Decline of a Foundation Tree Species Due to Invasive Insects Will Trigger Net Release of Soil Organic Carbon

Eastern U.S. forests are witnessing an ecologically disruptive decline in one of the region’s distinctive foundation tree species, the eastern hemlock (Tsuga canadenis). The exotic insect pests hemlock woolly adelgid (Adelges tsugae, HWA) and elongate hemlock scale (Fiorinia externa) have greatly altered many forest ecosystems previously dominated by this important evergreen conifer. The consequences for ecosystem processes are far reaching because hemlock is often replaced by deciduous tree species, such as black birch (Betula lenta), which have strongly divergent effects on forest floor microenvironments and nutrient cycling. We took advantage of an accidental experiment initiated by patch-level timber harvesting ~30 yr ago to investigate how the removal of hemlock, and its replacement by deciduous trees, has affected leaf litter characteristics, soil organic layer mass, C:N content, and soil respiration rates. We also contrasted these areas to nearby forest plots where deciduous B. lenta has been dominant for almost a century. The inclusion of healthy, intact hemlock stands in the design, and the close proximity of plots, allowed for a powerful space-for-time approach to detect ecosystem changes that are likely to occur across the broader landscape with HWA-induced hemlock loss in coming years. Three years of data collection from a series of plots in hemlock, young birch, and mature birch stands revealed dramatic differences in soil carbon pools and cycling. Between forests dominated by hemlock vs. mature birch, we saw a significant decrease in soil organic layer mass and in the C:N of the remaining organic material. Although hemlock and young birch stands showed no significant differences in soil respiration rates, mature birch stands had significantly higher soil respiration rates throughout the entire growing season, regardless of wet or dry years. Our results suggest that the carbon pool in the forest floor is likely to mobilize through greater decomposition with a 6.89 decline in soil organic layer C storage as hemlocks are replaced by deciduous trees, leading to a potential net release of ~4.5 tons C per hectare. We conclude that the ramifications of this change for carbon storage could be extensive, but may take decades to manifest

Spatial Characterization of Tumor-Infiltrating Lymphocytes and Breast Cancer Progression

Tumor-infiltrating lymphocytes (TILs) have been established as a robust prognostic biomarker in breast cancer, with emerging utility in predicting treatment response in the adjuvant and neoadjuvant settings. In this study, the role of TILs in predicting overall survival and progression-free interval was evaluated in two independent cohorts of breast cancer from the Cancer Genome Atlas (TCGA BRCA) and the Carolina Breast Cancer Study (UNC CBCS). We utilized machine learning and computer vision algorithms to characterize TIL infiltrates in digital whole-slide images (WSIs) of breast cancer stained with hematoxylin and eosin (H&E). Multiple parameters were used to characterize the global abundance and spatial features of TIL infiltrates. Univariate and multivariate analyses show that large aggregates of peritumoral and intratumoral TILs (forests) were associated with longer survival, whereas the absence of intratumoral TILs (deserts) is associated with increased risk of recurrence. Patients with two or more high-risk spatial features were associated with significantly shorter progression-free interval (PFI). This study demonstrates the practical utility of Pathomics in evaluating the clinical significance of the abundance and spatial patterns of distribution of TIL infiltrates as important biomarkers in breast cancer

Keratin 17 Modulates the Immune Topography of Pancreatic Cancer

BACKGROUND: The immune microenvironment impacts tumor growth, invasion, metastasis, and patient survival and may provide opportunities for therapeutic intervention in pancreatic ductal adenocarcinoma (PDAC). Although never studied as a potential modulator of the immune response in most cancers, Keratin 17 (K17), a biomarker of the most aggressive (basal) molecular subtype of PDAC, is intimately involved in the histogenesis of the immune response in psoriasis, basal cell carcinoma, and cervical squamous cell carcinoma. Thus, we hypothesized that K17 expression could also impact the immune cell response in PDAC, and that uncovering this relationship could provide insight to guide the development of immunotherapeutic opportunities to extend patient survival. METHODS: Multiplex immunohistochemistry (mIHC) and automated image analysis based on novel computational imaging technology were used to decipher the abundance and spatial distribution of T cells, macrophages, and tumor cells, relative to K17 expression in 235 PDACs. RESULTS: K17 expression had profound effects on the exclusion of intratumoral CD8+ T cells and was also associated with decreased numbers of peritumoral CD8+ T cells, CD16+ macrophages, and CD163+ macrophages (p \u3c 0.0001). The differences in the intratumor and peritumoral CD8+ T cell abundance were not impacted by neoadjuvant therapy, tumor stage, grade, lymph node status, histologic subtype, nor KRAS, p53, SMAD4, or CDKN2A mutations. CONCLUSIONS: Thus, K17 expression correlates with major differences in the immune microenvironment that are independent of any tested clinicopathologic or tumor intrinsic variables, suggesting that targeting K17-mediated immune effects on the immune system could restore the innate immunologic response to PDAC and might provide novel opportunities to restore immunotherapeutic approaches for this most deadly form of cancer

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Loss of a Foundation Species, Eastern Hemlock (Tsuga canadensis), May Lead to Biotic Homogenization of Fungal Communities and Altered Bacterial Abundance in the Forest Floor

Tsuga canadensis (Eastern Hemlock) is a key forest foundation species that is currently declining across the eastern US due to attack by exotic insect species, primarily Adelges tsugae (Hemlock Woolly Adelgid). In the northeastern US, declining Eastern Hemlock stands are typically replaced by fast-growing deciduous Betula lenta (Black Birch) trees, altering ecosystem processes and ecological communities. In this study, we used an approach that substituted space for time to explore how the forest floor\u27s soil organic horizon, macrofungal communities, and bacterial abundance might be altered following Eastern Hemlock replacement by Black Birch. We compared intact, mature Eastern Hemlock forest plots at 2 sites in western Massachusetts to adjacent areas of vigorous regeneration of young Black Birch triggered by logging activity at the sites ∼25-30 years ago. Forest-floor soil organic horizons were significantly thicker under Eastern Hemlock forests and exhibited a higher carbon:nitrogen (C:N) ratio, suggesting slower decomposition and greater accumulation of organic material under Eastern Hemlock compared to deciduous Black Birch canopies. Macrofungal communities emerging from the forest floor did not differ strongly in morphospecies richness between Eastern Hemlock and Black Birch plots. However, a greater number of rare fungi taxa, defined in this study as those represented by a single observation, were detected in Eastern Hemlock plots, and the composition of Black Birch plots was more homogenized and less variable. The abundance of bacterial colony forming units (CFUs) in the soil organic horizon appeared to follow a seasonal pattern of variation between mature Eastern Hemlock versus young Black Birch plots; CFUs were most abundant in Black Birch soils in the fall, potentially tracking input of new deciduous leaf litter, whereas Eastern Hemlock plots had higher CFU counts in the summer. The results of this study suggest that forest-floor characteristics will be substantially altered by the impending loss of Eastern Hemlock, associated macrofungal communities may become simplified and homogenized, and the timing of peak bacterial abundance in the forest floor might be shifted to the fall

Hyperbaric Oxygen Promotes Proximal Bone Regeneration and Organized Collagen Composition during Digit Regeneration

<div><p>Oxygen is critical for optimal bone regeneration. While axolotls and salamanders have retained the ability to regenerate whole limbs, mammalian regeneration is restricted to the distal tip of the digit (P3) in mice, primates, and humans. Our previous study revealed the oxygen microenvironment during regeneration is dynamic and temporally influential in building and degrading bone. Given that regeneration is dependent on a dynamic and changing oxygen environment, a better understanding of the effects of oxygen during wounding, scarring, and regeneration, and better ways to artificially generate both hypoxic and oxygen replete microenvironments are essential to promote regeneration beyond wounding or scarring. To explore the influence of increased oxygen on digit regeneration <i>in vivo</i> daily treatments of hyperbaric oxygen were administered to mice during all phases of the entire regenerative process. Micro-Computed Tomography (μCT) and histological analysis showed that the daily application of hyperbaric oxygen elicited the same enhanced bone degradation response as two individual pulses of oxygen applied during the blastema phase. We expand past these findings to show histologically that the continuous application of hyperbaric oxygen during digit regeneration results in delayed blastema formation at a much more proximal location after amputation, and the deposition of better organized collagen fibers during bone formation. The application of sustained hyperbaric oxygen also delays wound closure and enhances bone degradation after digit amputation. Thus, hyperbaric oxygen shows the potential for positive influential control on the various phases of an epimorphic regenerative response.</p></div

Coordinated single-cell tumor microenvironment dynamics reinforce pancreatic cancer subtype

Abstract Bulk analyses of pancreatic ductal adenocarcinoma (PDAC) samples are complicated by the tumor microenvironment (TME), i.e. signals from fibroblasts, endocrine, exocrine, and immune cells. Despite this, we and others have established tumor and stroma subtypes with prognostic significance. However, understanding of underlying signals driving distinct immune and stromal landscapes is still incomplete. Here we integrate 92 single cell RNA-seq samples from seven independent studies to build a reproducible PDAC atlas with a focus on tumor-TME interdependence. Patients with activated stroma are synonymous with higher myofibroblastic and immunogenic fibroblasts, and furthermore show increased M2-like macrophages and regulatory T-cells. Contrastingly, patients with ‘normal’ stroma show M1-like recruitment, elevated effector and exhausted T-cells. To aid interoperability of future studies, we provide a pretrained cell type classifier and an atlas of subtype-based signaling factors that we also validate in mouse data. Ultimately, this work leverages the heterogeneity among single-cell studies to create a comprehensive view of the orchestra of signaling interactions governing PDAC