Hiding from heat: The transcriptomic response of two clam species is modulated by behaviour and habitat.

Rising occurrence of extreme warming events are profoundly impacting ecosystems, altering their functioning and services with significant socio-economic consequences. Particularly susceptible to heatwaves are intertidal shellfish beds, located in estuarine areas already stressed by factors such as rainfall events, red tides, eutrophication, and pollution. In Galicia, Northwestern Spain, these beds support vital shellfisheries, featuring the native clam Ruditapes decussatus and the non-indigenous R. philippinarum. Over recent decades, these populations have experienced notable abundance shifts due to various anthropogenic impacts, including climate change. In this habitat, patches of the seagrass Zostera noltei that coexist with bare sand can act as thermal refuges for benthic organisms such as clams. To assess the impact of heatwaves on these ecosystems, a mesocosm experiment was conducted. Juveniles of both clam species in two habitat types-bare sand and sand with Z. noltei-were exposed to simulated atmospheric heatwaves during diurnal low tide for four consecutive days. Subsequent transcriptomic analysis revealed that high temperatures had a more pronounced impact on the transcriptome of R. philippinarum compared to R. decussatus. The habitat type played a crucial role in mitigating heat stress in R. philippinarum, with the presence of Z. noltei notably ameliorating the transcriptomic response. These findings have direct applications in shellfishery management, emphasizing the importance of preserving undisturbed patches of Z. noltei as thermal refuges, contributing to the mitigation of heatwave effects on shellfish populations.

Selective Killing of BRCA2-Deficient Ovarian Cancer Cells via MRE11 Blockade.

The MRE11 nuclease is essential during DNA damage recognition, homologous recombination, and replication. BRCA2 plays important roles during homologous recombination and replication. Here, we show that effecting an MRE11 blockade using a prototypical inhibitor (Mirin) induces synthetic lethality (SL) in BRCA2-deficient ovarian cancer cells, HeLa cells, and 3D spheroids compared to BRCA2-proficient controls. Increased cytotoxicity was associated with double-strand break accumulation, S-phase cell cycle arrest, and increased apoptosis. An in silico analysis revealed Mirin docking onto the active site of MRE11. While Mirin sensitises DT40 MRE11+/- cells to the Top1 poison SN-38, it does not sensitise nuclease-dead MRE11 cells to this compound confirming that Mirin specifically inhibits Mre11 nuclease activity. MRE11 knockdown reduced cell viability in BRCA2-deficient PEO1 cells but not in BRCA2-proficient PEO4 cells. In a Mirin-resistant model, we show the downregulation of 53BP1 and DNA repair upregulation, leading to resistance, including in in vivo xenograft models. In a clinical cohort of human ovarian tumours, low levels of BRCA2 expression with high levels of MRE11 co-expression were linked with worse progression-free survival (PFS) (p = 0.005) and overall survival (OS) (p = 0.001). We conclude that MRE11 is an attractive SL target, and the pharmaceutical development of MRE11 inhibitors for precision oncology therapeutics may be of clinical benefit.

Novel Approach to Measure Transepithelial Electrical Resistance in Intestinal Cells.

The technique electric cell-substrate impedance sensing (ECIS) can be used to detect and monitor the behavior of intestinal cells. The methodology presented was designed to achieve results within a short time frame, and it was tailored to use a colonic cancer cell line. Differentiation of intestinal cancer cells has previously been reported to be regulated by retinoic acid (RA). Here, colonic cancer cells were cultured in the ECIS array before being treated with RA, and any changes in response to RA were monitored after treatment. The ECIS recorded changes in impedance in response to the treatment and vehicle. This methodology poses as a novel way to record the behavior of colonic cells and opens new avenues for in vitro research.

Differentiated Epithelial Cells of the Gut.

The intestine is a prime example of self-renewal where stem cells give rise to progenitor cells called transit-amplifying cells which differentiate into more specialized cells. There are two intestinal lineages: the absorptive (enterocytes and microfold cells) and the secretory (Paneth cells, enteroendocrine, goblet cells, and tuft cells). Each of these differentiated cell types has a role in creating an "ecosystem" to maintain intestinal homeostasis. Here, we summarize the main roles of each cell type.

In Vitro and in Vivo Assays for Testing Retinoids Effect on Intestinal Progenitors' Lineage Commitments.

The intestine consists of epithelial cells surrounded by a complex environment as mesenchymal cells and the gut microbiota. With its impressive stem cell regeneration capability, the intestine is able to constantly replenish cells lost through apoptosis or abrasion by food passing through. Over the past decade, researchers have identified signaling pathways involved in stem cell homeostasis such as retinoids pathway. Retinoids are also involved in cell differentiation of healthy and cancer cells. In this study, we describe several approaches in vitro and in vivo to further investigate the effect of retinoids on stem cells, progenitors, and differentiated intestinal cells.

Intestinal Cell Differentiation and Phenotype in 2D and 3D Cell Culture Models.

Three-dimensional (3D) culture models are more physiologically relevant than two-dimensional (2D) cell culture models. 2D approaches cannot reproduce the complexity of the tumor microenvironment and are less able to translate biological insights; and drug response studies have many limitations to be extrapolated to the clinics. Here, we use the Caco-2 colon cancer cell line, which is an immortalized human epithelial cell line that under specific conditions can polarize and differentiate into a villus-like phenotype. We describe cell differentiation and cell growth in both 2D and 3D culture conditions, concluding that cell morphology, polarity, proliferation and differentiation are highly dependent on the type of cell culture system.

What's my hamburger meat made of?

Outreach activities give high school students an opportunity to better understand the techniques and strategies used by researchers. Here is an experience with high school students designed to familiarize them with genetic methodologies. Students have been challenged to discover whether restaurant beef burgers are made with female or male beef. This represents a didactic way to introduce students to genetic traceability methodologies and also to demonstrate the usefulness of these methodologies in relation to food safety and, more importantly, in sustaining consumer confidence. The exercise is planned to be conducted in a one-day laboratory session.

Challenges for Triple Negative Breast Cancer Treatment: Defeating Heterogeneity and Cancer Stemness.

The Triple Negative Breast Cancer (TNBC) subtype is known to have a more aggressive clinical course compared to other breast cancer subtypes. Targeted therapies for this type of breast cancer are limited and patients are mostly treated with conventional chemo- and radio-therapies which are not specific and do not target resistant cells. Therefore, one of the major clinical challenges is to find compounds that target the drug-resistant cell populations which are responsible for reforming secondary tumours. The molecular profiling of the different TNBC subtypes holds a promise for better defining these resistant cells specific to each tumour. To this end, a better understanding of TNBC heterogeneity and cancer stemness is required, and extensive genomic analysis can help to understand the disease complexity and distinguish new molecular drivers that can be targeted in the clinics. The use of persister cancer cell-targeting therapies combined with other therapies may provide a big advance to improve TNBC patients' survival.

Targeting Mre11 overcomes platinum resistance and induces synthetic lethality in XRCC1 deficient epithelial ovarian cancers.

Platinum resistance is a clinical challenge in ovarian cancer. Platinating agents induce DNA damage which activate Mre11 nuclease directed DNA damage signalling and response (DDR). Upregulation of DDR may promote chemotherapy resistance. Here we have comprehensively evaluated Mre11 in epithelial ovarian cancers. In clinical cohort that received platinum- based chemotherapy (n = 331), Mre11 protein overexpression was associated with aggressive phenotype and poor progression free survival (PFS) (p = 0.002). In the ovarian cancer genome atlas (TCGA) cohort (n = 498), Mre11 gene amplification was observed in a subset of serous tumours (5%) which correlated highly with Mre11 mRNA levels (p < 0.0001). Altered Mre11 levels was linked with genome wide alterations that can influence platinum sensitivity. At the transcriptomic level (n = 1259), Mre11 overexpression was associated with poor PFS (p = 0.003). ROC analysis showed an area under the curve (AUC) of 0.642 for response to platinum-based chemotherapy. Pre-clinically, Mre11 depletion by gene knock down or blockade by small molecule inhibitor (Mirin) reversed platinum resistance in ovarian cancer cells and in 3D spheroid models. Importantly, Mre11 inhibition was synthetically lethal in platinum sensitive XRCC1 deficient ovarian cancer cells and 3D-spheroids. Selective cytotoxicity was associated with DNA double strand break (DSB) accumulation, S-phase cell cycle arrest and increased apoptosis. We conclude that pharmaceutical development of Mre11 inhibitors is a viable clinical strategy for platinum sensitization and synthetic lethality in ovarian cancer.

Detecting Local Adaptation between North and South European Atlantic Salmon Populations.

Pollution and other anthropogenic effects have driven a decrease in Atlantic salmon (Salmo salar) in the Iberian Peninsula. The restocking effort carried out in the 1980s, with salmon from northern latitudes with the aim of mitigating the decline of native populations, failed, probably due to the deficiency in adaptation of foreign salmon from northern Europe to the warm waters of the Iberian Peninsula. This result would imply that the Iberian populations of Atlantic salmon have experienced local adaptation in their past evolutionary history, as has been described for other populations of this species and other salmonids. Local adaptation can occur by divergent selections between environments, favoring the fixation of alleles that increase the fitness of a population in the environment it inhabits relative to other alleles favored in another population. In this work, we compared the genomes of different populations from the Iberian Peninsula (Atlantic and Cantabric basins) and Scotland in order to provide tentative evidence of candidate SNPs responsible for the adaptive differences between populations, which may explain the failures of restocking carried out during the 1980s. For this purpose, the samples were genotyped with a 220,000 high-density SNP array (Affymetrix) specific to Atlantic salmon. Our results revealed potential evidence of local adaptation for North Spanish and Scottish populations. As expected, most differences concerned the comparison of the Iberian Peninsula with Scotland, although there were also differences between Atlantic and Cantabric populations. A high proportion of the genes identified are related to development and cellular metabolism, DNA transcription and anatomical structure. A particular SNP was identified within the NADP-dependent malic enzyme-2 (mMEP-2*), previously reported by independent studies as a candidate for local adaptation in salmon from the Iberian Peninsula. Interestingly, the corresponding SNP within the mMEP-2* region was consistent with a genomic pattern of divergent selection.

Dynamic changes in DNA methylation during seahorse (Hippocampus reidi) postnatal development and settlement.

INTRODUCTION: Most living marine organisms have a biphasic life cycle dependent on metamorphosis and settlement. These critical life-history events mean that a developmentally competent larva undergoes a range of coordinated morphological and physiological changes that are in synchrony with the ecological transition from a pelagic to a benthonic lifestyle. Therefore, transition from a pelagic to a benthonic habitat requires multiple adaptations, however, the underlying mechanisms regulating this process still remains unclear. Epigenetic regulation and specifically DNA methylation, has been suggested to be particularly important for organisms to adapt to new environments. Seahorses (Family Syngnathidae, Genus Hippocampus) are a fascinating group of fish, distinguished by their unique anatomical features, reproductive strategy and behavior. They are unique among vertebrate species due to their "male pregnancy", where males nourish developing embryos and larvae in a brood pouch until hatching and parturition occurs. After birth, free-swimming offspring are pelagic and subsequently they change into a demersal lifestyle. Therefore, to begin to address the question whether epigenetic processes could be involved in the transition from a planktonic to a benthonic lifestyle observed in seahorses, we studied global DNA methylation profiles in a tropical seahorse species (Hippocampus reidi) during postnatal development and settlement. RESULTS: We performed methylation-sensitive amplified polymorphism (MSAP) along with quantitative expression analysis for genes suggested to be involved in the methylation machinery at six age groups: 1, 5, 10, 20, 30 and 40 days after male's pouch release (DAR). Results revealed that the H. reidi genome has a significantly different DNA methylation profile during postnatal development and settlement on demersal habitats. Moreover, gene expression analysis showed up- and down-regulation of specific DNA methyltransferases (DNMTs) encoding genes. CONCLUSION: Our data show that the differences in the DNA methylation patterns seen among developmental stages and during the transition from a pelagic to a benthonic lifestyle suggest a potential for epigenetic regulation of gene expression (through DNA methylation) in this species. Therefore, epigenetic mechanisms could be necessary for seahorse settlement. Nevertheless, if these epigenetic mechanisms come from internal or if they are initiated via external environmental cues should be further investigated.

Biomaterials for intestinal organoid technology and personalized disease modeling.

Recent advances in intestinal organoid technologies have paved the way for in vitro recapitulation of the homeostatic renewal of adult tissues, tissue or organ morphogenesis during development, and pathogenesis of many disorders. In vitro modelling of individual patient diseases using organoid systems have been considered key in establishing rational design of personalized treatment strategies and in improving therapeutic outcomes. In addition, the transplantation of organoids into diseased tissues represents a novel approach to treat currently incurable diseases. Emerging evidence from intensive studies suggests that organoid systems' development and functional maturation depends on the presence of an extracellular matrix with suitable biophysical properties, where advanced synthetic hydrogels open new avenues for theoretical control of organoid phenotypes and potential applications of organoids in therapeutic purposes. In this review, we discuss the status, applications, challenges and perspectives of intestinal organoid systems emphasising on hydrogels and their properties suitable for intestinal organoid culture. We provide an overview of hydrogels used for intestinal organoid culture and key factors regulating their biological activity. The comparison of different hydrogels would be a theoretical basis for establishing design principles of synthetic niches directing intestinal cell fates and functions. STATEMENT OF SIGNIFICANCE: Intestinal organoid is an in vitro recapitulation of the gut, which self-organizes from intestinal stem cells and maintains many features of the native tissue. Since the development of this technology, intestinal organoid systems have made significant contribution to rapid progress in intestinal biology. Prevailing methodology for organoid culture, however, depends on animal-derived matrices and suffers from variability and potential risk for contamination of pathogens, limiting their therapeutic application. Synthetic scaffold matrices, hydrogels, might provide solutions to these issues and deepen our understanding on how intestinal cells sense and respond to key biophysical properties of the surrounding matrices. This review provides an overview of developing intestinal models and biomaterials, thereby leading to better understanding of current intestinal organoid systems for both biologists and materials scientists.

Genomic Instability Profiles at the Single Cell Level in Mouse Colorectal Cancers of Defined Genotypes.

The genomes of many human CRCs have been sequenced, revealing a large number of genetic alterations. However, the molecular mechanisms underlying the accumulation of these alterations are still being debated. In this study, we examined colorectal tumours that developed in mice with Apclox/lox, LSL-KrasG12D, and Tp53lox/lox targetable alleles. Organoids were derived from single cells and the spectrum of mutations was determined by exome sequencing. The number of single nucleotide substitutions (SNSs) correlated with the age of the tumour, but was unaffected by the number of targeted cancer-driver genes. Thus, tumours that expressed mutant Apc, Kras, and Tp53 alleles had as many SNSs as tumours that expressed only mutant Apc. In contrast, the presence of large-scale (>10 Mb) copy number alterations (CNAs) correlated strongly with Tp53 inactivation. Comparison of the SNSs and CNAs present in organoids derived from the same tumour revealed intratumoural heterogeneity consistent with genomic lesions accumulating at significantly higher rates in tumour cells compared to normal cells. The rate of acquisition of SNSs increased from the early stages of cancer development, whereas large-scale CNAs accumulated later, after Tp53 inactivation. Thus, a significant fraction of the genomic instability present in cancer cells cannot be explained by aging processes occurring in normal cells before oncogenic transformation.

High-content, targeted RNA-seq screening in organoids for drug discovery in colorectal cancer.

Organoids allow the recapitulation of intestinal homeostasis and cancerogenesis in vitro; however, RNA sequencing (RNA-seq)-based methods for drug screens are missing. We develop targeted organoid sequencing (TORNADO-seq), a high-throughput, high-content drug discovery platform that uses targeted RNA-seq to monitor the expression of large gene signatures for the detailed evaluation of cellular phenotypes in organoids. TORNADO-seq is a fast, highly reproducible time- and cost-effective ($5 per sample) method that can probe cell mixtures and their differentiation state in the intestinal system. We apply this method to isolate drugs that enrich for differentiated cell phenotypes and show that these drugs are highly efficacious against cancer compared to wild-type organoids. Furthermore, TORNADO-seq facilitates in-depth insight into the mode of action of these drugs. Our technology can easily be adapted to many other systems and will allow for more systematic, large-scale, and quantitative approaches to study the biology of complex cellular systems.

Exercise to explain X-chromosome inactivation in humans.

Distance learning requires the combined use of techniques because it is more complicated to keep the students' attention. This exercise is designed to explain the inactivation of the x-chromosome in humans and is intended to complement the theoretical explanations. It is estimated that it lasts two hours and makes use of different web resources. It is intended for students familiar with the use of BLAST tools.

Large-Scale Production of Recombinant Noggin and R-Spondin1 Proteins Required for the Maintenance of Stem Cells in Intestinal Organoid Cultures.

The presence of the proteins mouse R-Spondin1 (mRSpo1) and mouse Noggin (mNoggin) in a 3D-organoid culture allows for the maintenance of intestinal stem cells. Here, we describe a transient gene expression method for the production of these proteins from human embryo kidney 293 (HEK293) cells cultivated in suspension using orbitally shaken bioreactors. Plasmid DNA was delivered into cells using the cationic polymer polyethylenimine (PEI). The 7-day production cultures were performed in the presence of valproic acid (VPA), an enhancer of recombinant gene expression. Both proteins were secreted from the transfected cells. mRSpo1 was produced as a secreted Fc fusion protein (mRSpo1-Fc) and purified by protein A-based affinity chromatography. mNoggin was produced as a secreted histidine-tagged protein (mNoggin-His) and purified by immobilized metal affinity chromatography (IMAC). This transient transfection system supports a high production efficiency.

Single-Cell Studies of Intestinal Stem Cell Heterogeneity During Homeostasis and Regeneration.

Single-cell RNA-sequencing (scRNA-seq) provides a unique opportunity to study heterogeneous cell populations within tissues, including the intestinal epithelium, to gain detailed molecular insights into their biology. Many new putative markers of intestinal stem cells and their progeny have been described using single-cell transcriptomics, which has contributed to the identification of novel subpopulations of mature cell types and insight into their developmental trajectories. This approach has revealed tremendous cellular heterogeneity within the intestinal epithelium that is concordant with its diverse and multifaceted functions. We discuss the function of these subpopulations during tissue homeostasis, as well as putative subpopulations with inducible regenerative potential following tissue injury.

Specific Gene Expression in Lgr5+ Stem Cells by Using Cre-Lox Recombination.

Intestinal stem cells are responsible for tissue renewal. The study of stem cell properties has become a major challenge in the field. We describe here a method based on Cre recombinase inducible lentivirus vectors that permits delivery of transgenes, either for overexpression or knockdown, in primary stem cells that can be cultured in an 3D intestinal organoid system. This method is an excellent approach for genetic manipulation and can complement in vivo transgenic experiments.

An Intrasplenic Injection Model for the Study of Cancer Stem Cell Seeding Capacity.

In many tumor types, only a minor pool of cancer cells-the so-called cancer stem cells-is able to colonize distant organs and give rise to secondary tumors. In humans, the liver is one of the main target organs for many metastatic tumor types, including colorectal cancer. However, mouse tumour models only rarely spontaneously metastasize to the liver. Therefore, reliable in vivo experimental metastasis assays are crucial to study cell seeding capacity and the mechanisms controlling these metastatic stem cell properties. Here, we describe an intrasplenic injection model that mimics the process of liver metastasis occurring in cancer patients.

Identification of genomic regions regulating sex determination in Atlantic salmon using high density SNP data.

BACKGROUND: A complete understanding of the genetic basis for sexual determination and differentiation is necessary in order to implement efficient breeding schemes at early stages of development. Atlantic salmon belongs to the family Salmonidae of fishes and represents a species of great commercial value. Although the species is assumed to be male heterogametic with XY sex determination, the precise genetic basis of sexual development remains unclear. The complexity is likely associated to the relatively recent salmonid specific whole genome duplication that may be responsible for certain genome instability. This instability together with the capacity of the sex-determining gene to move across the genome as reported by previous studies, may explain that sexual development genes are not circumscribed to the same chromosomes in all members of the species. In this study, we have used a 220 K SNP panel developed for Atlantic salmon to identify the chromosomes explaining the highest proportion of the genetic variance for sex as well as candidate regions and genes associated to sexual development in this species. RESULTS: Results from regional heritability analysis showed that the chromosomes explaining the highest proportion of variance in these populations were Ssa02 (heritability = 0.42, SE = 0.12) and Ssa21 (heritability = 0.26, SE = 0.11). After pruning by linkage disequilibrium, genome-wide association analyses revealed 114 SNPs that were significantly associated with sex, being Ssa02 the chromosome containing a greatest number of regions. Close examination of the candidate regions evidenced important genes related to sex in other species of Class Actinopterygii, including SDY, genes from family SOX, RSPO1, ESR1, U2AF2A, LMO7, GNRH-R, DND and FIGLA. CONCLUSIONS: The combined results from regional heritability analysis and genome-wide association have provided new advances in the knowledge of the genetic regulation of sex determination in Atlantic salmon, supporting that Ssa02 is the candidate chromosome for sex in this species and suggesting an alternative population lineage in Spanish wild populations according to the results from Ssa21.