Decreased rates of cerebral protein synthesis measured in vivo in a mouse model of Tuberous Sclerosis Complex: unexpected consequences of reduced tuberin

Tuberous sclerosis complex (TSC ) is an autosomal dominant neurogenetic disorder affecting about 1 in 6000 people and is caused by mutations in either TSC 1 or TSC 2 . This disorder is characterized by increased activity of mammalian target of rapamycin complex 1 (mTORC 1), which is involved in regulating ribosomal biogenesis and translation initiation. We measured the effects of Tsc2 haploinsufficiency (Tsc2 ^+/? ) in 3‐month‐old male mice on regional rates of cerebral protein synthesis (rCPS ) by means of the in vivo L‐[1‐¹⁴C]leucine method. This quantitative autoradiographic method includes an estimate of the integrated specific activity of the tracer amino acid in brain tissue. The estimate accounts for recycling of unlabeled amino acids from tissue protein breakdown by means of a factor (λ) that was determined in control and Tsc2 ^+/? mice. The value of λ was higher in Tsc2 ^+/? mice, indicating that a greater fraction of leucine in the tissue precursor pool for protein synthesis is derived from the plasma compared to controls, consistent with reduced rates of protein degradation. We determined rCPS in freely moving, awake male Tsc2 ^+/? and control mice, and we used the determined values of λ in the calculation of rCPS . Unexpectedly, we found that rCPS were significantly decreased in 16 of the 17 brain regions analyzed in Tsc2 ^+/? mice compared to controls. Our results indicate a complex role of mTORC 1 in the regulation of cerebral protein synthesis that has not been previously recognized.

     

Maintaining transparency: a review of the developmental physiology and pathophysiology of two avascular tissues

The lens and cornea are transparent and usually avascular. Controlling nutrient supply while maintaining transparency is a physiological challenge for both tissues. During sleep and with contact lens wear the endothelial layer of the cornea may become hypoxic, compromising its ability to maintain corneal transparency. The mechanism responsible for establishing the avascular nature of the corneal stroma is unknown. In several pathological conditions, the stroma can be invaded by abnormal, leaky vessels, leading to opacification. Several molecules that are likely to help maintain the avascular nature of the corneal stroma have been identified, although their relative contributions remain to be demonstrated. The mammalian lens is surrounded by capillaries early in life. After the fetal vasculature regresses, the lens resides in a hypoxic environment. Hypoxia is likely to be required to maintain lens transparency. The vitreous body may help to maintain the low oxygen level around the lens. The hypothesis is presented that many aspects of the aging of the lens, including increased hardening, loss of accommodation (presbyopia), and opacification of the lens nucleus, are caused by exposure to oxygen. Testing this hypothesis may lead to prevention for nuclear cataract and insight into the mechanisms of lens aging. Although they are both transparent, corneal pathology is associated with an insufficient supply of oxygen, while lens pathology may involve excessive exposure to oxygen.     

Quantification of small cell numbers with a microchannel device

The ability to precisely quantify rare populations of cells has become an essential first step of many cell-based assays in stem cell research. Since current devices for cell quantification require relatively high cell concentrations and/or absolute cell numbers, we have developed a microchannel-based device, allowing precise quantification of limiting cell numbers/concentrations. We anticipate this device will serve as an important tool to overcome a practical obstacle in stem cell research.     

Pyridones as glucokinase activators: Identification of a unique metabolic liability of the 4-sulfonyl-2-pyridone heterocycle

A promising area of novel anti-diabetic therapy involves identification of small molecule activators of the glucokinase enzyme to reduce blood glucose and normalize glucose stimulated insulin secretion. Herein, we report the identification and optimization of a series of 4-sulfonyl-2-pyridone activators. The activators were evaluated for in vitro biochemical activation and pharmacokinetic properties. As part of these efforts, a unique metabolic liability of the 4-sulfonyl-2-pyridone ring system was identified wherein this heterocycle readily undergoes conjugation with glutathione under non-enzymatic conditions.     

Malaria vectors of Papua New Guinea

Understanding malaria transmission in Papua New Guinea (PNG) requires exact knowledge of which Anopheles species are transmitting malaria and is complicated by the cryptic species status of many of these mosquitoes. To identify the malaria vectors in PNG we studied Anopheles specimens from 232 collection localities around human habitation throughout PNG (using CO₂ baited light traps and human bait collections). A total of 22,970 mosquitoes were individually assessed using a Plasmodium sporozoite enzyme-linked immunosorbent assay to identify Plasmodiumfalciparum, Plasmodiumvivax and Plasmodiummalariae circumsporozoite proteins. All mosquitoes were identified to species by morphology and/or PCR. Based on distribution, abundance and their ability to develop sporozoites, we identified five species as major vectors of malaria in PNG. These included: Anophelesfarauti, Anopheleshinesorum (incriminated here, to our knowledge, for the first time), Anophelesfarauti 4, Anopheleskoliensis and Anophelespunctulatus. Anopheleslongirostris and Anophelesbancroftii were also incriminated in this study. Surprisingly, An. longirostris showed a high incidence of infections in some areas. A newly identified taxon within the Punctulatus Group, tentatively called An. farauti 8, was also found positive for circumsporozoite protein. These latter three species, together with Anopheleskarwari and Anophelessubpictus, incriminated in other studies, appear to be only minor vectors, while Anophelesfarauti 6 appears to be the major vector in the highland river valleys (>1500 m above sea level). The nine remaining Anopheles species found in PNG have been little studied and their bionomics are unknown; most appear to be uncommon with limited distribution and their possible role in malaria transmission has yet to be determined.     

Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells

Background

Highly pathogenic avian influenza (HPAI) H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease.

Aim

To study influenza A (H5N1) virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease.

Methods

We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces.

Results

We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our data suggests that viremia, secondary to, for example, gastro-intestinal infection, can potentially lead to infection of the lung. HPAI H5N1 virus was a more potent inducer of cytokines (e.g. IP-10, RANTES, IL-6) in comparison to H1N1 virus in alveolar epithelial cells, and these virus-induced chemokines were secreted onto both the apical and basolateral aspects of the polarized alveolar epithelium.

Conclusion

The predilection of viruses for different routes of entry and egress from the infected cell is important in understanding the pathogenesis of influenza H5N1 infection and may help unravel the pathogenesis of human H5N1 disease.     

Australia's Dengue Risk Driven by Human Adaptation to Climate Change

Background

The reduced rainfall in southeast Australia has placed this region''s urban and rural communities on escalating water restrictions, with anthropogenic climate change forecasts suggesting that this drying trend will continue. To mitigate the stress this may place on domestic water supply, governments have encouraged the installation of large domestic water tanks in towns and cities throughout this region. These prospective stable mosquito larval sites create the possibility of the reintroduction of Ae. aegypti from Queensland, where it remains endemic, back into New South Wales and other populated centres in Australia, along with the associated emerging and re-emerging dengue risk if the virus was to be introduced.

Methodology/Principal Findings

Having collated the known distribution of Ae. aegypti in Australia, we built distributional models using a genetic algorithm to project Ae. aegypti''s distribution under today''s climate and under climate change scenarios for 2030 and 2050 and compared the outputs to published theoretical temperature limits. Incongruence identified between the models and theoretical temperature limits highlighted the difficulty of using point occurrence data to study a species whose distribution is mediated more by human activity than by climate. Synthesis of this data with dengue transmission climate limits in Australia derived from historical dengue epidemics suggested that a proliferation of domestic water storage tanks in Australia could result in another range expansion of Ae. aegypti which would present a risk of dengue transmission in most major cities during their warm summer months.

Conclusions/Significance

In the debate of the role climate change will play in the future range of dengue in Australia, we conclude that the increased risk of an Ae. aegypti range expansion in Australia would be due not directly to climate change but rather to human adaptation to the current and forecasted regional drying through the installation of large domestic water storing containers. The expansion of this efficient dengue vector presents both an emerging and re-emerging disease risk to Australia. Therefore, if the installation and maintenance of domestic water storage tanks is not tightly controlled, Ae. aegypti could expand its range again and cohabit with the majority of Australia''s population, presenting a high potential dengue transmission risk during our warm summers.     

QTL analyses for seed iron and zinc concentrations in an intra-genepool population of Andean common beans (Phaseolus vulgaris L.)

Legumes provide essential micronutrients that are found only in low amounts in the cereals or root crops. An ongoing project at CIAT has shown that the legume common bean is variable in the amount of seed minerals (iron, zinc, and other elements), vitamins, and sulfur amino acids that they contain and that these traits are likely to be inherited quantitatively. In this study we analyzed iron and zinc concentrations in an Andean recombinant inbred line (RIL) population of 100 lines derived from a cross between G21242, a Colombian cream-mottled climbing bean with high seed iron/zinc and G21078, an Argentinean cream seeded climbing bean with low seed iron/zinc. The population was planted across three environments; seed from each genotype was analyzed with two analytical methods, and quantitative trait loci (QTL) were detected using composite interval mapping and single-point analyses. A complete genetic map was created for the cross using a total of 74 microsatellite markers to anchor the map to previously published reference maps and 42 RAPD markers. In total, nine seed mineral QTL were identified on five linkage groups (LGs) with the most important being new loci on b02 and other QTL on b06, b08, and b07 near phaseolin. Seed weight QTL were associated with these on b02 and b08. These Andean-derived QTL are candidates for marker-assisted selection either in combination with QTL from the Mesoamerican genepool or with other QTL found in inter and intra-genepool crosses, and the genetic map can be used to anchor other intra-genepool studies.