The drug-induced phenotypic landscape of colorectal cancer organoids.

Patient-derived organoids resemble the biology of tissues and tumors, enabling ex vivo modeling of human diseases. They have heterogeneous morphologies with unclear biological causes and relationship to treatment response. Here, we use high-throughput, image-based profiling to quantify phenotypes of over 5 million individual colorectal cancer organoids after treatment with >500 small molecules. Integration of data using multi-omics modeling identifies axes of morphological variation across organoids: Organoid size is linked to IGF1 receptor signaling, and cystic vs. solid organoid architecture is associated with LGR5 + stemness. Treatment-induced organoid morphology reflects organoid viability, drug mechanism of action, and is biologically interpretable. Inhibition of MEK leads to cystic reorganization of organoids and increases expression of LGR5, while inhibition of mTOR induces IGF1 receptor signaling. In conclusion, we identify shared axes of variation for colorectal cancer organoid morphology, their underlying biological mechanisms, and pharmacological interventions with the ability to move organoids along them.

A promising deep learning-assistive algorithm for histopathological screening of colorectal cancer.

Colorectal cancer is one of the most common cancers worldwide, accounting for an annual estimated 1.8 million incident cases. With the increasing number of colonoscopies being performed, colorectal biopsies make up a large proportion of any histopathology laboratory workload. We trained and validated a unique artificial intelligence (AI) deep learning model as an assistive tool to screen for colonic malignancies in colorectal specimens, in order to improve cancer detection and classification; enabling busy pathologists to focus on higher order decision-making tasks. The study cohort consists of Whole Slide Images (WSI) obtained from 294 colorectal specimens. Qritive's unique composite algorithm comprises both a deep learning model based on a Faster Region Based Convolutional Neural Network (Faster-RCNN) architecture for instance segmentation with a ResNet-101 feature extraction backbone that provides glandular segmentation, and a classical machine learning classifier. The initial training used pathologists' annotations on a cohort of 66,191 image tiles extracted from 39 WSIs. A subsequent application of a classical machine learning-based slide classifier sorted the WSIs into 'low risk' (benign, inflammation) and 'high risk' (dysplasia, malignancy) categories. We further trained the composite AI-model's performance on a larger cohort of 105 resections WSIs and then validated our findings on a cohort of 150 biopsies WSIs against the classifications of two independently blinded pathologists. We evaluated the area under the receiver-operator characteristic curve (AUC) and other performance metrics. The AI model achieved an AUC of 0.917 in the validation cohort, with excellent sensitivity (97.4%) in detection of high risk features of dysplasia and malignancy. We demonstrate an unique composite AI-model incorporating both a glandular segmentation deep learning model and a classical machine learning classifier, with excellent sensitivity in picking up high risk colorectal features. As such, AI plays a role as a potential screening tool in assisting busy pathologists by outlining the dysplastic and malignant glands.

Continuing fragmentation of a widespread species by geographical barriers as initial step in a land snail radiation on crete.

The phylogeographic structure of the land snail Xerocrassa mesostena on Crete inferred from AFLP markers and mitochondrial cox1 sequences can be explained by three mechanisms: gene flow restriction, population expansion and leptokurtic dispersal. Gene flow restriction by geographic barriers caused subdivision of the gene pool into distinct clusters. Population expansion was probably facilitated by deforestation of Crete in the postglacial. Newly available areas were colonized by leptokurtic dispersal, i.e. slow active expansion resulting in isolation by distance within the clusters and occasional long distance dispersal events that resulted in departures from the isolation by distance model. Less than one percent of the AFLP markers show correlations with environmental variables. Random phylogeographic breaks in the distribution of the mitochondrial haplotype groups indicate that single locus markers, especially mitochondrial DNA, might result in a misleading picture of the phylogeographic structure of a species. Restriction of gene flow between metapopulations caused by geographical barriers can interact with sexual selection resulting in the differentiation of these metapopulations into separate species without noticeable ecological differentiation. Evidence for gene flow between parapatrically distributed evolutionary units representing different stages of the speciation process suggests that the ongoing process of fragmentation of the X. mesostena complex might be an example for parapatric speciation. The lack of ecological differentiation between these units confirms theoretical predictions that divergent selection for local adaptation is not required for rapid speciation.

A comparison of DNA-based methods for delimiting species in a Cretan land snail radiation reveals shortcomings of exclusively molecular taxonomy.

We compared the results of different approaches for delimiting species based on single-locus DNA sequences with those of methods using binary multilocus data. As case study, we examined the radiation of the land snail genus Xerocrassa on Crete. Many of the methods based on mitochondrial sequences resulted in heavy under- or overestimations of the species number. The methods using AFLP data produced classifications with an on average higher concordance with the morphological classification than the methods based on mitochondrial sequences. However, the percentage of correct species classifications is low even with binary multilocus data. Gaussian clustering produced the classifications with the highest concordance with the morphological classification of all approaches applied in this study, both with single-locus sequences and with binary multilocus data. There are two general problems that hamper species delimitation, namely rarity and the hierarchical structure of biodiversity. Methods for species delimitation using genetic data search for clusters of individuals, but do not implement criteria that are sufficient to distinguish clusters representing species from other clusters. The success of morphological species delimitation results from the potential to focus on characters that are directly involved in the speciation process, whereas molecular studies usually rely on markers that are not directly involved in speciation. © The Willi Hennig Society 2011.

Pro-inflammatory effects of ochratoxin A on nasal epithelial cells.

Eosinophilic chronic rhinosinusitis (ECRS) largely consists of allergic fungal sinusitis, non-allergic fungal rhinosinusitis, aspirin-exacerbated ECRS, and superantigen-induced ECRS. The pathophysiology of ECRS is not completely understood, in particular, the role of mycotoxins remains unknown. The aim of this study was to evaluate the effects of one of the most widespread mycotoxin, ochratoxin A (OTA), on the release of pro-inflammatory cytokines such as interleukin-(IL)-6 and the CXC-chemokine IL-8 from nasal epithelial cell cultures (NEC) of subjects with and without ECRS. NEC (ECRS group: n = 16; controls: n = 7) were stimulated with OTA for 24 h. Protein concentrations of IL-6 and IL-8 levels were measured in NEC supernatants by ELISA prior and 24 h after addition of OTA. Baseline levels in the supernatants of NEC were 183.3 pg/ml for IL-6 and 384.6 pg/ml for IL-8. Stimulation with OTA induced a significant increase of IL-6 (p < 0.001) and IL-8 (p < 0.001) in both NEC of controls and ECRS, respectively. There were no significant differences between controls and ECRS. This is the first study evaluating the effects of a mycotoxin on epithelial airway cells. Our data show that the ubiquitous mycotoxin OTA has a strong pro-inflammatory effect on NEC resulting in the release of IL-6 and IL-8. Mycotoxins may promote inflammation in nasal mucosa.

Reconstructing the evolutionary history of the radiation of the land snail genus Xerocrassa on Crete based on mitochondrial sequences and AFLP markers.

BACKGROUND: A non-adaptive radiation triggered by sexual selection resulted in ten endemic land snail species of the genus Xerocrassa on Crete. Only five of these species and a more widespread species are monophyletic in a mitochondrial gene tree. The reconstruction of the evolutionary history of such closely related species can be complicated by incomplete lineage sorting, introgression or inadequate taxonomy. To distinguish between the reasons for the nonmonophyly of several species in the mitochondrial gene tree we analysed nuclear AFLP markers. RESULTS: Whereas six of the eleven morphologically delimited Xerocrassa species from Crete are monophyletic in the mitochondrial gene tree, nine of these species are monophyletic in the tree based on AFLP markers. Only two morphologically delimited species could not be distinguished with the multilocus data and might have diverged very recently or might represent extreme forms of a single species. The nonmonophyly of X. rhithymna with respect to X. kydonia is probably the result of incomplete lineage sorting, because there is no evidence for admixture in the AFLP data and the mitochondrial haplotype groups of these species coalesce deeply. The same is true for the main haplotype groups of X. mesostena. The nonmonophyly of X. franciscoi might be the result of mitochondrial introgression, because the coalescences of the haplotypes of this species with some X. mesostena haplotypes are shallow and there is admixture with neighbouring X. mesostena. CONCLUSION: The most likely causes for the nonmonophyly of species in the mitochondrial gene tree of the Xerocrassa radiation on Crete could be inferred using AFLP data by a combination of several criteria, namely the depth of the coalescences in the gene tree, the geographical distribution of shared genetic markers, and concordance with results of admixture analyses of nuclear multilocus markers. The strongly subdivided population structure increases the effective population size of land snail species and, thus, the likelihood of a long persistence of ancestral polymorphisms. Our study suggests that ancestral polymorphisms are a frequent cause for nonmonophyly of species with a strongly subdivided population structure in gene trees.

Sexual selection is involved in speciation in a land snail radiation on crete.

We investigated the importance of sexual selection in facilitating speciation in a land snail radiation on Crete. We used differences in the genitalia of the Cretan Xerocrassa species as potential indices of sexual selection. First, we rejected the hypothesis that differences in the genitalia of the Xerocrassa species can be explained by genetic drift using coalescent simulations based on a mitochondrial gene tree. Second, we showed that there is no evidence for the hypothesis that the differences in the genitalia can be explained by natural selection against hybrids under the assumption that this is more likely in geographically overlapping species pairs and clades. Third, we showed that there is a positive scaling between male spermatophore-producing organs and female spermatophore-receiving organs indicating sexual coevolution. The spermatophore enables the sperm to escape from the female gametolytic organ. Thus, the coevolution might be a consequence of sexual conflict or cryptic female choice. Finally, we showed that the evolution of differences in the length of the flagellum that forms the tail of the spermatophore is concentrated toward the tips of the tree indicating that it is involved in speciation. If speciation is facilitated by sexual selection, niches may remain conserved and nonadaptive radiation may result.

Nasal colonization with Staphylococcus aureus is not associated with the severity of symptoms or the extent of the disease in chronic rhinosinusitis.

OBJECTIVES: There is an increasing knowledge that the severity of perennial allergic rhinitis is associated with nasal carriage of Staphylococcus aureus (S. aureus). The aim of this study was to evaluate the rate of bacterial colonization with S. aureus in the nose of subjects with and without chronic rhinosinusitis (CRS) and to correlate these findings with the severity of symptoms and the extent of the disease. STUDY DESIGN AND SETTING: Open, prospective controlled trial. METHODS: 190 subjects with CRS and 42 subjects with septal deviation without sinusitis (control subjects) were included in this study. Swabs were taken endoscopically from the middle meatus and bacteria were cultured and identified. Airway symptoms were assessed by subjects in standardized questionnaires and frequencies of respiratory tract infections were noted. The rhinosinusitis extent was graded by CT scan assessment. Analysis of variance, chi-square test, and Pearson's correlation test were applied for statistical analyses. RESULTS: The S. aureus carriage rate was 25.5% in CRS and 31.4% in control subjects. Further facultative pathogens were cultured in 20.6% of subjects with CRS and in 8.5% of controls. 73.8% of S. aureus were ampicillin-resistant, multiresistant strains were cultured in 5.8%. Most airway symptoms and the frequencies of respiratory tract infections were significantly higher in the CRS group compared with control subjects. In post hoc comparison between the subgroups with and without S. aureus colonization, no significant differences were found between the extent of rhinosinusitis and the severity of airway symptoms. CONCLUSIONS: Facultative pathogens were detected in more than 40% of CRS patients without acute respiratory tract infections. In contrast to the findings in atopic dermatitis and perennial allergic rhinitis, the colonization rate with S. aureus is not increased and S. aureus carriage is not a risk factor for the severity or the extent of rhinosinusitis.