The evolutionary assembly of forest communities along environmental gradients: recent diversification or sorting of pre-adapted clades?

Recent studies have demonstrated that ecological processes that shape community structure and dynamics change along environmental gradients. However, much less is known about how the emergence of the gradients themselves shape the evolution of species that underlie community assembly. In this study, we address how the creation of novel environments leads to community assembly via two nonmutually exclusive processes: immigration and ecological sorting of pre-adapted clades (ISPC), and recent adaptive diversification (RAD). We study these processes in the context of the elevational gradient created by the uplift of the Central Andes. We develop a novel approach and method based on the decomposition of species turnover into within- and among-clade components, where clades correspond to lineages that originated before mountain uplift. Effects of ISPC and RAD can be inferred from how components of turnover change with elevation. We test our approach using data from over 500 Andean forest plots. We found that species turnover between communities at different elevations is dominated by the replacement of clades that originated before the uplift of the Central Andes. Our results suggest that immigration and sorting of clades pre-adapted to montane habitats is the primary mechanism shaping tree communities across elevations.

A case of incidental infection of Hepatitis E virus (HEV) genotype 1 in a domestic pig.

Hepatitis E virus (HEV) infection involving zoonotic genotypes is a public health problem in high-income and non-endemic developing countries. Herein we report the detection of a human genotype 1 (HEV-1) strain infecting a domestic pig, which is not considered a natural reservoir of this genotype. Viral load was quantified in stool by Real-Time qPCR and sequence analyses were performed. Infectivity of the HEV-1 strain was assesed by in vitro isolation in A549 cell line. Results suggest that certain epidemiological settings might favour accidental spillover infection and thus influence the host range restriction of HEV.

Historical Assembly of Andean Tree Communities.

Patterns of species diversity have been associated with changes in climate across latitude and elevation. However, the ecological and evolutionary mechanisms underlying these relationships are still actively debated. Here, we present a complementary view of the well-known tropical niche conservatism (TNC) hypothesis, termed the multiple zones of origin (MZO) hypothesis, to explore mechanisms underlying latitudinal and elevational gradients of phylogenetic diversity in tree communities. The TNC hypothesis posits that most lineages originate in warmer, wetter, and less seasonal environments in the tropics and rarely colonize colder, drier, and more seasonal environments outside of the tropical lowlands, leading to higher phylogenetic diversity at lower latitudes and elevations. In contrast, the MZO hypothesis posits that lineages also originate in temperate environments and readily colonize similar environments in the tropical highlands, leading to lower phylogenetic diversity at lower latitudes and elevations. We tested these phylogenetic predictions using a combination of computer simulations and empirical analyses of tree communities in 245 forest plots located in six countries across the tropical and subtropical Andes. We estimated the phylogenetic diversity for each plot and regressed it against elevation and latitude. Our simulated and empirical results provide strong support for the MZO hypothesis. Phylogenetic diversity among co-occurring tree species increased with both latitude and elevation, suggesting an important influence on the historical dispersal of lineages with temperate origins into the tropical highlands. The mixing of different floras was likely favored by the formation of climatically suitable corridors for plant migration due to the Andean uplift. Accounting for the evolutionary history of plant communities helps to advance our knowledge of the drivers of tree community assembly along complex climatic gradients, and thus their likely responses to modern anthropogenic climate change.

Cost-Efficient Sequence-Based Nonextensible Oligonucleotide in Real-Time PCR and High-Throughput Sequencing.

Molecular detection of disease-associated mutations, especially those with low abundance, is essential for academic research and clinical diagnosis. Certain variant detection methods reach satisfactory sensitivity and specificity in detecting rare mutations based on the introduction of blocking oligos to prevent the amplification of wild-type or unwanted templates, thus selectively amplifying and enriching the mutations. These blocking oligos usually suppress PCR amplification through the 3' chemical modifications, with high price, slow synthesis, and reduced purity. Herein, we introduce chemistry-free designs to block enzymatic extension during PCR by the steric hindrance from the secondary structures attached to the 3' end of the oligos (nonextensible oligonucleotide, NEO). We demonstrated that NEO efficiently prohibited the extension of both Taq and high-fidelity DNA polymerases. By further applying NEO as blockers in blocker displacement amplification (BDA) qPCR, multiplex BDA (mBDA) NGS, and quantitative BDA (QBDA) NGS methods, we showed that NEO blockers had performance comparable with previously validated chemical modifications. Comparison experiments using QBDA with NEO blockers and droplet digital PCR (ddPCR) on clinical formalin-fixed paraffin-embedded (FFPE) samples exhibited 100% concordance. Lastly, the ability of NEO to adjust plex uniformity through changes of PCR amplification efficiency was demonstrated in an 80-plex NGS panel.

Confirming putative variants at ≤ 5% allele frequency using allele enrichment and Sanger sequencing.

Whole exome sequencing (WES) is used to identify mutations in a patient's tumor DNA that are predictive of tumor behavior, including the likelihood of response or resistance to cancer therapy. WES has a mutation limit of detection (LoD) at variant allele frequencies (VAF) of 5%. Putative mutations called at ≤ 5% VAF are frequently due to sequencing errors, therefore reporting these subclonal mutations incurs risk of significant false positives. Here we performed ~ 1000 × WES on fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissue biopsy samples from a non-small cell lung cancer patient, and identified 226 putative mutations at between 0.5 and 5% VAF. Each variant was then tested using NuProbe NGSure, to confirm the original WES calls. NGSure utilizes Blocker Displacement Amplification to first enrich the allelic fraction of the mutation and then uses Sanger sequencing to determine mutation identity. Results showed that 52% of the 226 (117) putative variants were disconfirmed, among which 2% (5) putative variants were found to be misidentified in WES. In the 66 cancer-related variants, the disconfirmed rate was 82% (54/66). This data demonstrates Blocker Displacement Amplification allelic enrichment coupled with Sanger sequencing can be used to confirm putative mutations ≤ 5% VAF. By implementing this method, next-generation sequencing can reliably report low-level variants at a high sensitivity, without the cost of high sequencing depth.

Laparoscopic Roux-en-Y gastric bypass as a revision procedure after restrictive bariatric surgery.

BACKGROUND: Adjustable gastric banding (AGB) and vertical banded gastroplasty (VBG) have been extensively used to treat morbid obesity. Patients with insufficient weight loss or complications may require surgical revision. The laparoscopic Roux-en-Y gastric bypass (LRYGBP) is one of the most common procedures currently used for revision. The aim of the study was to analyze surgical outcomes of 30 consecutive patients who underwent revision to LRYGBP in a 2-year period. METHODS: The prospectively constructed database and the medical records of all patients undergoing revision to LRYGBP were reviewed. Demographics, surgical details, results, and complications were analyzed. RESULTS: There were 23 women and seven men with a mean age of 41.1+/-9.7 years (r=25-61). Mean body mass index (BMI) was 40.0+/-7.5 kg/m(2) (r=27.2-65.2). Initial operation was AGB in 24, VBG in five, and both in one patient. In ten patients, the band had been removed before revision, in 13 cases, band removal and LRYGBP were performed in one surgical intervention, and in two patients, it was performed in a two-step surgery. There were two conversions to open surgery. Five patients presented major surgical complications. Hospital stay averaged 5.1 days (r=3-25). Mean percent excess body weight loss at 6 and 12 months was 61.7+/-27.5 and 81.2+/-20.5 kg/m(2), respectively. Mean percent low body mass index at 6 and 12 months was 22.5+/-9.1 and 29.1+/-11.4 kg/m(2), respectively. CONCLUSIONS: LRYGBP as a revision procedure is feasible in most patients. Surgical complications are more frequent.

High frequency and extensive genetic heterogeneity of TTSuV1 and TTSuVk2a in PCV2- infected and non-infected domestic pigs and wild boars from Uruguay.

Torque teno sus virus (TTSuV) infection is common worldwide in both healthy and diseased swine and a relationship between this virus and a particular disease in pigs has not been established. This work aimed to investigate the presence of TTSuV1 and TTSuVk2a in Porcine Circovirus type 2 (PCV2)-infected and non-infected domestic pigs and free-living wild boars from Uruguay. Our data evidenced a high frequency of detection and a wide circulation of TTSuV among pig herds and wild boar populations. Furthermore, TTSuV1+TTSuVk2a co-infection was more frequent than single infections in domestic pigs. In addition, we thoroughly characterized at the molecular level TTSuV strains by extensive sequence data analysis. Our findings revealed an extremely high genetic heterogeneity among Uruguayan isolates. On the basis of detailed analyses, we proposed a more comprehensive criterion of TTSuV classification which would contribute to shedding light over the genetic diversity of these viruses worldwide. On the other hand, data obtained suggested that neither TTSuV1 nor TTSuVk2a frequency of infection or viral loads have any correlation with PCV2 infection, health status or age. The role of TTSuV during co-infection with other pathogens and the age-related dynamics of TTSuV infection are currently under debate. Therefore, taking into account the controversial epidemiological data regarding these viruses and their ubiquitous infection, a likely role as components of the host microbiota should be brought into discussion.

Molecular study of Porcine Circovirus type 2 in wild boars and domestic pigs in Uruguay from 2010 to 2014: Predominance of recombinant circulating strains.

Porcine Circovirus type 2 (PCV2) is a worldwide distributed pathogen and one of the most economically relevant swine infections. Four genotypes have been recognized and it is well known that PCV2a, PCV2b and PCV2d have a global distribution. However, the information about recombinant strains circulation and their influence in driving PCV2 evolution is a poorly studied area. In Uruguay, PCV2 associated symptoms began to be frequently observed in pigs from different farms since 2010. The main purpose of this study was to thoroughly investigate the molecular epidemiology of PCV2 in nationwide swine herds and free-living wild boars during the period 2010-2014, providing an extensive viral sequence dataset. Surprisingly, the findings revealed a predominance of recombinant strains circulation, evidencing for the first time in the field that PCV2 recombination can lead to the emergence of strains able to compete and potentially displace parental ones. In addition, the circulation of the genotypes PCV2d (29%), PCV2b (10.5%) and PCV2a (7.9%) were also observed. Since 2013, a high circulation of PCV2d was identified in the country and probably reflected the recent global scenario of the emergence of this genotype. In addition, fluctuations in the frequency of PCV2 infection in the period evaluated may suggest a limitation of biosecurity strategies implemented in Uruguay for the disease control, including the instability of vaccination practices. On the other hand, the sustained PCV2 infection observed in wild boar population and the similarity among circulating viral strains from these animals and domestic pigs, suggested that wild animals could serve as permanent reservoir of the disease. Altogether, this work put forward that many factors play a role in PCV2 heterogeneity including rapid viral spread and evolution, recombination, wide movement within national boundaries and multiples introduction events resulting of international trade. Continuous monitoring of viral epidemiology is needed to better understand the PCV2 population dynamics in Uruguay and the development of appropriate strategies are required for disease control.

Large-scale patterns of turnover and Basal area change in Andean forests.

General patterns of forest dynamics and productivity in the Andes Mountains are poorly characterized. Here we present the first large-scale study of Andean forest dynamics using a set of 63 permanent forest plots assembled over the past two decades. In the North-Central Andes tree turnover (mortality and recruitment) and tree growth declined with increasing elevation and decreasing temperature. In addition, basal area increased in Lower Montane Moist Forests but did not change in Higher Montane Humid Forests. However, at higher elevations the lack of net basal area change and excess of mortality over recruitment suggests negative environmental impacts. In North-Western Argentina, forest dynamics appear to be influenced by land use history in addition to environmental variation. Taken together, our results indicate that combinations of abiotic and biotic factors that vary across elevation gradients are important determinants of tree turnover and productivity in the Andes. More extensive and longer-term monitoring and analyses of forest dynamics in permanent plots will be necessary to understand how demographic processes and woody biomass are responding to changing environmental conditions along elevation gradients through this century.

Digestive stability and transport of norbixin, a 24-carbon carotenoid, across monolayers of Caco-2 cells.

Annatto is a natural pigment widely used in the food industry to add yellow to red colors to dairy and cereal products. Here, the in vitro bioaccessibility and potential bioavailability of norbixin, the abundant 24-carbon carotenoid in annatto, were investigated. Norbixin added to milk was highly stable during simulated digestion, and bile salts enhanced partitioning of this carotenoid in the aqueous fraction during the small intestinal phase of digestion. Apical uptake of norbixin by Caco-2 cells was proportional to the concentration in apical medium, but cellular content increased only slightly after 60 min. Transport of norbixin to the basolateral compartment was maximum at 120 min. Both all-trans and cis isomers of norbixin were present in cells and basolateral medium. The results suggest that ingested norbixin is stable during gastric and small intestinal phases of digestion and that both cis and all-trans isomers are bioavailable.