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Hydrodynamic stress shapes the flora and fauna that exist in wave-swept environments, alters species interactions, and can become the primary community structuring agent. Yet, hydrodynamics can be difficult to quantify because instrumentation is expensive, some methods are unreliable, and accurately measuring spatial and temporal differences can be difficult. Here, we explored the utility of barnacles as potential biological flow-indicators. Barnacles, nearly ubiquitous within estuarine environments, have demonstrated notable phenotypic plasticity in the dimensions of their feeding appendages (cirri) and genitalia in response to flow. In high flow, barnacles have shorter, stockier cirri with shorter setae; in low flow, barnacles have longer, thinner cirri with longer setae. By measuring the relative differences in cirral dimensions, comparative differences in flow among locations can be quantified. We tested our hypothesis that ivory barnacles (Amphibalanus eburneus) could be useful biological flow indicators in two experiments. First, we performed reciprocal transplants of A. eburneus between wave protected and wave exposed areas to assess changes in morphology over 4 weeks as well as if changes dissipated when barnacles were relocated to a different wave habitat. Then, in a second study, we transplanted barnacles into low (25 cm/s) environments that were largely free of waves and shielded half of the transplanted barnacles to lessen flow speed. In both experiments, barnacles had significant differences in cirral morphologies across high and low flow sites. Transplanting barnacles revealed phenotypic changes occur within two weeks and can be reversed. Further, ameliorating flow within sites did not affect barnacle morphologies in low flow but had pronounced effects in high flow environments, suggesting that flow velocity was the primary driver of barnacle morphology in our experiment. These results highlight the utility of barnacles as cheap, accessible, and biologically relevant indicators of flow that can be useful for relative comparisons of flow differences among sites.

Abstract Background The metabolic enzyme nicotinamide‐N‐methyltransferase (NNMT) is highly expressed in various cancer entities, suggesting tumour‐promoting functions. We systematically investigated NNMT expression and its metabolic interactions in clear cell renal cell carcinoma (ccRCC), a prominent RCC subtype with metabolic alterations, to elucidate its role as a drug target. Methods NNMT expression was assessed in primary ccRCC (n = 134), non‐tumour tissue and ccRCC‐derived metastases (n = 145) by microarray analysis and/or immunohistochemistry. Findings were validated in The Cancer Genome Atlas (kidney renal clear cell carcinoma [KIRC], n = 452) and by single‐cell analysis. Expression was correlated with clinicopathological data and survival. Metabolic alterations in NNMT‐depleted cells were assessed by nontargeted/targeted metabolomics and extracellular flux analysis. The NNMT inhibitor (NNMTi) alone and in combination with the inhibitor 2‐deoxy‐D‐glucose for glycolysis and BPTES (bis‐2‐(5‐phenylacetamido‐1,3,4‐thiadiazol‐2‐yl)ethyl‐sulfide) for glutamine metabolism was investigated in RCC cell lines (786‐O, A498) and in two 2D ccRCC‐derived primary cultures and three 3D ccRCC air–liquid interface models. Results NNMT protein was overexpressed in primary ccRCC (p = 1.32 × 10–16) and ccRCC‐derived metastases (p = 3.92 × 10–20), irrespective of metastatic location, versus non‐tumour tissue. Single‐cell data showed predominant NNMT expression in ccRCC and not in the tumour microenvironment. High NNMT expression in primary ccRCC correlated with worse survival in independent cohorts (primary RCC—hazard ratio [HR] = 4.3, 95% confidence interval [CI]: 1.5–12.4; KIRC—HR = 3.3, 95% CI: 2.0–5.4). NNMT depletion leads to intracellular glutamine accumulation, with negative effects on mitochondrial function and cell survival, while not affecting glycolysis or glutathione metabolism. At the gene level, NNMT‐depleted cells upregulate glycolysis, oxidative phosphorylation and apoptosis pathways. NNMTi alone or in combination with 2‐deoxy‐D‐glucose and BPTES resulted in inhibition of cell viability in ccRCC cell lines and primary tumour and metastasis‐derived models. In two out of three patient‐derived ccRCC air–liquid interface models, NNMTi treatment induced cytotoxicity. Conclusions Since efficient glutamine utilisation, which is essential for ccRCC tumours, depends on NNMT, small‐molecule NNMT inhibitors provide a novel therapeutic strategy for ccRCC and act as sensitizers for combination therapies.