Lake settlements, particularly crannogs, pose several contradictions—visible yet inaccessible, widespread yet geographically restricted, persistent yet vulnerable. To further our understanding, we developed the integrated use of palaeolimnological (scanning XRF, pollen, spores, diatoms, chironomids, Cladocera, microcharcoal, biogenic silica, SEM-EDS, stable-isotopes) and biomolecular (faecal stanols, bile acids, sedaDNA) analyses of crannog cores in south-west Scotland and Ireland. Both can be effective methods sets for revealing occupation chronologies and identifying on-crannog activities and practices. Strong results from sedaDNA and lipid biomarker analyses demonstrate probable on-site animal slaughter, food storage and possible feasting, suggesting multi-period, elite site associations, and the storage and protection of valuable resources.

OBJECTIVES/GOALS: Immunomodulatory drugs (IMiDs) are critical to multiple myeloma (MM) disease control. IMiDs act by inducing Cereblon-dependent degradation of IKZF1 and IKZF3, which leads to IRF4 and MYC downregulation (collectively termed the “Ikaros axis”). We therefore hypothesized that IMiD treatment fails to downregulate the Ikaros axis in IMiD resistant MM. METHODS/STUDY POPULATION: To measure IMiD-induced Ikaros axis downregulation, we designed an intracellular flow cytometry assay that measured relative protein levels of IKZF1, IKZF3, IRF4 and MYC in MM cells following ex vivo treatment with the IMiD Pomalidomide (Pom). We established this assay using Pom-sensitive parental and dose-escalated Pom-resistant MM cell lines before assessing Ikaros axis downregulation in CD38+CD138+ MM cells in patient samples (bone marrow aspirates). To assess the Ikaros axis in the context of MM intratumoral heterogeneity, we used a 35-marker mass cytometry panel to simultaneously characterize MM subpopulations in patient samples. Lastly, we determined ex vivo drug sensitivity in patient samples via flow cytometry. RESULTS/ANTICIPATED RESULTS: Our hypothesis was supported in MM cell lines, as resistant lines showed no IMiD-induced decrease in any Ikaros axis proteins. However, when assessed in patient samples, Pom treatment caused a significant decrease in IKZF1, IKZF3 and IRF4 regardless of IMiD sensitivity. Mass cytometry in patient samples revealed that individual Ikaros axis proteins were differentially expressed between subpopulations. When correlating this with ex vivo Pom sensitivity of MM subpopulations, we observed that low IKZF1 and IKZF3 corresponded to Pom resistance. Interestingly, most of these resistant populations still expressed MYC. We therefore assessed whether IMiD resistant MM was MYC dependent by treating with MYCi975. In 88% (7/8) of patient samples tested, IMiD resistant MM cells were sensitive to MYC inhibition. DISCUSSION/SIGNIFICANCE: While our findings did not support our initial hypothesis, our data suggest a mechanism where MYC expression becomes Ikaros axis independent to drive IMiD resistance, and resistant MM is still dependent on MYC. This suggests targeting MYC directly or indirectly via a mechanism to be determined may be an effective strategy to eradicate IMiD resistant MM.

The Eating Assessment in Toddlers FFQ (EAT FFQ) has been shown to have good reliability and comparative validity for ranking nutrient intakes in young children. With the addition of food items (n 4), we aimed to re-assess the validity of the EAT FFQ and estimate calibration factors in a sub-sample of children (n 97) participating in the Growing Up Milk – Lite (GUMLi) randomised control trial (2015–2017). Participants completed the ninety-nine-item GUMLi EAT FFQ and record-assisted 24-h recalls (24HR) on two occasions. Energy and nutrient intakes were assessed at months 9 and 12 post-randomisation and calibration factors calculated to determine predicted estimates from the GUMLi EAT FFQ. Validity was assessed using Pearson correlation coefficients, weighted kappa (κ) and exact quartile categorisation. Calibration was calculated using linear regression models on 24HR, adjusted for sex and treatment group. Nutrient intakes were significantly correlated between the GUMLi EAT FFQ and 24HR at both time points. Energy-adjusted, de-attenuated Pearson correlations ranged from 0·3 (fibre) to 0·8 (Fe) at 9 months and from 0·3 (Ca) to 0·7 (Fe) at 12 months. Weighted κ for the quartiles ranged from 0·2 (Zn) to 0·6 (Fe) at 9 months and from 0·1 (total fat) to 0·5 (Fe) at 12 months. Exact agreement ranged from 30 to 74 %. Calibration factors predicted up to 56 % of the variation in the 24HR at 9 months and 44 % at 12 months. The GUMLi EAT FFQ remained a useful tool for ranking nutrient intakes with similar estimated validity compared with other FFQ used in children under 2 years.

A sphere sinking through a chemical gradient drags fluid with it, deforming the gradient. The sphere leaves a trail of gradient enhancement that persists longer than the velocity disturbance in the Reynolds $10^{-2}\leqslant Re\leqslant 10^{2}$, Froude $10^{-1}\leqslant Fr\leqslant 10^{3}$ and Péclet $10^{2}<Pe\leqslant 10^{6}$ regime considered here. We quantify the enhancement of the gradient and the diffusive flux in the trail of disturbed chemical left by the passing sphere using a combination of numerical simulations and scaling analyses. When $Fr$ is large and buoyancy forces are negligible, dragged isosurfaces of chemical form a boundary layer of thickness $\unicode[STIX]{x1D6FF}_{\unicode[STIX]{x1D70C}}$ around the sphere with diameter $l$. We derive the scaling $\unicode[STIX]{x1D6FF}_{\unicode[STIX]{x1D70C}}/l\sim \mathit{Pe}^{-1/3}$ from the balance of advection and diffusion in the chemical boundary layer. The sphere displaces a single isosurface of chemical a maximum distance $\mathit{L}_{Def}$ that increases as $\mathit{L}_{Def}/l\sim l/\unicode[STIX]{x1D6FF}_{\unicode[STIX]{x1D70C}}\sim \mathit{Pe}^{1/3}$. Increased flux through the chemical boundary layer moving with the sphere is described by a Sherwood number, $Sh\sim l/\unicode[STIX]{x1D6FF}_{\unicode[STIX]{x1D70C}}\sim \mathit{Pe}^{1/3}$. The gradient enhancement trail extends much farther than $\mathit{L}_{Def}$ as displaced isosurfaces slowly return to their original positions through diffusion. In the reference frame of a chemical isosurface moving past the sphere, a new quantity describing the Lagrangian flux is found to scale as $\mathit{M}\sim (\mathit{L}_{Def}/l)^{2}\sim \mathit{Pe}^{2/3}$. The greater $\mathit{Pe}$ dependence of $\mathit{M}$ versus $Sh$ demonstrates the importance of the deformation trail for determining the total flux of chemical in the system. For $\mathit{Fr}\geqslant 10$, buoyancy forces are weak compared to the motion of the sphere and the preceding results are retained. Below $\mathit{Fr}=10$, an additional Froude dependence is found and $l/\unicode[STIX]{x1D6FF}_{\unicode[STIX]{x1D70C}}\sim Sh\sim Re^{1/6}Fr^{-1/6}Pe^{1/3}$. Buoyancy forces suppress gradient deformation downstream, resulting in $\mathit{L}_{Def}/l\sim Re^{-1/3}Fr^{1/3}Pe^{1/3}$ and $\mathit{M}\sim Re^{-1/3}Fr^{1/3}Pe^{2/3}$. The productivity of marine plankton – and therefore global carbon and oxygen cycles – depends on the availability of microscale gradients of chemicals. Because most plankton exist in the fluids regime under consideration, this work describes a new mechanism by which sinking particles and plankton can stir weak ambient chemical gradients a distance $\mathit{L}_{Def}$ and increase chemical flux in the trail by a factor of $\mathit{M}$.