance parameters g0 and g1 . Our modification of the USO model improved the goodness-of-fit and reduced bias, enabling robust estimation of conductance parameters at any irradiance. In addition, our modification revealed previously undetectable relationships between the stomatal slope parameter g1 and other leaf traits. We also observed nonlinear behavior between A and gsw in independent data sets that included data collected from attached and detached leaves, and from plants grown at elevated CO2 concentration. We propose that this empirical modification of the USO model can improve the measurement of gsw parameters and the estimation of plant and ecosystem-scale water and CO2  fluxes.

Diagnostic Value of MAML2 Rearrangements in Mucoepidermoid Carcinoma.

Mucoepidermoid carcinoma (MEC) is often seen in salivary glands and can harbor MAML2 translocations (MAML2+). The translocation status has diagnostic utility as an objective confirmation of the MEC diagnosis, for example, when distinction from the more aggressive adenosquamous carcinoma (ASC) is not straightforward. To assess the diagnostic relevance of MAML2, we examined our 5-year experience in prospective testing of 8106 solid tumors using RNA-seq panel testing in combinations with a two-round Delphi-based scenario survey. The prevalence of MAML2+ across all tumors was 0.28% (n = 23/8106) and the majority of MAML2+ cases were found in head and neck tumors (78.3%), where the overall prevalence was 5.9% (n = 18/307). The sensitivity of MAML2 for MEC was 60% and most cases (80%) were submitted for diagnostic confirmation; in 24% of cases, the MAML2 results changed the working diagnosis. An independent survey of 15 experts showed relative importance indexes of 0.8 and 0.65 for "confirmatory MAML2 testing" in suspected MEC and ASC, respectively. Real-world evidence confirmed that the added value of MAML2 is a composite of an imperfect confirmation test for MEC and a highly specific exclusion tool for the diagnosis of ASC. Real-world evidence can help move a rare molecular-genetic biomarker from an emerging tool to the clinic.

Detection of the metabolic response to drought stress using hyperspectral reflectance.

Drought is the most important limitation on crop yield. Understanding and detecting drought stress in crops is vital for improving water use efficiency through effective breeding and management. Leaf reflectance spectroscopy offers a rapid, non-destructive alternative to traditional techniques for measuring plant traits involved in a drought response. We measured drought stress in six glasshouse-grown agronomic species using physiological, biochemical, and spectral data. In contrast to physiological traits, leaf metabolite concentrations revealed drought stress before it was visible to the naked eye. We used full-spectrum leaf reflectance data to predict metabolite concentrations using partial least-squares regression, with validation R2 values of 0.49-0.87. We show for the first time that spectroscopy may be used for the quantitative estimation of proline and abscisic acid, demonstrating the first use of hyperspectral data to detect a phytohormone. We used linear discriminant analysis and partial least squares discriminant analysis to differentiate between watered plants and those subjected to drought based on measured traits (accuracy: 71%) and raw spectral data (66%). Finally, we validated our glasshouse-developed models in an independent field trial. We demonstrate that spectroscopy can detect drought stress via underlying biochemical changes, before visual differences occur, representing a powerful advance for measuring limitations on yield.

A best-practice guide to predicting plant traits from leaf-level hyperspectral data using partial least squares regression.

Partial least squares regression (PLSR) modelling is a statistical technique for correlating datasets, and involves the fitting of a linear regression between two matrices. One application of PLSR enables leaf traits to be estimated from hyperspectral optical reflectance data, facilitating rapid, high-throughput, non-destructive plant phenotyping. This technique is of interest and importance in a wide range of contexts including crop breeding and ecosystem monitoring. The lack of a consensus in the literature on how to perform PLSR means that interpreting model results can be challenging, applying existing models to novel datasets can be impossible, and unknown or undisclosed assumptions can lead to incorrect or spurious predictions. We address this lack of consensus by proposing best practices for using PLSR to predict plant traits from leaf-level hyperspectral data, including a discussion of when PLSR is applicable, and recommendations for data collection. We provide a tutorial to demonstrate how to develop a PLSR model, in the form of an R script accompanying this manuscript. This practical guide will assist all those interpreting and using PLSR models to predict leaf traits from spectral data, and advocates for a unified approach to using PLSR for predicting traits from spectra in the plant sciences.

The response of stomatal conductance to seasonal drought in tropical forests.

Stomata regulate CO2 uptake for photosynthesis and water loss through transpiration. The approaches used to represent stomatal conductance (gs ) in models vary. In particular, current understanding of drivers of the variation in a key parameter in those models, the slope parameter (i.e. a measure of intrinsic plant water-use-efficiency), is still limited, particularly in the tropics. Here we collected diurnal measurements of leaf gas exchange and leaf water potential (Ψleaf ), and a suite of plant traits from the upper canopy of 15 tropical trees in two contrasting Panamanian forests throughout the dry season of the 2016 El Niño. The plant traits included wood density, leaf-mass-per-area (LMA), leaf carboxylation capacity (Vc,max ), leaf water content, the degree of isohydry, and predawn Ψleaf . We first investigated how the choice of four commonly used leaf-level gs models with and without the inclusion of Ψleaf as an additional predictor variable influence the ability to predict gs , and then explored the abiotic (i.e. month, site-month interaction) and biotic (i.e. tree-species-specific characteristics) drivers of slope parameter variation. Our results show that the inclusion of Ψleaf did not improve model performance and that the models that represent the response of gs to vapor pressure deficit performed better than corresponding models that respond to relative humidity. Within each gs model, we found large variation in the slope parameter, and this variation was attributable to the biotic driver, rather than abiotic drivers. We further investigated potential relationships between the slope parameter and the six available plant traits mentioned above, and found that only one trait, LMA, had a significant correlation with the slope parameter (R2  = 0.66, n = 15), highlighting a potential path towards improved model parameterization. This study advances understanding of gs dynamics over seasonal drought, and identifies a practical, trait-based approach to improve modeling of carbon and water exchange in tropical forests.

Chronic rhinosinusitis in elderly patients is associated with an exaggerated neutrophilic proinflammatory response to pathogenic bacteria.

BACKGROUND: Potential effects of aging on chronic rhinosinusitis (CRS) pathophysiology have not been well defined but might have important ramifications given a rapidly aging US and world population. OBJECTIVE: The goal of the current study was to determine whether advanced age is associated with specific inflammatory CRS endotypes or immune signatures. METHODS: Levels of 17 mucus cytokines and inflammatory mediators were measured in 147 patients with CRS. Hierarchical cluster analysis was used to identify and characterize inflammatory CRS endotypes, as well as to determine whether age was associated with specific immune signatures. RESULTS: A CRS endotype with a proinflammatory neutrophilic immune signature was enriched in older patients. In the overall cohort patients 60 years and older had increased mucus levels of IL-1ß, IL-6, IL-8, and TNF-α when compared with their younger counterparts. Increases in levels of proinflammatory cytokines were associated with both tissue neutrophilia and symptomatic bacterial infection/colonization in aged patients. CONCLUSIONS: Aged patients with CRS have a unique inflammatory signature that corresponds to a neutrophilic proinflammatory response. Neutrophil-driven inflammation in aged patients with CRS might be less likely to respond to corticosteroids and might be closely linked to chronic microbial infection or colonization.

Terrestrial biosphere models may overestimate Arctic CO2 assimilation if they do not account for decreased quantum yield and convexity at low temperature.

How terrestrial biosphere models (TBMs) represent leaf photosynthesis and its sensitivity to temperature are two critical components of understanding and predicting the response of the Arctic carbon cycle to global change. We measured the effect of temperature on the response of photosynthesis to irradiance in six Arctic plant species and determined the quantum yield of CO2 fixation ( ϕCO2 ) and the convexity factor (θ). We also determined leaf absorptance (α) from measured reflectance to calculate ϕCO2 on an absorbed light basis ( ϕCO2.a ) and enabled comparison with nine TBMs. The mean ϕCO2.a was 0.045 mol CO2  mol-1 absorbed quanta at 25°C and closely agreed with the mean TBM parameterisation (0.044), but as temperature decreased measured ϕCO2.a diverged from TBMs. At 5°C measured ϕCO2.a was markedly reduced (0.025) and 60% lower than TBM estimates. The θ also showed a significant reduction between 25°C and 5°C. At 5°C θ was 38% lower than the common model parameterisation of 0.7. These data show that TBMs are not accounting for observed reductions in ϕCO2.a and θ that can occur at low temperature. Ignoring these reductions in ϕCO2.a and θ could lead to a marked (45%) overestimation of CO2 assimilation at subsaturating irradiance and low temperature.

From the Arctic to the tropics: multibiome prediction of leaf mass per area using leaf reflectance.

Leaf mass per area (LMA) is a key plant trait, reflecting tradeoffs between leaf photosynthetic function, longevity, and structural investment. Capturing spatial and temporal variability in LMA has been a long-standing goal of ecological research and is an essential component for advancing Earth system models. Despite the substantial variation in LMA within and across Earth's biomes, an efficient, globally generalizable approach to predict LMA is still lacking. We explored the capacity to predict LMA from leaf spectra across much of the global LMA trait space, with values ranging from 17 to 393 g m-2 . Our dataset contained leaves from a wide range of biomes from the high Arctic to the tropics, included broad- and needleleaf species, and upper- and lower-canopy (i.e. sun and shade) growth environments. Here we demonstrate the capacity to rapidly estimate LMA using only spectral measurements across a wide range of species, leaf age and canopy position from diverse biomes. Our model captures LMA variability with high accuracy and low error (R2  = 0.89; root mean square error (RMSE) = 15.45 g m-2 ). Our finding highlights the fact that the leaf economics spectrum is mirrored by the leaf optical spectrum, paving the way for this technology to predict the diversity of LMA in ecosystems across global biomes.

Leaf reflectance spectroscopy captures variation in carboxylation capacity across species, canopy environment and leaf age in lowland moist tropical forests.

Understanding the pronounced seasonal and spatial variation in leaf carboxylation capacity (Vc,max ) is critical for determining terrestrial carbon cycling in tropical forests. However, an efficient and scalable approach for predicting Vc,max is still lacking. Here the ability of leaf spectroscopy for rapid estimation of Vc,max was tested. Vc,max was estimated using traditional gas exchange methods, and measured reflectance spectra and leaf age in leaves sampled from tropical forests in Panama and Brazil. These data were used to build a model to predict Vc,max from leaf spectra. The results demonstrated that leaf spectroscopy accurately predicts Vc,max of mature leaves in Panamanian tropical forests (R2  = 0.90). However, this single-age model required recalibration when applied to broader leaf demographic classes (i.e. immature leaves). Combined use of spectroscopy models for Vc,max and leaf age enabled construction of the Vc,max -age relationship solely from leaf spectra, which agreed with field observations. This suggests that the spectroscopy technique can capture the seasonal variability in Vc,max , assuming sufficient sampling across diverse species, leaf ages and canopy environments. This finding will aid development of remote sensing approaches that can be used to characterize Vc,max in moist tropical forests and enable an efficient means to parameterize and evaluate terrestrial biosphere models.

Homoeostatic maintenance of nonstructural carbohydrates during the 2015-2016 El Niño drought across a tropical forest precipitation gradient.

Nonstructural carbohydrates (NSCs) are essential for maintenance of plant metabolism and may be sensitive to short- and long-term climatic variation. NSC variation in moist tropical forests has rarely been studied, so regulation of NSCs in these systems is poorly understood. We measured foliar and branch NSC content in 23 tree species at three sites located across a large precipitation gradient in Panama during the 2015-2016 El Niño to examine how short- and long-term climatic variation impact carbohydrate dynamics. There was no significant difference in total NSCs as the drought progressed (leaf P = 0.32, branch P = 0.30) nor across the rainfall gradient (leaf P = 0.91, branch P = 0.96). Foliar soluble sugars decreased while starch increased over the duration of the dry period, suggesting greater partitioning of NSCs to storage than metabolism or transport as drought progressed. There was a large variation across species at all sites, but total foliar NSCs were positively correlated with leaf mass per area, whereas branch sugars were positively related to leaf temperature and negatively correlated with daily photosynthesis and wood density. The NSC homoeostasis across a wide range of conditions suggests that NSCs are an allocation priority in moist tropical forests.

Spectroscopy can predict key leaf traits associated with source-sink balance and carbon-nitrogen status.

Approaches that enable high-throughput, non-destructive measurement of plant traits are essential for programs seeking to improve crop yields through physiological breeding. However, many key traits still require measurement using slow, labor-intensive, and destructive approaches. We investigated the potential to retrieve key traits associated with leaf source-sink balance and carbon-nitrogen status from leaf optical properties. Structural and biochemical traits and leaf reflectance (500-2400 nm) of eight crop species were measured and used to develop predictive 'spectra-trait' models using partial least squares regression. Independent validation data demonstrated that the models achieved very high predictive power for C, N, C:N ratio, leaf mass per area, water content, and protein content (R2>0.85), good predictive capability for starch, sucrose, glucose, and free amino acids (R2=0.58-0.80), and some predictive capability for nitrate (R2=0.51) and fructose (R2=0.44). Our spectra-trait models were developed to cover the trait space associated with food or biofuel crop plants and can therefore be applied in a broad range of phenotyping studies.

Terrestrial biosphere models underestimate photosynthetic capacity and CO2 assimilation in the Arctic.

Terrestrial biosphere models (TBMs) are highly sensitive to model representation of photosynthesis, in particular the parameters maximum carboxylation rate and maximum electron transport rate at 25°C (Vc,max.25 and Jmax.25 , respectively). Many TBMs do not include representation of Arctic plants, and those that do rely on understanding and parameterization from temperate species. We measured photosynthetic CO2 response curves and leaf nitrogen (N) content in species representing the dominant vascular plant functional types found on the coastal tundra near Barrow, Alaska. The activation energies associated with the temperature response functions of Vc,max and Jmax were 17% lower than commonly used values. When scaled to 25°C, Vc,max.25 and Jmax.25 were two- to five-fold higher than the values used to parameterize current TBMs. This high photosynthetic capacity was attributable to a high leaf N content and the high fraction of N invested in Rubisco. Leaf-level modeling demonstrated that current parameterization of TBMs resulted in a two-fold underestimation of the capacity for leaf-level CO2 assimilation in Arctic vegetation. This study highlights the poor representation of Arctic photosynthesis in TBMs, and provides the critical data necessary to improve our ability to project the response of the Arctic to global environmental change.

Assessing the feasibility of a multimodal liquid biopsy for the diagnosis of HPV-associated oropharyngeal squamous cell carcinoma.

OBJECTIVES: In this feasibility study, we explored the combined use of circulating tumor human papillomavirus (HPV) DNA (ctHPVDNA) and HPV serology as diagnostic tests for HPV-associated oropharyngeal squamous cell carcinoma (OPSCC). METHODS: Among patients with research-banked serum or plasma at diagnosis, IgG antibodies to oncoproteins from HPV types 16, 18, 31, 33, 35, 45, 52, and 58 were detected with multiplex serology. Positivity for HPV 16 was defined based on detection of combinations of anti-E6, E1, E2, and E7 and for other high-risk types on detection of anti-E6 and anti-E7. Circulating tumor HPV DNA was detected by custom digital droplet polymerase chain reaction (ddPCR) assays for HPV types 16, 18, 33, 35, and 45. p16 immunohistochemistry and high-risk HPV RNA in situ hybridization (ISH) using a cocktail of 18 high-risk HPV types were performed on tissue. RESULTS: Of 75 patients, 67 (89.3%) were HPV-associated (p16 and HPV RNA ISH positive) and 8 (10.7%) were HPV-independent. All 8 HPV-independent patients were seronegative and negative for ctHPVDNA (100% specificity). Serology was positive in 53 (79.1%) of 67 HPV-associated patients, while ddPCR was positive for ctHPVDNA in 59 (88.6%) of 67 HPV-associated patients. Requiring both tests to be positive resulted in a sensitivity of 50 (74.6%) of 67 while combining assays (either positive) improved sensitivity to 62 (92.6%) of 67. CONCLUSIONS: Compared to HPV RNA ISH, HPV serology and ctHPVDNA are sensitive and highly specific biomarkers for HPV-associated OPSCC at the time of presentation.

Virtual 3D Specimen Mapping in Head & Neck Oncologic Surgery.

OBJECTIVES: Virtual 3D specimen mapping of oncologic surgical specimens provides a visual record of the specimen and margin sampling sites which can be utilized in a variety of cancer care settings. Our objective was to perform a retrospective review of head and neck surgical oncology cases where the specimen was mapped post-operatively and to evaluate the utility of these 3D specimen maps amongst the multidisciplinary cancer care team. METHODS: A retrospective review of our 3D specimen model biorepository was performed. Surgical specimens were 3D scanned and then graphically annotated (or "mapped") during routine pathologic processing. The resulting 3D specimen maps were distributed to the multidisciplinary oncologic care team. Final margin status and any use of the 3D specimen maps were recorded. RESULTS: A total of 28 cases were included. Virtual 3D specimen maps were utilized by the cancer care team in 8 cases (29%), including 2 positive margin cases, 2 close margin cases, and 4 indeterminate margin cases. 3D specimen maps were used to visualize positive margin sites for pathologist-surgeon communication as a visual reference during tumor board discussions and to inform radiation treatment planning. CONCLUSION: Post-operative virtual 3D specimen mapping of oncologic specimens creates a permanent visual record of the specimen and the margins sampled and may serve as a beneficial tool for communication amongst the multidisciplinary cancer care team. LEVEL OF EVIDENCE: 4 Laryngoscope, 134:191-197, 2024.

Patterns of Major Frozen Section Interpretation Error: An In-Depth Analysis From a Complex Academic Surgical Pathology Practice.

OBJECTIVES: To establish baseline error rates due to misinterpretation and to identify scenarios in which major errors were most common and potentially preventable. METHODS: Our database was queried over a 3-year period for major discrepancies due to misinterpretation. These were stratified by histomorphologic setting, service, availability/type of prior material, and years of experience and subspecialization of the interpreting pathologist. RESULTS: The overall discordance rate between frozen section (FS) and final diagnoses was 2.9% (199/6,910). Seventy-two errors were due to interpretation, of which 34 (47.2%) were major. Major error rates were highest on the gastrointestinal and thoracic services. Of major discrepancies, 82.4% were rendered in subdisciplines outside those of the FS pathologist. Pathologists with fewer than 10 years' experience made more errors than those with more experience (55.9% vs 23.5%, P = .006). Major error rates were greater for cases without previous material compared to those with a prior glass slide (47.1% vs 17.6%, P = .009). Common histomorphologic scenarios in which disagreements were made involved discriminating mesothelial cells from carcinoma (20.6%) and accurately recognizing squamous carcinoma/severe dysplasia (17.6%). CONCLUSIONS: To improve performance and decrease future misdiagnoses, monitoring discordances should be a continuous component of surgical pathology quality assurance programs.