Detecting and Characterizing Genomic Signatures of Positive Selection in Global Populations

Natural selection is a significant force that shapes the architecture of the human genome and introduces diversity across global populations. The question of whether advantageous mutations have arisen in the human genome as a result of single or multiple mutation events remains unanswered except for the fact that there exist a handful of genes such as those that confer lactase persistence, affect skin pigmentation, or cause sickle cell anemia. We have developed a long-range-haplotype method for identifying genomic signatures of positive selection to complement existing methods, such as the integrated haplotype score (iHS) or cross-population extended haplotype homozygosity (XP-EHH), for locating signals across the entire allele frequency spectrum. Our method also locates the founder haplotypes that carry the advantageous variants and infers their corresponding population frequencies. This presents an opportunity to systematically interrogate the whole human genome whether a selection signal shared across different populations is the consequence of a single mutation process followed subsequently by gene flow between populations or of convergent evolution due to the occurrence of multiple independent mutation events either at the same variant or within the same gene. The application of our method to data from 14 populations across the world revealed that positive-selection events tend to cluster in populations of the same ancestry. Comparing the founder haplotypes for events that are present across different populations revealed that convergent evolution is a rare occurrence and that the majority of shared signals stem from the same evolutionary event.     

Deep Whole-Genome Sequencing of 100 Southeast Asian Malays

Whole-genome sequencing across multiple samples in a population provides an unprecedented opportunity for comprehensively characterizing the polymorphic variants in the population. Although the 1000 Genomes Project (1KGP) has offered brief insights into the value of population-level sequencing, the low coverage has compromised the ability to confidently detect rare and low-frequency variants. In addition, the composition of populations in the 1KGP is not complete, despite the fact that the study design has been extended to more than 2,500 samples from more than 20 population groups. The Malays are one of the Austronesian groups predominantly present in Southeast Asia and Oceania, and the Singapore Sequencing Malay Project (SSMP) aims to perform deep whole-genome sequencing of 100 healthy Malays. By sequencing at a minimum of 30× coverage, we have illustrated the higher sensitivity at detecting low-frequency and rare variants and the ability to investigate the presence of hotspots of functional mutations. Compared to the low-pass sequencing in the 1KGP, the deeper coverage allows more functional variants to be identified for each person. A comparison of the fidelity of genotype imputation of Malays indicated that a population-specific reference panel, such as the SSMP, outperforms a cosmopolitan panel with larger number of individuals for common SNPs. For lower-frequency (<5%) markers, a larger number of individuals might have to be whole-genome sequenced so that the accuracy currently afforded by the 1KGP can be achieved. The SSMP data are expected to be the benchmark for evaluating the value of deep population-level sequencing versus low-pass sequencing, especially in populations that are poorly represented in population-genetics studies.     

Life cycle assessment of cheese and whey production in the USA

Purpose

A life cycle assessment was conducted to determine a baseline for environmental impacts of cheddar and mozzarella cheese consumption. Product loss/waste, as well as consumer transport and storage, is included. The study scope was from cradle-to-grave with particular emphasis on unit operations under the control of typical cheese-processing plants.

Methods

SimaPro© 7.3 (PRé Consultants, The Netherlands, 2013) was used as the primary modeling software. The ecoinvent life cycle inventory database was used for background unit processes (Frischknecht and Rebitzer, J Cleaner Prod 13(13–14):1337–1343, 2005), modified to incorporate US electricity (EarthShift 2012). Operational data was collected from 17 cheese-manufacturing plants representing 24 % of mozzarella production and 38 % of cheddar production in the USA. Incoming raw milk, cream, or dry milk solids were allocated to coproducts by mass of milk solids. Plant-level engineering assessments of allocation fractions were adopted for major inputs such as electricity, natural gas, and chemicals. Revenue-based allocation was applied for the remaining in-plant processes.

Results and discussion

Greenhouse gas (GHG) emissions are of significant interest. For cheddar, as sold at retail (63.2 % milk solids), the carbon footprint using the IPCC 2007 factors is 8.60 kg CO₂e/kg cheese consumed with a 95 % confidence interval (CI) of 5.86–12.2 kg CO₂e/kg. For mozzarella, as sold at retail (51.4 % milk solids), the carbon footprint is 7.28 kg CO₂e/kg mozzarella consumed, with a 95 % CI of 5.13–9.89 kg CO₂e/kg. Normalization of the results based on the IMPACT 2002+ life cycle impact assessment (LCIA) framework suggests that nutrient emissions from both the farm and manufacturing facility wastewater treatment represent the most significant relative impacts across multiple environmental midpoint indicators. Raw milk is the major contributor to most impact categories; thus, efforts to reduce milk/cheese loss across the supply chain are important.

Conclusions

On-farm mitigation efforts around enteric methane, manure management, phosphorus and nitrogen runoff, and pesticides used on crops and livestock can also significantly reduce impacts. Water-related impacts such as depletion and eutrophication can be considered resource management issues—specifically of water quantity and nutrients. Thus, all opportunities for water conservation should be evaluated, and cheese manufacturers, while not having direct control over crop irrigation, the largest water consumption activity, can investigate the water use efficiency of the milk they procure. The regionalized normalization, based on annual US per capita cheese consumption, showed that eutrophication represents the largest relative impact driven by phosphorus runoff from agricultural fields and emissions associated with whey-processing wastewater. Therefore, incorporating best practices around phosphorous and nitrogen management could yield improvements.     

Evidence for the role of vacuolar soluble pyrophosphatase and inorganic polyphosphate in Trypanosoma cruzi persistence

Trypanosoma cruzi infection leads to development of a chronic disease but the mechanisms that the parasite utilizes to establish a persistent infection despite activation of a potent immune response by the host are currently unknown. Unusual characteristics of T. cruzi are that it possesses cellular levels of pyrophosphate (PP_i) at least 10 times higher than those of ATP and molar levels of inorganic polyphosphate (polyP) within acidocalcisomes. We characterized an inorganic soluble EF‐hand containing pyrophosphatase from T. cruzi (TcVSP) that, depending on the pH and cofactors, can hydrolyse either pyrophosphate (PP_i) or polyphosphate (polyP). The enzyme is localized to both acidocalcisomes and cytosol. Overexpression of TcVSP (TcVSP‐OE) resulted in a significant decrease in cytosolic PP_i, and short and long‐chain polyP levels. Additionally, the TcVSP‐OE parasites showed a significant growth defect in fibroblasts, less responsiveness to hyperosmotic stress, and reduced persistence in tissues of mice, suggesting that PP_i and polyP are essential for the parasite to resist the stressful conditions in the host and to maintain a persistent infection.     

Scots pine growing on forested mires in Abernethy Forest,Strathspey, Scotland

Summary

A substantial proportion of the Abernethy Forest Reserve has Scots pine (Pinus sylvestris) growing on the surfaces of a variety of mires. The hydrology of the mires has been affected by drainage and peat cutting but this area is unusual in having had a long period of protection from grazing by domestic stock. There are three main types of pine populations found on these mires. Woodland bog comprises predominantly bog vegetation with abundant pine seedlings due to the heavy seed rain from the surrounding woodland. Only a few very small trees survive, which are stunted, heavily diseased and have very low seed production. Wooded bog also comprises predominately bog vegetation but there are scattered mature trees of a moderate height with an open canopy. The trees are fertile and can form uneven aged stands with regeneration. Bog woodland is a predominantly woodland vegetation with tall, dense tree cover on deep peat. The trees are well grown with a dense canopy. A few remnants of bog vegetation remain in the ground flora although most have been replaced by woodland bryophytes and shrubs. Each of these three types is described and their development is discussed.     

Acute Effects of Capsaicin on Energy Expenditure and Fat Oxidation in Negative Energy Balance

Background

Addition of capsaicin (CAPS) to the diet has been shown to increase energy expenditure; therefore capsaicin is an interesting target for anti-obesity therapy.

Aim

We investigated the 24 h effects of CAPS on energy expenditure, substrate oxidation and blood pressure during 25% negative energy balance.

Methods

Subjects underwent four 36 h sessions in a respiration chamber for measurements of energy expenditure, substrate oxidation and blood pressure. They received 100% or 75% of their daily energy requirements in the conditions ‘100%CAPS’, ‘100%Control’, ‘75%CAPS’ and ‘75%Control’. CAPS was given at a dose of 2.56 mg (1.03 g of red chili pepper, 39,050 Scoville heat units (SHU)) with every meal.

Results

An induced negative energy balance of 25% was effectively a 20.5% negative energy balance due to adapting mechanisms. Diet-induced thermogenesis (DIT) and resting energy expenditure (REE) at 75%CAPS did not differ from DIT and REE at 100%Control, while at 75%Control these tended to be or were lower than at 100%Control (p = 0.05 and p = 0.02 respectively). Sleeping metabolic rate (SMR) at 75%CAPS did not differ from SMR at 100%CAPS, while SMR at 75%Control was lower than at 100%CAPS (p = 0.04). Fat oxidation at 75%CAPS was higher than at 100%Control (p = 0.03), while with 75%Control it did not differ from 100%Control. Respiratory quotient (RQ) was more decreased at 75%CAPS (p = 0.04) than at 75%Control (p = 0.05) when compared with 100%Control. Blood pressure did not differ between the four conditions.

Conclusion

In an effectively 20.5% negative energy balance, consumption of 2.56 mg capsaicin per meal supports negative energy balance by counteracting the unfavorable negative energy balance effect of decrease in components of energy expenditure. Moreover, consumption of 2.56 mg capsaicin per meal promotes fat oxidation in negative energy balance and does not increase blood pressure significantly.

Trial Registration

Nederlands Trial Register; registration number NTR2944     

Feasibility of Tomotherapy-Based Image-Guided Radiotherapy to Reduce Aspiration Risk in Patients with Non-Laryngeal and Non-Pharyngeal Head and Neck Cancer

Purpose

The study aims to assess the feasibility of Tomotherapy-based image-guided radiotherapy (IGRT) to reduce the aspiration risk in patients with non-laryngeal and non-hypopharyngeal cancer. A retrospective review of 48 patients undergoing radiation for non-laryngeal and non-hypopharyngeal head and neck cancers was conducted. All patients had a modified barium swallow (MBS) prior to treatment, which was repeated one month following radiotherapy. Mean middle and inferior pharyngeal dose was recorded and correlated with the MBS results to determine aspiration risk.

Results

Mean pharyngeal dose was 23.2 Gy for the whole group. Two patients (4.2%) developed trace aspiration following radiotherapy which resolved with swallowing therapy. At a median follow-up of 19 months (1–48 months), all patients were able to resume normal oral feeding without aspiration.

Conclusion and Clinical Relevance

IGRT may reduce the aspiration risk by decreasing the mean pharyngeal dose in the presence of large cervical lymph nodes. Further prospective studies with IGRT should be performed in patients with non-laryngeal and non-hypopharyngeal head and neck cancers to verify this hypothesis.     

?-Adrenergic Stimulation Increases RyR2 Activity via Intracellular Ca2+ and Mg2+ Regulation

Here we investigate how ß-adrenergic stimulation of the heart alters regulation of ryanodine receptors (RyRs) by intracellular Ca²⁺ and Mg²⁺ and the role of these changes in SR Ca²⁺ release. RyRs were isolated from rat hearts, perfused in a Langendorff apparatus for 5 min and subject to 1 min perfusion with 1 µM isoproterenol or without (control) and snap frozen in liquid N₂ to capture their phosphorylation state. Western Blots show that RyR2 phosphorylation was increased by isoproterenol, confirming that RyR2 were subject to normal ß-adrenergic signaling. Under basal conditions, S2808 and S2814 had phosphorylation levels of 69% and 15%, respectively. These levels were increased to 83% and 60%, respectively, after 60 s of ß-adrenergic stimulation consistent with other reports that ß-adrenergic stimulation of the heart can phosphorylate RyRs at specific residues including S2808 and S2814 causing an increase in RyR activity. At cytoplasmic [Ca²⁺] <1 µM, ß-adrenergic stimulation increased luminal Ca²⁺ activation of single RyR channels, decreased luminal Mg²⁺ inhibition and decreased inhibition of RyRs by mM cytoplasmic Mg²⁺. At cytoplasmic [Ca²⁺] >1 µM, ß-adrenergic stimulation only decreased cytoplasmic Mg²⁺ and Ca²⁺ inhibition of RyRs. The K_a and maximum levels of cytoplasmic Ca²⁺ activation site were not affected by ß-adrenergic stimulation.Our RyR2 gating model was fitted to the single channel data. It predicted that in diastole, ß-adrenergic stimulation is mediated by 1) increasing the activating potency of Ca²⁺ binding to the luminal Ca²⁺ site and decreasing its affinity for luminal Mg²⁺ and 2) decreasing affinity of the low-affinity Ca²⁺/Mg²⁺ cytoplasmic inhibition site. However in systole, ß-adrenergic stimulation is mediated mainly by the latter.     

Caregivers’ Attitudes towards HIV Testing and Disclosure of HIV Status to At-Risk Children in Rural Uganda

Caregivers of HIV-positive children were interviewed in the Mbarara and Isingiro districts of Uganda to identify current trends in practices related to HIV testing and the disclosure of HIV status to the child. A total of 28 caregivers of at least one HIV-positive child participated in semi-structured interviews exploring when and why they tested the child for HIV, when the child was informed of their positive status, and what the caregiver did to prepare themselves and the child for status disclosure. For a majority (96%) of respondents, the decision to test the child for HIV was due to existing illness in either the child or a relative. Other common themes identified included the existence of stigma in the caregivers’ communities and doubt that the children truly understood what was being explained to them when their status was disclosed. Most (65%) children were informed of their HIV status between the ages of 5 and 9, with the mean age of disclosure occurring at the age of 7. General provision of HIV information typically began at the same age as disclosure, and as many as two thirds (64%) of the caregivers sought advice from an HIV counsellor prior to disclosure. How a caregiver chose to prepare themselves and the child did not affect the caregiver’s perception of whether the disclosure experience was beneficial or not. These findings suggest that the HIV disclosure experience in Mbarara and Isingiro districts differs from current guidelines, especially with respect to age of disclosure, how caregivers prepare themselves and the child, and approaching disclosure as an ongoing process. The doubts expressed by caregivers regarding the child’s level of HIV understanding following the disclosure experience suggest the children may be insufficiently prepared at the time of the initial disclosure event. The findings also suggest that examining the content of pre-disclosure counselling and HIV education, and how health care professionals are trained to facilitate the disclosure process as important avenues for further research.     

Patterns of Genetic Variability in Island Populations of the Cane Toad (Rhinella marina) from the Mouth of the Amazon

The Amazonian coast has several unique geological characteristics resulting from the interaction between drainage pattern of the Amazon River and the Atlantic Ocean. It is one of the most extensive and sedimentologically dynamic regions of the world, with a large number of continental islands mostly formed less than 10,000 years ago. The natural distribution of the cane toad (Rhinella marina), one of the world’s most successful invasive species, in this complex Amazonian system provides an intriguing model for the investigation of the effects of isolation or the combined effects of isolation and habitat dynamic changes on patterns of genetic variability and population differentiation. We used nine fast-evolving microsatellite loci to contrast patterns of genetic variability in six coastal (three mainlands and three islands) populations of the cane toad near the mouth of the Amazon River. Results from Bayesian multilocus clustering approach and Discriminant Analyses of Principal Component were congruent in showing that each island population was genetically differentiated from the mainland populations. All F_ST values obtained from all pairwise comparisons were significant, ranging from 0.048 to 0.186. Estimates of both recent and historical gene flow were not significantly different from zero across all population pairs, except the two mainland populations inhabiting continuous habitats. Patterns of population differentiation, with a high level of population substructure and absence/restricted gene flow, suggested that island populations of R. marina are likely isolated since the Holocene sea-level rise. However, considering the similar levels of genetic variability found in both island and mainland populations, it is reliable to assume that they were also isolated for longer periods. Given the genetic uniqueness of each cane toad population, together with the high natural vulnerability of the coastal regions and intense human pressures, we suggest that these populations should be treated as discrete units for conservation management purposes.