Protein Distribution during Human Erythroblast Enucleation In Vitro

Enucleation is the step in erythroid terminal differentiation when the nucleus is expelled from developing erythroblasts creating reticulocytes and free nuclei surrounded by plasma membrane. We have studied protein sorting during human erythroblast enucleation using fluorescence activated cell sorting (FACS) to obtain pure populations of reticulocytes and nuclei produced by in vitro culture. Nano LC mass spectrometry was first used to determine the protein distribution profile obtained from the purified reticulocyte and extruded nuclei populations. In general cytoskeletal proteins and erythroid membrane proteins were preferentially restricted to the reticulocyte alongside key endocytic machinery and cytosolic proteins. The bulk of nuclear and ER proteins were lost with the nucleus. In contrast to the localization reported in mice, several key erythroid membrane proteins were detected in the membrane surrounding extruded nuclei, including band 3 and GPC. This distribution of key erythroid membrane and cytoskeletal proteins was confirmed using western blotting. Protein partitioning during enucleation was investigated by confocal microscopy with partitioning of cytoskeletal and membrane proteins to the reticulocyte observed to occur at a late stage of this process when the nucleus is under greatest constriction and almost completely extruded. Importantly, band 3 and CD44 were shown not to restrict specifically to the reticulocyte plasma membrane. This highlights enucleation as a stage at which excess erythroid membrane proteins are discarded in human erythroblast differentiation. Given the striking restriction of cytoskeleton proteins and the fact that membrane proteins located in macromolecular membrane complexes (e.g. GPA, Rh and RhAG) are segregated to the reticulocyte, we propose that the membrane proteins lost with the nucleus represent an excess mobile population of either individual proteins or protein complexes.     

CCDC65 Mutation Causes Primary Ciliary Dyskinesia with Normal Ultrastructure and Hyperkinetic Cilia

Background

Primary ciliary dyskinesia (PCD) is a genetic disorder characterized by impaired ciliary function, leading to chronic sinopulmonary disease. The genetic causes of PCD are still evolving, while the diagnosis is often dependent on finding a ciliary ultrastructural abnormality and immotile cilia. Here we report a novel gene associated with PCD but without ciliary ultrastructural abnormalities evident by transmission electron microscopy, but with dyskinetic cilia beating.

Methods

Genetic linkage analysis was performed in a family with a PCD subject. Gene expression was studied in Chlamydomonas reinhardtii and human airway epithelial cells, using RNA assays and immunostaining. The phenotypic effects of candidate gene mutations were determined in primary culture human tracheobronchial epithelial cells transduced with gene targeted shRNA sequences. Video-microscopy was used to evaluate cilia motion.

Results

A single novel mutation in CCDC65, which created a termination codon at position 293, was identified in a subject with typical clinical features of PCD. CCDC65, an orthologue of the Chlamydomonas nexin-dynein regulatory complex protein DRC2, was localized to the cilia of normal nasal epithelial cells but was absent in those from the proband. CCDC65 expression was up-regulated during ciliogenesis in cultured airway epithelial cells, as was DRC2 in C. reinhardtii following deflagellation. Nasal epithelial cells from the affected individual and CCDC65-specific shRNA transduced normal airway epithelial cells had stiff and dyskinetic cilia beating patterns compared to control cells. Moreover, Gas8, a nexin-dynein regulatory complex component previously identified to associate with CCDC65, was absent in airway cells from the PCD subject and CCDC65-silenced cells.

Conclusion

Mutation in CCDC65, a nexin-dynein regulatory complex member, resulted in a frameshift mutation and PCD. The affected individual had altered cilia beating patterns, and no detectable ultrastructural defects of the ciliary axoneme, emphasizing the role of the nexin-dynein regulatory complex and the limitations of certain methods for PCD diagnosis.     

The Impact of Syphilis Screening among Female Sex Workers in China: A Modelling Study

Background

In China, female sex workers (FSWs) are at high risk of syphilis infection, but are hard to reach for interventions. Point-of-care testing introduces opportunities for expanding syphilis control measures. Modelling is used to estimate the impact of using rapid tests to screen FSWs for syphilis. In other settings, modelling has predicted large rebounds in infectious syphilis following screening, which may undermine any impact achieved.

Methods

A deterministic syphilis transmission model among FSWs and clients was fitted to data from Yunnan Province (FSW syphilis prevalence = 7.5%), and used to estimate the impact of rapid syphilis testing and treatment for FSWs. Impact projections were compared for different model structures that included risk heterogeneity amongst FSWs, incoming syphilis infections amongst new FSWs and clients and re-infection from FSWs'' regular non-commercial partners. The rebound in syphilis prevalence after screening ceased was explored.

Results

All model structures suggest yearly syphilis screening could substantially reduce (by 72–88%) syphilis prevalence amongst FSWs in this setting over five years. However, incoming syphilis infections amongst new FSWs and clients or re-infections from regular non-commercial partners of FSWs can considerably reduce (>30%) the proportion of infections averted. Including heterogeneity in risk amongst FSWs had little effect upon the proportion of infections averted. In this setting, the rebound in syphilis prevalence after screening ceased is predicted to be slight, but it could be large in high prevalence settings.

Conclusions

Rapid test screening could dramatically reduce syphilis prevalence amongst hard-to-reach groups, but strategies to reduce re-infection from regular non-commercial partners are needed to maximise impact.     

Aerobic exercise alone results in clinically significant weight loss for men and women: Midwest exercise trial 2

Exercise is recommended by public health agencies for weight management; however, the role of exercise is generally considered secondary to energy restriction. Few studies exist that have verified completion of exercise, measured the energy expenditure of exercise, and prescribed exercise with equivalent energy expenditure across individuals and genders.

Objective:

The objective of this study was to evaluate aerobic exercise, without energy restriction, on weight loss in sedentary overweight and obese men and women.

Design and Methods:

This investigation was a randomized, controlled, efficacy trial in 141 overweight and obese participants (body mass index, 31.0 ± 4.6 kg/m²; age 22.6 ± 3.9 years). Participants were randomized (2:2:1 ratio) to exercise at either 400 kcal/session or 600 kcal/session or to a nonexercise control. Exercise was supervised, 5 days/week, for 10 months. All participants were instructed to maintain usual ad libitum diets. Because of the efficacy design, completion of ≥90% of exercise sessions was an a priori definition of per protocol, and these participants were included in the analysis.

Results:

Weight loss from baseline to 10 months for the 400 and 600 kcal/session groups was 3.9 ± 4.9 kg (4.3%) and 5.2 ± 5.6 kg (5.7%), respectively, compared with weight gain for controls of 0.5 ± 3.5 kg (0.5%) (P < 0.05). Differences for weight loss from baseline to 10 months between the exercise groups and differences between men and women within groups were not statistically significant.

Conclusions:

Supervised exercise, with equivalent energy expenditure, results in clinically significant weight loss with no significant difference between men and women.     

Global Epidemiology of Mental Disorders: What Are We Missing?

Background

Population-based studies provide the understanding of health-need required for effective public health policy and service-planning. Mental disorders are an important but, until recently, neglected agenda in global health. This paper reviews the coverage and limitations in global epidemiological data for mental disorders and suggests strategies to strengthen the data.

Methods

Systematic reviews were conducted for population-based epidemiological studies in mental disorders to inform new estimates for the global burden of disease study. Estimates of population coverage were calculated, adjusted for study parameters (age, gender and sampling frames) to quantify regional coverage.

Results

Of the 77,000 data sources identified, fewer than 1% could be used for deriving national estimates of prevalence, incidence, remission, and mortality in mental disorders. The two major limitations were (1) highly variable regional coverage, and (2) important methodological issues that prevented synthesis across studies, including the use of varying case definitions, the selection of samples not allowing generalization, lack of standardized indicators, and incomplete reporting. North America and Australasia had the most complete prevalence data for mental disorders while coverage was highly variable across Europe, Latin America, and Asia Pacific, and poor in other regions of Asia and Africa. Nationally-representative data for incidence, remission, and mortality were sparse across most of the world.

Discussion

Recent calls to action for global mental health were predicated on the high prevalence and disability of mental disorders. However, the global picture of disorders is inadequate for planning. Global data coverage is not commensurate with other important health problems, and for most of the world''s population, mental disorders are invisible and remain a low priority.     

Genetic architecture of apple fruit quality traits following storage and implications for genetic improvement

Accurate prediction of genetic potential and response to selection in breeding requires knowledge of genetic parameters for important selection traits. Data from breeding trials can be used to obtain estimates of these parameters so that predictions are directly relevant to the improvement program. Here, a factor allocation diagram was developed to describe the sampling design used to assess the quality of fresh and post-storage (2 months) fruit from advanced selection trial in an apple breeding program from which models for analyses were developed. Genetic variation was the largest source of variation for the fruit size, red colour type, proportion of red skin colour and lenticels, and instrumentally assessed fruit diameter, mass, puncture force and titratable acidity. In contrast, residual variation was the largest for fruit shape, juiciness, sweetness, aromatic flavour, eating and overall quality, and instrumental crispness. Genetic effects for traits were generally stable over fixed effects, except for a significant interaction with storage duration for firmness. Genetic correlations among traits were generally weak except between fruit mass (and diameter) and sensory size (0.98), titratable acidity and sensory acidity (0.97), puncture force and sensory firmness (0.96–0.90), crispness and juiciness (0.87), sweetness and aromatic flavour (0.84) and instrumental and sensory crispness (0.75). Predictions of the performance for seven commercial cultivars are presented. This study suggests that the Washington State apple production area can be treated as a single target environment and sufficient diversity exists to generate new elite cultivars. In addition, options for evaluating the efficiency of apple breeding are discussed.     

Cell‐to‐cell heterogeneity emerges as consequence of metabolic cooperation in a synthetic yeast community

Cells that grow together respond heterogeneously to stress even when they are genetically similar. Metabolism, a key determinant of cellular stress tolerance, may be one source of this phenotypic heterogeneity, however, this relationship is largely unclear. We used self‐establishing metabolically cooperating (SeMeCo) yeast communities, in which metabolic cooperation can be followed on the basis of genotype, as a model to dissect the role of metabolic cooperation in single‐cell heterogeneity. Cells within SeMeCo communities showed to be highly heterogeneous in their stress tolerance, while the survival of each cell under heat or oxidative stress, was strongly determined by its metabolic specialization. This heterogeneity emerged for all metabolite exchange interactions studied (histidine, leucine, uracil, and methionine) as well as oxidant (H₂O₂, diamide) and heat stress treatments. In contrast, the SeMeCo community collectively showed to be similarly tolerant to stress as wild‐type populations. Moreover, stress heterogeneity did not establish as sole consequence of metabolic genotype (auxotrophic background) of the single cell, but was observed only for cells that cooperated according to their metabolic capacity. We therefore conclude that phenotypic heterogeneity and cell to cell differences in stress tolerance are emergent properties when cells cooperate in metabolism.     

HPV Serology Testing Confirms High HPV Immunisation Coverage in England

Background

Reported human papillomavirus (HPV) vaccination coverage in England is high, particularly in girls offered routine immunisation at age 12 years. Serological surveillance can be used to validate reported coverage and explore variations within it and changes in serological markers over time.

Methods

Residual serum specimens collected from females aged 15–19 years in 2010–2011 were tested for anti-HPV16 and HPV18 IgG by ELISA. Based on these results, females were classified as follows: seronegative, probable natural infection, probable vaccine-induced seropositivity, or possible natural infection/possible vaccine-induced seropositivity. The proportion of females with vaccine-induced seropositivity was compared to the reported vaccination coverage.

Results

Of 2146 specimens tested, 1380 (64%) were seropositive for both types HPV16 and HPV18 and 159 (7.4%) positive for only one HPV type. The IgG concentrations were far higher for those positive for both HPV types than those positive for only one HPV type. 1320 (62%) females were considered to have probable vaccine-induced seropositivity. Among vaccine-induced seropositives, antibody concentrations declined with increasing age at vaccination and increasing time since vaccination.

Conclusions

The proportion of females with vaccine-induced seropositivity was closest to the reported 3-dose coverage in those offered the vaccination at younger ages, with a greater discrepancy in the older females. This suggests either some under-reporting of immunisations of older females and/or that partial vaccination (i.e. one- or two-doses) has provided high antibody responses in 13–17 year olds.     

Population structure and phylogeography of the Gentoo Penguin (Pygoscelis papua) across the Scotia Arc

Climate change, fisheries' pressure on penguin prey, and direct human disturbance of wildlife have all been implicated in causing large shifts in the abundance and distribution of penguins in the Southern Ocean. Without mark‐recapture studies, understanding how colonies form and, by extension, how ranges shift is challenging. Genetic studies, particularly focused on newly established colonies, provide a snapshot of colonization and can reveal the extent to which shifts in abundance and occupancy result from changes in demographic rates (e.g., reproduction and survival) or migration among suitable patches of habitat. Here, we describe the population structure of a colonial seabird breeding across a large latitudinal range in the Southern Ocean. Using multilocus microsatellite genotype data from 510 Gentoo penguin (Pygoscelis papua) individuals from 14 colonies along the Scotia Arc and Antarctic Peninsula, together with mitochondrial DNA data, we find strong genetic differentiation between colonies north and south of the Polar Front, that coincides geographically with the taxonomic boundary separating the subspecies P. p. papua and P. p. ellsworthii. Using a discrete Bayesian phylogeographic approach, we show that southern Gentoos expanded from a possible glacial refuge in the center of their current range, colonizing regions to the north and south through rare, long‐distance dispersal. Our findings show that this dispersal is important for new colony foundation and range expansion in a seabird species that ordinarily exhibits high levels of natal philopatry, though persistent oceanographic features serve as barriers to movement.