Time-resolved fate mapping identifies the intestinal upper crypt zone as an origin of Lgr5+ crypt base columnar cells.

In the prevailing model, Lgr5+ cells are the only intestinal stem cells (ISCs) that sustain homeostatic epithelial regeneration by upward migration of progeny through elusive upper crypt transit-amplifying (TA) intermediates. Here, we identify a proliferative upper crypt population marked by Fgfbp1, in the location of putative TA cells, that is transcriptionally distinct from Lgr5+ cells. Using a kinetic reporter for time-resolved fate mapping and Fgfbp1-CreERT2 lineage tracing, we establish that Fgfbp1+ cells are multi-potent and give rise to Lgr5+ cells, consistent with their ISC function. Fgfbp1+ cells also sustain epithelial regeneration following Lgr5+ cell depletion. We demonstrate that FGFBP1, produced by the upper crypt cells, is an essential factor for crypt proliferation and epithelial homeostasis. Our findings support a model in which tissue regeneration originates from upper crypt Fgfbp1+ cells that generate progeny propagating bi-directionally along the crypt-villus axis and serve as a source of Lgr5+ cells in the crypt base.

Hypoxia-Inducible Factor-1 Alpha Expression Is Predictive of Pathological Complete Response in Patients with Breast Cancer Receiving Neoadjuvant Chemotherapy.

To demonstrate the value of hypoxia-inducible factor-1α (HIF-1α) in predicting response in patients with breast cancer receiving standard neoadjuvant chemotherapy (NAC). METHODS: Ninety-five women enrolled in two prospective studies underwent biopsies for the histopathological diagnosis of breast carcinoma before receiving NAC, based on anthracyclines and taxanes. For expression of HIF-1α, EGFR, pAKT and pMAPK, tumor samples were analyzed by immunohistochemistry in tissues microarrays. Standard statistical methods (Pearson chi-square test, Fisher exact test, Kruskal-Wallis test, Mann-Whitney test and Kaplan-Meier method) were used to study the association of HIF-1α with tumor response, survival and other clinicopathologic variables/biomarkers. RESULTS: HIF-1α expression was positive in 35 (39.7%) cases and was significantly associated to complete pathological response (pCR) (p = 0.014). HIF-1α expression was correlated positively with tumor grade (p = 0.015) and Ki-67 expression (p = 0.001) and negativity with progesterone receptors (PR) (p = 0.04) and luminal A phenotype expression (p = 0.005). No correlation was found between HIF-1α expression and EGFR, pAKT and pMAPK. In terms of survival, HIF-1α expression was associated with a significantly shorter disease-free survival (p = 0.013), being identified as an independent prognostic factor in multivariate analysis. CONCLUSIONS: Overexpression of HIF-1α is a predictor of pCR and shorter DFS; it would be valuable to confirm these results in prospective studies.

Single-Cell Transcriptional Profiling of the Intestinal Epithelium.

Emerging single-cell technologies, like single-cell RNA sequencing (scRNA-seq), enable the study of heterogeneous biological systems at cellular resolution. By profiling the set of expressed transcripts in each cell, single-cell transcriptomics has allowed for the cataloging of the cellular constituents of multiple organs and tissues, both in health and disease. In addition, these technologies have provided mechanistic insights into cellular function, cell state transitions, developmental trajectories and lineage relationships, as well as helped to dissect complex, population-level responses to environmental perturbations. scRNA-seq is particularly useful for characterizing the intestinal epithelium because it is a dynamic, rapidly self-renewing tissue comprised of more than a dozen specialized cell types. Here we discuss the fundamentals of single-cell transcriptomics of the murine small intestinal epithelium. We review the principles of proper experimental design and provide methods for the dissociation of the small intestinal epithelium into single cells followed by fluorescence-activated cell sorting (FACS) and for scRNA-seq using the 10× Genomics Chromium platform.

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.

Induction of apoptosis via proteasome inhibition in leukemia/lymphoma cells by two potent piperidones.

PURPOSE: Previously, compounds containing a piperidone structure have been shown to be highly cytotoxic to cancer cells. Recently, we found that the piperidone compound P2 exhibits a potent anti-neoplastic activity against human breast cancer-derived cells. Here, we aimed to evaluate two piperidone compounds, P1 and P2, for their potential anti-neoplastic activity against human leukemia/lymphoma-derived cells. METHODS: Cytotoxicity and apoptosis induction were evaluated using MTS, annexin V-FITC/PI and mitochondrial membrane potential polychromatic assays to confirm the mode of action of the piperidone compounds. The effects of compound P1 and P2 treatment on gene expression were assessed using AmpliSeq analysis and, subsequently, confirmed by RT-qPCR and Western blotting. RESULTS: We found that the two related piperidone compounds P1 and P2 selectively killed the leukemia/lymphoma cells tested at nanomolar concentrations through induction of the intrinsic apoptotic pathway, as demonstrated by mitochondrial depolarization and caspase-3 activation. AmpliSeq-based transcriptome analyses of the effects of compounds P1 and P2 on HL-60 acute leukemia cells revealed a differential expression of hundreds of genes, 358 of which were found to be affected by both. Additional pathway analyses revealed that a significant number of the common genes were related to the unfolded protein response, implying a possible role of the two compounds in the induction of proteotoxic stress. Subsequent analyses of the transcriptome data revealed that P1 and P2 induced similar gene expression alterations as other well-known proteasome inhibitors. Finally, we found that Noxa, an important mediator of the activity of proteasome inhibitors, was significantly upregulated at both the mRNA and protein levels, indicating a possible role in the cytotoxic mechanism induced by P1 and P2. CONCLUSIONS: Our data indicate that the cytotoxic activity of P1 and P2 on leukemia/lymphoma cells is mediated by proteasome inhibition, leading to activation of pro-apoptotic pathways.

Intestinal Enteroendocrine Lineage Cells Possess Homeostatic and Injury-Inducible Stem Cell Activity.

Several cell populations have been reported to possess intestinal stem cell (ISC) activity during homeostasis and injury-induced regeneration. Here, we explored inter-relationships between putative mouse ISC populations by comparative RNA-sequencing (RNA-seq). The transcriptomes of multiple cycling ISC populations closely resembled Lgr5+ ISCs, the most well-defined ISC pool, but Bmi1-GFP+ cells were distinct and enriched for enteroendocrine (EE) markers, including Prox1. Prox1-GFP+ cells exhibited sustained clonogenic growth in vitro, and lineage-tracing of Prox1+ cells revealed long-lived clones during homeostasis and after radiation-induced injury in vivo. Single-cell mRNA-seq revealed two subsets of Prox1-GFP+ cells, one of which resembled mature EE cells while the other displayed low-level EE gene expression but co-expressed tuft cell markers, Lgr5 and Ascl2, reminiscent of label-retaining secretory progenitors. Our data suggest that the EE lineage, including mature EE cells, comprises a reservoir of homeostatic and injury-inducible ISCs, extending our understanding of cellular plasticity and stemness.

Activity of MM-398, nanoliposomal irinotecan (nal-IRI), in Ewing's family tumor xenografts is associated with high exposure of tumor to drug and high SLFN11 expression.

PURPOSE: To determine the pharmacokinetics and the antitumor activity in pediatric cancer models of MM-398, a nanoliposomal irinotecan (nal-IRI). EXPERIMENTAL DESIGN: Mouse plasma and tissue pharmacokinetics of nal-IRI and the current clinical formulation of irinotecan were characterized. In vivo activity of irinotecan and nal-IRI was compared in xenograft models (3 each in nu/nu mice) of Ewing's sarcoma family of tumors (EFT), neuroblastoma (NB), and rhabdomyosarcoma (RMS). SLFN11 expression was assessed by Affymetrix HuEx arrays, Taqman RT-PCR, and immunoblotting. RESULTS: Plasma and tumor concentrations of irinotecan and SN-38 (active metabolite) were approximately 10-fold higher for nal-IRI than for irinotecan. Two doses of NAL-IRI (10 mg/kg/dose) achieved complete responses maintained for >100 days in 24 of 27 EFT-xenografted mice. Event-free survival for mice with RMS and NB was significantly shorter than for EFT. High SLFN11 expression has been reported to correlate with sensitivity to DNA damaging agents; median SLFN11 mRNA expression was >100-fold greater in both EFT cell lines and primary tumors compared with NB or RMS cell lines or primary tumors. Cytotoxicity of SN-38 inversely correlated with SLFN11 mRNA expression in 20 EFT cell lines. CONCLUSIONS: In pediatric solid tumor xenografts, nal-IRI demonstrated higher systemic and tumor exposures to SN-38 and improved antitumor activity compared with the current clinical formulation of irinotecan. Clinical studies of nal-IRI in pediatric solid tumors (especially EFT) and correlative studies to determine if SLFN11 expression can serve as a biomarker to predict nal-IRI clinical activity are warranted.

[Assessing the profile and nutritional intake of an Ibero-American group of nutrition postgraduate students]. / Valoración del perfil e ingesta de nutrientes de un grupo de estudiantes iberoamericanos de postgrados en nutrición.

INTRODUCTION: Dietary habits have a great influence on people health of, and its study is extremely important to proof adaptation to current recommendations in order to develop nutritional education and/or supplementation policies adjusted to reality. MATERIAL AND METHODS: A descriptive cross study with the participation of postgraduate students. Participants were selected using non-probability accidental sampling accidental. Information was sent by e-mail, including a questionnaire on socio-demographic characteristics and a 3-day dietary record. We obtained a sample of n = 50 participants. The 3-day dietary records were evaluated with NutrIber software and the data obtained were treated using the statistical software SPSS 15. RESULTS AND DISCUSSION: The profile of the students was 80% females and 20% males, mean age 33.7 ± 9.32 years, and body mass index of 22.91 ± 4.07. All groups have an excessive consumption of proteins and lipids and carbohydrates deficit. At the macro-story is remarkable that no group covers iodine recommended nutrient intake, and that no group of women covers the recommendations for fiber, folic acid and iron intake. CONCLUSIONS: Further studies are needed to confirm the results found. However, it can be concluded that urban populations in Latin America are increasingly closer to the model of Western diet, rich in protein and fat and low in carbohydrates, fiber and micronutrients, which is indicative of poor diets in whole grains, fruits and vegetables.

Introducción: Los hábitos alimentarios tienen una gran influencia sobre la salud de las personas, por ello es de suma importancia profundizar en su estudio para comprobar su adaptación a las recomendaciones actuales y poder realizar políticas de educación nutricional y/o suplementación ajustadas a la realidad. Material y métodos: Estudio transversal descriptivo con la colaboración de estudiantes de postgrado. Los participantes se seleccionaron mediante muestreo no probabilístico accidental enviando vía mail la información del estudio, realizado mediante un cuestionario sobre sus características socio-demográficas y un registro dietético de 3 días. La muestra estuvo formada por n = 50 participantes. Los registros dietéticos de 3 días se valoraron con el software NutrIber y los datos obtenidos fueron tratados mediante el software estadístico SPSS 15. Resultados y discusión: El perfil de los estudiantes es de 80% mujeres y 20% hombres, con edad media de 33,7 ± 9,32 AÑOs, e índice de masa corporal de 22,91 ± 4,07. Todos los grupos presentan un consumo excesivo de proteínas y lípidos y deficitario en glúcidos. En cuanto a los micronutrientes es destacable que ningún grupo cubre las ingestas recomendadas de yodo, y que ningún grupo de mujeres cubre las recomendaciones de ingesta de fibra, ácido fólico ni hierro. Conclusiones: Son necesarios más estudios para confirmar los resultados hallado, sin embargo, se puede concluir que las poblaciones urbanas de Latinoamérica cada vez se acercan más al modelo de dieta occidental, rica en proteínas y grasas y pobre en glúcidos, fibra y micronutrientes, lo que es indicativo de dietas pobres en cereales integrales, frutas y verduras.

Valoración del perfil e ingesta de nutrientes de un grupo de estudiantes iberoamericanos de postgrados en nutrición / Assesing the profile and nutritional intakes of an ibero-american group of nutrition postgraduate students

Introducción: Los hábitos alimentarios tienen una gran influencia sobre la salud de las personas, por ello es de suma importancia profundizar en su estudio para comprobar su adaptación a las recomendaciones actuales y poder realizar políticas de educación nutricional y/o suplementación ajustadas a la realidad. Material y métodos: Estudio transversal descriptivo con la colaboración de estudiantes de postgrado. Los participantes se seleccionaron mediante muestreo no probabilístico accidental enviando vía mail la información del estudio, realizado mediante un cuestionario sobre sus características socio-demográficas y un registro dietético de 3 días. La muestra estuvo formada por n = 50 participantes. Los registros dietéticos de 3 días se valoraron con el software NutrIber y los datos obtenidos fueron tratados mediante el software estadístico SPSS 15. Resultados y discusión: El perfil de los estudiantes es de 80% mujeres y 20% hombres, con edad media de 33,7 ± 9,32 años, e índice de masa corporal de 22,91 ± 4,07. Todos los grupos presentan un consumo excesivo de proteínas y lípidos y deficitario en glúcidos. En cuanto a los micronutrientes es destacable que ningún grupo cubre las ingestas recomendadas de yodo, y que ningún grupo de mujeres cubre las recomendaciones de ingesta de fibra, ácido fólico ni hierro. Conclusiones: Son necesarios más estudios para confirmar los resultados hallado, sin embargo, se puede concluir que las poblaciones urbanas de Latinoamérica cada vez se acercan más al modelo de dieta occidental, rica en proteínas y grasas y pobre en glúcidos, fibra y micronutrientes, lo que es indicativo de dietas pobres en cereales integrales, frutas y verduras (AU)

Introduction: Dietary habits have a great influence on people health of, and its study is extremely important to proof adaptation to current recommendations in order to develop nutritional education and/or supplementation policies adjusted to reality. Material and methods: A descriptive cross study with the participation of postgraduate students. Participants were selected using non-probability accidental sampling accidental. Information was sent by e-mail, including a questionnaire on socio-demographic characteristics and a 3-day dietary record. We obtained a sample of n = 50 participants. The 3-day dietary records were evaluated with NutrIber software and the data obtained were treated using the statistical software SPSS 15. Results and discussion: The profile of the students was 80% females and 20% males, mean age 33.7 ± 9.32 years, and body mass index of 22.91 ± 4.07. All groups have an excessive consumption of proteins and lipids and carbohydrates deficit. At the macrostory is remarkable that no group covers iodine recommended nutrient intake, and that no group of women covers the recommendations for fiber, folic acid and iron intake. Conclusions: Further studies are needed to confirm the results found. However, it can be concluded that urban populations in Latin America are increasingly closer to the model of Western diet, rich in protein and fat and low in carbohydrates, fiber and micronutrients, which is indicative of poor diets in whole grains, fruits and vegetables (AU)

Mercury concentrations of fish, river water, and sediment in the Río Ramis-Lake Titicaca watershed, Peru.

This study reports the first set of data on the concentration of mercury in muscle tissue of several varieties of fish from Lake Titicaca, including the pejerrey (Basilichthyes bonariensis), the carachi (Orestias), and 2 types of indigenous catfish (Trichomycterus). Approximately 27% of the pejerrey and 75% of the carachi exceeded the US EPA fish tissue-based water quality criterion level of 0.30 microg g(-1). Mercury levels of pejerrey increased with fish size, although this relationship was less apparent for the smaller carachi. The pejerrey and carachi are important food fish for local residents. A synoptic sampling of the Río Ramis--the largest tributary to Lake Titicaca--was conducted in an attempt to determine if mercury releases from artisanal gold mining could be an important source of Hg contamination to Lake Titicaca. Although highly elevated concentrations of Hg and other heavy metals were documented in headwater streams near the mining centers of La Rinconada and Cecilia, the quantity of Hg entering Lake Titicaca that could be attributed to mining in the Ramis watershed was below the quantifiable limit in our July 2002 study. This does not diminish the localized threat to mercury exposure for the artisanal gold miners themselves, as well as their families. Further studies of mercury dynamics in Lake Titicaca are recommended, as well as in the rivers draining into the lake. It is probable that most of the downgradient transport of Hg and other trace metals from the headwater mining centers occurs as suspended sediment during seasonal periods of high-flow.