And yet, migration, population growth or mortality are not balanced among Cantabrian brown bear subpopulations. Reply to Blanco et al (2020).

In a recent paper, we presented new evidence and provided new insights on the status of Cantabrian brown bear subpopulations, relevant for this species conservation. Namely, we revealed the likely phylogeographic relation between eastern Cantabrian subpopulation and the historical Pyrenean population. We have also detected an asymmetric flow of alleles and individuals from the eastern to the western subpopulation, including seven first-generation male migrants. Based on our results and on those of previous studies, we called the attention to the fact that Eastern Cantabrian brown bears might be taking advantage of increased connectivity to avoid higher human pressure and direct persecution in the areas occupied by the eastern Cantabrian subpopulation. In reply, Blanco et al (2020) [11] have criticized our ecological interpretation of the data presented in our paper. Namely, Blanco and co-authors criticize: (1) the use of the exodus concept in the title and discussion of the paper; (2) the apparent contradiction with source-sink theory; (3) the apparent overlooking of historical demographic data on Cantabrian brown bear and the use of the expression of population decline when referring to eastern subpopulation. Rather than contradicting the long and growing body of knowledge on the two brown bear subpopulations, the results presented in our paper allow a new perspective on the causes of the distinct pace of population growth of the two brown bear subpopulations in the last decades. Here, we reply to the criticisms by: clarifying our ecological interpretation of the results; refocusing the discussion on how the new genetic data suggest that currently, the flow of individuals and alleles is stronger westward, and how it may be linked to direct persecution and killing of brown bears. We provide detailed data on brown bear mortality in the Cantabrian Mountains and show that neither migration, gene flow, population increase nor mortality are balanced among the two subpopulations.

Comparative genome sequence analysis of several species in the genus Tepidimonas and the description of a novel species Tepidimonas charontis sp. nov.

We performed high-quality genome sequencing of eight strains of the species of the genus Tepidimonas and examined the genomes of closely related strains from the databases to understand why Tepidimonas taiwanensis is the only strain of this genus that utilizes glucose and fructose for growth. We found that the assimilation of these hexoses by T. taiwanensis was due to the presence of two transporters that are absent in all other genomes of strains of members of the genus Tepidimonas examined. Some strains lack genes coding for glucokinase, but the Embden-Meyerhof-Parnas pathway appears to be otherwise complete. The pentose phosphate pathway has a complete set of genes, but genes of the Entner-Doudoroff pathway were not identified in the genomes of any of the strains examined. Genome analysis using average nucleotide identity (ANIb), digital DNA-DNA hybridization (dDDH), average amino acid identity (AAI) and phylogenetic analysis of 400 conserved genes was performed to assess the taxonomic classification of the organisms. Two isolates of the genus Tepidimonas from the hot spring at São Pedro do Sul, Portugal, designated SPSP-6T and SPSPC-18 were also examined in this study. These organisms are mixotrophic, have an optimum growth temperature of about 50 ºC, utilize several organic acids and amino acids for growth but do not grow on sugars. Distinctive phenotypic, 16S rRNA gene sequence and genomic characteristics of strains SPSP-6T and SPSPC-18 lead us to propose a novel species based on strain SPSP-6T for which we recommend the name Tepidimonas charontis sp. nov. (=CECT 9683T=LMG 30884T).

Paths for colonization or exodus? New insights from the brown bear (Ursus arctos) population of the Cantabrian Mountains.

Over the centuries, the geographical distribution of brown bear (Ursus arctos) across the Iberian Peninsula has been decreasing, with the species currently confined to North Iberia. The Cantabrian brown bear population is one of the smallest in Europe and is structured into two subpopulations, positioned along an east-west axis. Given the current critically endangered status of this population, it is essential to have a clear picture of its within-population genetic patterns and processes. We use a set of three molecular markers (mitochondrial DNA, autosomal microsatellites and sex markers) to clarify the genetic origins and assess the migration patterns and gene flow of the Cantabrian brown bear population. Our results reveal the presence of two different mitochondrial (matrilineal) haplotypes in the Cantabrian population, both belonging to European brown bear clade 1a. The two haplotypes are geographically structured between Eastern (haplotype CanE) and Western Cantabrian (haplotype CanW) subpopulations, which is consistent with the genetic structure previously identified using nuclear markers. Additionally, we show that CanE is closer to the historical Pyrenean (Pyr) haplotype than to CanW. Despite strong structuring at the levels of mtDNA and nuclear loci, there is evidence of bidirectional gene flow and admixture among subpopulations. Gene flow is asymmetrical and significantly more intense from the Eastern to the Western Cantabrian subpopulation. In fact, we only detected first generation male migrants from the Eastern to the Western Cantabrian subpopulation. These results suggest more intense migration from the smaller and more vulnerable Eastern Cantabrian subpopulation towards the larger and more stable Western Cantabrian subpopulation. These new insights are relevant for assessments of on-going conservation measures, namely the role of dispersal corridors and enhanced connectivity. We discuss the importance of complementary conservation measures, such as human-wildlife conflict mitigation and habitat improvement, for the conservation of a viable Cantabrian brown bear population.

Maximizing semen extraction from sanitary pads by chemical and shredding treatments.

Evidence of sexual aggression may be obtained from superabsorbent polymer (SAP) sanitary pads, which are used by forensic laboratories for semen evaluation. Semen can be extracted from their upper layers, which are free of SAPs. However, our previous results showed a need to optimize the protocol for semen analysis by considering its extraction from the lower core, often composed of sodium polyacrylate SAPs. SAPs generate a hydrogel, which traps the cellular components, hindering the possibility of obtaining cells and hence their genetic material. Simple filtration has been tried previously, but further maximization by application of a treatment has never been attempted. In this paper, we compare both chemical and physical shredding treatments for maximizing gel-trapped sperm and male cell DNA recaptures from hygienic superabsorbent substrates in sanitary pads, panty-liners or diapers. Our findings suggest that the lower core should be treated to induce a dewaterisation of the SAP hydrogels in order to maximize the extraction of bodily fluids.

RAD Sequencing and a Hybrid Antarctic Fur Seal Genome Assembly Reveal Rapidly Decaying Linkage Disequilibrium, Global Population Structure and Evidence for Inbreeding.

Recent advances in high throughput sequencing have transformed the study of wild organisms by facilitating the generation of high quality genome assemblies and dense genetic marker datasets. These resources have the potential to significantly advance our understanding of diverse phenomena at the level of species, populations and individuals, ranging from patterns of synteny through rates of linkage disequilibrium (LD) decay and population structure to individual inbreeding. Consequently, we used PacBio sequencing to refine an existing Antarctic fur seal (Arctocephalus gazella) genome assembly and genotyped 83 individuals from six populations using restriction site associated DNA (RAD) sequencing. The resulting hybrid genome comprised 6,169 scaffolds with an N50 of 6.21 Mb and provided clear evidence for the conservation of large chromosomal segments between the fur seal and dog (Canis lupus familiaris). Focusing on the most extensively sampled population of South Georgia, we found that LD decayed rapidly, reaching the background level by around 400 kb, consistent with other vertebrates but at odds with the notion that fur seals experienced a strong historical bottleneck. We also found evidence for population structuring, with four main Antarctic island groups being resolved. Finally, appreciable variance in individual inbreeding could be detected, reflecting the strong polygyny and site fidelity of the species. Overall, our study contributes important resources for future genomic studies of fur seals and other pinnipeds while also providing a clear example of how high throughput sequencing can generate diverse biological insights at multiple levels of organization.

Statistical approach for ATR-FTIR screening of semen in sexual evidence.

Genetic identification has revolutionized the Forensic Sciences, especially in sexual aggression cases. For the successful extraction of the genetic information of a criminal, a crucial step is the prior detection of bodily fluids on evidence. In this article, a method for non-destructive screening of semen samples is reported. Using chemometric tools, bodily fluids can be detected and differentiated without damaging the sample, by correlating the infrared spectra of sexual evidence with previously recorded spectra from undamaged stains of individual bodily fluids. In modern hospitals/laboratories, the proposed method would not require additional equipment/material nor specialized personnel. Furthermore, the method provides qualitative and reliable results, without requiring human interpretation. Therefore, the proposed method opens a door for a low-cost, fully automated and efficient system for non-destructive screening of semen, which could be easily and massively implemented.

Analysis of human bodily fluids on superabsorbent pads by ATR-FTIR.

Superabsorbent pads are composed of different layers with different grades of absorbent capacity, being the lower one the most absorbent layer. Due to their complexity, the analysis of bodily fluids on superabsorbent pads is certainly difficult. In this study, semen, vaginal fluid and urine stains placed on superabsorbent pads including sanitary napkins, panty-liners and diapers were non-destructively detected by Attenuated Total Reflectance (ATR) Fourier Transform Infrared spectroscopy (FTIR). In spite of the higher absorbent capacity of the lower layers, this technique was able to detect the three fluids on the upper layer of all pads, showing that bodily fluids are distributed within all layers. Additionally, mixtures of these bodily fluids prepared on superabsorbent pads and cotton were studied, since real forensic investigations involving sexual abuse cases usually deal with mixtures of these fluids. Due to their IR marked protein region (1800-1480cm-1), semen and vaginal fluid were easily distinguished from urine. However, since semen and vaginal fluid have both a high protein composition, that region of their IR signatures were quite similar, except for slight visual differences, that should be further analysed. Therefore, we propose ATR-FTIR as a suitable, presumptive, non-destructive and rapid approach to detect stains of human bodily fluids on the upper layer of superabsorbent pads from sexual crimes.

Modulation of the uptake of critical nutrients by breast cancer cells by lactate: Impact on cell survival, proliferation and migration.

This work aimed to characterize the uptake of folate and glucose by breast cancer cells and to study the effect of lactate upon the transport of these nutrients and upon cell viability, proliferation and migration capacity. Data obtained showed that: a) MCF7 cells uptake (3)H-folic acid ((3)H-FA) at physiological but not at acidic pH; b) T47D cells accumulate (3)H-FA and (14)C-5-methyltetrahydrofolate ((14)C-5-MTHF) more efficiently at acidic than at physiological pH; c) (3)H-deoxyglucose ((3)H-DG) uptake by T47D cells is sodium-independent, inhibited by cytochalasin B (CYT B) and stimulated by insulin. Regarding the effect of lactate, in T47D cells, acute (26 min) and chronic (24 h) exposure to lactic acid (LA) stimulated (3)H-FA uptake. Acute exposure to LA also stimulated (3)H-DG uptake and chronic exposure to LA significantly stimulated T47D cell migratory capacity. In conclusion, the transport of folates is strikingly different in two phenotypically similar breast cancer cell lines: MCF7 and T47D cells. Additionally, lactate seems to act as a signaling molecule which increases the uptake of nutrients and promotes the migration capacity of T47D cells.

The effect of oxidative stress upon the intestinal epithelial uptake of butyrate.

Our aim was to investigate the effect of oxidative stress upon butyrate uptake at the intestinal epithelial level. For this, IEC-6 cells were treated with tert-butylhydroperoxide 3000µM (tBOOH), which increased levels of oxidative stress biomarkers, while maintaining cellular viability. The effect of tBOOH upon uptake of [(14)C]butyrate ([(14)C]BT) (10µM) can be summarized as follows: (a) it caused a reduction in the intracellular accumulation of [(14)C]BT over time, (b) it strongly reduced total [(14)C]BT uptake but did not affect Na(+)-independent uptake of [(14)C]BT, and (c) it did not affect the kinetics of [(14)C]BT uptake at 37°C, but increased uptake at 4°C. Moreover, tBOOH increased the efflux of [(14)C]BT not mediated by breast cancer resistance protein. We thus conclude that tBOOH strongly inhibits Na(+)-coupled monocarboxylate cotransporter 1 (SMCT1)-mediated, but not H(+)-coupled monocarboxylate transporter (MCT1)-mediated butyrate uptake; moreover, it increases uptake and efflux of butyrate by passive diffusion. tBOOH did not affect the mRNA expression levels of MCT1 and SMCT1 nor their cell membrane insertion. Rather, its effect was dependent on extracellular signal regulated kinase 1/2 and protein tyrosine kinase activation and on the generation of reactive oxygen species by NADPH and xanthine oxidases and was partially prevented by the polyphenols quercetin and resveratrol. In conclusion, tBOOH is an effective inhibitor of SMCT1-mediated butyrate transport in non-tumoral intestinal epithelial cells. Given the important role played by butyrate in the intestine, this mechanism may contribute to the procarcinogenic and proinflammatory effect of oxidative stress at this level.

Inhibition of butyrate uptake by the primary bile salt chenodeoxycholic acid in intestinal epithelial cells.

Colorectal cancer (CRC) is one of the most common cancers worldwide. Epidemiological and experimental studies suggest that bile acids may play a role in CRC etiology. Our aim was to characterize the effect of the primary bile acid chenodeoxycholic acid (CDCA) upon(14) C-BT uptake in tumoral (Caco-2) and non-tumoral (IEC-6) intestinal epithelial cell lines. A 2-day exposure to CDCA markedly and concentration-dependently inhibited (14) C-BT uptake by IEC-6 cells (IC(50) = 120 µM), and, less potently, by Caco-2 cells (IC(50) = 402 µM). The inhibitory effect of CDCA upon (14) C-BT uptake did not result from a decrease in cell proliferation or viability. In IEC-6 cells: (1) uptake of (14) C-BT involves both a high-affinity and a low-affinity transporter, and CDCA acted as a competitive inhibitor of the high-affinity transporter; (2) CDCA inhibited both Na(+)-coupled monocarboxylate cotransporter 1 (SMCT1)- and H(+)-coupled monocarboxylate transporter 1 (MCT1)-mediated uptake of (14) C-BT; (3) CDCA significantly increased the mRNA expression level of SMCT1; (4) inhibition of (14) C-BT uptake by CDCA was dependent on CaM, MAP kinase (ERK1/2 and p38 pathways), and PKC activation, and reduced by a reactive oxygen species scavenger. Finally, BT (5 mM) decreased IEC-6 cell viability and increased IEC-6 cell differentiation, and CDCA (100 µM) reduced this effect. In conclusion, CDCA is an effective inhibitor of (14) C-BT uptake in tumoral and non-tumoral intestinal epithelial cells, through inhibition of both H(+) -coupled MCT1- and SMCT1-mediated transport. Given the role played by BT in the intestine, this mechanism may contribute to the procarcinogenic effect of CDCA at this level.

The effect of clotrimazole on energy substrate uptake and carcinogenesis in intestinal epithelial cells.

Clotrimazole has anticarcinogenic activity in several cell types. Our aims were to investigate the anticarcinogenic effect of clotrimazole in a tumoral intestinal epithelial (Caco-2) cell line, to compare it with the effect in a nontumoral intestinal epithelial cell line (IEC-6 cells), and to investigate inhibition of energy substrate uptake as a mechanism contributing to it. The effect of clotrimazole on cell proliferation, viability and differentiation, H-deoxyglucose (H-DG), H-O-methyl-glucose (H-OMG), and C-butyrate uptake, as well as mRNA expression levels of glucose transporters was assessed. In Caco-2 cells, clotrimazole decreased cellular viability and proliferation and increased cell differentiation. The effect on cell proliferation and viability was potentiated by rhodamine123. Clotrimazole also decreased cellular viability and proliferation in IEC-6 cells, but increased the cellular DNA synthesis rate and had no effect on cell differentiation. Exposure of Caco-2 cells to clotrimazole (10 µmol/l) for 1 and 7 days increased (by 20-30%) the uptake of H-DG and H-OMG, respectively, but had no effect on C-butyrate uptake. The effect on H-DG and H-OMG transport was maximal at 10 µmol/l, and the pharmacological characteristics of transport were not changed. However, clotrimazole changed the mRNA expression levels of the facilitative glucose transporter 2 and the Na-dependent glucose cotransporter. Clotrimazole exhibits comparable cytotoxic effects in tumoral and nontumoral intestinal epithelial cell lines. In Caco-2 cells, the cytotoxic effect of clotrimazole was strongly potentiated by the inhibition of oxidative phosphorylation. Moreover, stimulation of glucose uptake might be a compensation mechanism in response to the glycolysis inhibition caused by clotrimazole.

The short-chain fatty acid butyrate is a substrate of breast cancer resistance protein.

Colorectal cancer is one of the most common cancers worldwide. Butyrate (BT) plays a key role in colonic epithelium homeostasis. The aim of this work was to investigate the possibility of BT being transported by P-glycoprotein (MDR1), multidrug resistance proteins (MRPs), or breast cancer resistance protein (BCRP). Uptake and efflux of (14)C-BT and (3)H-folic acid were measured in Caco-2, IEC-6, and MDA-MB-231 cell lines. mRNA expression of BCRP was detected by RT-PCR. Cell viability, proliferation, and differentiation were quantified with the lactate dehydrogenase, sulforhodamine B, and alkaline phosphatase activity assays, respectively. In both IEC-6 cells and Caco-2 cells, no evidence was found for the involvement of either MDR1 or MRPs in (14)C-BT efflux from the cells. In contrast, several lines of evidence support the conclusion that BT is a substrate of both rat and human BCRP. Indeed, BCRP inhibitors reduced (14)C-BT efflux in IEC-6 cells, both BT and BCRP inhibitors significantly decreased the efflux of the known BCRP substrate (3)H-folic acid in IEC-6 cells, and BCRP inhibitors reduced (14)C-BT efflux in the BCRP-expressing MDA-MB-231 cell line. In IEC-6 cells, combination of BT with a BCRP inhibitor significantly potentiated the effect of BT on cell proliferation. The results of this study, showing for the first time that BT is a BCRP substrate, are very important in the context of the high levels of BCRP expression in the human colon and the anticarcinogenic and anti-inflammatory role of BT at that level. So, interaction of BT with BCRP and with other BCRP substrates/inhibitors is clearly of major importance.