Updated techniques and evidence for endoscopic ultrasound-guided tissue acquisition from solid pancreatic lesions.

Endoscopic ultrasound-guided tissue acquisition (EUS-TA), including fine-needle aspiration (EUS-FNA) and fine-needle biopsy (EUS-FNB), has revolutionized specimen collection from intra-abdominal organs, especially the pancreas. Advances in personalized medicine and more precise treatment have increased demands to collect specimens with higher cell counts, while preserving tissue structure, leading to the development of EUS-FNB needles. EUS-FNB has generally replaced EUS-FNA as the procedure of choice for EUS-TA of pancreatic cancer. Various techniques have been tested for their ability to enhance the diagnostic performance of EUS-TA, including multiple methods of sampling at the time of puncture, on-site specimen evaluation, and specimen processing. In addition, advances in next-generation sequencing have made comprehensive genomic profiling of EUS-TA samples feasible in routine clinical practice. The present review describes updates in EUS-TA sampling techniques of pancreatic lesions, as well as methods for their evaluation.

Parkinson's, where are we heading?

The prevalence of Parkinson's disease has rapidly increased over the last decade. This editorial discusses our current understanding of the pathophysiological basis for the condition, with a particular focus on the potential role of α-synuclein, and the consequent implications this has for both the development of new investigations and disease-modifying therapies. Specifically, the article discusses the development of a new diagnostic test for cerebrospinal fluid α-synuclein, the development of a new staging system for Parkinson's disease, which takes into account the α-synuclein, genetic and neuro-imaging status, and the results of two recently completed clinical trials, using monoclonal antibodies wherein α-synuclein is the principal target. We also discuss the increasing awareness of the importance of non-motor symptoms in Parkinson's disease including hyposmia, rapid eye movement sleep behaviour disorder, and autonomic and cognitive symptoms.

Optimizing forage harvest and the nutritive value of Italian ryegrass-based mixed forage cropping under northwestern Himalayan conditions.

The scarcity of high-quality forage has a significant influence on the productivity and profitability of livestock. Addressing this concern, an investigation was undertaken to assess the effects of distinct Italian ryegrass genotypes, namely, Punjab ryegrass-1, Kashmir collection, and Makhan grass, in conjunction with varying seeding ratios of Italian ryegrass to Egyptian clover. The seeding ratios considered were 100:0 (Italian ryegrass to Egyptian clover), 75:25, 50:50, and 25:75. All possible combinations of Italian ryegrass and Egyptian clover with seeding ratios were set up in a randomized complete block design and replicated thrice. Co-cultivating Italian ryegrass and Egyptian clover at a 75:25 seeding ratio yields the best yield benefit, as determined by the land equivalent ratio. It is noteworthy that in this configuration, real yield loss is higher for Egyptian clover and for Italian ryegrass when the seeding ratio is 25:75. The higher competitiveness of Italian ryegrass in comparison to Egyptian clover is highlighted by the competitive ratio. Notably, the nutritive parameter, crude protein yield, was significantly higher in the Makhan grass-based 50:50 and 75:25 seeding ratio. Results of the study ascertained the compatibility of grass-legume co-cultivation with significantly higher quantity and quality forage harvested under mixed cropping systems whereas Makhan grass as the superior and dominant genotype in comparison to Kashmir collection. The outcomes of this study revealed that the 100:0 seeding ratio, coupled with the Makhan grass genotype, exhibited superior performance in terms of cumulative forage harvest, dry matter accumulation, net returns, and benefit-cost ratio.

Intracranial seeding of pituitary neuroendocrine tumor: a case report.

Background: Pituitary neuroendocrine tumors (PitNETs) are predominantly benign, though a minority may exhibit invasive tendencies. A diagnosis of metastatic PitNETs, in the absence of malignant histology, hinges on the identification of craniospinal and/or systemic metastases. Only a minority of PitNETs exhibit intracranial seeding. Notably, craniotomy for PitNETs excision is a prominent catalyst for iatrogenic seeding. Case Description: This article presented a compelling case that 15 years following craniotomy for the resection of a somatotroph PitNET, a lesion emerged at the left frontal base within the ethmoid sinus. Subsequent post-operative pathology unveiled a mature plurihormonal pituitary specific transcription factor 1 (PIT-1)-lineage PitNET. Growth hormone (GH) levels decreased significantly from 22.8 ng/mL pre-operation to 2 ng/mL post-operative, and concurrently, prolactin (PRL) levels decreased from 26.7 ng/mL pre-operation to 4.5 ng/mL post-operation. Furthermore, in the follow-up examination conducted 5 months after the operation, both GH and PRL levels were found to be within the normal range for the patient. This robustly suggested that the initial surgical procedure played a key role in the development of the lesion. Conclusions: This underscores the paramount significance of strictly adhering to the non-tumor removal during craniotomy for PitNETs excision. Regardless of apparent complete resection on imaging, it remains imperative to conduct routine follow-up evaluations, encompassing both imaging studies and hormone level assessments.

Optimum seeding density and seedling age for the outstanding yield performance of Japonica rice using crop straw boards for seedling cultivation.

Crop straw boards, a novel nursery material, has proven effective for cultivating dense, young rice seedlings suitable for mechanized transplanting, thereby saving labor. However, under high-density nursery conditions, the biomass accumulation and yield formation in rice vary with different seedling ages, necessitating exploration of optimal seeding densities and seedling ages to achieve high yields. This study aims to determine the appropriate seeding densities and seedling ages using crop straw boards to maximize rice yield. Over two years, field studies were conducted using crop straw boards for rice cultivation at seeding densities of 150, 200, 250, 300, and 350 g/tray (labeled as D1, D2, D3, D4, and D5) and seedling ages of 10, 15, 20, and 25 days (labeled as A1, A2, A3, and A4).The results indicated that D4A2 significantly enhanced tiller number, dry matter accumulation, and photosynthetic capacity, resulting in a yield increase of 2.89% compared to the conventional method of D1A3. High-density and short-aged seedlings cultivated with crop straw boards can enhance rice yield by improving photosynthetic capacity and crop quality. This study emphasizes the importance of using crop straw boards for rice nursery practices, as well as selecting the appropriate seeding densities and seedling ages for optimizing rice production.

A Novel Method for the Early Detection of Single Circulating, Metastatic and Self-Seeding Cancer Cells in Orthotopic Breast Cancer Mouse Models.

BACKGROUND: Metastasis is the main cause of cancer-related deaths, but efficient targeted therapies against metastasis are still missing. Major gaps exist in our understanding of the metastatic cascade, as existing methods cannot combine sensitivity, robustness, and practicality to dissect cancer progression. Addressing this issue requires improved strategies to distinguish early metastatic colonization from metastatic outgrowth. METHODS: Luciferase-labelled MDA-MB-231, MCF7, and 4T1 breast cancer cells were spiked into samples from tumour-naïve mice to establish the limit of detection for disseminated tumour cells. Luciferase-labelled breast cancer cells (±unlabelled cancer-associated fibroblasts; CAFs) were orthotopically implanted in immunocompromised mice. An ex vivo luciferase assay was used to quantify tumour cell dissemination. RESULTS: In vitro luciferase assay confirmed a linear and positive correlation between cancer cell numbers and the bioluminescence detected at single cell level in blood, brain, lung, liver, and mammary fat pad samples. Remarkably, single luciferase-labelled cancer cells were detectable in all of these sites, as the bioluminescence quantified in the analysed samples was substantially higher than background levels. Ex vivo, circulating tumour cells, metastasis, and tumour self-seeding were detected in all samples from animals implanted with highly metastatic luciferase-labelled MDA-MB-231 cells. In turn, detection of poorly metastatic luciferase-labelled MCF7 cells was scarce but significantly enhanced upon co-implantation with CAFs as early as 20 days after the experiment was initiated. CONCLUSIONS: These results demonstrate the feasibility of using an ultrasensitive luciferase-based method to dissect the mechanisms of early metastatic colonization to improving the development of antimetastatic therapies.

Intracellular tau fragment droplets serve as seeds for tau fibrils.

Intracellular tau aggregation requires a local protein concentration increase, referred to as "droplets". However, the cellular mechanism for droplet formation is poorly understood. Here, we expressed OptoTau, a P301L mutant tau fused with CRY2olig, a light-sensitive protein that can form homo-oligomers. Under blue light exposure, OptoTau increased tau phosphorylation and was sequestered in aggresomes. Suppressing aggresome formation by nocodazole formed tau granular clusters in the cytoplasm. The granular clusters disappeared by discontinuing blue light exposure or 1,6-hexanediol treatment suggesting that intracellular tau droplet formation requires microtubule collapse. Expressing OptoTau-ΔN, a species of N-terminal cleaved tau observed in the Alzheimer's disease brain, formed 1,6-hexanediol and detergent-resistant tau clusters in the cytoplasm with blue light stimulation. These intracellular stable tau clusters acted as a seed for tau fibrils in vitro. These results suggest that tau droplet formation and N-terminal cleavage are necessary for neurofibrillary tangles formation in neurodegenerative diseases.

Human peritoneal fluid exerts ovulation- and nonovulation-sourced oncogenic activities on transforming fallopian tube epithelial cells.

Secretory cells in the fallopian tube fimbria epithelium (FTE) are regarded as the main cells of origin of ovarian high-grade serous carcinoma (HGSC). Ovulation is the main cause of FTE oncogenesis, which proceeds through a sequence of TP53 mutations, chromosomal instability due to Rb/cyclin E aberration, in situ carcinoma (STIC), and metastasis to the ovary and peritoneum (metastatic HGSC). Previously, we have identified multiple oncogenic activities of the ovulatory follicular fluid (FF), which exerts the full spectrum of transforming activity on FTE cells at different stages of transformation. After ovulation, the FF is transfused into the peritoneal fluid (PF), in which the FTE constantly bathes. We wondered whether PF exerts the same spectrum of oncogenic activities as done by FF and whether these activities are derived from FF. By using a panel of FTE cell lines with p53 mutation (FT282-V), p53/CCNE1 aberrations (FT282-CCNE1), and p53/Rb aberrations plus spontaneous transformation, and peritoneal metastasis (FEXT2), we analyzed the changes of different transformation phenotypes after treating with FF and PF collected before or after ovulation. Similar to effects exhibited by FF, we found that, to a lesser extent, PF promoted anchorage-independent growth (AIG), migration, anoikis resistance, and peritoneal attachment in transforming FTE cells. The more transformed cells were typically more affected. Among the transforming activities exhibited by PF treatment, AIG, Matrigel invasion, and peritoneal attachment growth were higher with luteal-phase PF treatment than with the proliferative-phase PF treatment, suggesting an ovulation source. In contrast, changes in anoikis resistance and migration activities were similar in response to treatment with PF collected before and after ovulation, suggesting an ovulation-independent source. The overall transforming activity of luteal-phase PF was verified in an i.p. co-injection xenograft mouse model. Co-injection of Luc-FEXT2 cells with either FF or luteal-phase PF supported early peritoneal implantation, whereas co-injection with follicular-phase PF did not. This study, for the first time, demonstrates that PF from ovulating women can promote different oncogenic phenotypes in FTE cells at different stages of malignant transformation. Most of these activities, other than anoikis resistance and cell migration, are sourced from ovulation.

Tail-dependence of masting synchrony results in continent-wide seed scarcity.

Spatial synchrony may be tail-dependent, meaning it is stronger for peaks rather than troughs, or vice versa. High interannual variation in seed production in perennial plants, called masting, can be synchronized at subcontinental scales, triggering extensive resource pulses or famines. We used data from 99 populations of European beech (Fagus sylvatica) to examine whether masting synchrony differs between mast peaks and years of seed scarcity. Our results revealed that seed scarcity occurs simultaneously across the majority of the species range, extending to populations separated by distances up to 1800 km. Mast peaks were spatially synchronized at distances up to 1000 km and synchrony was geographically concentrated in northeastern Europe. Extensive synchrony in the masting lower tail means that famines caused by beech seed scarcity are amplified by their extensive spatial synchrony, with diverse consequences for food web functioning and climate change biology.

A North American climate-masting-irruption teleconnection and its change under future climate.

Atmospheric variability can impact biological populations by triggering facultative migrations, but the stability of these atmosphere-biosphere connections may be vulnerable to climate change. As an example, we consider the leading mode of continental-scale facultative migration of Pine Siskins, where the associated ecological mechanism is changes in resource availability, with a mechanistic pathway of climate conditions affecting mast seeding patterns in trees which in turn drive bird migration. The three summers prior to pine siskin irruption feature an alternating west-east mast-seeding dipole in conifer trees with opposite anomalies over western and eastern North America. The climate driver of this west-east mast-seeding dipole, referred to as the North American Dipole, occurs during summer in the historical record, but shifts to spring in response to future climate warming during this century in a majority of global climate models. Identification of future changes in the timing of the climate driver of boreal forest mast seeding have broadly important implications for the dynamics of forest ecosystems.

Enhancing aromatic rice production through agronomic and nutritional management for improved yield and quality.

To meet the growing international demand for aromatic rice, this study, conducted at Uttar Banga Krishi Viswavidyalaya in Cooch Behar, West Bengal, aimed to enhance the yield and quality of the 'Tulaipanji' rice cultivar through advanced establishment methods and the use of organic nutrients over two years. The research tested three planting techniques: mechanical transplanting, wet direct seeding (using a drum seeder), and traditional methods, alongside four nutrient management strategies: vermicompost, farmyard manure, a mix of both, and conventional fertilizers. Findings revealed that mechanical transplanting significantly increased yield by over 31.98% and 71.05% compared to traditional methods and wet direct seeding, respectively. Using vermicompost alone as a nutrient source not only boosted yields by 21.31% over conventional fertilizers but also enhanced the rice's nutritional value and cooking quality. Moreover, soils treated with vermicompost showed higher dehydrogenase activity, indicating better soil health. Economically, mechanical transplanting with vermicompost was the most beneficial, yielding the highest net returns and benefit-cost ratios in both years studied. This approach presents a viable model for improving the sustainability of aromatic rice production globally, emphasizing the economic and environmental advantages of adopting mechanical planting techniques and organic fertilization methods.

Identification and characterization of TM4SF1+ tumor self-seeded cells.

Tumor self-seeding is a process whereby circulating tumor cells (CTCs) recolonize the primary tumor, which promotes tumor growth, angiogenesis, and invasion. However, the detailed nature and functions of tumor self-seeded cells (TSCs) have not been well defined due to challenges in tracking and isolating TSCs. Here, we report an accurate animal model using photoconvertible tagging to recapitulate the spontaneous process of tumor self-seeding and identify TSCs as a subpopulation of primary tumor cells with enhanced invasiveness and survival. We demonstrate transmembrane-4-L-six-family-1 (TM4SF1) as a marker of TSCs, which promotes migration, invasion, and anchorage-independent survival in cancer cells. By analyzing single-cell RNA sequencing datasets, we identify a potential TSC population with a metastatic profile in patients with cancer, which is detectable in early-stage disease and expands during cancer progression. In summary, we establish a framework to study TSCs and identify emerging cell targets with diagnostic, prognostic, or therapeutic potential in cancers.

Skin α-Synuclein Seeding Activity in Patients with Type 1 Gaucher Disease.

BACKGROUND: Patients with type 1 Gaucher disease (GD1) have a significantly increased risk of developing Parkinson's disease (PD). OBJECTIVE: The objective of this study was to evaluate skin α-synuclein (αSyn) seeding activity as a biomarker for GD1-related PD (GD1-PD). METHODS: This single-center study administered motor and cognitive examinations and questionnaires of nonmotor symptoms to adult patients with GD1. Optional skin biopsy was performed for skin αSyn seed amplification assay (αSyn SAA) using real-time quaking-induced conversion assay. RESULTS: Forty-nine patients were enrolled, and 36 underwent skin biopsy. Two study participants had PD. Ten participants were αSyn SAA positive (27.8%), 7 (19.4%) were intermediate, and 19 (52.8%) were negative. Positive αSyn seeding activity was observed in the single GD1-PD case who consented to biopsy. αSyn SAA positivity was associated with older age (p = 0.043), although αSyn SAA positivity was more prevalent in patients with GD1 than historic controls. CONCLUSIONS: Longitudinal follow-up is required to determine whether skin αSyn seeding activity can be an early biomarker for GD1-PD. © 2024 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Mast seeding is stronger in taller plants.

Introduction: Two economies of scale, predator satiation and pollination efficiency, have been proposed to explain the evolutionary mechanisms of mast seeding adopted by some long-lived plants. Plant height is strongly selected by pollination vectors and may also provide economies of scale; however, it remains unknown whether there is a close relationship between adult plant height and mast seeding intensity. Methods: Here, we analyzed mast seeding intensity of 158 plant species to test if adult plant height can select for mast seeding. Results: We show that mast seeding intensities are higher in taller plant species irrespective of phylogeny, life form, pollination vector, and type of Spermatophytes. We also show that anemophily rather than entomophily selects for taller plant species and higher mast seeding intensities. Discussion: The linear correlations and evolutionary links between adult plant height and mast seeding intensity provide evidence that mast seeding could have evolved as an adaptation to taller strategy of perennial plant species.

Ultrasensitive detection of aggregated α-synuclein using quiescent seed amplification assay for the diagnosis of Parkinson's disease.

BACKGROUND: Seed amplification assays (SAA) enable the amplification of pathological misfolded proteins, including α-synuclein (αSyn), in both tissue homogenates and body fluids of Parkinson's disease (PD) patients. SAA involves repeated cycles of shaking or sonication coupled with incubation periods. However, this amplification scheme has limitations in tracking protein propagation due to repeated fragmentation. METHODS: We introduced a modified form of SAA, known as Quiescent SAA (QSAA), and evaluated biopsy and autopsy samples from individuals clinically diagnosed with PD and those without synucleinopathies (control group). Brain biopsy samples were obtained from 14 PD patients and 6 controls without synucleinopathies. Additionally, skin samples were collected from 214 PD patients and 208 control subjects. Data were analyzed from April 2019 to May 2023. RESULTS: QSAA successfully amplified αSyn aggregates in brain tissue sections from mice inoculated with pre-formed fibrils. In the skin samples from 214 PD cases and 208 non-PD cases, QSAA demonstrated high sensitivity (90.2%) and specificity (91.4%) in differentiating between PD and non-PD cases. Notably, more αSyn aggregates were detected by QSAA compared to immunofluorescence with the pS129-αSyn antibody in consecutive slices of both brain and skin samples. CONCLUSION: We introduced the new QSAA method tailored for in situ amplification of αSyn aggregates in brain and skin samples while maintaining tissue integrity, providing a streamlined approach to diagnosing PD with individual variability. The integration of seeding activities with the location of deposition of αSyn seeds advances our understanding of the mechanism underlying αSyn misfolding in PD.

TDP-43 Promotes Amyloid-Beta Toxicity by Delaying Fibril Maturation via Direct Molecular Interaction.

Amyloid-ß (Aß) is a peptide that undergoes self-assembly into amyloid fibrils, which compose the hallmark plaques observed in Alzheimer's disease (AD). TAR DNA-binding protein 43 (TDP-43) is a protein with mislocalization and aggregation implicated in amyotrophic lateral sclerosis and other neurodegenerative diseases. Recent work suggests that TDP-43 may interact with Aß, inhibiting the formation of amyloid fibrils and worsening AD pathology, but the molecular details of their interaction remain unknown. Using all-atom discrete molecular dynamics simulations, we systematically investigated the direct molecular interaction between Aß and TDP-43. We found that Aß monomers were able to bind near the flexible nuclear localization sequence of the N-terminal domain (NTD) of TDP-43, adopting ß-sheet rich conformations that were promoted by the interaction. Furthermore, Aß associated with the nucleic acid binding interface of the tandem RNA recognition motifs of TDP-43 via electrostatic interactions. Using the computational peptide array method, we found the strongest C-terminal domain interaction with Aß to be within the amyloidogenic core region of TDP-43. With experimental evidence suggesting that the NTD is necessary for inhibiting Aß fibril growth, we also simulated the NTD with an Aß40 fibril seed. We found that the NTD was able to strongly bind the elongation surface of the fibril seed via extensive hydrogen bonding and could also diffuse along the lateral surface via electrostatic interactions. Our results suggest that TDP-43 binding to the elongation surface, thereby sterically blocking Aß monomer addition, is responsible for the experimentally observed inhibition of fibril growth. We conclude that TDP-43 may promote Aß toxicity by stabilizing the oligomeric state and kinetically delaying fibril maturation.

On the friendship paradox and inversity: A network property with applications to privacy-sensitive network interventions.

We provide the mathematical and empirical foundations of the friendship paradox in networks, often stated as "Your friends have more friends than you." We prove a set of network properties on friends of friends and characterize the concepts of ego-based and alter-based means. We propose a network property called inversity that quantifies the imbalance in degrees across edges and prove that the sign of inversity determines the ordering between ego-based or alter-based means for any network, with implications for interventions. Network intervention problems like immunization benefit from using highly connected nodes. We characterize two intervention strategies based on the friendship paradox to obtain such nodes, with the alter-based and ego-based strategy. Both strategies provide provably guaranteed improvements for any network structure with variation in node degrees. We demonstrate that the proposed strategies obtain several-fold improvement (100-fold in some networks) in node degree relative to a random benchmark, for both generated and real networks. We evaluate how inversity informs which strategy works better based on network topology and show how network aggregation can alter inversity. We illustrate how the strategies can be used to control contagion of an epidemic spreading across a set of village networks, finding that these strategies require far fewer nodes to be immunized (less than 50%, relative to random). The interventions do not require knowledge of network structure, are privacy-sensitive, are flexible for time-sensitive action, and only require selected nodes to nominate network neighbors.

Paternal and induced gut microbiota seeding complement mother-to-infant transmission.

Microbial colonization of the neonatal gut involves maternal seeding, which is partially disrupted in cesarean-born infants and after intrapartum antibiotic prophylaxis. However, other physically close individuals could complement such seeding. To assess the role of both parents and of induced seeding, we analyzed two longitudinal metagenomic datasets (health and early life microbiota [HELMi]: N = 74 infants, 398 samples, and SECFLOR: N = 7 infants, 35 samples) with cesarean-born infants who received maternal fecal microbiota transplantation (FMT). We found that the father constitutes a stable source of strains for the infant independently of the delivery mode, with the cumulative contribution becoming comparable to that of the mother after 1 year. Maternal FMT increased mother-infant strain sharing in cesarean-born infants, raising the average bacterial empirical growth rate while reducing pathogen colonization. Overall, our results indicate that maternal seeding is partly complemented by that of the father and support the potential of induced seeding to restore potential deviations in this process.

A Cell Pre-Wrapping Seeding Technique for Hydrogel-Based Tubular Organ-On-A-Chip.

Organ-on-a-chip (OOC) models based on microfluidic technology are increasingly used to obtain mechanistic insight into (patho)physiological processes in humans, and they hold great promise for application in drug development and regenerative medicine. Despite significant progress in OOC development, several limitations of conventional microfluidic devices pose challenges. First, most microfluidic systems have rectangular cross sections and flat walls, and therefore tubular/ curved structures, like blood vessels and nephrons, are not well represented. Second, polymers used as base materials for microfluidic devices are much stiffer than in vivo extracellular matrix (ECM). Finally, in current cell seeding methods, challenges exist regarding precise control over cell seeding location, unreachable spaces due to flow resistances, and restricted dimensions/geometries. To address these limitations, an alternative cell seeding technique and a corresponding workflow is introduced to create circular cross-sectioned tubular OOC models by pre-wrapping cells around sacrificial fiber templates. As a proof of concept, a perfusable renal proximal tubule-on-a-chip is demonstrated with a diameter as small as 50 µm, cellular tubular structures with branches and curvature, and a preliminary vascular-renal tubule interaction model. The cell pre-wrapping seeding technique promises to enable the construction of diverse physiological/pathological models, providing tubular OOC systems for mechanistic investigations and drug development.

Mast seeding in European beech (Fagus sylvatica L.) is associated with reduced fungal sporocarp production and community diversity.

Mast seeding is a well-documented phenomenon across diverse forest ecosystems. While its effect on aboveground food webs has been thoroughly studied, how it impacts the soil fungi that drive soil carbon and nutrient cycling has not yet been explored. To evaluate the relationship between mast seeding and fungal resource availability, we paired a Swiss 29-year fungal sporocarp census with contemporaneous seed production for European beech (Fagus sylvatica L.). On average, mast seeding was associated with a 55% reduction in sporocarp production and a compositional community shift towards drought-tolerant taxa across both ectomycorrhizal and saprotrophic guilds. Among ectomycorrhizal fungi, traits associated with carbon cost did not explain species' sensitivity to seed production. Together, our results support a novel hypothesis that mast seeding limits annual resource availability and reproductive investment in soil fungi, creating an ecosystem 'rhythm' to forest processes that is synchronized above- and belowground.