Single-cell technologies have described heterogeneity across tissues, but the spatial distribution and forces that drive single-cell phenotypes have not been well defined. Combining single-cell RNA ...and protein analytics in studying the role of stromal cancer-associated fibroblasts (CAFs) in modulating heterogeneity in pancreatic cancer (pancreatic ductal adenocarcinoma PDAC) model systems, we have identified significant single-cell population shifts toward invasive epithelial-to-mesenchymal transition (EMT) and proliferative (PRO) phenotypes linked with mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) signaling. Using high-content digital imaging of RNA in situ hybridization in 195 PDAC tumors, we quantified these EMT and PRO subpopulations in 319,626 individual cancer cells that can be classified within the context of distinct tumor gland “units.” Tumor gland typing provided an additional layer of intratumoral heterogeneity that was associated with differences in stromal abundance and clinical outcomes. This demonstrates the impact of the stroma in shaping tumor architecture by altering inherent patterns of tumor glands in human PDAC.
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•Cancer-associated fibroblasts contribute to pancreatic cancer heterogeneity•Cancer cells can have a double-positive phenotype: proliferation and invasion•High CAF abundance linked with DP cells enriched for MAPK and STAT3 co-signaling•Intra-tumoral gland types provide tissue heterogeneity linked with clinical outcome
Clinical outcomes for pancreatic cancer are impacted by intra-tumoral tissue architecture as defined by single-cell analyses and high content digital imaging.
The drivers of critical coronavirus disease 2019 (COVID-19) remain unknown. Given major confounding factors such as age and comorbidities, true mediators of this condition have remained elusive. We ...used a multi-omics analysis combined with artificial intelligence in a young patient cohort where major comorbidities were excluded at the onset. The cohort included 47 “critical” (in the intensive care unit under mechanical ventilation) and 25 “non-critical” (in a non-critical care ward) patients with COVID-19 and 22 healthy individuals. The analyses included whole-genome sequencing, whole-blood RNA sequencing, plasma and blood mononuclear cell proteomics, cytokine profiling, and high-throughput immunophenotyping. An ensemble of machine learning, deep learning, quantum annealing, and structural causal modeling were used. Patients with critical COVID-19 were characterized by exacerbated inflammation, perturbed lymphoid and myeloid compartments, increased coagulation, and viral cell biology. Among differentially expressed genes, we observed up-regulation of the metalloprotease
. This gene signature was validated in a second independent cohort of 81 critical and 73 recovered patients with COVID-19 and was further confirmed at the transcriptional and protein level and by proteolytic activity. Ex vivo ADAM9 inhibition decreased severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uptake and replication in human lung epithelial cells. In conclusion, within a young, otherwise healthy, cohort of individuals with COVID-19, we provide the landscape of biological perturbations in vivo where a unique gene signature differentiated critical from non-critical patients. We further identified
as a driver of disease severity and a candidate therapeutic target.
Scope
The differences between the baseline gut microbiota of patients who developed type 2 diabetes (T2D) consuming a low‐fat (LF) or a Mediterranean (Med) diet are explored and risk scores are ...developed to predict the individual risk of developing T2D associated with the consumption of LF or Med diet.
Methods and Results
All the patients from the CORDIOPREV study without T2D at baseline (n = 462) whose fecal sample are available, are included. Gut microbiota is analyzed by 16S sequencing and the risk of T2D after a median follow‐up of 60 months assessed by Cox analysis. Linear discriminant analysis effect size (LEfSe) analysis shows a different baseline gut microbiota in patients who developed T2D consuming LF and Med diets. A higher abundance of Paraprevotella, and lower Gammaproteobacteria and B. uniformis are associated with T2D risk when an LF diet is consumed. In contrast, higher abundances of Saccharibacteria, Betaproteobacteria, and Prevotella are associated with T2D risk when a Med diet is consumed.
Conclusion
The results suggest that different interactions between the microbiome and dietary patterns may partially determine the risk of T2D development, which may be used for selecting personalized dietary models to prevent T2D.
Gut microbiome may play a role in the different responses to dietary interventions. The results suggest that different interactions between the microbiome and dietary patterns may partially determine the risk of type 2 diabetes development according to the diet who is going to be consumed, which may be used for selecting personalized dietary models to prevent type 2 diabetes.
The incidence of type 2 diabetes mellitus (T2DM) is growing in Western countries. Nutritional interventions that promote high-quality dietary patterns could help reverse this trend. We aimed to ...evaluate whether changes in Nutrient-Rich Food Index 9.3 (NRF9.3) were related to the risk of developing T2DM in patients with coronary heart disease (CHD). The study was carried out in the context of two healthy dietary interventions (a Mediterranean and a low-fat diet). For this purpose, we evaluated all the patients in the CORDIOPREV study without T2DM at baseline. Data were obtained during the first 5 years of dietary intervention. The score was calculated using the Food Frequency Questionnaires at baseline and after 1 year of intervention. After 5 years of follow-up, 106 patients developed T2DM (incident-T2DM), while 316 subjects did not (non-T2DM). Total NRF9.3 score and changes during the first year of intervention were compared between incident-T2DM and non-T2DM. Incident-T2DM showed less improvement in NRF9.3 than non-T2DM (
= 0.010). In the multi-adjusted Cox proportional hazard study, patients with greater improvement in NRF9.3 had over 50% less risk of developing T2DM compared with the lowest tertile (HR 2.10, 95%, CI = 1.12-3.56). In conclusion, improved diet quality in terms of nutrient density after the dietary intervention was associated with a lower risk of T2DM in patients with CHD.
Background Type 2 diabetes mellitus (T2DM) is one of the most widely spread diseases, affecting around 90% of the patients with diabetes. Metabolomics has proven useful in diabetes research ...discovering new biomarkers to assist in therapeutical studies and elucidating pathways of interest. However, this technique has not yet been applied to a cohort of patients that have remitted from T2DM. Methods All patients with a newly diagnosed T2DM at baseline (n = 190) were included. An untargeted metabolomics approach was employed to identify metabolic differences between individuals who remitted (RE), and those who did not (non-RE) from T2DM, during a 5-year study of dietary intervention. The biostatistical pipeline consisted of an orthogonal projection on the latent structure discriminant analysis (O-PLS DA), a generalized linear model (GLM), a receiver operating characteristic (ROC), a DeLong test, a Cox regression, and pathway analyses. Results The model identified a significant increase in 12 metabolites in the non-RE group compared to the RE group. Cox proportional hazard models, calculated using these 12 metabolites, showed that patients in the high-score tercile had significantly (p-value < 0.001) higher remission probabilities (Hazard Ratio, HR, .sub.high versus low = 2.70) than those in the lowest tercile. The predictive power of these metabolites was further studied using GLMs and ROCs. The area under the curve (AUC) of the clinical variables alone is 0.61, but this increases up to 0.72 if the 12 metabolites are considered. A DeLong test shows that this difference is statistically significant (p-value = 0.01). Conclusions Our study identified 12 endogenous metabolites with the potential to predict T2DM remission following a dietary intervention. These metabolites, combined with clinical variables, can be used to provide, in clinical practice, a more precise therapy. Trial registration ClinicalTrials.gov, NCT00924937. Keywords: Diabetes, Insulin resistance, Prospective human study, Metabolomics
We aimed to identify a lipidic profile associated with type 2 diabetes mellitus (T2DM) development in coronary heart disease (CHD) patients, to provide a new, highly sensitive model which could be ...used in clinical practice to identify patients at T2DM risk.
This study considered the 462 patients of the CORDIOPREV study (CHD patients) who were not diabetic at the beginning of the intervention. In total, 107 of them developed T2DM after a median follow-up of 60 months. They were diagnosed using the American Diabetes Association criteria. A novel lipidomic methodology employing liquid chromatography (LC) separation followed by HESI, and detection by mass spectrometry (MS) was used to annotate the lipids at the isomer level. The patients were then classified into a Training and a Validation Set (60-40). Next, a Random Survival Forest (RSF) was carried out to detect the lipidic isomers with the lowest prediction error, these lipids were then used to build a Lipidomic Risk (LR) score which was evaluated through a Cox. Finally, a production model combining the clinical variables of interest, and the lipidic species was carried out.
LC-tandem MS annotated 440 lipid species. From those, the RSF identified 15 lipid species with the lowest prediction error. These lipids were combined in an LR score which showed association with the development of T2DM. The LR hazard ratio per unit standard deviation was 2.87 and 1.43, in the Training and Validation Set respectively. Likewise, patients with higher LR Score values had lower insulin sensitivity (P = 0.006) and higher liver insulin resistance (P = 0.005). The receiver operating characteristic (ROC) curve obtained by combining clinical variables and the selected lipidic isomers using a generalised lineal model had an area under the curve (AUC) of 81.3%.
Our study showed the potential of comprehensive lipidomic analysis in identifying patients at risk of developing T2DM. In addition, the lipid species combined with clinical variables provided a new, highly sensitive model which can be used in clinical practice to identify patients at T2DM risk. Moreover, these results also indicate that we need to look closely at isomers to understand the role of this specific compound in T2DM development. Trials registration NCT00924937.
Cardiovascular diseases (CVD), including coronary heart disease (CHD), display a higher prevalence in men than women. This study aims to evaluate the variations in the intestinal microbiota between ...men and women afflicted with CHD and delineate these against a non-CVD control group for each sex.
Our research was conducted in the framework of the CORDIOPREV study, a clinical trial which involved 837 men and 165 women with CHD. We contrasted our findings with a reference group of 375 individuals (270 men, 105 women) without CVD. The intestinal microbiota was examined through 16S metagenomics on the Illumina MiSeq platform and the data processed with Quiime2 software.
Our results showed a sex-specific variation (beta diversity) in the intestinal microbiota, while alpha-biodiversity remained consistent across both sexes. Linear discriminant analysis effect size (LEfSe) analysis revealed sex-centric alterations in the intestinal microbiota linked to CVD. Moreover, using random forest (RF) methodology, we identified seven bacterial taxa-g_UBA1819 (Ruminococcaceae), g_Bilophila, g_Subdoligranulum, g_Phascolarctobacterium, f_Barnesiellaceae, g_Ruminococcus, and an unknown genus from the Ruminococcaceae family (Ruminococcaceae incertae sedis)-as key discriminators between men and women diagnosed with CHD. The same taxa also emerged as critical discriminators between CHD-afflicted and non-CVD individuals, when analyzed separately by sex.
Our findings suggest a sex-specific dysbiosis in the intestinal microbiota linked to CHD, potentially contributing to the sex disparity observed in CVD incidence. Trial registration Clinical Trials.gov.Identifier NCT00924937.
Sensitive detection of off-target effects is important for translating CRISPR-Cas9 nucleases into human therapeutics. In vitro biochemical methods for finding off-targets offer the potential ...advantages of greater reproducibility and scalability while avoiding limitations associated with strategies that require the culture and manipulation of living cells. Here we describe circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), a highly sensitive, sequencing-efficient in vitro screening strategy that outperforms existing cell-based or biochemical approaches for identifying CRISPR-Cas9 genome-wide off-target mutations. In contrast to previously described in vitro methods, we show that CIRCLE-seq can be practiced using widely accessible next-generation sequencing technology and does not require reference genome sequences. Importantly, CIRCLE-seq can be used to identify off-target mutations associated with cell-type-specific single-nucleotide polymorphisms, demonstrating the feasibility and importance of generating personalized specificity profiles. CIRCLE-seq provides an accessible, rapid, and comprehensive method for identifying genome-wide off-target mutations of CRISPR-Cas9.
Broad use of CRISPR-Cas12a (formerly Cpf1) nucleases
has been hindered by the requirement for an extended TTTV protospacer adjacent motif (PAM)
. To address this limitation, we engineered an enhanced ...Acidaminococcus sp. Cas12a variant (enAsCas12a) that has a substantially expanded targeting range, enabling targeting of many previously inaccessible PAMs. On average, enAsCas12a exhibits a twofold higher genome editing activity on sites with canonical TTTV PAMs compared to wild-type AsCas12a, and we successfully grafted a subset of mutations from enAsCas12a onto other previously described AsCas12a variants
to enhance their activities. enAsCas12a improves the efficiency of multiplex gene editing, endogenous gene activation and C-to-T base editing, and we engineered a high-fidelity version of enAsCas12a (enAsCas12a-HF1) to reduce off-target effects. Both enAsCas12a and enAsCas12a-HF1 function in HEK293T and primary human T cells when delivered as ribonucleoprotein (RNP) complexes. Collectively, enAsCas12a provides an optimized version of Cas12a that should enable wider application of Cas12a enzymes for gene and epigenetic editing.
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