Site-specific Differences Research Articles

Land snail Δ47 thermometry using cultured and European natural populations of Clausilia pumila, Succinella oblonga and Trochulus hispidus

Clumped isotope composition (Δ47) of aragonitic shells of terrestrial molluscs allow the quantitative reconstructions of continental paleotemperatures from loess-paleosol records of the last 2.5 million years, but it needs testing the method under controlled laboratory conditions and using natural populations. In this study the stable carbon, oxygen and clumped isotope compositions of cultured individuals and field-collected specimens of three species (Clausilia pumila, Succinella oblonga and Trochulus hispidus), widespread in Quaternary loess deposits in Europe, were measured in the Debrecen (ATOMKI) and ETH Zürich laboratories. Our data of cultured snails demonstrate that shells of the three species record Δ47 signatures close to equilibrium conditions with mostly positive (C. pumila), both positive/negative (T. hispidus), and mostly negative (S. oblonga) offsets on the order of 0.0158 ± 0.0105 ‰ (mean offset±1 SD) compared to the Anderson et al. (2021) unified calibration. Calcification temperatures (TΔ47) derived from C. pumila shells were found to be in excellent agreement with chamber temperatures of 12, 18 and 24 °C, especially in the ATOMKI laboratory (mean T offset: 2.1 ± 2.2 °C, 1 SD), while the other two species exhibited larger discrepancies on the order of 5–8 °C (mean offset). Single shell-based mean TΔ47 values of field-collected molluscs indicated growth temperatures spanning values from the frost-free period to summer season, or even above summer maxima. The sites of natural populations, characterized by different climate conditions, could be grouped using the aridity index (AI) with values of 0.96–1.06 representing mean temperature of the frost-free period (TFFP), and AI ranging either from 0.85 to 0.91 or 1.14 to 1.51 reflecting mean summer season or July temperatures (TJJA/TJ). Multi-shell TΔ47, obtained by homogenizing the material of 3–5 shells of C. pumila and T. hispidus, reflect TJJA in most cases. We observed a dependence of the shell oxygen isotope compositions of the three species on oceanity/continentality with more negative/positive values characteristic for more continental/oceanic sites, likely due to moisture source distance and depletion of rainfall in 18O towards the continental interior. In addition, deviation of body fluid δ18O values (derived from δ18Oshell and Δ47shell) from seasonal precipitation δ18O is also controlled by an additional temperature effect, being independent of the climate zone of the collection site, but co-varying with the site-specific difference between single shell TΔ47 and TJJA.

584P Genomic and transcriptomic characterization of peritoneal, lung and liver metastases of colorectal carcinoma reveals site-specific differences

584P Genomic and transcriptomic characterization of peritoneal, lung and liver metastases of colorectal carcinoma reveals site-specific differences

Predictive Functional Profiling Reveals Putative Metabolic Capacities of Bacterial Communities in Drinking Water Resources and Distribution Supply in Mega Manila, Philippines

Assessing bacterial communities across water resources is crucial for understanding ecological dynamics and improving water quality management. This study examines the functional profiles of bacterial communities in drinking water resources in Mega Manila, Philippines, including Laguna Lake tributaries, pre-treatment plant sites, groundwater sources, and post-treatment plant sites. Using eDNA sequencing, flux balance analysis, and taxonomy-to-phenotype mapping, we identified metabolic pathways involved in nutrient metabolism, pollutant degradation, antibio- tic synthesis, and nutrient cycling. Despite site variations, there are shared metabolic pathways, suggesting the influence of common ecological factors. Site-specific differences in pathways like ascorbate, aldarate, and phenylalanine metabolism indicate localized environmental adaptations. Antibiotic synthesis pathways, such as streptomycin and polyketide sugar unit biosynthesis, were detected across sites. Bacterial communities in raw and pre-treatment water showed potential for pollutant degradation such as for endocrine-disrupting chemicals. High levels of ammonia-oxidizing and sulfate-reducing bacteria in pre- and post-treatment water suggest active nitrogen removal and pH neutralization, indicating a need to reassess existing water treatment approaches. This study underscores the adaptability of bacterial communities to environmental factors, as well as the importance of considering their functional profiles in assessing drinking water quality resources in urban areas.

Developmental changes in brain structure and function following exposure to oral LSD during adolescence

LSD is a hallucinogen with complex neurobiological and behavioral effects. Underlying these effects are changes in brain neuroplasticity. This is the first study to follow the developmental changes in brain structure and function following LSD exposure in periadolescence. We hypothesized LSD given during a time of heightened neuroplasticity, particularly in the forebrain, would affect cognitive and emotional behavior and the associated underlying neuroanatomy and neurocircuitry. Female and male mice were given vehicle, single or multiple treatments of 3.3 µg of LSD by oral gavage starting on postnatal day 51. Between postnatal days 90–120 mice were imaged and tested for cognitive and motor behavior. MRI data from voxel-based morphometry, diffusion weighted imaging, and BOLD resting state functional connectivity were registered to a mouse 3D MRI atlas with 139 brain regions providing site-specific differences in global brain structure and functional connectivity between experimental groups. Motor behavior and cognitive performance were unaffected by periadolescent exposure to LSD. Differences across experimental groups in brain volume for any of the 139 brain areas were few in number and not focused on any specific brain region. Multiple exposures to LSD significantly altered gray matter microarchitecture across much of the brain. These changes were primary associated with the thalamus, sensory and motor cortices, and basal ganglia. The forebrain olfactory system and prefrontal cortex and hindbrain cerebellum and brainstem were unaffected. The functional connectivity between forebrain white matter tracts and sensorimotor cortices and hippocampus was reduced with multidose LSD exposure. Does exposure to LSD in late adolescence have lasting effects on brain development? The bulk of our significant findings were seen through changes is DWI values across 74 brain areas in the multi-dose LSD group. The pronounced changes in indices of anisotropy across much of the brain would suggest altered gray matter microarchitecture and neuroplasticity. There was no evidence of LSD having consequential effects on cognitive or motor behavior when animal were evaluated as young adults 90–120 days of age. Neither were there any differences in the volume of specific brain areas between experimental conditions. The reduction in connectivity in forebrain white matter tracts with multidose LSD and consolidation around sensorimotor and hippocampal brain areas requires a battery of tests to understand the consequences of these changes on behavior.

Patient selection for nonoperating room anesthesia.

Given the rapid growth of nonoperating room anesthesia (NORA) in recent years, it is essential to review its unique challenges as well as strategies for patient selection and care optimization. Recent investigations have uncovered an increasing prevalence of older and higher ASA physical status patients in NORA settings. Although closed claim data regarding patient injury demonstrate a lower proportion of NORA cases resulting in a claim than traditional operating room cases, NORA cases have an increased risk of claim for death. Challenges within NORA include site-specific differences, limitations in ergonomic design, and increased stress among anesthesia providers. Several authors have thus proposed strategies focusing on standardizing processes, site-specific protocols, and ergonomic improvements to mitigate risks. Considering the unique challenges of NORA settings, meticulous patient selection, risk stratification, and preoperative optimization are crucial. Embracing data-driven strategies and leveraging technological innovations (such as artificial intelligence) is imperative to refine quality control methods in targeted areas. Collaborative efforts led by anesthesia providers will ensure personalized, well tolerated, and improved patient outcomes across all phases of NORA care.

Comparative analysis of the mutational landscape and evolutionary patterns of pancreatic ductal adenocarcinoma metastases in the liver or peritoneum

BackgroundPancreatic ductal adenocarcinoma (PDAC) often presents with liver or peritoneal metastases at diagnosis. Despite similar treatment approaches, patient outcomes vary between these metastatic sites. To improve targeted therapies for metastatic PDAC, a comprehensive analysis of the genetic profiles and evolutionary patterns at these distinct metastatic locations is essential. MethodsWe performed whole exome sequencing on 44 tissue samples from 27 PDAC patients, including primary tumours and matched liver or peritoneal metastases. We analysed somatic mutation profiles, signatures, and affected pathways for each group, and examined clonal evolution using subclonal architectures and phylogenetic trees. ResultsKRAS mutations remained the predominant driver alteration, with a prevalence of 89 % across all tumours. Notably, we observed site-specific differences in mutation frequencies, with KRAS alterations detected in 77.8 % (7/9) of peritoneal metastases and 87.5 % (7/8) of liver metastases. TP53 mutations exhibited a similar pattern, occurring in 55.6 % (5/9) of peritoneal and 37.5 % (3/8) of liver metastases. Intriguingly, we identified site-specific alterations in DNA repair pathway genes, including ATM and BRCA1, with distinct mutational profiles in liver versus peritoneal metastases. Furthermore, liver metastases demonstrated a significantly higher tumor mutational burden (TMB) compared to peritoneal metastases (median [IQR]: 2.14 [1.77–2.71] vs. 1.29 [1.21–1.69] mutations/Mb; P = 0.048). ConclusionsIn conclusion, metastasis of pancreatic cancer may be influenced by variables other than KRAS mutations, such as TP53. PDAC peritoneal and liver metastases may differ in potential therapeutic biomarkers. Further inquiry is needed on the biological mechanisms underlying metastasis and the treatment of diverse metastases.

Rational development of functional hydrophilic polymer to characterize site-specific glycan differences between bovine milk and colostrum

As vastly modified on secreted proteins, N-glycosylation is found on milk proteins and undergo dynamic changes during lactation, characterizing milk protein glycosylation would benefit the elucidation of glycosylation pattern differences between samples. However, their low abundance required specific enrichment. Herein, through rational design and controllable synthesis, we developed a novel multi-functional polymer for the isolation of protein glycosylation. It efficiently separated glycopeptides from complex background inferences with mutual efforts of hydrophilic interaction chromatography (HILIC), metal ion affinity and ion exchange. By fine-tuning Ca2+ as regulators of aldehyde hyaluronic acid (HA) conformation, the grafting density of HA was remarkably improved. Moreover, grafting Ti4+ further enhanced the enrichment performance. Application of this material to characterize bovine milk and colostrum proteins yields 479 and 611 intact glycopeptides, respectively. Comparative analysis unraveled the distinct glycosylation pattern as well the different distribution of glycoprotein abundances between the two samples, offering insights for functional food development.

Diversity, virulence and antibiotic resistance of Vibrio Harveyi clade species associated with bivalve aquaculture within marine protected areas

The aim of the present study was to investigate the diversity, distribution of virulence factor genes and antimicrobial resistance phenotypes of Vibrio Harveyi clade species from two distinct and spatially distant near-shore bivalve farming environments located in protected marine reserves of the eastern Adriatic. Tissues of farmed Mediterranean mussel Mytilus galloprovincialis and European flat oyster Ostrea edulis, seawater and sediment samples were collected during six bi-monthly sampling events conducted over one year from farming locations in Lim Bay and Mali Ston Bay, Croatia. Eight distinctive Harveyi subclades, namely, V. harveyi, V. alginolyticus, V. diabolicus, V. jasicida, V. parahaemolyticus, V. rotiferianus, V. campbellii and V. communis, were detected by Phylogenetic Multilocus Sequence Analysis (MLSA) based on rpoD, toxR and rctB concatenated genes sequences. Genomic fingerprinting of MLSA-classified samples revealed substantial diversity of environmental isolates. The virulence-related genes considered typical for the Harveyi clade and their homologues were represented in all subclades with varying frequencies. Of twelve antibiotics commonly used in human and veterinary medicine, the most frequent resistance phenotypes were resistance to ampicillin (90%), erythromycin (39%), imipenem (29%) and streptomycin (22%). >60% of isolates were resistant to at least two classes of antibiotics. Seasonal or site-specific differences were found in the proportion of isolates carrying virulence genes and displaying specific antibiotic resistance phenotype.This study is the first to provide a broader perspective on the properties of Vibrio Harveyi clade species associated with ecosystems under presumably low anthropogenic pressures, such as marine protected areas. New data on virulence and antibiotic resistance are presented of Harveyi clade species coexisting in different environmental compartments, including those that have been largely overlooked or understudied in the context of bivalve aquaculture. Taken together, these results highlight the importance of monitoring the presence of potentially pathogenic strains in the natural Vibrio population of bivalve farming areas to reduce the risk to seafood consumers.

Dual-Probe Activity-Based Protein Profiling Reveals Site-Specific Differences in Protein Binding of EGFR-Directed Drugs.

Comparative, dose-dependent analysis of interactions between small molecule drugs and their targets, as well as off-target interactions, in complex proteomes is crucial for selecting optimal drug candidates. The affinity of small molecules for targeted proteins is largely dictated by interactions between amino acid side chains and these drugs. Thus, studying drug-protein interactions at an amino acid resolution provides a comprehensive understanding of the drug selectivity and efficacy. In this study, we further refined the site-specific activity-based protein profiling strategy (ABPP), PhosID-ABPP, on a timsTOF HT mass spectrometer. This refinement enables dual dose-dependent competition of inhibitors within a single cellular proteome. Here, a comparative analysis of two activity-based probes (ABPs), developed to selectively target the epidermal growth factor receptor (EGFR), namely, PF-06672131 (PF131) and PF-6422899 (PF899), facilitated the simultaneous identification of ABP-specific binding sites at a proteome-wide scale within a cellular proteome. Dose-dependent probe-binding preferences for proteinaceous cysteines, even at low nanomolar ABP concentrations, could be revealed. Notably, in addition to the intrinsic affinity of the electrophilic probes for specific sites in targeted proteins, the observed labeling intensity is influenced by several other factors. These include the efficiency of cellular uptake, the stability of the probes, and their intracellular distribution. While both ABPs showed comparable labeling efficiency for EGFR, PF131 had a broader off-target reactivity profile. In contrast, PF899 exhibited a higher labeling efficiency for the ERBB2 receptor and bound to catalytic cysteines in several other enzymes, which is likely to disrupt their catalytic activity. Notably, PF131 effectively labeled ADP/ATP translocase proteins at a concentration of just 1 nm, and we found this affected ATP transport. Analysis of the effect of PF131 and its parent inhibitor Afatinib on murine translocase SLC25A4 (ANT1)-mediated ATP transport strongly indicated that PF131 (10 μM) partially blocked ATP transport. Afatinib was less efficient at inhibiting ATP transport by SLC25A4 than PF131, and the reduction of ATP transport by Afatinib was not significant. Follow-up analysis is required to evaluate the affinity of these inhibitors for ADP/ATP translocase SLC25A4 in more detail. Additionally, the analysis of different binding sites within the EGF receptor and the voltage-dependent anion channel 2 revealed secondary binding sites of both probes and provided insights into the binding poses of inhibitors on these proteins. Insights from the PhosID-ABPP analysis of these two ABPs serve as a valuable resource for understanding drug on- and off-target engagement in a dose- and site-specific manner.

Leveraging multi-site electronic health data for characterization of subtypes: a pilot study of dementia in the N3C Clinical Tenant.

To provide a foundational methodology for differentiating comorbidity patterns in subphenotypes through investigation of a multi-site dementia patient dataset. Employing the National Clinical Cohort Collaborative Tenant Pilot (N3C Clinical) dataset, our approach integrates machine learning algorithms-logistic regression and eXtreme Gradient Boosting (XGBoost)-with a diagnostic hierarchical model for nuanced classification of dementia subtypes based on comorbidities and gender. The methodology is enhanced by multi-site EHR data, implementing a hybrid sampling strategy combining 65% Synthetic Minority Over-sampling Technique (SMOTE), 35% Random Under-Sampling (RUS), and Tomek Links for class imbalance. The hierarchical model further refines the analysis, allowing for layered understanding of disease patterns. The study identified significant comorbidity patterns associated with diagnosis of Alzheimer's, Vascular, and Lewy Body dementia subtypes. The classification models achieved accuracies up to 69% for Alzheimer's/Vascular dementia and highlighted challenges in distinguishing Dementia with Lewy Bodies. The hierarchical model elucidates the complexity of diagnosing Dementia with Lewy Bodies and reveals the potential impact of regional clinical practices on dementia classification. Our methodology underscores the importance of leveraging multi-site datasets and tailored sampling techniques for dementia research. This framework holds promise for extending to other disease subtypes, offering a pathway to more nuanced and generalizable insights into dementia and its complex interplay with comorbid conditions. This study underscores the critical role of multi-site data analyzes in understanding the relationship between comorbidities and disease subtypes. By utilizing diverse healthcare data, we emphasize the need to consider site-specific differences in clinical practices and patient demographics. Despite challenges like class imbalance and variability in EHR data, our findings highlight the essential contribution of multi-site data to developing accurate and generalizable models for disease classification.

ADP-ribosylome analysis reveals homogeneous DNA-damage-induced serine ADP-ribosylation across wild-type and BRCA-mutant breast cancer cell lines

ADP-ribosylation (ADPr) signaling plays a crucial role in DNA damage response. Inhibitors against the main enzyme catalyzing ADPr after DNA damage, poly(ADP-ribose) polymerase 1 (PARP1), are used to treat patients with breast cancer harboring BRCA1/2 mutations. However, resistance to PARP inhibitors (PARPi) is a major obstacle in treating patients. To understand the role of ADPr in PARPi sensitivity, we use liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze ADPr in six breast cancer cell lines exhibiting different PARPi sensitivities. We identify 1,632 sites on 777 proteins across all cell lines, primarily on serine residues, with site-specific overlap of targeted residues across DNA-damage-related proteins across all cell lines, demonstrating high conservation of serine ADPr-signaling networks upon DNA damage. Furthermore, we observe site-specific differences in ADPr intensities in PARPi-sensitive BRCA mutants and unique ADPr sites in PARPi-resistant BRCA-mutant HCC1937 cells, which have low poly(ADP-ribose) glycohydrolase (PARG) levels and longer ADPr chains on PARP1.

Multi-resolution remote sensing for flark area detection in boreal aapa mires

ABSTRACT Peatlands have suffered significant degradation globally due to human impacts, which has increased the need to monitor the condition and changes in peatland ecosystems. With remote sensing, point-based in-situ observations can be upscaled to larger areas but there is a need to develop scalable monitoring methods. We predicted wet flark area extent, a key ecological indicator for patterned flark fens, in five sites in central Finland. Our primary aim was to test how the spatial and spectral resolution of different optical satellite image products (Landsat 8–9, Sentinel-2, PlanetScope) affect flark area coverage prediction. We also tested if there were seasonal or site-specific differences in prediction accuracy. Lastly, we upscaled the flark area coverage to entire mire areas. We used unmanned aerial vehicle (UAV)-derived flark area classification as a ground reference data to compare satellite sensors’ prediction accuracies. We predicted flark area coverage using spectral bands and indices as predictor variables using random forest regression. All sensors provided accurate results with some differences between Landsat (pseudo-R2 32−84%, root-mean squared error (RMSE) 10 − 18%), Sentinel-2 (R2 61−92%, RMSE 6−14%), and PlanetScope (R2 46 − 92%, RMSE 6 − 17%). The shortwave infrared bands of Landsat and Sentinel-2 did not increase the prediction accuracy. There were notable site-specific variations in prediction accuracy despite all the sites having typical open aapa mire wet flark – dry string patterns. With single-site models, the prediction accuracies were similar for early and late summer data, but when transferring the models to the other sites, performance significantly decreased, especially with the models using the late-summer imagery. Finally, we successfully upscaled the flark area coverage across entire mire areas. Our results demonstrate that the combination of UAV and satellite imagery can be used successfully to monitor peatland conditions and changes in them.

Mapping the geographical distribution of the mucosa-associated gut microbiome in GI-symptomatic children with autism spectrum disorder.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by cognitive, behavioral, and communication impairments. In the past few years, it has been proposed that alterations in the gut microbiota may contribute to an aberrant communication between the gut and brain in children with ASD. Consistent with this notion, several studies have demonstrated that children with ASD have an altered fecal microbiota compared with typically developing (TD) children. However, it is unclear where along the length of the gastrointestinal (GI) tract these alterations in microbial communities occur. In addition, the variation between specific mucosa-associated communities remains unknown. To address this gap in knowledge of the microbiome associated with ASD, biopsies from the antrum, duodenum, ileum, right colon, and rectum of children with ASD and age- and sex-matched TD children were examined by 16S rRNA sequencing. We observed an overall elevated abundance of Bacillota and Bacteroidota and a decreased abundance of Pseudomonadota in all GI tract regions of both male and female children with ASD compared with TD children. Further analysis at the genera level revealed unique differences in the microbiome in the different regions of the GI tract in children with ASD compared with TD children. We also observed sex-specific differences in the gut microbiota composition in children with ASD. These data indicate that the microbiota of children with ASD is altered in multiple regions of the GI tract and that different anatomic locations have unique alterations in mucosa-associated bacterial genera.NEW & NOTEWORTHY Analysis in stool samples has shown gut microbiota alterations in children with autism spectrum disorder (ASD) compared with typically developing (TD) children. However, it is unclear which segment(s) of the gut exhibit alterations in microbiome composition. In this study, we examined microbiota composition along the gastrointestinal (GI) tract in the stomach, duodenum, ileum, right colon, and rectum. We found site-specific and sex-specific differences in the gut microbiota of children with ASD, compared with controls.

Year-round monitoring of three water sources in Québec, Canada, reveals site-specific differences in conditions for Cryptosporidium and Giardia contamination.

Cryptosporidium and Giardia are protozoan parasites responsible for gastrointestinal illnesses in humans and in animal species. The main way these parasites are transmitted is by ingestion of their (oo)cysts in drinking water. Monitoring (oo)cysts in water sources is beneficial to evaluate the quality of raw water supplying treatment plants. Currently, the only standardized protocol to enumerate these parasites from water samples is United States Environmental Protection Agency (USEPA) Method 1623.1. With this method, we monitored three major water sources in Quebec over a year to assess temporal and geographical variations of these parasite (oo)cysts. These three water sources have independent watersheds despite being in the same region. We found a general pattern for Giardia, with high concentrations of cysts during cold and transition periods, and significantly lower concentrations during the warm period. Cryptosporidium's concentration was more variable throughout the year. Statistical correlations (Pearson's correlation coefficients) were established between the concentration of each parasite and various environmental parameters. The three study sites each showed unique factors correlating with the presence of both protozoa, supporting the idea that each water source must be seen as a unique entity with its own particular characteristics and therefore, must be monitored independently. Although some environmental parameters could be interesting proxies to the parasitic load, no parameter was strongly correlated throughout the whole sampling year and none of the parameters could be used as a single proxy for all three studies sources.

ASSESSING RISK FACTORS FOR OSTEOPOROSIS AND FRACTURES AT DIFFERENT SITES: LUMBAR VERTEBRAE VERSUS NECK OF FEMURS IN A LARGE COHORT. A CROSS-SECTIONAL STUDY

Osteoporosis can cause significant disability and cost to health services globally. We aim to compare risk fractures for both osteoporosis and fractures at the L1-L4 vertebrae (LV) and the neck of femurs (NOFs) in patients referred for DEXA scan in the North-West of England.Data was obtained from 31546 patients referred for DEXA scan in the North-West of England between 2004 and 2011. Demographic data was retrospectively analysed using STATA, utilising chi-squared and t-tests. Logistical models were used to report odds ratios for risk factors included in the FRAX tool looking for differences between osteoporosis and fracture risk at the LV and NOFs.In a study involving 2530 cases of LV fractures and 1363 of NOF fractures, age was significantly linked to fractures and osteoporosis at both sites, with a higher risk of osteoporosis at NOFs compared to LV. Height provided protection against fractures and osteoporosis at both sites, with a more pronounced protective effect against osteoporosis at NOFs. Weight was more protective for NOF fractures, while smoking increased osteoporosis risk with no site-specific difference. Steroids were unexpectedly protective for fractures at both sites, with no significant difference, while alcohol consumption was protective against osteoporosis at both sites and associated with increased LV fracture risk. Rheumatoid arthritis increased osteoporosis risk in NOFs and implied a higher fracture risk, though not statistically significant compared to LV. Results summarised in Table 1.Our study reveals that established osteoporosis and fracture risk factors impact distinct bony sites differently. Age and rheumatoid arthritis increase osteoporosis risk more at NOFs than LV, while height and steroids provide greater protection at NOFs. Height significantly protects LV fractures, with alcohol predicting them. Further research is needed to explore risk factors’ impact on additional bony sites and understand the observed differences’ pathophysiology.For any figures or tables, please contact the authors directly.

DNA metabarcoding as a tool for characterising the spatio-temporal distribution of planktonic larvae in the phylum Echinodermata

AbstractMetabarcoding is revolutionising the analysis of biodiversity in marine ecosystems, especially as it provides a means of detecting and identifying cryptic life stages in field samples. The planktonic larval stage of many species underpins the abundance and distribution of adult populations but is challenging to characterise given the small size of larvae and diffuse distributions in pelagic waters. Yet, planktonic larval dynamics are key to understanding phenomena observed in adult populations, such as the boom-and-bust dynamics exhibited by some echinoderms. Rapid changes in echinoderm population density can have significant effects on local benthic ecosystems. For example, outbreaks of the crown-of-thorns sea star (CoTS) on the Great Barrier Reef (GBR) have led to considerable declines in coral cover. Here, we used a DNA metabarcoding approach to investigate the spatio-temporal distribution and diversity of echinoderm larvae on the GBR, including CoTS. Generalised linear mixed models revealed that echinoderm larval richness, was significantly correlated with temporal variables (i.e. season and year) which is consistent with expected fluctuations in larval output based on adult spawning periodicity. However, neither site-specific differences in echinoderm larval richness, nor correlations between larval composition and environmental, temporal, or spatial variables were found. This study validates the utility of metabarcoding approaches for detecting and characterising echinoderm larvae, including CoTS, which could prove useful to future monitoring efforts. Our findings suggest that metabarcoding can be used to better understand the life history of planktonic larvae, and analyses combining environmental (e.g., temperature, nutrients) and oceanographic (e.g., currents) data could deliver valuable information on the factors influencing their spatio-temporal distributions.

Bioinspired and Photo-Clickable Thiol-Ene Bioinks for the Extrusion Bioprinting of Mechanically Tunable 3D Skin Models.

Bioinks play a fundamental role in skin bioprinting, dictating the printing fidelity, cell response, and function of bioprinted 3D constructs. However, the range of bioinks that support skin cells' function and aid in the bioprinting of 3D skin equivalents with tailorable properties and customized shapes is still limited. In this study, we describe a bioinspired design strategy for bioengineering double crosslinked pectin-based bioinks that recapitulate the mechanical properties and the presentation of cell-adhesive ligands and protease-sensitive domains of the dermal extracellular matrix, supporting the bioprinting of bilayer 3D skin models. Methacrylate-modified pectin was used as a base biomaterial enabling hydrogel formation via either chain-growth or step-growth photopolymerization and providing independent control over bioink rheology, as well as the mechanical and biochemical cues of cell environment. By tuning the concentrations of crosslinker and polymer in bioink formulation, dermal constructs were bioprinted with a physiologically relevant range of stiffnesses that resulted in strikingly site-specific differences in the morphology and spreading of dermal fibroblasts. We also demonstrated that the developed thiol-ene photo-clickable bioinks allow for the bioprinting of skin models of varying shapes that support dermis and epidermis reconstruction. Overall, the engineered bioinks expand the range of printable biomaterials for the extrusion bioprinting of 3D cell-laden hydrogels and provide a versatile platform to study the impact of material cues on cell fate, offering potential for in vitro skin modeling.

Abstract LB314: Organ-specific roles of CD83+ macrophages in breast cancer

Abstract The purpose of this study is to understand how heterogeneity in organ-specific immune infiltrates can lead to Triple Negative Breast Cancer (TNBC) progression and immunotherapies resistance in metastasis. Both innate and adaptive immune responses are essential for breast cancer progression. Using single-cell multiplexing technology on human and murine models of primary and metastatic breast cancers, we investigated the pattern of immune cell infiltration in bone and lung metastases in a temporal manner. The parallel profiling of metastasis sites revealed a strong involvement of the innate immunity in restraining tumor progression with an acute increase in Mrc1+ Cd83+ tumor-associated macrophages at the early stage of lung colonization. Interestingly, the increase was modest in bone metastasis and remained consistent overtime despite bone metastasis progression. In contrast, we saw an increase in the number of Ly6G+ Lrg1+ granulocytes. Profiling of paired TNBC patient samples from the primary and metastatic sites have shown an immunologically quiescent tumor microenvironment in metastasis as compared to the primary tumors. Hence, to further explore the role of Mrc1+ Cd83+ macrophages on therapeutic resistance, we profiled the orthotopic claudin-low TNBC models, T11 and T12, following their exposure to chemotherapy. While responsive tumors displayed a decreased infiltration of Mrc1+ Cd83+ macrophages, the opposite phenotype was observed in non-responders, suggesting their involvement in tumor progression. Overall, our results suggest differential responses in untreated lung/bone metastases and primary tumors indicating divergent roles for Cd83+ TAMs in restraining or promoting tumor progression, while also indicating some crosstalk between neutrophils and stromal cells that aid in bone metastatic progression. These site-specific differences in immune cell phenotype indicate that there is a great need to thoroughly characterize the tumor immune compartment in both the primary and the metastatic setting to improve treatment responses. Considering the inhibitory effect of macrophages on T cells in these models, this study highlights a tissue-specific reprogramming of immune cells in breast cancer metastasis. Citation Format: Swarnima Singh, Igor Bado. Organ-specific roles of CD83+ macrophages in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB314.

Abstract 3786: Genomic and transcriptomic characterization of peritoneal, lung and liver metastases of colorectal carcinoma reveals site-specific differences

Abstract Background: Metastasis in colorectal cancer (CRC) is a critical factor in patient outcomes, with 20% of patients affected at the time of diagnosis and up to 50% developing metachronous metastatic disease later on. Despite the growing importance of metastatic sites as independent predictors of overall survival (OS) and the ongoing development of new treatments, there has been limited effort to personalize systemic treatment based on specific metastatic locations. Aim: This study aims to compare the genomic landscape of different metastatic sites in CRC to detect novel diagnostic and therapeutic targets. Methods: DNA whole-genome-sequencing and RNA gene expression data from metastatic CRC samples were retrieved from the Hartwig Medical Foundation (HMF). Single nucleotide variants (SNVs), somatic copy number aberrations (SCNAs) and structural variants (SVs) were reported by HMF with their pipelines. In addition, focal amplifications and deletions were identified using GISTIC2 (v6.15.30). We included 464 microsatellite stable samples derived from liver metastases (n=369), peritoneal metastases (n=58) and lung metastases (n=37). We compared DNA mutation frequencies between tumors from different metastatic sites (two-sided Mann-Whitney tests) and performed differential gene expression and pathway enrichment analysis. Results: Liver metastases had the highest number of focal SCNAs per sample (median 33 vs 17 in peritoneum (p < 0.001); vs 18 in lung (p < 0.001)). Furthermore, we observed 39 site-specific SCNAs in liver, 9 in peritoneal and 15 in lung metastases. Of these, the most significant focal SCNAs were an amplification within 20q13.12 (79%) and deletion within 1p35.3 (64%) in peritoneal metastases and a deletion within 18q21.1 (84%) in lung metastases. Furthermore, peritoneal metastases displayed less genomic instability, featuring lower ploidy (median 2.65 vs 2.94 in liver (p=0.025); vs 3.15 in lung (p=0.025)) and lower aneuploidy scores (median 19 vs 26 in liver (p=0.016); vs 30 in lung (p=0.016)) while having no significant differences in tumor mutational burden or individual SNVs. Analysis of SVs revealed that peritoneal metastases exhibited fewer deletions (≥10kb; 1.3x fold change increase in liver and lung) and fewer duplications (≥1kb; 1.4x fold change increase in liver). Differential gene expression analysis uncovered higher expression of gene sets involved in the apical junction and the TGF-beta-signaling pathway in peritoneal metastases. Conclusion: Specific focal SCNA and differences in genomic instability exhibit an association with preferential organotropism in CRC metastasis and could offer potential targets for the development of new diagnostic and therapeutic approaches. Citation Format: Nerma Crnovrsanin, Soufyan Lakbir, Renske d. Wit, Steven L. Ketelaars, Niels F. Kok, Petur Snaebjornsson, Gerrit A. Meijer, Sanne Abeln, Remond J. Fijneman. Genomic and transcriptomic characterization of peritoneal, lung and liver metastases of colorectal carcinoma reveals site-specific differences [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3786.

Site-Specific Differences of Eligibility for Adjuvant Immunotherapy Among Urothelial Carcinoma Patients Treated With Radical Surgery: Results From a Multicenter Cohort Study

BackgroundThe CheckMate274 trial has reported enhanced disease-free survival rates in patients with stage pT3–4/ypT2–4 or pN+ urothelial carcinoma (UC) undergoing adjuvant nivolumab therapy. This study compares prognostic differences between urothelial carcinoma of the bladder (UCB) and upper tract urothelial carcinoma (UTUC). MethodsWe retrospectively analyzed data from 719 patients with UC who underwent radical surgery, stratifying to patients at stage pT3–4 and/or pN+ without neoadjuvant chemotherapy (NAC) or at ypT2–4 and/or ypN+ with NAC (potential candidates for adjuvant immunotherapy), and to those who were not candidates for adjuvant immunotherapy. We used Kaplan–Meier curves to assess oncological outcomes, particularly nonurothelial tract recurrence-free survival (NUTRFS), cancer-specific survival (CSS), and overall survival (OS). Risk factors were identified by Cox regression analysis. ResultsKaplan–Meier curves showed significantly lower NUTRFS, CSS, and OS for potential adjuvant immunotherapy candidates than for noncandidates in each UCB and UTUC group. NUTRFS, CSS, and OS did not differ significantly between adjuvant immunotherapy candidates with UBC or UTUC. Trends were similar among patients ineligible for adjuvant immunotherapy. Pathological T stage (pT3–4 or ypT2–4), pathological N stage, and lymphovascular invasion (LVI) were independent predictors of oncological outcomes on multivariate analysis. ConclusionThe criteria for adjuvant immunotherapy candidates from the CheckMate 274 trial can also effectively stratify UC patients after radical surgery. Substantial clinical significance is attached to LVI status as well as to pathological T and N status, suggesting that LVI status should be considered when selecting suitable candidates for adjuvant immunotherapy.