In vivo temporal resolution of acute promyelocytic leukemia progression reveals a role of Klf4 in suppressing early leukemic transformation.

Genome organization plays a pivotal role in transcription, but how transcription factors (TFs) rewire the structure of the genome to initiate and maintain the programs that lead to oncogenic transformation remains poorly understood. Acute promyelocytic leukemia (APL) is a fatal subtype of leukemia driven by a chromosomal translocation between the promyelocytic leukemia (PML) and retinoic acid receptor α (RARα) genes. We used primary hematopoietic stem and progenitor cells (HSPCs) and leukemic blasts that express the fusion protein PML-RARα as a paradigm to temporally dissect the dynamic changes in the epigenome, transcriptome, and genome architecture induced during oncogenic transformation. We found that PML-RARα initiates a continuum of topologic alterations, including switches from A to B compartments, transcriptional repression, loss of active histone marks, and gain of repressive histone marks. Our multiomics-integrated analysis identifies Klf4 as an early down-regulated gene in PML-RARα-driven leukemogenesis. Furthermore, we characterized the dynamic alterations in the Klf4 cis-regulatory network during APL progression and demonstrated that ectopic Klf4 overexpression can suppress self-renewal and reverse the differentiation block induced by PML-RARα. Our study provides a comprehensive in vivo temporal dissection of the epigenomic and topological reprogramming induced by an oncogenic TF and illustrates how topological architecture can be used to identify new drivers of malignant transformation.

TFIIIC Binding to Alu Elements Controls Gene Expression via Chromatin Looping and Histone Acetylation.

How repetitive elements, epigenetic modifications, and architectural proteins interact ensuring proper genome expression remains poorly understood. Here, we report regulatory mechanisms unveiling a central role of Alu elements (AEs) and RNA polymerase III transcription factor C (TFIIIC) in structurally and functionally modulating the genome via chromatin looping and histone acetylation. Upon serum deprivation, a subset of AEs pre-marked by the activity-dependent neuroprotector homeobox Protein (ADNP) and located near cell-cycle genes recruits TFIIIC, which alters their chromatin accessibility by direct acetylation of histone H3 lysine-18 (H3K18). This facilitates the contacts of AEs with distant CTCF sites near promoter of other cell-cycle genes, which also become hyperacetylated at H3K18. These changes ensure basal transcription of cell-cycle genes and are critical for their re-activation upon serum re-exposure. Our study reveals how direct manipulation of the epigenetic state of AEs by a general transcription factor regulates 3D genome folding and expression.

Liquidhope: methylome and genomic profiling from very limited quantities of plasma-derived DNA.

Analysis of the methylome of tumor cell-free deoxyribonucleic acid (DNA; cfDNA) has emerged as a powerful non-invasive technique for cancer subtyping and prognosis. However, its application is frequently hampered by the quality and total cfDNA yield. Here, we demonstrate the feasibility of very low-input cfDNA for whole-methylome and copy-number profiling studies using enzymatic conversion of unmethylated cysteines [enzymatic methyl-seq (EM-seq)] to better preserve DNA integrity. We created a model for predicting genomic subtyping and prognosis with high accuracy. We validated our tool by comparing whole-genome CpG sequencing with in situ cohorts generated with bisulfite conversion and array hybridization, demonstrating that, despite the different techniques and sample origins, information on cfDNA methylation is comparable with in situ cohorts. Our findings support use of liquid biopsy followed by EM-seq to assess methylome of cancer patients, enabling validation in external cohorts. This advance is particularly relevant for rare cancers like neuroblastomas where liquid-biopsy volume is restricted by ethical regulations in pediatric patients.

A set of accessible enhancers enables the initial response of breast cancer cells to physiological progestin concentrations.

Here, we report that in T47D breast cancer cells 50 pM progestin is sufficient to activate cell cycle entry and the progesterone gene expression program. At this concentration, equivalent to the progesterone blood levels found around the menopause, progesterone receptor (PR) binds only to 2800 genomic sites, which are accessible to ATAC cleavage prior to hormone exposure. These highly accessible sites (HAs) are surrounded by well-organized nucleosomes and exhibit breast enhancer features, including estrogen receptor alpha (ERα), higher FOXA1 and BRD4 (bromodomain containing 4) occupancy. Although HAs are enriched in RAD21 and CTCF, PR binding is the driving force for the most robust interactions with hormone-regulated genes. HAs show higher frequency of 3D contacts among themselves than with other PR binding sites, indicating colocalization in similar compartments. Gene regulation via HAs is independent of classical coregulators and ATP-activated remodelers, relying mainly on MAP kinase activation that enables PR nuclear engagement. HAs are also preferentially occupied by PR and ERα in breast cancer xenografts derived from MCF-7 cells as well as from patients, indicating their potential usefulness as targets for therapeutic intervention.

Rapid reversible changes in compartments and local chromatin organization revealed by hyperosmotic shock.

Nuclear architecture is decisive for the assembly of transcriptional responses. However, how chromosome organization is dynamically modulated to permit rapid and transient transcriptional changes in response to environmental challenges remains unclear. Here we show that hyperosmotic stress disrupts different levels of chromosome organization, ranging from A/B compartment changes to reduction in the number and insulation of topologically associating domains (TADs). Concomitantly, transcription is greatly affected, TAD borders weaken, and RNA Polymerase II runs off from hundreds of transcription end sites. Stress alters the binding profiles of architectural proteins, which explains the disappearance of local chromatin organization. These processes are dynamic, and cells rapidly reconstitute their default chromatin conformation after stress removal, uncovering an intrinsic organization. Transcription is not required for local chromatin reorganization, while compartment recovery is partially transcription-dependent. Thus, nuclear organization in mammalian cells can be rapidly modulated by environmental changes in a reversible manner.

Hormone-control regions mediate steroid receptor-dependent genome organization.

In breast cancer cells, some topologically associating domains (TADs) behave as hormonal gene regulation units, within which gene transcription is coordinately regulated in response to steroid hormones. Here we further describe that responsive TADs contain 20- to 100-kb-long clusters of intermingled estrogen receptor (ESR1) and progesterone receptor (PGR) binding sites, hereafter called hormone-control regions (HCRs). In T47D cells, we identified more than 200 HCRs, which are frequently bound by unliganded ESR1 and PGR. These HCRs establish steady long-distance inter-TAD interactions between them and organize characteristic looping structures with promoters in their TADs even in the absence of hormones in ESR1+-PGR+ cells. This organization is dependent on the expression of the receptors and is further dynamically modulated in response to steroid hormones. HCRs function as platforms that integrate different signals, resulting in some cases in opposite transcriptional responses to estrogens or progestins. Altogether, these results suggest that steroid hormone receptors act not only as hormone-regulated sequence-specific transcription factors but also as local and global genome organizers.

Multi-organ transcriptomic landscape of Ambystoma velasci metamorphosis.

Metamorphosis is a postembryonic developmental process that involves morphophysiological and behavioral changes, allowing organisms to adapt into a novel environment. In some amphibians, aquatic organisms undergo metamorphosis to adapt in a terrestrial environment. In this process, these organisms experience major changes in their circulatory, respiratory, digestive, excretory and reproductive systems. We performed a transcriptional global analysis of heart, lung and gills during diverse stages of Ambystoma velasci to investigate its metamorphosis. In our analyses, we identified eight gene clusters for each organ, according to the expression patterns of differentially expressed genes. We found 4064 differentially expressed genes in the heart, 4107 in the lung and 8265 in the gills. Among the differentially expressed genes in the heart, we observed genes involved in the differentiation of cardiomyocytes in the interatrial zone, vasculogenesis and in the maturation of coronary vessels. In the lung, we found genes differentially expressed related to angiogenesis, alveolarization and synthesis of the surfactant protein. In the case of the gills, the most prominent biological processes identified are degradation of extracellular matrix, apoptosis and keratin production. Our study sheds light on the transcriptional responses and the pathways modulation involved in the transformation of the facultative metamorphic salamander A. velasci in an organ-specific manner.

Duplications and Functional Convergence of Intestinal Carbohydrate-Digesting Enzymes.

Vertebrate diets and digestive physiologies vary tremendously. Although the contribution of ecological and behavioral features to such diversity is well documented, the roles and identities of individual intestinal enzymes shaping digestive traits remain largely unexplored. Here, we show that the sucrase-isomaltase (SI)/maltase-glucoamylase (MGAM) dual enzyme system long assumed to be the conserved disaccharide and starch digestion framework in all vertebrates is absent in many lineages. Our analyses indicate that independent duplications of an ancestral SI gave rise to the mammalian-specific MGAM, as well as to other duplicates in fish and birds. Strikingly, the duplicated avian enzyme exhibits similar activities to MGAM, revealing an unexpected case of functional convergence. Our results highlight digestive enzyme variation as a key uncharacterized component of dietary diversity in vertebrates.

Dietary adaptation to high starch involves increased relative abundance of sucrase-isomaltase and its mRNA in nestling house sparrows.

Dietary flexibility in digestive enzyme activity is widespread in vertebrates but mechanisms are poorly understood. When laboratory rats are switched to a higher carbohydrate diet, the activities of the apical intestinal α-glucosidases (AGs) increase within 6-12 h, mainly by rapid increase in enzyme transcription, followed by rapid translation and translocation to the intestine's apical, brush-border membrane (BBM). We performed the first unified study of the overall process in birds, relying on activity, proteomic, and transcriptomic data from the same animals. Our avian model was nestling house sparrows (Passer domesticus), which switch naturally from a low-starch insect diet to a higher starch seed diet and in whom the protein sucrase-isomaltase (SI) is responsible for all maltase and sucrase intestinal activities. Twenty-four hours after the switch to a high-starch diet, SI activity was increased but not at 12 h post diet switch. SI was the only hydrolase increased in the BBM, and its relative abundance and activity were positively correlated. Twenty-four hours after a reverse switch back to the lower starch diet, SI activity was decreased but not at 12 h post diet switch. Parallel changes in SI mRNA relative abundance were associated with the changes in SI activity in both diet-switch experiments, but our data also revealed an apparent diurnal rhythm in SI mRNA. This is the first demonstration that birds may rely on rapid increase in abundance of SI and its mRNA when adjusting to high-starch diet. Although the mechanisms underlying dietary induction of intestinal enzymes seem similar in nestling house sparrows and laboratory rodents, the time course for modulation in nestlings seemed half as fast compared with laboratory rodents. Before undertaking modulation, an opportunistic forager facing limited resources might rely on more extensive or prolonged environmental sampling, because the redesign of the intestine's hydrolytic capacity shortly after just one or a few meals of a new substrate might be a costly mistake.

Rapid and parallel changes in activity and mRNA of intestinal peptidase to match altered dietary protein levels in juvenile house sparrows (Passer domesticus).

Although dietary flexibility in digestive enzyme activity (i.e. reaction rate) is widespread in vertebrates, mechanisms are poorly understood. When laboratory rats are switched to a higher protein diet, the activities of apical intestinal peptidases increase within 15 h, in some cases by rapid increase in enzyme transcription followed by rapid translation and translocation to the intestine's apical, brush-border membrane (BBM). Focusing on aminopeptidase-N (APN), we studied intestinal digestive enzyme flexibility in birds, relying on activity and mRNA data from the same animals. Our model was nestling house sparrows (Passer domesticus), already known to modulate intestinal peptidase activity when switching between lower and higher protein diets. Twenty-four hours after a switch from an adequate, lower protein diet to a higher protein diet, APN activity was increased in both whole intestinal tissue homogenates and in isolated BBM, but not at 12 h post-diet switch. Twenty-four hours after a reverse switch back to the lower protein diet, APN activity was decreased, but not at 12 h post-diet switch. Changes in APN activity in both diet switch experiments were associated with parallel changes in APN mRNA. Although transcriptional changes seem to be an important mechanism underlying dietary modulation of intestinal peptidase in both nestling house sparrows and laboratory rodents, the time course for modulation in nestlings seemed slower (taking approximately twice as long) compared with laboratory rodents. It may be ecologically advantageous if nestlings biochemically restructure their gut in response to a sustained increase in insects and protein intake rather than one or a few lucky insect meals.

Adaptation of intestinal epithelial hydrolysis and absorption of dietary carbohydrate and protein in mammals and birds.

The small intestine of mammals and birds exhibits fascinating variation across taxa, body size, and life history features such as locomotion and diet. In the intestine's brush border membrane (BBM), hydrolases are more abundant than transporters in both mammals and birds, but there are differences among the groups in abundance of certain hydrolases and possibly in transporters. For example, mammals express two α-glucosidases, sucrase-isomaltase (SI) and maltase glucoamylase (MGAM), whereas songbirds we studied have only SI, and the chicken expresses SI plus another α-glucosidase that functions similarly to MGAM but is not a true ortholog. For intestinal absorption of sugars and amino acids, small fliers rely on a paracellular pathway to a greater extent than do nonflying mammals, which rely more on transporters. Possibly having evolved in fliers as compensation for lower intestinal nominal surface area (NSA), the fliers' reliance on paracellular absorption is supported by their greater villous surface enlargement that leads to more (per cm2 NSA) tight junctions and greater clearance of passively absorbed compounds. To match digestive capacity to nutrient load, a positive relationship is often observed between dietary intake of macronutrients and intestinal activity of the enzymes and transporters of their respective constituents. In enterocytes, rapid, fine-tuned adjustment to high dietary carbohydrate and protein involves rapid, specific correlated increase in activity and abundance of hydrolases and transporters in the BBM and increases in their mRNA.

Putative cis-regulatory drivers in colorectal cancer.

The cis-regulatory effects responsible for cancer development have not been as extensively studied as the perturbations of the protein coding genome in tumorigenesis. To better characterize colorectal cancer (CRC) development we conducted an RNA-sequencing experiment of 103 matched tumour and normal colon mucosa samples from Danish CRC patients, 90 of which were germline-genotyped. By investigating allele-specific expression (ASE) we show that the germline genotypes remain important determinants of allelic gene expression in tumours. Using the changes in ASE in matched pairs of samples we discover 71 genes with excess of somatic cis-regulatory effects in CRC, suggesting a cancer driver role. We correlate genotypes and gene expression to identify expression quantitative trait loci (eQTLs) and find 1,693 and 948 eQTLs in normal samples and tumours, respectively. We estimate that 36% of the tumour eQTLs are exclusive to CRC and show that this specificity is partially driven by increased expression of specific transcription factors and changes in methylation patterns. We show that tumour-specific eQTLs are more enriched for low CRC genome-wide association study (GWAS) P values than shared eQTLs, which suggests that some of the GWAS variants are tumour specific regulatory variants. Importantly, tumour-specific eQTL genes also accumulate more somatic mutations when compared to the shared eQTL genes, raising the possibility that they constitute germline-derived cancer regulatory drivers. Collectively the integration of genome and the transcriptome reveals a substantial number of putative somatic and germline cis-regulatory cancer changes that may have a role in tumorigenesis.

OneD: increasing reproducibility of Hi-C samples with abnormal karyotypes.

The three-dimensional conformation of genomes is an essential component of their biological activity. The advent of the Hi-C technology enabled an unprecedented progress in our understanding of genome structures. However, Hi-C is subject to systematic biases that can compromise downstream analyses. Several strategies have been proposed to remove those biases, but the issue of abnormal karyotypes received little attention. Many experiments are performed in cancer cell lines, which typically harbor large-scale copy number variations that create visible defects on the raw Hi-C maps. The consequences of these widespread artifacts on the normalized maps are mostly unexplored. We observed that current normalization methods are not robust to the presence of large-scale copy number variations, potentially obscuring biological differences and enhancing batch effects. To address this issue, we developed an alternative approach designed to take into account chromosomal abnormalities. The method, called OneD, increases reproducibility among replicates of Hi-C samples with abnormal karyotype, outperforming previous methods significantly. On normal karyotypes, OneD fared equally well as state-of-the-art methods, making it a safe choice for Hi-C normalization. OneD is fast and scales well in terms of computing resources for resolutions up to 5 kb.

Human Oocyte-Derived Methylation Differences Persist in the Placenta Revealing Widespread Transient Imprinting.

Thousands of regions in gametes have opposing methylation profiles that are largely resolved during the post-fertilization epigenetic reprogramming. However some specific sequences associated with imprinted loci survive this demethylation process. Here we present the data describing the fate of germline-derived methylation in humans. With the exception of a few known paternally methylated germline differentially methylated regions (DMRs) associated with known imprinted domains, we demonstrate that sperm-derived methylation is reprogrammed by the blastocyst stage of development. In contrast a large number of oocyte-derived methylation differences survive to the blastocyst stage and uniquely persist as transiently methylated DMRs only in the placenta. Furthermore, we demonstrate that this phenomenon is exclusive to primates, since no placenta-specific maternal methylation was observed in mouse. Utilizing single cell RNA-seq datasets from human preimplantation embryos we show that following embryonic genome activation the maternally methylated transient DMRs can orchestrate imprinted expression. However despite showing widespread imprinted expression of genes in placenta, allele-specific transcriptional profiling revealed that not all placenta-specific DMRs coordinate imprinted expression and that this maternal methylation may be absent in a minority of samples, suggestive of polymorphic imprinted methylation.

RhoA/ROCK pathway activity is essential for the correct localization of the germ plasm mRNAs in zebrafish embryos.

Zebrafish germ plasm is composed of mRNAs such as vasa and nanos and of proteins such as Bucky ball, all of which localize symmetrically in four aggregates at the distal region of the first two cleavage furrows. The coordination of actin microfilaments, microtubules and kinesin is essential for the correct localization of the germ plasm. Rho-GTPases, through their effectors, coordinate cytoskeletal dynamics. We address the participation of RhoA and its effector ROCK in germ plasm localization during the transition from two- to eight-cell embryos. We found that active RhoA is enriched along the cleavage furrow during the first two division cycles, whereas ROCK localizes at the distal region of the cleavage furrows in a similar pattern as the germ plasm mRNAs. Specific inhibition of RhoA and ROCK affected microtubules organization at the cleavage furrow; these caused the incorrect localization of the germ plasm mRNAs. The incorrect localization of the germ plasm led to a dramatic change in the number of germ cells during the blastula and 24hpf embryo stages without affecting any other developmental processes. We demonstrate that the Rho/ROCK pathway is intimately related to the determination of germ cells in zebrafish embryos.

Intestinal α-glycosidase transcriptional responses during development and diet adjustment in altricial birds.

We describe developmental changes in maltasic activity and its mRNA until adulthood, and in response to an increase in dietary starch. We studied house sparrows (Passer domesticus), which undergo a natural switch from insects to a starch-containing seed diet during development, and zebra finches (Taeniopygia guttata), which have a relatively fixed starchy seed diet during development. In zebra finches, in which maltasic activity increased with age but not with dietary starch, α-glycosidase (AG) mRNA was not affected by either age or dietary starch level. In house sparrow nestlings, in which maltasic activity increased with age and with added starch, AG mRNA was higher when birds were fed a diet with added starch but did not increase with age. These results are consistent with the idea that the apparent programmed developmental increase in maltasic activity is not mainly under transcriptional control of AG mRNA, whereas induction of maltasic activity by increased dietary starch is.

Absence of Maternal Methylation in Biparental Hydatidiform Moles from Women with NLRP7 Maternal-Effect Mutations Reveals Widespread Placenta-Specific Imprinting.

Familial recurrent hydatidiform mole (RHM) is a maternal-effect autosomal recessive disorder usually associated with mutations of the NLRP7 gene. It is characterized by HM with excessive trophoblastic proliferation, which mimics the appearance of androgenetic molar conceptuses despite their diploid biparental constitution. It has been proposed that the phenotypes of both types of mole are associated with aberrant genomic imprinting. However no systematic analyses for imprinting defects have been reported. Here, we present the genome-wide methylation profiles of both spontaneous androgenetic and biparental NLRP7 defective molar tissues. We observe total paternalization of all ubiquitous and placenta-specific differentially methylated regions (DMRs) in four androgenetic moles; namely gain of methylation at paternally methylated loci and absence of methylation at maternally methylated regions. The methylation defects observed in five RHM biopsies from NLRP7 defective patients are restricted to lack-of-methylation at maternal DMRs. Surprisingly RHMs from two sisters with the same missense mutations, as well as consecutive RHMs from one affected female show subtle allelic methylation differences, suggesting inter-RHM variation. These epigenotypes are consistent with NLRP7 being a maternal-effect gene and involved in imprint acquisition in the oocyte. In addition, bioinformatic screening of the resulting methylation datasets identified over sixty loci with methylation profiles consistent with imprinting in the placenta, of which we confirm 22 as novel maternally methylated loci. These observations strongly suggest that the molar phenotypes are due to defective placenta-specific imprinting and over-expression of paternally expressed transcripts, highlighting that maternal-effect mutations of NLRP7 are associated with the most severe form of multi-locus imprinting defects in humans.

House sparrow biomarkers as lead pollution bioindicators. Evaluation of dose and exposition length on hematological and oxidative stress parameters.

House sparrows (Passer domesticus) have been proposed as a key ecological indicator of urban pollution. Remarkably, we lack knowledge about the physiological effects of lead on this bird species. Therefore, this study was aimed to evaluate the effect of Pb on several physiological parameters in house sparrows exposed to environmental Pb concentrations. In a first experiment, birds were exposed to Pb sub-lethal doses (from 1.3 to 14.0 µg of Pb/g animal/day) during 5 days, which resulted in a dose response increase of blood Pb levels and decrease of blood ALAD activity. However, at the higher doses tested (> 7 µg of Pb/g animal/day) the blood ALAD activity inhibition (~82%) remained constant. Hematocrit and hemoglobin were significantly reduced only at the highest-doses, and the stress indicator, heterophils to lymphocyte (H/L) ratio, did not show apparent changes. In a second experiment, house sparrows were exposed to Pb in drinking water (12.3 ppm) during either 15 or 30 days. Pb concentration used in this study was enough to produce blood lead levels equivalents to those found recently in house sparrows inhabiting urban areas, reduced blood ALAD activity and inversion of the H/L ratio. Decreasing blood ALAD activities were correlated with increasing blood Pb levels. In addition, Pb exposure produced modification in the levels of hepatic antioxidant enzymes, increased GST activity and decreased CAT activity, without lipid peroxidation. In conclusion, our results suggest that blood ALAD activity is a reliable and sensitive biomarker for environmental Pb exposure in house sparrows, additionally chronic exposure produce physiological stress (H/L inversion) and small changes in antioxidant enzyme activity. Finally, this specie could be considered a bioindicator for monitoring the urban Pb contamination.

Lignans from Bursera fagaroides Affect In Vivo Cell Behavior by Disturbing the Tubulin Cytoskeleton in Zebrafish Embryos.

By using a zebrafish embryo model to guide the chromatographic fractionation of antimitotic secondary metabolites, seven podophyllotoxin-type lignans were isolated from a hydroalcoholic extract obtained from the steam bark of Bursera fagaroides. The compounds were identified as podophyllotoxin (1), ß-peltatin-A-methylether (2), 5'-desmethoxy-ß-peltatin-A-methylether (3), desmethoxy-yatein (4), desoxypodophyllotoxin (5), burseranin (6), and acetyl podophyllotoxin (7). The biological effects on mitosis, cell migration, and microtubule cytoskeleton remodeling of lignans 1⁻7 were further evaluated in zebrafish embryos by whole-mount immunolocalization of the mitotic marker phospho-histone H3 and by a tubulin antibody. We found that lignans 1, 2, 4, and 7 induced mitotic arrest, delayed cell migration, and disrupted the microtubule cytoskeleton in zebrafish embryos. Furthermore, microtubule cytoskeleton destabilization was observed also in PC3 cells, except for 7. Therefore, these results demonstrate that the cytotoxic activity of 1, 2, and 4 is mediated by their microtubule-destabilizing activity. In general, the in vivo and in vitro models here used displayed equivalent mitotic effects, which allows us to conclude that the zebrafish model can be a fast and cheap in vivo model that can be used to identify antimitotic natural products through bioassay-guided fractionation.

Small intestinal epithelial permeability to water-soluble nutrients higher in passerine birds than in rodents.

In the small intestine transcellular and paracellular pathways are implicated in water-soluble nutrient absorption. In small birds the paracellular pathway is quantitatively important while transcellular pathway is much more important in terrestrial mammals. However, there is not a clear understanding of the mechanistic underpinnings of the differences among taxa. This study was aimed to test the hypothesis that paracellular permeability in perfused intestinal segments is higher in passerine birds than rodents. We performed in situ intestinal perfusions on individuals of three species of passerine birds (Passer domesticus, Taeniopygia guttata and Furnarius rufus) and two species of rodents (Mus musculus and Meriones ungiculatus). Using radio-labelled molecules, we measured the uptake of two nutrients absorbed by paracellular and transcellular pathways (L-proline and 3-O-methyl-D-glucose) and one carbohydrate that has no mediated transport (L-arabinose). Birds exhibited ~2 to ~3 times higher L-arabinose clearance per cm2 epithelium than rodents. Moreover, paracellular absorption accounted for proportionally more of 3-O-methyl-D-glucose and L-proline absorption in birds than in rodents. These differences could be explained by differences in intestinal permeability and not by other factors such as increased retention time or higher intestinal nominal surface area. Furthermore, analysis of our results and all other existing data on birds, bats and rodents shows that insectivorous species (one bird, two bats and a rodent) had only 30% of the clearance of L-arabinose of non-insectivorous species. This result may be explained by weaker natural selection for high paracellular permeability in animal- than in plant-consumers. Animal-consumers absorb less sugar and more amino acids, whose smaller molecular size allow them to traverse the paracellular pathway more extensively and faster than glucose.